Inhibitor p21 in Synovial Fibroblasts Regulated by the Cyclin ...

IL-6 and Matrix Metalloproteinase-1 Are Regulated by the Cyclin-Dependent Kinase Inhibitor p21 in Synovial Fibroblasts This information is current as of May 29, 2013.

Harris Perlman, Kathleen Bradley, Hongtao Liu, Shawn 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; ; http://www.jimmunol.org/content/170/2/838

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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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,储 G. Kenneth Haines III,† Richard M. Pope*储

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heumatoid arthritis (RA)3 is an autoimmune disease characterized by infiltration of lymphocytes and macrophages, and hyperplasia of the synovial lining. In RA, synovial fibroblasts markedly increase in number, display a transformed phenotype, and invade and destroy adjacent cartilage (1). In contrast, osteoarthritis (OA) synovial fibroblasts do not hyperproliferate or invade articular cartilage in vivo (2). Furthermore, *Division of Rheumatology and †Department of Pathology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611; ‡Molecular Cardiology, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA 02118; § Section of Rheumatology, University of Illinois College of Medicine, Chicago, IL 60607; ¶Division of Rheumatology, Veterans Administration Chicago Healthcare System, Lakeside Division, Chicago, IL 60611; and 储Division of Rheumatology, Allergy and Immunology, University of California School of Medicine, La Jolla, CA 92093 Received for publication April 9, 2002. Accepted for publication November 8, 2002. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by an American Heart Association Grant in Aid award, an Arthritis National Research Foundation award, and National Institutes of Health Grant 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 Grants AR41492 and HL58695, funds from the Veterans Affairs Research Service, and the Gallagher Professorship for Arthritis Research (to A.E.K.); and by National Institutes of Health Grant AR45347 (to G.S.F.). 2 Address correspondence and reprint requests to Dr. Harris Perlman, Department of Molecular Microbiology and Immunology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104. E-mail address: [email protected] 3 Abbreviations used in this paper: RA, rheumatoid arthritis; Ad-␤-Gal, vector expressing ␤-galactosidase; Ad-Rb, adenovirus vector expressing Rb; cdk, cyclin-dependent kinase; JNK, c-Jun NH2-terminal kinase; MMP, matrix metalloproteinase; OA, osteoarthritis; Rb, retinoblastoma.

Copyright © 2003 by The American Association of Immunologists, Inc.

RA synovial fibroblasts spontaneously secrete numerous cytokines and matrix metalloproteinases (MMP) including IL-6, IL-8, MMP-1, and MMP-3, which may be regulated by NF-␬B and/or AP-1 (3–7). In culture, IFN-␥ inhibits TNF-␣-induced RA synovial fibroblast cell cycle activity and MMP-1 production (8), and IL-4 suppresses proliferation and MMP-1 and 3 mRNA expression (9), suggesting that RA synovial fibroblast proliferation and secretion of proinflammatory mediators may be linked. The mammalian cell cycle is divided into four stages, a growth phase (G0/G1), a synthesis phase (S), a second growth phase (G2), and mitosis (M). Progression through the different phases of the cell cycle is dependent on the activities of cyclin-dependent kinases (cdks) and their binding partners, cyclins. In G0/G1, hypophosphorylated retinoblastoma (Rb) is bound to the E2F transcription factor, thereby sequestering E2F and repressing its transcriptional activity (10, 11). Once Rb is phosphorylated, E2F is released to activate transcription of genes required for DNA synthesis during S phase (12). Recently, a new class of cell cycle regulatory proteins has been isolated, the cdk inhibitors. These inhibitors bind to cdk or cdk-cyclin complexes and inhibit kinase activity. The cdk inhibitors are grouped into two categories based on function and homology, although overexpression of any of the cdk inhibitors will induce G1 cell cycle arrest (13). The INK4 (inhibitors of cdk4) family of cdk inhibitors, including p15, p16, p18, and p19, bind to and inhibit cdk4 and cdk6. In contrast, the Cip/Kip (cdk2-interacting protein) family of cdk inhibitors (p21, p27 and p57) exhibits a broad specificity for cdks (13). The decision to progress through the cell cycle is determined by the relative stoichiometric levels of cdk-cyclins and their cognate inhibitors (14). 0022-1767/03/$02.00


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 fibroblasts. Here, we demonstrate a 50 ⴞ 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 inversely correlates with medium synovial lining thickness (r ⴝ ⴚ0.76; p < 0.02). The expression of p21 is also reduced in isolated 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 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.


analyzed for RA synovial tissue sections (n ⫽ 4), and 20 cells/area were examined for OA synovial tissue sections (n ⫽ 4).

Cell culture OA and RA synovial tissue samples were obtained from patients undergoing total joint replacement who met the American College of Rheumatology criteria (19, 20). Normal human synovial fibroblasts were obtained from arthroscopic knee biopsies (25). Isolated human and mouse synovial tissues were digested with collagenase, dispase, and DNase I, and singlecell suspensions were obtained (5, 26, 27). Synoviocytes were used from passages 3–9 in these experiments. A homogenous population was determined by flow cytometry (⬍1% CD11b, ⬍1% phagocytic, and ⬍1% Fc␥RII receptor positive) (22, 25, 28, 29). Human RA, OA, and normal synovial fibroblasts were cultured in 10% FBS/DMEM. For infections, cells were plated in growth medium (10% FBS) and allowed to attach before being transferred to low serum medium. Cultures were serumstarved in 0.5% FBS/DMEM for 24 – 48 h before infection. Cells were then counted, and cultures were incubated with a vector expressing ␤-galactosidase (Ad-␤-Gal) (30), Ad-p21 (30 –32), or an adenovirus vector expressing Rb (Ad-Rb) (33) for 12 h in low serum medium. At the end of the 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 stimulated for 48 h by the addition of medium containing 10% FBS/DMEM. Replication-defective adenovirus (Ad5) vectors were propagated in the 293 embryonic kidney cell line (American Type Culture Collection, Manassas, VA) and purified by ultracentrifugation through cesium chloride gradients. Plaque assay or serial dilutions assays were employed to determine the titers of viral stocks (34).

Western blot analysis Whole-cell extracts were prepared as previously described (30, 32) from uninfected and infected cultures. Extracts (25 ␮g) were analyzed by SDSPAGE on 12.5% polyacrylamide gels and transferred to Immobilon-P (Millipore 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 filters were then incubated with mouse anti-tubulin (Calbiochem, La Jolla, CA) Ab or rabbit anti-p21 Ab (Santa Cruz Biotechnology) at a concentration of 0.25– 0.4 ␮g/ml. All primary Abs were incubated overnight at 4°C 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 or anti-mouse secondary Ab (1/2000 dilution) conjugated to HRP (Amersham Pharmacia Biotech, Piscataway, NJ). Visualization of the immunocomplex was conducted by enhanced chemiluminescence (ECL Plus, Amersham Pharmacia Biotech).

Flow cytometry

Materials and Methods Immunohistochemistry Synovial tissue for immunohistochemistry was obtained at the time of arthroplasty from patients with the diagnosis of RA and from with OA. All patients met the American College of Rheumatology classification criteria for RA and OA, respectively (19, 20). Sections (5␮m) from synovial tissue fixed in methyl Carnoy were deparaffinized and blocked in 10% goat serum. Sections were incubated with rabbit anti-p21 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) or normal rabbit IgG (Sigma-Aldrich, St. Louis, MO). A biotinylated donkey anti-rabbit secondary Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) followed by alkaline phosphatase (BioGenex Laboratories, San Ramon, CA) conjugated to streptavidin was used to detect primary Ab complexes. Visualization was accomplished using the Vector blue alkaline phosphatase substrate kit (Vector Laboratories, Burlingame, CA), and counterstained was performed with phloxine. For dual immunohistochemistry, sections were incubated with mouse anti-CD68 Ab (DAKO, Carpenteria, CA) and rabbit anti-p21 Ab. The expression of p21 was detected as described above. To detect CD68 expression a biotinylated goat anti-rabbit secondary Ab (Jackson ImmunoResearch Laboratories), followed by peroxidase (BioGenex) conjugated to streptavidin, was used to detect primary Ab complexes. Visualization was accomplished using the 3-amino-9-ethyl-carbazole substrate kit (BioGenex). There was no counterstain in the dual-stained tissue sections. Specimens were examined and photographed on a Nikon ES400 microscope (Garden City, NY) equipped for phase contrast visualization. A pathologist blinded to the study (G.K.H.) determined the number of p21positive/CD68-negative cells in the synovial lining and the medium synovial lining thickness by analyzing multiple sections to avoid sampling error for each tissue as previously described (21–24). Fifty cells per area were

For cell cycle analysis and determination of subdiploid DNA content, cultures were harvested by trypsinization, fixed in 70% ethanol overnight, and stained with propidium iodide (Roche, Indianapolis, IN) as previously described (32). A gate was established to count 10,000 events for each sample. Flow cytometry was conducted at the Robert H. Lurie, Comprehensive Cancer Center, Flow Cytometry Core Facility, of Northwestern University Medical School (Chicago, IL).

EMSA 32

P-labeled oligonucleotides containing the AP-1 binding sequence from the MMP-1 promoter (35) was used as a probe. DNA binding reactions were performed by incubation for 20 min at room temperature in a final volume of 20 ␮l. The reaction mixture contained 100 mmol/L of NaCl, 20 mmol/l of HEPES, 1 mmol/l of EDTA, 4% glycerol, 5% (w/v) Ficoll, 0.25 ␮g of BSA, 1 ␮g of poly(dI-dC), 1 ng of 32P-labeled oligonucleotide, and 5–10 ␮g of the nuclear extract. Protein-DNA complexes were separated from free probe by electrophoresis on 5% polyacrylamide gels in 0.5⫻ Tris-borate-EDTA at 160 V for 2–3 h. Gels were dried onto 3M paper (Whatman, Maidstone, U.K.) and exposed to Kodak XAR film (Eastman Kodak, Rochester, NY). For supershift assays, 1–2 ␮l of a polyclonal Abs against c-Jun or c-Fos (Santa Cruz Biotechnology) was incubated with the nuclear extract on ice for 30 min before addition of the labeled oligonucleotide to the binding reaction.

RT-PCR RNA was isolated by the RNAzol B method (Tel-Test, Friendswood, TX) as described by the manufacturer. One microgram of total RNA was incubated in reaction buffer containing oligo(dT) primer, avian myeloblastosis virus reverse transcriptase, RNase inhibitor (recombinant RNAsin ribonuclease inhibitor), and dNTP mixture for 1 h at 42°C. The reaction was


The role of the cyclin-dependent kinase inhibitors in RA remains to be fully evaluated. RA and OA synovial fibroblasts and dermal fibroblasts express higher levels of p21 (15) following serum starvation, which induces quiescence, compared with serumstimulated cells. Moreover, overexpression of p21 or p16 by adenoviral-mediated delivery (Ad-p21) suppresses synovial fibroblast growth in vitro (15, 16). Furthermore, overexpression of p21 inhibits experimental arthritis development (16, 17). RT-PCR analysis of the Ad-p21-infected joints reveals a decrease in proinflammatory molecule expression, including IL-1␤, TNF-␣, and IL-6 mRNA (17). However, only a small percentage of cells in the joint are infected by replication-defective adenoviruses (18), suggesting that inhibiting synovial fibroblast proliferation may not account for the amelioration of experimental synovitis by Ad-p21. Further, the observed reduction in the cytokine profile following Ad-p21 suggests that p21 may have additional functional activities that inhibit the progression of arthritis. Here, we demonstrate a direct role for p21 in suppressing cytokine and MMP production. The expression of p21 is decreased in RA compared with OA synovial lining, and lower levels of p21 are observed in RA compared with OA synovial fibroblasts. Ectopic expression is accomplished by employing replication-defective adenovirus to express human p21. Ad-p21 inhibits S phase entry in RA and OA synovial fibroblasts, but only suppresses IL-6 and MMP-1 production in RA synovial fibroblasts. To confirm that p21 is essential for the regulation of IL-6, synovial fibroblasts isolated from p21-null mice demonstrate a 100-fold increase in IL-6 production compared with wild-type synovial fibroblasts. Restoration of p21, but not overexpression of Rb, decreases IL-6 production in p21-null synovial fibroblasts. Furthermore, AP-1, but not NF-␬B, activation is diminished in Ad-p21-transduced RA synovial fibroblasts. Additionally AP-1 activation is markedly enhanced in p21null synovial fibroblasts compared with wild-type cells, suggesting that p21 inhibits IL-6 and MMP-1 synthesis through inhibition of the AP-1 transcription factor. These data suggest a novel role for p21 in suppressing proinflammatory cytokine and MMP production, independent of cell cycle inhibition.

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840 stopped by incubation at 94°C for 5 min. Semiquantitative PCR was performed using primers for human IL-6 (forward, 5⬘-ATGAACTCCTTCTC CACAAGCGC-3⬘; reverse, 5⬘-GAAGAGCCCTCAGGCTGGACTG-3⬘) (6), MMP-1 (forward, 5⬘-ATTTCTCCGCTTTTCAACTT-3⬘; reverse, 5⬘ATGCACAGCTTTCCTCCACT-3⬘), and G3PDH (Clontech, Palo Alto, CA). Mouse primers were the following: IL-6 (forward, 5⬘-GACAAAG CCAGAGTCCTTCAGAGAG-3⬘; reverse, 5⬘-CTAGGTTTGCCGAGTA GATCTC-3⬘) (36) and MMP-3 (forward, 5⬘-CTCCAACACTATGGAG CT-3⬘; reverse, 5⬘-TCCAGGTGCATAGGCATG-3⬘) (37). Cycling conditions included one initial denaturation cycle for 5 min at 94°C; 28 cycles of amplification for 2 min at 72°C, 1 min at 94°C, and 1 min at 50°C; and a final extension phase consisting of one cycle of 10 min at 72°C.

ELISA

Statistics Results were expressed as the mean ⫾ SE. Differences between groups were analyzed using unpaired two-tailed Student’s t test. Wilcoxon ranksum tests were performed by a statistician (E.S.) on RA and non-RA synovial fibroblasts.

Results Expression of p21 is reduced in RA RA is an autoimmune disease characterized by increased proliferation of synovial fibroblasts leading to hyperplasia of the synovial lining. Cell cycle activity is suppressed by p21, and synovial fibroblasts exhibit increased proliferation in RA; therefore, we examined whether p21 expression is decreased in RA synovial tissue. Indeed, a decrease in the percentage of p21-postive cells (blue nuclei per total cell number) was detected in the RA (Fig. 1A) compared with OA synovial lining (Fig. 1, A and D). The synovial lining thickness was also greater in RA compared with OA synovial tissue. The decrease in p21 was specific to the RA synovial

FIGURE 2. Immunoblot analysis of p21. RA and OA synovial tissue samples were obtained from patients undergoing total joint replacement who met the American College of Rheumatology criteria (19, 20). Isolated human synovial tissues were digested with collagenase, dispase, and DNase I, and single-cell suspensions were obtained (5, 26, 27). Synoviocytes were used from passages 3–9 in these experiments. A homogenous population was determined by flow cytometry (⬍1% CD11b, ⬍1% phagocytic, and ⬍1% Fc␥RII receptor positive) (22, 25, 28, 29). Whole-cell extracts were prepared from serum-starved quiescent (0.5% FBS) and serum-stimulated (10% FBS) RA and OA synovial fibroblasts (SF). Immunoblot analysis was performed with an Ab to p21 or tubulin. The immunoblot is representative of three experiments. Q, serum-starved cells; S, serum-stimulated cells.

lining, since the endothelial and smooth muscle cells lining the blood vessel walls in both RA and OA synovial tissue were p21 positive (compare Fig. 1, B and E). These data suggest that the reduced percentage of p21-positive cells in RA synovial lining is unique and is not due to a general decrease in p21-positive cells in multiple other cell types. A 50 ⫾ 10% reduction in the percentage of p21-positive (blue nuclei) per CD68-negative (clear cytoplasm) cells was observed in RA compared with OA synovial lining cells (compare Fig. 1, C and F). Furthermore, there was an inverse correlation between p21-positive synovial fibroblasts in the synovial lining and medium synovial lining thickness (r ⫽ ⫺0.76; p ⬍ 0.02). Collectively, these data suggest that p21 expression is reduced in RA compared with OA synovial lining. Similar to the RA synovial lining fibroblasts, cultured serumstarved (Q) RA-synovial fibroblasts exhibited decreased p21 expression compared with OA synovial fibroblasts (Fig. 2, compare lanes 1 and 3 to lane 5). The expression of p21 remained weaker in proliferating (S) RA compared with OA synovial fibroblasts (Fig. 2, compare lanes 2 and 4 to lane 6), similar to a previously 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.


For human IL-6, pro-MMP-1, and mouse IL-6, sandwich ELISAs were performed according to the manufacturer’s instructions, employing commercially available kits (R&D Systems, Minneapolis, MN). The sensitivity for human IL-6 is 18.8 pg/ml, that for human pro-MMP-1 is 0.4 ng/ml, and that for mouse IL-6 is 31.3 pg/ml. IL-6 production ranged from 700 – 4,755 pg/ml in RA synovial fibroblasts, from 1,175–11,540 pg/ml in OA synovial fibroblasts, and from 590 –1,321 pg/ml in normal synovial fibroblasts. The range of pro-MMP-1 production ranged from 2.7–23.0 ng/ml in RA synovial fibroblasts, from 1.3–35 ng/ml in OA synovial fibroblasts, and from 0.4 –13.0 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.

p21 REGULATION OF IL-6 AND MMP


1 and further suggest that p21 expression may be reduced in RA compared with OA synovial fibroblasts. Ad-p21 induces cell cycle arrest in RA synovial fibroblasts To determine the significance of decreased p21 expression in RA synovial fibroblasts, we ectopically expressed p21 in RA and OA synovial fibroblasts (not shown) and analyzed for cell cycle activity. p21 was introduced into cells using replication-defective E1⫺ E3⫺ adenovirus vector (Ad-p21), which readily and uniformly infects synovial fibroblasts (22). Ad-␤-Gal was used as a negative control. As shown in Fig. 3, 66% of the Ad-␤-gal-infected RA synovial fibroblasts cells were in G0/G1, whereas 92% of the Adp21-infected RA synovial fibroblasts cells were in G0/G1. At the same time, 14% of the Ad-␤-gal-infected cells were in S phase compared with only 2% of the Ad-p21-infected cells (Fig. 3). Similar data were observed employing OA synovial fibroblasts (not shown). These data indicate that RA and OA synovial fibroblasts infected with Ad-p21 arrest in the G0/G1 phase of the cell cycle. Suppression of spontaneous IL-6 and MMP-1 production by p21 in RA synovial fibroblasts RA synovial fibroblasts not only display an increased growth rate, but they also spontaneously secrete IL-6 and MMP-1. Therefore,

FIGURE 5. Suppression of IL-6 and MMP-1 mRNA accumulation by p21 in RA synovial fibroblasts. RA synovial fibroblasts were infected Ad␤-Gal or Ad-p21. RNA was isolated and used to perform semiquantitative RT-PCR for IL-6, MMP-1, and G3PDH. The data are representative of three individuals. Twenty-eight cycles were employed for the PCR, which was within the linear range of IL-6, MMP-1, and G3PDH.

we examined the effects of Ad-p21 infection on IL-6 and MMP-1 secretion by RA and non-RA synovial fibroblasts. Synovial fibroblasts were infected with Ad-␤-Gal or Ad-p21, and supernatants were isolated 72 h following infection. Ad-p21 reduced IL-6 and MMP-1 secretion in RA synovial fibroblasts by 53 ⫾ 11% ( p ⬍ 0.0022) and 84 ⫾ 8% ( p ⬍ 0.0079), respectively (Fig. 4A). In contrast, Ad-p21 had no effect on IL-6 and MMP-1 production in non-RA synovial fibroblasts (OA and normal synovial fibroblasts, n ⫽ 11; Fig. 4B). Ad-I␬B␣ infection, which suppresses NF-␬B activation (6, 38), inhibited IL-6 and MMP-1 in all RA, OA, and normal synovial fibroblast cultures examined (not shown), similar to published studies (6, 39). The Ad-I␬B data indicate that unlike the disease type-specific inhibition of IL-6 and MMP-1 by p21, the ectopic expression of I␬B␣ inhibits cytokine and MMP-1 production regardless of the origin of the cell type. Down-regulation of IL-6 and MMP-1 mRNA by p21 To characterize the mechanism for the reduction of IL-6 and MMP-1, IL-6 and MMP-1 mRNA accumulation in Ad-␤-Gal- and Ad-p21-infected RA synovial fibroblasts was examined by semiquantitative RT-PCR. Ad-p21 decreased IL-6 and MMP-1 accumulation compared with Ad-␤-Gal-infected cells (Fig. 5). These data suggest that p21 suppresses IL-6 and MMP-1 at the transcriptional level. The p21-deficient synovial fibroblasts exhibit enhanced IL-6 and MMP-3 protein synthesis To further define the role of p21 in suppressing IL-6 and MMP-1 expression, we examined synovial fibroblasts isolated from wildtype and p21-deficient (p21⫺/⫺) 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 ⫽ 8) and non-RA synovial fibroblasts (n ⫽ 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 ⫾ SE and were compared for statistical significance by Wilcoxon rank-sum test. The p value is vs Ad-␤-Gal-infected cultures.


FIGURE 3. Ad-p21 induces G0/G1 cycle arrest in RA synovial fibroblasts. Seventy-two-hour serum-starved (0.5% FBS) cultures were transduced with Ad-␤-Gal or Ad-p21 (500 multiplicity of infection virus particles determined by serial dilution assay) for 12 h, after which the virus 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 for 48 h to allow cell cycle progression. Cells were fixed in 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry. Values represent the mean ⫾ SE and were compared for statistical significance by Wilcoxon rank-sum test. ⴱ, p ⬍ 0.05 vs Ad-p21-infected cultures. No cell death was apparent even at 96 h postinfection in all cultures (not shown).

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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

FIGURE 6. Loss of p21 induces IL-6 production and increases IL-6 and MMP-3 mRNA in murine synovial fibroblasts. A, p21⫺/⫺ synovial fibroblasts display a marked increase in IL-6 production. Supernatants from wild-type (n ⫽ 4) and p21-null knee (p21 KO) synovial fibroblasts (n ⫽ 10) were analyzed for IL-6 production by ELISA. Values represent the mean ⫾ SE and were compared for statistical significance by unpaired two-tailed Student’s t test. The p values are vs p21-deficient cells. B, Enhanced expression of IL-6 and MMP-3 mRNA in p21⫺/⫺ synovial fibroblasts. RNA was isolated from wild-type and p21-null synovial fibroblasts and was analyzed for IL-6, MMP-3, and G3PDH expression by semiquantitative RT-PCR. The data are representative of three experiments.

Overexpression of p21, but not Rb, restores IL-6 and MMP-3 to basal levels in p21-null synovial fibroblasts The p21-deficient synovial fibroblasts were transduced with Ad␤-Gal or Ad-p21 to document that reconstitution of p21 is sufficient to suppress IL-6 and MMP expression. Ad-Rb, which suppresses the cell cycle, was used to examine whether cell cycle arrest is required for IL-6 and MMP-3 reduction. Restoration of p21 by Ad-p21 suppressed IL-6 production in p21⫺/⫺ synovial fibroblasts (Fig. 7A). In contrast, Ad-Rb infection of p21-deficient synovial fibroblasts had no effect on IL-6 secretion. Similar to the ELISA data, Ad-p21 reduced IL-6 and MMP-3 mRNA accumu-

Discussion Overexpression of p21 or p16 ameliorates experimental arthritis in multiple animals models and is associated with reduced IL-1␤, TNF-␣, and IL-6 mRNA (15–17). However, replication-defective adenoviruses delivered to the whole synovium nonspecifically infect lymphocytes, macrophages, fibroblasts, osteoclasts, and neutrophils. We demonstrated that ⬍15% of the total cells in the synovium are infected by standard replication-defective adenoviruses (18). Collectively, these data suggest that inhibition of proliferation through p21 overexpression may not totally account for the observed reduced synovitis in experimental models of arthritis (15–17). Thus, in addition to preventing cell cycle progression, p21 may inhibit the expression of pro-inflammatory mediators. Here, we demonstrate a direct role for p21 in suppressing spontaneous IL-6 and MMP-1 production in RA synovial fibroblasts. In contrast, the overexpression of p21 has no effect on IL-6 or MMP-1 production in normal and OA synovial fibroblasts. Although it is unlikely, replication-defective adenoviruses may infect 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 ⫾ 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 p21⫺/⫺ 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.


examined, because to date the murine orthologue of human MMP-1 has not been reported. Therefore, MMP-3 was examined in all experiments using mouse cells. Wild-type knee synovial fibroblasts spontaneously expressed low of levels of IL-6 (61 ⫾ 27 pg/ml). In stark contrast, p21-deficient synovial fibroblast expressed high levels of IL-6 (6178 ⫾ 615 pg/ml; p ⬍ 0.00001) as measured by ELISA (Fig. 6A). In accordance with the ELISA data, IL-6 and MMP-3 mRNA accumulation was lower in the wild-type compared with the p21-deficient synovial fibroblasts (Fig. 6B). These data suggest that intact p21 is required to suppress IL-6 and MMP-3 expression.

AP-1 has been shown to regulate IL-6 and MMP-1 expression (40 – 43). To further characterize the mechanism of suppression mediated by Ad-p21, EMSAs were employed. Ad-p21 markedly decreased the binding of AP-1 to DNA compared with Ad-␤-Galinfected RA-synovial fibroblasts (Fig. 8A). In contrast, Ad-p21 did not inhibit the binding of CEBP/␤ or NF-␬B to DNA in RA synovial fibroblasts (not shown). Since ectopic expression of p21 reduced AP-1 DNA-binding activity (Fig. 8B), we examined AP-1 DNA-binding activity in p21-deficient compared with wild-type synovial fibroblasts. AP-1 DNA-binding activity was increased in p21⫺/⫺ compared with wild-type synovial fibroblasts. Both antic-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 of c-Jun/c-Fos heterodimers. These data suggest that p21 suppresses AP-1 DNA-binding activity and subsequent IL-6 and MMP-1 expression.


843

FIGURE 8. Decreased activation of AP-1 by p21 in RA and mouse synovial fibroblasts. Nuclear extracts were prepared from RA (A; n ⫽ 3) and mouse synovial fibroblasts (B; n ⫽ 2) that were untreated (⫺) 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, 200 – 400 ng of anti-c-Jun or c-Fos Ab was incubated with the nuclear extract before the addition of the labeled oligonucleotide.

promoting proinflammatory molecule production. Furthermore, cFos and c-Jun expression correlates with synovial hyperplasia in RA synovial tissue (62), and increased AP-1 DNA-binding activity is observed in RA compared with OA synovial tissue (63, 64). Addition of AP-1 oligonucleotides ameliorates joint destruction in experimental arthritis models (42). Thus, AP-1 is integral for the pathogenesis of RA. We showed that p21 inhibits the JNK/AP-1 pathway, indicating that p21 may inhibit IL-6 and MMP-1 through the suppression of JNK/AP-1. Ectopic p21 expression reduces JNK activity (not shown) and AP-1 activation in RA synovial fibroblasts, which corresponds to decreased IL-6 and MMP-1 mRNA and protein. Furthermore, p21-deficient synovial fibroblasts display elevated AP-1 DNA binding compared with wildtype cells. Anti-c-Fos and anti-c-Jun Ab markedly reduced binding to the AP-1 oligonucleotide employing p21-null synovial fibroblast nuclear extracts. Collectively, these data suggest that p21 may suppress IL-6 and MMP-1 through inhibition of JNK/AP-1 pathway (Fig. 9). Recent investigations have demonstrated that mice deficient for p21 exhibit lupus-like disease at 9 mo of age (65, 66). However, it

FIGURE 9. Schematic of transformation of a normal synovial fibroblast to the RA synovial fibroblast. In the normal fibroblast p53 induces p21, which suppresses proliferation through the inhibition of cyclin/cdk and proliferating cell nuclear Ag (PCNA). The p21 also inhibits JNK activity and subsequent AP-1 activation. However, in RA synovial fibroblasts, p53 is inactive, leading to a decrease in p21 expression. In the absence of p21, cyclin/cdk and PCNA are able to activate the cell cycle. The loss of p21 removes the inhibitory block on JNK, freeing JNK to phosphorylate AP-1. The now active AP-1 enhances proliferation and induces the transcription of IL-6 and MMP-1, leading to an inflammatory response.


However, equal viral titers of Ad-p21 are employed in RA, normal, and OA synovial fibroblasts, which induces similar levels of protein expression (not shown) and equal inhibition of the cell cycle in the G0/G1 phase (Fig. 3). One potential explanation may be that the levels of p21 in normal and OA synovial fibroblasts may be at a maximum, and any increase in the expression of p21 has no effect on the additional functions of p21, including cytokine and MMP production. Alternatively, a factor that is present only in RA synovial fibroblasts may be required for p21-mediated suppression of IL-6 and MMP-1. Collectively, these data demonstrate a novel function for p21 in RA synovial fibroblasts and suggest that upregulating p21 in the synovium, particularly in the synovial fibroblast population, may be therapeutically beneficial for RA. The expression of p21 was down-regulated in RA synovial lining fibroblasts in vivo and in vitro compared with OA. One of the potential regulators of p21 is p53 (44), a transcription factor that inhibits cell cycle activity and/or induces apoptosis in response to stress or damage (45, 46). Although p53 expression is markedly increased in RA synovial tissue compared with normal or OA synovial tissue (47, 48), somatic mutations in p53 were also reported in the RA synovium (49 –52). These data suggest that a subset of synovial fibroblasts may possess an inactive form of p53. Overexpression 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 wildtype 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 (53–56, 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 MMP-1 is unclear. c-Jun NH2-terminal kinase (JNK), which phosphorylates the c-Jun component of AP-1 and enhances its activity by 30-fold (59, 60), is increased in RA compared with OA synovial fibroblasts (28). JNK-1- or JNK-2-deficient synovial fibroblasts fail to express constitutive MMP-3 or -13 expression (5). Furthermore, p21 binds to and inhibits JNK activity (61). Inhibition of JNK and subsequently decreased AP-1 activity prevents cartilage and bone destruction in the rat model of adjuvant-induced arthritis (5). Thus, JNK activation of AP-1 may be essential for

844 is unknown whether p21-null mice develop spontaneous arthritis or enhanced/accelerated experimental arthritis. Thus, these data suggest a vital role for p21 in suppressing proinflammatory cytokines and MMPs, which contribute to the development of autoimmune disease.

p21 REGULATION OF IL-6 AND MMP

26.

27.

References 28.

29. 30.

31. 32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42. 43.

44.

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48.

49.

50.

51.

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