Macrophage Migration Inhibitory Factor Up-regulates Expression of Matrix Metalloproteinases in Synovial Fibroblasts of Rheumatoid Arthritis
2000; Elsevier BV; Volume: 275; Issue: 1 Linguagem: Inglês
10.1074/jbc.275.1.444
ISSN1083-351X
AutoresShin Onodera, Kiyoshi Kaneda, Yuka Mizue, Yoshikazu Koyama, Mami Fujinaga, Jun Nishihira,
Tópico(s)Macrophage Migration Inhibitory Factor
ResumoNeutral matrix metalloproteinases (MMPs) are responsible for the pathological features of rheumatoid arthritis (RA) such as degradation of cartilage. We herein show the up-regulation of MMP-1 (interstitial collagenase) and MMP-3 (stromelysin) mRNAs of cultured synovial fibroblasts retrieved from rheumatoid arthritis (RA) patients in response to macrophage migration inhibitory factor (MIF). The elevation of MMP-1 and MMP-3 mRNA was dose-dependent and started at 6 h post-stimulation by MIF, reached the maximum level at 24 h, and was sustained at least up to 36 h. Interleukin (IL)-1β mRNA was also up-regulated by MIF. These events were preceded by up-regulation of c-jun and c-fos mRNA. Tissue inhibitor of metalloproteinase (TIMP)-1, a common inhibitor of these proteases, was slightly up-regulated by MIF. Similarly, mRNA up-regulation of MMP-1 and MMP-3 was observed in the synovial fibroblasts of patients with osteoarthritis. However, their expression levels were much lower than those of RA synovial fibroblasts. The mRNA up-regulation by MIF was inhibited by the tyrosine kinase inhibitors genestein and herbimycin A, as well as the protein kinase C inhibitors staurosporine and H-7. On the other hand, the inhibition was not seen after the addition of the cyclic AMP-dependent kinase inhibitor, H-8. The mRNA up-regulation of MMPs was also inhibited by curcumin, an inhibitor of transcription factor AP-1, whereas interleukin-1 receptor antagonist, an IL-1 receptor antagonist, failed to inhibit the mRNA up-regulation. Considering these results, it is suggested that 1) MIF plays an important role in the tissue destruction of rheumatoid joints via induction of the proteinases, and 2) MIF up-regulates MMP-1 and MMP-3 via tyrosine kinase-, protein kinase C-, and AP-1- dependent pathways, bypassing IL-1β signal transduction. Neutral matrix metalloproteinases (MMPs) are responsible for the pathological features of rheumatoid arthritis (RA) such as degradation of cartilage. We herein show the up-regulation of MMP-1 (interstitial collagenase) and MMP-3 (stromelysin) mRNAs of cultured synovial fibroblasts retrieved from rheumatoid arthritis (RA) patients in response to macrophage migration inhibitory factor (MIF). The elevation of MMP-1 and MMP-3 mRNA was dose-dependent and started at 6 h post-stimulation by MIF, reached the maximum level at 24 h, and was sustained at least up to 36 h. Interleukin (IL)-1β mRNA was also up-regulated by MIF. These events were preceded by up-regulation of c-jun and c-fos mRNA. Tissue inhibitor of metalloproteinase (TIMP)-1, a common inhibitor of these proteases, was slightly up-regulated by MIF. Similarly, mRNA up-regulation of MMP-1 and MMP-3 was observed in the synovial fibroblasts of patients with osteoarthritis. However, their expression levels were much lower than those of RA synovial fibroblasts. The mRNA up-regulation by MIF was inhibited by the tyrosine kinase inhibitors genestein and herbimycin A, as well as the protein kinase C inhibitors staurosporine and H-7. On the other hand, the inhibition was not seen after the addition of the cyclic AMP-dependent kinase inhibitor, H-8. The mRNA up-regulation of MMPs was also inhibited by curcumin, an inhibitor of transcription factor AP-1, whereas interleukin-1 receptor antagonist, an IL-1 receptor antagonist, failed to inhibit the mRNA up-regulation. Considering these results, it is suggested that 1) MIF plays an important role in the tissue destruction of rheumatoid joints via induction of the proteinases, and 2) MIF up-regulates MMP-1 and MMP-3 via tyrosine kinase-, protein kinase C-, and AP-1- dependent pathways, bypassing IL-1β signal transduction. rheumatoid arthritis osteoarthritis protein kinase C activator protein 1 glyceroaldehyde-3-phosphate dehydrogenase interleukin-1β macrophage migration inhibitory factor matrix metalloproteinase tissue inhibitor of matrix metalloproteinases interleukin-1 receptor antagonist nonessential amino acids fetal calf serum Eagle's minimum essential medium polymerase chain reaction enzyme-linked immunosorbent assay base pair tetradecanoylphorbolacetate TPA-responsive element tumor necrosis factor-α Degradation of extracellular matrix components is often seen as a typical pathological characteristic of rheumatoid arthritis (RA)1 and osteoarthritis (OA) (1Krane S.M. Simon L.S. Med. Clin. N. Am. 1986; 70: 263-284Crossref PubMed Scopus (64) Google Scholar). The tissue degradation is thought to be largely mediated by neutral metalloproteinases (MMPs) (2Evanson J.M. Jeffrey J.J. Krane S.M. J. Clin. Invest. 1968; 47: 2639-2651Crossref PubMed Scopus (121) Google Scholar, 3Evanson J.M. Jeffrey J.J. Krane S.M. Science. 1967; 158: 499-502Crossref PubMed Scopus (129) Google Scholar, 4Dayer J.M. Russel R.G.G. Krane S.M. Science. 1977; 195: 181-183Crossref PubMed Scopus (193) Google Scholar, 5Okada Y. Nagase H. Harris Jr., E.D. J. Biol. Chem. 1986; 261: 14245-14255Abstract Full Text PDF PubMed Google Scholar). MMPs are mainly produced by synovial fibroblasts (6Werb Z. Kelly W.N. Harris Jr., E.D. Rubby S. Sledge C.B. Textbook of Rheumatology. W. B. Saunders Co., Philadelphia1989: 300-321Google Scholar), in which MMP-1 (interstitial collagenase) is considered to be the rate-limiting enzyme in collagenolysis and elicits degradation of collagen types I, II, III, and X (7Welgus H.G. Jeffrey J.J. Eisen A.Z. J. Biol. Chem. 1981; 256: 9511-9515Abstract Full Text PDF PubMed Google Scholar). Similarly, MMP-3 (stromelysin-1) is capable of degrading various components of the extracellular matrix, including cartilage aggrecan, and types II, IV, IX, and XI collagen (8Wu J.J. Lark M.W. Chun L.E. Eyre D.R. J. Biol. Chem. 1991; 266: 5625-5628Abstract Full Text PDF PubMed Google Scholar) and, moreover, has the potential to activate interstitial procollagenase (9Murphy G. Cockett M. I. Stephens P. E. Smith B. J. Docherty A.J.P. Biochem. J. 1987; 248: 265-268Crossref PubMed Scopus (394) Google Scholar, 10He C. Wilhelm S.M. Pentland A.P. Marmel B.L. Grant G.A. Eisen A.Z. Goldberg G.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2632-2636Crossref PubMed Scopus (523) Google Scholar) and progelatinase-B (pro-MMP-9) (11Ogata Y. Enghild J.J. Nagase H. J. Biol. Chem. 1992; 267: 3581-3584Abstract Full Text PDF PubMed Google Scholar). In this context, MMP-1 and MMP-3 are regarded to be responsible in large part for the connective tissue degradation in RA (6Werb Z. Kelly W.N. Harris Jr., E.D. Rubby S. Sledge C.B. Textbook of Rheumatology. W. B. Saunders Co., Philadelphia1989: 300-321Google Scholar). MMP-1 and MMP-3 are produced by synovial lining cells of fibroblasts and infiltrating macrophages, and their mRNA levels are greater in RA than in OA (12Firestein G.S. Paine M.M. Littman B.H. Arthritis & Rheum. 1991; 34: 1094-1105Crossref PubMed Scopus (228) Google Scholar, 13Gravallese E.M. Darling J.M. Ladd A.L. Katz J.N. Glimcher L.H. Arthritis & Rheum. 1991; 34: 1076-1084Crossref PubMed Scopus (189) Google Scholar, 14McCachren S.S. Arthritis & Rheum. 1991; 34: 1085-1093Crossref PubMed Scopus (195) Google Scholar). On the other hand, tissue inhibitor of metalloproteinase (TIMP)-1, which is also released from synovial fibroblasts, is a glycoprotein that forms a 1:1 stoichiometric complex with MMP-1 and MMP-3 (15Cawstone T.E. Barret A.J. Salvesen G. Proteinase Inhibitors. Elsevier Science Publishers B.V., Amsterdam1986: 589-610Google Scholar). A number of reports showed that the proteolytic activities of MMPs in connective tissues are regulated by TIMPs (TIMP-1 and TIMP-2), which contribute to the suppression of excessive tissue degradation by MMPs. Macrophage migration inhibitory factor (MIF) was initially identified as a soluble factor in culture medium of activated T cells (16Bloom B.R. Bennet B. Science. 1966; 153: 80-82Crossref PubMed Scopus (1267) Google Scholar, 17David J.R. Proc. Natl. Acad. Sci. U. S. A. 1966; 56: 72-77Crossref PubMed Scopus (1089) Google Scholar); however, its precise biological function long remained unelucidated. Following the cloning of MIF cDNA (18Weiser W.Y. Temple P.A. Witec-Giannoti J.S. Remold H.G. Clark S.C. David J.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7522-7526Crossref PubMed Scopus (324) Google Scholar), previously unrecognized biological functions of MIF have been revealed. MIF is released as a hormone by the anterior pituitary gland in endotoxic shock (19Bernhagen J. Calandra T. Mitchell R.A. Martin S.B. Tracy K.J. Voelter W. Manogue K.R. Cerami A. Bucala R. Nature. 1993; 365: 756-759Crossref PubMed Scopus (930) Google Scholar, 20Nishino T. Bernhagen J. Shiiki H. Calandra T. Dohi K. Bucala R. Mol. Med. 1995; 1: 781-788Crossref PubMed Google Scholar) and as a proinflammatory cytokine and glucocorticoid-induced immunomodulator mainly produced by macrophages in response to a variety of inflammatory stimuli (21Calandra T. Bernhagen J. Mitchell R.A. Bucala R. J. Exp. Med. 1994; 179: 1895-1902Crossref PubMed Scopus (888) Google Scholar). In terms of arthritis, it was reported that an anti-MIF antibody suppressed inflammatory responses in a mouse model of type II collagen-induced arthritis (22Mikulowska A. Metz C.N. Bucala R. Holmdahl R. J. Immunol. 1997; 158: 5514-5517PubMed Google Scholar). We report herein for the first time that MIF up-regulates mRNAs of MMP-1 and MMP-3 in synovial fibroblasts obtained from RA patients. Moreover, we evaluated the signal transduction pathway of MIF with regard to the up-regulation of MMPs. The present results will shed light on the novel pathological mechanism of tissue destruction in rheumatoid joints and should give a further insight into the regulatory mechanism of the production of MMPs by synovial fibroblasts. The following materials were obtained from commercial sources. Collagenase, staurosporine, genestein, and herbimycin A were purchased from Wako (Osaka, Japan); H-7 and H-8 were from Seikagaku Kogyo (Tokyo, Japan); interleukin-1 receptor antagonist (IL-1ra) was from Anapure Bioscientific (Beijing, China); Eagle's minimum essential medium (MEM) was from ICN Biomedicals (Aurora, Ohio); fetal calf serum (FCS) was from HyClone (Logan, UT); nonessential amino acids (NEAA) were from Life Technologies, Inc.; Isogen RNA extraction kit and GenePure were from Nippon Gene (Toyama, Japan); Biotrack MMP-1 assay kit and Hybond N nylon membrane were from Amersham Pharmacia Biotech; Ex-Taq DNA polymerase, DNA random primer labeling kit, and c-fos cDNA probe were from Takara (Kyoto, Japan); curcumin was from Nakarai Tesque (Kyoto, Japan), and pT7 vector was from CLONTECH (Palo Alto, CA). All other chemicals were of analytical grade. cDNA of c-jun was a kind gift from Dr. M. Sakai of the Department of Biochemistry, Hokkaido University School of Medicine. Recombinant human MIF was expressed in Escherichia coliBL21/DE3 (Novagen, Madison, WI) and purified as described (23Nishihira J. Kuriyama T. Sakai M. Nishi S. Ohki S. Hikichi K. Biochim. Biophys. Acta. 1995; 1247: 159-162Crossref PubMed Scopus (73) Google Scholar). It contained less than 1 pg of endotoxin per μg of protein as determined by the chromogenic Limulus amoebocyte assay (BioWhittaker, Walkerville, MD). Synovial fibroblasts were isolated from knee biopsies of patients with RA or OA at the time of total joint replacement surgery. The study was conducted according to Declaration of Helsinki principles. Synovial tissues were minced and digested in 0.2% collagenase in MEM containing 5% FCS and 100 μmNEAA for 6 h at 37 °C. After centrifugation and washing, cells were resuspended in MEM supplemented with 10% FCS and NEAA in 100-mm culture dishes in a humidified 5% CO2 atmosphere at 37 °C. After 48 h, nonadherent cells were removed, and adherent cells were trypsinized with 0.25% trypsin/EDTA and were successively passaged. Purity of the cells was >95% fibroblast-like cells as confirmed by microscopic analysis. To examine the effect of MIF on the mRNA expression of MMPs, cultured synovial fibroblasts of RA and OA patients were used with and without passages. After reaching confluence (10–14 days after initial plating), the primary RA and OA fibroblasts (synoviocytes) without passage were rinsed with PBS and challenged with 1, 10, and 100 ng/ml and 1 and 10 μg/ml MIF in 10 ml of serum-free MEM containing NEAA for 12 h. To examine the influence of repeated passages in response to MIF in RA synovial fibroblasts, the same procedure was performed on the 3rd- and 7th-passage RA synovial fibroblasts obtained from the same patient. For the time course study, parallel cultures of 3rd-passage RA synovial fibroblasts were serum-starved for 12 h, then treated simultaneously with 1 μg/ml MIF, and harvested at indicated intervals after stimulation. As controls, 4 sets of the 3rd-passage RA synovial fibroblasts and 2 sets of the 3rd-passage OA synovial fibroblasts were used. The cells were harvested and subjected to Northern blot analysis. In all the cell culture experiments, 30 μg of polymyxin B/ml was added to the culture medium. Human complete coding cDNA for human MMP-1 (2.05 kilobase pairs) in a pSP64 vector was purchased from the American Type Culture Collection. The templates of human MMP-3, TIMP-1, IL-1β, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA for Northern blot analyses were obtained by reverse transcription-polymerase chain reaction (PCR) from a human cDNA library of human synovial fibroblasts. 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Holt J.T. Matrisian L.M. Science. 1988; 242: 1424-1427Crossref PubMed Scopus (211) Google Scholar, 38Kerr L.D. Miller D.B. Martrisian L.M. Cell. 1990; 61: 267-278Abstract Full Text PDF PubMed Scopus (375) Google Scholar, 39Lafyatis R. Kim S.J. Angel P. Roberts A.B. Sporn M.B. Karin M. Wilder R.L. Mol. Endocrinol. 1990; 4: 973-980Crossref PubMed Scopus (129) Google Scholar, 40Nicholson R.C. Mader S. Nagpal S. Leid M. Rochette Egly C. Chambon P. EMBO J. 1990; 9: 4443-4454Crossref PubMed Scopus (316) Google Scholar, 41Takahashi N. Nishihira J. Sato Y. Kondo M. Ogawa H. Ohshima T. Une Y. Todo S. Mol. Med. 1998; 4: 707-714Crossref PubMed Google Scholar, 42Lambert C.A. Lapiere C.M. Nusgens B.V. J. Biol. Chem. 1998; 273: 23143-23149Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 43Fini M.E. Strissel K.J. Girard M.T. Mays J.W. Rinehart W.B. J. Biol. Chem. 1994; 269: 11291-11298Abstract Full Text PDF PubMed Google Scholar, 44Calandra T. Bernhagen J. Metz C.N. Spiegel L.A. 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Nature. 1990; 344: 678-682Crossref PubMed Scopus (908) Google Scholar, 53Vincenti M.P. Coon C.I. White L.A. Barchowsky A. Brinckerhoff C.E. Arthritis & Rheum. 1996; 39: 574-582Crossref PubMed Scopus (47) Google Scholar, 54Subdeck B.D. Parks W.C. Welgus H.G. Pentland A.P. J. Biol. Chem. 1994; 269: 30022-30029PubMed Google Scholar, 55Callaghan M.M. Lovis R.M. Rammohan C. Lu Y. Pope R.M. J. Leukocyte Biol. 1996; 59: 125-132Crossref PubMed Scopus (25) Google Scholar, 56Uehara Y. Fukazawa H. Methods Enzymol. 1991; 201: 370-379Crossref PubMed Scopus (222) Google Scholar) and antisense primer 5′-CTGGGTACAGCTCTCTTTAGGAA-3′ (881–903) (GenBankTMaccession number X02532); GAPDH (1024 bp), sense primer 5′-CGGGATCCATGGGGAAGGTGAAGGTC-3′ (59–78) and antisense primer 5′-CGGGATCCTTACTCCTTGGTGGCCAT-3′ (1051–1070) (GenBankTMaccession number M33197). Each PCR product was separated by agarose gel purification, purified by GenePure, and subcloned into pT7 plasmid vector by TA cloning. The subcloned plasmids were transformed into DH5α-competent cells. After amplification, each insert was prepared by restriction enzyme digestion, checked by a sequencing analyzer (Applied Biosystems Inc., 377A), and used as a template for Northern blot analysis. Total cellular RNA was isolated from RA and OA synovial fibroblasts using an Isogen RNA extraction kit according to the manufacturer's protocols. RNA was quantitated by spectrophotometry, and equal amounts of RNA (10 μg) from control and test samples were loaded on a formaldehyde-agarose gel. The gel was stained with ethidium bromide to visualize RNA standards, and the RNA was transferred onto a nylon membrane. Fragments obtained by restriction enzyme treatments forMMP-1, MMP-3, TIMP-1, IL-1β, c-jun, c-fos, and GAPDH were labeled with [α-32P]dCTP using a DNA random primer labeling kit. Hybridization was carried out at 42 °C for 24–48 h. Post-hybridization washes were performed in 0.1% SDS, 0.2× SSC (1× SSC: 0.15 m NaCl, 0.015 m sodium citrate) at 65 °C. The radioactive bands were visualized by autoradiography on Kodak X-AR5 film and quantitatively analyzed using the NIH Image system. Multiple autoradiographic data were examined to ensure that the results reflected those produced in the linear range of the film. The results were standardized with respect to GAPDH mRNA levels. Comparison of ethidium bromide-stained gels with corresponding GAPDH mRNA levels showed that the GAPDH mRNA levels reflected total RNA loaded onto gels. MIF was unexpectedly found to be an isomerase, convertingd-2-carboxy-2,3-dihydroxyindole-5,6-quinone (d-dopachrome) to 5,6-dihydroxyindole-2-carboxylic acid, in which the N-terminal proline functions as a catalytic base (24Rosengren E. Bucala R. Aman P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar, 25Bendrat Y. Al-Abed Y. Callaway D.J.E. Peng T. Calandra T. Mets C.N. Bucala R. Biochemistry. 1997; 36: 15356-15362Crossref PubMed Scopus (143) Google Scholar). To examine the biological link between isomerase activity and biological functions, cDNA of the P1A mutant of MIF was prepared using a site-directed mutagenesis technique as described previously (26Nishihira J. Fujinaga M. Kuriyama T. Suzuki M. Sugimoto H. Nakagawa A. Tanaka I. Sakai M. Biochem. Biophys. Res. Commun. 1998; 243: 538-544Crossref PubMed Scopus (34) Google Scholar). In brief, the sense primer designed was 5′-CATATGGGCATGTTCATCGTA-3′ (1Krane S.M. Simon L.S. Med. Clin. N. Am. 1986; 70: 263-284Crossref PubMed Scopus (64) Google Scholar, 2Evanson J.M. Jeffrey J.J. Krane S.M. J. Clin. Invest. 1968; 47: 2639-2651Crossref PubMed Scopus (121) Google Scholar, 3Evanson J.M. Jeffrey J.J. Krane S.M. Science. 1967; 158: 499-502Crossref PubMed Scopus (129) Google Scholar, 4Dayer J.M. Russel R.G.G. Krane S.M. Science. 1977; 195: 181-183Crossref PubMed Scopus (193) Google Scholar, 5Okada Y. Nagase H. Harris Jr., E.D. J. Biol. Chem. 1986; 261: 14245-14255Abstract Full Text PDF PubMed Google Scholar, 6Werb Z. Kelly W.N. Harris Jr., E.D. Rubby S. Sledge C.B. Textbook of Rheumatology. W. B. Saunders Co., Philadelphia1989: 300-321Google Scholar, 7Welgus H.G. Jeffrey J.J. Eisen A.Z. J. Biol. Chem. 1981; 256: 9511-9515Abstract Full Text PDF PubMed Google Scholar, 8Wu J.J. Lark M.W. Chun L.E. Eyre D.R. J. Biol. Chem. 1991; 266: 5625-5628Abstract Full Text PDF PubMed Google Scholar, 9Murphy G. Cockett M. I. Stephens P. E. Smith B. J. Docherty A.J.P. Biochem. J. 1987; 248: 265-268Crossref PubMed Scopus (394) Google Scholar, 10He C. Wilhelm S.M. Pentland A.P. Marmel B.L. Grant G.A. Eisen A.Z. Goldberg G.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2632-2636Crossref PubMed Scopus (523) Google Scholar, 11Ogata Y. Enghild J.J. Nagase H. J. Biol. Chem. 1992; 267: 3581-3584Abstract Full Text PDF PubMed Google Scholar, 12Firestein G.S. Paine M.M. Littman B.H. Arthritis & Rheum. 1991; 34: 1094-1105Crossref PubMed Scopus (228) Google Scholar, 13Gravallese E.M. Darling J.M. Ladd A.L. Katz J.N. Glimcher L.H. Arthritis & Rheum. 1991; 34: 1076-1084Crossref PubMed Scopus (189) Google Scholar, 14McCachren S.S. Arthritis & Rheum. 1991; 34: 1085-1093Crossref PubMed Scopus (195) Google Scholar, 15Cawstone T.E. Barret A.J. Salvesen G. Proteinase Inhibitors. Elsevier Science Publishers B.V., Amsterdam1986: 589-610Google Scholar, 16Bloom B.R. Bennet B. Science. 1966; 153: 80-82Crossref PubMed Scopus (1267) Google Scholar, 17David J.R. Proc. Natl. Acad. Sci. U. S. A. 1966; 56: 72-77Crossref PubMed Scopus (1089) Google Scholar, 18Weiser W.Y. Temple P.A. Witec-Giannoti J.S. Remold H.G. Clark S.C. David J.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7522-7526Crossref PubMed Scopus (324) Google Scholar) containing the 5′-end sequence encoding an alanine instead of a proline after the initiating methionine with an NdeI restriction site, and the antisense primer was 5′-GGATCCTTAGGCGAAGGTGGAGTT-3′ (331–348) containing a 3′-end sequence identical to the wild-type cDNA with a BamHI restriction site. After PCR, the product was subcloned into pT7 vector. The P1A mutant MIF was expressed and purified as described previously (23Nishihira J. Kuriyama T. Sakai M. Nishi S. Ohki S. Hikichi K. Biochim. Biophys. Acta. 1995; 1247: 159-162Crossref PubMed Scopus (73) Google Scholar). The overall protein structure of P1A examined by crystallography was well conserved in comparison with wild-type MIF, consistent with a previous report (25Bendrat Y. Al-Abed Y. Callaway D.J.E. Peng T. Calandra T. Mets C.N. Bucala R. Biochemistry. 1997; 36: 15356-15362Crossref PubMed Scopus (143) Google Scholar, 27Suzuki M. Sugimoto H. Nakagawa A. Tanaka I. Nishihira J. Sakai M. Nat. Struct. Biol. 1996; 3: 259-266Crossref PubMed Scopus (188) Google Scholar). Both 3rd-passage RA and OA synovial fibroblasts were used to investigate the effect of MIF on the protein levels of MMP-1. After reaching confluence (10–14 days after initial plating), the cells were trypsinized and then plated on a 24-well culture dish at the number of 4 × 104cells per well in 500 μl of MEM containing 10% FCS and NEAA. After 48 h, the medium was replaced with 300 μl of serum-free MEM containing NEAA and various doses of MIF (0, 1, 10, and 100 ng/ml and 1 and 10 μg/ml). After 48 h, the supernatants were collected and subjected to ELISA for MMP-1. For the time course study, we used a procedure similar to the dose-response study in the presence of 1 μg/ml MIF with regard to the 3rd-passage RA synovial fibroblasts, and we obtained aliquots at the indicated times up to 36 h. MMP-1 was assayed by a sandwich enzyme-linked immunosorbent assay (ELISA) using a Biotrack MMP-1 assay kit according to the manufacturer's protocol. The minimal sensitivity of the assay system was 6.25 ng/ml, and good linearity was observed up to 100 ng/ml. By this ELISA system, all forms of MMP, including pro-MMP-1, MMP-1, and MMP-1 complexed with TIMP-1, could be measured. To investigate the signal transduction pathways leading to the up-regulation of MMP mRNA by MIF, 3rd-passage RA synovial fibroblasts were used. After reaching confluence, the cells were treated with or without MIF (1 μg/ml) at 30 min after the addition of genestein (0, 10, and 100 μm), herbimycin A (0, 1, and 10 μm), staurosporine (0, 10, and 100 nm), H-7 (0, 1, and 10 μm), H-8 (0, 1.5, and 15 μm), curcumin (0, 1, and 10 μm), or IL-1ra (0, 10, 100, and 1000 ng/ml) in serum-free medium. After 12 h, the cells were harvested and subjected to Northern blot analysis for MMP-1 and MMP-3 mRNA. Statistical analysis was performed using analysis of variance and Fisher's Protected Least Significant Difference as a post hoc test. In both primary RA and OA synovial fibroblasts, MMP-1 mRNA was markedly up-regulated in a dose-dependent manner in response to MIF ranging from 1 ng/ml to 10 μg/ml for 12-h treatment (Fig. 1, A and B). With regard to MMP-3, its mRNA level was already high in RA fibroblasts, which might have been due to self-induction as often seen in the primary culture without passage, and no significant change was seen with MIF stimulation, whereas a significant increase was observed in OA fibroblasts. On the other hand, the TIMP-1 mRNA level was slightly increased in RA fibroblasts but not in OA fibroblasts. IL-1β mRNA was also up-regulated in both RA and OA fibroblasts, but the extent of up-regulation was much higher in RA than in OA. In the 3rd-passage RA synovial fibroblasts, both MMP-1 and MMP-3 mRNA were coordinately expressed in a dose-dependent manner, reaching the maximal level at 10 μg/ml of MIF, whereas TIMP-1 mRNA was not up-regulated (data not shown). On the other hand, the minimum dose of MIF for the induction of MMP-3 mRNA was 10 μg/ml in the 7th-passage RA synovial fibroblasts, and up-regulation of MMP-1 and TIMP-1 mRNA was not observed at any dose up to 10 μg/ml MIF (data not shown), which might have been due to cellular aging. To compare the mRNA levels of MMPs between RA and OA, mRNA levels on unstimulated and stimulated 3rd-passage fibroblasts of RA and OA synovial tissues were also assessed by Northern blot analysis. The basal and MIF-stimulated expression levels of MMP-1 and MMP-3 mRNAs were much higher in RA fibroblasts than in OA fibroblasts (Fig. 2). It is of note that TIMP-1 mRNA was markedly expressed in both RA and OA synovial fibroblasts. MMP-1 mRNA expression of 3rd-passage RA synovial fibroblasts was found to increase at 6 h post-stimulation with MIF (1 μg/ml) and reached the maximum at 24 h (Fig. 3 A). The enhanced mRNA level was sustained for at least 36 h. Similarly, MMP-3 mRNA started to increase at 3–6 h post-stimulation, but the magnitude of the increase was relatively small compared with that of MMP-1. As for TIMP-1, its
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