FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2068058635> ?p ?o ?g. }

• W2068058635 endingPage "7874" @default.
• W2068058635 startingPage "7865" @default.
• W2068058635 abstract "Neutral matrix metalloproteinases (MMPs) play an important role in bone matrix degradation accompanied by bone remodeling. We herein show for the first time that macrophage migration inhibitory factor (MIF) up-regulates MMP-13 (collagenase-3) mRNA of rat calvaria-derived osteoblasts. The mRNA up-regulation was seen at 3 h in response to MIF (10 μg/ml), reached the maximum level at 6–12 h, and returned to the basal level at 36 h. MMP-13 mRNA up-regulation was preceded by up-regulation of c-jun and c-fos mRNA. Tissue inhibitor of metalloproteinase (TIMP)-1 and MMP-9 (92-kDa type IV collagenase) were also up-regulated, but to a lesser extent. The MMP-13 mRNA up-regulation was significantly suppressed by genistein, herbimycin A and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Similarly, a selective mitogen-activated protein kinase (MAPK) kinase (MEK)1/2 inhibitor (PD98059) and c-jun/activator protein (AP)-1 inhibitor (curcumin) suppressed MMP-13 mRNA up-regulation induced by MIF. The mRNA levels of c-junand c-fos in response to MIF were also inhibited by PD98059. Consistent with these results, MIF stimulated phosphorylation of tyrosine, autophosphorylation of Src, activation of Ras, activation of extracellular signal-regulated kinases (ERK) 1/2, a MAPK, but not c-Jun N-terminal kinase or p38, and phosphorylation of c-Jun. Osteoblasts obtained from calvariae of newborn JunAA mice, defective in phosphorylation of c-Jun, or newborn c-Fos knockout (Fos−/−) mice, showed much less induction of MMP-13 with the addition of MIF than osteoblasts obtained from wild-type or littermate control mice. Taken together, these results suggest that MIF increases the MMP-13 mRNA level of rat osteoblasts via the Src-related tyrosine kinase-, Ras-, ERK1/2-, and AP-1-dependent pathway. Neutral matrix metalloproteinases (MMPs) play an important role in bone matrix degradation accompanied by bone remodeling. We herein show for the first time that macrophage migration inhibitory factor (MIF) up-regulates MMP-13 (collagenase-3) mRNA of rat calvaria-derived osteoblasts. The mRNA up-regulation was seen at 3 h in response to MIF (10 μg/ml), reached the maximum level at 6–12 h, and returned to the basal level at 36 h. MMP-13 mRNA up-regulation was preceded by up-regulation of c-jun and c-fos mRNA. Tissue inhibitor of metalloproteinase (TIMP)-1 and MMP-9 (92-kDa type IV collagenase) were also up-regulated, but to a lesser extent. The MMP-13 mRNA up-regulation was significantly suppressed by genistein, herbimycin A and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Similarly, a selective mitogen-activated protein kinase (MAPK) kinase (MEK)1/2 inhibitor (PD98059) and c-jun/activator protein (AP)-1 inhibitor (curcumin) suppressed MMP-13 mRNA up-regulation induced by MIF. The mRNA levels of c-junand c-fos in response to MIF were also inhibited by PD98059. Consistent with these results, MIF stimulated phosphorylation of tyrosine, autophosphorylation of Src, activation of Ras, activation of extracellular signal-regulated kinases (ERK) 1/2, a MAPK, but not c-Jun N-terminal kinase or p38, and phosphorylation of c-Jun. Osteoblasts obtained from calvariae of newborn JunAA mice, defective in phosphorylation of c-Jun, or newborn c-Fos knockout (Fos−/−) mice, showed much less induction of MMP-13 with the addition of MIF than osteoblasts obtained from wild-type or littermate control mice. Taken together, these results suggest that MIF increases the MMP-13 mRNA level of rat osteoblasts via the Src-related tyrosine kinase-, Ras-, ERK1/2-, and AP-1-dependent pathway. matrix metalloproteinase activator protein 1 Dulbecco's modified Eagle's medium extracellular signal-regulated kinase fetal calf serum glyceroaldehyde-3-phosphate dehydrogenase interleukin c-Jun N-terminal kinase monoclonal antibody mitogen-activated protein mitogen-activated protein kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase minimal essential medium macrophage migration inhibitory factor nonessential amino acid(s) phosphate-buffered saline 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine parathyroid hormone Ras-binding domain tissue inhibitor of matrix metalloproteinases tetradecanoyl phorbol acetate TPA-responsive element Matrix metalloproteinases (MMPs)1 are a family of proteolytic enzymes, including collagenases, gelatinases, and stromelysins (1Mauviel A. J. Cell. Biochem. 1993; 53: 288-295Crossref PubMed Scopus (397) Google Scholar, 2Matrisian L.M. Hogan B.L.M. Curr. Top. Dev. Biol. 1990; 24: 219-259Crossref PubMed Scopus (200) Google Scholar, 3Freije J.M.P. Diez-Itza I. Balvin M. Sanchez L.M. Blasco R. Tolivia J. Lopez-Otin C. J. Biol. Chem. 1994; 269: 16766-16773Abstract Full Text PDF PubMed Google Scholar). Collagenases cleave fibrillar collagens at neutral pH and play an important role in matrix remodeling. Collagenases are largely categorized into three classes: collagenases 1, 2, and 3, which are mainly secreted from fibroblasts and osteoblasts, neutrophils, and breast carcinoma cells, respectively (1Mauviel A. J. Cell. Biochem. 1993; 53: 288-295Crossref PubMed Scopus (397) Google Scholar, 3Freije J.M.P. Diez-Itza I. Balvin M. Sanchez L.M. Blasco R. Tolivia J. Lopez-Otin C. J. Biol. Chem. 1994; 269: 16766-16773Abstract Full Text PDF PubMed Google Scholar). In rodents, rat osteoblasts and rat osteosarcoma cells have the potential to express collagenase 3 such as MMP-13, but not MMP-1 (4Partridge N.C. Jeffrey J.J. Ehlich L.S. Titelbaum S.L. Fliszar C. Welgus H.G. Kahn A.J. Endocrinology. 1987; 120: 1956-1962Crossref PubMed Scopus (170) Google Scholar, 5Quinn C.O. Scott D.K. Brinckerhoff C.E. Matrisian L.M. Jeffrey J.J. Partridge N.C. J. Biol. Chem. 1990; 265: 22342-22347Abstract Full Text PDF PubMed Google Scholar, 6Varghese S. Delany A.M. Liang L. Gabbitas B. Jeffrey J.J. Canalis E. Endocrinology. 1996; 137: 431-437Crossref PubMed Scopus (47) Google Scholar). With regard to collagenase production, several hormones and cytokines stimulate MMP-1 synthesis in human and MMP-13 in rat osteoblasts (4Partridge N.C. Jeffrey J.J. Ehlich L.S. Titelbaum S.L. Fliszar C. Welgus H.G. Kahn A.J. Endocrinology. 1987; 120: 1956-1962Crossref PubMed Scopus (170) Google Scholar, 7Civitelli R. Hruska K. Jeffrey J. Kahn A. Avioli L. Partridge N.C. Endocrinology. 1989; 124: 2928-2933Crossref PubMed Scopus (57) Google Scholar, 8Varghese S. Ramsby M.L. Jeffrey J.J. Canalis E. Endocrinology. 1995; 136: 2156-2162Crossref PubMed Google Scholar). From the data available to date, it is considered that most molecules potentially inducing bone resorption often stimulate MMP-1 production in humans and MMP-13 production in rodents. In this context, it is conceivable that MMP-13 may play a pivotal role in bone remodeling in rats. Macrophage migration inhibitory factor (MIF) was originally identified as a soluble factor in culture medium of activated-T cells (9Bloom B.R. Bennet B. Science. 1966; 153: 80-82Crossref PubMed Scopus (1274) Google Scholar, 10David J.R. Proc. Natl. Acad. Sci. U. S. A. 1966; 56: 72-77Crossref PubMed Scopus (1092) Google Scholar); however, its precise biological function was largely unknown for nearly 30 years. Following the cloning of human MIF cDNA (11Weiser 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 (325) Google Scholar), an array of novel biological functions of this protein has been reported (12Bucala R. FASEB J. 1996; 7: 19-24Google Scholar, 13Nishihira J. J. Interferon Cytokine Res. 2000; 20: 751-762Crossref PubMed Scopus (172) Google Scholar). MIF is released as a hormone by the anterior pituitary gland in endotoxin shock (14Bernhagen 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 (931) Google Scholar) and a glucocorticoid-induced immunomodulator released from macrophages in response to a variety of inflammatory stimuli (15Calandra T. Bernhagen J. Mitchell R.A. Bucala R. J. Exp. Med. 1994; 179: 1895-1902Crossref PubMed Scopus (890) Google Scholar). Regarding the potential role of MIF in induction of MMPs, we revealed that MIF could stimulate MMP-1 and MMP-3 mRNA expression in synovial fibroblasts obtained from rheumatoid arthritis patients (16Onodera S. Kaneda K. Mizue Y. Koyama Y. Fujinaga M. Nishihira J. J. Biol. Chem. 2000; 275: 444-450Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), suggesting its pathological role for direct destruction of joint tissues in autoimmune disease. In a previous study, we demonstrated that mouse osteoblasts expressed high amounts of MIF (17Onodera S. Suzuki K. Matsuno T. Kaneda K. Kuriyama T. Nishihira J. Immunology. 1996; 89: 430-435Crossref PubMed Scopus (60) Google Scholar); however, its pathophysiological role in the bone tissues remains to be elucidated. We herein report for the first time that MIF enhances the MMP-13 mRNA level in osteoblasts obtained from newborn rat calvariae. Following this discovery, we further investigated the signal transduction pathway of MIF in the event of MMP-13 mRNA up-regulation to elucidate its intracellular mechanism of action. The following materials were obtained from commercial sources. Collagenase, staurosporine, genistein, and herbimycin A were purchased from Wako (Osaka, Japan); H-7 and H-8 from Seikagaku Kogyo (Tokyo, Japan); 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) from Calbiochem (La Jolla, CA); cycloheximide, indomethacin, and tyrphostin A25 from Sigma; [γ-32P]ATP and [32P]orthophosphate (8 mCi/ml, carrier-free) from PerkinElmer Life Sciences (Wilmington, DE); Dulbecco's modified Eagle's medium (DMEM) and parathyroid hormone (PTH) from ICN Biomedicals (Aurora, OH); fetal calf serum (FCS) from HyClone (Logan, UT); α-MEM and nonessential amino acids (NEAA) from Invitrogen; Isogen RNA extraction kit and GenePure from Nippon Gene (Toyama, Japan); Hybond N nylon membrane and ECL Western blotting detection system from Amersham Biosciences, Inc.; horseradish peroxidase-conjugated antibody from Bio-Rad; nonimmune mouse IgG plus Pansorbin cells from Calbiochem-Novabiochem (La Jolla, CA); Ex-Taq DNA polymerase and DNA random primer labeling kit from Takara (Kyoto, Japan); curcumin from Nakarai Tesque (Kyoto, Japan); pT7 vector from CLONTECH (Palo Alto, CA); anti-mouse Src monoclonal antibody (mAb) (GD11; mouse IgG1) and Ras activation assay kit from Upstate Biotechnology (Lake Placid, NY); antibodies for phosphorylated-form mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, from New England Biolabs (Beverly, MA), and anti-phosphotyrosine mAb (PY20; mouse IgG1) from ICN ImmunoBiologicals (Costa Mesa, CA); mouse anti-rat MMP-13 (collagenase-3) monoclonal antibody from Chemicon (Temecula, CA); anti-c-Jun (H-79) from Santa Cruz Biotechnology (Santa Cruz, CA), and Micro BCA protein assay reagent kit from Pierce. All other chemicals were of analytical grade. Recombinant rat MIF was expressed in Escherichia coliBL21/DE3 (Novagen, Madison, WI) and purified as described (18Nishihira J. Kuriyama T. Sakai M. Nishi S. Ohki S. Hikichi K. Biochim. Biophys. Acta. 1995; 1247: 159-162Crossref PubMed Scopus (74) Google Scholar). It contained less than 1 pg of endotoxin/μg of protein, as determined by the chromogenic Limulus amoebocyte assay (BioWhittaker, Walkersville, MD). Rat calvarial osteoblasts were prepared by serial collagenase digestion as described previously (19Wong G. Cohn D.V. Nature. 1974; 252: 713-715Crossref PubMed Scopus (198) Google Scholar). In brief, newborn rat calvariae (day 1) were removed from soft tissues and digested with 0.1% collagenase and 0.2% dispase in DMEM containing 100 μm NEAA for 10 min at 37 °C five times (fractions 1–5), of which fractions 3–5 were used. After centrifugation and washing with the medium, cells were resuspended in DMEM 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 harvested after treatment with 0.25% trypsin/EDTA and were successively passaged. Rat synovial fibroblasts were prepared by 0.2% collagenase digestion of rat knee synovial tissues. The murine osteoblastic cell line MC3T3-E1 was purchased from RIKEN Cell Bank (Tsukuba, Japan) and cultured in α-MEM supplemented with 10% FCS. Rat osteosarcoma cell line UMR106 was purchased from the American Type Culture Collection (ATCC) (Rockville, MD), and cultured in Eagle's MEM supplemented with 10% FCS and NEAA. Rat articular chondrocytes were obtained by collagenase digestion of minced articular cartilage tissues from the femoral chondyle, femoral head, and humeral head of 8-week-old rats. Rat skin fibroblasts were obtained by harvesting outgrowing cells from explant cultures of minced skin tissues from newborn rats. All of these cells were cultured in DMEM supplemented with 10% FCS and NEAA. To examine the effect of MIF on the expression of MMP mRNA, production of MIF protein, phosphorylation of tyrosine, Src kinase activity, MAPK activity, and Ras activation, cultured rat osteoblasts of the third passages were used throughout the experiment. After reaching confluence (10–14 days after initial plating), the osteoblasts were rinsed with phosphate-buffered saline (PBS), serum-starved for 24 h, and challenged with 100 ng/ml, 1 μg/ml, and 10 μg/ml MIF for 6 h in 10 ml of serum-free DMEM containing NEAA. Rat PTH (10−9m and 10−8m) and tetradecanoyl phorbol acetate (TPA) (10−6m) were used as positive controls. Two sets of the third-passage rat synovial fibroblasts, MC3T3-E1 cells, UMR106 cells, second-passage rat articular chondrocytes, and second-passage rat skin fibroblasts were used as control cells. For the time-course study, parallel cultures of rat osteoblasts were treated simultaneously with or without 10 μg/ml MIF, and harvested at indicated intervals after stimulation. Following this, the cells were subjected to Northern blot analysis to evaluate mRNA levels of MMP-13, TIMP-1, and type I (α) procollagen (col(I)α (1Mauviel A. J. Cell. Biochem. 1993; 53: 288-295Crossref PubMed Scopus (397) Google Scholar)), and also to immunoblot analysis and in vitro kinase assay. To exclude possible endotoxin contamination in recombinant MIF, the effect of heat-denatured recombinant MIF treated at 65 °C for 1 h was also assessed. Then neutralizing effect of an anti-MIF monoclonal antibody (IgG1) on MIF-induced MMP up-regulation was also evaluated. The anti-MIF monoclonal antibody was prepared by immunizing mice with recombinant rat MIF as described elsewhere (20Mizue Y. Nishihira J. Miyazaki T. Fujiwara S. Chida M. Nakamura K. Kikuchi K. Mukai M. Int. J. Mol. Med. 2000; 5: 397-403PubMed Google Scholar). To evaluate involvement of c-Jun phosphorylation in the signaling pathway of MIF, calvarial osteoblasts retrieved from newborn JunAA mice were also examined. In JunAA mice, serine residues 63 and 73 of the endogenous Jun allele were substituted with alanine residues (21Behrens A. Sibilia M. Wagner E.F. Nat. Genet. 1999; 21: 326-329Crossref PubMed Scopus (594) Google Scholar). JunAA mice were a kind gift from Dr. E. F. Wagner (Research Institute of Molecular Pathology, Vienna, Austria). Calvarial osteoblasts derived from native strain C57BL/6 mice were used as wild-type osteoblasts for positive controls. We also assessed the involvement of c-Fos in the signaling pathways of MIF using calvarial osteoblasts retrieved from newborn mice that were homozygous for the Fos mutation (Fos−/−) and littermate wild type (Fos+/+) mice as controls. Mice that were heterozygous for Fos mutation (Fos+/−) (B6, 129S-Fostm1Pa female and male mice) were purchased from the Jackson Laboratory (Bar Harbor, ME). After mating, the genotypes of the newborn mice obtained were determined by reverse transcription-polymerase chain reaction (PCR) analysis of the lysed tail from each of them. These murine calvarial osteoblasts were retrieved in a manner similar to rat osteoblasts, and were cultured in α-MEM supplemented with 10% FCS. To investigate the signal transduction pathways regarding up-regulation of MMP-13 mRNA in response to MIF, effects of various reagents on rat osteoblasts were tested. After reaching confluence following serum starvation for 24 h, the cells were challenged with MIF (10 μg/ml) 30 min after the addition of cycloheximide (3.6 μm), indomethacin (10 μm), genistein (10 and 100 μm), herbimycin A (1 and 10 μm), PP2 (1, 10, and 50 μm), tyrphostin A25 (10 and 100 μm), staurosporine (10 and 100 nm), H-7 (1 and 10 μm), H-8 (1.5 and 15 μm), PD98059 (5 and 25 μm), SB203580 (1, 10, and 20 μm), or curcumin (1 and 10 μm) in serum-free medium. After 6 h, the cells were harvested and subjected to Northern blot analysis for MMP-13 mRNA. Rat c-jun cDNA and rat col(I)α (1Mauviel A. J. Cell. Biochem. 1993; 53: 288-295Crossref PubMed Scopus (397) Google Scholar) cDNA probes were kindly provided by Dr. M. Sakai (Department of Biochemistry, Hokkaido University Graduate School of Medicine) and Dr. M. Shibanuma (Department of Microbiology, Showa University School of Pharmaceutical Sciences), respectively. The templates for Northern blot analyses of rat MMP-13, MMP-2, MMP-9, TIMP-1, c-fos, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA were obtained by reverse transcription-PCR from a cDNA library of rat synovial fibroblasts. Preparation of each template was as follows: MMP-13 (424 bp), sense primer 5′-GCGGGAATCCTGAAGAAGTCTAC-3′ (143–165) and antisense primer 5′-TTGGTCCAGGAGGAAAAGCG-3′ (547–566) (GenBank™ M60616); MMP-2 (217 bp), sense primer 5′-GCTGATACTGACACTGGTACTG-3′ (1642–1663) and antisense primer 5′-CAATCTTTTCCGGGAGCTC-3′ (1840–1858) (GenBank™U65656); MMP-9 (280 bp), sense primer 5′-AAGGATGGTCTACTGGCAC-3′ (553–571) and antisense primer 5′-AGAGATTCTCACTGGGGC-3′ (815–832) (GenBank™ U24441); TIMP-1 (413 bp), sense primer 5′-CAGATATCCGGTTCGCCTACACC-3′ (149–171) and antisense primer 5′-CAGGCAAGGTGACGGGACTGGAAGC-3′ (539–561) (GenBank™ L29512); c-fos (897 bp), sense primer 5′-CTGAAAGAGAAGGAAAAACTGGA-3′ (693–715) and antisense primer 5′-TGGCTCACATGCTACTAACTACC-3′ (1567–1589) (GenBank™ X06769); GAPDH (983 bp), sense primer 5′-TGAAGGTCGGTGTCAACGGATTTGGC-3′ (35–60) and antisense primer 5′-CATGTAGGCCATGAGGTCCACCAC-3′ (994–1017) (GenBank™ M17701). Each PCR product was separated by 1% agarose gel, purified by GenePure, and subcloned into a 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 (ABI 377A), and used as a probe for Northern blot analysis. Total RNA was isolated from rat osteoblasts using an Isogen RNA extraction kit according to the manufacturer's protocols. RNA was quantitated using a spectrophotometer, and equal amounts of RNA (20 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 for MMP-13, MMP-2, MMP-9, TIMP-1, col(I)α (1Mauviel A. J. Cell. Biochem. 1993; 53: 288-295Crossref PubMed Scopus (397) Google Scholar), c-jun, c-fos, and GAPDH were labeled with [α-32P]deoxycytidine triphosphate using a DNA random primer labeling kit. Hybridization was carried out at 42 °C for 24–48 h. Post-hybridization washing was performed in 0.1% SDS, 0.2× standard saline citrate (SSC) (1× SSC: 0.15 m NaCl, 0.015m sodium citrate) at 65 °C for 15 min. The radioactive bands were visualized by autoradiography on Kodak X-AR5 film and quantitatively analyzed using the NIH Image system. The results were normalized by GAPDH mRNA levels. Cells (1 × 106 cells) were disrupted with a Polytron homogenizer (Kinematica, Lucerne, Switzerland). The protein concentrations of the cell homogenates were quantitated using a Micro BCA protein assay reagent kit. Equal amounts of homogenates were dissolved in 20 μl of Tris-HCl, 50 mm(pH 6.8), containing 2-mercaptoethanol (1%), sodium dodecyl sulfate (SDS) (2%), glycerol (20%), and bromphenol blue (0.04%), and heated at 100 °C for 5 min. The samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described (22Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10480) Google Scholar) and transferred electrophoretically onto a nitrocellulose membrane. The membranes were blocked with 1% nonfat dry milk in PBS, probed with 1 μg/ml anti-phosphotyrosine mAb or anti-Src mAb, and reacted with the goat anti-mouse IgG Ab coupled with horseradish peroxidase. The resultant complexes were processed for detection by enhanced chemiluminescence using ECL Western blotting detection system according to the manufacturer's protocol. Moreover, we performed immunoblot analysis not only for the MMP-13 protein level in response to MIF, but also for three distinct MAPKs to further elucidate their involvement in the signal transduction of MIF for expression of MMP-13. Immunoblot analysis was carried out as described above except for the use of antibodies against rat MMP-13 and MAPKs, including ERK1/2, JNK, and p38. The immune complex kinase assay was performed as described previously (23Iwabuchi K. Hatakeyama S. Takahashi A. Ato M. Okada M. Kajino Y. Kajino K. Ogasawara K. Takami K. Nakagawa H. Onoé K. Eur. J. Immunol. 1997; 27: 742-749Crossref PubMed Scopus (24) Google Scholar). In brief, cells were lysed, precleared by incubating with nonimmune mouse IgG plus Pansorbin cells and Protein A-Sepharose 4B overnight. The supernatant was incubated with the anti-mouse Src mAb (10 μg) and then with 20 μl of Protein A-Sepharose 4B for 2 h. These procedures were performed at 4 °C. The beads were incubated in a kinase buffer containing 370 kBq of [γ-32P]ATP at 30 °C for 10 min. Thereafter, the beads were washed, boiled for 5 min, and subjected to SDS-PAGE. The gel was dried, exposed to an imaging plate for 30 min, and analyzed with a BAS2000 imaging system (Fuji Film, Tokyo, Japan). Primary osteoblasts were transfected with a recombinant adenovirus carrying a kinase-defective rat C-terminal Src family kinase (Csk). The recombinant virus contains the cytomegalovirus immediate early enhancer, chicken β-actin promotor, and rabbit β-globin poly(A) signal and AxCATcsk (K222R), in which the lysine 222 is replaced by an arginine residue, as described previously (24Miyazaki T. Takayanagi H. Isshiki M. Takahashi T. Okada M. Fukui Y. Oda H. Nakamura K. Hirai H. Kurokawa T. Tanaka S. J. Bone Miner. Res. 2000; 15: 41-51Crossref PubMed Scopus (34) Google Scholar). The control virus Ax1w1 contains no foreign genes. The third-passage rat primary osteoblasts were infected with the recombinant adenovirus as follows. The cells were cultured with small amounts of DMEM containing recombinant adenovirus for 1 h at 37 °C at an indicated multiplicity of infection. Then, more than 10 volumes of medium with 10% FCS was added to the cells. After 48 h of the infection, the cells were washed, serum-starved for 24 h, and treated with MIF (10 μg/ml) for 6 h. Then they were collected and subjected to Northern blot analysis. Rat osteoblasts were labeled as described previously (25Kasuga M. White M.F. Kahn C.R. Methods Enzymol. 1985; 109: 609-621Crossref PubMed Scopus (55) Google Scholar). Briefly, 2.0 × 106 cells were washed twice with 50 mm HEPES containing 0.1% bovine serum albumin. The cells were propagated for 15 min in phosphate-free medium (Irvine Scientific, Santa Ana, CA), labeled with 0.5 mCi of [32P]orthophosphate for 3 h at 37 °C, and then treated with MIF (10 μg/ml) at the indicated intervals. After labeling, the cells were washed twice with cold PBS containing 20 mm sodium pyrophosphate, 100 mm sodium fluoride, 4 mm EDTA, and then solubilized for 10 min on ice in RIPA lysis buffer containing 20 mm sodium pyrophosphate, 100 mm sodium fluoride, 4 mm EDTA, and 2 mm phenylmethylsulfonyl fluoride. The cell lysates were cleared by ultracentrifugation at 100,000 × g for 45 min. Then the supernatants were subjected to immunoprecipitation with an anti-c-Jun antibody and Protein G-Sepharose, and to SDS-PAGE. The gel was dried, exposed to an imaging plate for 30 min, and analyzed with a BAS2000 imaging system. The activation of Ras was evaluated using a Ras activation assay kit according to the manufacturer's protocol. Briefly, rat primary osteoblasts (2.0 × 106cells) were serum-starved for 24 h, treated with MIF (10 μg/ml) at the indicated intervals, washed twice with cold PBS, and lysed with 1 ml of Mg2+ Lysis/Wash Buffer (MLB). Then the lysate was precleared by glutathione-agarose, 10 μg of Raf-1 Ras-binding domain (RBD)-conjugated agarose was added, and it was incubated at 4 °C for 30 min. Raf-1 RBD-conjugated agarose is a glutathioneS-transferase fusion protein, corresponding to the human Ras binding domain (residues 1–149) of Raf-1, provided bound to glutathione-agarose; it specifically binds to and precipitates Ras-GTP from cell lysates. After washing the beads three times with MLB, they were suspended in 2× Laemmli sample buffer, subjected to SDS-PAGE and immunoblot analysis using 1 μg/ml anti-Ras mAb as a primary antibody, and visualized using the ECL Western blotting detection system. In rat calvaria-derived osteoblasts, up-regulation of MMP-13 mRNA at 6 h after the challenge with MIF was slight at the dose of 1 μg/ml, but it became apparent at the dose of 10 μg/ml (12.8-fold increase compared with the level of nonstimulated control) (Fig.1 a). The induction of MMP-13 by MIF was much higher than that by PTH (10−9 to 10−8m) (2.1-fold increase at the dose of 10−8m), a representative physiological inducer of MMP-13. TPA (10−6m), a positive control, most strongly enhanced MMP-13 mRNA (25.2-fold increase at the dose of 10−6m). The TIMP-1 mRNA level was also up-regulated, but to a much lesser extent, at doses ranging from 0.1 μg/ml to 10 μg/ml. The type I procollagen (col(I)α (1Mauviel A. J. Cell. Biochem. 1993; 53: 288-295Crossref PubMed Scopus (397) Google Scholar)) mRNA level was higher at the basal level and was essentially unchanged with addition of MIF. Moreover, we assessed the responsiveness of MMP-2 and MMP-9 mRNA to MIF. MMP-9 mRNA was significantly up-regulated, similar to MMP-13 but to a lesser extent (4.5-fold increase compared with the level of the nonstimulated control at 10 μg/ml MIF), whereas MMP-2 mRNA was slightly increased. To compare expression levels of MMP-13, TIMP-1, and col(I)α (1Mauviel A. J. Cell. Biochem. 1993; 53: 288-295Crossref PubMed Scopus (397) Google Scholar) among different cell types in response to MIF, rat primary osteoblasts, rat primary synovial fibroblasts, murine MC3T3-E1 cells, and rat UMR-106 cells were examined. MMP-13 mRNA was up-regulated by MIF (10 μg/ml) in rat primary osteoblasts and in rat synovial fibroblasts (Fig. 1 b). It was minimally changed in MC3T3-E1 cells and UMR-106 cells, in which the basal expression level of MMP-13 in UMR-106 was very high. On the other hand, TIMP-1 mRNA was slightly up-regulated by MIF in rat primary osteoblasts, but not changed in synovial fibroblasts, MC3T3-E1 cells, or UMR-106 cells. The mRNA level of col(I)α (1Mauviel A. J. Cell. Biochem. 1993; 53: 288-295Crossref PubMed Scopus (397) Google Scholar) was essentially not affected by MIF stimuli in all these cells. Furthermore, we examined mRNA expression levels in rat articular chondrocytes and rat skin fibroblasts. MMP-13 mRNA was up-regulated in the articular chondrocytes in response to MIF, but not in skin fibroblasts (Fig. 1 c). A slight increase of TIMP-1 mRNA in articular chondrocytes was also observed. To assess the structure-specific action of MIF protein, we treated MIF at 65 °C for 1 h. The potential of MIF for induction of MMP-13 mRNA was significantly reduced by the heat treatment (data not shown). We also assessed the neutralizing effect of an anti-MIF monoclonal antibody on MMP-13 mRNA expression. Addition of 50 μg/ml antibody 30 min prior to addition of MIF significantly suppressed the induction of MMP-13 and TIMP-1 mRNAs, though the neutralizing effect was incomplete (Fig. 1 d). Nonimmune mouse IgG (IgG1) minimally affected the expression of MMP-13 mRNA. We performed immunoblot analysis of MMP-13 at the protein level to confirm the results regarding the mRNA level. We found that the MMP-13 protein level was increased in response to MIF (10 μg/ml) by 24 h treatment as seen for the PTH (10−8m) and TPA (10−6m) treatment used as positive controls (Fig. 2). MMP-13 mRNA expression of rat primary osteoblasts increased at 3 h after stimulation in response to MIF (10 μg/ml), reached the maximal level at 6–12 h (13.4-fold increase compared with the level of time 0), and returned to the unstimulated basal level at 36 h (Fig.3). The mRNA level of TIMP-1 was slightly up-regulated in response to MIF at 3 h after stimulation and then decreased. The mRNA level of col(I)α (1Mauviel A. J. Cell. Biochem. 1993; 53: 288-295Crossref PubMed Scopus (397) Google Scholar) was essentially unchanged throughout the time course. Prior to the up-regulation of the MMP-13 mRNA level in response to MIF, transient increases of c-jun and c-fos mRNA levels were observed. The c-jun mRNA was transiently up-regulated at 1 h after stimulation, and then decreased to the basal level at 3 h. The c-fos mRNA was markedly up-regulated at 30 min after stimulation, and immediately returned to the basal level at 1 h. No significant changes of MMP-13 and TIMP-1 mRNA levels were observed in the absence of MIF (data not shown). To examine whether the effect of MIF on rat MMP-13 mRNA levels de" @default.
• W2068058635 created "2016-06-24" @default.
• W2068058635 creator A5008370626 @default.
• W2068058635 creator A5018373846 @default.
• W2068058635 creator A5021932087 @default.
• W2068058635 creator A5036304615 @default.
• W2068058635 creator A5055584432 @default.
• W2068058635 creator A5064546023 @default.
• W2068058635 creator A5069803785 @default.
• W2068058635 date "2002-03-01" @default.
• W2068058635 modified "2023-10-14" @default.
• W2068058635 title "Macrophage Migration Inhibitory Factor Up-regulates Matrix Metalloproteinase-9 and -13 in Rat Osteoblasts" @default.
• W2068058635 cites W1481444615 @default.
• W2068058635 cites W1491979926 @default.
• W2068058635 cites W1532387473 @default.
• W2068058635 cites W1583835478 @default.
• W2068058635 cites W1668309971 @default.
• W2068058635 cites W1965503681 @default.
• W2068058635 cites W1969191130 @default.
• W2068058635 cites W1973645860 @default.
• W2068058635 cites W1975055683 @default.
• W2068058635 cites W1975085160 @default.
• W2068058635 cites W1976197008 @default.
• W2068058635 cites W1985063935 @default.
• W2068058635 cites W1986298697 @default.
• W2068058635 cites W1986837253 @default.
• W2068058635 cites W1986862345 @default.
• W2068058635 cites W1994174424 @default.
• W2068058635 cites W1999157780 @default.
• W2068058635 cites W2001634895 @default.
• W2068058635 cites W2003152672 @default.
• W2068058635 cites W2005402970 @default.
• W2068058635 cites W2010189280 @default.
• W2068058635 cites W2011553184 @default.
• W2068058635 cites W2020134388 @default.
• W2068058635 cites W2024525376 @default.
• W2068058635 cites W2027059345 @default.
• W2068058635 cites W2028773046 @default.
• W2068058635 cites W2036834425 @default.
• W2068058635 cites W2037447928 @default.
• W2068058635 cites W2037761234 @default.
• W2068058635 cites W2040780377 @default.
• W2068058635 cites W2048983596 @default.
• W2068058635 cites W2049310294 @default.
• W2068058635 cites W2055235179 @default.
• W2068058635 cites W2056832798 @default.
• W2068058635 cites W2062824264 @default.
• W2068058635 cites W2063301799 @default.
• W2068058635 cites W2067458000 @default.
• W2068058635 cites W2068378027 @default.
• W2068058635 cites W2068886704 @default.
• W2068058635 cites W2071226610 @default.
• W2068058635 cites W2073535369 @default.
• W2068058635 cites W2074701159 @default.
• W2068058635 cites W2076413757 @default.
• W2068058635 cites W2080035405 @default.
• W2068058635 cites W2083328029 @default.
• W2068058635 cites W2085524313 @default.
• W2068058635 cites W2090895449 @default.
• W2068058635 cites W2113190937 @default.
• W2068058635 cites W2145828270 @default.
• W2068058635 cites W2146058212 @default.
• W2068058635 cites W2146107565 @default.
• W2068058635 cites W2156537658 @default.
• W2068058635 cites W2171943084 @default.
• W2068058635 cites W2184993925 @default.
• W2068058635 cites W2192686175 @default.
• W2068058635 cites W987547262 @default.
• W2068058635 doi "https://doi.org/10.1074/jbc.m106020200" @default.
• W2068058635 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11751895" @default.
• W2068058635 hasPublicationYear "2002" @default.
• W2068058635 type Work @default.
• W2068058635 sameAs 2068058635 @default.
• W2068058635 citedByCount "152" @default.
• W2068058635 countsByYear W20680586352012 @default.
• W2068058635 countsByYear W20680586352013 @default.
• W2068058635 countsByYear W20680586352014 @default.
• W2068058635 countsByYear W20680586352015 @default.
• W2068058635 countsByYear W20680586352016 @default.
• W2068058635 countsByYear W20680586352017 @default.
• W2068058635 countsByYear W20680586352018 @default.
• W2068058635 countsByYear W20680586352019 @default.
• W2068058635 countsByYear W20680586352020 @default.
• W2068058635 countsByYear W20680586352021 @default.
• W2068058635 countsByYear W20680586352022 @default.
• W2068058635 countsByYear W20680586352023 @default.
• W2068058635 crossrefType "journal-article" @default.
• W2068058635 hasAuthorship W2068058635A5008370626 @default.
• W2068058635 hasAuthorship W2068058635A5018373846 @default.
• W2068058635 hasAuthorship W2068058635A5021932087 @default.
• W2068058635 hasAuthorship W2068058635A5036304615 @default.
• W2068058635 hasAuthorship W2068058635A5055584432 @default.
• W2068058635 hasAuthorship W2068058635A5064546023 @default.
• W2068058635 hasAuthorship W2068058635A5069803785 @default.
• W2068058635 hasBestOaLocation W20680586351 @default.
• W2068058635 hasConcept C104177226 @default.
• W2068058635 hasConcept C106487976 @default.
• W2068058635 hasConcept C109523444 @default.

• next