Glycoprotein130 (Gp130)/interleukin-6 (IL-6) signalling in osteoclasts promotes bone formation in periosteal and trabecular bone

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Glycoprotein130 (Gp130) / Interleukin-6 (IL-6) signalling in osteoclastspromotes bone formation in periosteal and trabecular bone

Rachelle W. Johnson, Narelle E. McGregor, Holly J. Brennan, BlessingCrimeen-Irwin, Ingrid J. Poulton, T. John Martin, Natalie A. Sims

PII: S8756-3282(15)00316-6DOI: doi: 10.1016/j.bone.2015.08.005Reference: BON 10832

To appear in: Bone

Received date: 17 March 2015Revised date: 27 July 2015Accepted date: 4 August 2015

Please cite this article as: Johnson Rachelle W., McGregor Narelle E., Brennan Holly J.,Crimeen-Irwin Blessing, Poulton Ingrid J., Martin T. John, Sims Natalie A., Glycopro-tein130 (Gp130) / Interleukin-6 (IL-6) signalling in osteoclasts promotes bone formationin periosteal and trabecular bone, Bone (2015), doi: 10.1016/j.bone.2015.08.005

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Glycoprotein130 (Gp130) / Interleukin-6 (IL-6) signalling in osteoclasts promotes

bone formation in periosteal and trabecular bone

Rachelle W Johnson1, Narelle E McGregor

1, Holly J Brennan

1, Blessing Crimeen-

Irwin1, Ingrid J Poulton

1, T John Martin

1,2, Natalie A Sims

1,2

1St. Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia

2University of Melbourne, Department of Medicine at St. Vincent’s Hospital, Fitzroy,

Victoria, Australia

Corresponding author:

Natalie A Sims

9 Princes St

Fitzroy, Victoria 3122

Australia

Email: [email protected]

Phone: +613-9288-2555

Fax: +613-9416-2676

Funding sources: This work was supported by a National Health and Medical

Research Council (Australia) Project Grant 1002728. N.A.S. is supported by a

National Health and Medical Research Council (Australia) Senior Research

Fellowship.

Key words: osteoclast, cortical, bone formation, glycoprotein130

COI statement: The authors have no conflicts of interest to report.

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Abstract

Interleukin-6 (IL-6) and interleukin-11 (IL-11) receptors (IL-6R and IL-11R,

respectively) are both expressed in osteoclasts and transduce signal via the

glycoprotein130 (gp130) co-receptor, but the physiological role of this pathway is

unclear. To determine the critical roles of gp130 signaling in the osteoclast, we

generated mice using cathepsin K Cre (CtskCre) to disrupt gp130 signalling in

osteoclasts. Bone marrow macrophages from CtskCre.gp130f/f

mice generated more

osteoclasts in vitro than cells from CtskCre.gp130w/w

mice; these osteoclasts were also

larger and had more nuclei than controls. While no increase in osteoclast numbers

was observed in vivo, osteoclasts on trabecular bone surfaces of CtskCre.gp130f/f

mice

were more spread out than in control mice, but had no functional defect detectable by

serum CTX1 levels or trabecular bone cartilage remnants. However, trabecular

osteoblast number and mineralising surfaces were significantly lower in male

CtskCre.gp130f/f

mice compared to controls, and this was associated with a

significantly lower trabecular bone volume at 12 weeks of age. Furthermore,

CtskCre.gp130f/f

mice exhibited greatly suppressed periosteal bone formation at this

age, indicated by significant reductions in both double-labeled surface and mineral

apposition rate. By 26 weeks, CtskCre.gp130f/f

mice exhibited narrower femora, with

lower periosteal and endocortical perimeters than CtskCre.gp130w/w

controls. Since

IL-6 and IL-11R global knockout mice exhibited a similar reduction in femoral width,

we also assessed periosteal bone formation in those strains, and found bone forming

surfaces were reduced to a similar extent in male IL-6 null mice. These data suggest

that IL-6/gp130 signalling in the osteoclast is not essential for normal bone resorption

in vivo, but maintains both trabecular and periosteal bone formation in male mice by

promoting osteoblast activity.

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Introduction

The glycoprotein 130 (gp130) co-receptor subunit transduces signals in response to

interleukin-6 (IL-6) family cytokines, and is expressed in all cells, including the cells

of the skeleton [1]. IL-6 family cytokines have been proposed to contribute to the

bone destruction associated with estrogen deficiency [2, 3], colitis [4], inflammatory

arthritis [5, 6], multiple myeloma [7], Paget’s disease [8] and Gorham’s disease [9].

Many members of this cytokine family, including IL-6, interleukin-11 (IL-11),

oncostatin M (OSM), leukemia inhibitory factor (LIF), and cardiotrophin-1 (CT-1),

stimulate osteoclast formation by promoting production of receptor activator of NFκB

ligand (RANKL) in osteoblasts [10-12]. However, osteoclast numbers were

dramatically elevated in mice with systemic deletion of gp130, [13, 14].

Systemic deletion of individual IL-6 family cytokines or cytokine-specific receptors

revealed varying influences on bone resorption. For example, while osteoclast

formation in LIF knockout mice was normal in remodelling bone, osteoclasts at the

base of the growth plate hypertrophic zone were larger and more numerous

throughout life, resulting in low trabecular bone mass specific to that region [15]. This

regional phenotype recapitulated a high level of osteoclastogenesis observed at the

growth plate in gp130, LIF receptor (LIFR), and CT-1 null neonate mice [14, 16, 17].

In contrast, OSM receptor (OSMR) and IL-11 receptor (IL-11R) null mice

demonstrated fewer osteoclasts because of decreased osteoblast support of

osteoclastogenesis [18, 19]. Despite these effects, and the known role of osteoblasts in

supporting osteoclast formation in response to IL-6 family cytokines in vitro [10-12,

20], deletion of gp130 throughout the osteoblast lineage or in osteocytes did not result

in any change in osteoclast number or function [21]. These data suggest a role for

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gp130 signaling in osteoclast formation and activity independent of the osteoblast

lineage that remains poorly understood.

While OSMR and LIFR have not been detected in osteoclasts [18, 22] precluding any

action of OSM, CT-1, LIF or CNTF in these cells, in vitro studies suggest some direct

effects of IL-6 signalling on mature osteoclasts. Both IL-6 receptor (IL-6R) and IL-

11R have been identified in mature osteoclasts [23, 24]. IL-11 appears to have no

direct effect on osteoclast activity or survival [25], and while IL-6 treatment of mature

bone marrow-derived osteoclasts increased resorption of dentine, an effect that was

blocked by an IL-6R neutralizing antibody [23], this data was not corrected for the

number of osteoclasts present, and was not reproduced by others using similar

systems [26, 27]. Although it is generally accepted that the major osteoclastogenic

influence of IL-6 is mediated by RANKL production by osteoblast lineage cells [10-

12, 20] there are in vitro circumstances in which IL-6 directly stimulates osteoclast

precursors to form osteoclasts [28, 29] or may partially facilitate RANKL or TNFα-

induced osteoclastogenesis [26], and other conditions where IL-6 inhibits RANKL-

induced osteoclastogenesis [30]. The relevance of these conflicting in vitro findings to

osteoclastogenesis and bone resorption in vivo is not known.

To determine the role of gp130 signalling in the osteoclast lineage in normal bone

growth and remodelling, we assessed the bone phenotype of mice with cathepsin-K-

driven gp130 deletion. We detected reduced trabecular bone volume and impaired

bone formation on both trabecular and periosteal bone surfaces in adult male mice,

but no change in osteoclast number or resorptive capacity in vivo. This suggests that

the major physiological function of IL-6:gp130 signaling in osteoclasts is the

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generation of signals that maintain osteoblast formation and function on trabecular

and cortical surfaces.

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Methods

Animals. All animal procedures were conducted with approval of the St. Vincent’s

Health Melbourne Animal Ethics Committee. CtskCre mice backcrossed to C57BL/6

were obtained from Dr Shigeaki Kato [31]. Floxed gp130 mice backcrossed onto

C57BL/6 were obtained from Rodger McEver (Oklahoma Medical Research

Foundation) [32]. Mice hemizygous for the CtskCre transgene were crossed with a

gp130 flox mouse in which the transmembrane domain was flanked by loxP sites,

resulting in ablation of intracellular gp130 signalling, as previously reported [33]. For

all experiments, Cre+ hemizygous wildtype littermates or cousins were used as

controls. IL-11Rα1−/− [34] and IL-6−/− mice [35] were obtained from Dr Matthias

Ernst. Compound IL-11Rα1−/−:IL-6−/− mice were generated by crossing IL-6−/−

mice with IL-11Rα1+/− mice as previously described [19]. Both male and female

mice were analysed, as indicated below.

Semi-quantitative real-time PCR (qPCR). RNA extraction, cDNA synthesis and

qPCR assays were performed as previously described [21]. Briefly, RNA was

extracted from primary osteoblasts lysed in TRIzol (Qiagen, Australia), and cDNA

was prepared using random hexamers (AffinityScript, Integrated Biosciences,

Australia) according to the manufacturer’s protocol. Real-time RT-PCR was

performed using the Stratagene MX3000P (Agilent Technologies) as previously

described[21]. Gp130 (Il6st) and Hmbs primer sets were designed using Primer Blast

(NCBI) as previously described [21]. Il6st primers: F –

AGAAGCCATAGTCGTGCCTGTGT, R – AAAGCAGAACAAGACGCCCAGCA.

Hmbs primers: F - TCATGTCCGGTAACGGCG, R –

CACTCGAATCACCCTCATCTTTG.

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In vitro osteoclast formation and Western Blotting. Osteoclasts were generated

from CtskCre.gp130w/w

and CtskCre.gp130f/f

mice from flushed bone marrow (mouse

bone marrow, MBM) or from non-adherent L-cell conditioned medium (LCM)-

treated bone marrow macrophage precursors (bone marrow macrophages, BMM)

stimulated for 7 days with M-CSF (30ng/ml, R&D Systems, Minneapolis, MN, USA)

and RANKL (100ng/ml, Oriental Yeast Company, Japan) in vitro as previously

described [36].

For Western Blots, osteoclasts were generated from 8 week old C57BL/6 mice as

above for 10 days; BMM precursors were treated for the same duration, but with M-

CSF alone. Cells were starved of serum for 3 hours and then treated with IL-6 +/-

soluble IL-6R at the concentrations indicated. Western membranes were

immunoprobed for phospho-Y(705)STAT3, phospho-S(727)STAT3 and total STAT3

(all from Cell Signaling Technologies; Danvers, MA, USA) and pan actin

(NeoMarkers; Fremont CA, USA), as previously described, using a 4-12% gradient

gel and iBlot transfer apparatus (Life Technologies, Grand Island NY, USA) [37].

Histomorphometry and microCT. Trabecular and cortical histomorphometry were

performed on undecalcified methacrylate-embedded samples of the proximal tibial

metaphysis of CtskCre.gp130w/w

and CtskCre.gp130f/f

littermates, and WT, IL-6, IL-

11R and IL-6/IL-11R null mice as previously described [19]. For dynamic

histomorphometry, calcein was injected 10 and 3 days prior to sacrifice (20mg/kg)

into 12 week-old mice. MicroCT was performed using the SkyScan 1076 system

(Bruker-microCT, Kontich, Belgium) on distal femoral specimens from 6, 12 and 26

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week-old CtskCre.gp130w/w

and CtskCre.gp130f/f

mice as previously described [21].

Briefly, images were acquired using the following settings: 9μm voxel resolution,

0.5mm aluminium filter, 50kV voltage and 100μA current, 2600 ms exposure time,

rotation 0.5o, frame averaging =1. Images were reconstructed and analysed using

SkyScan software NRecon (version 1.6.3.3), DataViewer (version 1.4.4) and CT

Analyser (version 1.12.0.0). Femoral trabecular analysis region of interest (ROI) was

determined by identifying the distal end of the femur and calculating 15% of the total

femur length towards the femora mid-shaft, where we then analysed an ROI of 12.6%

of the total femur length.

Serum biochemistry. Serum collected from fasting 12 week-old male and female

CtskCre.gp130w/w

and CtskCre.gp130f/f

mice was assayed for CTX1 and serum N-

terminal propeptide of type I collagen (P1NP) (both Immunodiagnostic Systems

Limited, Bolton, Tyne & Wear, UK) as per the manufacturer’s instructions.

Statistics. All graphs are mean + standard error of the mean. For in vitro osteoclast

formation, data represents 3 independent experiments, each using pooled long-bone

marrow from n=2 mice. N/genotype and sex is as reported in figure legends, and

statistical significance was determined using ANOVA analysis in Prism (Graphpad).

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Results

Effects of CtskCre-driven gp130 deletion on in vitro osteoclast formation

A significant knockdown of gp130 mRNA was observed in CtskCre.gp130f/f

bone

marrow macrophages (BMM) treated with RANKL by qPCR (Fig. 1A); since these

cultures are a mixture of differentiating and mature osteoclasts, as well as

macrophages, it is not surprising that decrease of gp130 was significant but

incomplete. Mouse bone marrow (MBM) and BMM isolated from CtskCre.gp130f/f

mice both generated significantly more osteoclasts/well when compared to marrow

from CtskCre.gp130w/w

littermates (Fig. 1B,C). Osteoclasts from CtskCre.gp130f/f

mice were also larger, and had a higher proportion of osteoclasts with >10 nuclei than

from CtskCre.gp130w/w

precursors (Fig. 1D), suggesting that gp130 signalling in

committed osteoclasts may limit their formation and fusion in vitro.

CtskCre-driven gp130 deletion increases femoral length

No detectable bone phenotype was observed in neonate CtskCre.gp130f/f

bones when

assessed by microCT or histology (data not shown). However, CtskCre.gp130f/f

female mice showed significantly greater femoral length at 12 weeks of age compared

to their w/w littermates (Fig. 2A), while femora from male mice were significantly

longer than their w/w littermates at both 12 and 26 weeks of age (Fig. 2B). To

determine whether this could be due to more extensive chondrocyte proliferation in

the absence of gp130 in CtskCre-expressing cells, the proximal tibial growth plate

width was measured in 6 week-old mice and found to be significantly greater in male

mice (Fig. 2C,D), although no statistically significant changes were detected in either

the hypertrophic or proliferating zones alone. Since growth plate width and bone

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length are determined by chondrocyte differentiation, this mild phenotype may relate

to low levels of cathepsin K expression in growth plate chondrocytes, as previously

reported [38]. Taken together with the early growth plate closure in mice with

attenuated STAT3 and enhanced ERK signalling downstream of gp130 [39] this

suggests that ERK signalling downstream of gp130 in cathepsin K positive cells

limits longitudinal bone growth. This is consistent with a previous report of reduced

bone growth in IL-6 overexpressing mice [40] that suggests a direct inhibitory effect

of IL-6 on bone growth.

Effects of CtskCre-driven gp130 deletion on trabecular structure and

remodelling

Trabecular bone structure was assessed by ex vivo microCT in 6, 12, and 26 week-old

mice. CtskCre.gp130f/f

male mice had a 25% lower trabecular bone volume and

significantly lower trabecular number at 12 weeks compared to CtskCre.gp130w/w

controls (Fig. 3A,B). There was no significant difference in trabecular thickness at

any of these ages (Fig. 3C). No significant difference in trabecular bone volume was

detected between female CtskCre.gp130f/f

and CtskCre.gp130w/w

controls (Fig. 3A-C).

Histomorphometric analysis revealed no alteration in osteoclast number in 12 week-

old male or female CtskCre.gp130f/f

mice (Fig. 3D), but osteoclasts were significantly

more spread out on the bone surface in both male and female CtskCre.gp130f/f

mice

(Fig. 3E). However, this increase in osteoclast length did not appear to relate to a

detectable change in osteoclast activity, as indicated by cartilage remnants in the

trabecular bone (Fig. 3F), or serum CTX1 levels (Fig. 3G). Surprisingly, although

gp130 deletion was directed to the osteoclast lineage, 12 week old male and female

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CtskCre.gp130f/f

mice both showed significantly fewer osteoblasts than their

littermate controls (Fig. 3H). Furthermore, mineralising surface was reduced in male

CtskCre.gp130f/f

mice, but not in female CtskCre.gp130f/f

mice (Fig. 3I). No

significant alteration in serum P1NP levels was detected in 12 week old male mice

(mean+SEM: CtskCre.gp130w/w: 38.2±4.6; CtskCre.gp130f/f: 34.7±3.2ng/ml). This

suggests that a low level of bone formation in the presence of normal bone resorption

causes the trabecular osteopenia of male CtskCre.gp130f/f

mice.

CtskCre-driven gp130 deletion reduces cortical growth

To determine whether the low level of bone formation in CtskCre.gp130f/f

mice was

restricted to trabecular bone, we assessed periosteal bone formation rate in these mice

and found that it was significantly lower in CtskCre.gp130f/f

male mice compared to

CtskCre.gp130w/w

controls (Fig. 4A). Bone formation rate is determined by both the

rate of mineralisation, which was significantly reduced on the periosteum in the

CtskCre.gp130f/f

male mice (Fig. 4B), and the extent of bone surface on which

mineralisation occurs (mineralising surface). Mineralising surface traditionally reports

the sum of double-labelled surface and half the single-labelled surface [41]. Double-

labelled surface forms when bone mineralisation occurs during the full extent of time

between administration of both labels. Single-labelled surface reflects the presence of

bone mineralised when only one dose was administered, or areas where osteoclasts

have resorbed labelled bone. Since the areas of periosteal growth are not undergoing

remodelling, we divided the mineralising surface into its separate components to

assess only the extent of bone surface that was growing throughout the entire

interlabel period (double-labelled surface). Periosteal double-labelled mineralising

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surface was significantly lower in both male and female CtskCre.gp130f/f

mice (Fig.

4C). In male mice, the lower mineral appositional rate, and smaller double-labelled

surface was associated with significantly smaller cortices by 26 weeks of age,

indicated by lower endocortical and periosteal perimeters in CtskCre.gp130f/f

compared to CtskCre.gp130w/w

males (Fig. 4D,E).

Global IL-6 knockout mice also exhibit impaired periosteal bone formation

Since osteoclasts do not express receptor subunits known to heterodimerise with

gp130 (OSMR and LIFR) [18, 22], any role of gp130 signalling in osteoclasts would

be restricted to cytokines capable of acting through gp130 homodimers complexed

with IL-11 and/or IL-6. Osteoclasts express both IL-11R [24] and IL-6R subunits [23]

and IL-6 signalling may also occur in osteoclasts through soluble IL-6R trans-

signalling. We previously reported that male mice with systemic deletion of IL-6 had

a lower periosteal mineral apposition rate than controls, but no significant reduction in

trabecular bone formation [19]. These data suggest that the low level of periosteal

growth in CtskCre.gp130f/f

mice could be explained by disrupted IL-6 signalling in

the CtskCre.gp130f/f

osteoclast. We therefore examined archived samples from our

earlier study to assess periosteal bone formation in mice with systemic deletion of IL-

6 and IL-11R. Systemic IL-6 deletion, but not IL-11R deletion, resulted in a

significantly lower level of periosteal double-labelled surface in male mice compared

to wild type controls (Fig. 5A), similar to that observed in CtskCre.gp130f/f

male

mice, suggesting that IL-6, and not IL-11, is the cytokine that promotes periosteal

growth via gp130 signalling in the osteoclast.

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IL-6 stimulates STAT3 tyrosine phosphorylation in primary osteoclasts

To determine whether IL-6 directly influences the osteoclast via either the membrane

bound IL-6R, or through trans-signalling via soluble IL-6 receptor (sIL-6R), we

treated C57BL/6 osteoclasts, generated by treating bone marrow macrophages

(BMM) with RANKL and M-CSF with IL-6 and the sIL-6R. Since osteoclast

precursors are always present, even in mature primary osteoclast cultures, we also

assessed the response of osteoclast precursors (BMM + M-CSF) cultured at the same

time. In both osteoclasts and their precursors, IL-6 stimulated phosphorylation of

tyrosine 705, but not serine 727 (Figure 5B). Addition of sIL-6R slightly augmented

the STAT3 tyrosine 705 phosphorylation response, but only in BMM precursors

treated with the highest dose of IL-6. This confirms that osteoclasts and their

precursors are respond to IL-6 primarily through classical membrane-bound IL-6R

signalling.

We also assessed mRNA levels of previously described coupling factors Ctf1,

Sema4d, Sphk1 and Spns2 in osteoclasts treated with IL-6 (10ng/ml) for 24 hours. At

that time point, we observed no significant changes (data not shown), suggesting that

osteotransmitters released in response to IL-6 treatment may be novel and discovering

these will require a more thorough investigation beyond the scope of this study.

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Discussion

Osteoclasts have two functions that modify the structure of bone: their defining

function is their ability to resorb bone, but they also stimulate the action of bone

forming osteoblasts. This activity is attributed to the release of activities that couple

the resorptive activity of osteoclasts with subsequent bone formation by osteoblasts

during remodelling in the “Basic Multicellular Unit” (BMU), and thereby maintain

bone mass. These factors are either produced by the osteoclasts themselves, or

released during resorption of the bone matrix, and many have been proposed to date

[42-45]. Here we report that CtskCre-directed genetic deletion of gp130 in osteoclasts

is capable of separating the osteoclast’s resorptive function from its coupling activity.

While resorption in vivo was unchanged by this genetic manipulation, bone formation

was reduced. Bone formation was reduced, not only on trabecular surfaces, but also

on the growing periosteum, where osteoclasts are not present. We therefore suggest

that gp130 signalling, most likely transduced by IL-6, induces osteoclasts to release

signals that are transmitted through the cortical bone to periosteal osteoblasts.

Although gp130 deletion was targeted to the osteoclast lineage with CtskCre, the most

profound phenotype observed in vivo was a low level of bone formation. In trabecular

bone this resulted in a transient lowering of trabecular bone volume in male mice.

Ctskgp130f/f

male mice develop a normal trabecular network by 6 weeks of age, but

do not reach the same peak trabecular bone mass as Ctskgp130w/w

male mice. This

suggests that the low level of bone formation in these mice is required for the accrual

of peak bone mass due to remodelling of the trabecular network developed by 6

weeks of age. The normal age-related decline in trabecular bone mass until 26 weeks

occurs. This suggests that there is a deficiency in coupling factor activity from gp130-

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deficient osteoclasts required for remodelling and consolidation of the trabecular

network during early adulthood. Factors that contribute to coupling may be secreted

by osteoclasts or released by the bone matrix, and may either stimulate or inhibit bone

formation [42, 44]. Since resorptive activity of Ctsk.gp130f/f

osteoclasts was not

reduced in vivo, the coupling activity responsible for the reduced bone formation on

trabecular bone surfaces is unlikely to be one released from the bone matrix.

Dissociation between the osteoclast’s resorptive and coupling activities has previously

been reported in a number of mouse models with disrupted bone resorption. For

example, in osteoclast-rich osteopetrotic mice, high numbers of non-resorbing

osteoclasts have been suggested to release sufficient coupling activity to allow bone

formation to continue in the absence of resorption [46-49]. Our study indicates that it

is possible for coupling activity of the osteoclast to be reduced without any change in

resorptive activity in vivo.

The term “coupling” refers to mechanisms that equate the activities of bone resorption

and formation, occurring sequentially on the same surface (i.e. within the BMU)

during bone remodelling. Our data suggests that there is also activity, promoted by

IL-6 in the osteoclast that stimulates bone formation in bone that has not recently

been resorbed by osteoclasts (i.e. modelling). We could not detect TRAP+ osteoclasts

intercalated with osteoblasts on the periosteal surfaces on which bone formation was

measured in either w/w or Ctsk.gp130f/f

mice at 6, 12 or 26 weeks of age (data not

shown), consistent with previous observations that osteoclasts are rarely found on the

diaphyseal periosteum after early stages of development [50]. The possibility that

endosteal osteoclasts can influence periosteal growth is supported by earlier work

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showing restored periosteal growth in osteoclast-deficient osteopetrotic RANKL null

mice when lymphocytes expressing RANKL were provided and induced osteoclast

formation on endocortical, but not periosteal bone surfaces [51]. Further evidence for

osteoclast - derived activity that can influence bone formation on the periosteum

comes from an osteoclast-specific cathepsin K null mouse, an osteoclast-rich

osteopetrotic mouse model that showed increased periosteal diameter [49]. This

mechanism of action may be relevant to the effect of cathepsin K inhibition by

Odanacatib of increasing periosteal bone formation in the monkey [52]. These

findings are consistent with the suggestion that factors released by endosteal

osteoclasts can signal to the osteoblast lineage on the periosteal surface.

To influence periosteal osteoblasts, the osteoclast-secreted factors might act locally on

the osteocytes closest to the osteoclast, with the signal passing through the osteocyte

network to the periosteal surface. An example of this is the way that CT-1 inhibits

osteocytic sclerostin production [17]. Alternatively, osteoclast-derived factors

themselves may pass through the lacunar-canalicular system as soluble factors, and

then act directly on the periosteal osteoblast to stimulate bone formation (represented

schematically in Fig 5). To distinguish this activity from coupling factors that act

during BMU-based remodelling, we suggest the term “osteotransmitter” for inhibitory

or stimulatory signals that originate from endosteal osteoclasts and influence

osteoblasts on the periosteal surface. It is possible that some overlap will be found in

the identity of the proteins that carry out these two roles.

Of all gp130 dependent ligand-specific receptors, only IL-6R and IL-11R are

expressed in osteoclasts [23, 24]. When germline deficient mice for either of these

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signalling pathways have been studied, only the IL-6 null mice demonstrate a

phenotype of reduced femoral width, with no significant alteration in trabecular bone

formation [19], suggesting that the osteotransmitters lacking in the Ctsk.gp130f/f

mouse may depend on IL-6 stimulation. Since in vitro studies to investigate the

specific defect in Ctsk.gp130f/f

osteoclasts that results in reduced osteoblast function

would be confounded by the increased level of osteoclastogenesis from Ctsk.gp130f/f

precursors in culture, we assessed STAT3 phosphorylation in IL-6-treated C57BL/6

osteoclasts, and carried out a limited assessment of known coupling factors which

may also act as osteotransmitters. STAT3 phosphorylation was robustly increased in

both osteoclasts, and their precursors by IL-6, both in the presence and absence of

sIL-6R. However, we could detect no significant alteration in mRNA levels of

cardiotrophin-1, Semaphorin 4d, sphingosine 1 kinase, or spinster homologue 2

(Spns2) induced by IL-6 (10ng/ml) in these cultures (data not shown), suggesting that

these previously described coupling factors [17, 53-55] are not the osteotransmitters

regulated by IL-6. Identifying such factors would require microarray or RNA-Seq

analysis and further testing in vitro and in vivo.

We report here that IL-6 null mice also exhibit reduced periosteal mineralising

surface, recapitulating the phenotype observed in the Ctsk.gp130f/f

mouse. This is

consistent with our previous work identifying that an IL-6-dependent pathway is

involved in coupling of bone formation to an increased level of resorption in the

osteopenic gp130Y757F

mice [39]. Thus we propose that IL-6 acts directly on the

committed osteoclast to promote release of osteotransmitters that stimulate osteoblast

differentiation and activity on the periosteal surface. Since IL-11R, but not IL-6 null

mice exhibit impaired bone formation on trabecular bone surfaces and this was not

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cell autonomous to the osteoblast lineage [19], the gp130-dependent stimulus of

coupling factor activity in trabecular bone is most likely to be IL-11 (see Figure 5).

The Ctsk.gp130f/f

phenotype of reduced bone formation on both cortical and

trabecular surfaces was most pronounced in male mice, as was the low level of

periosteal mineralising surface in IL-6 KO mice [19]. The periosteal perimeter of 26

week old male Ctsk.gp130f/f

mice was also not significantly greater than female

control or Ctsk.gp130f/f

mice. This suggests that the testosterone-specific pathways

that stimulate periosteal growth that are responsible for greater bone width in male

mice compared to females, and in all male mammals, may depend on IL-6/gp130

signalling in the osteoclast.

gp130 signalling in osteoclasts appears to also limit osteoclast size and continued

fusion. The greater osteoclast size observed in Ctsk.gp130f/f

mice both in vivo and in

vitro, and the higher nuclear number observed in vitro is consistent with increased

osteoclast size observed in mice with germline deletions in LIF, LIFR and CT-1 [16,

17, 56]. This had suggested that these cytokines are the key gp130 family members

that limit osteoclast precursor fusion, although the effect of LIF on osteoclast size was

restricted to those osteoclasts resorbing calcified cartilage [15]. However since the

LIFR, through which both LIF and CT-1 act, is not expressed in mature osteoclasts

[22], it is unlikely that these are the key IL-6 family members lacking in the

Ctsk.gp130f/f

model. Since IL-6 treatment reduces osteoclast size of mature

osteoclasts in the absence of osteoblasts [26], it is likely that the large osteoclasts of

Ctsk.gp130f/f

mice result from a lack of IL-6 mediated inhibition of osteoclast size.

Since the increased numbers of osteoclasts formed by Ctsk.gp130f/f

precursors in

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culture conditions was not reproduced in vivo, compensatory mechanisms in the mice

that normalise osteoclast numbers must exist. Increased osteoclast size was observed

both in vitro and in vivo. In vitro this was associated with increased osteoclast fusion

and greater nuclear number, a parameter that could not be reliably assessed in vivo.

The increase in osteoclast size in vivo could relate to increased osteoclast fusion, but

may also relate to a change in osteoclast attachment to the bone surface, as observed

in Src [57] and Pyk2 [47] deficient osteoclasts. However, unlike those osteoclasts we

observed no osteopetrosis resulting from defective resorption in Ctsk.gp130f/f

mice.

Does the increased osteoclast size observed in Ctsk.gp130f/f

mice relate to the

secondary reduction in bone formation? We previously reported a dramatic increase

in osteoclast size and reduced bone formation in both the osteoclast-rich osteopetrotic

Clc-7 null mouse [58] and the more mild osteoclast-rich osteopetrotic CT-1 null

mouse [17], suggesting that these observations may be linked. However, the

osteopetrotic Src [46] and Pyk2 null mice [47] have large osteoclasts with high levels

of bone formation so this conclusion cannot be generalised.

We propose the following model for those activities of osteoclasts that require

endogenous gp130 signalling in vivo, illustrated in Figure 5. IL-6 and possibly IL-11

signals, mediated by gp130 in osteoclasts, limit the spreading of osteoclasts on the

bone surface. In addition IL-11 actions on trabecular, and possibly endocortical,

osteoclasts promote release of one or more osteoclast-derived coupling factors that act

on mesenchymal stem cells (MSC) and/or committed osteoblast progenitors to

promote osteoblast differentiation (MSC) and trabecular bone formation. IL-6 signals

in the osteoclast to promote the release of osteotransmitters that promote bone

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formation on the periosteal surface. The transmission of these signals from the

endosteal space to the periosteal surface may occur through the osteocyte network,

since osteoclasts are not found adjacent to the periosteal surfaces on which bone

formation occurs. In the Ctsk.gp130f/f

mouse, reduced stimulation of coupling factor

and osteotransmitter release results in impaired bone formation on both trabecular and

periosteal surfaces.

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Figure Legends

Figure 1: Confirmation of reduced gp130 mRNA in osteoclasts formed from

CtskCre.gp130f/f

(f/f) BMM compared to CtskCre.gp130w/w

(w/w) and increased

osteoclast (OC) formation by mouse bone marrow (MBM) and bone marrow

macrophages (BMM) derived from f/f mice compared to w/w. A: gp130 (Il6st)

mRNA levels in osteoclasts formed from w/w and f/f BMM, differentiated for 7 days

in the presence of RANKL and M-CSF; B,C: Number of osteoclasts formed from

w/w and f/f precursors, and representative images taken from the same culture in the

centre of the well. D: Number of nuclei per osteoclast. Data is mean±SEM from n=3

independent cultures each from 2 animals, pooled. **, p<0.01; ***, p<0.001; ****,

p<0.0001 vs w/w.

Figure 2: Increased femoral length and growth plate width in CtskCre.gp130f/f

(f/f) mice compared to CtskCre.gp130w/w

(w/w). Femoral length in 6, 12 and 26

week old female (A) and male (B) mice. C: Total growth plate width in male and

female w/w and f/f mice at 6 weeks of age, split into hypertrophic and proliferating

zones; D: representative images of growth plates in 6 week old mice showing that

there is no major disturbance; scale bar=50μm. Data is mean±SEM from n=5-9 mice

per group. *, p<0.05; ***, p<0.001 vs w/w.

Figure 3: MicroCT analysis of trabecular bone, and histomorphometric analysis

of bone formation and resorption in CtskCre.gp130f/f

(f/f) mice compared to

CtskCre.gp130w/w

(w/w) mice. A-C shows microCT analysis of femora from 6, 12,

and 26 week (wk) old male and female mice. Shown are trabecular bone volume

(BV/TV), trabecular number (TbN) and trabecular thickness (TbTh). D-G: analysis of

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osteoclasts in proximal tibiae of 12 week old mice. Shown are osteoclast number /

bone perimeter (NOc/BPm), average osteoclast length (OcL), cartilage volume per

unit bone volume (CtgV/BV) and serum levels of CTX-1, as well as representative

TRAP stained images to show the increased osteoclast length (black arrows indicate

osteoclasts); scale bar=40μm. H-I shows trabecular osteoblast number (NOb/BPm)

and trabecular mineralizing surface (MS/BS) from 12 week old mice. Data is

mean±SEM from n=5-9 mice per group; analysis of 12 week old mice is from n=6-8.

*, p<0.05; ***, p<0.001; vs w/w.

Figure 4: Periosteal growth is significantly impaired in 12 week old male

CtskCre.gp130f/f

(f/f) mice compared to CtskCre.gp130w/w

(w/w) mice, resulting

in reduced periosteal perimeter by 26 weeks of age. Shown are (A) Periosteal bone

formation rate (PsBFR/BS), (B) periosteal mineral appositional rate (PsMAR), (C)

percentage of tibial periosteal bone surface with either no label (unlabeled), single

label or double label, and representative images of the region measured; scale bar =

50μm. D-E: Endocortical and periosteal perimeter of femoral bones measured by

microCT at 26 weeks of age. Data is mean±SEM from n=6-9 mice per group; **,

p<0.01; ***, p<0.001; vs w/w.

Figure 5: Periosteal mineralising surface is low in male IL-6 null mice, and IL-6

stimulates STAT3 phosphorylation in cultured osteoclasts and their precursors.

A: Periosteal mineralising surfaces in 16 week old WT, IL-6, IL-11R and IL-6/IL-

11R null mice; Data is mean±SEM from n=6-12 mice per group. B: STAT3

phosphorylation (pSTAT3) at tyrosine 705 (Y705) and serine 727 (S727) in response

to IL-6 treatment with and without addition of the soluble IL-6 receptor (sIL-6R), in

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bone marrow macrophage (BMM) osteoclast precursors and osteoclasts generated by

addition of M-CSF and RANKL. Total STAT3 (tSTAT3) and pan actin, as a loading

control, are also shown.

Figure 6: Key roles of gp130 in osteoclasts in trabecular and cortical bone: IL-6

(green) and possibly IL-11 signals, mediated by gp130 in osteoclasts, limit the

spreading of osteoclasts on the bone surface. In addition IL-11 (pink) actions on

trabecular osteoclasts promotes release of one or more osteoclast-derived coupling

factors (blue dotted lines) that act on mesenchymal stem cells (MSC) and/or

committed osteoblast progenitors to promote osteoblast differentiation (MSC). IL-6

signals in osteoclasts to promote the release of osteotransmitters (red dotted line) that

promote bone formation on the periosteal surface. The transmission of these signals

from the endosteal space to the periosteal surface may occur through the osteocyte

network, since osteoclasts are not found adjacent to the periosteal surfaces on which

bone formation occurs. In the Ctsk.gp130f/f

mouse, reduced stimulation of coupling

factor and osteotransmitter release results in impaired bone formation on both

trabecular and periosteal surfaces.

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Figure 1

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Figure 2

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Figure 4

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Figure 5

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Highlights (for review)

The role of gp130 signalling in osteoclasts was investigated using CtskCre driven deletion of gp130.

CtskCre.gp130f/f mice showed no change in bone resorption or osteoclast numbers in vivo.

CtskCre.gp130f/f mice showed impaired bone formation on periosteal and trabecular surfaces.

Similar observations in IL-6 null mice suggest IL-6 acts on osteoclasts to stimulate release of osteotransmitters in cortical bone.