ARTHRITIS & RHEUMATISMVol. 50, No. 1, January 2004, pp 78–87DOI 10.1002/art.11482© 2004, American College of Rheumatology

Retinoblastoma Suppression of Matrix Metalloproteinase 1,but Not Interleukin-6, Through a p38-Dependent Pathway in

Rheumatoid Arthritis Synovial Fibroblasts

Kathleen Bradley,1 John C. Scatizzi,1 Stefano Fiore,2 Eli Shamiyeh,3 Alisa E. Koch,3

Gary S. Firestein,4 Laura L. Gorges,1 Kevin Kuntsman,3 Richard M. Pope,3 Terry L. Moore,1

Jiahuai Han,5 and Harris Perlman1

Objective. Rheumatoid arthritis (RA) is charac-terized by increased synovial lining cellularity, inflam-mation, and destruction of cartilage and bone. Duringthe pathogenesis of RA, synovial fibroblasts reenter thecell cycle and multiply in number. RA synovial fibro-blasts express high levels of the MAP kinase p38, whichmay contribute to the production of interleukin-6 (IL-6)and matrix metalloproteinases (MMPs). IL-6 andMMP-1 promote inflammation and joint destruction,respectively. Taken together, these findings indicatethat in RA the enhanced cell cycle activity and produc-tion of IL-6 and MMP-1 may be linked. Therefore, wesought to determine if the tumor suppressor geneproduct retinoblastoma (Rb), a negative regulator ofcell cycle activity, inhibits IL-6, MMP-1, and p38 in RAsynovial fibroblasts.

Methods. RA and non-RA synovial fibroblastswere examined by enzyme-linked immunosorbent assay(ELISA) for the relative expression of inactive hyper-phosphorylated Rb (inactive Rb/total Rb). Ectopic Rbexpression was mediated by infection with a replication-defective adenovirus that expresses Rb (Ad-Rb). A con-trol replication-defective adenovirus that expresses�-galactosidase (Ad-�-gal) was used. Cell cycle activitywas determined by flow cytometry. IL-6 and MMP-1expression was examined by real-time polymerase chainreaction and ELISA. Expression and activation of p38were determined by kinase assays and ELISA. Theactivity of p38 was enhanced by infecting RA synovialfibroblasts with a replication-defective adenovirus thatexpresses a constitutively active form of MAPK kinase 3(Ad-CA-MKK3), an upstream activator of p38.

Results. Quiescent RA, compared with non-RAsynovial fibroblasts, displayed a 200% (P < 0.02) in-crease in the inactive Rb isoform. Proliferating RAsynovial fibroblasts exhibited a 60% (P < 0.12) increasein the inactive Rb isoform compared with non-RAsynovial fibroblasts. Increased levels of the active Rbisoform inhibited cell cycle progression and suppressedIL-6 and MMP-1 secretion in RA synovial fibroblasts,although the steady-state levels of IL-6 and MMP-1messenger RNA remained unchanged. However, Rboverexpression had no effect on spontaneous or IL-1�–induced production of IL-6 or MMP-1 in non-RA syno-vial fibroblasts. Ectopic Rb expression reduced theactivity of p38. Ad-CA-MKK3 infection in RA synovialfibroblasts increased p38 phosphorylation, and MMP-1but not IL-6 secretion. In contrast, Rb overexpressioninhibited Ad-CA-MKK3–mediated phosphorylation ofp38 and subsequent increase in MMP-1.

Conclusion. Rb-mediated suppression of IL-6 and

Dr. Fiore’s work was supported by NIH grant AR-45931. Dr.Koch’s work was supported by NIH grants AR-41492 and HL-58695,funds from the Veterans Administration Research Service, and aGallagher Professorship for Arthritis Research. Dr. Firestein’s workwas supported by NIH grant AR-45347. Dr. Pope’s work was sup-ported by NIH grants AR-43642 and AR-30692 and a grant from theArthritis Foundation. Dr. Perlman’s work was supported by grantsfrom the American Heart Association, the Arthritis National ResearchFoundation, and the NIH (grants AR-02147 and AR-050250).

1Kathleen Bradley, BA, John C. Scatizzi, BS, Laura L.Gorges, BA, Terry L. Moore, MD, Harris Perlman, PhD: Saint LouisUniversity, School of Medicine, St. Louis, Missouri; 2Stefano Fiore,MD: College of Medicine, University of Illinois at Chicago; 3EliShamiyeh, MS, Alisa E. Koch, MD, Kevin Kuntsman, BA, Richard M.Pope, MD: Northwestern University, The Feinberg School of Medi-cine, Chicago, Illinois; 4Gary S. Firestein, MD: School of Medicine,University of California, San Diego; Jiahuai Han, PhD: The ScrippsResearch Institute, La Jolla, California.

Address correspondence and reprint requests to Harris Perl-man, PhD, Saint Louis University, School of Medicine, Department ofMolecular Microbiology and Immunology, 1402 South Grand Boule-vard, St. Louis, MO 63104. E-mail: perlmanh@slu.edu.

Submitted for publication April 25, 2003; accepted in revisedform October 1, 2003.

78

MMP-1 occurs at a posttranscriptional level. However,Ad-Rb reduction of MMP-1 but not IL-6 requires inhi-bition of the p38 pathway. These results suggest that Rbnegatively regulates p38 activation, leading to decreasedMMP-1 secretion in RA synovial fibroblasts.

Synovial lining fibroblasts are one of the keycellular contributors to the progression of rheumatoidarthritis (RA). RA synovial fibroblasts display a trans-formed phenotype, proliferate, and then invade anddestroy adjacent cartilage (1). In contrast, normal orosteoarthritis (OA) synovial fibroblasts do not exhibitenhanced proliferation or invade articular cartilage invivo (2). In culture, RA synovial fibroblasts spontane-ously secrete one of the most abundant cytokines in thejoint, interleukin-6 (IL-6) (3). In addition, they producethe degradative enzyme collagenase (matrix metallopro-teinase 1 [MMP-1]), which is partially responsible for thedestruction of cartilage and bone. The expression of IL-6and MMP-1 correlates with disease activity (4–7). Defi-ciencies in IL-6 (8–11) or MMP (12) prevent and/orreduce the severity of experimental arthritis, indicatingthat targeting these 2 inflammatory molecules may be apotential therapy. IL-6 and MMP-1 are activated by aplethora of molecules, including the MAP kinase p38,which induces IL-6 and MMP-1 synthesis in a variety ofcell types, including RA synovial fibroblasts (13–18).Furthermore, p38 expression is enhanced in RA com-pared with OA synovial lining (19), and suppression ofp38 ameliorates experimental arthritis (20). These datasuggest that reducing p38, which may result in decreasedIL-6 and MMP-1 production, is sufficient to prevent thedevelopment of experimental arthritis.

The role of cell cycle regulators in RA is un-known. Somatic mutations in the tumor suppressor p53,which also induces cell cycle arrest, are observed in theRA synovium (21–24). Overexpression of plasmids en-coding the p53 mutants found in RA increases IL-6transcription (25,26) and MMP-1 transcription (27,28),which are normally suppressed by wild-type p53. Acyclin-dependent protein kinase inhibitor (CDKI), p21,which is an inhibitor of cyclin (E or A)/CDK-2 activity,is decreased in RA, but not in OA, synovial fibroblasts(29). Moreover, restoration of p21 ameliorates experi-mental arthritis development (30,31). Another CDKI,p16, which functions to suppress CDK-4 or -6/cyclin D,is increased in RA compared with OA synovial fibro-blasts (32) and at sites of articular cartilage destructionin the SCID mouse RA model of arthritis (33). Overex-pression of p16 suppresses experimental arthritis devel-opment (30,32). These data suggest that CDKIs may be

differentially expressed in RA, but overexpression ofeither p21 or p16 inhibits experimental arthritis devel-opment.

One of the central functions of CDKI is to preventthe phosphorylation of retinoblastoma (Rb), resulting in itsinactivation, which to date has not been investigated in RA.In the G0/G1 phase of the cell cycle, hypophosphorylatedRb (active Rb) is bound to the E2F transcription factor,thereby sequestering E2F and repressing its transcriptionalactivity (34). Rb is phosphorylated by CDK(4/6)/cyclin Dand cyclin (E/A)/CDK-2, releasing E2F to activate thetranscription of genes required for DNA synthesis duringthe S phase (35). The CDKs for cyclins A and B areactivated in the S and G2 phases, and maintain Rb in itshyperphosphorylated, inactive state until the cell exits fromthe cell cycle.

In the present study, we examined the expressionof the inactive isoform of Rb in RA and non-RAsynovial fibroblasts. The hyperphosphorylated, inactiveisoform of Rb was enhanced in both quiescent andproliferating RA compared with non-RA synovial fibro-blasts. Overexpression of the active isoform of Rb, usinga replication-defective adenovirus that expresses humanRb (Ad-Rb), increased the percent of RA synovialfibroblasts in the G0/G1 phase of the cell cycle com-pared with cells infected with a control replication-defective adenovirus that expresses �-galactosidase (Ad-�-gal). Ad-Rb inhibited IL-6 and MMP-1 production inRA but not in non-RA synovial fibroblasts. Ad-Rb hadno effect on IL-6 and MMP-1 messenger RNA (mRNA)levels compared with control infected RA synovial fibro-blasts. Ad-Rb markedly suppressed p38 kinase activitycompared with Ad-�-gal–infected cells. Adenoviral-mediated infection of a constitutively active MAPKkinase (MKK-3) (Ad-CA-MKK3), an upstream activatorof p38 kinase, increased p38 activity and MMP-1 pro-duction in the Ad-�-gal–infected cells. In contrast,Ad-Rb suppressed p38 activity and MMP-1 productioneven in the presence of Ad-CA-MKK3. However, Ad-CA-MKK3 had no effect on spontaneous IL-6 produc-tion in RA synovial fibroblasts. These data suggest thatin normal synovial fibroblasts, Rb functions to suppressMMP-1 by inhibiting the activation of p38.

MATERIALS AND METHODS

Cell culture. Synovial tissue samples were obtainedfrom patients undergoing total joint replacement who met theAmerican College of Rheumatology (formerly, the AmericanRheumatism Association) criteria for OA or RA (36,37).Normal human synovial fibroblasts were obtained from arthro-scopic knee biopsy samples (38). Isolated human synovial

RETINOBLASTOMA INHIBITS MMP-1 BY SUPPRESSING p38 ACTIVITY 79

tissue was digested with collagenase, dispase, and DNase I, andsingle-cell suspensions were obtained (39–41). A homoge-neous population was determined by flow cytometry (�1%CD11b, �1% phagocytic, and �1% Fc� receptor type IIpositive) (38,42–44). Human RA, OA, and normal synovialfibroblasts, passages 3–10, were cultured in 10% fetal bovineserum (FBS)/Dulbecco’s modified Eagle’s medium (DMEM).For infections, cells were plated in growth medium (10% FBS)and allowed to attach before being transferred to 0.5% FBS/DMEM (low serum) for 24–48 hours prior to infection. Cellswere then counted and cultures were infected at 500 multiplic-ities of infection (MOI) with Ad-�-gal (45), Ad-Rb (46), orAd-p21 (29) for 12 hours in low-serum medium. At the end ofthe infection period, the virus was removed by washing withphosphate buffered saline and returned to low-serum mediumfor an additional 12 hours. The cultures were then stimulatedfor 48 hours by the addition of medium containing 10%FBS/DMEM or 10% FBS/DMEM and 1 ng/ml of recombinanthuman IL-1� (R&D Systems, Minneapolis, MN). For dualinfections, RA synovial fibroblasts were infected with 500 MOIof Ad-CA-MKK3 (17,18) and 300 MOI of Ad-�-gal or Ad-Rb.Replication-defective adenovirus (Ad5) vectors were propa-gated in the 293 embryonic kidney cell line (American TypeCulture Collection, Rockville, MD) and purified by ultracen-trifugation through cesium chloride gradients. Plaque assay orserial dilution assays were used to determine the titers of viralstocks (47).

Flow cytometry. For cell cycle analysis and subdiploidDNA content, cultures were harvested by trypsinization, fixedin 70% ethanol overnight, and stained with propidium iodide(Roche Biochemical, Indianapolis, IN), as previously described(48). A gate was established to count 10,000 events for eachsample. Flow cytometry was conducted at the Robert H. LurieComprehensive Cancer Center, Flow Cytometry Core Facilityof the Northwestern University Medical School.

Polymerase chain reaction (PCR). RNA was isolatedby the RNAzol B method (Tel-Test, Friendswood, TX), asdescribed by the manufacturer. One microgram of total RNAwas incubated in reaction buffer containing oligo(dT) primer,avian myeloblastosis virus reverse transcriptase, RNase inhib-itor (recombinant RNasin ribonuclease inhibitor), and dNTPmixture for 1 hour at 42°C. The reaction was stopped byincubation at 94°C for 5 minutes. Semiquantitative PCR wasperformed using real-time PCR primers for human IL-6,MMP-1, and hypoxanthine guanine phosphoribosyltransferase(HPRT). Cycling conditions were as follows: 1 initial denatur-ation cycle for 5 minutes at 94°C, 28 cycles of amplification for2 minutes at 72°C, 1 minute at 94°C, and 1 minute at 50°C, anda final extension phase consisting of 1 cycle for 20 minutes at72°C. Quantitative real-time PCR was performed on an ABIPrism 7700 sequence detection system (Perkin Elmer, Em-eryville, CA). Amplification of HPRT was used for samplenormalization, and quantitation of gene expression relative toHPRT was calculated by the ��CT method. Primer and probesets for HPRT (49), IL-6 (50), and MMP-1 (51) have beenpreviously described.

Enzyme-linked immunosorbent assay (ELISA). HumanIL-6 and pro–MMP-1 sandwich ELISAs (R&D Systems) andRb, phospho-RbpSpT249/252, p38, and phospho-p38pTpY180/182

ELISAs (BioSource International, Camarillo, CA) were per-formed according to the instructions of the manufacturer. Thesensitivity for human IL-6 is 18.8 pg/ml, pro–MMP-1 is 0.4

ng/ml, Rb is 62.5 pg/ml, phospho-RbpSpT249/252 is 1.6 units/ml,p38 is 312 pg/ml, and phospho-p38pTpY180/182 is 6.3 units/ml.The optical densities were measured with a Microplate Versa-Max reader (Molecular Devices, Sunnyvale, CA) or a Bio-Rad680 (Bio-Rad, Richmond, CA) reader. All data were normal-ized by cell number or total protein in each sample.

Statistical analysis. Results were expressed as themean � SEM. Differences between groups were analyzedusing Student’s unpaired 2-tailed t-test. Wilcoxon’s rank sumtests of differences between RA and non-RA fibroblasts wereperformed by a statistician (ES).

RESULTS

Enhancement of inactive Rb in RA synovialfibroblasts. RA synovial fibroblasts, unlike OA or nor-mal synovial fibroblasts, undergo anchorage-independent growth in vitro and express a number ofproto-oncogenes, such as c-myc, or c-fos, characteristicof transformed cells (52–54). The expression of CDKI,p21, and p16 (which function to block cyclin/CDKphosphorylation of Rb) has been examined in RAsynovial fibroblasts (29,32). However, the expression ofRb or its role in RA is unclear. Total Rb and hyperphos-phorylated Rb (pSpT249/252) (55,56), indicative of theinactive isoform, were measured by ELISA using proteinextracts isolated from quiescent (Q) or proliferating (P)RA or non-RA synovial fibroblasts (OA and normalsynovial fibroblasts). The quiescent RA synovial fibro-blasts (n � 11) displayed a 200% increase (P � 0.02) inthe relative inactive Rb isoform (inactive/total Rb) com-pared with non-RA synovial fibroblasts (n � 15) (Figure1). The RA compared with non-RA synovial fibroblastsgrowing in the logarithmic phase exhibited a 60% in-crease (P � 0.12) in the levels of inactive Rb (Figure 1).There was no difference in the expression of total Rb inthe RA compared with non-RA synovial fibroblasts.These data suggest that Rb is inactivated in RA com-pared with non-RA synovial fibroblasts.

Suppression of cell cycle activity by Ad-Rb. Toincrease the active Rb isoform, Rb was ectopicallyexpressed by using a replication-defective adenovirusthat expressed Rb complementary DNA (cDNA). Infec-tion with Ad-Rb resulted in G0/G1 arrest (45,46), inhi-bition of E2F transcription activation (46), and de-creased CDK activity (57). As a control, Ad-�-gal wasused for all experiments that required adenoviral infec-tion. Ninety-one percent of the Ad-Rb–infected synovialfibroblasts were in the G0/G1 phase of the cell cycle,even 48 hours post–serum stimulation (P � 0.05), while75% of the Ad-�-gal–infected synovial fibroblasts werein the G0/G1 phase (Figure 2). These data are consistentwith the results of previous studies, which demonstrate

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that overexpression of Rb suppresses cell cycle progres-sion (46,58–60).

Reduction of spontaneous IL-6 and MMP-1 se-cretion by Rb. We recently demonstrated that p21overexpression inhibits IL-6 and MMP-1 synthesis in RAbut not in non-RA synovial fibroblasts (61). Therefore,we examined IL-6 and MMP-1 production in RA and in

non-RA synovial fibroblasts following infection withAd-�-gal or Ad-Rb. The average expression of IL-6 inRA (n � 8), OA (n � 8), and normal (n � 3) synovialfibroblasts was 3,327 � 563 pg/ml, 5,286 � 1,383 pg/ml,and 1,682 � 527 pg/ml, respectively. The mean � SEMexpression of MMP-1 in RA, OA, and normal synovial

Figure 1. Increase of inactive retinoblastoma (Rb) in rheumatoidarthritis (RA) synovial fibroblasts. Protein lysates from 48–72-hourserum-starved (Q) and proliferating (P) RA and non-RA synovialfibroblasts were isolated and analyzed for total Rb and phospho-RbpSpT249/252 expression by an enzyme-linked immunosorbent assay(ELISA)–based method. The data obtained from the ELISA werestandardized to the protein concentration in each lysate. Values arethe mean and SEM. P values were determined by Wilcoxon’s rank sumtest.

Figure 2. Induction of G0/G1 cycle arrest by an adenovirus thatexpresses retinoblastoma (Ad-Rb). Quiescent rheumatoid arthritissynovial fibroblasts were transduced with an adenovirus that expresses�-galactosidase (Ad-�-gal) or Ad-Rb, and 24-hour postinfection cellswere stimulated in 10% fetal bovine serum/Dulbecco’s modifiedEagle’s medium. Cell cycle was analyzed by flow cytometry 48 hourspost–serum stimulation. Values are the mean and SEM. � � P � 0.05versus Ad-Rb–infected cultures, by Student’s t-test. No cell death wasapparent in any culture, even at 96 hours postinfection (data notshown).

Figure 3. Retinoblastoma (Rb)–expressing adenovirus reducesinterleukin-6 (IL-6) and matrix metalloproteinase 1 (MMP-1) inrheumatoid arthritis (RA) synovial fibroblasts but not in non-RAsynovial fibroblasts. A and B, RA synovial fibroblasts (n � 8) and Cand D, non-RA synovial fibroblasts (n � 11) were transduced with anadenovirus that expresses �-galactosidase (�-gal) or Rb. Supernatantswere collected 72 hours following infection and analyzed for IL-6 andMMP-1 production by enzyme-linked immunosorbent assay. For eachexperiment, the amount of IL-6 and MMP-1 was standardized to thecell number. Values are the mean and SEM. P values versus Ad-�-gal–infected cultures were determined by Wilcoxon’s rank sum test.

Figure 4. Effect of Rb-expressing adenovirus on IL-1�–induced IL-6and MMP-1 production in non-RA synovial fibroblasts. Non-RAsynovial fibroblasts (n � 5) were transduced with �-gal or Rb andstimulated with 1 ng/ml of IL-1�. Supernatants were collected 72 hoursfollowing infection and analyzed for IL-6 and MMP-1 production byenzyme-linked immunosorbent assay. For each experiment, theamount of IL-6 and MMP-1 was standardized to the cell number.Values are the mean and SEM. See Figure 3 for definitions.

RETINOBLASTOMA INHIBITS MMP-1 BY SUPPRESSING p38 ACTIVITY 81

fibroblasts was 10 � 3 ng/ml, 13 � 4 ng/ml, and 9 � 4ng/ml, respectively. Ad-Rb reduced IL-6 and MMP-1secretion in RA synovial fibroblasts (Figures 3A and B),by 59 � 18% (P � 0.08) and 89 � 3% (P � 0.008),respectively. In contrast, Ad-Rb had no effect on spon-taneous (Figures 3C and D) or IL-1�–induced (Figures4A and B) IL-6 and MMP-1 production in non-RA

synovial fibroblasts. IL-1�–treated non-RA synovial fi-broblasts produced 34 � 10 ng/ml of IL-6 and 89 � 27ng/ml of MMP-1. Overexpression of Rb inhibited thespontaneous production of IL-6 and MMP-1 in RAsynovial fibroblasts, while Rb failed to suppress sponta-neous and IL-1�–induced IL-6 and MMP-1 expressionin non-RA synovial fibroblasts.

Rb has no effect on IL-6 and MMP-1 mRNA. Tocharacterize the mechanism for the reduction ofMMP-1, the steady-state mRNA levels in Ad-�-gal– andAd-Rb–infected RA synovial fibroblasts were examinedby real-time reverse-transcriptase PCR. OA and normalsynovial fibroblasts were not examined, since Rb over-expression had no inhibitory effect on spontaneous orIL-1�–induced IL-6 or MMP-1 production. In contrastto p21 overexpression (61), no differences were ob-served in IL-6 and MMP-1 mRNA accumulation inAd-�-gal–infected compared with Ad-Rb–infected syno-vial fibroblasts (Figure 5A). Moreover, quantitativePCR (n � 5) confirmed that the relative number of IL-6(Figure 5B) and MMP-1 (Figure 5C) transcripts re-mained unchanged in the Ad-Rb–infected compared

Figure 5. Effect of Rb on IL-6 and MMP-1 mRNA accumulation. RAsynovial fibroblasts (n � 5) were infected with Ad-�-gal, Ad-Rb, orAd-p21. RNA was isolated and used to perform A, reversetranscriptase–polymerase chain reaction (PCR), or B and C, real-timePCR for B, IL-6 or C, MMP-1. For real-time PCR, the y-axis shows therelative amounts of IL-6 or MMP-1 transcripts after correction byhypoxanthine guanine phosphoribosyltransferase (HPRT), as de-scribed in Materials and Methods. Values are the mean and SEM. Pvalues were determined by Student’s t-test. NS � not significant (seeFigure 3 for other definitions).

Figure 6. Suppression of p38 activity by Rb. A, Nonradioactive kinaseassays were performed. RA synovial fibroblasts infected with theindicated adenovirus were lysed and 100 �g of protein was incubatedwith glutathione S-transferase (GST)–phospho-p38 fusion proteinbeads. Following 3 washes, the GST-phospho-p38 beads were incu-bated with activating transcription factor 2 (ATF-2), fusion protein19-96, and ATP. Samples were then boiled in loading buffer, loaded onsodium dodecyl sulfate–polyacrylamide gels, transferred toImmobilon-P membrane, and immunoblotted using anti-phospho–ATF-2 (Thr71) antibody. The data are representative of studies donewith fibroblasts from 2 individuals. Immunoblot with anti-tubulinantibody demonstrates equal loading for the kinase assay. B, RAsynovial fibroblasts were treated as described above. Protein lysateswere analyzed for phosphorylated p38 (pTpY180/182) and p38 (non-phosphorylated and phosphorylated) by an enzyme-linked immu-nosorbent assay–based method. The amount of phosphorylated p38was divided by the total p38 content in each lysate and this ratio wasstandardized to the protein concentration in each lysate. The data arerepresentative of studies done with fibroblasts from 4 individuals withRA, performed in duplicate or triplicate. Values are the mean andSEM. P value was determined by Student’s t-test. IP � immunopre-cipitation; IB � immunoblot (see Figure 3 for other definitions).

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Figure 7. Failure of constitutively active mitogen-activated protein kinase kinase 3 (CA-MKK-3) to rescue p38 activity in Ad-Rb–infected RAsynovial fibroblasts. A, Ad-Rb inhibits phosphorylation of p38 by CA-MKK-3. Triplicate cultures of RA synovial fibroblasts were infected with eitherAd-�-gal, Ad-�-gal/Ad-Rb, Ad-�-gal/Ad-CA-MKK-3, or Ad-Rb/Ad-CA-MKK-3. Forty-eight hours following serum stimulation, protein lysateswere analyzed for phosphorylated p38 (pTpY180/182) and p38 (nonphosphorylated and phosphorylated) by an enzyme-linked immunosorbentassay–based method. The amount of phosphorylated p38 was divided by the total p38 content in each lysate and this ratio was standardized to theprotein concentration in each lysate. The data are representative of 2 experiments. B, Ad-Rb suppresses CA-MKK-3–induced MMP-1 secretion.Supernatants from the infected cells as described above were analyzed for MMP-1 expression. C, Lack of effect of CA-MKK-3 on IL-6 secretion.Supernatants from the infected cells as described above were analyzed for IL-6 expression. Values are the mean and SEM. P values were determinedby Student’s t-test. See Figure 3 for other definitions.

RETINOBLASTOMA INHIBITS MMP-1 BY SUPPRESSING p38 ACTIVITY 83

with Ad-�-gal–infected cells, while Ad-p21 suppressedIL-6 and MMP-1 relative transcripts (Figures 5B and C).These data suggest that Rb suppressed IL-6 and MMP-1at a posttranscriptional level.

Inhibition of p38 kinase activity by Rb. To fur-ther characterize Ad-Rb–mediated suppression ofMMP-1, p38 kinase assays (Cell Signaling Technology,Beverly, MA) were used. The p38 is known to activateMMP-1 synthesis in a posttranscriptional manner(17,18). Ad-Rb markedly reduced p38 kinase activity, asdemonstrated by reduced phosphorylation of the acti-vating transcription factor 2 substrate (Figure 6A). Ad-ditionally, lysates from Ad-�-gal– and Ad-Rb–infectedRA synovial fibroblasts (n � 4) were measured for p38and phosphorylated p38 expression by ELISA. Ad-Rbinhibited p38 activation (phosphorylated p38/total p38)by a mean � SEM of 33.3 � 9% compared withAd-�-gal–infected cells (Figure 6B). These data suggestthat Ad-Rb may inhibit IL-6 or MMP-1 through thesuppression of p38.

Prevention of phosphorylation of p38 in thepresence of constitutively active MKK-3 by Ad-Rb.Overexpression of p38 (Ad-p38) failed to increase p38activity in Ad-�-gal–infected cells compared with mock-infected cells (data not shown). Therefore, Ad-CA-MKK3 (15,17,18,62–65) was used to restore p38 activity.Coinfection of RA synovial fibroblasts with Ad-CA-MKK3 and Ad-�-gal resulted in a 50% increase in p38phosphorylation compared with Ad-�-gal–infected cells(Figure 7A). However, Ad-Rb suppressed p38 activationin Ad-�-gal– or Ad-CA-MKK3–coinfected RA synovialfibroblasts (Figure 7A). Furthermore, Ad-Rb inhibitedMMP-1 (Figure 7B) production in Ad-�-gal– or Ad-CA-MKK3–coinfected RA synovial fibroblasts comparedwith Ad-�-gal alone or Ad-�-gal– and Ad-CA-MKK3–infected cells. In contrast, Ad-CA-MKK3 had no effecton IL-6 expression and was unable to rescue Ad-Rb–mediated reduction of IL-6 (Figure 7C). These datademonstrate a novel role for Rb in regulating p38activity. Moreover, an MKK-3–p38 pathway may not berequired for IL-6 expression. Thus, in RA synovialfibroblasts, Rb functions to suppress p38 activation,thereby reducing MMP-1 expression.

DISCUSSION

Synovial hyperplasia is a general feature of RA,and is partially attributed to an increase in the number ofsynovial fibroblasts. However, few studies have exam-ined cell cycle regulatory proteins in RA. Mutations inp53, which is normally a suppressor of cell cycle through

the induction of p21 (66,67), are observed in RA syno-vial tissue (23). Expression of p21 is reduced in culturedRA compared with OA synovial fibroblasts (29). More-over, there is a 50% decrease in the number of p21-positive synovial fibroblasts in RA compared with OAsynovial tissue (29). In contrast to p21, p16 is inducedonly in RA, but not in OA or normal synovial fibroblasts(32). Overexpression of p21 or p16 suppresses experi-mental arthritis development (30,32). Both p21 and p16function to maintain Rb in its active hypophosphory-lated state (68), yet no studies have examined Rb statusin RA.

We demonstrate herein an increase in the rela-tive inactive Rb isoform in quiescent and proliferatingRA compared with non-RA synovial fibroblasts. Al-though only cultured RA synovial and non-RA synovialfibroblasts were examined, inactive Rb expression wasobserved in RA synovial tissue compared with non-RAsynovial tissue. A recent study demonstrated that p16 islocalized to the Ki-67–negative synovial fibroblasts in-vading articular cartilage in the RA-SCID mouse model(33). Since the relative total Rb expression (total Rb/total protein) is unchanged in RA compared withnon-RA synovial fibroblasts, these data suggest that theactive isoform is being converted to an inactive statethrough an unknown mechanism. The observed decreasein p21 (29) may lead to an increase in cyclin/CDK-2kinase activity, resulting in hyperphosphorylated Rb.Collectively, these data suggest that Rb may be differ-entially regulated in RA compared with non-RA syno-vial fibroblasts.

Overexpression of p21 inhibits IL-6 and MMP-1in RA synovial fibroblasts, indicating that cell cycleinhibitory factors suppress multiple proinflammatorycytokines or MMPs in RA (61). Rb expression has beenlinked to the expression of IL-6. Fifty percent of patients

Figure 8. Schematic of Rb function in RA. Rb suppresses p38 bydirectly inhibiting mitogen-activated protein kinase kinase 3 (MKK-3)or MKK-6 activation or by blocking p38 activation downstream ofMKK-3 or MKK-6 activation. Rb-mediated inhibition of p38 results indecreased MMP-1 production. However, the mechanism of Rb-mediated suppression of IL-6 is unknown. See Figure 3 for otherdefinitions.

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with multiple myeloma, a disease in which IL-6 iscritical, display a loss of Rb expression (69,70). Further-more, the expression of IL-6 was inversely correlatedwith the loss of Rb in blasts from patients with acutemyeloblastic leukemia (71). Reduction of Rb in Rb-positive blasts by antisense treatment induced the ex-pression of IL-6 (71,72). These data indicate an inversecorrelation between Rb expression and IL-6 productionin cancer. IL-6 is also abundantly expressed in RAsynovial tissue (3), and deficiencies in IL-6 preventand/or reduce the severity of experimental arthritis(8–11). Ad-Rb inhibits IL-6 secretion and, similar toMMP-1, the steady-state levels of IL-6 mRNA areunaffected by Ad-Rb. In contrast to MMP-1, overex-pression of Ad-CA-MKK3 fails to increase IL-6 produc-tion. Although in RA synovial fibroblasts, p38 has beenimplicated in IL-1�–induced IL-6 expression (13), theconstitutive expression of IL-6 may be regulated by ap38-independent pathway (Figure 8).

The steady-state mRNA levels of MMP-1 wereunchanged in the Ad-Rb–infected synovial fibroblastscompared with control-infected cultures. MMP-1 ex-pression is induced through the NF-�B, activator protein1 (AP-1), or p38 signal transduction pathways (73). InRA synovial fibroblasts, Ad-Rb markedly reduces p38activity, but has no inhibitory effect on NF-�B or AP-1DNA binding activity (data not shown), suggesting thatblocking of p38 is required for Rb-mediated suppressionof MMP-1. Since Rb had no effect on MMP-1 or IL-6 innon-RA synovial fibroblasts, the ability of Rb to inhibitp38 activity was not examined in non-RA synovialfibroblasts. Furthermore, p38 promotes transcriptionand mRNA stability, and/or enhances translation (14).In human foreskin fibroblasts, p38 has been shown to berequired for MMP-1 transcription (16), while in normalskin fibroblasts, it is necessary for the stabilization ofMMP-1 mRNA (15). However, our data suggest thatp38 may regulate MMP-1 at the posttranscriptionallevel.

Previous studies have shown that p38 phosphor-ylates MAPKAPK-2, which differentially regulates cyto-kine expression. MAPKAPK-2 promotes IL-6 mRNAstability in lipopolysaccharide (LPS)–treated macro-phages (74,75), while MAPKAPK-2 enhances LPS-induced tumor necrosis factor � at a posttranscriptional/mRNA stability level (74,75). Translation is alsoenhanced by p38 through the activation of a subunit ofthe eukaryotic initiation factor 4e (eIF4e), which specif-ically recognizes and interacts with the 5� cap structureof mRNA (76–78). Although to date no direct work hasshown that MAPKAPK-2 or eIF4e enhances MMP-1

synthesis, it is possible that Ad-Rb–mediated suppres-sion of p38 may affect the activation of eIF4e orMAPKAPK-2 and subsequently inhibit MMP-1 produc-tion in RA synovial fibroblasts.

Overexpression of Ad-CA-MKK3 increasesMMP-1 production in control-infected RA synovial fi-broblasts, while Ad-CA-MKK3 fails to enhance p38activation or MMP-1 production in Ad-Rb–infectedcells. These data indicate that Rb interacts with p38 andthereby prevents its phosphorylation by Ad-CA-MKK3(Figure 8). Alternatively, Rb may bind directly toMKK-3 and block its ability to phosphorylate p38 (Fig-ure 8). Recent investigations showed that Rb binds toc-Jun N-terminal kinase (79,80) but not p38 in a humancancer cell line (80), suggesting that in RA synovialfibroblasts Rb may not act directly on p38. However, it ispossible that the interaction between Rb and p38 may becell-type specific. Collectively, these data demonstrate anovel role for Rb in suppressing p38 activation andsubsequent MMP-1 expression.

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