Inhibitor P21 in Synovial Fibroblasts Regulated by the Cyclin-Dependent Kinase IL-6 and Matrix Metalloproteinase-1

IL-6 and Matrix Metalloproteinase-1 Are Regulated by the Cyclin-Dependent Kinase Inhibitor p21 in Synovial Fibroblasts

This information is current as Harris Perlman, Kathleen Bradley, Hongtao Liu, Shawn of September 23, 2021. Cole, Eli Shamiyeh, Roy C. Smith, Kenneth Walsh, Stefano Fiore, Alisa E. Koch, Gary S. Firestein, G. Kenneth Haines III and Richard M. Pope J Immunol 2003; 170:838-845; ;

doi: 10.4049/jimmunol.170.2.838 Downloaded from http://www.jimmunol.org/content/170/2/838

References This article cites 66 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/170/2/838.full#ref-list-1 http://www.jimmunol.org/

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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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-6 and Matrix Metalloproteinase-1 Are Regulated by the Cyclin-Dependent Kinase Inhibitor p21 in Synovial Fibroblasts1

Harris Perlman,2* Kathleen Bradley,* Hongtao Liu,* Shawn Cole,* Eli Shamiyeh,* Roy C. Smith,‡ Kenneth Walsh,‡ Stefano Fiore,§ Alisa E. Koch,*¶ Gary S. Firestein,ʈ G. Kenneth Haines III,† Richard M. Pope*ʈ

During the pathogenesis of rheumatoid arthritis (RA), the synovial fibroblasts increase in number and produce proinflammatory cytokines and matrix metalloproteinases (MMPs) that function to promote inflammation and joint destruction. Recent investigations have suggested that cell cycle activity and inflammation may be linked. However, little is known about the mechanisms responsible for the coordinate regulation of proliferation and the expression of proinflammatory molecules in RA synovial fibro- Downloaded from blasts. Here, we demonstrate a 50 ؎ 10% decrease in the expression of p21, a cell cycle inhibitor, in the synovial fibroblast population from RA compared with osteoarthritis (OA) synovial tissue. Moreover, p21 positivity in the synovial fibroblasts ؊0.76; p < 0.02). The expression of p21 is also reduced in isolated؍ inversely correlates with medium synovial lining thickness (r RA synovial fibroblasts compared with OA synovial fibroblasts. Adenovirus-mediated delivery of p21 (Ad-p21) arrests both RA and OA synovial fibroblasts in the G0/G1 phase of the cell cycle without inducing cytotoxicity. However, the spontaneous production of IL-6 and MMP-1 is suppressed only in the Ad-p21-infected RA synovial fibroblasts, indicating a novel role for p21 in http://www.jimmunol.org/ RA. Analyses of p21-deficient mouse synovial fibroblasts reveal a 100-fold increase in IL-6 protein and enhance IL-6 and MMP-3 mRNA. Restoration of p21, but not overexpression of Rb, which also induces G0/G1 cell cycle arrest, decreases IL-6 synthesis in p21-null synovial fibroblasts. Furthermore, in RA synovial fibroblasts the ectopic expression of p21 reduces activation of the AP-1 transcription factor. Additionally, p21-null synovial fibroblasts display enhanced activation of AP-1 compared with wild-type synovial fibroblasts. These data suggest that alterations in p21 expression may activate AP-1 leading to enhanced proinflammatory cytokine and MMP production and development of autoimmune disease. The Journal of Immunology, 2003, 170: 838–845.

3 heumatoid arthritis (RA) is an autoimmune disease RA synovial fibroblasts spontaneously secrete numerous cytokines by guest on September 23, 2021 characterized by infiltration of lymphocytes and macro- and matrix metalloproteinases (MMP) including IL-6, IL-8, R phages, and hyperplasia of the synovial lining. In RA, MMP-1, and MMP-3, which may be regulated by NF-␬B and/or synovial fibroblasts markedly increase in number, display a trans- AP-1 (3Ð7). In culture, IFN-␥ inhibits TNF-␣-induced RA syno- formed phenotype, and invade and destroy adjacent cartilage (1). vial fibroblast cell cycle activity and MMP-1 production (8), and In contrast, osteoarthritis (OA) synovial fibroblasts do not hyper- IL-4 suppresses proliferation and MMP-1 and 3 mRNA expression proliferate or invade articular cartilage in vivo (2). Furthermore, (9), suggesting that RA synovial fibroblast proliferation and secretion of proinflammatory mediators may be linked. *Division of Rheumatology and †Department of Pathology, Northwestern University, The mammalian cell cycle is divided into four stages, a growth ‡ Feinberg School of Medicine, Chicago, IL 60611; Molecular Cardiology, Whitaker phase (G0/G1), a synthesis phase (S), a second growth phase (G2), Cardiovascular Institute, Boston University School of Medicine, Boston, MA 02118; and mitosis (M). Progression through the different phases of the ¤Section of Rheumatology, University of Illinois College of Medicine, Chicago, IL 60607; ¶Division of Rheumatology, Veterans Administration Chicago Healthcare cell cycle is dependent on the activities of cyclin-dependent System, Lakeside Division, Chicago, IL 60611; and ʈDivision of Rheumatology, Al- kinases (cdks) and their binding partners, cyclins. In G0/G1,hy- lergy and Immunology, University of California School of Medicine, La Jolla, CA 92093 pophosphorylated retinoblastoma (Rb) is bound to the E2F trans- Received for publication April 9, 2002. Accepted for publication November 8, 2002. cription factor, thereby sequestering E2F and repressing its transcriptional activity (10, 11). Once Rb is phosphorylated, E2F is The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance released to activate transcription of genes required for DNA syn- with 18 U.S.C. Section 1734 solely to indicate this fact. thesis during S phase (12). Recently, a new class of cell cycle 1 This work was supported by an American Heart Association Grant in Aid award, an regulatory proteins has been isolated, the cdk inhibitors. These Arthritis National Research Foundation award, and National Institutes of Health Grant inhibitors bind to cdk or cdk-cyclin complexes and inhibit kinase AR02147 (to H.P.); by National Institutes of Health Grants AR43642 and AR30692 and a grant from the Arthritis Foundation (to R.M.P.); by National Institutes of Health activity. The cdk inhibitors are grouped into two categories based Grants AR41492 and HL58695, funds from the Veterans Affairs Research Service, on function and homology, although overexpression of any of the and the Gallagher Professorship for Arthritis Research (to A.E.K.); and by National Institutes of Health Grant AR45347 (to G.S.F.). cdk inhibitors will induce G1 cell cycle arrest (13). The INK4 2 Address correspondence and reprint requests to Dr. Harris Perlman, Department of (inhibitors of cdk4) family of cdk inhibitors, including p15, p16, Molecular Microbiology and Immunology, St. Louis University School of Medicine, p18, and p19, bind to and inhibit cdk4 and cdk6. In contrast, the 1402 South Grand Boulevard, St. Louis, MO 63104. E-mail address: [email protected] Cip/Kip (cdk2-interacting protein) family of cdk inhibitors (p21, p27 3 Abbreviations used in this paper: RA, rheumatoid arthritis; Ad-␤-Gal, vector ex- and p57) exhibits a broad specificity for cdks (13). The decision to ␤ pressing -galactosidase; Ad-Rb, adenovirus vector expressing Rb; cdk, cyclin-de- progress through the cell cycle is determined by the relative stoichi- pendent kinase; JNK, c-Jun NH2-terminal kinase; MMP, matrix metalloproteinase; OA, osteoarthritis; Rb, retinoblastoma. ometric levels of cdk-cyclins and their cognate inhibitors (14).

The role of the cyclin-dependent kinase inhibitors in RA re- analyzed for RA synovial tissue sections (n ϭ 4), and 20 cells/area were mains to be fully evaluated. RA and OA synovial fibroblasts and examined for OA synovial tissue sections (n ϭ 4). dermal fibroblasts express higher levels of p21 (15) following se- Cell culture rum starvation, which induces quiescence, compared with serum- stimulated cells. Moreover, overexpression of p21 or p16 by OA and RA synovial tissue samples were obtained from patients undergoing total joint replacement who met the American College of Rheuma- adenoviral-mediated delivery (Ad-p21) suppresses synovial fibro- tology criteria (19, 20). Normal human synovial fibroblasts were obtained blast growth in vitro (15, 16). Furthermore, overexpression of p21 from arthroscopic knee biopsies (25). Isolated human and mouse synovial inhibits experimental arthritis development (16, 17). RT-PCR tissues were digested with collagenase, dispase, and DNase I, and single- analysis of the Ad-p21-infected joints reveals a decrease in pro- cell suspensions were obtained (5, 26, 27). Synoviocytes were used from ␤ ␣ passages 3Ð9 in these experiments. A homogenous population was deter- inflammatory molecule expression, including IL-1 , TNF- , and mined by flow cytometry (Ͻ1% CD11b, Ͻ1% phagocytic, and Ͻ1% IL-6 mRNA (17). However, only a small percentage of cells in the Fc␥RII receptor positive) (22, 25, 28, 29). Human RA, OA, and normal joint are infected by replication-defective adenoviruses (18), sug- synovial fibroblasts were cultured in 10% FBS/DMEM. For infections, gesting that inhibiting synovial fibroblast proliferation may not cells were plated in growth medium (10% FBS) and allowed to attach before being transferred to low serum medium. Cultures were serum- account for the amelioration of experimental synovitis by Ad-p21. starved in 0.5% FBS/DMEM for 24Ð48 h before infection. Cells were then Further, the observed reduction in the cytokine profile following counted, and cultures were incubated with a vector expressing ␤-galacto- Ad-p21 suggests that p21 may have additional functional activities sidase (Ad-␤-Gal) (30), Ad-p21 (30Ð32), or an adenovirus vector express- that inhibit the progression of arthritis. ing Rb (Ad-Rb) (33) for 12 h in low serum medium. At the end of the Here, we demonstrate a direct role for p21 in suppressing cy- infection period the virus was removed by washing with PBS and returned to low serum medium for an additional 12 h. The cultures were then stim- Downloaded from tokine and MMP production. The expression of p21 is decreased in ulated for 48 h by the addition of medium containing 10% FBS/DMEM. RA compared with OA synovial lining, and lower levels of p21 are Replication-defective adenovirus (Ad5) vectors were propagated in the 293 observed in RA compared with OA synovial fibroblasts. Ectopic embryonic kidney cell line (American Type Culture Collection, Manassas, expression is accomplished by employing replication-defective ad- VA) and purified by ultracentrifugation through cesium chloride gradients. Plaque assay or serial dilutions assays were employed to determine the enovirus to express human p21. Ad-p21 inhibits S phase entry in titers of viral stocks (34). RA and OA synovial fibroblasts, but only suppresses IL-6 and

MMP-1 production in RA synovial fibroblasts. To confirm that p21 Western blot analysis http://www.jimmunol.org/ is essential for the regulation of IL-6, synovial fibroblasts isolated Whole-cell extracts were prepared as previously described (30, 32) from from p21-null mice demonstrate a 100-fold increase in IL-6 pro- uninfected and infected cultures. Extracts (25 ␮g) were analyzed by SDS- duction compared with wild-type synovial fibroblasts. Restoration PAGE on 12.5% polyacrylamide gels and transferred to Immobilon-P (Mil- of p21, but not overexpression of Rb, decreases IL-6 production in lipore Corp., Bedford, MA) by semidry blotting. Filters were blocked for 1 h at room temperature in PBS/0.2% Tween 20/5% nonfat dry milk. The p21-null synovial fibroblasts. Furthermore, AP-1, but not NF-␬B, filters were then incubated with mouse anti-tubulin (Calbiochem, La Jolla, activation is diminished in Ad-p21-transduced RA synovial fibro- CA) Ab or rabbit anti-p21 Ab (Santa Cruz Biotechnology) at a concentra- blasts. Additionally AP-1 activation is markedly enhanced in p21- tion of 0.25Ð0.4 ␮g/ml. All primary Abs were incubated overnight at 4¡C null synovial fibroblasts compared with wild-type cells, suggesting in PBS/0.2% Tween 20/2% nonfat dry milk. Filters were washed in PBS/

0.2% Tween 20/2% nonfat dry milk and incubated with donkey anti-rabbit by guest on September 23, 2021 that p21 inhibits IL-6 and MMP-1 synthesis through inhibition of or anti-mouse secondary Ab (1/2000 dilution) conjugated to HRP (Amer- the AP-1 transcription factor. These data suggest a novel role for sham Pharmacia Biotech, Piscataway, NJ). Visualization of the immuno- p21 in suppressing proinflammatory cytokine and MMP produc- complex was conducted by enhanced chemiluminescence (ECL Plus, Am- tion, independent of cell cycle inhibition. ersham Pharmacia Biotech). Flow cytometry For cell cycle analysis and determination of subdiploid DNA content, cul- Materials and Methods tures were harvested by trypsinization, fixed in 70% ethanol overnight, and Immunohistochemistry stained with propidium iodide (Roche, Indianapolis, IN) as previously described (32). A gate was established to count 10,000 events for each sam- Synovial tissue for immunohistochemistry was obtained at the time of ar- ple. Flow cytometry was conducted at the Robert H. Lurie, Comprehensive throplasty from patients with the diagnosis of RA and from with OA. All Cancer Center, Flow Cytometry Core Facility, of Northwestern University patients met the American College of Rheumatology classification criteria Medical School (Chicago, IL). for RA and OA, respectively (19, 20). Sections (5␮m) from synovial tissue fixed in methyl Carnoy were deparaffinized and blocked in 10% goat se- EMSA

rum. Sections were incubated with rabbit anti-p21 Ab (Santa Cruz Bio- 32 technology, Santa Cruz, CA) or normal rabbit IgG (Sigma-Aldrich, St. P-labeled oligonucleotides containing the AP-1 binding sequence from Louis, MO). A biotinylated donkey anti-rabbit secondary Ab (Jackson Im- the MMP-1 promoter (35) was used as a probe. DNA binding reactions munoResearch Laboratories, West Grove, PA) followed by alkaline phos- were performed by incubation for 20 min at room temperature in a final ␮ phatase (BioGenex Laboratories, San Ramon, CA) conjugated to strepta- volume of 20 l. The reaction mixture contained 100 mmol/L of NaCl, 20 vidin was used to detect primary Ab complexes. Visualization was mmol/l of HEPES, 1 mmol/l of EDTA, 4% glycerol, 5% (w/v) Ficoll, 0.25 ␮ ␮ 32 accomplished using the Vector blue alkaline phosphatase substrate kit g of BSA, 1 g of poly(dI-dC), 1 ng of P-labeled oligonucleotide, and 5Ð10 ␮g of the nuclear extract. Protein-DNA complexes were separated (Vector Laboratories, Burlingame, CA), and counterstained was performed ϫ with phloxine. For dual immunohistochemistry, sections were incubated from free probe by electrophoresis on 5% polyacrylamide gels in 0.5 with mouse anti-CD68 Ab (DAKO, Carpenteria, CA) and rabbit anti-p21 Tris-borate-EDTA at 160 V for 2Ð3 h. Gels were dried onto 3M paper (Whatman, Maidstone, U.K.) and exposed to Kodak XAR film (Eastman Ab. The expression of p21 was detected as described above. To detect ␮ CD68 expression a biotinylated goat anti-rabbit secondary Ab (Jackson Kodak, Rochester, NY). For supershift assays, 1Ð2 l of a polyclonal Abs ImmunoResearch Laboratories), followed by peroxidase (BioGenex) con- against c-Jun or c-Fos (Santa Cruz Biotechnology) was incubated with the jugated to streptavidin, was used to detect primary Ab complexes. Visu- nuclear extract on ice for 30 min before addition of the labeled oligonu- alization was accomplished using the 3-amino-9-ethyl-carbazole substrate cleotide to the binding reaction. kit (BioGenex). There was no counterstain in the dual-stained tissue sec- RT-PCR tions. Specimens were examined and photographed on a Nikon ES400 microscope (Garden City, NY) equipped for phase contrast visualization. A RNA was isolated by the RNAzol B method (Tel-Test, Friendswood, TX) pathologist blinded to the study (G.K.H.) determined the number of p21- as described by the manufacturer. One microgram of total RNA was in- positive/CD68-negative cells in the synovial lining and the medium syno- cubated in reaction buffer containing oligo(dT) primer, avian myeloblas- vial lining thickness by analyzing multiple sections to avoid sampling error tosis virus reverse transcriptase, RNase inhibitor (recombinant RNAsin ri- for each tissue as previously described (21Ð24). Fifty cells per area were bonuclease inhibitor), and dNTP mixture for1hat42¡C. The reaction was 840 p21 REGULATION OF IL-6 AND MMP stopped by incubation at 94¡C for 5 min. Semiquantitative PCR was performed using primers for human IL-6 (forward, 5Ј-ATGAACTCCTTCTC CACAAGCGC-3Ј; reverse, 5Ј-GAAGAGCCCTCAGGCTGGACTG-3Ј) (6), MMP-1 (forward, 5Ј-ATTTCTCCGCTTTTCAACTT-3Ј; reverse, 5Ј- ATGCACAGCTTTCCTCCACT-3Ј), and G3PDH (Clontech, Palo Alto, CA). Mouse primers were the following: IL-6 (forward, 5Ј-GACAAAG CCAGAGTCCTTCAGAGAG-3Ј; reverse, 5Ј-CTAGGTTTGCCGAGTA GATCTC-3Ј) (36) and MMP-3 (forward, 5Ј-CTCCAACACTATGGAG CT-3Ј; reverse, 5Ј-TCCAGGTGCATAGGCATG-3Ј) (37). Cycling conditions FIGURE 2. Immunoblot analysis of p21. RA and OA synovial tissue included one initial denaturation cycle for 5 min at 94¡C; 28 cycles of ampli- samples were obtained from patients undergoing total joint replacement fication for 2 min at 72¡C, 1 min at 94¡C, and 1 min at 50¡C; and a final who met the American College of Rheumatology criteria (19, 20). Isolated extension phase consisting of one cycle of 10 min at 72¡C. human synovial tissues were digested with collagenase, dispase, and ELISA DNase I, and single-cell suspensions were obtained (5, 26, 27). Synovio- cytes were used from passages 3Ð9 in these experiments. A homogenous For human IL-6, pro-MMP-1, and mouse IL-6, sandwich ELISAs were population was determined by flow cytometry (Ͻ1% CD11b, Ͻ1% phago- performed according to the manufacturer’s instructions, employing com- cytic, and Ͻ1% Fc␥RII receptor positive) (22, 25, 28, 29). Whole-cell mercially available kits (R&D Systems, Minneapolis, MN). The sensitivity extracts were prepared from serum-starved quiescent (0.5% FBS) and se- for human IL-6 is 18.8 pg/ml, that for human pro-MMP-1 is 0.4 ng/ml, and rum-stimulated (10% FBS) RA and OA synovial fibroblasts (SF). Immu- that for mouse IL-6 is 31.3 pg/ml. IL-6 production ranged from 700Ð4,755 pg/ml in RA synovial fibroblasts, from 1,175Ð11,540 pg/ml in OA synovial noblot analysis was performed with an Ab to p21 or tubulin. The immu- fibroblasts, and from 590Ð1,321 pg/ml in normal synovial fibroblasts. The noblot is representative of three experiments. Q, serum-starved cells; S, serum-stimulated cells. range of pro-MMP-1 production ranged from 2.7Ð23.0 ng/ml in RA syno- Downloaded from vial fibroblasts, from 1.3Ð35 ng/ml in OA synovial fibroblasts, and from 0.4Ð13.0 ng/ml in normal synovial fibroblasts. The ODs were read by a Microplate VersaMax reader (Molecular Devices, Menlo Park, CA). All data were normalized by cell number. lining, since the endothelial and smooth muscle cells lining the blood vessel walls in both RA and OA synovial tissue were p21 Statistics positive (compare Fig. 1, B and E). These data suggest that the Ϯ reduced percentage of p21-positive cells in RA synovial lining is

Results were expressed as the mean SE. Differences between groups http://www.jimmunol.org/ were analyzed using unpaired two-tailed Student’s t test. Wilcoxon rank- unique and is not due to a general decrease in p21-positive cells in sum tests were performed by a statistician (E.S.) on RA and non-RA sy- multiple other cell types. A 50 Ϯ 10% reduction in the percentage novial fibroblasts. of p21-positive (blue nuclei) per CD68-negative (clear cytoplasm) cells was observed in RA compared with OA synovial lining cells Results (compare Fig. 1, C and F). Furthermore, there was an inverse Expression of p21 is reduced in RA correlation between p21-positive synovial fibroblasts in the syno- RA is an autoimmune disease characterized by increased prolifer- vial lining and medium synovial lining thickness (r ϭϪ0.76; p Ͻ ation of synovial fibroblasts leading to hyperplasia of the synovial 0.02). Collectively, these data suggest that p21 expression is re-

lining. Cell cycle activity is suppressed by p21, and synovial fi- duced in RA compared with OA synovial lining. by guest on September 23, 2021 broblasts exhibit increased proliferation in RA; therefore, we ex- Similar to the RA synovial lining fibroblasts, cultured serum- amined whether p21 expression is decreased in RA synovial tissue. starved (Q) RA-synovial fibroblasts exhibited decreased p21 ex- Indeed, a decrease in the percentage of p21-postive cells (blue pression compared with OA synovial fibroblasts (Fig. 2, compare nuclei per total cell number) was detected in the RA (Fig. 1A) lanes 1 and 3 to lane 5). The expression of p21 remained weaker compared with OA synovial lining (Fig. 1, A and D). The synovial in proliferating (S) RA compared with OA synovial fibroblasts lining thickness was also greater in RA compared with OA syno- (Fig. 2, compare lanes 2 and 4 to lane 6), similar to a previously vial tissue. The decrease in p21 was specific to the RA synovial published study (15). These data are consistent with those in Fig.

FIGURE 1. The expression of p21 in RA synovial tissue. Parafﬁn-embedded, methanol-ﬁxed synovial tissues were examined by immunohistochemistry, analyzed with anti-p21 Ab (blue), and counterstained with phloxine (red). The top panels are RA synovial tissue (A–C), and the bottom panels are OA synovial tissue (D–F). For dual staining, synovial tissues were stained for p21 (blue) and CD68 (brown-red; C and F). No counterstain was used in the dual-stained sections. SL, synovial lining; BV, blood vessel. Panels shown are a representative of four RA and four OA synovial tissues. Arrowheads in C and F denote the p21/CD68-positive cells. The Journal of Immunology 841

FIGURE 5. Suppression of IL-6 and MMP-1 mRNA accumulation by p21 in RA synovial fibroblasts. RA synovial fibroblasts were infected Ad- ␤ FIGURE 3. Ad-p21 induces G0/G1 cycle arrest in RA synovial fibro- -Gal or Ad-p21. RNA was isolated and used to perform semiquantitative blasts. Seventy-two-hour serum-starved (0.5% FBS) cultures were trans- RT-PCR for IL-6, MMP-1, and G3PDH. The data are representative of duced with Ad-␤-Gal or Ad-p21 (500 multiplicity of infection virus par- three individuals. Twenty-eight cycles were employed for the PCR, which ticles determined by serial dilution assay) for 12 h, after which the virus was within the linear range of IL-6, MMP-1, and G3PDH. was removed, and cultures were returned to low serum (0.5% FBS) medium for an additional 12 h. Growth medium (10% FBS) was then added we examined the effects of Ad-p21 infection on IL-6 and MMP-1 for 48 h to allow cell cycle progression. Cells were fixed in 70% ethanol, secretion by RA and non-RA synovial fibroblasts. Synovial fibro- stained with propidium iodide, and analyzed by flow cytometry. Values blasts were infected with Ad-␤-Gal or Ad-p21, and supernatants Downloaded from Ϯ represent the mean SE and were compared for statistical significance by were isolated 72 h following infection. Ad-p21 reduced IL-6 and Ͻ ء Wilcoxon rank-sum test. , p 0.05 vs Ad-p21-infected cultures. No cell MMP-1 secretion in RA synovial fibroblasts by 53 Ϯ 11% ( p Ͻ death was apparent even at 96 h postinfection in all cultures (not shown). 0.0022) and 84 Ϯ 8% ( p Ͻ 0.0079), respectively (Fig. 4A). In contrast, Ad-p21 had no effect on IL-6 and MMP-1 production in 1 and further suggest that p21 expression may be reduced in RA non-RA synovial fibroblasts (OA and normal synovial fibroblasts, compared with OA synovial fibroblasts. n ϭ 11; Fig. 4B). Ad-I␬B␣ infection, which suppresses NF-␬B http://www.jimmunol.org/ activation (6, 38), inhibited IL-6 and MMP-1 in all RA, OA, and Ad-p21 induces cell cycle arrest in RA synovial fibroblasts normal synovial fibroblast cultures examined (not shown), similar To determine the significance of decreased p21 expression in RA to published studies (6, 39). The Ad-I␬B data indicate that unlike synovial fibroblasts, we ectopically expressed p21 in RA and OA the disease type-specific inhibition of IL-6 and MMP-1 by p21, the synovial fibroblasts (not shown) and analyzed for cell cycle activ- ectopic expression of I␬B␣ inhibits cytokine and MMP-1 produc- ity. p21 was introduced into cells using replication-defective E1Ϫ tion regardless of the origin of the cell type. E3Ϫ adenovirus vector (Ad-p21), which readily and uniformly in- fects synovial fibroblasts (22). Ad-␤-Gal was used as a negative Down-regulation of IL-6 and MMP-1 mRNA by p21 by guest on September 23, 2021 control. As shown in Fig. 3, 66% of the Ad-␤-gal-infected RA To characterize the mechanism for the reduction of IL-6 and ␤ synovial fibroblasts cells were in G0/G1, whereas 92% of the Ad- MMP-1, IL-6 and MMP-1 mRNA accumulation in Ad- -Gal- and p21-infected RA synovial fibroblasts cells were in G0/G1.Atthe Ad-p21-infected RA synovial fibroblasts was examined by semi- same time, 14% of the Ad-␤-gal-infected cells were in S phase quantitative RT-PCR. Ad-p21 decreased IL-6 and MMP-1 accu- compared with only 2% of the Ad-p21-infected cells (Fig. 3). Sim- mulation compared with Ad-␤-Gal-infected cells (Fig. 5). These ilar data were observed employing OA synovial fibroblasts (not data suggest that p21 suppresses IL-6 and MMP-1 at the transcrip- shown). These data indicate that RA and OA synovial fibroblasts tional level. infected with Ad-p21 arrest in the G0/G1 phase of the cell cycle. The p21-deficient synovial fibroblasts exhibit enhanced IL-6 and Suppression of spontaneous IL-6 and MMP-1 production by p21 MMP-3 protein synthesis in RA synovial fibroblasts To further define the role of p21 in suppressing IL-6 and MMP-1 RA synovial fibroblasts not only display an increased growth rate, expression, we examined synovial fibroblasts isolated from wild- but they also spontaneously secrete IL-6 and MMP-1. Therefore, type and p21-deficient (p21Ϫ/Ϫ) mice. However, MMP-1 was not

FIGURE 4. Ad-p21 reduces IL-6 and MMP-1 in RA synovial fibroblasts (A), but not in non-RA synovial fibroblasts (B). RA synovial fibroblasts (n ϭ 8) and non-RA synovial fibroblasts (n ϭ 11) were transduced with Ad-␤-Gal or Ad-p21. Supernatants were collected at 72 h following infection and were analyzed for IL-6 and MMP-1 production by ELISA. Values represent the mean Ϯ SE and were compared for statistical significance by Wilcoxon rank-sum test. The p value is vs Ad-␤-Gal-infected cultures. 842 p21 REGULATION OF IL-6 AND MMP

lation compared with wild-type levels (compare Fig. 7B with Fig. 6B). Again, IL-6 and MMP-3 mRNA levels remained unchanged following Ad-Rb infection (Fig. 7B). Collectively, these data demonstrate that p21 suppresses IL-6 and MMP synthesis in synovial ﬁbroblasts, independently of cell cycle arrest.

Suppression of the activation of AP-1 by p21 AP-1 has been shown to regulate IL-6 and MMP-1 expression (40Ð43). To further characterize the mechanism of suppression FIGURE 6. Loss of p21 induces IL-6 production and increases IL-6 and mediated by Ad-p21, EMSAs were employed. Ad-p21 markedly MMP-3 mRNA in murine synovial fibroblasts. A, p21Ϫ/Ϫ synovial fibro- decreased the binding of AP-1 to DNA compared with Ad-␤-Gal- blasts display a marked increase in IL-6 production. Supernatants from infected RA-synovial fibroblasts (Fig. 8A). In contrast, Ad-p21 did wild-type (n ϭ 4) and p21-null knee (p21 KO) synovial fibroblasts (n ϭ not inhibit the binding of CEBP/␤ or NF-␬B to DNA in RA sy- 10) were analyzed for IL-6 production by ELISA. Values represent the novial fibroblasts (not shown). Since ectopic expression of p21 Ϯ mean SE and were compared for statistical significance by unpaired reduced AP-1 DNA-binding activity (Fig. 8B), we examined AP-1 two-tailed Student’s t test. The p values are vs p21-deficient cells. B, En- Ϫ Ϫ DNA-binding activity in p21-deficient compared with wild-type hanced expression of IL-6 and MMP-3 mRNA in p21 / synovial fibro- synovial fibroblasts. AP-1 DNA-binding activity was increased in blasts. RNA was isolated from wild-type and p21-null synovial fibroblasts Ϫ/Ϫ and was analyzed for IL-6, MMP-3, and G3PDH expression by semiquan- p21 compared with wild-type synovial fibroblasts. Both anti- Downloaded from titative RT-PCR. The data are representative of three experiments. c-Fos and c-Jun Abs decreased the amount of bound oligonucleotide (Fig. 8B) in p21-deficient synovial fibroblasts, suggesting that the AP-1 complex in the p21-null synovial fibroblasts consists examined, because to date the murine orthologue of human of c-Jun/c-Fos heterodimers. These data suggest that p21 sup- MMP-1 has not been reported. Therefore, MMP-3 was examined presses AP-1 DNA-binding activity and subsequent IL-6 and in all experiments using mouse cells. Wild-type knee synovial fi- MMP-1 expression. broblasts spontaneously expressed low of levels of IL-6 (61 Ϯ 27 http://www.jimmunol.org/ pg/ml). In stark contrast, p21-deficient synovial fibroblast ex- Discussion pressed high levels of IL-6 (6178 Ϯ 615 pg/ml; p Ͻ 0.00001) as Overexpression of p21 or p16 ameliorates experimental arthritis in measured by ELISA (Fig. 6A). In accordance with the ELISA data, multiple animals models and is associated with reduced IL-1␤, IL-6 and MMP-3 mRNA accumulation was lower in the wild-type TNF-␣, and IL-6 mRNA (15Ð17). However, replication-defective compared with the p21-deficient synovial fibroblasts (Fig. 6B). adenoviruses delivered to the whole synovium nonspecifically in- These data suggest that intact p21 is required to suppress IL-6 and fect lymphocytes, macrophages, fibroblasts, osteoclasts, and neu- MMP-3 expression. trophils. We demonstrated that Ͻ15% of the total cells in the

synovium are infected by standard replication-defective adenovi- by guest on September 23, 2021 Overexpression of p21, but not Rb, restores IL-6 and MMP-3 to ruses (18). Collectively, these data suggest that inhibition of pro- basal levels in p21-null synovial fibroblasts liferation through p21 overexpression may not totally account for The p21-deficient synovial fibroblasts were transduced with Ad- the observed reduced synovitis in experimental models of arthritis ␤-Gal or Ad-p21 to document that reconstitution of p21 is suffi- (15Ð17). Thus, in addition to preventing cell cycle progression, cient to suppress IL-6 and MMP expression. Ad-Rb, which sup- p21 may inhibit the expression of pro-inflammatory mediators. presses the cell cycle, was used to examine whether cell cycle Here, we demonstrate a direct role for p21 in suppressing sponta- arrest is required for IL-6 and MMP-3 reduction. Restoration of neous IL-6 and MMP-1 production in RA synovial fibroblasts. In p21 by Ad-p21 suppressed IL-6 production in p21Ϫ/Ϫ synovial contrast, the overexpression of p21 has no effect on IL-6 or fibroblasts (Fig. 7A). In contrast, Ad-Rb infection of p21-deficient MMP-1 production in normal and OA synovial fibroblasts. Al- synovial fibroblasts had no effect on IL-6 secretion. Similar to the though it is unlikely, replication-defective adenoviruses may infect ELISA data, Ad-p21 reduced IL-6 and MMP-3 mRNA accumu- RA synovial fibroblasts more readily than OA synovial fibroblasts.

FIGURE 7. Restoration of p21, but not Rb overexpression, reduces IL-6 and MMP in p21-deficient synovial fibroblasts. A, Ad-Rb has no effect on IL-6 production in p21-null synovial fibroblasts. The p21-null synovial fibroblasts were infected with the indicated adenovirus (200 multiplicity of infection), and IL-6 was measured 48 h following infection. Values represent the mean Ϯ SE and were compared for statistical significance by unpaired two-tailed Student’s t test. The p values are vs Ad-␤-Gal-infected cultures. The data are representative of four experiments. B, Ad-p21 reduces IL-6 and MMP in p21Ϫ/Ϫ synovial fibroblasts. RNA was isolated from infected p21-null synovial fibroblasts, which were analyzed for IL-6, MMP-3, and G3PDH expression by semiquantitative RT-PCR. The data are representative of two experiments. The Journal of Immunology 843

FIGURE 8. Decreased activation of AP-1 by p21 in RA and mouse synovial fibroblasts. Nuclear extracts were prepared from RA (A; n ϭ 3) and mouse synovial fibroblasts (B; n ϭ 2) that were untreated (Ϫ) or infected with the Ad-␤-Gal (BG) or Ad-p21 (p21). 32P-labeled oligonucleotides containing the AP-1 binding sequence from the MMP-1 promoter (35) were used as a probe. Protein:DNA complexes were separated from free probe by electrophoresis on 5% polyacrylamide gels, dried, and exposed to film. For supershift assays, 200Ð400 ng of anti-c-Jun or c-Fos Ab was incubated with the nuclear extract before the addition of the labeled oligonucleotide.

However, equal viral titers of Ad-p21 are employed in RA, normal, promoting proinflammatory molecule production. Furthermore, c- Downloaded from and OA synovial fibroblasts, which induces similar levels of pro- Fos and c-Jun expression correlates with synovial hyperplasia in tein expression (not shown) and equal inhibition of the cell cycle RA synovial tissue (62), and increased AP-1 DNA-binding activity in the G0/G1 phase (Fig. 3). One potential explanation may be that is observed in RA compared with OA synovial tissue (63, 64). the levels of p21 in normal and OA synovial fibroblasts may be at Addition of AP-1 oligonucleotides ameliorates joint destruction in a maximum, and any increase in the expression of p21 has no experimental arthritis models (42). Thus, AP-1 is integral for the

effect on the additional functions of p21, including cytokine and pathogenesis of RA. We showed that p21 inhibits the JNK/AP-1 http://www.jimmunol.org/ MMP production. Alternatively, a factor that is present only in RA pathway, indicating that p21 may inhibit IL-6 and MMP-1 through synovial fibroblasts may be required for p21-mediated suppression the suppression of JNK/AP-1. Ectopic p21 expression reduces of IL-6 and MMP-1. Collectively, these data demonstrate a novel JNK activity (not shown) and AP-1 activation in RA synovial fi- function for p21 in RA synovial fibroblasts and suggest that up- broblasts, which corresponds to decreased IL-6 and MMP-1 regulating p21 in the synovium, particularly in the synovial fibro- mRNA and protein. Furthermore, p21-deficient synovial fibroblast population, may be therapeutically beneficial for RA. blasts display elevated AP-1 DNA binding compared with wild- The expression of p21 was down-regulated in RA synovial lin- type cells. Anti-c-Fos and anti-c-Jun Ab markedly reduced binding ing fibroblasts in vivo and in vitro compared with OA. One of the to the AP-1 oligonucleotide employing p21-null synovial fibro- potential regulators of p21 is p53 (44), a transcription factor that blast nuclear extracts. Collectively, these data suggest that p21 by guest on September 23, 2021 inhibits cell cycle activity and/or induces apoptosis in response to may suppress IL-6 and MMP-1 through inhibition of JNK/AP-1 stress or damage (45, 46). Although p53 expression is markedly pathway (Fig. 9). increased in RA synovial tissue compared with normal or OA sy- Recent investigations have demonstrated that mice deficient for novial tissue (47, 48), somatic mutations in p53 were also reported p21 exhibit lupus-like disease at 9 mo of age (65, 66). However, it in the RA synovium (49Ð52). These data suggest that a subset of synovial fibroblasts may possess an inactive form of p53. Over- expression of plasmids encoding the p53 mutants found in RA synovium suppresses wild-type p53 function, as indicated by the induction of known p53-regulated genes, including IL-6 (53, 54) and MMP-1 (55, 56), which are normally inhibited by wild-type p53. Additionally, mice deficient in p53 display enhanced experimental arthritis, elevated levels of IL-6 in the synovium, and increased spontaneous MMP-13 expression compared with wild- type mice (57). Thus, p21-deficeint synovial fibroblasts behave similarly to the RA synovial fibroblasts that lack p53 or express the mutant forms of p53. Furthermore, ectopic expression of p21 or overexpression of wild-type p53 reduces IL-6 and MMP-1 expression in RA synovial fibroblasts (53Ð56, 58). Collectively, these data suggest that p21 may be one of the nodal points through which p53 inhibits cytokines and MMPs (Fig. 9). The mechanism of p21-mediated down-regulation of IL-6 and FIGURE 9. Schematic of transformation of a normal synovial fibroblast MMP-1 is unclear. c-Jun NH2-terminal kinase (JNK), which phos- phorylates the c-Jun component of AP-1 and enhances its activity to the RA synovial fibroblast. In the normal fibroblast p53 induces p21, by 30-fold (59, 60), is increased in RA compared with OA syno- which suppresses proliferation through the inhibition of cyclin/cdk and proliferating cell nuclear Ag (PCNA). The p21 also inhibits JNK activity vial fibroblasts (28). JNK-1- or JNK-2-deficient synovial fibro- and subsequent AP-1 activation. However, in RA synovial fibroblasts, p53 blasts fail to express constitutive MMP-3 or -13 expression (5). is inactive, leading to a decrease in p21 expression. In the absence of p21, Furthermore, p21 binds to and inhibits JNK activity (61). Inhibi- cyclin/cdk and PCNA are able to activate the cell cycle. The loss of p21 tion of JNK and subsequently decreased AP-1 activity prevents removes the inhibitory block on JNK, freeing JNK to phosphorylate AP-1. cartilage and bone destruction in the rat model of adjuvant-induced The now active AP-1 enhances proliferation and induces the transcription arthritis (5). Thus, JNK activation of AP-1 may be essential for of IL-6 and MMP-1, leading to an inflammatory response. 844 p21 REGULATION OF IL-6 AND MMP is unknown whether p21-null mice develop spontaneous arthritis human synovial fibroblasts and enhances synthesis of tissue inhibitors of metal- or enhanced/accelerated experimental arthritis. Thus, these data loproteinases. J. Immunol. 164:2660. 26. Koch, A. E., S. L. Kunkel, L. A. Harlow, B. Johnson, H. L. Evanoff, suggest a vital role for p21 in suppressing proinflammatory cyto- G. K. Haines, M. D. Burdick, R. M. Pope, and R. M. Strieter. 1992. 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