TGF-β1 induces epithelial-mesenchymal transition but not myofibroblast transdifferentiation in primary cultures of human epithelial renal tubular cells

British Society for Matrix Biology Meeting, London,18–19 September 2003

The molecular basis of fibrosis

The autumn 2003 meeting of BSMB was held at Imperial

College, London, and had as its them e the molecular basis

of fibrosis, with a particular emphasis on the kidney. It was

also a special meeting held to mark the retirement of Professor

Roger Mason. The meeting was organized by Professor John

Couchman and Dr Graham Riley and was generously spon-

sored by the National Kidney Research Foundation, The Well-

come Trust and Imperial College. Additional support was also

provided by the following companies: Applied Biosystems,

Bio-Rad, BMG Laboratory Technologies, Camlab, Fisher,

Invitrogen, Jencons PLS, Kendro, Labtech International, Pro-

mega, Qiagen, Quidel, R & D Systems, VWR International

and Wiley publishing. There were 132 delegates registered for

the meeting, comprising 79 members of the society (includ-

ing 15 graduate students) and 53 nonmembers (including 16

graduate students). There were 17 invited speakers, with three

from the USA, five from the EU and five from the UK.

Jean Schwarzbauer (Princeton University, USA) opened the

meeting by describing her recent findings concerning the

events that control fibronectin (Fn) matrix assembly. The

assembly of Fn matrix fibrils is influenced by extracellular

factors such as availability of Fn and other matrix molecules,

and intracellular signalling pathways mediated by integrin

receptors. Jean’s group has investigated two kinases down-

stream of integrin receptors, focal adhesion kinase (FAK) and

pp60-Src. Mouse fibroblasts lacking FAK were found to

assemble reduced amounts of Fn fibrils. Src family kinases

phosphorylate FAK, and it was found that fibroblasts without

Src family kinases (SYF cells) or wild-type cells treated with an

Src inhibitor (PP1) also lack Fn matrix. The extracellular

influence of tenascin-C was also discussed. In fibroblasts on a

3D Fn matrix, FAK was constitutively phosphorylated,

whereas in the presence of tenascin-C, FAK is transiently

activated, and there is a reduction in the assembly of Fn matrix

fibrils. Other aspects of integrin–Fn interactions were also

described, including the stimulation of Fn matrix formation

by dexamethasone in the tumourgenic cell line HT1080.

Karl Kadler (University of Manchester) summarized

findings on the molecular and cellular basis of collagen fibril-

logenesis. The talk focussed on the sorting and secretion of

type-I collagen in organ cultures of embryonic chick tendon. It

was found that around 50% of newly synthesized type I pro-

collagen is cleaved to collagen type I inside the cell with

around 50% cleaved in the ECM. In a particularly striking

3D model created using serial section reconstructions and

immunoEM, it was shown that cross-striated nascent collagen

fibrils are enclosed within tube-like secretory vesicles within

cells and are secreted via plasma membrane protusions known

as nozzles. It was proposed that the nucleation phase of col-

lagen fibrillogenesis takes place in secretory vesicles, whilst the

propagation phase occurs after secretion of early fibres into

tunnel-shaped extracellular spaces through nozzles.

John Couchman (Imperial College, London) talked about

recent work with the basement membrane molecule entactin-

1, also known as nidogen-1. Entactin often originates from the

mesechymal compartment as opposed to other major com-

ponents that are synthesized by epithelia. The renal epithelial

cell line MDCK does not express entactin-1, and the effect of

recombinant entactin-1 expression in these cells was investi-

gated. MDCK cells expressing entactin-1 constitutively or

under tetracycline-responsive regulation (Ent-1 cells) adopt a

flattened phenotype as opposed to the normal, cuboidal phe-

notype and exhibit altered integrin expression profiles and

increased expression of Fn. These results indicate that entac-

tin-1 binding to aVb3 integrin can lead to Fn matrix assembly,

and that Fn ligation through aVb3 integrin promotes a

flattened phenotype with prominent microfilament bundles.

The relevance of this mechanism was discussed with respect

to carcinomas and epithelial–mesenchymal transitions.

Andrew Chantry (University of East Anglia, Norwich) dis-

cussed the regulation of TGF-b-/Smad-signalling pathways.

Their work focuses on decorin-mediated Ca2þ-dependent

phosphorylation and its regulation of Smads and TGF-b.

Their work shows that decorin disrupts the TGF-b-/Smad-

dependent transcriptional events in human mesangial cells

through phosphorylation of Smad-2 at serine-240 by Ca2þ

activation of Cam kinase II. Decorin induces serine-240 phos-

pho-Smad hetero-oligomerization with Smad-4 in the nucleus,

independent of TGF-b receptor activation. Part 2 of the talk

covered work on MMP/TIMP expression in Smad-3 and

Smad-4 knockout cells. The response of Smad-3 and Smad-4

knockout and wild-type cells to treatment with TGF-b was ana-

lysed. TIMP-1 was induced by TGF-b and is therefore a TGF-b-

dependent gene. TIMP-3 was completely Smad-dependent.

Int. J. Exp. Path. (2004), 85, A1–A44

� 2004 Blackwell Publishing Ltd A1

Wild-type cells behave differently between Smad-3 and Smad-

4. Adenoviral expression of Smad-4 in Smad-4 knockout cells

recovers TGF-b-induced ADAM-12 expression. This is preli-

minary data and the work is ongoing. Part 3 of the talk focused on

identification of novel therapeutic targets within the TGF-b path-

way. These include ubiquitin C-terminal hydrolases to deubiqui-

tinate or remove mono or poly ubiquitin side chains, targeted

ubiquitinatination by Smurfs leading to Smad degradation, and

Smad-3 interacts with ubiquitin-c-hydrolyase-37 (UCH37) and

effects the transcriptional response from a Smad-binding element

reporter. The findings may help development of novel approaches

in treatment of fibrotic diseases.

Nadia Wahab (Imperial College, London) discussed the role

of connective tissue growth factor (CTGF) in mediating the

pathogenesis of diabetic nephropathy. Chronic hyperglycae-

mia is the primary cause of the development of mesangial cell

dysfunction and diabetic nephropathy. CTGF levels are

increased markedly in the glomeruli of patients with diabetic

nephropathy and in experimental animal models. TGF-b is a

major fibrogenic growth factor implicated with the pathogen-

esis of renal scarring. Overexpression in transgenic mice leads

to sclerosis. CTGF expression enhances the phosphorylation

and nuclear transfer of activated TGF-b receptors Smad-2 and

Smad-3 following formation of a complex with S4. They

localize at the nucleus where they act in co-operation with

co-activators and co-receptors as transcription factors regulat-

ing gene expression. Smad-7 is an inhibitor of TGF-bsignalling. It binds to Smad-2 and Smad-3 and inhibits phos-

phorylation and therefore limits the effects of TGF-b. Work

carried out in this lab has found that CTGF suppresses expres-

sion of Smad-7 within 2 h of addition to mesangial cells. This

inhibition was found to be due to a CTGF-related increase in

TIEG protein levels. TIEG is a repressor of Smad-7 and there-

fore may allow continuous activation of TGF-b. These

findings suggest that CTGF mediates TGF-b-dependent

increase in expression of ECM molecules in mesangial cells

leading to fibrosis under diabetic conditions.

Liliana Schaefer (University of Muenster, Germany) dis-

cussed the anti-fibrotic properties of decorin and biglycan in

renal disease and how it is more than an inhibitor of TGF-b.

Decorin is involved in assembly of matrix suprastructures,

control of cell growth, differentiation and death, and it modu-

lates cytokine activities. Based on studies carried out in this

lab, two mechanisms for modulation of TGF-b-mediated fibro-

sis by decorin were described; (1) increased quantities of dec-

orin/TGF-b complexes are removed via glomerular capillaries

or the urinary tract and (2) along with collagen-I, decorin

forms complexes with TGF-b and can sequester it in the

ECM therefore withdrawing the TGF-b from its receptors.

Wild-type (þ/þ) and decorin knockout (–/–) mice models

with unilateral ureteral obstruction (UUO) were used to ana-

lyse decorin and urinary tract removal of decorin/TGF-b com-

plexes and their effect on development of tubulointerstitial

fibrosis. Decorin (–/–) UUO mice developed enhanced apopto-

sis prior to TGF-b up-regulation. Enhanced inflammation was

a consequence of TGF-b activity and there was an increased

rate of collagen-I degradation in decorin (–/–) UUO mice,

suggesting that decorin protects collagen fibrils against proteo-

lytic digestion. An increase in TGF-b leads to an increase in

biglycan after 7 days. Decorin (–/–) UUO kidneys were more

atropic than (þ/þ) kidneys. Biglycan (–/–) UUO mice were also

analysed. There was a loss of mechanical stability of renal

tissue in biglycan deficiency and a decrease of fibrillin 1 in the

glomerulus. Biglycan was found to protect the survival of

mesangial cells.

Josef Pfeilshifter (Klinikum der Johann Wolfgang Goehte-

Universitat, Germany) presented the post-transcriptional regu-

lation of matrix metalloproteinases (MMPs) through mRNA

destabilization in vitro. MMPs are important in the turnover or

ECM and are thus major players in many inflammatory pro-

cesses in the kidney. The process in which nitric oxide (NO)

inhibits the mRNA stabilizing ELAV-like protein HuR and

hence reduces the half-life of MMP-9 mRNA was described.

NO action is attributed to the 3´-untranslated region (UTR) of

MMP-9 mRNA. MMP-9 is synthesized and secreted in high

levels by glomerular mesenchymal cells (MCs) upon treatment

with proinflammatory cytokine interleukin (IL)-1b. Dr Pfeilshif-

ter described the switch mechanism of MMP-9 regulation by

NO and O2–. NO and O2

– differentially modulate cytokine-

induced MMP-9 glatinolytic content and steady state mRNA

levels, where NO attenuates MMP-9 expression and O2–

amplifies it as a response to cytokine induction. The O2– effect

occurs via the activation of different MAPK pathways. Future

work will concentrate on confirmation of these results in vivo.

Detlef Schlondorff (Ludwig Maximilians University,

Germany) presented the recently characterized expression of

chemokines and their receptive receptors during progressive

renal fibrosis after UUO in the mouse which is widely used as a

model for this disease. This is important because there is evidence

from human renal biopsy studies that the expression of some

chemokine receptors on the surface of infiltrating leucocytes is

important in the progression of renal fibrosis. The attempt to

block the CCR1 chemokine receptor using nonpeptide antago-

nist BX471 in order to analyse the effect on leucocyte infiltration

and thus on renal fibrosis after UUO was also described. UUO

kidneys from BX471-treated mice had 40–60% reduction in

interstitial macrophage and lymphocyte infiltrate compared to

controls as well as a marked reduction in CCR1 and CCR5

mRNA levels, of CD8þ/CCR5þ T cells, and of markers of

renal fibrosis such as mRNA and protein expression of

A2 British Society for Matrix Biology Meeting

� 2004 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 85, A1–A44

collagen-I, interstitial volume and interstitial fibroblasts. This

suggests a new therapeutic strategy for reducing cellular infiltra-

tion and renal fibrosis by blockade of chemokine receptors.

Jill Norman (Royal Free and University College Medical

School, London) presented evidence gathered from in vitro

and in vivo studies showing the role of hypoxia in the patho-

genesis and progression of renal scarring and its possible con-

tribution to the fibrotic process in other tissues. A known

mechanism for transmission of glomerular injury to the tubu-

lointerstitium is via protein/macromolecules that pass through

the compromised glomerular basement membrane. These

macromolecules induce tubular epithelial cell injury leading

to recruitment of inflammatory cells and interstitial cell acti-

vation. However, some diseases with heavy proteinuria do not

progress, suggesting the presence of other mechanisms of

transmission. The chronic hypoxia hypothesis described by

Dr Norman proposes endothelial cell injury leading to inter-

stitial hypoxia as an alternative mechanism for activation of

interstitial cells. Cortical microvascular pO2 was measured in

a rat model of progressive fibrosis and was shown to decline

with time of disease. This fall preceded histological accumula-

tion of ECM. In vitro, the effect of hypoxia was consistent

with increased accumulation of ECM. Collagen-I and TIMP-1

were used as representatives of changes in ECM synthesis and

turnover in order to investigate hypoxia-induced gene expres-

sion. Changes in expression of TIMP-1 and COL1A-1 were

shown to be independent of hypoxia-induced growth factors;

instead there is a direct transcriptional effect of low oxygen

levels on the two genes.

Professor Charles Pusey (Imperial College, London) opened

the last session of the day with a comprehensive talk on regula-

tion of inflammation and scarring in glomerulonephritis. He

presented results from studies using a model of crescentic glom-

erulonephritis in the Wistar-Kyoto (WKY) rat induced by the

administration of a rabbit anti-rat GBM antibody. The effects

of treatment with an anti-tumour necrosis factor-a (anti-TNF-

a) antibody and anti-inflammatory cytokines were determined.

If administered from day 0, anti-TNF-a antibody prevented

kidney disease, and if administered from day 4, it reduced

inflammation. The anti-inflammatory cytokines IL-4 and IL-11

were also shown to reduce inflammation. In addition, blockade

of the integrin very late antigen-4 (VLA-4) prevented acute

inflammation and also reduced scarring; however, blockade of

VLA-1 did not have the same effect on inflammation but did

reduce scarring. Altogether, his results indicated that anti-TNF-a

therapywas the most successful,whichwas furtherbacked-upbya

pilot study on 15 anti-neutrophil cytoplasmic antibody (ANCA)-

associated systemic vasculitis patients treated with infliximab.

Meguid El-Nahas (University of Sheffield, UK) outlined the

role of tissue transglutaminase in renal fibrosis. He gave an

overview of the transglutaminase gene family and the role

and function of tissue transglutaminase and highlighted its

involvement in scarring and fibrosis. Results presented

included light and confocal microscopy showing an increase

of tissue transglutaminase and its cross-link product exores-

sion in the renal interstitium and glomerular component of

diseased human kidneys. He also presented investigations into

the inhibition of tissue transglutaminase in a subtotally

nephrectomized (SNX) rat model and showed that inhibitors

injected in the rat kidney from 90 days reduced scarring and

fibrosis and preserved clearance of creatinine and therefore

kidney function.

George Bou-Gharios (Hammersmith Hospital, London)

gave a presentation on the promoter and enhancer sequences

of the type-I collagen gene col-I-a2(1). Type-I collagen is

expressed in different cell lineages of common origin, and

George presented work that investigated how its temporal

and spatial expression is determined. Data presented from

work using transgenic mice showed a far-upstream enhancer

which directs expression of collagen-I during development and

in response to tissue injury. Further analysis by deletion studies

looked at the minimal functional region required for the

enhancer effect. He showed regions that were identified as

needed for essential function of the enhancer and as tissue-

specific elements. Comments were also made on the different

rearrangements of the enhancer elements between human and

mouse species.

Kathleen Flanders (Wakayama Medical University, Japan)

discussed Smad-3 as a mediator of the fibrotic response. She

discussed her group’s work on radioprotection and protection

against tubulointerstitial fibrosis following UUO in Smad-3–/–

and Smad-3þ/þ mice. Smad-3–/– mice appear to be resistant to

ionizing radiation. The effect of radiation on Smad-3–/– and

Smad-3þ/þ UUO mice was analysed when the skin flank of

each mouse was exposed to 30 Gy of localized g-irradiation.

The group used histology and gene expression at time points

post irradiation to analyse this. Smad-3–/– mice showed less

inflammation in response to irradiation compared to Smad-

3þ/þ mice 6 weeks post irradiation. But Smad-3–/– did show

increased number of neutrophils compared to Smad-3þ/þ skin

at 6–8 h post irradiation. Smad-3–/– dermal fibroblasts did not

migrate but did differentiate in response to TGF-b. Also,

piciosirius red staining of the flank at 6 weeks post Gy irradi-

ation shows an increase in scarring in Smad-3þ/þ compared to

Smad-3–/– mice. Smadþ/þ but not Smad–/– fibroblasts showed

an increase in TGF-b and CTGF mRNA following irradiation

and TGF-b treatment. Primary culture systems of Smad-3þ/þ

renal tubular epithelial cells underwent phenotypic changes when

treated with TGF-b; this did not occur in Smad-3–/– mice. Smad-

3–/– mice maintain normal renal architecture and reverse ETM

British Society for Matrix Biology Meeting A3


14 days after UUO compared to Smad-3þ/þ mice, where the

kidneys are enlarged with an influx of mononuclear cells, collagen

and TGF-b.

Christos Chatziantoniou (Tenon Hospital, Paris, France)

described the mechanisms by which vasoconstrictors such as

angiotensin-II and endothelin induce renal vascular and glom-

erular fibrosis in experimental hypertension, by using trans-

genic mice expressing reporter genes controlled by the

collagen-I gene promoter. The talk was a summary of a num-

ber of papers from his laboratory. Angiotensin-II was found to

induce renal fibrosis via an endothelin-mediated activation of

the collagen-I gene. This fibrinogenic mechanism involves

activation of TGF-b and tyrosine kinase growth factor recep-

tors, and MAPK/ERK and Rho-signalling pathways. Protec-

tion of renal vasculature was observed during treatment with

the receptor antagonists AT1 or ETA/B, apparent in the inhib-

ition of collagen-I gene activation independently of systolic

blood pressure levels. To introduce a relevance to clinical

medicine, they then looked at regression (as opposed to pre-

vention) of renal fibrosis by treating animals with angiotensin

and/or endothelin after the occurrence of fibrotic lesions. A

regression was observed, which appeared to be due to inhibi-

tion of collagen-I synthesis and the activation of collagen-

degrading enzymes such as MMP-2 and MMP-9.

Michael Ryan (University College Dublin) presented emer-

ging evidence for the involvement of epithelial–mesenchymal

transdifferentiation (EMT) in tubuloinsterstitial fibrosis. EMT

is the loss of epithelial and the gain of mesenchymal character-

istics by cells. It is a normal physiological process in develop-

ment and wound healing, but it may be involved in tissue

fibrosis and in early stages of transformation, invasion and

metastatis of carcinoma cells in the adult. Dr Ryan described

the developed models of renal cell transdifferentiation whereby

epithelial tubular cells (HK-2 cells) were exposed to various

factors and indices of EMT were investigated. Suppression

substractive hybridization or Affymetrix gene microarrays

were used to analyse altered gene expression. TGF-b, CTGF,

HMGB-1, b-catenin translocation to nucleus, PKC-b and Ras

family of small GTPases were shown to have a possible involve-

ment in EMT. Understanding of EMT through the investigation

of the various molecular switches and their effectors may lead to

the development of therapeutic targets for renal fibrosis.

Frank Strutz (Georg-August University) presented the recent

findings of the potential role of integrin-linked kinase (ILK) in

the mediation of epithelial–mesenchymal transition. ILK,

which is stimulated by TGF-b1, mediates the effects of integ-

rins and their function in cell–matrix interactions. ILK sense

and anti-sense cDNA were overexpressed in murine tubular

epithelial cells. This resulted in the down-regulation of epithe-

lial markers and up-regulation of mesenchymal markers such

as FSP-1 and vimentin. Another observed effect was the dra-

matic increase in Fn sythesis and the stimulation of cell migra-

tion. On the other hand, a negative effect on apoptosis

was noted. Strutz also described the inhibitory effect of

BMP-7 on TGF-b1-mediated mesenchymalization. BMP-7

had a positive effect on E-cadherin expression and a negative

effect on Smad-mediated EMT. Moreover, BMP-7 reversed

interstitial fibrosis and tubular atrophy in various animal

models suggesting a novel therapeutic approach to chronic

progressive renal disease through the inhibition of EMT.

Aled Phillips (University of Wales College of Medicine,

Cardiff) discussed the regulation of proximal tubular cell

(PTC) phenotype and function by TGF-b. The group has

looked at how TGF-b effects cell–cell contact, cell phenotype,

migration and matrix degradation. By using light microscopy

on cultured PTC cell lines, TGF-b was shown to cause cells to

lose their epithelial phenotype and gain a fibroblastic pheno-

type, along with the loss of cell–cell contact, rearrangement of

the actin cytoskeleton, formation of stress fibres and focal

adhesions. Cytochalasin-D-mediated disruption of the actin

cytoskeleton prevented phenotypic changes associated with

the addition of TGF-b. TGF-b-mediated functional changes

showed an increase in collagen-III and collagen-IV. Transient

transfection with Smad-2/-4 or Smad-3/-4 expression vectors

showed that phenotypic changes are not Smad-related. Repla-

cement of TGF-b with 1% FCS reversed the phenotypic

change to epithelial cells, demonstrating that the cells were

not permanently differentiated. TGF-b-mediated phenotypic

changes, including a decrease in expression of E-cadherin

and cytokeratin and an increase in expression of a-SMA,

were augmented by interstitial collagens as shown when cells

were grown on tissue culture dishes coated with collagen-I and

collagen-III, where TGF-b had no such effect by itself. TGF-badded to cells grown on type-IV collagen had no greater effect

than TGF-b alone. In conclusion, a combination of the pro-

fibrotic cytokine TGF-b and exposure to certain interstitial

ECM components, after damage to the tubular basement mem-

brane, is involved in regulation of PTC phenotype and function.

Function follows form: how fibronectin matrixarchitecture controls cell behaviour

Jean E. SchwarzbauerDepartment of Molecular Biology, Princeton University,

Princeton, NJ, USA

Introduction Fibronectin (FN) matrix assembly is a tightly

regulated stepwise process that is initiated by interactions

between FN and cell-surface integrin receptors. Assembly is



affected by extracellular factors including availability of FN

and the presence of other matrix molecules. In turn, FN

matrix activates intracellular signalling pathways via integrin

receptors, and these signals regulate cell adhesion, migration

and survival. Two kinases immediately downstream of integ-

rins are focal adhesion kinase (FAK) and pp60-Src. Our

results show that activation of these kinases regulates accu-

mulation of FN matrix fibrils and that this process is affected

by tenascin-C, an ECM protein that modulates cell inter-

actions with FN.

Methods FN assembly was monitored by indirect immu-

nofluorescence and by immunoblotting of deoxycholate

(DOC)-insoluble matrix material with anti-FN antibodies.

Wild-type mouse fibroblasts and cells lacking FAKs or Src

family kinases (SYF cells) were used. Kinase and phosphatase

inhibitors were used to regulate enzyme activities.

Results Mouse fibroblasts lacking FAK assemble significantly

reduced amounts of FN fibrils and DOC-insoluble matrix. FAK is

phosphorylated by Src family kinases and fibroblasts lacking Src

family kinases (SYF cells) or cells treated with PP1, an inhibitor of

SYF kinases, also lack FN matrix. The effects of Src activity are

most dramatic during the early stages of de novo assembly by

fibroblasts implicating this kinase in the initiation process. While

FN stimulates FAK, tenascin-C, an ECM protein that is expressed

at sites of cell movement, has the opposite effect on FAK activity.

In fibroblasts on a 3D FN matrix, FAK is constitutively phos-

phorylated. In contrast, FAK is only transiently activated in the

presence of tenascin-C. Concomitant with the absence of FAK

phosphorylation is an inability to assemble FN matrix fibrils.

Conclusion Our results show that FAKs and Src kinases, which

lie downstream of integrins, are essential for efficient initiation of

FN matrix assembly. The effects of tenascin-C on FN matrix

and FAK phosphorylation suggest that this protein limits the

extent of matrix deposition by regulating FAK activity. Thus,

extracellular and intracellular events co-operate to control FN

matrix assembly.

References

Midwood K.S. & Schwarzbauer J.E. (2002) Tenascin-C modulates

matrix contraction via focal adhesion kinase- and Rho-mediated

signaling pathways. Mol. Biol. Cell 13, 3601–3613.

Wierzbicka-Patynowski I. & Schwarzbauer J.E. (2002) Regula-

tory role for Src PI3-kinase in initiation of fibronectin matrix

assembly. J. Biol. Chem. 277, 19703–19708

Wierzbicka-Patynowski I. & Schwarzbauer J.E. (2003) The

ins and outs of fibronectin matrix assembly. J. Cell Sci. 116,

3269–3276.

The ins and outs of extracellular matrix assembly

Karl E. KadlerWellcome Trust Centre for Cell-Matrix Research, University of

Manchester, Manchester, UK

Introduction To a rough approximation, the adult human

comprises 2–5 kg of cells. The remaining mass originates

from the extracellular matrix (ECM). The ECM is highly

organized, which is surprising considering the paucity of

cells and the fact that the ECM comprises some of the largest

and most insoluble macromolecules encoded by the genome.

The high level of supramolecular order of the ECM is parti-

cularly evident in tendon in which millimetre-long collagen

fibrils lie parallel to each other and to the tendon-long axis

(Canty & Kadler 2002). The work in our laboratory is

focused on understanding the molecular and cellular basis of

collagen fibrillogenesis. The work is directly relevant to under-

standing the secretion and assembly of large proteins, tissue

homeostasis and embryonic development, as well as under-

standing the aetiology of heritable and acquired diseases of

connective tissue including fibrosis.

Materials and methods A multidisciplinary approach is

being used including protein biochemistry, recombinant pro-

tein expression, immunofluorescence, immunoEM and serial

section reconstruction from electron micrographs.

Results Using pulse-chase in organ cultures of embryonic

chick tendon we show that approximately 50% of newly synthe-

sized type-I procollagen is secreted to the ECM where it is

cleaved to type-I collagen, and approximately 50% of the pro-

collagen is cleaved to collagen inside the cells. ImmunoEM

shows collagen in specialized secretory vesicles, which bud

from the trans-face of the Golgi apparatus. Serial section recon-

struction of E13 chick metatarsal tendon and E15.5 mouse tail

tendon shows cross-striated nascent collagen fibrils, which are

approximately 1mm in length and identical to collagen early

collagen fibrils (Graham et al. 2000), enclosed within tube-like



secretory vesicles. The nascent fibrils are secreted via nozzles

(plasma membrane protrusions) into tunnel-shaped spaces

between the cells. Bone morphogenetic protein-1 (ProBMP-1) is

cleaved to active BMP-1 in the TGN by dibasic convertases

(Graham et al. 2000) (e.g. furin) and thereby initiates the assem-

bly of collagen fibrils. Further studies have identified the minimal

structure of procollagen C-proteinase activity of BMP-1

(Leighton & Kadler 2003).

Discussion In vitro studies in the 1980s established that col-

lagen fibrillogenesis resembles a crystallization process, in that it

proceeds by distinct nucleation and propagation phases. How-

ever, there was no obvious mechanism for how the two phases

occurred separately in vivo. Our recent studies show that the

nucleation phase occurs in specialized secretory vesicles. The

early fibrils are deposited in the ECM via nozzles which are long

protrusions of the plasma membranes. The propagation phase for

fibril assembly occurs in the ECM. Our studies have direct rele-

vance to the assembly of other matrix polymers, e.g. fibrillin and

type-VI collagen, as well as understanding the molecular basis of

ECM assembly in diseases such as fibrosis and wound healing.

References

Canty E.G. & Kadler K.E. (2002) Collagen fibril biosynthesis in

tendon: a review and recent insights. Comp. Biochem. Physiol.

Part A 133 (4), 979–985.

Graham H.K. et al. (2000) Identification of collagen fibril fusion

during vertebrate tendon morphogenesis. The process relies on

unipolar fibrils and is regulated by collagen–proteoglycan

interaction. J. Mol. Biol. 295, 891–902.

Hartigan N. et al. (2003) J. Biol. Chem. 278, 18045–18049.

Leighton M. & Kadler K.E. (2003) Paired basic/furin-like

proprotein convertase cleavage of proBMP-1 in the trans-Golgi

network. J. Biol. Chem. 278, 18478–18484.

Basement membrane and tubular epithelial cellbehaviour

John R. Couchman,* Yashi Mahalingam* and

Anna C. Erickson†

*Division of Biomedical Sciences, Imperial College London,

London, UK; †Lawrence Berkeley Laboratory, University of

California, Berkeley, CA, USA

Basement membranes are known to be important regulators of

epithelial behaviour and differentiation, and many biological

properties have been ascribed to their major components. The

major components include a number of laminins, type-IV col-

lagen, perlecan and agrin. However, all basement membranes

contain entactin-1 and/or entactin-2 (also known as nidogen-1

and nidogen-2), about which rather less is known. It is recog-

nized, however, that the mesenchymal compartment is often the

source of basement membrane entactin, unlike many of the

other major components that are synthesized by epithelia. The

well characterized renal epithelial cell line, MDCK is no excep-

tion, expressing neither entactin-1 or entactin-2. In experiments

to determine whether basement membrane assembly in this cell

line could be enhanced by recombinant expression of entactin-1

in these cells, some surprising findings resulted.

MDCK expressing entactin-1 constitutively or under

tetracycline-responsive regulation (Ent-1 cells) adopted a dis-

tinct morphology, being flattened, and with large number of

microfilament bundles on the basal side of the cell. Control

cells adopted a normal epithelial, more cuboidal phenotype,

with circumferential microfilament bundles. Tight junction

assembly appeared unaffected. Instead of enhanced basement

membrane assembly, Ent-1 cells showed both decreased base-

ment membrane matrix accumulation and enhanced fibronectin

expression at mRNA and protein levels. Integrin receptor

profiles measured by FACS analysis were altered, Ent-1 cells

having decreased levels of b1-, b4-, a6- and a3-integrin subu-

nits, while the a2-subunit pairing with b1 to provide the major

collagen receptor was unchanged. The distribution of many

integrin subunits was also altered. Others have reported that

aVb3 and a3b1 integrins can serve as receptors for entactin-1,

and consistent with our findings, it has been reported that

entactin-1 binding to a3b1 integrin can lead to fibronectin

matrix assembly, perhaps through a direct interaction between

entactin-1 and fibronectin. In addition, adhesion of MDCK cells

to entactin-1 was mediated by a3b1 integrin, with no apparent

role for a6-integrins. Our experiments with cell adhesion assays

and recombinant aVb3 suggested a major role for this integrin

in MDCK cell interactions with fibronectin, as expected, but no

clear role in adhesion to full-length entactin-1.

It therefore appears that MDCK cell expression of entactin-

1 is followed by binding to a3b1 integrin, which then pro-

motes fibronectin matrix assembly. Our hypothesis is that

entactin-1 signals through a3b1 integrin, to regulate fibronec-

tin expression at the transcriptional level. Fibronectin ligation

by aVb3 integrin promotes the flattened phenotype with

prominent microfilament bundles. Under these circumstances,

it appears that the important role of a3b1 integrin in basement

membrane assembly is diverted. This may be relevant not only

to some carcinomas where epithelial entactin-1 expression has

been observed, but also to epithelial–mesenchymal transitions

that occur in kidney development and extracellular matrix

deposition in fibrosis and scarring disease.



Novel mechanisms for regulating the TGF-b-/Smad-signalling pathway

Stephen Wicks, Katherine Haros, Marjorie

Maillard and Andrew ChantrySchool of Biological Sciences, University of East Anglia, Norwich,

UK

Introduction Aberrant transforming growth factor-b (TGF-

b) signalling is responsible for a number of developmental

disorders, human cancers and fibrotic disease. Signalling is

accomplished via cell-surface serine/threonine kinase receptors

and intracellular Smad transcription factors. A key regulatory

step in the pathway involves phosphorylation of Smads by

Ca2þ-dependent kinases such as Cam kinase II (Wicks et al.

2000; Abdel-Wahab et al. 2001). In addition, specific ubiqui-

tination by Smurfs and other ubiquitin ligases mediates the

proteosomal degradation of Smads. The aims of this study

were to functionally analyse the regulation of Smads by phos-

phorylation in response to decorin-mediated Ca2þ mobiliza-

tion and to use modified yeast-2 hybrid systems for the

identification of novel Smad regulatory proteins.

Methods Phosphorylation sites in Smad-2 were mapped in vitro

using Smad-2–GST fusion proteins incubated with purified kinases

in the presence of 32P-g-ATP. Sites were confirmed by site-directed

mutagenesis, and phosphopeptide-specific antisera were used to

study in vivo phosphorylation in decorin-treated human mesangial

cells. Yeast-2 hybrid screening was performed using Smad31�240 as

bait and a mouse brain cDNA library. Interactions were then

verified by co-expression and co-immunoprecipitation of epitope-

tagged proteins in HEK-293 cells.

Results In this study, we provide evidence that decorin, a

naturally occurring inhibitor of the TGF-b-dependent fibrotic

response, can disrupt glucose- and TGF-b/Smad-dependent

transcriptional events in human mesangial cells through a

mechanism that involves an increase in Ca2þ signalling, the

activation of Cam kinase II and ensuing phosphorylation of

Smad-2 at serine-240. We show that decorin also induces

serine-240 phospho-Smad hetero-oligomerization with Smad-

4 and the nuclear localization of this complex, independently

of TGF-b receptor activation. Thus, in human mesangial cells,

the mechanism of decorin-mediated inhibition of TGF-b sig-

nalling may involve activation of Ca2þ signalling, the subse-

quent phosphorylation of Smad-2 at a key regulatory site and

the sequestration of Smad-4 in the nucleus. In other studies, we

have used two-hybrid screening approaches to identify Smad-

specific regulatory proteins. Data will be presented on a novel

interaction between Smad-3 and a ubiquitin c-termninal

hydrolase that is likely to compete with Smurf-mediated ubi-

quitination and subsequent Smad turnover.

Conclusions In summary, we conclude that an important

mechanism by which decorin can play a functional role in regulat-

ing TGF-b signalling is through decorin-induced Ca2þ signalling

and subsequent inhibition of the Smad pathway. In addition, we

have identified a novel interaction that occurs between the Smad-3

transcription factor and a ubiquitin C-terminal hydrolase that

could lead to stabilization of the Smad-3 protein and potentiation

of TGF-b signalling by reversal of ubiquitin-mediated proteosomal

degradation. Overall, our present findings may lead to the devel-

opment of new approaches in the treatment of fibrotic diseases.

References

Abdel-Wahab N. et al. (2001) Decorin suppresses TGFb-induced

plasminogen activator inhibitor-1 in human mesangial cells

through a mechanism that involves Ca2þ-dependent phosphor-

ylation of Smad-2 at serine-240. Biochem. J. 362, 643–649.

Wicks S.J. et al. (2000) Inactivation of smad-transforming growth

factor beta signaling by Ca2þ-calmodulin-dependent protein

kinase II. Mol. Cell Biol. 20, 8103–8111.

The role of CTGF in mediating the pathogenesisof diabetic nephropathy

Nadia WahabDepartment of Cell and Molecular Biology, Imperial College

School of Medicine, London, UK

Diabetes is the most common cause of end-stage renal disease

throughout the developed world. The complications rather

than the primary disease seem to largely determine the quality

of life of patients with diabetes. Diabetic nephropathy (DN)

leading to renal failure is one common complication. The

disease is characterized by glomerular basement membrane

thickening and expansion of the mesangium due to the accu-

mulation of extracellular matrix (ECM) proteins secreted by

mesangial cells (MCs). However, the molecular mechanisms

mediating dysfunction of MCs and thus the pathogenesis of

this disease are not fully understood. Connective tissue growth

factor (CTGF; CCN2) expression increases markedly in the

glomeruli of patients with DN and in diabetic glomerulo-

sclerosis in experimental animals. CTGF is rapidly induced



by and appears to mediate at least some of the fibrogenic

actions of TGF-b. However, the molecular mechanisms by

which CTGF functions are not elucidated.

Smad proteins are the main signalling transducers down-

stream from TGF-b receptors. Upon activation of the TGF-breceptors, Smad-2 and Smad-3 are phosphorylated and form a

complex with Smad-4. Smad complexes then translocate into

the nucleus where they, in co-operation with co-activators and

co-repressors, act as transcription factors regulating gene

expression. Smad-7 is an intracellular antagonist for TGF-bsignalling. It associates with activated TGF-b receptors and

hinders the activation of Smad-2 and Smad-3. Thus the expres-

sion level of Smad-7 is a major determinant for TGF-btranscriptional responsiveness. Moreover, Smad-7 is rapidly

induced by TGF-b family members and provides a negative

feedback loop to control TGF-b activity.

We have found that CTGF rapidly suppresses the expression

of Smad-7 to an undetectable level within 2 h of the addition

of CTGF to MCs. This CTGF-dependent Smad-7 inhibition is

mainly due the transactivation of the TIEG transcription fac-

tor which is known to bind to a specific element in the Smad-7

promoter and suppress its transcription. These findings suggest

that the marked CTGF-dependent decrease in Smad-7 expres-

sion will, undoubtedly, exaggerate the magnitude of TGF-bresponses in cells.

In summary, our data indicate that TGF-b and CTGF work

in concert to promote the pathogenesis of fibrosis.

Decorin in renal inflammation and fibrosis –more than an inhibitor of TGF-bLiliana SchaeferDepartment of Internal Medicine, University of Muenster, Muenster,

Germany

Introduction Decorin, a small leucine-rich proteoglycan,

participates in extracellular matrix assembly and influences

cell behaviour by interacting with signalling membrane

receptors and TGF-b. Treatment with decorin has been

shown to have beneficial effects in an acute model of

mesangioproliferative glomerulonephritis due to interac-

tion with TGF-b. The underlying mechanisms, however,

remain unclear because upon complex formation with

TGF-b, the cytokine’s activity may become increased,

decreased or not influenced at all. Hence, the objectives of

the present study were twofold: firstly, to provide evidence

that decorin influences the course and final outcome of

renal inflammation and secondly, to find anti-fibrotic

mechanisms of decorin both related and unrelated to the

regulation of TGF-b activity.

Results Based on our studies in human diabetic nephropa-

thy, we postulate two regulatory mechanisms by which dec-

orin modulates TGF-b-mediated fibrosis: (1) increased

quantities of glomerular decorin are synthesized and form

complexes with TGF-b, which then are removed via glomer-

ular capillaries or the urinary tract, and (2) in the presence of

type-I collagen, decorin is able to sequester TGF-b in the

extracellular matrix, thereby withdrawing the cytokine

from its cell-surface receptors. Furthermore, we have com-

pared the evolution of tubulointerstitial fibrosis in wild-type

(WT) and decorin–/– mice in a model of unilateral ureteral

obstruction. Without obstruction, kidneys from decorin–/–

mice did not differ in any aspect from their WT counterparts.

However, already 12 h after obstruction, decorin–/– animals

showed lower levels of p27KIP1 and soon thereafter a more

pronounced up-regulation and activation of initiator and

effector caspases, followed by enhanced apoptosis of tubular

epithelial cells. At later stages, a higher increase of TGF-b1

became apparent. After 7 days, there was a 15-fold transient

up-regulation of the related proteoglycan biglycan, which

was mainly caused by the appearance of biglycan-expressing

mononuclear cells. Other small proteoglycans showed no

similar response. Owing to enhanced degradation of type-I

collagen and increased tubular epithelial cell apoptosis, end-

stage kidneys from decorin–/– animals were more atrophic

than WT kidneys.

Conclusion These data suggest that decorin exerts beneficial

effects on renal inflammation, primarily by influencing the

expression of a key cyclin-dependent kinase inhibitor, thereby

limiting the degree of apoptosis and tubular atrophy. In later

stages, anti-fibrotic effects of decorin are based on the regula-

tion of TGF-b1 expression, mononuclear cell infiltration and

collagen turnover.

References

Schaefer L. et al. (2001) Small proteoglycans in human diabetic

nephropathy: Discrepancy between glomerular expression and

protein accumulation of decorin, biglycan, lumican and

fibromodulin. FASEB J. 15, 559–561.

Schaefer L. et al. (2002) Absence of decorin adversely influences

tubulointerstitial fibrosis of the obstructed kidney by enhanced

apoptosis and increased inflammatory reaction. Am. J. Pathol.

160, 1181–1191.



Matrix-metabolizing enzymes as prime targetsof redox regulation by nitric oxide

Josef Pfeilschifter, El Sayed Akool and Wolfgang

EberhardtPharmazentrum Frankfurt, Klinikum der Johann Wolfgang

Goehte-Universitat Frankfurt, Frankfurt, Germany

Introduction Dysregulation of extracellular matrix (ECM)

turnover is an important feature of many inflammatory processes

in the kidney. Rat mesangial cells (MCs) express high levels of

inducible nitric oxide synthase (iNOS) and matrix metalloprotei-

nase-9 (MMP-9) in response to interleukin-1b (IL-1b) (Pfeilschif-

ter & Schwarzenbach 1990; Eberhardt et al. 2000a). The aim of

our studies was to elucidate the mechanisms of NO-dependent

modulation of MMP-9 expression.

Methods The expression level of MMP-9 was detected by

zymography and by Northern blot analysis, respectively. The

influence of NO on mRNA stability was measured by use of

actinomycin-D and by Luciferase reporter gene assays using

heterologous MMP-9 reporter gene constructs. The effects of

NO on MMP-9 transcripts were monitored by in vitro degra-

dation assays and RNA-binding activity determined by elec-

trophoretic mobility shift assays (EMSA).

Results MMP-9 (gelatinase-B) is a metalloproteinase that is

synthesized and secreted in high levels by the glomerular MCs

upon treatment with the proinflammatory cytokine IL-1bmainly via an increase in MMP-9 transcription (Eberhardt

et al. 2000a). The increase in MMP-9 expression by cytokines

is partially due to generation of superoxide and involves the

activation of different MAPK pathways (Eberhardt et al.

2000b). Furthermore, we demonstrate that NO substantially

inhibits the cytokine-induced expression of MMP-9 without

affecting MMP-9 enzyme activity (Eberhardt et al. 2000a). By

using actinomycin-D, we found that NO reduces the half-life

of MMP-9 mRNA (Akool et al. 2003). The reduction of

mRNA stability is attributable to the 3´-untranslated region

of MMP-9 since introduction of this region confered a negative

NO responsiveness to an otherwise nonresponding heterolo-

gous MMP-9 reporter luciferase gene (Akool et al. 2003). By

EMSA, we found that cytoplasmic fractions display the con-

situtive RNA binding of complexes containing the ELAV-like

protein HuR (HuA). Moreover, the RNA binding to three

putative AU-rich elements (AREs) is strongly attenuated by

different NO donors (Akool et al. 2003). Correspondingly,

the decay of MMP-9 transcripts was accelerated by cytoplas-

mic extracts from NO-treated MCs as shown by in vitro

degradation assay (Akool et al. 2003).

Conclusion NO negatively regulates cytokine-induced

MMP-9 expression by a post-transcriptional mechanism

which involves inhibition of the mRNA stabilizing ELAV-

like protein HuR.

References

Akool E.S. et al. (2003) Nitric oxide increases the decay of

MMP-9 mRNA by inhibiting the expression of mRNA-

stabilizing factor HuR. Mol. Cell Biol. 23, 4901–4916.

Eberhardt W. et al. (2000a) Nitric oxide modulates expression

of matrix metalloproteinase-9 in rat mesangial cells. Kidney

Int. 57, 59–69.

Eberhardt W. et al. (2000b) Amplification of IL-1b-induced

MMP-9 expression by superoxide in rat glomerular mesangial

cells is mediated by increased activities of NF-kB and AP-1 and

involves activation of the mitogen-activated protein kinase

pathways. J. Immunol. 165, 5788–5797.

Pfeilschifter J. & Schwarzenbach H. (1990) Interleukin 1 and

tumor necrosis factor stimulate cGMP formation in rat renal

mesangial cells. FEBS Lett. 273, 185–187.

The role of chemokines in renal fibrosis

Detlef SchlondorffLudwig Maximilians University, Munich, Germany

In the kidney, tubulointerstitial fibrosis is the main predictor

for the progression to end-stage renal disease (Becker &

Hewitson 2000). Renal fibrosis is characterized by a mixed

tubulointerstitial leucocytic cell infiltrate, fibroblast prolifera-

tion, increased matrix production leading to tubular cell

necrosis, and apoptosis (Zeisberg M. et al. 2001). During

this process, infiltrating macrophages and lymphocytes are a

major source of inflammatory mediators such as cytokines,

nitric oxide and growth factors. Inhibition of leucocyte

infiltration may reduce production of such mediators and

may therefore be an option to halt progressive renal fibrosis

and to prevent or to delay end-stage renal disease.

The leucocytic cell infiltrate is triggered by locally secreted

chemokines (Zlotnik & Yoshie 2000). There is increasing

evidence from human renal biopsy studies that the expression



of certain chemokine receptors on the surface of leucocytes

infiltrating the tubulointerstitium plays an important role in

the progression of renal disease. Unilateral ureteral obstruc-

tion (UUO) is a widely used model for progressive renal fibro-

sis that is independent of hypertension or systemic immune

disease. We have recently characterized the expression of

chemokines and their respective receptors during pro-

gressive renal fibrosis after UUO in the mouse (Vielhauer

et al. 2001).

Based on these data, we hypothetized that blocking the

chemokine receptor CCR1 using the nonpeptide antagonist

BX471 could reduce leucocyte infiltration and renal fibrosis

after UUO. Pharmacokinetics in vivo and effectiveness of

BX471 in vitro were first established. UUO kidneys from

BX471-treated mice (day 0–10 and day 6–10) revealed a

40–60% reduction of interstitial macrophage and lymphocyte

infiltrate compared to controls. A marked reduction of mRNA

for CCR1 and CCR5 in UUO kidneys was noted of

BX471-treated mice, and FACS analysis showed a comparable

reduction of CD8þ/CCR5þ T cells. Markers of renal fibrosis

such as interstitial fibroblasts, interstitial volume, mRNA and

protein expression for collagen-I were all significantly reduced

by BX471 treatment compared to vehicle controls. By con-

trast, treatment from day 0–5 only was ineffective (Anders

et al. 2002).

In summary, blockade of CCR1 substantially reduces cell

accumulation and renal fibrosis after UUO. Most interestingly,

late onset of treatment is also effective. We therefore conclude

that CCR1 blockade may represent a new therapeutic strategy

for reducing cellular infiltration and renal fibrosis as major

factors in the progression to end-stage renal failure.

References

Anders H.-J. et al. (2002) A chemokine receptor CCR-1

antagonist reduces renal fibrosis after unilateral ureter ligation.

J. Clin. Invest. 109, 251–259.

Becker G.J. & Hewitson T.D. (2000) The role of tubulointerstitial

injury in chronic renal failure. Curr. Opin. Nephrol. Hyper-

tens. 9, 133–138.

Vielhauer V. et al. (2001) Obstructive nephropathy in the mouse:

progressive fibrosis correlates with tubulointerstitial chemokine

expression and accumulation of CC-chemokine receptor-2

and – 5 positive leukocytes. J. Am. Soc. Nephrol. 12,

1173–1187.

Zeisberg M. et al. (2001) Renal fibrosis: an update. Curr. Opin.

Nephrol. Hypertens. 10, 315–320.

Zlotnik A. & Yoshie O. (2000) Chemokines: a new classification

system and their role in immunity. Immunity 12, 121–127.

The role of hypoxia in fibrosis

Jill NormanCenter for Nephrology, Royal Free and University College

Medical School, London, UK

Although many fibrotic renal diseases are glomerular in origin, it

is tubulointerstitial involvement that best predicts the onset of

progressive scarring. This raises the question of how glomerular

injury is transmitted to the tubulointerstitium. One mechanism

is via proteins/macromolecules that pass through the comprom-

ised glomerular basement membrane to induce tubular epithe-

lial cell injury with subsequent recruitment of inflammatory cells

and activation of interstitial cells. However, some diseases with

low proteinuria progress while others with heavy proteinuria do

not, suggesting there must be other mechanisms. An alternative

route to tubulointerstitial involvement may be via endothelial

cell injury, leading to interstitial hypoxia which could activate

interstitial cells, an idea that was formalized as The Chronic

Hypoxia Hypothesis. To substantiate this hypothesis, it was

necessary to demonstrate that (1) hypoxia occurs in progressive

scarring in vivo, and (2) hypoxia can induce fibrogenic changes

in interstitial fibroblasts, which in fibrosis undergo proliferation,

differentiation to a myofibroblastic phenotype [characterized by

expression of a-smooth muscle actin (SMA)] and accumulate

extracellular matrix (ECM). In vivo evidence that hypoxia

occurs in progressive renal scarring was obtained from measure-

ments of cortical microvascular pO2 in a rat model of progres-

sive fibrosis which showed that microvascular pO2 declined

with time of disease with the initial fall preceding histological

accumulation of ECM. In vitro, hypoxia (1% O2) stimulated

renal fibroblast proliferation, promoted myofibroblast differen-

tiation and increased expression of mRNAs for ECM proteins

(collagen-I, collagen-III and fibronectin), TIMP-1 and TIMP-3

but suppressed levels of MMP-1 mRNA, consistent with

increased accumulation of ECM. Investigations into the

mechanisms of hypoxia-induced gene expression focused on

collagen-I (COL1A1) and TIMP-1 as representative of changes

in ECM synthesis and turnover. Although hypoxia up-regulated

expression of a number of growth factors including TGF-b,

neutralizing antibody and conditioned medium transfer experi-

ments indicated that changes in expression of TIMP-1 and

COL1A1 were independent of hypoxia-induced growth factors

and suggested a direct transcriptional effect of low oxygen on

the two genes. In transient transfections, both the TIMP-1 and

COL1A1 promoters were hypoxia-inducible. Many hypoxia-

inducible genes are regulated by binding of hypoxia-inducible

factor-1 (HIF-1) to hypoxia response elements (HRE). Deletion

analysis of the TIMP-1 promoter identified an HRE within the



proximal promoter. Mutation of the HIF-1 consensus-binding

site in the HRE abrogated the hypoxic induction of the promo-

ter, suggesting hypoxic regulation of this gene is mediated by

HIF-1. In contrast, although the COL1A1 promoter contained a

cluster of putative HIF-1-binding sites, COL1A1 promoter

activity was found to be HIF-independent occurring via an

HRE between �220 and þ115. DNAse-I footprinting revealed

increased binding of proteins to the COL1A1 promoter in

hypoxia. A combined Southwestern-Proteomics approach was

used to identify these proteins amongst which Sp1 showed the

largest (7.3-fold) increase in binding in hypoxia vs. normoxia.

Sequence scanning revealed three potential Sp1-binding sites in

the proximal COL1A1 promoter (�123/�114, �93/�84 and

�64/�54). EMSAs and antibody supershifts demonstrated

increased binding to all three sites in hypoxia and identified

Sp1 in the DNA–protein complexes. Exposure of cells to mithra-

mycin which inhibits Sp1 binding to DNA abolished the

hypoxic induction of the COL1A1 promoter. Mutation of the

Sp1 sites revealed a dominant role for the �123/�114 site in

both basal and inducible COL1A1 promoter activity. Hypoxia

had no effect on Sp1 mRNA levels but increased Sp1 protein and

phosphorylation. These data identify Sp1 as a critical mediator

of hypoxia-induced COL1A1 transcription. Taken together, in

vivo and in vitro studies suggest an important role for hypoxia in

the pathogenesis of progressive renal scarring potentially setting

up a self-perpetuating process of tissue destruction. Hypoxia

may also be an important contributor to the fibrotic process in

other organs.

Models and mechanisms of renal epithelial–mesenchymal transdifferentiation

T. Mcmorrow, J. Lynch, E. Campbell, C. Slattery

and M.P. RyanDepartment of Pharmacology, Conway Institute of Biomolecular

and Biomedical Research, University College Dublin, Belfield,

Dublin, Ireland

Epithelial–mesenchymal transdifferentiation (EMT) is defined

as the loss of epithelial and the gain of mesenchymal charac-

teristics by cells. It is a normal physiological process in devel-

opment and wound healing. However, in the adult, EMT may

be involved in tissue fibrosis and in the early stages of trans-

formation, invasion and metastasis of carcinoma cells. Tubu-

lointerstitial fibrosis is a common feature of inflammatory

diseases leading to progressive renal disease. Renal fibrosis is

characterized by interstitial fibroblast activation that is

believed to play a central role in the pathogenesis of renal

intestitial fibrosis. Although the exact origin of these myofibo-

blasts remains uncertain, emerging evidence suggests that they

be derived from tubular epithelial cells by an EMT process.

We have developed in our laboratory a number of models of

renal cell transdifferentiation whereby human renal epithelial

tubular cells (HK-2 cells) transdifferentiate into mesenchymal

or fibroblast-like cells following exposure to either (1) super-

natant from activated peripheral blood mononuclear cells, (2)

transforming growth factor-b (TGF-b) or (3) cyclosporine A.

Indices of EMT, which were investigated included (1) cell

morphology, (2) cell migratory ability, (3) fibronectin produc-

tion, (4) a-smooth muscle actin expression, (5) loss of E-cad-

herin and (6) b-catenin translocation to the nucleus. Possible

intracellular signalling mechanisms in these models were

investigated. Altered gene expression was analysed by either

suppression substractive hybridization or Affymetrix gene

microarrays.

Evidence from these models indicate that the following may

play a role in EMT: (1) TGF-b, (2) connective tissue growth

factor (CTGF), (3) high mobility group protein (HMGB-1), (4)

b-catenin translocation to the nucleus, (5) protein kinase C-b(PKC-b) and (6) Ras family of small GTPases.

The investigation of the complex regulation of molecular

switches and their effectors may lead to a better understanding

of the process of EMT and therefore ultimately the develop-

ment of novel therapeutic targets for renal fibrosis.

Regulation of epithelial–mesenchymaltransformation in vitro

F. StrutzDepartment of Nephrology and Rheumatology, Georg-August

University Gottingen, Gottingen, Germany

Epithelial–mesenchymal transformation is a critical event for

the recruitment of renal fibroblasts in renal fibrogenesis. We

have demonstrated that cytokines such as TGF-b1 or FGF-2

may induce this process in vitro [Kidney Int 61 (5), 2002].

Moreover, our group did demonstrate the influence of the

surrounding extracellular matrix composition on the cellular

differentiation state [Am J Pathol 159 (4), 2001]. Whereas the

tubular basement membrane did have a stabilizing function for

tubular epithelial cells, disruption of the tubular basement mem-

brane and/or cultivation of tubular epithelial cells on interstitial

collagens resulted in the acquisition of a mesenchymal phenotype.

Recently, we have demonstrated the potential role of

integrin-linked kinase (ILK) in the mediation of epithelial–

mesenchymal transition. Integrins are essentially involved in

cell–matrix interaction and its effects are mediated by ILK

which is also stimulated by TGF-b1. We overexpressed ILK



sense and anti-sense cDNA in two murine tubular epithelial

cells by stable transfection. We analysed the effects of ILK

overexpression on cellular differentiation markers, matrix

synthesis, secretion of matrix metalloproteinases, proliferation

and apoptosis. Stable transfections were confirmed by AKT

and b-catenin measurements as well as b-catenin nuclear

localization (by confocal microscopy). Overexpression of

ILK resulted in robust down-regulation of epithelial markers

and an up-regulation of mesenchymal markers vimentin and

FSP-1. ILK-overexpressing tubular epithelial cells resulted in a

robust increase of fibronectin synthesis and secretion. Migra-

tion was stimulated by a factor of 2.3±0.2 as determined

by migration assay. There was only a minor effect on prolifer-

ation; however, apoptosis rate was reduced considerably.

We conclude that integrin-linked kinase plays an integral

part in epithelial–mesenchymal transformation. Finally, we

have found very strong inhibitory effects of bone morpho-

genetic protein (BMP)-7 on TGF-b1-mediated mesenchymali-

zation (Nat Med 2003). BMP-7 reintroduced E-Cadherin

expression, one of the master genes of epithelial cells, and

prevented Smad-mediated epithelial–mesenchymal transfor-

mation. In addition, addition of BMP-7 was able to reverse

interstitial fibrosis and tubular atrophy in a number of animal

models indicating that the mechanisms of EMT and its inhibi-

tion may provide a novel therapeutic approach to chronic

progressive renal disease.

Regulation of PTC phenotype and function byTGF-b1: implications for transdifferentiation

Ya-Chung Tian and Aled PhillipsInstitute of Nephrology, University of Wales College of Medicine,

Cardiff, Wales, UK

Introduction There is now increasing evidence that proximal

tubular cells (PTCs) contribute to renal interstitial fibrosis by

alteration of matrix turnover and by the generation of pro-fibrotic

cytokines such as TGF-b1. Recent studies suggest that, through a

process of transdifferentiation, the PTCs are one source of the

interstitial myofibroblasts that directly drive the fibrotic process.

The aim of this work was to examine the role and mechanism by

which TGF-b1 may regulate PTC phenotype and function.

Methods Experiments were performed using both primary-

cultures of PTC and the human PTC cell line HK2. All experi-

ments were performed on growth-arrested cells in the absence

of serum.

Results TGF-b1 altered cell phenotype, assessed by light

microscopy, with cells appearing elongated and spindle-

shaped. This was associated with loss of cell–cell contact

and rearrangement of the actin cytoskeleton, increased for-

mation of stress fibres and focal adhesions. Disruption of the

actin cytoskeleton with cytochalasin-D prevented phenotypic

alterations following addition of TGF-b1. Transient transfec-

tion with Smad-2/-4 or Smad-3/-4 expression vectors did

not alter cell phenotype. Previously, we have demonstrated

b-catenin translocation to PTC nuclei and its association with

Smad proteins following addition of TGF-b1, suggesting the

possibility that TGF-b1 may modulate Wnt signalling. Wnt-

responsive Xtwn-reporter construct was, however, silent in

response to TGF-b1. Similarly, a second Wnt-/LEF-1-regulated

element Toplflash, which does not contain Smad-binding sites,

was insensitive to TGF-b1 signalling. In contrast, phenotypic

changes in response to TGF-b1 were abrogated by inhibitors of

the RhoA downstream target ROCK, which also prevented loss

of cell–cell contact and adherens junction disassembly. Removal

of TGF-b1 and addition of 1% FCS, however, reverted cell

phenotype to a typical cobblestone epitheliod appearance, sug-

gesting that TGF-b1 did not result in terminal PTC transdiffer-

entiation. Cells grown on tissue culture dishes coated with either

type-I or type-III collagen also acquired an elongated fibroblastic

phenotype; this effect was exaggerated by the addition of TGF-

b1. In contrast to the cells stimulated with TGF-b1 alone, fol-

lowing stimulation by both TGF-b1 and exposure to interstitial

collagens, cell phenotype was stable in that it was not reversed

upon removal of TGF-b1 and addition of FCS. Addition of TGF-

b1 to cells grown on type-IV collagen had no greater effect than

TGF-b1 alone. Addition of TGF-b1 alone had little effect on the

expression of a-SMA. In contrast, cells grown on either type-I or

type-III collagen, following addition of TGF-b1, demonstrated

marked increased expression of a-SMA, which appeared to be

incorporated into the cell cytoskeleton. Similarly, the combina-

tion of interstitial collagen (either type-I or type-III) and TGF-b1

had synergistic effect on the relocation and down-regulation of

the epithelial markers E-cadherin and cytokeratin. Finally, the

results demonstrated synergistic effects of coating with intersti-

tial collagen (either type-I or type-III), on cell ‘fibroblastic’ cell

function as assessed by cell migration and by the synthesis of

type-III and type-IV collagen.

Conclusion The results of these in vitro experiments suggest

that terminal transdifferentiation of proximal tubular epithelial

cells is the result of a combination of the effects of the pro-fibrotic

cytokine TGF-b1 and exposure of the cells to components of the

interstitial extra-cellular matrix to which the cells are not exposed

in the absence of damage to the tubular basement membrane.



Smad-3 as a mediator of the fibrotic response

Kathleen Flanders,* Misako Sato,† Akira

Ooshima,† Angelo Russo‡ and Anita Roberts*

*Laboratory of Cell Regulation and Carcinogenesis, National

Cancer Institute, Bethesda, MD, USA; †Department of

Pathology, Wakayama Medical University, Wakayama, Japan;‡Radiation Biology Branch, National Cancer Institute, Bethesda,

MD, USA

Introduction Smad-3, a key cytoplasmic mediator of trans-

forming growth factor-b (TGF-b) signalling, mediates many of

its inflammatory and fibrotic effects in vivo (Roberts et al.

2001). Smad-3 null mice are protected against cutaneous injury

induced by ionizing irradiation (Flanders et al. 2002). Here, we

report on our continuing studies on radioprotection as well as

protection against tubulointerstitial fibrosis following unilateral

ureteral obstruction (UUO) in Smad-3 null mice.

Methods For radioprotection studies, the flank skin of Smad-

3þ/þ wild-type (WT) and Smad-3–/– knockout (KO) mice was

exposed to 30 Gy of localized g-irradiation and analysed for

histology and gene expression at various times post irradiation.

In the UUO model, the right proximal ureter of WT and KO

mice was ligated, and 1–2 weeks later kidneys were analysed for

inflammation, fibrosis and gene expression.

Results Six weeks after exposure to irradiation, skin from KO

mice shows less epidermal acanthosis and influx of mast cells,

macrophages and neutrophils than skin of WT mice. Paradoxi-

cally, at 6–8 h post irradiation, KO skin shows a significantly

greater number of neutrophils. Irradiated KO skin also exhibits

less immunoreactive TGF-b, fewer myofibroblasts and less scar-

ring than does WT. Smad-3 null dermal fibroblasts do not respond

to the chemotactic effects of TGF-b and show less induction of

fibrogenic cytokines when treated with irradiation plus TGF-bcompared to WT cells. Following UUO, normal kidney archi-

tecture is preserved in KO mice, while kidneys from WT mice

are enlarged with an influx of mononuclear cells and increased

expression of collagen and TGF-b1. Additionally, renal tubules

in obstructed kidneys of KO mice remain positive for E-cadherin

without expression of a-smooth muscle actin, while the opposite

expression pattern is seen in obstructed kidneys of WT mice.

TGF-b treatment of primary cultures of WT renal tubular

epithelial cells results in a phenotypic change from a cobblestone

pattern to a spindle-shaped fibroblastic appearance, while KO

cells treated with TGF-b maintain their original appearance.

Conclusion Smad-3 plays an important role in mediating

pathogenic inflammation and fibrosis in several model systems

and is also essential for TGF-b1-induced epithelial–mesenchy-

mal transition in renal tubular epithelial cells. Inhibitors of the

Smad-3 pathway may have clinical applications in the treat-

ment of a number of fibrotic conditions.

References

Flanders K.C. et al. (2002) Mice lacking Smad3 are protected

against cutaneous injury induced by ionizing radiation.

Am. J. Pathol. 160, 1057–1068.

Roberts A.B. et al. (2001) Smad3-a major player in signal

transduction pathways leading to fibrogenesis. Chest 120,

43S–47S.

Transgenics, knockouts and fibrosis

Christos ChatziantoniouInserm U489, Tenon Hospital, Paris, France

Our team has contributed in the elucidation of renal fibrotic

mechanisms by identifying several critical steps of the molecular

and cellular events leading to the development of renal vascular

and glomerular fibrosis. In particular, using transgenic mice exp-

ressing reporter genes under the control of collagen-I gene pro-

moter, we examined the mechanisms by which vasoconstrictors

such as angiotensin-II and endothelin induce renal vascular and

glomerular fibrosis in experimental hypertension (NO deficiency

model) (Chatziantoniou et al. 1998; Boffa et al. 1999; Tharaux

et al. 1999, 2000; Fakhouri et al. 2001; Flamant et al. 2002).

The major findings were: (1) angiotensin-II induced renal

fibrosis through an endothelin-mediated activation of col-

lagen-I gene; (2) this activation was an early phenomenon (pre-

ceding blood pressure increase) occurring locally to renal

resistance vessels; (3) this fibrogenic process involved activation

of TGF-b, tyrosine-kinase growth factor receptors, MAPK/ERK

and Rho kinase-signalling pathways and (4) a complete protec-

tion of the renal vasculature was observed during treatment

with an AT1 or ETA/B receptor antagonist, owing to inhibition

of collagen-I gene activation independently of systolic blood

pressure levels.

In these studies, the receptor antagonists were administered

in a preventive way (simultaneously to hypertension); and thus

these observations are consistent with protection against the pro-

gressionrather thanregressionof renalfibrosis.Wehaveaddressed

the latter issue in subsequent experiments in which angiotensin

and/or endothelin receptor antagonism was introduced after the



appearance of fibrotic lesions (Flamant et al. 2002; Boffa et al.

2001). The treated animals displayed a less severe degree of

glomerular lesions compared with those at the beginning of the

treatment, thus suggesting that renal vascular fibrosis regressed.

This regression appeared to be due to a dual mechanism: inhibi-

tion of collagen-I synthesis and activation of systems degrading

collagen such as metalloproteinase-2 and metalloproteinase-9.

References

Boffa J.J. et al. (1999) Angiotensin II activates collagen type I gene

in the renal vasculature of transgenic mice during inhibition

of nitric oxide synthesis: evidence for an endothelin-mediated

mechanism. Circulation 100, 1901–1908.

Boffa J.J. et al. (2001) Regression of renal vascular fibrosis by

endothelin receptor antagonism. Hypertension 37, 490–496.

Boffa J.J. et al. (2003) Regression of renal vascular and

glomerular fibrosis: role of angiotensin II receptor antagonism

and matrix metalloproteinases. J. Am. Soc. Nephrol. 14, 1132–

1144.

Chatziantoniou C. et al. (1998) Nitric oxide inhibition induces

early activation of type I collagen gene in renal resistance

vessels and glomeruli in transgenic mice. Role of endothelin.

J. Clin. Invest. 101, 2780–2789.

Fakhouri F. et al. (2001) Angiotensin II activates collagen type I

gene in the renal cortex and aorta of transgenic mice through

interaction with endothelin and TGF-b. J. Am. Soc. Nephrol.

12, 2701–2710.

Flamant M. et al. (2002) Epidermal growth factor receptor trans-

activation mediates the tonic and fibrogenic effects of

endothelin in the aortic wall of transgenic mice. FASEB J. 17,

327–329.

Tharaux P.L. et al. (1999) Vascular endothelin-1 gene expression

and synthesis and effect on renal type I collagen synthesis and

nephroangiosclerosis during nitric oxide synthase inhibition in

rats. Circulation 99, 2185–2191.

Tharaux P.L. et al. (2000) Angiotensin II activates collagen I gene

through a mechanism involving the MAP/ER kinase pathway.

Hypertension 36, 330–336.

Regulation of inflammation and scarring inglomerulonephritis

Charles D PuseyDepartment of Renal Medicine, Imperial College, London, UK

Glomerulonephritis is the commonest cause of end-stage renal

failure worldwide. In most cases, it is caused by deposition or

formation of immune complexes within the glomerulus.

This leads to engagement of inflammatory mechanisms which

often lead to scarring. Although the molecular mechanisms

involved can be studied in vitro, it is important to determine

their role in vivo before progressing to clinical trials. We are

studying a model of crescentic glomerulonephritis in the WKY

rat induced by administration of rabbit anti-rat GBM antibody.

In this model of nephrotoxic nephritis, glomerular cellularity

peaks at day 4, followed by crescent formation peaking at

around 2 weeks and then by scarring which leads to renal fail-

ure by about 6 weeks. We have examined the role of cytokines

and adhesion molecules in vivo in this model.

Blockade of TNF-a using a monoclonal antibody was effective

in prevention of disease, if administered from day 0, and in redu-

cing inflammation, if administered from day 4. Perhaps, more

importantly, it could also reduce scarring and improve renal func-

tion when administered from the time of peak crescent formation

at day 14. As an alternative to blocking pro-inflammatory cyto-

kines, we have found that the administration of anti-inflammatory

cytokines is another effective approach. Both interleukin-4 (IL-4)

and IL-11 could prevent disease when started from day 0, and IL-4

was also shown to reduce inflammation when administered later

in the course of disease.

The development of nephrotoxic nephritis has been shown

to be leucocyte-dependent, and, in this model, monocytes/

macrophages are the predominant leucocyte. Blockade of

VLA-4 using antibodies to the a4-integrin chain was effective

both in preventing acute inflammation and also in reducing

scarring and slowing the progression to renal failure. How-

ever, this effect did not appear to depend on a reduction in

macrophage infiltration of the glomerulus. Blockade of VLA-1

using antibodies to the a1-integrin chain did not have a sign-

ificant effect on acute inflammation but was effective in redu-

cing scarring and preventing renal failure. This might be due to

inhibition of the migration or activation of macrophages in the

extravascular tissues.

Based on our experimental work, we (in collaboration with

others) have recently performed a pilot study of the use of anti-

TNF antibody in 30 patients with systemic vasculitis. Inflix-

imab was used as additional therapy at the time of acute

presentation or as the only change in therapy at the time of

relapse or in grumbling disease. Results from this pilot study

suggest that TNF blockade is an effective approach in most

patients with renal vasculitis. A randomized controlled trial is

therefore being planned.

The use of our animal model of crescentic glomerul-

onephritis has demonstrated the effectiveness in vivo of

blocking pro-inflammatory cytokines, administering anti-

inflammatory cytokines or blocking adhesion molecules.

These approaches are worth investigating in patients with

glomerulonephritis.



Tissue-Transglutaminase and the developmentof renal fibrosis

A.F. El-Koraie,* T. Johnson,* N.M. Baddour†,

E.H. Elkashef‡ and A.M El-Nahas*

*Sheffield Kidney Institute, Northern General Hospital Trust,

Sheffield, UK; †Department of Pathology; ‡Nephrology Unit,

Alexandria Medical School, Alexandria, Egypt

In a previous experimental work, we have found that tissue-

Transglutaminase (tTg) may contribute to the development of

fibrosis in renal disease, through stabilization of laid down col-

lagen fibrils. However, tTg has never been studied in human

kidney diseases. We investigated the intensity of tTg expression

as well as its cross-link product (C/L) by immunohistochemistry,

in renal biopsies from 70 patients with different forms of glomer-

ulonephritis and five controls. Using a monoclonal anti-human

tTg antibody and a polyclonal anti-human C/L antibody and

applying this in three different techniques using both light and

confocal fluroscent microscopy, where by the help of the con-

focal microscopy, the emission of the positivity could be easily

and accurately estimated, while the parrafin sections allowed

clear determination of the pattern of distribution of the staining.

There was an increase in the intensity of both tTg and its C/L

product expression in the renal interstitium (both in the extra-

cellular matrix and the tubules) as well as the glomerular com-

ponent (of mesangial distribution) of diseased kidneys. In

schistosomal glomerulonephritis (n5 30) the mean tTg was

68.18± 46.27%, and the mean C/L expression was

16.11± 10.27%. In a second group (n5 40) with various aetiol-

ogies for glomerulonephritis (but with comparable pathology to

the first group), the mean tTg expression was 56.50± 49.83%,

and the mean C/L expression was 12.40±13.97%, whereas in

the control group (n5 5), the mean tTg was 6.11±2.27%, and

the mean C/L expression was 3.61±1.20%. Both tTg expres-

sion and C/L expression were significantly different from the

control group (P5 0.003 for tTg and P5 0.005 for C/L), but

no significant difference was detected between individual

nephropathies. There was a very high positive correlation

between the intensity of tTg and the intensity of C/L immuno-

staining in all nephropathies (r5 0.974, P5 0.000).

Both tTg and C/L immunostaining was postively correlated

with blood urea (r5 0.671 and 0.684, P5 0.000, group I;

r5 0.733and0.740,P5 0.000,group II; for tTgandC/L, respec-

tively)andserumcreatinine(r5 0.618and0.618,P5 0.000,group

I; r5 0.579, P5 0.019, r5 0.586, P5 0.018, group II; for tTg

and C/L, respectively) in the two studied groups, but they were

notcorrelatedwithmeanarterialbloodpressurenorwiththe level

of 24 h proteinuria. Also, both of the tTg and the C/L were very

strongly correlated with interstitial fibrosis, as well as glomerular

sclerosis (r5 0.887, P5 0.000 for tTg, r5 0.892, P5 0.000 for

C/Lin group I; r5 0.919, P5 0.000 for tTg, r5 0.901, P5 0.000

for C/Lin group II), as well as glomerular sclerosis (r5 0.735,

P5 0.000 for tTg, r5 0.747, P5 0.000 for C/Lin group I;

r5 0.810,P5 0.000fortTg,r5 0.823,P5 0.000forC/Lingroup

II). Furthermore, there were significant correlations with inter-

stitial a-smooth muscle actin (a marker of activated fibroblasts/

myofibroblasts) (r5 0.844 and 0.0824, P5 0.000, for tTg and

r5 0.826 and 0.835, P5 0.000 for C/L, for each of group I and

group II, respectively). Again there were significant correlations

withinterstitialapoptosis (r5 0.733and0.931,P5 0.000fortTg

and r5 0.746 and 0.956, P5 0.000 for C/L, for each of group I

and group II, respectively). Whereas most of these correlation

were found to be dependant on the interstitial part of the tTg, the

glomerular tTgexpressionwasnot correlatedwithanyparameter

except for the degreeofmesangialhypercellularity (r5 0.710and

0.942, P5 0.000, for group I and II, respectively) as well as the

glomerular vimentin expression (r5 0.771 and 0.661, P5 0.000

for group I and II, respectively) On running simple and multiple

regression, the intensity of interstitial tTg expression was

found to be a very powerful determinant of interstitial fibrosis

(as evidenced by interstitial trichrome) (R25 95%, P5 0.000).

In conclusion, tTg seems to be one of the important contri-

butors to the development of renal fibrosis in human glomer-

ulonephritis and may be responsible in part for the stability of

the collagen fibrils and their resistance to degradation.

Emodin ameliorates glucose-induced fibronectinsynthesis in human peritoneal mesothelial cellsby inhibiting PKC-a activation

Susan Yung,* Jack Leung,* Ryan Tsang,* Zhi

Hong Liu,† Lei Shi Li† and Tak Mao Chan*

*Department of Medicine, University of Hong Kong, Queen Mary

Hospital, Hong Kong; †Research Institute of Nephrology, Nanjing

University School of Medicine, China

Introduction Peritoneal dialysis (PD) is recognized as an

effective treatment for patients with end-stage renal failure.

Conventional PD solutions are unphysiological, as they con-

tain bio-incompatible concentrations of glucose to provide

the osmotic drive (Popovich et al. 1978) Thus, with increas-

ing duration on PD, the peritoneum undergoes progressive

structural and functional deterioration. Diabetiform altera-

tions of the peritoneum, for example, the reduplication of the

basement membrane, and increased matrix accumulation and

deposition within the submesothelium are commonly

observed in PD patients. These changes are mediated in part



through activation of PKC and induction of TGF-b1 (Ha et al.

2001). Emodin (3-methyl-1,6,8 trihydroxyanthraquinone)

has previously been demonstrated to reduce cell proliferation

and fibronectin synthesis in cultured mesangial cells (Kuo

et al. 2001; Yao et al. 1994). How emodin modulates glu-

cose-induced fibronectin synthesis in human peritoneal

mesothelial cells (HPMCs) has not been elucidated and thus

constitutes the aim of this study.

Materials and methods Confluent growth-arrested HPMCs

were cultured under physiological (5 mm) or elevated (30 mm)

d-glucose concentrations in the presence or absence of emodin

(20mg/ml) for periods up to 72 h. PKC-a activation in HPMCs

cultured under control and experimental conditions was deter-

mined by Western blot analysis of cytosolic and cell membrane

extracts. Synthesis of fibronectin and TGF-b1 was investigated

by a combination of ELISAs, immunohistochemistry and

Western blot analysis. Mannitol was used as the hexose control.

Results Exposure of HPMCs to 30 mm d-glucose resulted in

the activation of PKC-a commencing 12 h after stimulation, as

demonstrated by the translocation of PKC-a from the cytosol to

the cell membrane. Maximum induction of PKC-a was observed

48 h after stimulation and increased 1.4-fold and 3.7-fold in the

cytosolic and membrane fractions, respectively, compared to

5 mm d-glucose. Activation of PKC-a was accompanied by the

induction of TGF-b1 secretion in a time-dependent manner

(3.72± 0.29 and 4.30±0.50pg/mg cellular protein at 24 h and

48h, respectively, for 30mm d-glucose compared to 2.13± 0.23

and 2.65± 0.32pg/mg cellular protein at 24h and 48h, respec-

tively, for 5mm d-glucose, P< 0.001 at both time points) and

increased fibronectin synthesis. Whilst emodin had no effect on

constitutive PKC-a, TGF-b1 or fibronectin synthesis, emodin

abrogated elevated glucose stimulation of PKC-a, and decreased

TGF-b1 secretion and fibronectin synthesis to basal levels.

Discussion Our findings demonstrate that emodin can ame-

liorate the undesirable effects of concentrated glucose on

HPMCs through the suppression of PKC-a activation. This

study suggests that emodin may have therapeutic potential in

the prevention or treatment of glucose-induced structural and

functional abnormalities in the peritoneal membrane.

References

Ha H., Yu M.R. & Lee H.B. (2001) High glucose-induced

PKC activation mediates TGF-b1 and fibronectin syn-

thesis by peritoneal mesothelial cells. Kidney Int. 59, 463–470.

Kuo Y.C. et al. (2001) Immune responses in human mesangial

cells regulated by emodin from Polygonum hypoleucum Ohwi.

Life Sci. 68, 1271–1286.

Popovich R.P., Moncrief J.W., Nolph K.D. (1978) Continuous

ambulatory peritoneal dialysis. Artif. Organs 2, 84–86.

Yao J., Li L.S., Zhou H. (1994) Inhibition of fibronectin synthesis

in cultured human mesangial cells by emodin. Chin. J. Nephrol.

Dial. Transplant 3, 349–352.

The human hyaluronan synthase genes: putativemediators of renal fibrosis

Timothy Bowen, Jamie Monslow, Malcolm Davies

and John D. WilliamsInstitute of Nephrology, University of Wales College of Medicine,

Heath Park, Cardiff, Wales, UK

Introduction The glycosaminoglycan hyaluronan (HA) is a key

component of the vertebrate extracellular matrix and is synthe-

sized by the HA synthase (HAS) enzymes HAS1, HAS2 and HAS3

at the plasma membrane. Accumulating evidence emphasizes the

relevance of HA metabolism in clinical nephrological processes

such as renal fibrosis (Strutz 2001) and peritoneal mesothelial

wound healing (Yung et al. 2000). In the present study, the geno-

mic sequences and organization of the genes encoding the human

HAS isoforms were deduced, in silico, from reference cDNA and

genomic sequence data and subsequently supported by in vitro

data. The region immediately upstream of each HAS gene was

then screened for promoter activity.

Materials and methods The in silico methods used in the

present study have been described in detail elsewhere

(Williams et al. 2002). The programmes BLAST and BLAST2

were used to identify genomic DNA sequences containing

the reference cDNA sequences for HAS1, HAS2 and HAS3.

Intron/exon boundary sequences were confirmed by sequen-

cing of PCR products from genomic DNA, and each PCR-

amplified promoter region was cloned and analysed by luciferase

assay (Hoogendoorn et al. 2003).

Results The HAS1 gene comprised five exons, with the trans-

lation start site situated 9 bp from the 3´-end of of exon 1. In

contrast, the genomic structures for HAS2 and both HAS3

variants spanned four exons, exon 1 forming a discrete

5´-untranslated region and the translation start site located at

nucleotide 1 of exon 2. Luciferase analysis of approximately



500 bp of the flanking genomic sequence of each HAS gene

showed constitutive promoter activity.

Discussion Excessive HA synthesis in the renal cortex is

characteristic of pathological processes such as the inflamma-

tion and early matrix expansion that precede renal fibrosis

(Strutz 2001). The regulation of expression of the human

HAS genes is therefore of great interest. We have used in

vitro and in silico methods to deduce the genomic structures

of the human HAS genes. Furthermore, we have demonstrated

that the sequences immediately upstream of these genomic loci

are transcriptionally active. We are continuing with more

detailed analysis of these promoters, the data from which

will help to evaluate the role of the HAS genes in renal fibrosis.

References

Hoogendoorn B. et al. (2003) Functional analysis of human

promoter polymorphisms. Hum. Mol. Genet. 15, 2249–2254.

Strutz. F. (2001) Potential methods to prevent interstitial fibrosis in

renal disease. Expert Opin. Investig. Drugs 10, 1989–2001.

Williams N.M. et al. (2002) Determination of the genomic

structure and mutation screening in schizophrenic individuals

for five subunits of the N-methyl-D-aspartate glutamate

receptor. Mol. Psychiatry 7, 508–514.

Yung S., Thomas G.J., Davies M. (2000) Induction of hyaluronan

metabolism after mechanical injury of human peritoneal mesothe-

lial cells in vitro. Kidney Int. 58, 1953–1962.

Bone morphogenetic protein-1 cleavesprodecorin in vitro and in cellulo

Hanane Gouizi, Laure Guarrigue-Antar, Susan

Richardson and Karl KadlerWellcome Trust Centre for Cell Matrix Research, University of


Introduction Decorin is a ubiquitously distributed small leu-

cine-rich protein (SLRP) with pivotal roles in collagen fibrillo-

genesis, regulation of cell matrix deposition as well as cell

growth, adhesion and migration. It is expressed in the proform

but found mainly in the processed form in various tissues.

However, the role of propeptide of prodecorin is still unclear.

A possible function is in the trafficking of prodecorin to

specific compartment(s) in the cell where it may bind/interact

with other ECM molecules prior to secretion. BMP-1 is a

metalloproteinase that is responsible for the processing of

various ECM molecules including procollagens and prolysy-

loxidase. Probiglycan, a SLRP with 57% homology to prode-

corin, has also been shown to be a substrate of BMP-1, which

led to the hypothesis that prodecorin could also be processed

by the same enzyme. This would emphasize the important role

of BMP-1 in the deposition of the ECM.

Materials and methods To aid detection, Flag-tag

(DYKDDDDK) was introduced into the prodecorin sequence

immediately 5´ of the propeptide sequence. This was achieved

via a two-step PCR reaction. The triple-alanine mutant was

generated using the same method replacing the putative BMP-

1 cleavage site EDE (30–32) with AAA residues. Transient and

stable transfectants were generated in HT1080 human fibro-

blast cells. These were chosen because of their ability to

express and secrete the mutant prodecorin. Purification of

decorin was achieved using anion exchange chromatography.

BMP-1-Flag was expressed in, and purified from, transfected

293-EBNA cells.

Results BMP-1 was assayed for cleavage of prodecorin in vitro.

Addition of BMP-1 to the assay resulted in removal of the prodo-

main. Addition of EDTA restores detection of prodecorin by anti-

Flag antibody. HT1080 cells doubly transfected with Flag-tagged

prodecorin and BMP-1-Flag secreted decorin in the mature form.

Control cells secreted uncleaved prodecorin. HT1080 cells doubly

transfected with Flag-tagged triple-alanine prodecorin mutant and

BMP-1-Flag did not show cleavage of prodecorin. It was observed

that the GAG-chain addition to this mutant was affected by the

mutation.

Discussion BMP-1 cleaves prodecorin in vitro and in cellulo.

The cleaved bond is possibly with the E-DE (30–32) sequence, but

this will be confirmed by cleavage of prodecorin and sequencing of

the mature protein. These residues are also important in the initia-

tion and stability of the glycosaminoglycan chain of decorin,

because mutation of these results in production of mainly ungly-

canated prodecorin. This suggests a possible role of the prodomain

in the initiation of the GAG chain.

References

Danielson K.G. et al. (1997) Targeted disruption of decorin leads

to abnormal collagen fibril morphology and skin fragility. J.

Cell Biol. 136 (3), 729–743.

Hausser H. et al. (1994) Selective inactivity of TGFb/decorin

complexes. FEBS Lett. 353, 243–245.

Scott I.C. et al. (2000) Bone morphogenetic protein 1 processes

probiglycan. J. Biol. Chem. 275 (39), 30504–30511.



Proteomics of tendon ECM assembly

Susan H. Richardson, Dave J. Thornton and Karl

E. KadlerWellcome Trust Centre for Cell-Matrix Research, School of

Biological Sciences, University of Manchester, Manchester, UK

Introduction The ability of tendon to transduce tensile force to

the skeleton relies on 10s of 1000s of long (>100 microns) extra-

cellular collagen fibrils that are in parallel register with the long

axis of the tendon. Electron microscopy studies of developing

mouse-tail tendon show that at E13.5, the tissue is predominantly

composed of cells with little or no matrix. By E15.5, intracellular

fibrils can be seen. The long collagen fibrils are deposited one-by-

one by ‘finger-like’ projections of the plasma membrane into

hexagonally packed bundles of the fibrils. Moreover, the fibrils

have a precise interfibrillar spacing of approximately 30nm. The

aim of this work was to identify proteins whose patterns of expres-

sion change during tail development in order to understand better

the molecular basis of intracellular collagen fibril assembly, tubule

trafficking and supramolecular organization of the fibrils in the

embryonic mouse tendon.

Materials and methods Fresh embryonic tail tissue was har-

vested and proteins extracted into buffer containing urea,

thiourea, detergent and DTT (DL-dithiothreitol). Proteins

(approximately 100mg) were first separated by isoelectrofocus-

ing on 24 cm, linear immobilized pH gradient strips (pH 4–7).

Strips were reduced with 1% DTT and alkylated with 5%

iodoacetamide before second dimension separation by SDS-

PAGE on an 11% gel. Gels were stained with either 0.1%

colloidal Coomassie Blue or silver nitrate.

Coomassie-stained gels were used for MS analysis. Protein

spots of interest were excised from the 2D gel, destained,

reduced, alkylated and digested overnight with trypsin. The

resulting peptides were then extracted and analysed by LC-

MS/MS. The data were used to search against SWISSPROT

and Tremblnew public protein databases.

Results We have established reproducible extraction

conditions and separation methods to obtain 2D gels

containing >500 protein ‘spots’ that are visible by Coomassie

Blue staining.

Discussion Our approach is to extract proteins from whole

embryonic tail from both E13.5 and E15.5 tissues, separate the

proteins by 2D-gel electrophoresis, highlight key changes in

the spot profile and identify these proteins by trypsin digestion,

q-TOF mass spectrometry and database matching. A surprising

observation was the abundance of intracellular proteins

involved in protein folding and trafficking. A poster will be

presented that lists the first proteins that we have identified.

Identification, expression and tissue distributionof the three rat lysyl hydroxylase isoforms

D.K. Mercer, P.F. Nicol, C. Kimbembe and

S.P. RobinsRowett Research Institute, Bucksburn Aberdeen, UK

Introduction Lysyl hydroxylases (LHs) (procollagen-lysine

2-oxoglutarate 5-dioxygenase; PLOD) catalyse the hydroxyla-

tion of lysine residues during the post-translational modifica-

tion of collagenous proteins.

Results In this poster, we describe the first identification and

cloning of LH isoforms 2 and 3 from the rat, including both

LH2-splice variants (LH2a and LH2b). The rat LHs are

expressed in almost all tissue and cell types examined, indicat-

ing a probable lack of tissue specificity for LH function. All

LH isoforms were stably transfected into CHO-K1 cells, and

this represents the first example of recombinant LH produc-

tion in a eukaryotic cell line. Expression and production of all

LH isoforms led to an increase in total collagen synthesis. LH1

and LH2a expression and production led to an increase in

total pyridinium cross-link production.

Discussion Evidence that LH2a possesses telopeptide lysyl

hydroxylase activity, previously thought to be a novel

enzyme, is presented.

References

Mercer D.K. et al. (2003) Identification, expression and tissue

distribution of the three rat lysyl hydroxylase isoforms.

Biochem. Biophys. Res. Commun. 307, 803–809.

The role of acetylation in Timp-1 regulation

David A. Young, Dylan R. Edwards and Ian

M. ClarkSchool of Biological Sciences, University of East Anglia, Norwich,

Norfolk, UK

Introduction Aberrant matrix turnover, mediated by a

family of proteases known as matrix metalloproteinases

(MMPs), is involved in a number of pathologies including



rheumatoid arthritis and osteoarthritis, tumour invasion and

metastasis, and liver fibrosis. The active forms of all of the

MMPs are inhibited by a family of specific inhibitors, the

tissue inhibitors of metalloproteinases (TIMPs). As inhibition

represents a major level of control of MMP activity, a detailed

knowledge of the mechanisms controlling TIMP gene expres-

sion is therefore important in developing therapies for these

diseases. Histone acetyl transferases (HATs) play a crucial role

in gene regulation via acetylation of both histones (to loosen

nucleosomal structure and to recruit accessory factors) and

transcription factors. Here, we describe the effects of a histone

deacetylase inhibitor (Trichostatin A – TSA) on the TGF-b1

and Phorbol ester (PMA) induction of Timp-1.

Materials and methods RNA isolation, TaqMan� real-time

RT-PCR, cell culture, transient transfections and reporter gene

assays along with electrophoretic mobility shift assays (EMSA)

were performed essentially as described (Young et al. 2002).

Results In murine C3H10T1/2 cells both TGF-b1 and PMA

induce Timp-1 expression in a protein synthesis-dependent

manner as measured by Northern blotting and TaqMan�

real-time RT-PCR. TSA superinduces PMA-induced Timp-1

expression but represses TGF-b-induced Timp-1 expression.

A TSA dose–response experiment further demonstrates that

TGF-b and PMA stimulate Timp-1 gene expression via dif-

ferent mechanisms. The effects of TGF-b, PMA and TSA on

Timp-1 expression can be reiterated in transient transfection

studies with Timp-1 promoter containing reporter con-

structs. Deletion of the proximal AP-1 motif from the

promoter demonstrates that this sequence is critical for

TGF-b1-induced reporter expression, while PMA appears to

act via other sequences/mechanisms.

Discussion Repression of deacetylation by TSA differentially

affects the TGF-b or PMA induction of Timp-1. These results

will help elucidate the different molecular mechanisms by which

these factors regulate the expression of Timp-1 and other genes.

Furthermore, the effects of acetylation on TGF-b induction of

Timp-1 may act as a paradigm for other cytokine-induced genes.

This work was funded and supported by the Arthritis

Research Campaign (ARC) and the Dunhill Medical Trust.

References

Young D.A. et al. (2002) Identification of an initiator-like element

essential for the expression of the tissue inhibitor of metallo-

proteinases-4 (Timp-4) gene. Biochem. J. 364, 89–99.

The role of glutamate signalling in rheumatoidarthritis

S.L. Flood, V.C. Duance and D.J. MasonConnective Tissue Biology Laboratories, School of Biosciences,

Cardiff University, Museum Avenue, Cardiff, UK

Introduction Rheumatoid arthritis (RA) is characterized by

joint inflammation and destruction mediated by enhanced

secretion of degradative enzymes and cytokines. Inflammation

of the joint is accompanied by elevated levels of glutamate

within the synovial fluid (McNearney et al. 2000). Glutamate

has been shown to modulate bone cell phenotype (Chenu et al.

1998). We hypothesize that the elevated glutamate in synovial

fluid that accompanies RA can induce phenotypic changes

associated with synovial joint destruction.

Methods The mRNA expression of glutamate receptors and

transporters was investigated in tissues of the rat knee joint by

RT-PCR to determine which cell types are potentially responsive

to glutamate. To determine whether extracellular glutamate affects

synoviocyte phenotype, we have investigated the effect of different

glutamate concentrations on human, primary, RA synoviocytes’

enzyme and cytokine expression. Matrix metalloproteinases

(MMPs) and tissue inhibibitors of MMPs (TIMPs) protein expres-

sion was determined by zymography, and IL-6 release by RA

synoviocytes was measured using an IL-6 ELISA (R & D systems).

Results RT-PCR has revealed glutamate transporter (GLAST-

1) and ionotropic and metabotropic receptor mRNA expression

in several tissues of the rat knee joint. Pro-MMP2, TIMP1

and TIMP2 activity were all increased in the presence of

high extracellular glutamate concentrations and in the

presence of glutamate transporter inhibitors. The presence of

glutamate transporter inhibitors also increased production of the

cytokine IL-6 but only at low glutamate concentrations.

Discussion Our data show that many cells of the synovial

joint have potential for responding to glutamate signals. Pri-

mary synoviocytes from RA patients increase MMP and TIMP

expression in response to high extracellular glutamate and in

the presence of glutamate transporter inhibitors which also

increase IL-6 production. We postulate therefore that the high

levels of glutamate present in RA synovial fluid are having a pro-

inflammatory effect on synoviocytes. This may be mediated

through the cytokine IL-6, the synovial fluid levels of which

correlate with RA disease activity. We have previously shown



that IL-6 treatment of RA synoviocytes induces the glutamate

transporter (GLAST-1) expression therefore indicating a feed-

back mechanism between IL-6 and GLAST-1 expression. We

are currently investigating the effect of ionotropic glutamate

receptor antagonists on the pheontype of RA synoviocytes

to elucidate the specific signalling mechanisms involved.

References

Chenu C. et al. (1998) Glutamate receptors are expressed by bone

cells and are involved in bone resorption. Bone 22 (4), 295–299.

Flood S.L. et al. (2002) XVIIIth Federation of European

Connective Tissue Societies Meeting, Brighton, UK. [Abstract 39].

McNearney T. et al. (2000) Excitatory amino acid profiles of

synovial fluid from patients with arthritis. J. Rheumatol. 27 (3),

739–745.

Discovery of potent and selective inhibitors ofprocollagen C-proteinase for the treatment offibrotic disorders

R.P. Butt,* J.P. Huggins,* D. Greiling,*

B. Hopkins,* S. Gaboardi,* D. Winslow,*

M. Ronald,* S. Lewis,* S. Ward,* E. Levett,*

J. Owen,* F. Burslem,* M. Collis,* S. Bailey,†

P.V. Fish,* G. Whitlock,* S. Billotte,* K. James,*

A. Mcelroy* and J. Blagg*

*Tissue Repair Group, Discovery Biology; †Chemistry, PGRD,

Sandwich, Kent, UK

Introduction Fibrosis is a component of many tissue pathol-

ogies leading to loss of normal tissue function, primarily due to

excessive collagen deposition. Collagen is deposited following

cleavage of the C- and N- terminal peptides from the pro-

collagen molecule. The cleavage of the globular C-peptide by

PCP reduces solubility of the fibrillar collagen molecule, result-

ing in deposition of insoluble collagen. Increased insoluble

collagen deposition is a feature of all organ fibroses, with

inhibition of this process, a key potential anti-fibrotic mechan-

ism. The aim of this work was to discover potent and selective

PCP inhibitors as experimental, topically applied, anti-fibrotic

drugs for clinical evaluation.

Materials and methods PCP was cloned from human

osteosarcoma cells and enzymatic activity demonstrated

using a PCP-specific peptide cleavage assay. Activities were

confirmed by measuring cleavage of [3H]C-peptide from

type-I pro-collagen. A cell-based fibroplasias model was

employed to demonstrate compound efficacy using collagen

deposition, liberated C-peptide and histological endpoints.

The activities of PCP inhibitors in fibroblast and epithelial

in vitro cell proliferation and migration assays, and selectiv-

ity vs. a panel of MMPs were also determined.

Results

Assay UK-383,367

NH

HOO

O N

NNH2

O

UK-421,045

NH

N

O N

O

HONH SMe

O

O

Inhibition of peptide cleavage (Ki, nM) 33.3±1.92 8.7± 2.9

Inhibition of pro-collagen cleavage (IC50, nM) 27.5±3.2 3.4± 1.0

Maximal inhibition of collagen deposition in fibroplasia model 75.5% @10mM 76.0% @10mM

Maximal inhibition of C-peptide liberation (IC50 approximately 2 mM) (IC50 approximately 1mM)

n.d. 72.2% @10mM

(IC50 approximately 1 mM)

Effect on fibroblast proliferation/migration No effect No effect

Selectivity vs. MMPs (fold vs. PCP Ki in peptide substrate assay) MMP-1,-2,-9,13,-14 >2273 >10,000

MMP-3 1684 9,000

TACE >500 >5,000

All values mean±SEM; n�3 replicate experiments. Compounds: UK-383,367 and UK-421,045. MMP, matrix metalloproteinase; PCP,

procollagen C-proteinase.



Discussion In summary, we have identified and character-

ized potent and selective inhibitors of PCP for progression to

clinical studies for investigation as a treatment paradigm for

fibrotic disease.

Gene expression and matrix proteinase activityin osteochondrosis dessicans

C.L. Curtis, C. Wilson, R. Williams, C. Dent and

B. CatersonConnective Tissue Biology Laboratories, School of Biosciences,

University of Wales College of Medicine, Cardiff, UK

Introduction Osteochondrosis dessicans (OCD) is a disorder

of unknown aetiology where often a fragment of cartilage and

subchondral bone separates from the articular surface. Previous

studies have shown histological changes in glycosaminoglycan

content in OCD cartilage compared to normal cartilage (Koch

et al. 1997). It has also been shown in equine OCD cartilage

that there is excessive degradation of type-II collagen compared

to normal cartilage (Laverty et al. 2002). The present study was

undertaken to examine the gene expression in human OCD

cartilage compared to its normal autologous articular cartilage

and human osteoarthritic (OA) cartilage.

Methods Cartilage from five OCD patients (18–34 years) was

obtained at the time of surgery. Pieces of cartilage were either

snap-frozen (in preparation for RNA isolation) or the proteo-

glycans extracted with 4 m GuHCl. Total RNA was isolated

from the cartilage using RNeasy minicolumns and reagents

(Qiagen) according to the manufacturer’s protocol. RT-PCR

was performed using an RNA PCR kit (Perkin-Elmer) using a

number of oligonucleotide primers. GuHCl-extracted proteo-

glycan fragments were analysed using Western blotting with a

number of antibodies to aggrecan metabolites, collagen meta-

bolites and the small leucine-rich proteoglycans.

Results and discussion When OCD cartilage was compared

to normal and human OA cartilage, there was an increase

in aggrecan, collagen type-II and collagen type-X RNA expres-

sion. There was no change in RNA expression of link protein

or type-I collagen. The RNA expression of the aggrecanases

(ADAMTS enzymes) was also different in the three different

cartilage samples. Neither ADAMTS-1, -4 or -5 was present

in the normal cartilage. In contrast, in the OCD cartilage,

there was expression of both ADAMTS-1 and -4, whereas in

the OA cartilage, there was expression of ADAMTS-4 and -5.

In the case of MMP RNA expression, MMP-3 was decreased

and MMP-13 increased in OCD cartilage compared to both

normal and OA samples. In addition, the expression of all

three TIMP isoforms was increased in the OCD cartilage.

Although inflammatory components are not expected in

OCD pathology, expressions of inflammatory mediators such

as COX-2, IL-1-a and TNF-a were all increased in the OCD

cartilage when compared to normal, but expression of

these mRNAs in the OA cartilage was higher. Analysis of

proteoglycan fragments in the OCD cartilage by Western

blotting showed the presence of aggrecan fragments contain-

ing the G1 domain, interglobular domain and the C-terminal

neoepitope generated by aggrecanase cleavage. There was also

immunoreactivity for biglycan and link protein.

Conclusion These results suggest that the phenotypic expres-

sion of chondrocytes at the site of the OCD lesion are mark-

edly different from ‘normal’ articular cartilage and also

pathological OA cartilage. Interestingly, the expression pat-

terns of matrix proteinases and their natural inhibitors were

also markedly different in OCD cartilage, again suggesting

that there are specific biochemical expression patterns in

OCD pathology, which may potentially be biomarkers of the

disease process. Further studies are necessary to elucidate how

the differences in gene expression and matrix protease activity

may be involved in the aetiology of OCD.

References

Koch S. et al. (1997) Cartilage and bone morphology in

osteochondritis dissecans. Knee Surg. Sports Traumatol.

Arthrosc. 5 (1), 42–45.

Laverty S. et al. (2002) Excessive degradation of type II collagen

in articular cartilage in equine osteochondrosis. J. Orthop. Res.

20 (6), 1282–1289.

Novel strategies for enhancing tissue integrationin cartilage repair

L.C. Davies, B. Caterson and V.C. DuanceConnective Tissue Biology Laboratories, School of Biosciences,

Cardiff University, Cardiff, UK

Introduction Articular cartilage is unable to initiate a spon-

taneous repair response when injured due to its avascular and

aneural properties. Within adult cartilage, chondrocytes are

entrapped within an extensive extracellular matrix and are

unable to migrate to sights of injury to regulate tissue repair.



Injury to this tissue therefore inevitably leads to degeneration

of the cartilage and the development of degenerative diseases

such as osteoarthritis.

The surgical technique of autologous chondrocyte transplanta-

tion (ACT) was developed for the treatment of full-thickness

cartilage defects (Brittberg et al. 1994). Implantation of chondro-

cytes into the defect site repairs the injury site with a mixture of

fibrocartilaginous and hyaline-like tissue that poorly integrates

with the existing cartilage and frequently degenerates with time.

In this current study, we have developed an in vitro model to

investigate methods for enhancing this integration and the devel-

opment of a more biomechanically stable repair tissue.

Materials and methods Bovine articular cartilage explants

from the metacarpalphalangeal joint were experimentally

injured using a stainless steel trephine and cultured for a period

of 28 days. Autologous chondrocytes in an agarose suspension

were injected into the interface region at the injury site. Media

was collected and analysed for proteoglycan and collagen con-

tent using the DMMB and hydroxyproline assays, respectively.

Matrix metalloproteinase (MMP) expression was also analysed

using zymography and an adapted collagen fibril assay.

Results Morphological analyses indicate attempts at repair

and integration within both control and experimental treat-

ment groups, although the presence of autologous chondro-

cytes appeared to amplify this repair response. Although not

statistically significant, considerable differences in proteogly-

can release between injured explants and the intact control

group were seen. Collagen release into the media was only

seen at day 28 within experimental cultures. An up-regulation

of MMP-2 and MMP-9 was seen within the experimental

cultures compared to the controls. Preliminary data also

suggest up-regulation of collagenases in the experimental

group when compared to controls.

Discussion As seen with clinical ACT treatment, the pre-

sence of autologous chondrocytes appears to enhance repair

and integration attempts; however, morphologically, this

repair tissue appears to be fibrocartilaginous. Further analysis

will establish whether the repair tissue is true hyaline cartilage

and monitor the synthesis and turnover of macromolecules

within the established culture system.

References

Brittberg M. et al. (1994) Treatment of deep cartilage defects

in the knee with autologous chondrocyte transplantation.

N. Engl. J. Med. 331 (14), 889–895.

Effects of n-3 polyunsaturated fatty acids onCOX-2 and PGE2 protein levels in articularcartilage chondrocytes

S. Hurst, C.L. Curtis, S.G. Rees, J.L. Harwood and

B. CatersonConnective Tissue Biology Laboratories, School of Biosciences,


Introduction Previous studies within our laboratory have

shown that supplementation with n-3 polyunsaturated fatty

acids (PUFAs), but not other fatty acids, has a beneficial effect

on reducing the expression and activity of degradative and

inflammatory factors known to cause damage and destruction

of cartilage in arthritic diseases (Curtis et al. 2000, 2002).

Cyclooxygenase (COX), also known as prostaglandin H

synthase, catalyses the rate-limiting step in the formation of

inflammatory prostaglandins (PGs) (Hla & Neilson 1992). PG

synthesis requires conversion of arachidonic acid to PGH2 by

either the constitutive COX-1 or by COX-2, which is induced

by inflammatory and mitogenic stimuli (Smith et al. 2000).

Prostaglandin E2 (PGE2) is produced from PGH2 and has been

shown to have a number of functions including both anti- and

pro-inflammatory actions (Christman et al. 1991). The aim of

this project was to investigate the effects of n-3 PUFAs on

cyclooxygenase-2 and prostaglandin E2 protein levels in

articular cartilage chondrocytes.

Materials and methods Articular cartilage was obtained both

from 7-day-old bovine metacarpo-metatarsophalangeal joints

and from human patients undergoing total knee replacement

surgery for osteoarthritis (Llandough Hospital, S.Wales, UK).

Both explant and monolayer cultures were set up in DMEM

with or without 10–300 mg/ml n-3 [eicosapentaenoic acid

(EPA)] or n-6 [arachidonic acid (AA)] PUFAs (minimum 8 h,

at 37 ˚C, in 5%CO2) and in the absence or presence of IL-1

(10 ng/ml) for a further 4 days.

Results Total RNA was extracted and RT-PCR performed

using oligonucleotide primers specific to COX-2 (Invitrogen,

UK). COX-2 mRNA was found to be absent or only present at

very low levels in both bovine and human control cultures.

After treatment with IL-1, this expression greatly increased.

However, supplementation of IL-1-treated cultures with n-3

PUFA (EPA) resulted in a loss of COX-2 mRNA expression. In

contrast, supplementation with n-6 PUFA (AA) had no effect.

Western blot analysis, using a polyclonal antibody specific to

COX-2 (Santa Cruz Biotechnology Inc., USA), showed that

COX-2 protein was absent in all control samples and the IL-1

induction of bovine COX-2 protein could also be reduced



when supplemented with n-3 PUFAs but not n-6 PUFAs. Using

a commercially available PGE2 ELISA Immunoassay kit, it

was possible to analyse the PGE2 levels present in explant

media from bovine or human samples. In one example, in a 74-

year-oldfemalepatient,PGE2proteinwasverylowinthecartilage

cultured without IL-1 treatment or PUFA supplementation (con-

trol). When the cartilage was treated with IL-1, there was a huge

inductionofPGE2levelsbyasmuchas60fold.This induction is in

turnreducedgreatlywiththe supplementationofn-3PUFA(EPA)

but not with n-6 PUFA (AA).

Discussion It has long been accepted that COX-2 plays an

important role in inflammation. COX-2 has been found in

joints affected by arthritic diseases (Hla & Neilson 1992) and

in cultures induced by IL-1. The current study has shown that

both the IL-1-induced COX-2 message and protein levels can be

reduced with supplementation by n-3 PUFAs but not n-6 PUFAs.

This work has also shown that with a decrease in COX-2

protein levels, there is also a corresponding decrease in prosta-

glandin E2 levels caused by n-3 PUFA supplementation.

References

Christman J.W. et al. (1991) Paradoxical regulation by PGE-2 on

release of neutrophil chemoattractants by rat bone marrow

macrophages. Prostaglandins 41, 251–262.

Curtis C.L. et al. (2000) n-3 fatty acids specifically modulate

catabolic factors involved in articular cartilage degradation.

J. Biol. Chem. 275, 721–724.

Curtis C.L. et al. (2002) Pathologic indicators of degradation and

inflammation in human osteoarthritic cartilage are abrogated

by exposure to n-3 fatty acids. Arthritis Rheum. 46 (6), 1544–

1553.

Hla T. & Neilson K. (1992) Human cyclooxygenase-2 cDNA.

Proc. Natl. Acad. Sci. USA 89, 7384–7388.

Smith W.L. et al. (2000) Cyclooxygenases: structural, cellular,

and molecular biology. Annu. Rev. Biochem. 69, 145–182.

Expression profiling of metalloproteinases andinhibitors in cartilage

Lara Kevorkian,* David A. Young,* Clare

Darrah,† Simon T. Donell†, Lee Shepstone,‡ Sarah

Porter,* Sarah Brockbank,§ Dylan R. Edwards,*

Andrew E. Parker§ and Ian M. Clark*

*School of Biological Sciences and Medicine, University of East

Anglia, Norwich; †Institute of Orthopaedics, Norfolk & Norwich

University Hospital, Norwich; School of Biological Sciences and‡Medicine, University of East Anglia, Norwich; §Respiratory &

Inflammation Research, AstraZeneca, Cheshire, UK

Objective To profile the expression of all known members

of the matrix metalloproteinase (MMP), a disintegrin and

metalloproteinase with thrombospondin motifs (ADAMTS),

and tissue inhibitor of metalloproteinases (TIMPs) gene families

in normal cartilage and that from patients with osteoarthritis

(OA).

Methods Human cartilage was obtained from femoral

heads at joint replacement for either osteoarthritis or follow-

ing fracture to the neck of femur. Total RNA was purified

and expression of genes assayed using quantitative real-time

PCR.

Results Several members of the above gene families were

regulated in OA. Genes increasing in expression in OA were:

at P < 0.001, MMP-13, MMP-28, ADAMTS-16; at P < 0.01,

MMP-9, MMP-16, ADAMTS-2, ADAMTS-14 and at

P < 0.05, MMP-2, TIMP-3, ADAMTS-12. Genes decreasing

in expression in OA were: at P < 0.001, MMP-1, MMP-3,

ADAMTS-1; at P < 0.01, MMP-10, TIMP-1, ADAMTS-9

and at P < 0.05, TIMP-4, ADAMTS-5, ADAMTS-15. Correl-

ation analysis revealed that groups of genes across the gene

families are co-expressed in cartilage.

Conclusion This is the first comprehensive expression profile

of all known MMP, ADAMTS and TIMP genes in cartilage.

Patterns of expression provide a foundation on which to under-

stand mechanisms of gene regulation in OA and potentially for

refining the specificity of anti-proteolytic therapies.

ADAMTS-4 and ADAMTS-5 sequestration andactivity in chondrocyte-agarose cultures

Alison J. Rees, Chris B. Little, Bruce Caterson and

Clare E. HughesUniversity of Wales Cardiff, Cardiff, UK

Introduction A primary event in the destruction of cartilage

in arthritic diseases is the loss of aggrecan from the extra-

cellular matrix of articular cartilage. During aggrecan break-

down, cleavage sites are utilized, which reside within the IGD

of the aggrecan core protein. The Asn341–Phe342 bond

is cleaved by members of the MMP family, whereas the

second of the two cleavage sites, the Glu373–Ala374 bond, is

cleaved by the aggrecanases which are all members of the



A disintegrin and metalloproteinase with thrombospondin

motifs (ADAMTS) family. Both ADAMTS-4 and -5 have

been shown to readily cleave aggrecan at this so-called aggre-

canase site (Tortorella et al. 1999; Abbaszade et al. 1999;

Sandy et al. 2000). An in vitro model of cartilage degradation

has also shown that these enzymes are responsible for the loss

of aggrecan from explant cultures of articular cartilage sti-

mulated with IL-1-a or TNF-a (Tortorella et al. 2001). Both

ADAMTS-4 and -5 are thought to be synthesized as inactive

zymogens that are activated via removal of their propeptide

domain by the Golgi enzyme furin. The secreted active

ADAMTS-4 and -5 have predicted molecular weights of 67.9

and 73.6 kDa, respectively. The objective of this study was to

monitor ADAMTS-4 and -5 secretion and sequestration in the

extracellular matrix of chondrocyte-agarose cultures.

Methods Porcine articular chondrocytes were isolated and

embedded in agarose (Hughes et al. 1997) before preculture

in DMEMþ 50 mg/ml gent. with 10% FBS and 25 mg/ml

Phos.C for 21 days. The plates were washed, then cultured in

serum-free DMEM with or without IL-1-a for 96 h. GAG

release to the medium was measured using the DMMB assay.

Media samples were analysed by Western blotting for aggre-

can metabolites using mAb BC-3 to recognize the aggrecanase-

generated neoepitope ARGSV. The presence of ADAMTS-4 in

the media was analysed using the mAb anti-TS-4N, which

recognizes the metalloproteinase domain, and commercially

available polyclonal antibodies to the pro- and spacer domains

of ADAMTS-4. The presence of ADAMTS-5 was detected using

commercially available polyclonal antibodies to the pro- and

spacer domains of ADAMTS-5. Agarose plugs were extracted

in detergent buffer and analysed by Western blotting for

ADAMTS-4 and -5 using the same monoclonal and polyclonal

antibodies.

Results In control cultures, only 20–30% of the total GAG

was released into the medium after 96 h of culture. In contrast,

80–90% of the total GAG was released in cultures exposed to IL-1.

Western blot analysis showed aggrecanase-generated aggrecan

metabolites in the IL-1-treated cultures but none in the control

cultures. Sequestered forms of both ADAMTS-4 and -5 are pre-

sent in the matrix prior to treatment in serum-free conditions,

and following treatment with or without IL-1 for 96 h, there are

no differences in the high molecular weight isoforms of the

enzymes sequestered in the matrix. Western blots of partially

purified media samples showed no differences in the zinc

chelator-bound isoforms of either ADAMTS-4 or -5 between

control and IL-1-treated cultures. However, the predominant

heparin sepharose-bound isoforms of ADAMTS-4 and -5 co-

migrate at approximately 37 kDa. Each of the heparin-bound

37-kDa isoforms of ADAMTS-4 and -5 are detected in increased

amounts in IL-1a-treated cultures compared to controls.

Discussion The increased amounts of the 37-kDa isoforms

of both ADAMTS-4 and -5 in the IL-1-treated cultures suggest

a role for these smaller isoforms in the increased aggrecanase

activity seen in the IL-1-treated cultures compared to controls.

This study has identified multiple isoforms of putative aggreca-

nase activity that could be responsible for increased aggrecan

catabolism that leads to cartilage degradation in arthritis.

References

Abbaszade A. et al. (1999) Cloning and characterization of

ADAMTS11, an aggrecanase from the ADAMTS family.

J.Biol. Chem. 274, 23443–23450.

Hughes C. et al. (1997) Utilization of a recombinant substrate

rAgg1 to study the biochemical properties of aggrecanase in cell

culture systems. J. Biol. Chem. 272, 20269–20274.

Sandy J. et al. (2000) Versican V1 proteolysis in human aorta in

vivo occurs at the Glu441-Ala442 bond, a site that is cleaved by

recombinant ADAMTS-1 and ADAMTS-4. J. Biol. Chem. 276,

13372–13378.

Tortorella M. et al. (1999) Purification and cloning of aggreca-

nase-1: a member of the ADAMTS family of proteins. Science

284, 1664–1666.

Tortorella M. et al. (2001) The role of ADAM-TS4 (aggrecanase-

1) and ADAM-TS5 (aggrecanase-2) in a model of cartilage

degradation. Osteoarthritis Cartilage 9, 539–552.

TGF-b promotes the formation of pyridinolinecross-links in fibrosis via the induction of LH2expression

Annemarie J. Van Der Slot,* Anne-Marie

Zuurmond,* David J. Abraham† and Ruud

A. Bank*

*TNO Prevention and Health, Department Biomedical Research,

Leiden, The Netherlands,†Royal Free and University College

Medical School, Centre of Rheumatology, London, UK

Introduction The hallmark of fibrosis is an excessive accu-

mulation of collagen, a process in which TGF-b plays an

important role. The deposited collagen shows an increase in



pyridinoline cross-links, due to overhydroxylation of lysine

residues within the telopeptides. As we have found that the

enzyme responsible for the hydroxylation of the telepeptide

lysine residues is lysyl hydroxylase-2 (LH2), it was examined

whether LH2 is increased in fibrotic lesions. TGF-b is a key

mediator in fibrosis, and therefore its effect on the formation

of pyridinolines was examined.

Material and methods Using real-time PCR, LH2 mRNA

expression was measured in fibroblasts cultured from the

fibrotic skin of systemic sclerosis (SSc) patients. Furthermore,

the amount of pyridinoline cross-links was analysed in the

matrix deposited by fibroblasts stimulated with TGF-b.

Results Elevated LH2 mRNA expression levels were found

in SSc fibroblasts, which result in increased amounts of pyri-

dinoline cross-links. Furthermore, increased pyridinoline

levels were found in collagen deposited by fibroblasts stimu-

lated with TGF-b, which was a consequence of increased

LH2 mRNA levels.

Conclusion These data demonstrate for the first time that

during fibrotic processes, TGF-b plays a key role in the formation

of pyridinoline cross-links via the induction of LH2 expression.

Regulation of the small GTPase RhoA bysyndecan-4 and integrins in focal adhesionformation

Atsuko Yoneda, Athanassios Dovas, Hinke A.B.

Multhaupt and John R. CouchmanCell & Molecular Biology Section, Division of Biomedical

Sciences, Imperial College London, London, UK

Introduction The transmembrane heparan sulfate proteogly-

can, syndecan-4, and integrins are important receptors for focal

adhesion (FA) formation on fibronectin (FN) substrates. The

small GTPase RhoA is also known to regulate FA and

stress fiber formation. It has been suggested that syndecan-4 and

integrins co-operatively regulate the assembly of FA in a Rho-

dependent manner, but the mechanism is unclear. Here, we

examined the function of RhoA and the Rho effector kinases

ROCKs in syndecan-4 signalling on the process of FA formation

and the possible mechanism by which syndecan-4 may regulate

RhoA activity.

Methods Primary rat embryonic fibroblasts (REFs) were

seeded on FN or ‘RGD’-containing integrin-binding domain

of FN and lysed at various time points. The amount of active

form of RhoA in each lysate was analysed by pull-down

experiments.

Results and discussion The relative activities of RhoA showed

one peak in the process of FA formation on FN, whereas no peak

was obtained on the integrin-binding domain. The one peak of

RhoA activity on integrin-binding domain was restored by add-

ition of heparin-binding domain into medium. These results sug-

gested that a signal through syndecan-4 link to the Rho pathway.

Both ROCK-I and -II isozymes were present in REF cell lysates

and each could be specifically immunoprecipitated. The ROCK

kinase activities in immunoprecipitates were analysed using

GST-myosin light chain as a substrate. The amount of ROCK-I

and-IIactivitieschangedthrough theadhesionprocessonFNand

appeared to be independently regulated. Therefore, one or both

ROCKsmaybedownstream ofa syndecan-4-mediated signalling

response through RhoA. The core protein of syndecan-4 can

directly bind to and activate PKC-a. We found that PKC-a could

phosphorylate Rho-Guanine Nucleotide Dissociation Inhibitor

(GDI) in vitro. It has been suggested that PKC-a-mediated phos-

phorylation of Rho GDI stimulates GDI dissociation, thereby

resultinginRhoactivation. It ispossible that syndecan-4regulates

Rho/ROCK pathway through PKC-a activation on the processof

FA formation.

TGF-b1 induces epithelial–mesenchymaltransition but not myofibroblasttransdifferentiation in primary cultures ofhuman epithelial renal tubular cells

Franca Anglani,* Monica Forino,* Luisa Murer,†

Manuela Dalla Vella,† Dorella Del Prete,* Monica

Ceol,* Giovanni Gambaro* and Angela D’Angelo*

*Laboratory of Molecular Biology, Division of Nephrology,

Department of Medical and Surgical Sciences; †Department of

Pediatrics, University of Padua, Padova, Italy

Introduction Although the origin of renal interstitial myofi-

broblasts is still a matter of debate (Powell et al. 1999), emer-

ging evidences suggest that they may derive from tubular

epithelial cells. Since the well-known mesenchymal origin of

human tubular epithelial cells (HUTECs), our working

hypothesis is that in renal fibrogenesis, fibrogenetic cytokines

could reactivate the mesenchymal program [epithelial–

mesenchymal transition (EMT)], switched-off during renal

nephrogenesis, leading to differentiation into MF. TGF-b1 is



a key modulator of the myofibroblast phenotype in fibroblasts

during the process of wound healing and in mesangial cells in

culture. TGF-b1 is also able to induce EMT in a variety of cell

types. However, most of these studies including the recent one

on SV40-transformed HKC-8 human tubular cells (Yang &

Liu 2001) have been carried out in immortalized cells.

Materials and methods Primary HUTEC cultures were estab-

lished from histologically normal human renal cortexes, obtained

from surgical biopsies performed following informed consent in

paediatric patients undergoing surgery because of extrinsic pye-

louretheral obstruction. HUTECs were cultured for 4 and 6 days

on plastic or type-I collagen-coated plates with or without 1, 5, 10

and 50 ng/ml TGF-b1. Time-course experiments (24 h to 6 days)

with 1 ng/ml TGF-b1 were also performed on primary human

dermal fibroblasts, used as control mesenchymal cells, and

HUTECs. Control conditions were represented by cells main-

tained for 4 and 6 days in 1% serum without TGF-b1. The

EMT process was monitored by morphology and immunophe-

notyping for a-SMA, cytokeratin 8–18, E-cadherin, vimentin and

Ki67. Quantitative comparative RT/PCR evaluated the expres-

sion of collagen-III and -V, fibronectin, tenascin, MMP-2, CTGF,

E-cadherin and cadherin 11 genes. TGF-b1 regulation of a-SMA

was investigated at both transcriptional and translational

(Western blot) levels.

Results and discussion TGF-b1-driven EMT was documented

morphologically, biochemically and molecularly. HUTEC mor-

phology changes following TGF-b1 exposure were already evi-

dent at 24 h and with as little as 1 ng/ml of TGF-b1; HUTECs

acquired a spindle shape with front-end/back-end polarity, like

fibroblasts. The transition was characterized by drastic up-reg-

ulation of all the mesenchymal markers studied, including

CTGF, and down-regulation of cytokeratin and E-cadherin

expression. These phenomena were dose-dependent and

favuored by growth on collagen-I. TGF-b1 treatment did not

induce MF conversion, because it induces neither de novo

expression of �-SMA gene nor the myofibroblast phenotype.

We demonstrate that the TGF-b1-driven EMT is characterized

by the re-appearance of developmental gene networks which, in

incomplete or uncoordinate fashion, could contribute to the

pathogenesis of renal fibrosis.

References

Powell D.W. et al. (1999) Myofibroblasts. I: paracrine

cells important in health and diseases. Am. J. Physiol. 277,

C1–C19.

Yang J. & Liu Y. (2001) Dissection of key events in tubular

epithelial to myofibroblast transition and its implications in

renal interstitial fibrosis. Am. J. Pathol. 159, 1465–1475.

Mesangial matrix-activated mononuclear cellsexpress functional scavenger receptors andaccumulate intracellular lipid

Enam Rahman,* Ravinder S. Chana,† Xiong Z.

Ruan,* Stephen H. Powis,* Zac Varghese* and

David C. Wheeler*

*Centre for Nephrology and the Department of Medicine, Royal

Free & University College Medical School, London; †Department

of Cell Physiology and Pharmacology, University of Leicester,

Leicester, UK

Introduction Monocyte recruitment into the mesangium and

foam cell formation are recognized features of glomerular

injury. External signals encountered by these infiltrating cells

may determine their behaviour and thereby potentially influ-

ence disease outcomes. Our previous studies indicate that

activation of monocytes by mesangial matrix stimulates the

production of a variety of mediators including inflammatory

cytokines and matrix degrading enzymes.

Methods Using expression of peroxisome proliferator acti-

vator receptor-g (PPAR-g) and scavenger receptor (ScR) as

differentiation markers, we examined whether matrix activa-

tion was associated with the expression of monocyte charac-

teristics usually associated with a macrophage phenotype.

THP-1 mononuclear cells were incubated for 7 days with

500 mg/ml solublized matrix extracted from cultured human

mesangial cells.

Results Using phorbol methyl ester (PMA) (125 nm) and albu-

min (500mg/ml) as positive and negative controls, respectively,

we demonstrated that matrix activation of monocytes led to

intracellular lipid accumulation as demonstrated by oil red O

staining. Matrix activation was also associated with a

concentration-dependent increase in the expression of both ScR

and PPAR-g mRNA and a corresponding increase in PPAR-gprotein expression on Western blotting. The presence of func-

tional ScR was confirmed using FACS analysis in

which incubation of matrix-activated monocytes with

Dil-labelled acetylated low-density lipoprotein (LDL) led to an

increase in mean fluorescent intensity of 373% (P < 0.001) as

compared to albumin (100%) and PMA (423%). This could be

inhibited by addition of excess unlabelled ligand, suggesting



specific binding to the ScR. Furthermore, incubation of LDL with

mesangial matrix in the absence of cells led to enhanced electro-

phoretic mobility of recovered lipoprotein on agarose gel. A

similar shift was seen when LDL was incubated with Cu2þ, a

powerful lipoprotein oxidant.

Discussion These results demonstrate that interactions

with mesangial matrix induce expression of monocyte charac-

teristics associated with a macrophage phenotype and promote

oxidation of LDL, thereby converting it to an ScR ligand. Such

observations may help to explain foam cell formation in the

mesangium in the context of glomerular disease.

Decorin affects endothelial cells by interactingwith IGF-I and its receptor

Elke SchonherrMatrix Biology & Tissue Repair Research Unit, University of

Wales College of Medicine Dental School, Cardiff, UK

Introduction Endothelial cells (ECs) undergoing angiogenesis

start to synthesize decorin, a member of the family of small

proteoglycans with leucine-rich repeats. Previously, we could

show that decorin synthesis in ECs leads to capillary formation

and survival of ECs in an angiogenesis model (Schonherr et al.

1999). In this model, decorin enhanced the phosphorylation of

protein kinase B (Akt) and subsequently induced the cyclin-

dependent kinase inhibitor p21 (Schonherr et al. 2001).

Materials and methods Cell culture: ECs of the permanent cell

line EA.hy 926 were cultured on collagen type-I-coated dishes in

Waymouth MAB 87/3 medium containing 1% heat-inactivated

fetal calf serum and antibiotics. Decorin purified from fibroblast

cultures and/or the respective inhibitors were added.Binding studies: Decorin and IGF-I were labelled with

125I-iodine. (1) Decorin was run on an SDS-PAGE and transferred

to nitrocellulose. Blots were probed with 125I-IGF-I and exposed

to X-ray films. (2) A Sepharose CL-4B gel filtration column

was equilibrated with or without decorin containing buffer and125I-IGF-I (500.000 cpm) was applied. The elution profiles were

monitored. (3) IGF-receptor was immunoprecipitated from

EA.hy 926 cells with the antibody (sc-713) against the b-chain

of the receptor. The immune complex was incubated with

labelled or unlabelled decorin or IGF-I (as indicated). Immune

complexes without receptor were used as controls.

Results To analyse how decorin activates Akt, inhibitors of

different receptor tyrosine kinases were tested. The EGF-recep-

tor inhibitor tyrphostin AG1478 (10mm) had no effect on dec-

orin-induced Akt posphorylation, but preincubation with

tyrphostin AG1024 (10mm), an inhibitor of the insulin and the

IGF-I receptor tyrosine kinases, inhibited Akt phophorylation

and p21 expression. Combined addition of IGF-I and decorin to

ECs in culture had an additive effect on Akt phosphorylation.

Binding experiments with 125I-IGF-I to decorin immobilized on

nitrocellulose revealed that both the core protein and the pro-

teoglycan bind 125I-IGF-I. Binding of 125I-IGF-I to decorin in

solution showed the dose-dependent formation of a high mol-

ecular weight complex that eluted in the included volume of the

column. Immunoprecipitated IGF-I receptor bound 125I–IGF-I

as well as 125I-decorin. In addition, 125I-IGF-I bound to the

receptor could be displaced by unlabelled decorin.

Discussion These results suggest that decorin can bind to

IGF-I and to its receptor and that this interaction enhances

Akt phosphorylation in ECs. The effect of decorin on the IGF-I

receptor could be mediated by direct interaction. However, a

further possibility is that decorin couples trace amounts of

IGF-I from the medium or the collagen on the dishes to a

larger complex, which can more effectively activate the IGF-I

receptor. These possibilities as well as the role of IGF binding

proteins will need further investigation.

References

Schonherr E. et al. (1999) Paracrine or virus-mediated induction

of decorin expression by endothelial cells contributes to tube

formation and prevention of apoptosis in collagen lattices. Eur.

J. Cell Biol. 78, 44–55.

Schonherr E. et al. (2001) Decorin-mediated signal transduction

in endothelial cells. Involvement of Akt/protein kinase B in up-

regulation of p21 (WAF1/CIP1) but not p27 (KIP1). J. Biol.

Chem. 276, 40687–40692.

Domain deletions outside of the catalyticdomain of drosophila tolloid result in loss ofprotease activity

E.G. Canty and K.E. KadlerWellcome Trust Centre for Cell Matrix Research, University of


Introduction The tolloid (TLD)-related zinc metalloproteinases

serve multiple functions in development, being involved in both

extracellular matrix assembly and TGF-b signalling. Signalling by



bone morphogenetic proteins BMPs, which form a subgroup of

the TGF-b superfamily, is a highly regulated process which

involves cleavage of extracellular BMP antagonists by TLD-like

(TLL) proteases. In vertebrates, BMP-1 and mTLL-1 cleave the

BMP antagonist chordin to release free BMPs, while in inverte-

brates, TLD cleaves the BMP antagonist short gastrulation

(SOG) to release decapentaplegic (DPP). Mammalian TLD

(mTLD), TLL-1 and TLL-2 share the same domain structure

as drosophila TLD, while BMP-1 is a shorter splice variant of

mTLD which lacks the EGF-2, CUB-4 and CUB-5 domains.

Material and methods Site-directed mutagenesis was used to

produce TLD constructs lacking various domains. These were

subcloned into the vector pAc5.1V5His, along with wild-type

TLD and full-length SOG. Transient transfections in droso-

phila S2 cells were used to produce recombinant proteins, and

aliquots of medium containing the appropriate recombinant

proteins were dialysed or desalted as required. In vitro digests

of SOG by TLD and the TLD mutants were performed over-

night at 25 ˚C in the presence of recombinant DPP (R & D

Systems). Digests were analysed by SDS Page and Western

blotting, and full-length SOG and SOG fragments were

detected using an anti-V5 antibody (Invitrogen).

Results Wild-type TLD was able to cleave SOG at three

distinct sites as previously reported (Marques et al. 1997).

A ‘BMP1-like’ TLD construct, truncated after the CUB3

domain, was efficiently secreted by S2 cells but was unable

to cleave SOG. Wild-type TLD was also found to have

enhanced activity in the presence of calcium ions, indicating

that it is a calcium-dependent protease. Removal of the puta-

tive calcium-binding domains from TLD (EGF1 and EGF2)

resulted in a decreased amount of enzyme present in the

medium of the transfected S2 cells and less (DEGF1) or

abolished (DEGF2 and double deletion) protease activity.

Discussion These preliminary findings indicate that the EGF-

2, CUB-4 and CUB-5 domains are required for cleavage of SOG

by TLD. This is in direct contrast to the cleavage of chordin

which is actually cleaved most efficiently by the short-splice

variant BMP-1 (Scott et al. 1999). In vitro cleavage of SOG

also requires the presence of DPP, whereas chordin is cleaved

in the absence of exogenous BMPs. It is possible that these

domains could be involved in interactions with the substrate or

alternatively with DPP itself. The putative calcium-binding

domains, EGF1 and EGF2, appear to be required both for

secretion and stability of the protein as well as for protease

activity. The mechanism whereby calcium augments the pro-

tease activity of TLD is unknown, but it could aid the binding

of enzyme to substrate or create specific conformational changes

in the enzyme to increase activity.

References

Marques G. et al. (1997) Production of a DPP activity gradient in

the early Drosophila embryo through the opposing actions of

the SOG and TLD proteins. Cell 91 (3), 417–426.

Scott I.C. et al. (1999) Mammalian BMP-1/Tolloid-related

metalloproteinases have differential enzymatic activities and

distributions of expression relevant to patterning and skeleto-

genesis. Dev Biol. 213 (2), 283–300.

NADPH oxidases and MMP-9/TIMP-1 balance inhuman glomeruli: putative links to diabeticnephropathy

C. Millet,*† C. Trocme,* S. Vergnaud,*

S. Papacatzis,* J.L. Descotes,‡ F. Morel* and

P. Zaoui*†

*GREPI EA 2938 J. Fourier University Enzymology Laboratory;†Nephrology DUNE Department; ‡Urology DUNE Department,

CHU Grenoble, Grenoble Cedex, France

Study aim Glomerular basement membrane thickening, the

hallmark of diabetic nephropathy, is thought to be related to an

enhanced oxidative stress and reduced matrix proteolysis. Our

study concerned the mRNA and protein expression of NADPH

oxidase (NOX) components, MMP-2, MMP-9 and TIMP-1 in

freshly isolated human glomeruli as well as enzymatic activities

and their modulation by glucose, H2O2 and angiotensin-2.

Material and methods NOX, cytosolic and membrane-bound

associated proteins and mRNA were analysed by RT-PCR and

Western blotting after glomerular extraction. Oxidase activity

was identified by cytochrome c reduction and chemilumines-

cence. Gelatinases and inhibitors were semiquantitatively

assessed by RT-PCR, gelatin zymography and ELISA in a

model of glomerular conditioned survival.

Results NOX-2, NOX-4 and membrane-bound and cytoso-

lic factors could be observed in freshly extracted glomeruli

(RNAþ protein). p40phox, p67phox and p47phox molecular

weights were increased compared to their phagocytic counter-

parts advocating for specific glomerular analogues, and a slight

specific oxidase activity was retrieved in isolated glomeruli.



Also, mRNA coding for MMP-2, -9 and TIMP-1, -2 were

detected. High glucose concentrations (25 mm) reduced

TIMP-1 release in glomerular survival media and MMP-2

activity in glomerular extracts. On the opposite, angiotensin-2

significantly induced MMP-2 and -9 activities in the survival

media as well as H2O2 in glomerular extracts, while addition

of 25 mm glucose blunted these findings.

Conclusion Glomerular matrix remodelling, the backbone of

renal fibrosis in diabetic patients, could be induced by H2O2

from specific glomerular NADPH oxidases under the influence

of extra-cellular glucose and angiotensin-2 and could partici-

pate in the control of MMP activities.

Up-regulated lactate, TGF-b and collagensynthesis in the ischaemic skin of patientswith peripheral vascular disease

S.J Dalton,*† C.V, Whiting,† D.C. Mitchell* and

J.F. Tarlton†

*Department of Vascular Surgery, Southmead Hospital, Bristol;†Matrix Biology Research Group, Division of Molecular and

Cellular Biology, Bristol University, Bristol, UK

Introduction We have previously shown that dermal

hypoxia alters collagen turnover in chronically ischaemic

skin; however, no mechanism for this has been determined.

In cultured human dermal fibroblasts, hypoxia causes an up-

regulation of collagen synthesis, probably mediated by TGF-

b (Falanga et al. 2002) in the presence of increased lactate.

Here, we examine whether these processes are present in vivo

in chronically ischaemic skin.

Materials and methods Paired biopsies of uninjured skin

were harvested at below knee amputation from 16 patients

with a history of peripheral vascular disease (PVD), following

the quantification of ischaemia, using the ankle brachial

pressure index (ABPI) and by lactate measurement. Non-

ischaemic samples were taken proximally from the amputa-

tion resection margin and ischaemic samples from a

predetermined distal site. Site-matched biopsies were taken

for control at total knee replacement and varicose vein opera-

tions. Lactate levels were measured using enzymatic

determination (Sigma, UK), collagen type-I synthesis was

determined by immunoassay for released C-terminal propep-

tide (PICP) (Prolagen C, Quidel) and TGF-b by ELISA. TGF-

b RI and RII were localized using immunohistochemistry.

Results The ABPI in all patients with PVD was <0.4 indicating

severe ischaemia. Levels of lactate were elevated in the ischaemic

tissue of these patients when compared to nonischaemic samples

(P < 0.001), and an up-regulation of collagen type-I synthesis was

demonstrated in the ischaemic samples (P < 0.01). Levels of TGF-

b were also raised (P < 0.05). TGF-b RI and RII were expressed

on dermal fibroblasts, keratinocytes and endothelial cells.

Discussion Increased lactate levels resulting from hypoxic

metabolism have been demonstrated in skin flaps of animal

models (Hoopes & Im 1978); however, lactate levels in human

tissue, as a direct assessment of chronic ischaemia, have not

previously been reported. Hypoxia, lactate and TGF-bhave been shown to stimulate collagen synthesis in vitro (Falanga

et al. 2002; Cerbon-Ambriz et al. 1991) but not in vivo in PVD.

These findings are consistent with the hypothesis that chronic

hypoxia leads to changes in the ECM of uninjured but ischaemic

skin and may predispose it to dermal failure.

References

Cerbon-Ambriz J., Cerbon-Solorzano J., Rojkind M. (1991)

Regulation of collagen production in freshly isolated cell

populations from normal and cirrhotic rat liver, effect of

lactate. Hepatology 13, 551–556.

Falanga V., Zhou L. & Yufit T. (2002) Low oxygen tension

stimulates collagen synthesis and COL1A1 transcription

through the action of TGF-beta1. J. Cell Physiol. 191, 42–50.

Hoopes J.E. & Im M.J. (1978) Skin flap necrosis in guinea pigs:

limitation of glucose supply and accumulation of lactate. Plast.

Reconstr. Surg. 61, 748–752.

The importance of elastin and fibulin-5 on spinedevelopment: a study of elastin KO and fibulin-5KO on the development of vertebral body andintervertebral disc

Jing Yu,* Sean Mcleans,† Hiromi Yanagisawa,‡

C. Peter Winlove,§ Sally Roberts,{ Robert P.

Mecham† and Jill Urban*

*Laboratory of Physiology, Oxford University, Oxford, UK;†Department of Cell Biology and Physiology, Washington

University School of Medicine; ‡Department of Molecular

Biology, University of Texas Southwestern Medical Center, USA;§School of Physics, Exeter University, Exeter, UK; ¶Centre for

Spinal Studies, RJAH Orthopaedic Hospital, Oswestry, UK

Introduction Idiopathic scoliosis is the most common scolio-

sis and generally develops during juvenile or adolescent



growth spurt (Stehbens 2003). It was reported that elastic fibre

system might play a role in some idiopathic scoliosis patients

(Hadley-Miller et al. 1994). Indeed, transgenic mice with

defects in elastic fibre system (including elastin null and

fibulin-5 null) appear to develop severe kyphoscoliosis. The

aim of this study was to understand how such defects exert

their effect on the spinal column.

Materials and methods Newborn elastin KO and 16-week-old

fibulin-5 KO mice spines were fixed in 10% formalin. Paraffin-

embedded sections (20mm) were stained with haematoxylin &

eosin (H&E) and alcian blue after the sections were dewaxed and

rehydrated. The sections were examined by light microscopy.

Results H&E staining revealed that those newborn elastin

KO mice seem to have a delayed ossification in vertebral

bodies compared to that of wild-type. Also, cell morphology

in IVD appears very much different. Cells in outer annular

(OA) and endplate regions are much round-like comparing

fibroblast-like cells in wild-type. In addition, GAG

expressions showed by alcian blue staining appear much

sparse and irregular in the matrix of IVD in newborn

elastin KO mice. Fibulin-5 KO mice (16 weeks) seem to

have many cell clusters or clones in the growth plate,

which is an indication of abnormal growth. Our results

reveal the importance of elastin and fibulin-5 on the devel-

opment of spine.

Discussion Kypho-scoliosis is a spinal deformity. Several differ-

ent spinal tissues, e.g. muscle, vertebrae, IVD and ligament, are

involved in the stability and load carriage of the spinal column.

Some gene defects in these load-bearing structures can lead

to the scoliotic deformity, e.g. elastin null, fibulin-5 null, col-

lagen-II null (Aszodi et al. 1998), perlecan null (Costell et al.

1999) and LTBP3-null (Dabovic et al. 2002) as well as a

mutant in a muscle-specific protein (Blanco et al. 2001), but

other defects [such as collagen-IX KO’s (Kimura et al. 1996)]

do not. The relationship between defects in these structures

and development of scoliotic is still unclear.

References

Aszodi A. et al. (1998) Collagen II is essential for the removal of

the notochord and the formation of intervertebral discs. J. Cell

Biol. 143 (5), 1399–1412.

Blanco G. et al. (2001) The kyphoscoliosis (ky) mouse is deficient

in hypertrophic responses and is caused by a mutation in a

novel muscle-specific protein. Hum. Mol. Genet. 10 (1), 9–16.

Costell M. et al. (1999) Perlecan maintains the integrity of

cartilage and some basement membranes. J. Cell Biol. 147 (5),

1109–1122.

Dabovic B. et al. (2002) Bone abnormalities in latent TGF-[beta]

binding protein (LTBP)-3-null mice indicate a role for LTBP-3

in modulating TGF-[beta] bioavailability. J. Cell Biol. 156 (2),

227–232.

Hadley-Miller N. et al. (1994) The potential role of the elastic

fiber system in adolescent idiopathic scoliosis. J. Bone Joint

Surg. Am. 76 (8), 1193–1206.

Kimura T. et al. (1996) Progressive degeneration of articular

cartilage and intervertebral discs. An experimental study in

transgenic mice bearing a type IX collagen mutation. Int.

Orthop. 20 (3), 177–181.

Stehbens W.E. (2003) Pathogenesis of idiopathic scoliosis

revisited. Exp. Mol. Pathol. 74, 49–60.

The isoprostane 8-iso-PGF2a suppresses monocyteadhesion to microvascular endothelial cells: a rolein limiting inflammation and fibrosis?

Anila Kumar, Edward Kingdon and Jill NormanCentre for Nephrology, Department of Medicine, Royal Free &

University College Medical School, London, UK

Introduction Isoprostanes, produced in vivo by nonenzy-

matic free radical-induced lipid peroxidation, are recognized mar-

kers of oxidative stress with elevated serum and urine levels of 8-

iso-PGF2a (iP) reported in a variety of fibrotic diseases including

end-stage renal disease, systemic sclerosis and atherosclerosis. It

has been suggested that iP may also have pathogenic functions.

Many fibrotic diseases are characterized by early perivascular

inflammatory infiltrates, and inflammation has been shown to

be a critical component of the fibrotic process. Adhesion of

monocytes to the capillary endothelium is an initiating event in

inflammation and, in line with a proposed pathological role

for iP, we hypothesized that iP might stimulate monocyte

adhesion and thus promote fibrosis.

Methods Human monocytes (U937 or THP-1) were added to

confluent MECs and the number of adhered monocytes meas-

ured colorimetrically or by cell counting.

Results In dose–response assays (10�12�10�5 M),

iP > 10�7 M inhibited monocyte adhesion (by 76% P < 0.01)

to quiescent or proliferating human and rat renal MECs. In

contrast, iP stimulated U937 adhesion to HUVEC (as reported

by Leitinger et al. FASEB J 15,1254, 2001) demonstrating

diverse effects of iP on different ECs. iP had no effect on



viability (trypan blue exclusion), proliferation (3H-thymidine

incorporation) or apoptosis (Annexin V) of U937 or MECs.

Inhibition of adhesion was dependent on preincubation (min-

imum 1–2 h) of iP; simultaneous addition of monocytes and iP

to HMEC or treatment of U937 had no effect; suggesting that

inhibition is due to iP-induced changes in HMEC. These

changes appear to be independent of de novo protein synthe-

sis. Furthermore, iP, added either before or after TNF-a,

blocked TNF-a-induced monocyte adhesion. The inhibitory

effect of iP was mimicked by a thromboxane receptor (TxR)

agonist (U46619, 10 mm) and blocked by a receptor antagonist

(SQ29548, 10 mm) indicating a TxR-mediated process. Experi-

ments using signal transduction pathway inhibitors

(SB203580, curcumin and PD98059) implicate p38 and JNK,

but not ERK, in iP-induced suppression of monocyte adhesion.

In addition to a direct effect, conditioned medium (CM) trans-

fer experiments suggest that iP induces a secondary mediator

which also suppresses monocyte adhesion but via an alterna-

tive mechanism which is TxR-independent and dependent on

new protein synthesis. This pathway also involves activation

of p38 but is only partially dependent on JNK.

Conclusion The data show that iP can suppress the

attachment of monocytes to MECs via two independent

pathways indicating an unexpected, potentially protective

effect of iP in the microvasculature. Characterization of the

mechanisms and mediators involved in this process may

provide novel targets for intervention in inflammation and

subsequent fibrosis.

Fluorescence lifetime imaging of articularcartilage

C.B. Talbot,* M.J. Lever,* R.K.P. Benninger,†

J. Mcginty,† J. Requejo-Isidro,† D.S. Elson,†

P.M.W. French,† A. Sandison,‡ A.L. Wallace,‡

H. Nagase,‡ Y. Itoh,‡ J. Saklatvala‡ and

T. Vincent‡*Department of Bioengineering; †Department of Physics, Imperial

College London; ‡Imperial College School of Medicine, London, UK

Introduction Fluorescence lifetime imaging (FLIM) provides a

contrast parameter for tissue components independent of wave-

length, spectrum or polarization. It has been shown that contrast

between autofluorescent matrix components such as collagen and

elastin is available through FLIM (Siegel et al. 2003; Dowling et al.

1998), which is not clear using other fluorescent techniques. The

lifetime of collagen has been studied whilst in solution; however,

research is ongoing to quantify its fluorescence whilst in tissue. The

aim of this study was to investigate the contrast available by

applying fluorescence lifetime imaging (FLIM) to articular carti-

lage, which has extracellular matrix containing collagen type-II

and proteoglycan.

Materials and methods A time-gated FLIM system was used

to analyse the articular cartilage from unstained sections of

human femurs as well as from the knee joints young and old

sheep. The sections were also observed under a conventional

fluorescence microscope, and for the human femurs, adjacent

H&E-stained sections were also obtained. Details of the FLIM

system can be found elsewhere (Dowling et al. 1998), with the

laser source being tuneable, allowing excitation over a range

350–600 nm. Fluorescence half-lives were obtained using single

exponentials applied to the intensity decay data.

Results It was found that younger cartilage was generally

less fluorescent than older. For example, when exciting at

401 nm and observing above 450 nm, it was found that the

young sheep samples provided a signal to noise ratio too low

to obtain lifetime data, whereas other samples fluoresced

brightly. It was also found that within the matrix, FLIM

provided contrast unavailable with conventional fluorescence

microscopy and, in the case of human femurs, with H&E

stains.

Discussion In the study of collagen, it is believed that the cross-

linkages are responsible for fluorescence (Richards-Kortum &

Sevick-Muraca 1996). Because the cross-linkages increase with

age, the fluorescence intensity is also expected to increase. The

lesser amount of fluorescence from the younger samples there-

fore suggests that the dominant fluorophore in cartilage is col-

lagen. The contrast obtained with FLIM in the cartilage indicates

nonuniformity in the matrix structure.

Acknowledgements This work was supported by the EPSRC,

BBSRC, Kentech Instruments, a DTI Beacon award and a

Wellcome Trust Showcase award.

References

Dowling K. et al. (1998) Fluorescence lifetime imaging with

picosecond resolution for biomedical applications. Optics Lett.

23 (10), 810–812.

Richards-Kortum R. & Sevick-Muraca E. (1996) Quantitative

optical spectroscopy for tissue diagnosis. Annu. Rev. Phys.

Chem. 47, 555–606.



Siegel J. et al. (2003) Studying biological tissue with fluorescence

lifetime imaging: microscopy, endoscopy and complex decay

profiles. Appl. Optics 42 (16), 2995–3004.

Factors affecting oxygen concentrationgradients across articular cartilage

Shengda Zhou,* Zhanfeng Cui* and Jill P.G.

Urban†

*Department of Engineering Science; †Physiology Laboratory,

Oxford University, Oxford, UK

Introduction Although it is commonly stated that oxygen con-

centrations fall to below 1% oxygen in the deep zone of articular

cartilage, the evidence for this is sketchy and there is little informa-

tion on the oxygen concentrations found in cartilage in vivo. The

oxygen levels could be important as a number of studies have

showed that low oxygen concentrations affect cartilage metab-

olism adversely (Grimshaw & Mason 2000; Lee & Urban 1997).

As an initial step in determining oxygen concentration profiles

across cartilage, we have measured the rate of oxygen consump-

tion by chondrocytes. We then used this information to calculate

the effect of various factors, such as presence of serum, cell density

and perfusion from the subchondral bone on oxygen gradients

across the joint.

Methods Chondrocytes were isolated from the metacarpal–

phalangeal joint of adult steers and cultured for 7 days in algi-

nate beads. The rate of oxygen consumption was measured in a

respiration chamber using an oxygen electrode. The effect of

oxygen tension, time in culture and serum addition on oxygen

consumption was investigated. This data was used to predict the

oxygen tension profiles across articular cartilage. The variation

in oxygen tension with distance across the joint was predicted by

solving a one-dimensional reaction–diffusion equation using a

finite-difference method. The effect of influx of oxygen from the

subchondral bone on oxygen profiles was estimated.

Results Oxygen consumption rates were significantly (2.5

fold) greater on the first day after isolation; on the second

and subsequent days of culture, rates were similar to those

reported by others (c. 10 nmol. million cells�1 h�1) (Bywaters

1937). The presence of serum increased the rate by around

50%. The consumption rate was relatively independent of

oxygen tension between 5 and 21% oxygen, but below 5%

oxygen, consumption rates fell in a concentration-dependent

manner. Calculations of the oxygen profile across cartilage

showed that the concentration was sensitive to consumption

rate and was also critically dependent on the extent of supply

from the subchondral bone. Calculations also showed that

growth factor addition led to a fall in oxygen tension through-

out the tissue.

Discussion Here, we show that oxygen concentration

profiles across cartilage are affected by cellular metabolic

rates; addition of serum or growth factors for instance, by

increasing consumption rates, can lower oxygen concentra-

tions within the tissue significantly. Transport from the sub-

chondral bone can also strongly affect the profile of oxygen

across cartilage. Although its influence is usually ignored,

there is anatomical and MRI evidence (Bashir et al. 1997)

that this transport route is significant. Changes known to occur

in the subchondral bone in osteoarthritis could alter transport

into cartilage via this route and hence affect concentrations of

oxygen and other important nutrients across the joint.

References

Bashir A. et al. (1997) Glycosaminoglycan in articular cartilage:

in vivo assessment with delayed Gd (DTPA) (2-) -enhanced MR

imaging. Radiology 205, 551–558.

Bywaters E. (1937) Metabolism of joint tissues. J. Pathol.

Bacteriol. 44, 247–268.

Grimshaw M.J. & Mason R.M. (2000) Bovine articular

chondrocyte function in vitro depends upon oxygen tension.

Osteoarthritis Cartilage 8, 386–392.

Lee R.B. & Urban J.P. (1997) Evidence for a negative Pasteur

effect in articular cartilage. Biochem. J. 321, 95–102.

A novel keratanase-generated keratan sulphateantibody and its applications

B.C. Kerr, C.E. Hughes, A. Hayes and B. CatersonConnective Tissue Biology Laboratories, Department of

Bioscience, Cardiff University, Wales, UK

Introduction The sequencing of the genome has provided us

with important information regarding the primary structure of

many matrix proteins. This in turn has lead to advances in studies

of the functions of post-translational modifications on connective

tissueproteoglycans (PGs).Changes inGAGstructurewithageing

and disease have been well documented (Thonar et al. 1986;

Brown et al. 1998). However, little is known about the exact

sites of and differential substitution of GAGs on the aggrecan

core protein and how these substitutions facilitate normal func-

tionor the changes seenwithdisease.The CS : KS ratioof substitu-



tion change significantly, with KS levels increasing with age and

decreasing with the onset of disease. Objective was to produce

monoclonal antibody (MAb) reagents to keratanase (k’ase) gen-

erated stub epitopes, that couldbe used to help identify, character-

ize and quantify sites of KS substitution on PGs, providing the

potential to determine how the arrangement of such substitutions

change with development, ageing and pathology.

Methods Bovine Nasal Cartilage aggrecan (BNC A1D1) was

trypsin digested, generating a range of glycosaminoglycan

(GAG) fragments. The sample was then subjected to anion-

exchange and size exclusion chromatography to separate

KS from CS fragments. Fractions collected were analysed by

SDS-PAGE and Western blotting. Fractions positive for KS

were pooled and kinase digested to expose the KS stub anti-

gens. Immunization and fusions were carried out as previously

described (Nieduszynski et al. 1990). Initial screenings were

carried out using ELISA. Briefly, 96-well microtitre plates were

coated with the immunizing antigen overnight at 37 ˚C. The

plates were then blocked prior to the addition of hybridoma

media for 1–2 h at 37 ˚C. Binding was detected using an alkaline

phosphatase-conjugated secondary antibody for 1 h at 37 ˚C

prior to the addition of the substrate. Positive wells were further

screened by ELISA and SDS-PAGE using the immunizing anti-

gen, chondroitinase-digested BNC and an A1D1 BNC prepara-

tion to establish the kinase stub specificity of the hybridomas.

Further screenings by Western blotting was carried out on posi-

tive hybridomas selected. Antigens used included keratanase-

digested bovine corneal KS-PGs, keratanase-II-digested KS-PGs

and a nonkeratanase-digested corneal KS-PG sample.

Results Screening: Screening identified two positive hybri-

domas, B-KS-I and B-KS-II, which were specific for kinase-

generated KS stub. On screening, these antigens showed

reactivity specifically for kinase-digested BNC abc core, with

no reactivity to the nonkinased linear KS GAG epitopes.

Reactivity to kinase-digested corneal KS-PGs indicated that

the MAbs generated were indeed to a stub structure in the

KS chain and not to some linkage region epitope, amino acid

sequence or oligosaccharide present on the core protein.Application: Immunohistochemistry utilizing B-KS-I was

used to localize KS in a range of tissues along side anti-KS 5D4.

In human articular cartilage engineered grafts, labelling showed

B-KS-I and 5D4 to have broadly overlapping labelling patterns

for KS; however, label for B-KS-I had a much more restricted

and subtle tissue distribution than that of antibody 5D4.

Discussion These new KS stub MAbs have potential to be

used in many different areas of research. They may be used in

analysis of trypsin-digested purified aggrecan from cattle joints

of different ages to determine sites of KS substitution, which

remain common or change with development and ageing.

They may also be used in analysis of cartilage explant culture

metabolites to assess KS substitution on the aggrecan fragments

generated after stimulation of these cultures with cytokines such

as IL-1 or TNF-a. Collectively it will provide important new

information on the changing pattern of KS substitution in con-

nective tissue PGs with development, ageing and the onset of

pathology.

References

Brown G.M. et al. (1998) Human aggrecan keratan sulfate

undergoes structural changes during adolescent development.

J. Biol. Chem. 273, 26408–26414.

Nieduszynski I.A. et al. (1990) There are two major types of

skeletal keratan sulphates. Biochem. J. 271, 243–245.

Thonar E.J.M. et al. (1986) Articular Cartilage Biochemistry

273–283.

Changes in tendon extracellular matrixcomposition with age

E. Blain,* Y. Zhang,* D. Aeschlimann,†

B. Caterson* and V. Duance*

*CTBL, School of Biosciences, Cardiff University; †Matrix

Biology & Tissue Repair Research Unit, Dental School,

University of Wales College of Medicine, Cardiff, UK

Introduction A major aspect of the normal ageing process is the

loss of suppleness of tissues; skin becomes wrinkled and there is a

decline in joint flexibility. These gradual changes predominantly

result due to long-term alterations in the extracellular matrices

(ECMs) of structures including skin, tendons/ligaments, bones,

cartilage and blood vessels. ECMs are generally remodelled during

an individual’s life, but for tendons/ligaments this occurs at a slow

rate. Parameters such as slow turnover, long-term post-transla-

tional modifications and extensive cell–matrix interactions are

three aspects of ECM biology, which influence the mechanisms

of ageing. Ageing of tendon/ligament is a major problem affecting

the mobility of an increasingly ageing population, and age-related

changes lead to numerous musculoskeletal pathologies in old age.

Therefore, the initial objective of these studies was to compare the

biochemical composition of young vs. old tendon, with an aim

of elucidating the mechanisms controlling the ageing process in

tendon ECM.



Materials and methods Tail tendons were dissected from 2-, 9-,

12- or 22-month-old C57/Blacks, and both protein and RNA were

analysed for differences in ECM tendon composition with age

(n5 4). Proteoglycans were extracted in 4 m guanidine hydro-

chloride and sulphated glycosaminoglycans measured using

the DMMB assay. Collagens were extracted by acid hydrolysis

and total collagen measured using the hydroxyproline

assay; additionally, the ratio of collagen types in the ageing tail

tendons was analysed by Western blotting with antibodies raised

against collagen types I, III and V. The degree of tissue hydration

was determined by measuring the water content of the tendons.

Matrix metalloproteinase-2 (MMP-2) and MMP-9 expression

and activation were detected by gelatin substrate zymography,

and the presence of their inhibitors was assessed by reverse zymo-

graphy. To assess age changes at the transcriptional level, RNA

wasextracted from tendons, labelled withbiotinand cDNA arrays

(Affymetrix) performed.

Results The results indicate that there is a change in tendon

matrix composition with increasing age (Table 1). There is a

significant loss in tissue hydration and a reduction in the

amount of sulphated GAGs present in the 22-month tendons

compared with young tissue, whilst the amount of collagen

present does not significantly alter.

Analysis of MMP expression by gelatin zymography demon-

strated that there was a significant loss of MMP-9 expression in

the tendons of 9- and 12-month-old animals. MMP-9 was only

evident in the 2- and 22-month-old tendons indicative of develop-

mental turnover and a remodelling response, respectively. MMP-2

expression and activation was evident in all tendon ages analysed,

as were the MMP inhibitors – TIMP-1 and TIMP-2.

Discussion The data demonstrate that during the ageing pro-

cess, the composition of tendon ECM is modified. This may

compromise the response of the tissue to application of mechan-

ical loads hence the increased incidences of musculoskeletal inju-

ries, e.g. tendon/ligament sprains in elderly people. The reduction

in both water and sGAGs, and the increased expression of MMP-

9 may be responsible for changes observed in biomechanical

properties where the tendons become more brittle and less able

to withstand load. Understanding changes in the biochemical

composition may allow us to manipulate cell activity and ulti-

mately ECM structure and function to combat these age-related

effects and the musculoskeletal pathologies incurred.

The role of the cytoskeleton in articularcartilage chondrocyte homeostasis

Emma J. Blain, Sophie J. Gilbert and

Victor C. DuanceCTBL, School of Biosciences, Cardiff University, Museum

Avenue, Cardiff, UK

Introduction The aetiology of osteoarthritis (OA) is unknown

although abnormal loading of the joint is a contributory factor.

We have demonstrated previously that a cyclic, compressive

load (0.5 MPa, 1Hz) applied to immature articular cartilage

induces a significant increase in the expression and activation

of MMP-2 and MMP-9 (Blain et al. 2001). Using differential

RNA display, we identified a mechanically regulated gene –

thymosin �4 (Blain et al. 2002). The primary function of thy-

mosin �4 is in the sequestration of filamentous actin (F-actin).

Therefore, we hypothesize that the mechanical induction of

matrix degradation, i.e. the up-regulation of MMP gene expres-

sion, is initiated via the actin cytoskeleton, whether directly or

indirectly remains to be elucidated. Thus, the objective of this

study was to determine whether the actin cytoskeleton, in addi-

tion to the tubulin and vimentin cytoskeletal networks are

involved in the signalling pathways involved in chondrocyte

MMP regulation.

Materials and methods Primary chondrocytes, isolated from

7-day-old bovine calves, were seeded at a density of 1 · 106

cells/ml, and individual cytoskeletal elements were disrupted

with 10 mm cytochalasin-D (for F-actin), 10 mm colchicine (for

tubulin) or 5 mm acrylamide (for vimentin) for 1–7 days.

Amounts of sulphated glycosaminoglycan (sGAG) were deter-

mined using the DMMB assay, and total collagen content was

assessed using the hydroxyproline assay. MMP activity was

measured using gelatin substrate zymography and the amounts

of their inhibitors, the TIMPs, assessed by reverse zymography.

Results We have demonstrated that disruption of the cytoske-

letal elements can affect cartilage chondrocyte homeostasis.

There was a significant decrease in sGAG release for all three

cytoskeletal disruption treatments when compared to untreated

controls (P < 0.01). There was a reduction in total collagen

released from the cells which was significant after 7 days in

Table 1

2 months 9 months 12 months 22 months

sGAG (mg/mg)* 49.6± 8.7 — 40.1± 4.6 28.9± 0.9

Collagen (mg/mg)† 48.4± 5.3 39.9±3.4 40.2± 4.7 47.2± 4.3

Water content (%) 80.4± 3.9 74.6±2.4 74.5± 3.8 69.3± 2.8

*mg/mg wet weight tissue.†mg/mg dry weight tissue.



actin-disrupted (P5 0.012) and vimentin-disrupted cells

(P5 0.05). No collagen was detected in tubulin-disrupted cells

at any time point, which may be due to a decrease in cell

viability. Interestingly, at days 3 and 7, both tubulin and vimen-

tin disruption abrogated synthesis of pro-MMP-2 and signifi-

cantly reduced the amount of MMP-2 activation (P < 0.01).

Actin disruption significantly enhanced both synthesis and activa-

tion of MMP-2 (P < 0.02). In comparison, TIMP-1 expression

was also abrogated in cells without a functioning tubulin or

vimentin network, whereas in actin-disrupted cells, there was a

reduction in TIMP-1 compared to untreated controls (P < 0.001).

Discussion Clearly disruption of the cytoskeletal networks can

affect cartilage chondrocyte homeostasis. Thymosin �4 has been

shown to induce MMP activity in our cell culture system which

may be directly attributable to F-actin depolymerization. Addi-

tion of cytochalasin-D to chondrocytes revealed an increase in

MMP-2 synthesis/activation and reduced TIMP-1 expression

implicating the actin cytoskeleton in this process, whether directly

or indirectly remains to be determined. We are currently using

antibodies that recognize key signalling intermediates, i.e.

FAK125, p38 kinase and ERK 1/2 to assess the involvement of

these molecules in events proceeding cytoskeletal disruption and

prior to the mediation of MMP expression. We are also starting to

investigate the mechanisms involved in abrogation of MMP

synthesis after tubulin and vimentin disruption in chondrocytes.

Elucidation of the role that the three cytoskeletal elements play

in cartilage homeostasis will enable us to fully appreciate their

functions in cartilage tissue turnover and dysregulation in disease.

This work is supported by the EU 5th Framework and ARC

(Grant No. D0600).

References

Blain E. et al. (2001) Up-regulation of matrix metalloproteinase

expression and activation following cyclical compressive

loading of articular cartilage in vitro. Arch. Biochem. Biophys

396, 49–55.

Blain E. et al. (2002) The effect of thymosin beta4 on articular

cartilage chondrocyte matrix metalloproteinase expression.

Biochem. Soc. Trans. 30, 879–882.

PRG-4/SZP N- and C-terminal domains: cloning,expression and characterization

A.R.C. Jones,* C.E. Hughes,* S.D. Wainwright,*

C.R. Flannery† and B. Caterson*

*Connective Tissue Biology Laboratories, Cardiff University,

Cardiff, UK; †Wyeth Research, Cambridge, MA, USA

Introduction Proteoglycan-4 (PRG-4), also known as

superficial zone protein/proteoglycan (SZP), is an approxi-

mately 345-kDa mucinous proteoglycan that has been

detected in a variety of tissues including cartilage, tendon,

bone, heart and liver (Ikegawa et al. 2000). In the synovial

joint, PRG-4 is specifically synthesized by chondrocytes

located in the superficial zone of articular cartilage and by

some surface-lining cells of the synovium (Schumacher et al.

1994). Sequence analyses have shown that the N- and C-

terminal vitronectin-like domains of PRG-4 may impart

interesting functions relevant to synovial joint metabolism

(Merberg et al. 1993; Flannery et al. 1999). The objective

of this study was to investigate these potential functions,

facilitated by the production of PRG-4 N- and C-terminal

domains as recombinant proteins.

Methods cDNAs for the human N-terminal (exons 2–5) and

bovine C-terminal (exons 7–12) domains of PRG-4 were

obtained by RT-PCR and cloned into the expression vector

pMT-BiP for inducible, secreted expression in Drosophila S2

cells. Proteins were purified using FLAG-M2 antibody affinity

chromatography and visualized by SDS-PAGE and Western

blotting with PRG-4-specific antibodies. The heparin-binding

properties of recombinant proteins were investigated using

heparin affinity chromatography. The interactions of recombi-

nant PRG-4 domains with human plasminogen activator-inhi-

bitor (PAI)-1 and bovine type-II collagen were assayed using

standard ELISA techniques.

Results Stable cell lines have been generated that express human

N-terminal and bovine C-terminal PRG-4 domains. In both cases,

two proteins have been purified, possibly due to a splice mechan-

ism by the expression system. N-terminal sequence data and Wes-

tern blotting indicate that the two species in each case could

represent full-length and truncated proteins. Analyses of the two

PRG-4 N-terminal domain species have confirmed the presence of

a predicted heparin-binding domain and indicate that the mole-

cule can bind to PAI-1, with binding activity localized towards its

two somatomedin B domains. The somatomedin B domain of

vitronectin is known to bind PAI-1 (Seiffert 1997). Analyses of

the two PRG-4 C-terminal species have demonstrated

self-association under nonreducing conditions and binding to

heparin and PAI-1.

Discussion The exact role of PRG-4 in the synovial joint is

yet to be elucidated. However, these results point towards the

interaction of the N- and C-terminal domains of PRG-4 with

structural molecules such as type-II collagen and heparin, and



functional molecules such as PAI-1, a serpin that is involved in

the fibrinolytic cascade and cell adhesion. These properties are

in addition to the well-documented boundary lubricating

activity of the central mucinous region of PRG-4.

References

Flannery C.R. et al. (1999) Articular cartilage superficial zone

protein (SZP) is homologous to megakaryocyte stimulating

factor precursor and Is a multifunctional proteoglycan with

potential growth-promoting, cytoprotective, and lubricating

properties in cartilage metabolism. Biochem. Biophys. Res.

Commun. 254, 535–541.

Ikegawa S.L. et al. (2000) Isolation, characterization and

mapping of the mouse and human PRG4 (proteoglycan 4)

genes. Cytogenet. Cell Genet. 90, 291–297.

Merberg D.M. et al. (1993) In: Biology of Vitronectin and Their

Receptors (eds. Preissner K.T. et al.) pp. 45–52.

Schumacher B.L. et al. (1994) A novel proteoglycan synthesized

and secreted by chondrocytes of the superficial zone of articular

cartilage. Arch. Biochem. Biophys. 311, 144–152.

Seiffert D. (1997) The glycosaminoglycan binding site governs

ligand binding to the somatomedin B domain of vitronectin.

J. Biol. Chem. 272, 9971–9978.

Matrix-degrading enzyme synthesis by cellsisolated from the canine cranial cruciateligament

V.M. Anderson, A. Vaughan-Thomas and J.F. InnesConnective Tissue Research Group, Department of Veterinary

Clinical Science, University of Liverpool, Crown Street, Liverpool,

UK

Introduction Cruciate ligament disease or injury is common

in humans and dogs. There is some evidence to indicate that

both its incidence and the degree of tissue pathology may vary

between breeds and in relation to body weight, age, gender

and body condition. In order to investigate a direct role of

endocrine influences on pathology, we have attempted to

establish a model system using isolated canine cranial cruciate

ligament (CCL) cells in culture. In this preliminary study, we

isolated CCL cells and characterized the matrix metalloprotei-

nase (MMP) activities expressed by these cells in culture.

MMPs contribute to the extracellular matrix turnover of con-

nective tissue, and an imbalance between matrix synthesis and

degradation leads to changes in the matrix, which may com-

promise ligament integrity and strength.

Materials and methods CCLs were collected from animals

following euthanasia for reasons other than joint disease.

Ligament cells were isolated using collagenase digestion and

maintained in culture in DMEM/F12 containing 10%(v/v)

fetal bovine serum. Early passage number cells were incubated

in medium containing reduced serum concentrations [1%

(v/v)] in order to determine levels of MMP production.

Gelatinase activities (MMP-2 and MMP-9) in the media of

cultured cells were determined using gelatin zymography. The

identification of MMP was confirmed by using EDTA to inhi-

bit gelatinolytic activity.

In order to determine whether the cells express collagenase

activities, a fluorogenic substrate assay was used in which cell-

conditioned medium was treated with APMA prior to incubation

with thesubstrate (MCA-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2).

Hormones (relaxin, leptin, growth hormone and oestrogen)

were added at near physiological serum levels, where known,

to 12-well plates after cells were 100% confluent, and medium

samples were analysed 72 h after treatment.

Results Pro-MMP-2 was the predominant gelatinase activity

expressed by canine cruciate ligament cells in culture. The active

forms of both MMP-2 and MMP-9 were observed at very low

levelsornotatall,differencesbeingobservedbetweenanimals.The

volumes of medium samples analysed were adjusted such that the

levels of MMP detected were within the range of activities that

yielded a linear densitometric response curve. Our preliminary

results show no change in levels of pro and active forms of

MMP-2 and MMP-9 in cell culture after treatment with the

hormones, oestradiol, relaxin, growth hormone and leptin.

Collagenase activity was detected in the ligament cell-conditioned

mediumusingthefluorogenicsubstrate,andthevolumeofmedium

used for assay was adjusted to lie within the standard curve. Our

preliminary data suggest that hormonal treatment may modulate

the production of collagenase activity. However, these findings

require confirmation.

Discussion These results show that pro-MMP-2 is the pre-

dominant gelatinase activity synthesized by cultured ligament

cells. Previous studies have shown that this is also the pred-

ominant gelatinase activity present in extracts of canine cru-

ciate tissue. We have also shown that CCL cells express a

collagenase activity in culture. Therefore, we intend to use the

CCL cell-culture system as a model to investigate the effects of

systemic and local endocrine influences on the matrix degrada-

tive cascades. This model will allow us to obtain information on

the roles of hormones in pathological processes prior to liga-

ment rupture in vivo.

References

Comerford E. (2002) University of Bristol: PhD Thesis.



Matrix remodelling in the superficial digitalflexor tendon of the horse

A. Vaughan-Thomas, V.M. Anderson, A. Kipar,

J.F. Innes and P.D. CleggConnective Tissue Research Group, Departments of Veterinary

Clinical Science and Veterinary Pathology, University of

Liverpool, Liverpool, UK

Introduction Injuries to the superficial digital flexor tendon

(SDFT) are very common in the horse. The macroscopic

degeneration of the tendon that is often apparent is associated

with an altered matrix composition indicating substantial

tissue-remodelling activity.

The matrix metalloproteinases (MMPs) are responsible for

degrading matrix components during tissue remodelling or

turnover. There is evidence that mediators of angiogenesis,

particularly vascular endothelial growth factor (VEGF), regu-

late the activities of gelatinases, and it is likely that tendon

overuse or injury leads to disruption of tissue homeostasis,

leading to hypoxia and VEGF up-regulation. In this study, we

investigated the presence and levels of MMP-2 and MMP-9

and VEGF within both grossly normal and pathological tendon

tissue. Furthermore, we investigated the presence of cartilage

oligomeric matrix protein (COMP) within tendon.

Materials and methods SDFT samples were collected post

euthanasia. From tendons showing no gross pathology, sam-

ples were taken along the length of the SDFT and pulverized in

a liquid nitrogen-cooled mill. Tendon tissue in which a central

degenerate core lesion was observed was sampled and pro-

cessed identically. Extracts of soluble protein in SDS-PAGE

sample buffer were analysed by gelatin zymography for the

presence of MMP-2 and MMP-9 and also by Western blot

analysis for the presence of VEGF isoforms and COMP. SDS-

PAGE followed by coomassie-blue staining was used to obtain

a profile of the proteins extracted, and casein substrate gel

zymography was used for anaylsis of serum-associated proteo-

lytic activities.

Results In the nonpathological tendon samples, no significant

differences were observed along the length of the tendon. Basically,

protein profiles, MMP-2 activity, activities co-migrating with

serum-derived plasminogen and VEGF isoforms were constant.

Pro-MMP-2 was the predominant gelatinase activity but active

MMP-2 was also present. The main immunoreactive band

detected using a polyclonal antiserum to VEGF had an apparent

Mr of 16.5kDa. Analysis of the pathological tendon samples

revealed less consistent expression of these molecules and activities.

The protein profile showed an apparent absence of some serum-

derived soluble proteins in some areas, with raised levels of both

pro and active MMP-2. An activity co-migrating with an active

MMP-9 standard was also observed. Intact subunits of COMP

were observed in all samples of pathological tissue. The presence of

VEGF within the pathological tissue is being investigated.

Discussion These results indicate that mediators of tissue

remodelling are present both within normal and pathological

SDFT. However, it is apparent that there may be imbalance

between some protein components and enzyme activities

within the pathological tendon. We will undertake further

studies to determine whether our findings are common to

further samples. This study should aid our understanding of the

mechanisms that underlie SDFT pathology in the horse.

References

Birch H.L. et al. (1998) Macroscopic ‘degeneration’ of equine

superficial digital flexor tendon is accompanied by a change in

extracellular matrix composition. Equine Vet. J. 30 (6), 534–

539.

Riley G.P. et al. (2002) Matrix metalloproteinase activities and

their relationship with collagen remodelling in tendon pathol-

ogy. Matrix Biol. 21, 185–195.

Controllers of apoptosis in herniated anddiseased intervertebral disc

J. Menage,* A. Wojcik,† M. Krajewski,‡ J.C. Reed,‡

S.M. Eisenstein,* W.E.B. Johnson* and S. Roberts*

*Centre for Spinal Studies, RJAH Orthopaedic Hospital,

Oswestry, Shropshire, UK; †Burnham Institute, La Jolla, CA,

USA; ‡Hinchingbrooke Hospital, Cambridge, UK

Introduction Cell death in intervertebral disc is common,

and this may account for the degeneration of discs occurring

relatively early in life in comparison to other connective tis-

sues. Apoptosis, or programmed cell death, provides an

efficient mechanism for degrading and disposing of dying

cells, minimizing the chance of an inflammatory response;

necrosis, in contrast, is uncontrolled and more damaging.

The type of cell death in intervertebral disc is not well studied.

Disruption of the control system for apoptosis could well play

a role in disc diseases, resulting in premature cell death and

malfunctioning of the tissue. We have studied the presence of

three proteins, members of the Bcl-2 family, which are either pro-

or anti-apoptotic, in herniated and diseased intervertebral disc.



Materials and methods Thirty-seven intervertebral discs

from 36 patients aged 16–71 years (with spondylolisthesis,

low back pain or disc herniations) were immunostained for

the presence of members of the Bcl-2 family of proteins: BAK,

which is pro-apoptotic and Bcl-X and Bcl-2, which are anti-

apoptotic. Polyclonal antibodies were raised against synthetic

peptides corresponding to the human proteins. Indirect immu-

nohistochemistry was carried out labelling with DAB. Con-

trols were performed using antibody preabsorbed with the

antigen in place of the primary antibody. The prevalence of

positively stained cells was scored on a scale of 0–3.

Results There was staining of some cells for all the markers

studied in all but three samples. Most samples had approxi-

mately 30% of cells staining positively. Staining was generally

greater for cells in clusters (more commonly found in degenerate

discs) than single cells, particularly for BAK. All antigens were

present in the same cluster of cells, sometimes possibly produced

by the same cells. There was a positive correlation between

staining for all three members of the Bcl-2 family, both apopto-

sis-promoting and -inhibiting.

Discussion This preliminary study suggests that members of

the Bcl-2 family play a role in regulating cell death via apop-

tosis, at least in diseased human intervertebral discs. Apoptosis

of prolapsed disc tissue may be the least deleterious way for

the tissue to ‘self destruct’. Both pro- and anti-apoptotic mar-

kers are present in the same region, which at first glance may

appear surprising. However, Bcl-2 and Bcl-x promote apopto-

sis on homodimerization. One manner in which BAK inhibits

apoptosis is by binding to Bcl-2 and Bcl-x and preventing this

occurring. In addition, the presence of pro- and anti-apoptotic

members of the Bcl family may simply indicate that regulatory

pathways of cell survival or death have been initiated within

the cells. The fate of these cells may well depend on relative

levels of expression or activity of these pro- or anti-apoptotic

factors within a given cell. Hence, the production and interaction

of various members of these multigene families within the apop-

totic pathway in intervertebral disc remains to be clarified.

Altered patterns of gene expression inendothelial cells in scleroderma

S.L. Howat, D. Abraham and J.D. PearsonCentre for Cardiovascular Biology & Medicine, King’s College

London and Rheumatology Research Unit, Royal Free & UC

Medical School, London, UK

Introduction Scleroderma (systemic sclerosis, SSc) presents

clinically as fibrosis of the skin but also involves fibrosis of blood

vessels and internal organs, causing damage and complications

that in the most severe forms lead to organ failure and death. The

earliest detectable structural feature of SSc pathology appears to

be endothelial cell damage. This leads to an inflammatory

response with adhesion of leucocytes to the blood vessel walls,

emigration and accumulation in the tissue. Paracrine factors

secreted from activated endothelial cells have been implicated in

the consequential fibroblast dysfunction and excessive deposition

of extracellular matrix in lesional tissues (Denton et al. 1996).

Activated lesional fibroblasts, which maintain their variant

phenotype in culture for several passages, in turn, act upon the

endothelial cells causing a ‘cross-talk’ which perpetuates the SSc

phenotype of both endothelial cells and fibroblasts. The aim of this

work was to use an in vitro approach to identify the altered pattern

of gene expression in endothelial cells induced by co-culture with

SSc lesional fibroblasts, which may reflect the phenotypic changes

in endothelial cells in SSc.

Materials and methods Human dermal microvascular

endothelial cells (HMEC-1, Ribeiro et al. 1995) were co-cultured

with dermal fibroblasts obtained from biopsies of lesional areas of

the skin of patients with scleroderma or normal demal fibroblasts.

Co-cultures were carried out in six-well transwell tissue culture

plates. All cells were grown to confluence then fibroblasts with

their conditioned medium were co-cultured with HMEC-1 for 2,

4, 6, 24 or 48 h. Total RNA was extracted from HMEC-1, reverse

transcribed, and expressed genes were identified using AtlasTM

Nylon cDNA Expression Arrays (human broad range 1.2) (Clon-

tech). Arrays were imaged using a Typhoon 9210 phosphorimager

(Molecular Dynamics), and images were analysed using the Atlas-

ImageTM 2.0 software. Selected mRNA levels were subsequently

measured by real-time PCR using a LightCyclerTM (Roche).

Results The human broad range 1.2 array has 1176 cDNAs

plus nine housekeeping cDNAs and negative control cDNAs.

The overall pattern of gene expression in HMEC-1

co-cultured with normal dermal fibroblasts (control)

and HMEC-1 co-cultured with lesional SSc fibroblasts

(diseased) was similar. In total, 32–49% of cDNAs were detec-

tably expressed of which between 5 and 10% of cDNAs were

apparently differentially expressed in diseased relative to control.

Ratios of diseased: normal ranged from 11.7 to 0.09. Six genes of

potential interest were selected from the 6-h array and measured

by real-time PCR. Array results (at 6 h) showed that the leucocyte

function-associated molecule 1 alpha chain and caspase 10

were up-regulated, and MMP-11 was down-regulated; connec-

tive tissue growth factor (CTGF), plasminogen activator

inhibitor-1 (PAI-1) and endothelin-2 (ET-2) were expressed at

similar levels in diseased and normal samples. When measured by

real-time PCR at all co-culture time points, these genes were



found to be either unchanged or down-regulated in HMEC-1 co-

cultured with SSc fibroblasts.

Discussion We have identified the pattern of expression of 1176

cDNAs in HMEC-1 co-cultured with normal fibroblasts or with

those from patients with SSc. These contain candidate genes which

may be responsible for early vascular abnormalities in SSc. In

future experiments, we will study the role of these candidate

genes using in vitro models of the pathology of SSc.

References

Denton C.P. et al. (1996) Scleroderma fibroblast phenotype is

modulated by endothelial cell co-culture. J. Rheumatol. 23,

633–638.

Ribeiro M.J. et al. (1995) Hemostatic properties of the SV-40

transfected human microvascular endothelial cell line (HMEC-

1). A representative in vitro model for microvascular endothe-

lium. Thromb. Res. 79, 153–161.

Investigating the PKR-signalling pathway in thearticular joint

S.J. Gilbert, V.C. Duance and D.J. MasonConnective Tissue Biology Laboratories, School of Biosciences,


Introduction Our previous studies have shown that the protein

kinase, PKR and its activator, PACT are involved in TNF-a signal-

ling in articular cartilage (Gilbert et al. 2002). The sphingolipid

ceramide is known to play an important role in signal transduction

of TNF-a and is a potent apoptotic agent. The PKR pathway is

also known to be activated by ceramide (Ruvolo et al. 2001).

Recent studies have shown that ceramide stimulates proteoglycan

degradation and mRNA expression of MMP-1, MMP-3 and

MMP-13 in rabbit cartilage suggesting a role for this second

messenger in cartilage degradation (Sabatini et al. 2000). In the

current study, we investigated the role of ceramide, TNF-a and

PKR in bovine articular cartilage degradation. In addition, we

treated human primary synoviocytes with TNF and investigated

whether PKR is activated and if NFkB, a transcription factor

downstream of PKR, is translocated to the nucleus.

Materials and methods Bovine articular cartilage explants

were stimulated with C2-ceramide (50 mm) or TNF-a (100 ng/

ml) for 24 h. To inhibit the activation of PKR, 2-aminopurine

(10 mm) was added to duplicate cultures 1 h prior to the addi-

tion of treatments. Media was collected and MMPs analysed

by gelatin zymography. Proteoglycan release was measured by

the DMMB assay and cell viability determined by the

Cytotox� assay. Human primary synoviocytes were stimu-

lated with TNF-a (10 ng/ml) for 30 min, and phosphorylated

PKR and the Rel A p65 subunit of NFkB were detected by

immunofluorescence.

Results C2-ceramide treatment resulted in a significant

release of both pro and active MMP-2. Explants incubated

with the PKR inhibitor, 2-aminopurine, prior to TNF-a or C2-

ceramide treatment resulted in a marked reduction in

both MMP-2 and MMP-9 activation. A significant increase

in proteoglycan release was observed following treatment

with TNF-a and C2-ceramide, which was significantly inhib-

ited by 2-aminopurine. A significant loss of cell viability was

observed when explants were treated with C2-ceramide, which

was also found to be regulated by a PKR-dependant pathway.

Studies using antisense oligonucleotides to PACT and PKR are

currently being undertaken to confirm these results.

An increase in phosphorylation of PKR and NFkB translo-

cation was observed in synoviocytes treated with TNF.

Discussion Our data has shown that PKR potentially is an

important mediator of degradative and death pathways in

chondrocytes. Collectively, these results suggest a novel role

for the PKR pathway in the turnover of articular cartilage and

support our hypothesis that PKR and PACT are implicated in

the cartilage loss that occurs in arthritic disease.

References

Gilbert S.J. et al. (2002) TNF-a upregulates PACT and increases

phosphorylation of PKR and eIF2-a in articular chondrocytes.

Biochem. Soc. Trans. 30 (6), 886–889.

Ruvolo P.P. et al. (2001) Ceramide regulates protein synthesis by

a novel mechanism involving the cellular PKR activator RAX.

J. Biol. Chem. 276 (15), 11754–11758.

Sabatini M. et al. (2000) Effects of ceramide on apoptosis,

proteoglycan degradation, and matrix metalloproteinase

expression in rabbit articular cartilage. Biochem. Biophys.

Res. Commun. 267, 438–444.

Characterization of the promoter in ADAMTS-5(aggrecanase-2)

M.R. Heming, S.D. Wainwright, B. Caterson and

C.E. HughesCardiff University, Wales, UK

Introduction Loss of aggrecan metabolites from the cartilage

matrix into the surrounding synovial fluid have allowed for



identification of two major cleavage sites within aggrecan:

Asn341–Phe342 and Glu373–Ala374 peptide bonds. Proteolysis

at the Glu373–Ala374 bond has been specifically attributed to

ADAMTS-4 and ADAMTS-5. Genetic reporters are com-

monly used in cell biology to study mechanisms regulating

gene expression. Firefly luciferase is widely used as such a

reporter for the immediate availability of reporter activity,

the sensitivity of the assay, and quickness and ease of use. By

making time-controlled cuts in the 5´-noncoding region of

ADAMTS-4 and -5 and inserting the deletions into the genetic

reporters, which are then transfected into a suitable cell line,

activity can be measured. The project aim was to identify the

cis and trans acting factors in the 5´-noncoding regions that

regulate ADAMTS-5 expression using the Luciferase Assay

System (Promega, Madison, WI, USA).

Methods A 3-kb fragment containing the 5´-flanking region

of ADAMTS-5 was subcloned into pGL3-Basic (Promega), a

luciferase reporter vector lacking a eukaryotic promoter.

Truncations have also been made to the full-length construct

using Erase-a-Base System (Promega) at restriction enzyme

sites SacI at 11 bp from the origin and NheI at 21 bp from

the origin, resulting in 200–2300 bp deletions on the TS-5

vector. Adherent kidney embryonic fibroblast-like cells

(PEAK) were grown in RPMI 1640 media containing supple-

ments (Gibco Invitrogen Corporation) at 37 ˚C in 5% CO2.

Cells were transfected using Fugene-6 Reagent (Roche) as

recommended by the manufacturer. 1.5· 105 cells were plated

out in 12-well plates and grown overnight. The next day,

serum media was removed and RPMI 1640 media containing

only glutamate was added. DNA complexes were prepared by

mixing 97 ml of serum-free medium, 3 ml of Fugene-6, and 2 mg

of DNA from Control, Basic, or TS-5 full constructs and

selected truncations and incubated for 30 min at RT. Hundred

microlitres of the complex was pipetted directly into each well.

After a 4–5-h incubation, 100ml of FBS was added to each well

and left overnight at 37 ˚C in 5% CO2. The next morning, the

cells were harvested and washed twice in PBS, and resus-

pended in ·1 Passive Lysis Buffer. About 20 ml of the cell lysate

was assayed for luciferase activity as described in the manu-

facturer’s protocol (Promega).

Results and discussion Full-length and seven deletion frag-

ments (numbering downstream from the ATG start codon) span-

ning the 5´-noncoding region were selected from a library of 41

deletions prepared by the Erase-a-Base System: 3b.4 (�2322 bp),

4a.5 (�2110 bp), 5a.2 (1810 bp), 4a.2 (1427 bp), t3a.3

(�718 bp), 2a.1 (�433 bp) and 4a.1 (�278 bp). Firstly, an

increase in activity occurs with 256 base pairs deleted from the

full-length vector (3b.4). With control considered 100% of activ-

ity, the full-length vector produces greater than that amount

indicating TS5 vector as having double the activity. A greater

increase in activity (·6 that of the control) between the full length

and 3b.4 suggests the presence of a suppressor somewhere in the

first 256 base pairs of the 5´-noncoding region. Activity then

drops between 3b.4 and 4a.5. However, activity is still greater

than that produced by the full length. A levelling-off occurs at

5a.2, which continues until a sudden decrease at t3a.3 that is

maintained in 2a.1 and 4a.1. Loss of activity between 4a.2 and

t3a.3 is linked to loss of a TATA box at 1753–59 bp. There is a

total of seven motifs existing in the first few hundred base pairs

that could be possible suppressors including an H1 conserved site

at 248–254 base pairs. By following the same methods using

selected deletion fragments within �2565 to �2322, it will be

possible to identify which motif is responsible for suppressing

activity in this cell line. Similar experiments have been carried out

in isolated articular chondrocytes.

Identification of cell markers of the nucleus andannulus of bovine intervertebral discs

Barry K. Derham and Jill UrbanUniversity Laboratory of Physiology, Oxford, UK

Introduction The intervertebral disc consists of three regions,

the nucleus pulpous and the inner and outer annulus containing

cells with individual phenotypes. However, no molecular markers

are known to discriminate between the various cell types. Mole-

cular markers would help identify the cell types through develop-

ment of the disc, ageing of the disc, cell localization and in

pathological states such as degenerative disc disease and scoliosis.

Here, we present data revealing major difference between the cell

types using SDS-PAGE and mass spectrometry. Such differences

will help to develop molecular markers to identify the cell types

using immunoblotting and immunohistochemistry.

Methods Intervertebral discs were isolated from bovine

tails and separated into three distinct regions of the

nucleus, inner and outer annulus. The isolated regions

were separately digested with collagenase and peptidase

overnight. The remaining cell suspensions were sieved

through a filter to remove large particles and then exten-

sively washed. The cells were then separated into mem-

brane and supernatant fractions by incubation with Triton

followed by centrifugation. SDS-PAGE analysis using a

variety of acylamide gels of the various fractions revealed



bands of interest that where then cut from the gel, digested with

trypsin and analysed by mass spectrometry. The peptide mass/

charge data were collected and then compared with a databank of

known peptide masses to give a composite protein mass.

Results Analysis of the membrane and supernatant fractions of

the cells from the three distinct regions in the disc by SDS-PAGE

revealeduniqueproteinpatternsbetweentheregionsandfractions.

A large broad band from the membrane of nucleus cells was ana-

lysed by mass spectrometry that revealed strong matches for myo-

sin and clathrin and a match for basement membrane collagen-IV.

Acontrolbandfromtheouterannulusalsorevealedastrongmatch

for myosin and to a lesser extent basement membrane collagen

type-IV. A strong SDS-PAGE band from the membrane fraction of

the outer annulus revealed a mass spectrometry match to actin.

Analysis of the corresponding supernatant fraction revealed a

strong match to actin, whereas a band of similar molecular weight

from the inner annulus revealed another myosin chain.

Conclusion The differences revealed in the protein profile of

cells from the three regions of disc and the identification of

prominent proteins demonstrate that these differences can be

used to identify molecular markers. SDS-PAGE and mass spec-

trometry analysis of significant bands showed strong matches in

the membrane fraction for myosin and clathrin, and also base-

ment membrane collagen-IV. By using immunoblotting and

immunohistochemistry, it will be possible to follow the devel-

opment, ageing and pathology of the three cell types and will

hopefully be extended to finding markers that characterize the

individual stages of the degenerative changes. This study will

also include genechip technology to identify at the RNA level

the difference between the three cell types.

This work was funded by the EU EURODISC project

(QLK6-CT-2002–02582).

Type-X collagen interacts with the smallleucine-rich proteoglycans decorin and biglycan

S. Hancock, A.P.L. Kwan and V.C. DuanceConnective Tissue Biology Laboratories, School of Biosciences,


Introduction Type-X collagen is expressed by hypertrophic

chondrocytes in the epiphyseal growth plate. The 59-kDa a-

chain consists of a 45-kDa triple-helical domain flanked by two

noncollagenous regions, a large C-terminal domain termed NC1

and a smaller N-terminal domain termed NC2. The restricted

distribution of type-X collagen within the growth plate indicates

a potential role during the process of endochondral ossification.

Type-X collagen may form a hexagonal lattice-like matrix, per-

missive to vascular invasion and mineralization.

Decorin and biglycan are small leucine-rich proteoglycans,

which are usually substituted with one or two glycosamino-

glycan (GAG) chains, respectively. Their 40-kDa protein

cores contain N-terminal GAG attachment site(s), several

central leucine-rich repeats and a disulphide-bonded loop at

the C-terminal. They are ubiquitously expressed and are

found in many connective tissues, including skin, cartilage

and bone. They are known to interact with many proteins

including fibrillar collagens. The molecular interactions of

type-X collagen with decorin and biglycan have been investi-

gated in vitro. Characterizing these interactions may elucidate

the precise role of these complexes in the hypertrophic cartilage

matrix.

Materials and methods To investigate the interactions of type-

X collagen with decorin and biglycan, solid phase assays, includ-

ing competitive assays and surface plasmon resonance were used.

Proteins used during the investigation included type-X collagen

purified from embryonic chick tibial hypertrophic chondrocytes,

pepsin-treated type-X collagen, human recombinant NC1

domain of type-X collagen, human recombinant decorin and

biglycan purified from bovine cartilage.

Results Type-X collagen interacts with biglycan and decorin

in solid phase assays and surface plasmon resonance, using the

BIAcore 3000 system. The interactions occur primarily via the

NC1 domain of type-X collagen and are not dependent on the

presence of the GAG chains on the proteoglycans. Dissociation

constants have been calculated and indicate high affinity binding.

Results from competitive binding assays indicate that decorin

and biglycan bind to the same site on type-X collagen. Rotary

shadowing is currently being used to confirm interactions and to

locate the interaction sites better.

Discussion Interactions between type-X collagen and other

matrix components may be required for the assembly of the

hypertrophic cartilage matrix and to maintain its integrity.

Within the growth plate, type-X collagen interactions with

decorin and biglycan may have potential roles in regulation or

maintenance of the type-X collagen hexagonal network and/or

presentation of growth factors, e.g. TGF-b known to be import-

ant in endochondral ossification.



Modulation of the expression of connectivetissue growth factor (CTGF) by alterations of thecytoskeleton

Christian Ott,* Angela Graness,* Klaudia Giehl†

and Margarete Goppelt-Struebe*

*Medizinische Klinik IV, Universitat Erlangen-Nurnberg; †Institut

fur Pharmakologie und Toxikologie, Universitat Ulm, Germany

Introduction In closing wounds or developing fibrotic tissue,

fibroblasts are exposed to mechanical stress, which leads to

alterations in cell morphology and reorganization of the cyto-

skeleton. The impact of morphological changes on gene expres-

sion was investigated in the present study. As a model system,

we used a human renal fibroblast cell line and studied the

expression of connective tissue growth factor (CTGF). CTGF

is a downstream mediator of TGF-b mediating many of the pro-

fibrotic actions of this growth factor and has also been shown to

be up-regulated by static or dynamic pressure. The molecular

mechanisms regulating CTGF expression related to stress are

not yet known.

Materials and methods A human renal fibroblast cell line

was kindly provided by Dr Muller, Gottingen, Germany.

These cells express CTGF upon treatment with soluble sti-

muli (Heusinger-Ribeiro et al. 2001; Eberlein et al. 2001).

CTGF expression was detected by northern and Western blot

analysis. The actin cytoskeleton was visualized by rhodamine

phalloidin staining, and microtubules were detected by immuno-

cytochemistry.

Results Low concentrations of the microtubule disrupting

agents nocodazole and colchicine strongly up-regulated CTGF

mRNA and protein expression in the human renal fibroblast cell

line TK173. The up-regulation was prevented by stabilization of

the microtubules by paclitaxel (taxol). As a consequence of micro-

tubule disruption, the small GTPase RhoA was activated and the

actin stress fibers were stabilized. Both effects were related to

CTGF induction: interference with RhoA signalling by simvasta-

tin, toxin B and Y27632 prevented up-regulation of CTGF. The

important role of RhoA was supported by an increased CTGF

expression upon overexpression of constitutively active RhoA.

Direct disassembly of the actin cytoskeleton by latrunculin B

interfered with colchicine-mediated up-regulation of CTGF

expression. Disassembly of actin fibers by cytochalasin D unex-

pectedly increased CTGF expression. This indicated that the con-

tent of F-actin per se was not the major determinant for CTGF

gene expression. It has been shown, however, that cytochalasin D

sequesters G-actin, whereas latrunculin increases the level of G-

actin. Our data are thus in accordance with an inverse correlation

between G-actin levels and CTGF expression.

Discussion These data link alterations in the microtubule and

actin cytoskeleton to the expression of CTGF. Recently,

decreased levels of G-actin were observed in vascular smooth

muscle cells in response to increased vascular pressure (Cipolla

et al. 2002). Our findings thus provide a molecular basis for the

observation that CTGF is up-regulated in cells exposed to

mechanicalstress.

References

Cipolla M.J. et al. (2002) Pressure-induced actin polymerization

in vascular smooth muscle as a mechanism underlying

myogenic behavior. FASEB J. 16, 72–76.

Eberlein M. et al. (2001) Rho-dependent inhibition of the

induction of connective tissue growth factor (CTGF) by

HMG Co-A reductase inhibitors (statins). Br. J.Pharmacol.

133, 1172–1180.

Heusinger-Ribeiro J. et al. (2001) Lysophosphatidic acid-induced

expression of connective tissue growth factor in human renal

fibroblasts: Regulatory role of RhoA and cAMP. J. Am. Soc.

Nephrol. 12, 1853–1861.

Homophilic complex formation is prerequisitefor MT1-MMP to degrade type-I collagen on thecell surface

Yoshifumi Itoh,* Noriko Ito,* Hideaki Nagase*

and Motoharu Seiki†*Kennedy Institute of Rheumatology, Imperial College London,

London, UK; †Institute of Medical Science, University of Tokyo,

Tokyo, Japan

Introduction MT1-MMP degrades a wide variety of extra-

cellular matrix macromolecules and some cell-surface proteins

such as CD44, transglutaminase and av-integrin, and activates

zymogen of MMP-2 and MMP-13. Among these activities, the

collagenolytic activity is one of the most important functions

of the enzyme in biology, as the phenotypes of MT1-MMP

gene knockout mice, such as abnormal skeletal development

and various connective tissue abnormalities, are thought to be

attributed to the lack of cellular collagenase activity. We have

previously shown that MT1-MMP forms homophilic complex

through its haemopexin (HPX) domain facilitating the activa-

tion of proMMP-2 (Itoh et al. 2001). In this report, we present

data that collagen-degrading activity of MT1-MMP also

requires homophilic complex formation.



Methods COS7 cells were cultured on type-I collagen film

and transfected with MT1-MMP and its mutant gene to look

at collagen degradation activity of these gene products.

Results Co-expression of the catalytic domain-deleted mutant

of MT1-MMP (MT1dCat) with the wild-type enzyme inhibited

its homophilic complex formation as well as its ability to acti-

vate proMMP-2 on the cell surface, and MT1dCat also inhibited

cell-surface collagenolytic activity of the wild-type MT1-MMP.

A chimeric MT1-MMP mutant (MT-MMP13) whose catalytic

domain, hinge region and HPX domain were replaced with

those derived from collagenase-3 (MMP-13) did not degrade

collagen though MT-MMP13 was expressed and maintained its

proteolytic activity against gelatin on the cell surface. When an

interface for homophilic complex formation, the HPX domain

derived from MT1-MMP, was further added to the chimeric

enzyme (MT-MMP13-HPXmt1), it degraded collagen. Co-

expression of MT1dCat and MT-MMP13-HPXmt1 also inhib-

ited the collagenolytic activity.

Discussion Homphilic complex formation is prerequisite

for the expression of collagenase activity on the cell surface.

Presumably, each MT1-MMP in the complex has different

roles; one unwinds triple-helical structure of collagen locally

and the other cuts peptide bond.

References

Itoh Y. et al. (2001) Homophilic complex formation of MT1-

MMP facilitates proMMP-2 activation on the cell surface and

promotes tumor cell invasion. EMBO J. 20, 4782–4793.

Role of the EGF-like domains in mammaliantolloid (mTLD) secretion and procollagenC-proteinase activity

Laure Garrigue-Antar and Karl E. KadlerWellcome Trust for Cell-Matrix Research, School of Biological

Sciences, University of Manchester, Manchester, UK

Introduction Bone morphogenetic protein (BMP)-1 and its

larger splice variant mammalian tolloid (mTLD) belong to

the tolloid group of astacin-like metalloproteinases that are

fundamental to tissue patterning and extracellular matrix

assembly. BMP-1 and mTLD exhibit similar substrate specifi-

city in vitro; however, BMP-1 is a much better procollagen

C-proteinase than mTLD. mTLD consists of a prodomain

(which is cleaved by a furin-like enzyme) (Leighton & Kadler

2003), a zinc metalloproteinase domain and a C-terminal

part comprising five CUB domains thought to be important for

protein–protein interactions (Hartigan et al. 2003), and two

EGF-like domains, which in other proteins are involved in

calcium ion binding. BMP-1 lacks the most C-terminal two

CUB domains and one EGF-like domain. mTLD activity is

known to be calcium ion dependent, as demonstrated for

the chick homologue (Hojima et al. 1985). In our current

work, we are studying the role of the EGF-like domains in the

secretion and procollagen C-proteinase activity of mTLD,

and the contribution that these domains made to calcium ion

dependency.

Materials and methods We designed proteins lacking EGF1,

EGF2 or both. NotI sites were introduced by PCR at the

borders of the EGF domain of a cDNA clone encoding a V5-His

mTLD. Restriction enzyme digestion was used to delete individual

domains. The mutant constructs in pCEP4 were stably transfected

into 293-EBNA cells. Expression was analysed by Western blot.

The wild-type and the mutant enzymes were purified on a nickel

ion column, and their activity was determined by cleavage of type-I

procollagen in the presence or absence of 5mm CaCl2.

Results We showed that (1) the mTLD proteins lacking

EGF1, EGF2 or EGF1þEGF2 were poorly secreted into the

culture medium compared to mTLD and (2) the EGF deletion

mutants remained calcium ion dependent, but some differences

were seen. Most notably, the DEGF2 and DEGF1þDEGF2

mutants were found to be better C-proteinases than the wild-

type enzyme in the presence of calcium ions.

Conclusion From these preliminary data, we concluded that

(1) the EGF domains are necessary for efficient secretion (2)

both EGF1 and EGF2 domains contribute to the calcium ion

dependency of mTLD and (3) the EGF2 domain might be a

Ca2þ-activated hinge that ‘swings’ the CUB-4 and CUB-5

domains away from the active site. The ?EGF2 mTLD might

be expected to have an open conformation, thereby making it a

better C-proteinase than the wild-type enzyme, and (?4) Ca2þ

ions are bound by other domains in mTLD and not only by the

EGF-like domains.

References

Hartigan N. et al. (2003) Bone morphogenetic protein (BMP) -1:

identification of the minimal domain structure for procollagen

C-proteinase activity. J. Biol. Chem. 278, 18045–18049.



Hojima Y. et al. (1985) Type I procollagen carboxyl-terminal

proteinase from chick embryo tendons. Purification and

characterization. J. Biol. Chem. 260, 15996–16003.

Leighton M. & Kadler K.E. (2003) Paired basic/Furin-like proprotein

convertase cleavage of pro-BMP-1 in the trans-Golgi network.

J. Biol. Chem. 278, 18478–18484.

Age-related changes in the glycosaminoglycansof human meniscal aggrecan

Meneerah Al-Jafary, Thomas N. Huckerby and

Robert M. LauderDepartment of Biological Sciences, Lancaster University, UK

Introduction Meniscal damage and degradation, which are

strongly correlated with subsequent OA, have been identified

in approximately 60% of people over 60 years of age. Age-

related changes in articular cartilage glycosaminoglycans

(GAGs) have been described, and used to facilitate the study

of pathology-related changes (Plass et al. 1998). However,

such data do not yet exist for the meniscus.

Materials and methods Undamaged human menisci were

obtained following leg amputations, and the vascular and avas-

cular zones of each lateral and medial meniscus were extracted

into 4 m GuHCl. Aggrecan was recovered in the A1 fraction

following CsCl density gradient centrifugation, and the relative

abundance of chondroitin, dermatan and keratan sulphates

(CS, DS and KS) was examined by NMR spectroscopy at

400 MHz and 43 ˚C.

Results Human meniscal aggrecan was shown to contain CS,

DS and KS, and our data show age-related changes in the

relative abundance of these GAGs. The change was similar

for medial and lateral menisci and for the vascular and avas-

cular zones within these.

The KS abundance in aggrecan from young menisci (<15 years)

was found to be 15–20% of the total GAGs. However, in older

samples, it comprised only 7–12% of the GAGs.

We have confirmed the presence of DS in human meniscal

aggrecan and show that the abundance of DS gradually falls

from approximately 16% at 10 years to 2–4% at 75 years.

There is some variability between humans, although the

trend is clear and for each human there is good agreement

between medial and lateral menisci and vascular and avascular

locations.

The levels of CS comprise the remainder of the GAG

attached to aggrecan and contribute the remainder of the

GAG abundance. This can be seen to increase from 67 to

72% at 10 years to approximately 90% at 75 years.

Discussion Our data show a clear age-related change in the

relative abundance CS, DS and KS from human meniscal

aggrecan. The data show a decrease in the abundance of KS

and DS and a concomitant increase in CS levels. These obser-

vations differ from those widely seen for articular cartilage, in

which the levels of CS are seen to fall with age.

We have confirmed that DS is a component of human menis-

cal aggrecan in agreement with previous work (McNicol &

Roughley 1980). However, previously reported levels of DS,

approximately 20%, are those found only in younger menisci.

Absolute levels of these GAGs have not yet been deter-

mined, and hence the mechanisms which bring about this

relative increase in CS with age may include either changes

in biosynthetic output and/or widespread GAG loss in which

KS and DS loss increases with age.

References

McNicol D. & Roughley P.J. (1980) Extraction and characterisation

of proteoglycan from human meniscus. Biochem. J. 185, 705–713.

Plass A.H.K. et al. (1998) Glycosaminoglycan sulfation in human

osteoarthritis. J. Biol. Chem. 273, 12642–12649.



TGF-β1 induces epithelial-mesenchymal transition but not myofibroblast transdifferentiation in primary cultures of human epithelial renal tubular cells

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