Inflammation-Induced Chondrocyte Hypertrophy Is Driven by Receptor for Advanced Glycation End Products

This information is current as Denise L. Cecil, Kristen Johnson, John Rediske, Martin of September 28, 2021. Lotz, Ann Marie Schmidt and Robert Terkeltaub J Immunol 2005; 175:8296-8302; ; doi: 10.4049/jimmunol.175.12.8296 http://www.jimmunol.org/content/175/12/8296 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Inflammation-Induced Chondrocyte Hypertrophy Is Driven by Receptor for Advanced Glycation End Products1

Denise L. Cecil,* Kristen Johnson,* John Rediske,‡ Martin Lotz,§ Ann Marie Schmidt,† and Robert Terkeltaub2*

The multiligand receptor for advanced glycation end products (RAGE) mediates certain chronic vascular and neurologic degen- erative diseases accompanied by low-grade inflammation. RAGE ligands include S100/calgranulins, a class of low-molecular-mass, calcium-binding polypeptides, several of which are chondrocyte expressed. Here, we tested the hypothesis that and RAGE signaling modulate osteoarthritis (OA) pathogenesis by regulating a shift in chondrocyte differentiation to hypertrophy. We analyzed human cartilages and cultured human articular chondrocytes, and used recombinant human S100A11, soluble RAGE, and previously characterized RAGE-specific blocking Abs. Normal human knee cartilages demonstrated constitutive RAGE and Downloaded from S100A11 expression, and RAGE and S100A11 expression were up-regulated in OA cartilages studied by immunohistochemistry. CXCL8 and TNF-␣ induced S100A11 expression and release in cultured chondrocytes. Moreover, S100A11 induced cell size increase and expression of type X collagen consistent with chondrocyte hypertrophy in vitro. CXCL8-induced, IL-8-induced, and TNF-␣-induced but not retinoic acid-induced chondrocyte hypertrophy were suppressed by treatment with soluble RAGE or RAGE-specific blocking Abs. Last, via transfection of dominant-negative RAGE and dominant-negative MAPK kinase 3, we

demonstrated that S100A11-induced chondrocyte type X collagen expression was dependent on RAGE-mediated p38 MAPK http://www.jimmunol.org/ pathway activation. We conclude that up-regulated chondrocyte expression of the RAGE ligand S100A11 in OA cartilage, and RAGE signaling through the p38 MAPK pathway, promote inflammation-associated chondrocyte hypertrophy. RAGE signaling thereby has the potential to contribute to the progression of OA. The Journal of Immunology, 2005, 175: 8296–8302.

ow-grade chronic inflammation, mediated partly by the proteinase 13 (MMP-13), and promotion of pathologic calcifica- effects on chondrocytes of cartilage-expressed and syno- tion (8–10). OA cartilages typically develop foci of maturation of L vium-expressed cytokines including IL-1␤, TNF-␣, (IL- cells to hypertrophic differentiation (10). 8)/CXCL8, and (growth-related oncogene ␣)/CXCL1 appear to A growing body of evidence has implicated the receptor for contribute to the progression of osteoarthritis (OA)3 (1–8). Re- advanced glycation end products (RAGE) in certain chronic arte- by guest on September 28, 2021 cently, we linked the induction of altered chondrocyte differenti- rial, renal, and neurologic degenerative conditions associated with ation to chemokine-induced inflammation, by demonstrating that low-grade tissue inflammation (11–14). RAGE is a broadly ex- CXCL8 and CXCL1, both of which are up-regulated in OA car- pressed 45-kDa transmembrane bearing a 43-aa cytosolic tilage (6), stimulated chondrocyte hypertrophic differentiation in tail, and three extracellular domains (i.e., the ligand-binding V vitro (8). Chondrocyte hypertrophy can contribute to the progres- (VЈ) domain, and two C domains) that confer RAGE membership sion of OA via effects including dysregulation of matrix repair in the Ig superfamily (11–14). RAGE is a cognate receptor for four through reduced expression of collagen II and aggrecan, increased distinct classes of ligands (11–14), including S100/calgranulins, a expression of type collagen X, up-regulation of matrix metallo- family of Ͼ20 low-molecular-mass (ϳ10–14 kDa), acidic that form homodimers, heterodimers, and larger multimers, and bind calcium via two internal EF-hand motifs in each monomer *Veterans Affairs Medical Center, Department of Medicine, University of California, (14–16). Certain individual S100/calgranulins (, S100A2, San Diego, CA 92161; †Department of Surgery, College of Physicians and Surgeons, S100A4, ) have been reported to be expressed by chondro- Columbia University, New York, NY 10032; ‡Novartis Institutes for Biomedical Research, Cambridge, MA 02139; and §The Scripps Research Institute, La Jolla, CA cytes (17–19), and intense S100 immunoreactivity is a marker of 92037 chondrogenic differentiation (20). In addition, concentrations of Received for publication May 12, 2005. Accepted for publication September certain calgranulins in diseased joint fluids reach nanomolar con- 23, 2005. centrations (21, 22). The costs of publication of this article were defrayed in part by the payment of page Recently, RAGE expression was demonstrated in human artic- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ular cartilage and observed to be more robust in aging and OA 1 This work was supported by a Merit Review grant from Department of Veterans cartilages (18). Furthermore, IL-1 and fibronectin fragments in- Affairs (to R.T.) and by support from the National Institutes of Health (including duced chondrocyte RAGE expression and micromolar S100B in- Grants P01AGO7996, HL077360-01, and AR049366-01A2). duced MMP-13 in chondrocytes mediated by RAGE in vitro (18). 2 Address correspondence and reprint requests to Dr. Robert Terkeltaub, Veterans In this study, we examined the potential role in articular chondro- Affairs Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161. E-mail address: [email protected] cyte hypertrophy of RAGE, with particular focus on RAGE inter- 3 Abbreviations used in this paper: OA, osteoarthritis; MMP, matrix metalloprotein- action with S100A11 (S100C, calgizzarin) (15, 16). Our results ase; RAGE, receptor for advanced glycation end products; ATRA, all-trans retinoic implicate up-regulated S100A11 expression, and RAGE-depen- acid; sRAGE, soluble RAGE; DN, dominant negative; MKK, MAPK kinase; AGE, advanced glycation end products; HMGB-1, high-mobility group box-1; TG2, trans- dent and p38 MAPK-dependent signaling, in inflammatory regu- glutaminase 2. lation of chondrocyte hypertrophic differentiation in OA cartilage.

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 8297

Materials and Methods and DN-RAGE were further verified by SDS-PAGE/Western blotting anal- Reagents ysis (26), using the aforementioned murine monoclonal anti-RAGE Ab. Unless otherwise indicated, Western blotting in this study analyzed ali- All-trans retinoic acid (ATRA), and human recombinant CXCL8 and quots of 30-␮g protein obtained from whole-cell lysates, or precipitated TNF-␣ were from R&D Systems, and rabbit polyclonal Abs to type X from conditioned medium using 15% TCA, followed by separation via collagen were from Calbiochem. mAb to RAGE was from Chemicon In- 10% SDS-PAGE. The Western blots used HRP-conjugated secondary Abs, ternational. Soluble human RAGE (sRAGE) and rabbit polyclonal RAGE- as described (26). Immunoreactive products were detected using ECL. Pri- specific functional blocking Abs were as previously characterized (23). mary and secondary Ab dilutions were 1/2000. Abs to type X collagen Human RAGE and dominant-negative (DN) RAGE cDNA in the vector were from Calbiochem, to p38 and phosphorylated p38 were from Cell pCDNA4-V5His (Invitrogen Life Technologies) were as previously de- Signaling, and to ␣- were from Sigma. HRP-conjugated goat anti- scribed (23, 24). DN-MAPK kinase 3 (MKK3) (25) was generously pro- rabbit IgG and anti-mouse IgG were obtained from Santa Cruz vided by Dr. J. Han (The Scripps Research Institute, La Jolla, CA). Unless Biotechnology. otherwise indicated, all other reagents were obtained from Sigma-Aldrich. Flow cytometric analyses Immunohistochemistry for RAGE and S100A11 Human articular chondrocytes (1 ϫ 106) were permeabilized using the BD Mouse polyclonal Abs recognizing the human S100A11-specific peptide Cytofix/Cytoperm kit (BD Pharmingen). Where indicated, the cells were CHDSFLKAVPSQKRT, which is expressed by S100A11 but not by other incubated for 60 min at 4°C with murine monoclonal anti-RAGE or murine S100/calgranulins, were generated by Zymed Laboratories. All research IgG isotype control. Washed cells were incubated with FITC-conjugated involving human subjects was performed under institutionally approved Ј goat F(ab )2 anti-mouse IgG for 60 min at 4°C. Fluorescence was detected protocols. As previously described (26), defined specimens of normal and using a FACSCalibur apparatus (BD Biosciences) with data analyzed using OA human articular cartilage (age ranges, 22–32 and 66–83, respectively) CellQuest software (Purdue University, West Lafayette, IN). were taken as full-thickness blocks. For immunohistologic analyses of Downloaded from RAGE and S100A11, frozen sections (5 ␮m) were fixed using 4% para- formaldehyde for 20 min. After washing with PBS, the sections were in- Preparation of recombinant soluble S100A11 and “dot-blot” cubated with 1% Triton X-100 and microwaved for 1 min, blocked with immunoblotting studies 10% goat serum for 20 min, and incubated for 24 h at 4oC with mouse anti-RAGE or anti-S100A11 Abs. The primary Ab was detected via the Human S100A11 cDNA was purchased from American Type Culture Col- avidin-biotin conjugate method applied according to manufacturer’s in- lection and was subcloned into pcDNA4/HisMax (Invitrogen Life Tech- ϫ 7 structions using the Sigma Rabbit ExtrAvidin Peroxidase Staining kit. Per- nologies). Aliquots of 6 10 HEK 293 cells were transfected with N- oxidase activity was detected using the Sigma Fast DAB staining kit, ac- terminal His-tagged S100A11 cDNA using Superfect (Qiagen), according http://www.jimmunol.org/ cording to manufacturer’s instructions. to manufacturer’s protocol. S100A11 protein was isolated using the Pro- Bond Purification System (Invitrogen Life Technologies) under native con- Chondrocyte isolation, culture, transfection, stimulation and ditions. S100A11 protein was then dialyzed using the Slide-A-Lyzer 3,500 agonists used, and SDS-PAGE/Western blotting MWCO dialysis cassette (Pierce Biotechnology) in PBS overnight at 4°C. SDS-PAGE/Western blotting was performed to confirm purity. LPS was Chondrocytes were obtained from the medial and lateral condyles, the pa- undetectable (Ͻ0.025 endotoxin U/ml) in rS100A11 by the Limulus ame- tellar groove and the tibial plateau of normal human knee articular carti- bocyte lysate assay (BioWhittaker). For “dot-blot” immunoblotting, 250 ng lages (donor age range, 22–62), by previously described methods (26). For of S100A11, human recombinant S100B, and S100A12 were directly spot- the studies described, chondrocytes were carried in monolayer culture in ted onto nitrocellulose and allowed to dry for 1 h. The blots were blocked DMEM/high glucose supplemented with 10% FCS, 1% L-glutamine, 100 in 5% milk for 2 h, then probed with the anti-peptide Ab to S100A11 for ␮ by guest on September 28, 2021 U/ml penicillin, and 50 g/ml streptomycin at 37°C with 5% CO2. For 18 h, and with secondary Ab as described above for Western blots. Where experiments in which first passage was stimulated with agonist serum sup- indicated, we mixed the Abs to S100A11 with a 5-fold molar excess of the plementation in the medium was changed to 1% from 10%, and unless immunogenic S100A11 polypeptide and incubated for 18 h at 4°C before otherwise indicated, cells were treated with 10 ng/ml CXCL8 and TNF-␣, applying this mixture to the blot. 10 nM ATRA, and 100 ng/ml S100A11. In transfection studies, aliquots of 1.0 ϫ 106 human chondrocytes in 100-mm culture dishes were grown for 18 h in DMEM/high glucose sup- Results plemented with 10% FCS. Then, transfections were performed using an RAGE and S100A11 expression in OA cartilage in situ electroporator from Amaxa, according to the manufacturer’s protocol for chondrocytes. Transfection efficiency, assessed as a control in each exper- S100/calgranulin sequences contain several highly conserved ele- iment via ␤-galactosidase transfection and staining, was consistently be- ments (16). Hence, for these studies, we generated murine poly- tween 70 and 80% in human chondrocytes. Forced expression of RAGE clonal Abs to the C-terminal 15-aa peptide of the calgranulin

FIGURE 1. Specificity of the murine anti-peptide polyclonal S100A11. A, Sequence specificity of S100A11 C-terminal peptide sequence. The schematic of structural domains of S100A11 highlights a 15-aa peptide C-terminal to the second canonical S100/calgranulin EF hand used as the immunogen to make murine polyclonal Abs (please refer to Table I). B, S100A11 Ab specificity verified by “dot-blot” immunoblotting. Human S100A11, S100B, and S100A12 (250 ng each) were directly applied, as indicated, to nitrocellulose for immunoblotting, as described in Materials and Methods, and using the anti-peptide S100A11 Ab. Lane 4 contained 250 ng of S100A11 but was probed with the anti-peptide S100A11 Abs after preadsorption with 5-fold molar excess to Abs of the unique immunogen S100A11 C-terminal peptide described above. 8298 RAGE AND CHONDROCYTE HYPERTROPHY

RAGE ligand S100A11, a region C-terminal to the canonical EF- hand domain (Fig. 1A) and that was determined to have a unique sequence by BLAST analysis, including comparison aligned C ter- mini of other S100/calgranulins (Table I). “Dot-blot” immunoblot- ting confirmed specificity of the Ab for S100A11 (Fig. 1B). Immunohistochemical analyses revealed low-level constitutive expression of RAGE and S100A11 in normal human knee articular cartilages (Fig. 2). We confirmed (18) that chondrocyte RAGE expression appeared up-regulated in OA knee cartilages, particu- larly so in the deep zone but also as detected in chondrocytes in the superficial zone (Fig. 2). S100A11 expression was markedly in- creased in all zones of OA cartilages (Fig. 2). S100A11 became particularly abundant in the pericellular matrix of OA chondro- cytes (Fig. 2), suggesting that chondrocytes actively secreted S100A11 in the course of OA.

Induction of S100A11 in cultured human articular chondrocytes Cultured normal human knee articular chondrocytes were stimu- lated for 24 h with the known inducers of chondrocyte hypertrophy Downloaded from CXCL8 and ATRA (8, 9, 26). We also stimulated chondrocytes with TNF-␣, a modulator of OA pathogenesis (1, 2, 7). As as- sessed by flow cytometry, RAGE was expressed by 59 Ϯ 9% of normal articular chondrocytes (n ϭ 6), with no detectable, signif- icant effects on RAGE expression of CXCL8, ATRA, or TNF-␣

(Fig. 3A). Concurrent analysis of the normal articular chondrocytes http://www.jimmunol.org/ by SDS-PAGE/Western blotting revealed that S100A11, which re- mained below limits of detection in both unstimulated and stimu- lated cell lysates (not shown), was both robustly induced and re- FIGURE 2. Comparison of expression of RAGE and S100A11 in nor- mal and OA human knee articular cartilages. Frozen sections of normal and leased into the conditioned medium by chondrocytes in response to ␣ OA human knee articular cartilages were stained with murine monoclonal CXCL8, ATRA, and TNF- (Fig. 3B). S100A11, like certain other anti-RAGE Ab and with murine anti-peptide polyclonal Abs specific for calgranulins, is known to form covalently bonded dimers and mul- S100A11, as described in Materials and Methods. Magnifications of ϫ25, ϫ63, and ϫ116 are shown. The normal mouse serum control is shown at ϫ25 magnification. These data are representative of three normal donors and three OA donors and demonstrate up-regulation of RAGE and, even by guest on September 28, 2021 Table I. Comparison of the aligned C-terminal amino acid sequences of S100A11 and 14 other S100/calgranulinsa more so, S100A11 in OA cartilages, as discussed in the text.

timers and this process optimizes binding to RAGE (16, 27, 28). Whereas S100A11 isolated from 293 cells was detectable only as a monomer of ϳ11 kDa, S100A11 in conditioned medium of chondrocytes treated with exogenous S100A11, or with CXCL8, ATRA, and TNF-␣, appeared partially multimeric, with particu- larly large multimers detected in CXCL8-treated cells (Fig. 3B).

S100A11-induced chondrocyte hypertrophy mediated by RAGE Because the release of S100A11 was inducible in normal chon- drocytes, we next tested for the potential of S100A11 to regulate chondrocyte differentiation. We observed that ϳ10 nM S100A11 (100 ng/ml) induced type X collagen expression in human articular chondrocytes after 5 days in culture (Fig. 4A). Dose-response stud- ies indicated detectable induction of type X collagen by 100 ng/ml S100A11 but not 0.1–10 ng/ml S100A11 (data not shown). Chon- drocytes coincubated with sRAGE, which lacks the transmem- brane and cytosolic tail domains of RAGE (23), demonstrated at- tenuated type X collagen expression in response to 100 ng/ml S100A11 (Fig. 4A). Because sRAGE has the potential to affect the binding of RAGE ligands to receptors other than RAGE, we ad- ditionally studied the effects of chondrocyte treatment with 20 ␮g/ml previously described rabbit polyclonal blocking Abs spe- a Alignment of C-terminal amino acid sequences of the indicated S100/calgranu- cific for RAGE (23). We used conditions under which 20 ␮g/ml lins by BLAST analysis is shown. The C-terminal 15-aa peptide sequence of nonimmune rabbit IgG was confirmed to not suppress the induc- S100A11 (in bold italics) was verified to be unique not only to calgranulins but also other proteins by BLAST analysis, and this peptide was the immunogen in generating tion of type X collagen (data not shown). Under these conditions, murine polyclonal S100A11-specific Abs as described in Materials and Methods. the RAGE-specific blocking Abs were observed to suppress The Journal of Immunology 8299 Downloaded from

FIGURE 4. Effects of soluble S100A11 on type X collagen expression and chondrocyte size mediated by RAGE. First-passage normal human chondrocytes (donor ages, 22–62) (5 ϫ 105 cells/six-well dish) were stim- ulated with S100A11 in the presence or absence of 1 ␮g/ml sRAGE or 20 ␮g/ml RAGE-specific blocking Abs, where indicated. A, SDS-PAGE/ Western blotting analysis for type X collagen expression was performed FIGURE 3. RAGE expression and inducible S100A11 secretion in cul- http://www.jimmunol.org/ using cell lysates at 5 days in culture. B, Flow cytometry measurements of tured human articular chondrocytes. A, RAGE expression. First-passage forward and side scatter were performed on the chondrocytes after 24 h of human knee articular chondrocytes (5 ϫ 105 cells/six-well dish) from six stimulation. These data are representative of results from eight normal normal donors (age range, 29–62), were prepared as described in Mate- human donors. rials and Methods, were studied for RAGE expression by flow cytometric analysis. The panels, taken from studies of one normal donor representative of all the donors, depict binding of isotype control IgG (open histogram) RAGE-specific blocking Abs (Fig. 5, A and C, respectively). Con- and additionally of anti-RAGE IgG without stimulation or in response to comitantly, we discovered that TNF-␣ induced chondrocyte type X CXCL8, TNF-␣, and ATRA for 24 h (solid histograms). In normal chon- collagen expression and that TNF-␣ did so in a manner attenuated drocytes, constitutive RAGE expression was detected in 59 Ϯ 9% of cells, by sRAGE or RAGE-specific blocking Abs (Fig. 5B). by guest on September 28, 2021 and there were no significant changes in the proportions of RAGE-express- ing chondrocytes in response to CXCL8, TNF-␣, or ATRA, as seen in the S100A11 signaling through RAGE to induce p38 MAPK kinase representative results from the single donor demonstrated here. B, pathway activation and type X collagen S100A11 expression and multimerization in chondrocytes. SDS-PAGE/ We previously demonstrated that p38 MAPK pathway activation Western blotting analysis was performed on 1 ␮g of human recombinant S100A11 isolated from transfected 293 cells as described in Materials and was essential for transduction of chondrocyte hypertrophy in re- Methods (left side of figure), and on 30-␮g aliquots of protein precipitated sponse to CXCL8 and CXCL1 (8). Hence, we concluded the study from conditioned medium of first-passage normal human knee chondro- cytes (right side of figure) (donor ages, 29–62). The chondrocytes were prepared under the same conditions as for A above, and stimulated for 24 h by addition of S100A11, CXCL8, TNF-␣, or ATRA, at which time con- ditioned media were studied. Results are representative of those from six different normal human donors, as in A.

S100A11-induced chondrocyte type X collagen expression (Fig. 4A). Next, we validated induction of chondrocyte hypertrophy by examining the chondrocytes for S100A11-induced size increase. Within 24 h, Ͼ20% of chondrocytes increased detectably beyond the resting size range in response to S100A11, as assessed by increase in forward scatter via flow cytometry analysis (Fig. 4B). These changes in chondrocyte size in response to S100A11 were also inhibited by both sRAGE and RAGE-specific blocking Abs (Fig. 4B). FIGURE 5. sRAGE and anti-RAGE inhibit CXCL8- and TNF-␣- but RAGE mediation of inflammatory cytokine-induced but not not ATRA-induced type X collagen expression. Primary normal human ATRA-induced chondrocyte hypertrophy knee articular chondrocytes (donor ages, 29–62) (5 ϫ 105 cells/six-well dish) were stimulated with CXCL8 (A), ATRA (B), or TNF-␣ (C)inthe We confirmed (8, 9, 26) the induction of cultured articular chon- presence or absence of 1 ␮g/ml sRAGE or 20 ␮g/ml anti-RAGE. SDS- drocyte hypertrophy by CXCL8 and ATRA, and we concurrently PAGE and Western blotting analysis for type X collagen were performed observed that type X collagen expression induction by CXCL8 but on cell lysates at 5 days in culture. These data are representative of results not ATRA was attenuated by coincubation with sRAGE or with from six normal human donors. 8300 RAGE AND CHONDROCYTE HYPERTROPHY by assessing the specific role of RAGE signaling via the p38 MAPK pathway in S100A11-induced type X collagen expression. We observed that S100A11 stimulated rapid p38 phosphorylation in human chondrocytes (Fig. 6A). S100A11-induced p38 activa- tion was inhibited by transfection of chondrocytes with a DN con- struct of MKK3 (DN-MKK3), a specific inhibitor of the highly selective p38 pathway activation inducer MKK3 (25) (Fig. 6B). DN-MKK3 also inhibited type X collagen expression induction by S100A11 (Fig. 6B). Because RAGE is not the sole plasma mem- brane receptor for S100/calgranulins (16, 29, 30), we assessed the FIGURE 7. Schematic of RAGE signaling in the induction of chondro- effects of specific inhibition of RAGE signal transduction using cyte hypertrophy. This schematic depicts the induction by CXCL8 and TNF-␣ of secretion of S100A11 (and likely other chondrocyte-expressed DN RAGE (DN-RAGE). Transient transfection of DN-RAGE at- RAGE ligands not limited to calgranulins). The paradigm further depicts tenuated both S100A11-induced p38 phosphorylation and type X subsequent RAGE signaling critically transduced by p38 MAPK pathway collagen expression in human articular chondrocytes (Fig. 6C). activation as a central event in chondrocyte hypertrophy associated with low-grade inflammation in the OA joint articular cartilage. Discussion In this study, we demonstrated marked up-regulation of expression of the RAGE ligand S100A11 within human OA cartilage. S100/ calgranulins exert a variety of physiologic effects on cell function, observations (18). S100/calgranulins bind not only RAGE but also Downloaded from mediated partly by calcium binding and calmodulin-like activities, CD36 (30). S100/calgranulin-mediated cellular effects via binding complex formation with selected and other proteins, and to cell surface proteoglycans also have been demonstrated (30). intracellular translocation (15, 16, 19, 22). For example, S100A11 However, using an established approach based on parallel results has been implicated in regulatory effects of TGF␤ on cell growth, of treatment with sRAGE or with function-blocking Abs to block via induction by TGF␤ of protein kinase C-mediated S100A11 RAGE ligand-induced signaling (23), we demonstrated that both phosphorylation and nuclear S100A11 translocation (31). But, in- S100A11-induced chondrocyte size increase and expression of http://www.jimmunol.org/ creasingly, certain S100 proteins have been observed to be se- type X collagen were RAGE mediated. creted and to exert extracellular effects, illustrated by proinflam- The prior observation that the chemokines CXCL8 and CXCL1 matory effects on leukocyte recruitment by S100A8, and S100A9 induce cultured articular chondrocyte hypertrophy (8) suggested a (12, 32), and by proinflammatory RAGE-dependent effects of novel mechanistic linkage between low-grade joint inflammation S100A12 (extracellular newly identified RAGE binding protein) and dysregulated chondrocyte differentiation within OA cartilage on endothelial cell and macrophage cytokine release and adhesion (1–7). An unexpected finding in this study was that CXCL8- and (14). In this study, we demonstrated that CXCL8, TNF-␣, and TNF-␣-induced type X collagen expression also were inhibited via ATRA induced chondrocytes to release S100A11. Moreover, we sRAGE and RAGE-specific blocking Abs. This result suggested observed that exogenous nanomolar rS100A11 was sufficient to that S100A11, and likely other RAGE ligands induced by CXCL8 by guest on September 28, 2021 promote chondrocyte hypertrophy, as evidenced by both type X and TNF-␣, were critical downstream mediators of chondrocyte collagen expression and cell size increase. hypertrophy in response to IL-8 and TNF-␣, as schematized in Fig. In this study, we observed that RAGE was constitutively ex- 7. Importantly, under conditions in which RAGE mediated pressed by normal articular chondrocytes in situ, with the qualita- CXCL8- and TNF-␣-induced chondrocyte hypertrophy and in tive appearance of up-regulated chondrocyte expression of RAGE which ATRA induced S100A11, we observed no suppression of by chondrocytes in OA cartilage, confirming recently published ATRA-induced expression of type X collagen expression. We speculate that chondrocyte hypertrophy in response to the growth and differentiation mediator ATRA is driven by more redundant mechanisms (9) than is the hypertrophy response to the inflamma- tory cytokines CXCL8 and TNF-␣. Using transfection of exquisitely selective DN inhibitors of RAGE and of p38 MAPK pathway activation, we demonstrated that activation of the p38 MAPK pathway via RAGE-dependent signaling was essential for transduction of chondrocyte hypertro- phy in response to S100A11. We previously observed that CXCL8-induced p38 MAPK pathway activation and chondrocyte hypertrophy (8) are critically mediated by stimulation of Pit-1 ex- pression and increased sodium-dependent phosphate cotrans-

porter-mediated Pi uptake (26). Hence, it will be of interest to test potential roles of Pit-1 expression, and Pi uptake in S100A11- induced and RAGE-mediated chondrocyte hypertrophy. FIGURE 6. S100A11 signals through RAGE to induce p38 activity. Pri- Significantly, RAGE ligands of the advanced glycation end 6 mary normal human chondrocytes (donor ages, 60–72) (1 ϫ 10 cells/ products (AGE) class accumulate in tissues in association with 10-cm dish) were stimulated with S100A11 for the indicated time. SDS- both normal aging and diabetes mellitus (11, 12) and also form by PAGE and Western blotting analysis for phosphorylated p38 was posttranslational modification of collagen and other extracellular performed on cell lysate. A, Primary human chondrocytes (5 ϫ 105 cells/ six-well dish) were transfected with the indicated construct as described in matrix proteins in association with cartilage aging and OA (33). Materials and Methods. The cells were stimulated with S100A11 for 15 AGE recognition by RAGE is a central mediator of diabetic mi- min and SDS-PAGE/Western blotting was performed for phosphorylated crovascular, renal, and neuropathologic complications (11, 12). p38 (B) and type X collagen (C). These data are representative of results Moreover, AGE-induced effects on chondrocytes have been linked from three to five different human donors. to decreased proteoglycans and collagen synthesis in vitro (33). The Journal of Immunology 8301

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Polyamine depletion inhibits NF-␬B binding to DNA and interleukin-8 production in human chondrocytes inhibitor of cell growth, and regulator of cell senescence and ap- stimulated by tumor necrosis factor-␣. J. Cell. Physiol. 204: 956–963. Downloaded from optosis (37, 38). Because p38 pathway activation, but not activa- 8. Merz, D., R. Liu, K. Johnson, and R. A. Terkeltaub. 2003. IL-8/CXCL8 and tion of ERK1/2 or JNK, is central to CXCL8-induced chondrocyte growth-related oncogene ␣/CXCL1 induce chondrocyte hypertrophic differenti- ation. J. Immunol. 171: 4406–4415. hypertrophy (8), we limited mechanistic studies here to the in- 9. Johnson, K. A., D. van Etten, N. Nanda, R. M. Graham, and R. A. Terkeltaub. volvement of p38 pathway activation in chondrocyte hypertrophic 2003. Distinct transglutaminase 2-independent and transglutaminase 2-dependent differentiation mediated by nanomolar S100A11. The current pathways mediate articular chondrocyte hypertrophy. J. Biol. Chem. 278: 18824–18832. study did not assess potential effects of S100A11-induced chon- 10. Kirsch, T., B. Swoboda, and H. Nah. 2000. Activation of II and V http://www.jimmunol.org/ drocyte activation of ERK1/ERK2 pathway and NF-␬B, which expression, terminal differentiation, mineralization and in human os- were recently reported to modulate chondrocyte activation by teoarthritic cartilage. Osteoarthritis Cartilage 8: 294–302. 11. Ramasamy, R., S. J. Vannucci, S. Shi Du Yan, K. Herold, S. Fang Yan, and S100B added at micromolar concentration (18). We did not mon- A. Marie Schmidt. 2005. Advanced glycation end products and RAGE: a com- itor for potential constitutive or stimulated chondrocyte release of mon thread in aging, diabetes, neurodegeneration, and inflammation. Glycobiol- endogenous sRAGE (39) or for potential function-regulating ef- ogy 15: 16R–28R. 12. Cooper, M. E. 2004. 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Mesenchymal chondrosarcoma of the retroperi- motes chondrocyte hypertrophic differentiation and thereby may toneum: report of a case diagnosed by fine needle aspiration biopsy with immu- nohistochemical, electron microscopic demonstration of S-100 protein in undif- factor into contributory effects of low-grade inflammation to OA ferentiated cells. Acta Cytol. 39: 1237–1243. progression. 21. Foell, D., M. Frosch, C. Sorg, and J. Roth. 2004. Phagocyte-specific calcium- binding S100 proteins as clinical laboratory markers of inflammation. Clin. Chim. Acta 344: 37–51. 22. Foell, D., and J. Roth. 2004. Proinflammatory S100 proteins in arthritis and Acknowledgments autoimmune disease. Arthritis Rheum. 50: 3762–3771. 23. Miyata, T., O. Hori, J. Zhang, S. D. Yan, L. Ferran, Y. Iida, and A. M. Schmidt. We gratefully acknowledge Jacquie Quach (The Scripps Research Institute, 1996. The receptor for advanced glycation end products (RAGE) is a central La Jolla, CA) for technical help with collection of chondrocyte samples and ␤ mediator of the interaction of AGE- 2-microglobulin with human mononuclear also gratefully acknowledge the other staff of the Tissue Collection Core of phagocytes via an oxidant-sensitive pathway: implications for the pathogenesis of the National Institutes of Health-sponsored Program Project on Cartilage dialysis-related amyloidosis. J. Clin. Invest. 98: 1088–1094. and Aging for assistance with cartilage sample acquisition and handling. 24. Arumugam, T., D. M. Simeone, A. M. Schmidt, and C. D. Logsdon. 2004. S100P stimulates cell proliferation and survival via receptor for activated glycation end products (RAGE). J. Biol. Chem. 279: 5059–5065. 25. Han, Q., J. Leng, D. Bian, C. Mahanivong, K. A. Carpenter, Z. K. Pan, J. Han, and S. Huang. 2002. Rac1-MKK3-p38-MAPKAPK2 pathway promotes uroki- Disclosures nase plasminogen activator mRNA stability in invasive breast cells. The authors have no financial conflict of interest. J. Biol. Chem. 277: 48379–48385. 8302 RAGE AND CHONDROCYTE HYPERTROPHY

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