Tumor Necrosis Factor Promotes Runx2 Degradation through Up-regulation of Smurf1 and Smurf2 in Osteoblasts
2005; Elsevier BV; Volume: 281; Issue: 7 Linguagem: Inglês
10.1074/jbc.m509430200
ISSN1083-351X
AutoresHiroyuki Kaneki, Ruolin Guo, Di Chen, Zhenqiang Yao, Edward M. Schwarz, Ying E. Zhang, Brendan F. Boyce, Lianping Xing,
Tópico(s)Bone and Joint Diseases
ResumoTumor necrosis factor (TNF) plays an important role in the pathogenesis of inflammatory bone loss through stimulation of osteoclastic bone resorption and inhibition of osteoblastic bone formation. Compared with the well established role of TNF in osteoclastogenesis, mechanisms by which TNF inhibits osteoblast function have not been fully determined. Runx2 is an osteoblast-specific transcription factor whose steady-state protein levels are regulated by proteasomal degradation, mediated by the E3 ubiquitin ligases, Smurf1 and Smurf2. We hypothesized that TNF inhibits osteoblast function through Smurf-mediated Runx2 degradation. We treated C2C12 and 2T3 osteoblast precursor cell lines and primary osteoblasts with TNF and found that TNF, but not interleukin-1, significantly increased Smurf1 and Smurf2 expression. TNF increased the degradation of endogenous or transfected Runx2 protein, which was blocked by treating cells with a proteasomal inhibitor or by infecting cells with small interfering (si)RNA against Smurf1 or Smurf2. TNF inhibited the expression of bone morphogenetic protein and transforming growth factor-β signaling reporter constructs, and the inhibition of each was blocked by Smurf1 siRNA and Smurf2 siRNA, respectively. Overexpression of Smurf1 and/or Smurf2 siRNAs prevented the inhibitory effect of TNF on Runx2 reporter. Consistent with these in vitro findings, bones from TNF transgenic mice or TNF-injected wild type mice had increased Smurf1 and decreased Runx2 protein levels. We propose that one of the mechanisms by which TNF inhibits bone formation in inflammatory bone disorders is by promoting Runx2 proteasomal degradation through up-regulation of Smurf1 and Smurf2 expression. Tumor necrosis factor (TNF) plays an important role in the pathogenesis of inflammatory bone loss through stimulation of osteoclastic bone resorption and inhibition of osteoblastic bone formation. Compared with the well established role of TNF in osteoclastogenesis, mechanisms by which TNF inhibits osteoblast function have not been fully determined. Runx2 is an osteoblast-specific transcription factor whose steady-state protein levels are regulated by proteasomal degradation, mediated by the E3 ubiquitin ligases, Smurf1 and Smurf2. We hypothesized that TNF inhibits osteoblast function through Smurf-mediated Runx2 degradation. We treated C2C12 and 2T3 osteoblast precursor cell lines and primary osteoblasts with TNF and found that TNF, but not interleukin-1, significantly increased Smurf1 and Smurf2 expression. TNF increased the degradation of endogenous or transfected Runx2 protein, which was blocked by treating cells with a proteasomal inhibitor or by infecting cells with small interfering (si)RNA against Smurf1 or Smurf2. TNF inhibited the expression of bone morphogenetic protein and transforming growth factor-β signaling reporter constructs, and the inhibition of each was blocked by Smurf1 siRNA and Smurf2 siRNA, respectively. Overexpression of Smurf1 and/or Smurf2 siRNAs prevented the inhibitory effect of TNF on Runx2 reporter. Consistent with these in vitro findings, bones from TNF transgenic mice or TNF-injected wild type mice had increased Smurf1 and decreased Runx2 protein levels. We propose that one of the mechanisms by which TNF inhibits bone formation in inflammatory bone disorders is by promoting Runx2 proteasomal degradation through up-regulation of Smurf1 and Smurf2 expression. Tumor necrosis factor (TNF) 2The abbreviations used are: TNF, tumor necrosis factor; ALP, alkaline phosphatase; BMP, bone morphogenetic protein; CMV, cytomegalovirus; E3, ubiquitin-protein isopeptide ligase; IL-1, interleukin-1; Luc, luciferase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OC, osteocalcin; PBS, phosphate-buffered saline; RANKL, receptor activator NF-κB ligand; RT, reverse transcription; siRNA, small interfering RNA; Smurf, Smad ubiquitin regulatory factor; Tg, transgenic; TGF-β, transforming growth factor-β; TRAF, TNF receptor-associated factor; wt, wild type. is a major contributor to pathologic bone loss through stimulation of osteoclastic bone resorption and inhibition of osteoblastic bone formation. In patients with rheumatoid arthritis, TNF and other cytokines are overproduced in inflamed joints by various cells infiltrating the synovial membrane. This leads to severe local erosion of cartilage and bone, periarticular osteopenia, as well as systemic osteoporosis (1.Goldring S.R. Gravallese E.M. Curr. Opin. Rheumatol. 2000; 12: 195-199Crossref PubMed Scopus (172) Google Scholar, 2.Srivastava S. Weitzmann M.N. Cenci S. Ross F.P. Adler S. Pacifici R. J. Clin. Invest. 1999; 104: 503-513Crossref PubMed Scopus (224) Google Scholar). Under these conditions, osteoblasts do not catch up with the accelerated bone resorption, indicating impaired osteoblast function (3.Nair S.P. Williams R.J. Henderson B. Rheumatology. 2000; 39: 821-834Crossref PubMed Scopus (46) Google Scholar). The inhibitory effects of TNF on bone formation in vitro were first described in 1987 in neonatal rat calvarial organ cultures (4.Canalis E. Endocrinology. 1987; 121: 1596-1604Crossref PubMed Scopus (106) Google Scholar). Subsequent studies demonstrated that TNF inhibits recruitment of osteoblast progenitors, reduces expression of genes produced by mature osteoblasts, and promotes osteoblast apoptosis through nuclear factor-κB signaling pathway (5.Li Y.P. Stashenko P. J. Immunol. 1992; 148: 788-794PubMed Google Scholar, 6.Taichman R.S. Hauschka P.V. Inflammation. 1992; 16: 587-601Crossref PubMed Scopus (90) Google Scholar, 7.Kitajima I. Soejima Y. Takasaki I. Beppu H. Tokioka T. Maruyama I. Bone. 1996; 19: 263-270Crossref PubMed Scopus (116) Google Scholar, 8.Jilka R.L. Weinstein R.S. Bellido T. Parfitt A.M. Manolagas S.C. J. Bone Miner. Res. 1998; 13: 793-802Crossref PubMed Scopus (471) Google Scholar, 9.Gilbert L. He X. Farmer P. Boden S. Kozlowski M. Rubin J. Nanes M.S. Endocrinology. 2000; 141: 3956-3964Crossref PubMed Scopus (0) Google Scholar). However, compared with our understanding of the role of TNF in osteoclast biology, little is known of the molecular mechanisms that mediate the effect of TNF on osteoblast inhibition. To date, the most important mechanistic finding of TNF-mediated osteoblast inhibition was the demonstration that TNF decreases Runt-related gene 2 (Runx2) expression and its DNA binding activity in osteoblasts (10.Gilbert L. He X. Farmer P. Rubin J. Drissi H. van Wijnen A.J. Lian J.B. Stein G.S. Nanes M.S. J. Biol. Chem. 2002; 277: 2695-2701Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). This is partially through suppression of Runx2 gene transcription and destabilization of Runx2 mRNA through the TNF receptor 1 signaling pathway (10.Gilbert L. He X. Farmer P. Rubin J. Drissi H. van Wijnen A.J. Lian J.B. Stein G.S. Nanes M.S. J. Biol. Chem. 2002; 277: 2695-2701Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 11.Abbas S. Zhang Y.H. Clohisy J.C. Abu-Amer Y. 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In the past several years, ubiquitin-mediated proteasomal degradation has been implicated in the regulation of bone morphogenetic protein (BMP)-2 and transforming growth factor-β (TGF-β) signaling pathways in various cell types (13.Izzi L. Attisano L. Oncogene. 2004; 23: 2071-2078Crossref PubMed Scopus (216) Google Scholar, 14.Datto M. Wang X.F. Cell. 2005; 121: 2-4Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). We and others have demonstrated that the E3 ubiquitin ligase, Smad ubiquitin regulatory factor (Smurf)1, regulates osteoblast differentiation by promoting proteasomal degradation of the BMP signaling protein, Smad1 and Smad5, and of the osteoblast transcription factor, Runx2 (15.Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. Nature. 1999; 400: 687-693Crossref PubMed Scopus (688) Google Scholar, 16.Ying S.X. Hussain Z.J. Zhang Y.E. J. Biol. Chem. 2003; 278: 39029-39036Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 17.Zhao M. Qiao M. Oyajobi B.O. Mundy G.R. Chen D. J. Biol. Chem. 2003; 278: 27939-27944Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 18.Zhao M. Qiao M. Harris S.E. Oyajobi B.O. Mundy G.R. Chen D. J. Biol. Chem. 2004; 279: 12854-12859Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Smurf2, a closely related homolog of Smurf1, was shown to reduce the steady-state protein levels of Smad1 and 2, but not Smad3 and 4, in Smurf2-transfected cells (19.Lin X. Liang M. Feng X.H. J. Biol. Chem. 2000; 275: 36818-36822Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 20.Zhang Y. Chang C. Gehling D.J. Hemmati-Brivanlou A. Derynck R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 974-979Crossref PubMed Scopus (427) Google Scholar). Ectopic expression of Smurf1 in 2T3 osteoblast precursors and C2C12 myoblast/osteoblast precursors induces the proteasomal degradation of Smad1 and Runx2 proteins, leading to inhibition of osteoblast differentiation (17.Zhao M. Qiao M. Oyajobi B.O. Mundy G.R. Chen D. J. Biol. Chem. 2003; 278: 27939-27944Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Smurf1 blocks BMP-induced osteogenic conversion of C2C12 cells and facilitates their myogenic differentiation by inducing degradation of Smad5 (16.Ying S.X. Hussain Z.J. Zhang Y.E. J. Biol. Chem. 2003; 278: 39029-39036Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In vitro, Smurf1 also targets a member of the Rho family of small GTPases, RhoA, for ubiquitination and degradation (21.Wang H.R. Zhang Y. Ozdamar B. Ogunjimi A.A. Alexandrova E. Thomsen G.H. Wrana J.L. Science. 2003; 302: 1775-1779Crossref PubMed Scopus (445) Google Scholar, 22.Zhang Y. Wang H.R. Wrana J.L. Cell Cycle. 2004; 3: 391-392Crossref PubMed Google Scholar). In vivo, overexpression of Smurf1 in osteoblasts by the osteoblast-specific type I collagen (Col1a1) promoter leads to reduction in osteoblast proliferation and activity. Col1a1-Smurf1 transgenic mice have decreased bone formation rates and decreased Runx2 protein expression in osteoblasts (18.Zhao M. Qiao M. Harris S.E. Oyajobi B.O. Mundy G.R. Chen D. J. Biol. Chem. 2004; 279: 12854-12859Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Smurf1 knock-out (Smurf1-/-) mice were generated recently. Although they survive to adulthood, they exhibit an age-dependent increase in bone mass (23.Yamashita M. Ying S.X. Zhang G.M. Li C. Cheng S.Y. Deng C.X. Zhang Y.E. Cell. 2005; 121: 101-113Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Interestingly, osteoblasts from Smurf1-/- mice have normal levels of the BMP receptors, Smad1, 2, 3, and 5, and Runx2, all of which have been defined previously as targets of Smurf1. Because Smurf1 and Smurf2 possess overlapping functions, it is possible that Smurf2 compensates for the loss of Smurf1 in these knock-out animals (19.Lin X. Liang M. Feng X.H. J. Biol. Chem. 2000; 275: 36818-36822Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 20.Zhang Y. Chang C. Gehling D.J. Hemmati-Brivanlou A. Derynck R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 974-979Crossref PubMed Scopus (427) Google Scholar, 24.Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1103) Google Scholar). Currently, the role of Smurf2 in osteoblast function in vivo and the role of Smurf E3 ligases in the pathogenesis of bone diseases remain poorly understood. Additionally, there is little information on the regulation of Smurfs expression under physiological and pathological conditions. To explore the molecular mechanisms of TNF-mediated osteoblast inhibition, we tested the hypothesis that TNF inhibits osteoblastic bone formation by up-regulating Smurf E3 ligases that degrade Runx2 protein. We found that TNF increased Smurf1 and Smurf2 expression in osteoblasts, leading to enhanced ubiquitination and degradation of Runx2 protein. This TNF-induced Runx2 degradation was reversed by proteasome inhibitors and by knocking down endogenous Smurf1 or Smurf2 using small interfering RNA (siRNA) against Smurf1 or Smurf2. Bones from TNF-overexpressing mice exhibited increased Smurf1 and decreased Runx2 protein levels. Taken together, our findings point to a novel molecular mechanism of TNF inhibition of osteoblasts, which involves post-transcriptional regulation of protein function through Smurf E3 ligase-mediated proteasomal degradation. Animals—TNF transgenic (Tg) mice in a CBA × C57BL/6 background (3647 TNF-Tg line) were obtained from Dr. G. Kollias. C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). The Institutional Animal Care and Use Committee approved all animal studies. Antibodies—Monoclonal antibodies specific for FLAG and β-actin were purchased from Sigma. Anti-Runx2 monoclonal antibody was from MBL (Woburn, MA). Anti-ubiquitin monoclonal antibody was from Santa Cruz (Santa Cruz, CA). Anti-Smurf1 polyclonal antibody was from Abgent (San Diego, CA). Cell Culture and Transfection Conditions—C2C12 myoblast/osteoblast precursors were cultured in Dulbecco's modified Eagle's medium, and 2T3 osteoblast precursors were cultured in α-minimal essential medium supplemented with 1% penicillin-streptomycin (all from Invitrogen) and 10% fetal calf serum (JRH Biosciences, Lenexa, KS). When cells were grown to 90% confluence, the cDNA expression plasmid, pCMV-FLAG-tagged Runx2 (F-Runx2) and/or a pCMV-Myc-tagged Smurf1 (M-Smurf1) were transiently transfected into the cells using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. Total amounts of transfected plasmids in each group were equalized by the addition of empty vector. After transfection (24 h), the cells were cultured further in the presence and absence of murine TNF (R&D Systems) and subjected to reverse transcription (RT)-PCR or Western blot analysis. Bone Nodule Formation—Bone marrow cells were flushed from the tibiae and femur of wild type (wt) and TNF-Tg mice, and the cells were seeded at a density of 2 × 106/ml. The cells were cultured in 37 °C with a humidified 5% CO2 atmosphere. When the cells reached confluence (day 0), the medium was changed to an osteoblast-inducing medium (α-minimal essential medium-supplemented 10% fetal calf serum with 100 μg/ml l-ascorbic acid and 5 mm β-glycerophosphate) with or without 40 ng/ml BMP-2, and the medium was changed twice a week. After an 18-day incubation, the cells were fixed with 10% formalin and stained by the von-Kossa method. The area of mineralized bone nodules was determined under light microscopy by point counting, as described previously (25.Boyce B.F. Aufdemorte T.B. Garrett I.R. Yates A.J. Mundy G.R. Endocrinology. 1989; 125: 1142-1150Crossref PubMed Scopus (318) Google Scholar). Quantitative Real Time RT-PCR—Cells were homogenized using 1 ml of TRIzol reagent (Invitrogen), and total RNA was extracted according to the manufacturer's protocol. cDNA was synthesized using 20 μl of reverse transcription reaction solution containing 1 μg of total RNA, 10 mm Tris-HCl buffer (pH 8.3), 50 mm KCl, 5 mm MgCl2, 1 mm deoxynucleoside triphosphates, 2.5 μm random hexamers, 20 units RNase inhibitor, and 50 units of Moloney murine leukemia virus reverse transcriptase (all from Roche Applied Science). Quantitative real time PCR amplifications were performed in an iCycler real time PCR machine using iQ SYBR Green supermix (both from Bio-Rad Laboratories) according to the manufacturer's instruction. The sequences of primer sets for Smurf1, Smurf2, alkaline phosphatase (ALP), osteocalcin (OC), and β-actin mRNAs, target sites on mRNAs and product sizes by PCR are shown in Table 1. To minimize the background of products amplified from genomic DNAs, these primers were designed to exist on two different exons. The quantity of Smurf1, Smurf2, ALP, and OC mRNA in each sample was normalized using the CT (threshold cycle) value obtained for the β-actin mRNA amplifications.TABLE 1Sequences of primers used in the real time PCRGenesGenBank™ accession numberSequences of primersaF, forward primer; R, reverse primerTarget sites on genesProduct sizesbpSmurf1NM029438F: 5′-AGTTCGTGGCCAAATAGTGG-3′687-78599R: 5′-GTTCCTTCGTTCTCCAGCAG-3′Smurf2NM 025481F: 5′-GTGAAGAGCTCGGTCCTTTG-3′1041-1154114R: 5′-AGAGCCGGGGATCTGTAAAT-3′ALPAF285233F: 5′-CGGGACTGGTACTCGGATAA-3′550-706157R: 5′-ATTCCACGTCGGTTCTGTTC-3′OCAH004426F: 5′-CTTGGTGCACACCTAGCAGA-3′638-824186R: 5′-CTCCCTCATGTGTTGTCCCT-3′β-actinNM 001101F: 5′-AGATGTGGATCAGCAAGCAG-3′1134-1251118R: 5′-GCGCAAGTTAGGTTTTGTCA-3′a F, forward primer; R, reverse primer Open table in a new tab Western Blot Analysis—Cells were washed with cold phosphate-buffered saline (PBS), and whole cell lysates were prepared by the addition of M-PER mammalian protein extraction reagent (Pierce) containing a protease inhibitor mixture (Roche Applied Science). Twenty μg of protein was loaded per lane and separated on a 10% polyacrylamide gel, followed by transfer to a nitrocellulose membrane (Bio-Rad) by electroblotting. Membrane was blocked for nonspecific binding in 3% nonfat dry milk and followed by incubation with an antibody at 4 °C. After membrane was washed, the blots were probed with a horseradish peroxidase-conjugated secondary antibody (Bio-Rad) and visualized by an enhanced chemiluminescence system (Amersham Biosciences) according to the manufacturer's instructions. Ubiquitination of Runx2—2T3 cells were incubated in medium containing 7.5 ng/ml TNF for 72 h in the presence of PBS or 0.1 mm MG132 (Calbiochem) for the last 12 h of TNF treatment. For the immunoprecipitation, cell lysate was incubated with anti-Runx2 antibody overnight at 4 °C followed by the addition of protein G-agarose (Roche Applied Science) overnight at 4 °C. The immunoprecipitates were washed with 50 mm Tris-HCl buffer (pH 8.0), containing 150 mm NaCl, 1% Nonidet P-40, 0.05% deoxycholate, and 0.1% SDS, resuspended in 1 × reducing sample buffer, and subjected to Western blot analysis with an anti-ubiquitin antibody. The same membrane was stripped and reprobed for Runx2. siRNA and Virus Infection—Platinum-E cells were transfected with a retrovirus vector (pRetro-H1G) that encodes Smurf1 or Smurf2 siRNA, or an empty vector using FuGENE 6 reagent (Roche Applied Science). The sequences of Smurf1 and Smurf2 siRNAs are shown in Table 2. After 2 days, viral supernatants were harvested and filtered using a 0.45-μm membrane filter. 2T3 cells were infected with virus supernatant in the presence of Polybrene. After 4 h, 2 ml of α-minimal essential medium was added to the cells to dilute Polybrene. The cells were cultured for an additional 48 h in α-minimal essential medium containing 10% fetal bovine serum. The cells were then used for RNA extraction or Western blot analysis.TABLE 2Sequences of siRNA used in the infectionTarget genesaParentheses indicate GenBank™ accession numbersSequences of siRNAbsiRNAs are designated to encode two complementary sequences of 19 nucleotides homologous to a segment of Smurf1 or Smurf2 mRNA (underlined) separated by a nine-nucleotide space (small characters), and have a terminator signal (TTTTTTC) at a 3′ terminusTarget sites on mRNASmurfl (NM 029438)5′–GATTCGAACCTTGCAAAGAAAGAC ttcaagagaGTCTTTCTTTGCAAGGTTCTTTTTTC–3′372–390Smurf2 (NM 025481)5′–GATTCGACCAACAGCAACAGCAAG ttcaagagaCTTGCTGTTGCTGTTGGTCTTTTTTC–3′1199–1217a Parentheses indicate GenBank™ accession numbersb siRNAs are designated to encode two complementary sequences of 19 nucleotides homologous to a segment of Smurf1 or Smurf2 mRNA (underlined) separated by a nine-nucleotide space (small characters), and have a terminator signal (TTTTTTC) at a 3′ terminus Open table in a new tab Luciferase Assay—2T3 cells were transfected with the BMP, 12×SBE-OC-Luc (17.Zhao M. Qiao M. Oyajobi B.O. Mundy G.R. Chen D. J. Biol. Chem. 2003; 278: 27939-27944Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) or the TGF-β, p3TP-Lux (18.Zhao M. Qiao M. Harris S.E. Oyajobi B.O. Mundy G.R. Chen D. J. Biol. Chem. 2004; 279: 12854-12859Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), signaling reporter construct using Lipofectamine 2000 transfection reagent. After 6 h, cells were treated with 10 ng/ml TNF for 48 h, followed by a 24-h incubation in the presence or absence of 50 ng/ml BMP-2 or 2 ng/ml TGF-β (both from R&D Systems). For determination of the effect of TNF on Runx2 expression, 2T3 cells were cotransfected with a Runx2 expression vector, F-Runx2, and the Runx2 reporter construct, 6×OSE2-OC-pGL3 (17.Zhao M. Qiao M. Oyajobi B.O. Mundy G.R. Chen D. J. Biol. Chem. 2003; 278: 27939-27944Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) followed by a 48-h TNF treatment. Cell lysates were extracted, and luciferase activity was measured using a Dual Luciferase Reporter Assay System (Promega) and normalized by Renilla luciferase activity. Caspase-3 Assay—2T3 cells were treated with TNF (2.5, 5, and 7.5 ng/ml) for 24, 48, and 72 h and then lysed in a buffer containing 1% Nonidet P-40, 200 mm NaCl, 20 mm Tris-HCl (pH 7.4), 10 μg/ml leupeptin, and aprotinin (0.27 mm trypsin inhibitor/ml). Caspase-3 activity was determined by incubation of cell lysate (containing 25 μg of total protein) with 50 μm fluorogenic substrate, N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Calbiochem) in 200 μl of 10 mm HEPES (pH 7.4), containing 220 mm mannitol, 68 mm sucrose, 2 mm NaCl, 2.5 mm KH2PO4, 0.5 mm EGTA, 2 mm MgCl2, 5 mm pyruvate, 0.1 mm phenylmethylsulfonyl fluoride, and 1 mm dithiothreitol. The release of fluorescent 7-amino-4-methylcoumarin was measured by spectrofluorometry (excitation/emission, 499/521 nm). MTT Cell Viability Assay—Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) dye reduction assay according to the method of Green et al. (26.Green L.M. Reade J.L. Ware C.F. J. Immunol. Methods. 1984; 70: 257-268Crossref PubMed Scopus (522) Google Scholar). Briefly, after cells in 96-well plates were treated with various concentrations of TNF for 24, 48, or 96 h, 10 μl of MTT was added for 3 h, and the absorbance was read at 540 nm. Cell viability was calculated as the ratio of optical densities in wells with and without TNF. Statistics Analysis—All data are represented as the mean ± S.E. Comparisons of results were performed by paired Student's t tests, accepting p < 0.05 as the criterion of significance. All experiments were repeated at least twice with similar results. TNF-Tg Mice Have Reduced Osteoblast Function—The TNF transgenic mouse is a well established animal model of rheumatoid arthritis, which exhibits polyarthritis because of chronic exposure to low levels of TNF (27.Keffer J. Probert L. Cazlaris H. Georgopoulos S. Kaslaris E. Kioussis D. Kollias G. EMBO J. 1991; 10: 4025-4031Crossref PubMed Scopus (1341) Google Scholar). At 3-4 months of age, TNF-Tg mice develop a moderate to severe form of rheumatoid arthritis-like joint inflammation and destruction. This is characterized by chronic inflammation, local bone and cartilage erosion, and increased circulating TNF levels (27.Keffer J. Probert L. Cazlaris H. Georgopoulos S. Kaslaris E. Kioussis D. Kollias G. EMBO J. 1991; 10: 4025-4031Crossref PubMed Scopus (1341) Google Scholar, 28.Li P. Schwarz E.M. O'Keefe R.J. Ma L. Looney R.J. Ritchlin C.T. Boyce B.F. Xing L. Arthritis Rheum. 2004; 50: 265-276Crossref PubMed Scopus (184) Google Scholar). Apart from these well described features, animals develop general osteoporosis as show in Fig. 1A. Trabecular bone is markedly reduced in the metaphysis of long bones of TNF-Tg mice, compared with wt littermates (Fig. 1B). To examine whether osteoblast function is altered in TNF-Tg mice, bone marrow stromal cells were isolated from 4-month-old TNF-Tg mice and wt littermates. Cells were cultured in osteoblast differentiation medium to form mineralized bone nodules. Compared with wt mice, cells from TNF-Tg mice formed significantly fewer and smaller nodules under basal conditions and in the presence of BMP-2 (Fig. 1, C and D), indicating reduced osteoblast function. TNF Increases Smurf1 Expression, Runx2 Degradation, and Ubiquitination of Runx2 Protein—Smurf1 is a negative regulator of the BMP signaling pathway and inhibits osteoblast function by promoting Runx2 degradation (17.Zhao M. Qiao M. Oyajobi B.O. Mundy G.R. Chen D. J. Biol. Chem. 2003; 278: 27939-27944Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). To determine whether TNF affects Smurf1 expression, C2C12 myoblast/osteoblast precursor cells, 2T3 osteoblast precursors, and primary wt calvarial preosteoblasts were treated with PBS or 2.5-7.5 ng/ml TNF. Smurf1 expression was measured by real time RT-PCR at 24, 48, and 72 h. The highest dose of TNF (7.5 ng/ml) significantly increased Smurf1 mRNA levels at 24 h in both cell lines, with the highest indication being observed at 72 h. At this time, the lowest dose of TNF (2.5 ng/ml) also had increased Smurf1 mRNA expression (Fig. 2A). TNF-induced Smurf1 expression was similar in 2T3 and C2C12 cells. No significant increase in Smurf1 level was observed when the cells were treated with 7.5 ng/ml TNF for 2, 4, 8, and 12 h (data not shown). Consistent with these mRNA results, TNF increased Smurf1 protein expression in a dose-dependent manner after 24 h (Fig. 2B). Similarly, TNF also increased Smurf1 expression in primary preosteoblasts. TNF increased Smurf1 mRNA levels in these cells by 4-5-fold over the PBS-treated cells at 48 h (the ratio of Smurf1/β-actin in TNF-treated group versus that from PBS group: 4.7 ± 0.14, p < 0.002). To determine the specificity of TNF for Smurf1, C2C12 and 2T3 cells were treated with IL-1 or receptor activator NF-κB ligand (RANKL) that activate intracellular signaling pathways similar to those activated by TNF. They did not alter Smurf1 mRNA abundance (Fig. 2C). These doses of IL-1 and RANKL (10 ng/ml) stimulate osteoclast formation from osteoclast precursors (data not shown). To examine whether TNF-induced apoptosis is associated with increased Smurf1 expression, 2T3 cells were treated with various doses of TNF for 24, 48, and 72 h. Apoptosis was determined by measuring caspase-3 activity and cell viability by MTT assay. Smurf1 mRNA expression was examined by real time PCR in the same samples. At the doses (2.5-7.5 ng/ml) that TNF increased Smurf1 expression (data not shown), osteoblasts were morphological normal with normal caspase-3 activity (Fig. 2D). In contrast, cells treated with 10 and 20 ng/ml TNF induced cell apoptosis, and dead cells were detached from the culture plates (data not shown). To determine whether TNF induces Runx2 degradation, C2C12 or 2T3 cells were cotransfected with FLAG-tagged-Runx2 (F-Runx2) and/or Myc-tagged-Smurf1 (M-Smurf1) expression vectors or an empty vector in the presence of TNF. F-Runx2 expression was detected by Western blot analysis using an anti-FLAG antibody. As a positive control, Smurf1 overexpression decreased F-Runx2 protein levels. Similar to Smurf1 overexpression, TNF significantly reduced F-Runx2 protein levels in a dose-dependent manner (Fig. 2E). Smurf1 induces Runx2 degradation by increasing its ubiquitination (17.Zhao M. Qiao M. Oyajobi B.O. Mundy G.R. Chen D. J. Biol. Chem. 2003; 278: 27939-27944Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). If TNF-induced Runx2 degradation is mediated by Smurf1, we should be able to detect increased ubiquitinated-Runx2 complexes in TNF-treated cells. To test this hypothesis, 2T3 cells were treated with TNF in the presence and absence of the proteasomal inhibitor MG132, and endogenous Runx2 protein was immunoprecipitated with an anti-Runx2 antibody and followed by Western blot analysis using an anti-ubiquitin antibody. MG132 treatment revealed small amounts of ubiquitinated Runx2 in vehicle-treated cells. These were greatly increased in the presence of TNF (Fig. 3), indicating that TNF induces ubiquitination of Runx2 protein, leading to its rapid breakdown through proteasomal degradation. TNF-induced Runx2 Degradation Is Dependent on Smurf1 and Smurf2—To determine whether TNF-induced Runx2 degradation is dependent on Smurf1, 2T3 cells were infected first with retroviral supernatant containing double-stranded siRNA specific for Smurf1 to knock down endogenous Smurf1. They were then transfected with F-Runx2 in the presence of TNF. Smurf1 siRNA decreased TNF-induced Smurf1 mRNA by 95% and reduced Smurf1 expression in PBS-treated cells to almost undetectable levels (Fig. 4A). Without changing the expression of Smurf2 mRNA (Fig. 4B), Smurf1 siRNA partially blocked TNF-mediated inhibition of ALP mRNA expression compared with the empty vector control (Fig. 4C) and reduced TNF-induced Runx2 degradation by 30% (by a densitometric analysis, Fig. 4D). Interestingly,
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