Functional Interplay between Interleukin-1 Receptor and Elastin Binding Protein Regulates Fibronectin Production in Coronary Artery Smooth Muscle Cells

EXPERIMENTAL CELL RESEARCH 225, 122–131 (1996)ARTICLE NO. 0163

Functional Interplay between Interleukin-1 Receptor and ElastinBinding Protein Regulates Fibronectin Production

in Coronary Artery Smooth Muscle Cells

ALEKSANDER HINEK,1 SILVANA MOLOSSI, AND MARLENE RABINOVITCH

Division of Cardiovascular Research, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8; andDepartment of Pathology and Department of Pediatrics, University of Toronto, Toronto, Ontario, Canada M5G 1X8

vascular injury contribute to the genesis and progres-We have previously shown that free galactosugars sion of intimal lesions in atherosclerosis and allograft

and N-acetylgalactosamine glycosaminoglycans, e.g., arteriopathy [1, 2]. These interactions result in the syn-chondroitin sulfate (CS), release the 67-kDa elastin thesis and release of a wide range of cytokines andbinding protein (EBP) from arterial smooth muscle growth factors and in alterations of the extracellularcell (SMC) surfaces. This disrupts cell contact with matrix [3–5]. Rapid changes in the composition of ex-elastin, impairs assembly of new elastic fibers, and in- tracellular matrix further influence the synthetic andcreases fibronectin production, all of which promote migratory behavior of both vascular and inflammatorySMC migration and intimal thickening. The present cells [6, 7]. Among the numerous alterations in matrixstudy uncovered a mechanism regulating fibronectin components, increased and modified expression of fi-production in vascular myocytes related to a func- bronectin (FN) has been related to vascular pathobiol-tional interplay between EBP and the interleukin-1 re-

ogy. The accumulation of FN, its EIIIA spliced variant,ceptor type I. We showed that CS-induced shedding ofand of FN-derived peptides has been observed in earlythe EBP or internalization of this receptor after satu-lesions of atherosclerosis [8, 9] and related to pheno-ration with elastin-derived peptides (k-elastin, k-El)typic changes in vascular cells [10, 11], to their migra-stimulated fibronectin production in cultures of coro-tion [12], as well as to trafficking of leukocytes acrossnary artery SMC to a level observed with recombinantthe vessel wall [13–16]. Expression of FN can be regu-interleukin (IL)-1b. Upregulation of fibronectin by CSlated by a variety of factors implicated in develop-or k-El was abolished by a soluble IL-1 receptor antag-

onist, and synergistic stimulation of fibronectin pro- mental and pathological processes [17].duction occurred when CS or k-El was added with IL- Our group has shown that leukocytic infiltration of1b. Immunohistochemistry showed that EBP and IL-1 donor coronary arteries in cardiac allografts was ac-receptor type I codistributed on surfaces of unstimu- companied by increased expression of interleukin-1blated coronary artery SMC, while CS- and k-El-depen- (IL-1b) and accumulation of FN in the subendotheliumdent removal of EBP from the cell surface increased and inner media [11, 15, 16, 18–20]. In vitro studiesbinding of radiolabeled IL-1b to CA SMC. We propose further documented that allograft coronary artery en-a unique interaction between both receptors in which

dothlial and smooth muscle cells synthesize FN in largeunoccupied EBP interferes with IL-1b binding. Con-amounts when compared with host (nonallograft) cells,versely, increased accumulation of N-acetylgalactos-and this is regulated by endogenously produced IL-1b.amine glycosaminoglycans or elastin-derived peptidesOther studies from our group showed that treatmentin the vascular wall may unmask IL-1 receptor type Iof cultured vascular smooth muscle cells (SMC) withand increase binding of the cytokine and consequentN-acetylgalactosamine glycosaminoglycans (GAGs),upregulation of fibronectin production. q 1996 Academic

chondroitin sulfate (CS), and dermatan sulfate (DS),Press, Inc.

or with free galactosugars causes upregulation of FNsynthesis [21]. This occurs after shedding of the 67-INTRODUCTIONkDa elastin binding protein (EBP), a galactolectin ho-

Interactions between endothelium, smooth muscle mologous to the alternatively spliced variant of b-ga-cells, and activated leukocytes recruited to the site of lactosidase that normally resides on cell surfaces and

is capable of interacting with elastin and laminin [22–1 To whom correspondence and reprint requests should be ad- 25]. Binding of galactosugar-containing moieties to thedressed at Division of Cardiovascular Research, The Hospital for

lectin site of the EBP alters its molecular folding, whichSick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8,Canada. Fax: (416) 813-7480. results in discharge of the matrix ligands and release

1220014-4827/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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123CONTROL OF FIBRONECTIN SYNTHESIS IN SMC

duced by SMC. The amount of radiolabeled FN was normalized forof the receptor from the cell surface. We therefore spec-DNA content determined from each culture using Hoescht Reagentulated that a high concentration of galacto-GAGs,H3313 (Calbiochem), as previously described [28]. The fact that

which could remove this peripheral cell membrane pro- Western immunoblots with polyclonal anti-FN antibody (ICN, Costatein, may be involved in the mechanism controlling FN Mesa, CA) of the flow-through and washes from the gelatin 4B–

Sepharose columns did not detect any 220-kDa FN or significantproduction by vascular SMC.amounts of degradation products indicates that k-El and CS presentIn the present report, we have shown that culturedin the conditioned media do not interfere with FN binding to thecoronary artery smooth muscle cells (CA SMC) upregu- gelatin 4B–Sepharose and that FN can be quantitatively recovered

late production of FN, not only after CS-dependent from media and cell extracts.shedding of the EBP from their surfaces, but also after Assessment of fibronectin mRNA and mRNA stability. Culturesinternalization of this receptor triggered by saturation of CA SMC (101 106 cells/dish), maintained for 24 h in the presence

or absence of CS and k-El, were washed in PBS, and total RNAwith k-elastin (k-El), a preparation of soluble elastin-was extracted according to the methods used by Chomczynski andderived peptides which may mimic those released afterSacchi [29]. A purified 1.5-kb human FN cDNA (Telios Pharmaceu-proteolytic elastin degradation in arterial disease [26].ticals, La Jolla, CA) was labeled with [32P]d-CTP using a random

Chondroitin sulfate- and k-El-treated CA SMC stimu- primer kit (Amersham), and probes with specific activities of ú109

late FN production to an extent similar to that caused cpm/mg were recovered following Sephadex G-50 column chroma-tography. Prehybridization and hybridizations were performed forby recombinant IL-1b. We implicated IL-1b in the24 h at 427C using standard techniques. Autoradiographs of RNAmechanism causing upregulation of FN in response toblot hybridizations were analyzed by quantitative densitometry.both CS and k-El, since this effect is prevented by the Values were standardized to mRNA levels of glyceraldehyde-3-

soluble IL-1 receptor antagonist and since SMC pre- phosphate dehydrogenase assessed with labeled 1.2-kb cDNAtreated with CS or k-El were characterized by loss of GAPD probe (ATCC, Rockville, MD).

To assess the stability of mRNA for FN, confluent cultures of CAcell surface EBP and increased binding of radiolabeledSMC (10 1 106 cells/dish), maintained for 24 h in the presence orIL-1b.absence of CS or k-El, were additionally treated with actinomycin D(10 mg/ml) for 1, 2, 4, 6, 12, and 24 h. At the end of each time period,the total RNA was isolated from triplicate cultures and NorthernMATERIALS AND METHODSblots were assessed and standardized as described above.

Since preparations of CS and k-El isolated from animal tissues byCell cultures. Smooth muscle cells were harvested from explantsthe commercial suppliers were used in relatively high concentrationsof the tunica media taken from coronary arteries (CA) of young York-compared to that of recombinant IL-1b, we tested them for possibleshire piglets. The cells were passaged by trypsinization and main-IL-1b contamination. Western immunoblots with a specific anti-IL-tained in Medium 199 supplemented with 20 mM Hepes, 1% antibiot-1b antibody did not reveal any immunoreactive species in 2-mg sam-ics/antimycotics, and 10% fetal bovine serum (FBS) (all purchasedples of both reagents when resolved on SDS–PAGE under reducingfrom GIBCO, Burlington, Ontario, Canada). Confluent second pas-conditions (data not shown). To detect any general or toxic effect ofsage cells, immuno-positive with monoclonal antibody to smooththe tested reagents, total protein synthesis in cultures maintainedmuscle actin (Enzo, New York), were used in all experiments de-for 48 h in the presence and absence of 400 mg/ml of CS, 1 mg/ml ofscribed below.k-El, and 100 ng/ml of recombinant human IL-1b (both in mediumProduction of fibronectin by CA SMC. Confluent cultures (10 1with 5% FBS and in serum-free medium) was also determined in106 cells/dish) were maintained in Medium 199 (serum-free or con-triplicate after radiolabeling with 2 mCi/ml of [3H]leucine (Amer-taining 5% FBS) for 48 h in the presence or absence of 400 mg/ml ofsham), as previously described [23].CS (Sigma, St. Louis, MO), 1 mg/ml of k-El (Elastin Products, Pacific,

Immunocytochemistry. To illustrate the deposition of FN, con-MO), 100 ng/ml of recombinant human IL-1b (Upjohn, Kalamazoo,fluent cultures of CA SMC were treated with the above-mentionedMI), and 40 ng/ml of soluble IL-1b receptor antagonist (RA) (R andreagents for 48 h, and then fixed in 100% cold methanol for 30 minD Systems, Minneapolis, MN). The above-mentioned reagents wereat 0207C, blocked with 1% BSA in PBS, and incubated with a MBused either alone or in combinations, as described in the figure leg-140 monoclonal antibody to cell-associated human FN (Chemicon,ends.Temecula, CA) (5 mg/ml diluted 1:500). The immunoreaction wasAfter the first 24 h of incubation, 20 mCi/ml of [35S]methioninethen visualized with goat anti-mouse fluorescein-conjugated (GAM-(Amersham, Oakville, ON) was added to each sample and culturesFITC) immunoglobulin (ZYMED, San Francisco, CA), diluted 1:100.were kept for another 24 h. After this period, the culture mediumThe nuclei were counterstained with red propidium iodide (Sigma).was removed and stored in the presence of proteinase inhibitors

For the simultaneous immunofluorescent localization of the cell(2 mM PMSF, 2 mM EACA, 2 mM benzamidine, 10 mM N-ethylma-surface EBP and IL-1 receptor type I, cultured CA SMC were quicklyleamide, 2 mM leupeptin, and 10 mM EDTA, all from Sigma). Thefixed for 2 min with 0.5% paraformaldehyde (nonpermeabilized),cell layers were extracted overnight at 47C with 3 M guanidine HCL,blocked with 0.5 M glycine, 5% normal goat serum, and 1% BSA in10 mM Hepes, 0.1 M dithiotreitol (DTT), and 0.5% octyl-b-glucosidePBS and then incubated with the affinity-purified polyclonal anti-(OBG), in the presence of proteinase inhibitors. After centrifugationbody (anti-S-GAL) raised to a synthetic peptide, reflecting the elastinof insoluble material, the supernatant was dialyzed exhaustively atbinding domain of the 67-kDa EBP (5 mg/ml, diluted 1:200) [24] and47C against 0.1 M sodium bicarbonate, pH 8, containing proteinasewith the monoclonal antibody to the IL-1 receptor type I (Genzyme,inhibitors. Fibronectin was then extracted separately from the sam-Cambridge, MA). The cells were then stained with goat anti-rabbitples of media and cell layers using 1 ml gelatin 4B–Sepharose col-fluorescein-conjugated (GAR-FITC) and goat anti-mouse rhodamine-umns [27], eluted, and resolved by SDS–PAGE. The 220-kDa bandsconjugated (GAM-RITC) secondary antibodies (ZYMED), both di-were dissected from the gels and counted by liquid scintillation spec-luted 1:100. To compare the cell surface distribution of EBP andtrometry as previously described [16, 21]. The relatively large volumeIL-1 receptors with other ‘‘nonrelated’’ receptors, the cells were alsoof 4B-Sepharose present in each column (Pharmacia, Uppsala, Swe-immunostained with polyclonal antibody to EGF receptor.den) excluded the possibility that FN present in the FBS added to

the medium would compete for binding with radiolabeled FN pro- To illustrate CS-depending shedding of the EBP from the cell sur-


124 HINEK, MOLOSSI, AND RABINOVITCH

face, cultures of SMC preincubated for 15 min with 10 mg/ml of CS labeled IL-1b and with 1 mg/ml of CS or k-El in the presence orabsence of the IL-1 soluble receptor antagonist (50 ng/ml). Cells werewere lightly fixed for 2 min with 0.5% paraformaldehyde (nonperme-

abilized) and then immunostained with polyclonal anti-S-GAL and then washed to remove unbound cytokine (as mentioned above) andresuspended in 1 ml of phosphate-buffered saline, pH 8.3, containingwith the monoclonal antibody to the IL-1 receptor type I, as men-

tioned above. To establish whether binding of the elastin-derived 1 mM MgCl2 and 300 mg/ml of disuccinimidyl suberate (DSS) chemi-cal crosslinker (Pierce Chemical Co., Rockford, IL). After 15 min atpeptides to the cell surface EBP might affect cell surface distribution

of this elastin receptor or cause its internalization, SMC cultured on 47C, the DSS-treated cells were rinsed with 2 mM glycine in PBS toblock the crosslinker activity and lysed on ice for 15 min with 1%coverslips were incubated for 15 min at 47C in the presence of 1 mg/

ml of k-El and with 2 mg/ml of the F(ab*)2 fragments of anti-P-Gal NP-40 in 50 mM TBS, pH 8, containing proteinase inhibitors. Aftercentrifugation at 15,000g for 15 min, the supernatants were collectedimmunoglobulin which recognizes EBP without blocking its elastin-

binding domain [24]. The coverslips were then washed twice with for analysis by SDS–PAGE under unreduced conditions and by auto-radiography. To establish the identity of the crosslinked complex,the cold medium and transferred to 377C for 5, 10, 20, and 30 min.

At the end of each incubation period, the cells were extensively samples of cell extract resolved on polyacrylamide gel under unre-duced conditions were also transferred to immobilon membrane andwashed in PBS containing 0.5% sodium azide, fixed/permeabilized

with 100% methanol, and washed twice for 10 min with heat-inacti- immunoblotted with antibodies to IL-1b and with antibody to IL-1receptor type I.vated 3% normal goat serum and 1% bovine serum albumin. Immu-

noreactivity was then visualized after incubation with fluorescein- Analysis of data. In all biochemical studies, mean and standardlabeled F(ab*)2 fragments of GAR secondary antibody and counter- deviations were calculated, and statistical analyses were carried outstaining with propidium iodide. The parallel cultures incubated at by ANOVA followed by the Duncan test of multiple comparisons.47C with k-El were also treated with polyclonal anti-elastin antibodyto trace the fate of this external ligand after cell transfer to 377C.

RESULTSMetabolic labeling and immunoprecipitation of IL-1b. To estab-

lish whether endogenous IL-1b production might be regulated byUpregulation of Fibronectin Production by Galacto-CS and k-El, metabolic labeling of CA SMC cultures followed by

immunoprecipitation with the polyclonal antibody to porcine IL-1b GAGs and k-Elwas carried out. The cells (5 1 106 cells/dish) were preincubated in

Our present experiments confirmed the previouslyserum-free Medium 199 for 4 h, kept for 2 h in the presence orabsence of 400 mg/ml of CS and 1 mg/ml of k-El, and labeled with 5 reported CS-related increased synthesis of FN [21] andmCi/ml of [3H]proline (Amersham) for 6 h at 377C. Cells were then additionally showed that treatment of CA SMC withscraped into the medium and lysed in the presence of proteinase elastin-derived peptides (k-El) also leads to upregula-inhibitors after the addition of 10 ml of NP-40, 5 mg of sodium deoxy-

tion of FN production (Fig. 1). The Northern blots de-cholate to each 1-ml sample. After centrifugation at 20,000g for 15tecting steady state levels of FN mRNA and experi-min, the supernatants were precleaned by 30-min incubation with

50 ml of protein A–Sepharose beads, then incubated with polyclonal ments aimed at assessing mRNA stability showing noantibody to porcine IL-1b (Cytokine Science, Boston, MA) for 1 h at significant differences when control, galacto-GAGs,47C and precipitated by mixing with 50 ml of protein A–Sepharose

and k-El-treated cultures were compared (Fig. 2) sug-beads (Pharmacia). The samples were then quantitatively assessedgested that the mechanism of increased FN productionin a Beckman LS 8000 scintillation counter. The identity of labeled

IL-1b was confirmed by SDS–PAGE followed by autoradiography. is likely due to posttranscriptional and even posttrans-lational regulation.Binding of 125I-labeled IL-1b to CA SMC. Recombinant IL-1b (1

mg) was suspended in 20 ml of 0.1 M potassium phosphate (pH 7.4)containing 0.02% Nonidet P-40 and labeled with 0.5 mCi of 125I- Production of Fibronectin in Cultures of CA SMC IsBolton Hunter reagent (Amersham) according to the manufacturer’s Upregulated by IL-1binstructions. Iodinated cytokine was separated from unbound radio-activity by a P-2 column (Bio-Rad) and aliquoted. Coronary artery Further biochemical assessments showed that, in CASMC were then scraped from the culture dishes, washed, resus-

SMC cultures kept in medium with 5% FBS, both CSpended in ice-cold binding buffer consisting of RPMI 1640 (GIBCO),(400 mg/ml) and k-El (1 mg/ml) increased FN in theand supplemented with 0.5% BSA, 25 mM Hepes, and 0.05% sodium

azide, pH 7.2. Triplicate samples of CA SMC (1 1 106 cells/sample) conditioned medium (Fig. 1A), as well as cell- and ex-were then transferred to siliconized test tubes, incubated at 47C for tracellular matrix-associated FN (Fig. 1B) to the same2 h with various concentrations of 125I-labeled IL-1b, ranging from level as exogenous recombinant IL-1b (100 ng/ml).0.25 to 2 nM. To assess nonspecific binding, duplicate samples were

Cells maintained in serum-free medium displayed aalso incubated with 125I-labeled IL-1b in the presence of 200 nMproportionally similar increase in secreted FN in re-unlabeled IL-1b. At the end of each incubation period, cells were

washed twice for 5 min in medium containing 200 nM of nonlabeled sponse to CS, k-El, and IL-1b, but the absolute levelsrecombinant IL-1b. Then cell-bound radioactivity was separated of FN (cpm/mg DNA) were approximately 20% lowerfrom unbound 125I-labeled IL-1b by centrifuging the cells through a than in serum-containing cultures (data not shown).10:1 ratio of silicon:paraffin oil mixture. Cells were solubilized in 0.5

We next showed that stimulation of FN productionml of 0.5 N NaOH for 30 min, and cell-associated radioactivity waswith CS or k-El was likely mediated through endoge-determined by a Beckman 4000 g spectrometer. The influence of CS

and k-El on the 125I-labeled IL-1b binding efficiency to CA SMC was nous IL-1b, since this effect was blocked with the IL-also assessed when triplicate samples (1 1 106 cells/ml) were incu- 1 receptor antagonist (40 ng/ml). The specificity of thebated for different periods of time ranging from 5 min to 2 h at 47C soluble IL-1 receptor antagonist was confirmed by inhi-with 1.5 nM of 125I-labeled IL-1b in the presence or absence of various

bition of the effect of recombinant IL-1b (Figs. 1A andconcentrations of CS (0.01, 0.1, and 1 mg/ml) and k-El (1 mg/ml).1B). The IL-1 receptor antagonist did not affect theTo establish further the specificity of IL-1b binding to its cell sur-

face receptors, CA SMC were incubated for 2 h with 1.5 nM 125I- basal level of FN produced by unstimulated cells, but



The total protein synthesis measured by [3H]leucineincorporation increased linearly during the 48-h timeperiod in both serum-free and serum-containing cul-tures treated with concentrations as high as 400 mg/mlof CS, 1 mg/ml of k-El, and 100 ng/ml of Il-1b, indicat-ing that these agents were well tolerated by culturedSMC (data not shown).

EBP and IL-1 Receptors Codistribute on the CellSurfaces of CA SMC

Since our biochemical studies indicated that factorswhich remove or occupy the cell surface EBP influenceIL-1b-dependent stimulation of FN production, a closeassociation between the EBP and the IL-1 receptor waspostulated. We first established by immunostainingwith the respective antibodies that EBP and IL-1 recep-tors are generously distributed on the entire cell sur-

FIG. 1. Influence of CS (400 mg/ml), k-El (1 mg/ml), IL-1b (100ng/ml), and IL-1 receptor antagonist (RA) (40 ng/ml) on productionof secreted and unassembled (A) and cell- and matrix-associated fi-bronectin (B) in 48-h cultures of confluent CA SMC assessed by incor-poration of [35S]methionine into 220-kDa proteins isolated by gela-tin–Sepharose affinity. Values of mean { SD from three differentexperiments were statistically compared to the control (*Põ 0.001).

abolishes the stimulatory effect of CS, k-El, and IL-1b.Moreover, an additive increase in FN production wasobserved in cultures treated with a combination of CSor k-El and recombinant IL-1b.

Immunohistochemical studies with monoclonal anti-body recognizing cell- and matrix-associated fibronec-tin supported our biochemical observations. Theyshowed that CA SMC in the presence of CS, k-El, orIL-1b (Fig. 3C, 3E, and 3G, respectively) deposited sub-stantially more FN, which appeared as a network ofthick interconnected fibers, when compared with cells FIG. 2. Densitometry of Northern blots detecting the mRNA formaintained in control medium alone (Fig. 3A). These FN in SMC cultures normalized for glyceraldehyde dehydrogenase

(GAPD) mRNA does not reveal significant differences when compar-studies also showed that addition of the soluble IL-1bing control, CS-treated (400 mg/ml) and k-El-treated (1 mg/ml) cul-receptor antagonist abolished the stimulatory effect oftures (A). FN/GAPD mRNA stability assessed at two 24-h timeCS, k-El, and IL-1b (Fig. 3 D, F and H respectively) on courses following addition of actinomycin D (10 mg/ml) as described

the deposition of cell-associated FN but did not affect under Materials and Methods. There are also no significant differ-ences when comparing control, CS-, and k-El-treated cultures (B).the control pattern of distribution (Fig. 3B).



face of CA SMC with a similar filamentous and punctu- SMC revealed a single radiolabeled complex whichruns as a 97-kDa band on SDS–PAGE, suggesting thatate patterns (Figs. 4A and 4B, respectively). The mono-

clonal antibodies to the ‘‘unrelated’’ cell surface radiolabeled cytokine (molecular weight 17-kDa) boundto its 80-kDa receptor type I (Fig. 6, lane A). The ap-receptor for EGF also demonstrated fairly ubiquitous

immunostaining with a punctuate but not a filamen- pearance of the labeled 97-kDa species could be com-pletely blocked by the addition of an excess of unlabeledtous pattern on CA SMC surfaces (Fig. 4C). The double

immunostaining of nonpermeabilized CA SMC incu- IL-1b (Fig. 6, lane B). Cells treated with 1 mg/ml ofCS and k-El displayed enhanced amounts of 97-kDabated with a polyclonal antibody (anti-S-GAL) recog-

nizing the EBP and with a monoclonal antibody to IL- complex, suggesting an increase in binding of 125I-IL-1b to the cell surface (Figs. 6C and 6D, respectively).1 receptor demonstrated numerous overlapping areas

of immunostaining, which suggested the close proxim- Addition of a high concentration of soluble IL-1 recep-tor antagonist which competes with its cell surfaceity of these receptors on the cell surface (Fig. 4D). We

also showed that cells preincubated with CS lose most counterpart completely blocked binding of radiolabeledcytokine to CA SMC in the presence of 1 mg/ml of CSof their EBP, while the IL-1 receptors were maintained

(Fig. 4E). or k-El (Figs. 6E and 6F, respectively). The lack of anyother labeled bands diminished the likelihood that cellThe labeling of CA SMC surface EBP at 47C with

F(ab*)2 fragments of anti-P-Gal (which also recognize surface-attached aggregates of k-El or CS nonspecifi-cally bind IL-1b. The identity of the 97-kDa species asthe EBP) confirmed the filamentous and punctuate dis-

tribution of the EBP on the surface of CA SMC (Fig. a complex of cytokine and its receptor was confirmedby Western blots with antibodies to IL-1b and to IL-14F). The immediate incubation of cells with CS and

anti-P-Gal confirmed shedding of EBP from the cell receptor type I (Fig. 6, bottom, lanes B and C, respec-tively). We also showed that the synergistic effect be-surfaces (Fig. 4G), while binding of the elastin-derived

peptides to the cell surface EBP caused quick (2–5 min) tween CS or k-El and exogenous IL-1b is not relatedto increased synthesis of endogenous IL-1b. Metabolicclustering of the k-El–EBP complexes labeled with

anti-P-Gal and their subsequent internalization within labeling of CA SMC, followed by immunoprecipitationwith a polyclonal antibody to porcine IL-1b, showed10–20 min (Fig. 4H). Cell surface clusters formed after

exposure to k-El and internalized complexes were also that control, CS-, and k-El-treated cultures (media /cell lysates) contain equal amounts of radiolabeled pro-positively traced with an anti-elastin antibody (data

not shown). Distribution of IL-1 receptor remained the tein (data not shown), which excluded the possibilityof upregulation of endogenous IL-1b under the influ-same as before exposure to k-El.ence of CS or k-El.

EBP Interferes with IL-1b Binding to SMC Surfaces

DISCUSSIONWe further established that 125I-labeled recombinantIL-1b at a 1.5 nM concentration can saturate all bind-ing sites on CA SMC after a 1-h incubation at 47C and A physiologically significant interaction between cell

surface receptors and their potential ligands can takethat its binding can be competitively abolished by theaddition of excess unlabeled IL-1b (Fig. 5A). Moreover, place only when the permissive conformation of the

ligand coincides with the expression of the active cellwe have demonstrated that removal or saturation ofthe endogenous EBP does increase and/or accelerate surface receptor [30]. The functional status of the cell

surface receptors can be influenced by numerous fac-binding of IL-1b to its cell surface receptor (Fig. 5B).The time course of binding 125I-labeled IL-1b at 47C tors in the pericellular compartment [31, 32]. Proteo-

lytic enzymes, divalent cations, cell surface proteogly-documented that CS-treated CA SMC deprived of cellsurface EBP show a rapid and dose-dependent increase cans, and soluble ECM degradation products can either

stabilize, saturate, or remove receptor molecules fromin binding of radiolabeled cytokine when comparedwith control cells. Cells exposed to k-El (which satu- the cell surface [10, 33–35]. It has been shown that

enzymatic degradation of the ECM may be necessaryrates the EBP and causes its internalization) alsobound more radioactive cytokine, but the effect was to unmask the avb3 integrin [36] or the cell surface

receptors for TGF-b [37] and thrombin [38].visible only after 30 min of exposure.Crosslinking of 125I-labeled recombinant IL-1b to CA Our present report offers another paradigm, that un-

FIG. 3. Representative photomicrographs of cultured CA SMC maintained for 48 h in control medium (A) or in media containing RA(B), CS (C), CS and RA (D), k-El (E), k-El and RA (F), IL-1b (G), and IL-1b and RA (G) following immunostaining with monoclonal antibodyrecognizing cell- and matrix-associated fibronectin followed by fluorescein-conjugated secondary antibody. Nuclei were counterstained witha red propidium iodide. Original magnification, 1001.


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viously reported finding [21] that N-acetylgalactos-amine-containing GAGs, such as CS and DS, whichcause rapid shedding of the EBP from cell surfaces,upregulate production of FN by vascular SMC. Resultsof our present experiments indicate that saturation ofthe EBP with k-El and its subsequent internalizationalso upregulate FN production.

Since IL-1b is a potent inducer of FN production inCA SMC [20, 39], we directed our attention to a possibleinteraction between IL-1 receptors and the EBP. Chon-droitin sulfate-dependent shedding of the EBP fromCA SMC surfaces or saturation of the EBP with k-Elstimulated FN production to the same extent as wasobserved with exogenous IL-1b. Since the stimulatoryeffects of IL-1b, as well as CS and k-El, were abolishedin the presence of the soluble IL-1 receptor antagonist[40], we concluded that signaling pathways leading toincreased FN production in CS- and k-El-treated cellswere in fact activated through the cell surface receptorsfor IL-1. This was further suggested in experimentsshowing an additive effect of CS and IL-1b or k-El andIL-1b in inducing FN production. Results of theseassays were consistent with the immunostaining show-ing proportionally similar effects of IL-1b, CS, and k-El on net deposition of FN. In our experiments, weassessed the net production of FN over a 24-h period.The results suggest, but do not prove, that CS- and k-El-dependent accumulation of metabolically labeledFN is directly related to its increased synthesis. We

FIG. 5. (A) Triplicate samples of CA SMC (1 1 106 cells/sample) are fully aware, however, that the stimulating effectswere incubated with various concentrations of 125I-IL-1b at 47C for of CS and k-El on the net production of FN might also2 h. Specificity of binding was confirmed by competition with 200 be exercised by their possible influence on other pro-nM unlabeled IL-1b. (B) CA SMC (1 1 106 cells/sample) incubated

cesses, i.e., protein stability, fiber assembly, or inhibi-at 47C with 1.5 nM 125I IL-1b and various concentrations of CS (0.01–tion of proteolytic enzymes responsible for FN degrada-1 mg/ml) show a time- and dose-dependent increase in binding of

radioactive cytokine compared to control cells. A CS-dependent in- tion. Results of our experiments suggest that upregula-crease in 125I-IL-1b binding is visible after 5 min of incubation, while tion of FN production following CS or k-El treatment islonger exposure to k-El is needed for a significant increase in binding

due to a posttranscriptional event unrelated to mRNAof labeled IL-1b. The data have been corrected for nonspecific bindingstability. It may be related to increased efficiency ofof radioactive IL-1b by subtraction of values measured in the pres-

ence of 200 mM unlabeled IL-1b. translation of mRNA and/or to posttranslational modi-fications of the newly synthesized FN, allowing for ef-fective processing and secretion. This is consistent withresults obtained after stimulation of endothelial cellsmasking certain cell surface receptors can also be ac-with IL-1b [19].complished by nonproteolytic removal of another,

It has been previously shown that the functional sta-closely located cell surface protein. Our data further-more provide insight into the mechanism of the pre- tus of the IL-1 type I receptor can be affected by the

FIG. 4. (Top) Double immunostaining of nonpermeabilized CA SMC incubated with anti-S-GAL polyclonal antibody to EBP (detectedwith fluorescein-conjugated secondary antibody (A) and with monoclonal antibody to IL-1 receptor (detected with rhodamine) (B). (C)Immunostaining with a monoclonal antibody to cell surface receptor for EGF. Original magnification, 2501. (Middle) Double-exposedmicrograph of CA SMC (D) shows yellow-colored overlapping immunostaining for EBP and for IL-1 receptor. Cells preincubated with CSlose most of their EBP (detected with fluorescein) while the IL-1 receptors (detected with rhodamine) were maintained (E). Originalmagnification, 10001. (Bottom) The external labeling of CA SMC EBP at 47C with F(ab*)2 fragments of anti-P-Gal (which also recognizethe EBP) showed the filamentous and punctuate distribution of the EBP on the cell surface (F). Incubation of CA SMC with CS resultedin shedding of cell surface EBP (G). Cells preincubated with elastin-derived peptides at 47C and then incubated at 377C demonstrateinternalization of the EBP detected with anti-P-Gal (H). Original magnification, 4001.



pearance of the EBP. Since CS or k-El treatment doesnot upregulate expression of endogenous IL-1b, it ispossible that even the basal levels of this cytokine pro-duced by cultured SMC may be sufficient to triggerupregulation of FN after removal of the EBP. The con-cept of ‘‘masking’’ of IL-1 cell surface receptors by theEBP was strongly supported by binding studies. Cellstreated with CS and k-El, even at low concentrations,bound significantly more labeled IL-1b than control(untreated) cells. The effect of CS was quite rapid andeven a 5-min incubation was enough to evoke a visibleeffect. Longer incubations (30 min) were required todemonstrate the stimulatory effect of k-El. In addition,cross-linking experiments showed that radioactive IL-1b specifically binds to a single protein on the cell sur-face (presumably the 80-kDa IL-1 receptor type I) anda 97-kDa cross-linked complex of both proteins is pro-duced. The identity of the 97-kDa species as a molecu-lar complex of IL-1b and its receptor was confirmed bythe Western blots with the respective antibodies. Most

FIG. 6. CA SMC incubated for 2 h at 47C with 1.5 nM 125I-IL-1bimportant, our autoradiographs suggest that increas-in the presence of CS or k-El and in the presence or absence of IL-ing amounts of the 97-kDa species were extracted from1 soluble receptor antagonist were exposed to a DSS crosslinker,

lysed, and analyzed by SDS–PAGE in noneduced conditions followed cells treated with CS and k-El.by autoradiography and Western blots. (Top) Representative autora- In summary, our study illustrates a unique cell ma-diographs show that 125I-IL-1b is present in the 97-kDa protein com- trix interaction in which removal or conformationalplex fixed by protein crosslinker. Cells incubated in the presence of

changes of a cell surface matrix receptor (EBP) un-1 mg/ml of CS (lane C) and with 1 mg/ml of k-El (lane D) demonstratemasks an adjacent cytokine receptor, allowing accessan increase in 125I-IL-1b-containing species compared to control cells

(lane A). Formation of radiolabeled 97-kDa protein complex is of its ligand (IL-1b) and induction of an intracellularblocked in the presence of high concentration (200 nM ) of unlabeled signaling process by which a posttranscriptional mech-IL-1b (lane B). Addition of 50 ng/ml of IL-1 receptor antagonist to anism leads to a net increase in the production of meta-the incubation medium competitively decreases the specific binding

bolically labeled FN. Our observations indicate that aof 125I-IL-1b to cells stimulated with 1 mg/ml of CS (lane E) or withdynamic interplay between different cell surface recep-1 mg/ml of k-El (lane F). (Bottom) Western immunoblots show that

the 97-kDa species is immunoreactive with both the antibody to IL- tors may be controlled by components of the ECM or by1b (lane B) and the antibody to IL-1 receptor type I (lane C). Lane their degradation products and further define anotherA shows MW markers. level of complexity in an interaction between SMC and

three diverse matrix components: CS, elastin, and FN.Our observations would appear relevant to the mech-

anisms facilitating SMC migration into the subendo-dynamics of synthesis, by recycling, and by their lateralmovement in the plane of plasma membrane [41]. It thelium observed in vascular diseases. Since SMC mi-

gration is accompanied by increased accumulation ofhas also been established that IL-1 type I receptor mol-ecules may form dimers, which may explain the two N-acetylgalactosamine-containing GAGs, especially

CS and DS, and by accumulation of nonassembled andclasses of receptor binding affinity [42]. Since the opti-mal binding of IL-1 may be affected by dimerization of partially degraded elastin [26, 46–48], we speculate

that those conditions which cause removal of the EBPreceptors [43], it is conceivable that removal of the EBPmay affect lateral mobility and clustering of IL-1 type may permit an unrestricted cellular response to IL-1b.

This in turn may lead to accumulation of FN, whichI receptor molecules, increasing their avidity for theavailable IL-1b. On the other hand, the association be- facilitates SMC and leukocyte migration associated

with the development of vascular lesions [13– 16]. Thetween EBP and IL-1 type I receptor may also preventthe constant binding of IL-1 to this receptor and its EBP and IL-1 receptor type 1 are also present on sur-

faces of leukocytes, macrophages, fibroblasts, and chon-subsequent downregulation, which has been also de-scribed [44, 45]. drocytes. It is therefore conceivable that the functional

interplay between these two cell surface receptors mayThe possibility of a close interaction between bothcell surface receptors was substantiated in immunohis- also contribute to the pathobiology of inflammatory dis-

eases.tochemical studies in which we first localized the EBPand IL-1 receptor type I on cell surfaces of unstimu-lated CA SMC and later showed the CS-induced disap- This work was supported by a Program Grant from the Medical



Research Council of Canada (PG 12351). A.H. and M.R. are Career 23. Hinek, A., Mecham, R. P., Keeley, F. W., and Rabinovitch, M.(1991) J. Clin. Invest. 88, 2083–2094.Investigators of the Heart and Stroke Foundation of Ontario. The

authors are grateful to Dr. John Callahan from the Division of Neuro- 24. Hinek, A., Rabinovitch, M., Keeley, F. W., and Callahan, J.sciences, Hospital for Sick Children, Toronto for a generous gift of (1993) J. Clin. Invest. 91, 1198–1205.anti-S-GAL antibody, and to Dr. Irit Lax from New York University 25. Hinek, A. (1994) Cell Adhesion Commun. 2, 185–193.Medical Center for a gift of anti-EGF receptor antibody. We also

26. Oho, S., and Rabinovitch, M. (1994) Am. J. Pathol. 145, 202–thank Darinka Sakac and Mike Starr for skillful technical help and210.Susy Taylor for excellent secretarial assistance.

27. Wrana, J., Maeno, M., Hawrylyshyn, B., Yao, K., Domenicucci,C., and Sodek, J. (1988) J. Cell Biol. 106, 915–924.

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Received December 8, 1995Revised version received February 20, 1996


Functional Interplay between Interleukin-1 Receptor and Elastin Binding Protein Regulates Fibronectin Production in Coronary Artery Smooth Muscle Cells

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