Bcl-2 Positively Regulates Sox9-dependent Chondrocyte Gene Expression by Suppressing the MEK-ERK1/2 Signaling Pathway
2005; Elsevier BV; Volume: 280; Issue: 34 Linguagem: Inglês
10.1074/jbc.m502751200
ISSN1083-351X
AutoresRieko Yagi, Denise L. McBurney, Walter E. Horton,
Tópico(s)interferon and immune responses
ResumoBcl-2 is an anti-apoptotic protein that has recently been shown to regulate other cellular functions. We previously reported that Bcl-2 regulates chondrocyte matrix gene expression, independent of its anti-apoptotic function. Here, we further investigate this novel function of Bcl-2 and examine three intracellular signaling pathways likely to be associated with this function. The present study demonstrates that the activity of Sox9, a master transcription factor that regulates the gene expression of chondrocyte matrix proteins, is suppressed by Bcl-2 small interference RNA in the presence of caspase inhibitors. This effect was attenuated by prior exposure of chondrocytes to an adenoviral vector expressing sense Bcl-2. In addition, the down-regulation of Bcl-2, Sox9, and chondrocyte-specific gene expression by serum withdrawal in primary chondrocytes was reversed by expressing Bcl-2. Inhibition of the protein kinase Cα and NFκB pathways had no effect on the maintenance of Sox9-dependent gene expression by Bcl-2. In contrast, whereas the MEK-ERK1/2 pathway negatively regulated the differentiated phenotype in wild type chondrocytes, inhibition of this pathway reversed the loss of differentiation markers and fibroblastic phenotype in Bcl-2-deficient chondrocytes. In conclusion, the present study identifies a specific signaling pathway, namely, MEK-ERK1/2, that is downstream of Bcl-2 in the regulation of Sox9-dependent chondrocyte gene expression and phenotype. Bcl-2 is an anti-apoptotic protein that has recently been shown to regulate other cellular functions. We previously reported that Bcl-2 regulates chondrocyte matrix gene expression, independent of its anti-apoptotic function. Here, we further investigate this novel function of Bcl-2 and examine three intracellular signaling pathways likely to be associated with this function. The present study demonstrates that the activity of Sox9, a master transcription factor that regulates the gene expression of chondrocyte matrix proteins, is suppressed by Bcl-2 small interference RNA in the presence of caspase inhibitors. This effect was attenuated by prior exposure of chondrocytes to an adenoviral vector expressing sense Bcl-2. In addition, the down-regulation of Bcl-2, Sox9, and chondrocyte-specific gene expression by serum withdrawal in primary chondrocytes was reversed by expressing Bcl-2. Inhibition of the protein kinase Cα and NFκB pathways had no effect on the maintenance of Sox9-dependent gene expression by Bcl-2. In contrast, whereas the MEK-ERK1/2 pathway negatively regulated the differentiated phenotype in wild type chondrocytes, inhibition of this pathway reversed the loss of differentiation markers and fibroblastic phenotype in Bcl-2-deficient chondrocytes. In conclusion, the present study identifies a specific signaling pathway, namely, MEK-ERK1/2, that is downstream of Bcl-2 in the regulation of Sox9-dependent chondrocyte gene expression and phenotype. Bcl-2 is a well known anti-apoptotic protein (1Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4789) Google Scholar). Accumulating evidence suggests that Bcl-2 regulates other cellular behavior, in addition to or independent of apoptosis (2Biroccio A. Candiloro A. Mottolese M. Sapora O. Albini A. Zupi G. Del Bufalo D. FASEB J. 2000; 14: 652-660Crossref PubMed Scopus (118) Google Scholar, 3Chen D.F. Schneider G.E. Martinou J.C. Tonegawa S. Nature. 1997; 385: 434-439Crossref PubMed Scopus (428) Google Scholar, 4Harada H. Mitsuyasu T. Seta Y. Maruoka Y. Toyoshima K. Yasumoto S. J. Oral Pathol. Med. 1998; 27: 11-17Crossref PubMed Scopus (48) Google Scholar, 5Haughn L. Hawley R.G. Morrison D.K. von Boehmer H. Hockenbery D.M. J. Biol. Chem. 2003; 278: 25158-25165Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 6Hilton M. Middleton G. Davies A.M. Curr. Biol. 1997; 7: 798-800Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 7Iervolino A. Trisciuoglio D. Ribatti D. Candiloro A. Biroccio A. Zupi G. Del Bufalo D. FASEB J. 2002; 16: 1453-1455Crossref PubMed Scopus (113) Google Scholar, 8Lee L. Irani K. Finkel T. Mol. Genet. Metab. 1998; 64: 19-24Crossref PubMed Scopus (21) Google Scholar, 9Matsuzaki Y. Nakayama K. Nakayama K. Tomita T. Isoda M. Loh D.Y. Nakauchi H. Blood. 1997; 89: 853-862Crossref PubMed Google Scholar, 10Middleton G. Pinon L.G. Wyatt S. Davies A.M. J. Neurosci. 1998; 18: 3344-3350Crossref PubMed Google Scholar, 11Trisciuoglio D. Iervolino A. Candiloro A. Fibbi G. Fanciulli M. Zangemeister-Wittke U. Zupi G. Del Bufalo D. J. Biol. Chem. 2004; 279: 6737-6745Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). For example, the growth and regeneration of retinal or trigeminal axons is promoted by Bcl-2 (3Chen D.F. Schneider G.E. Martinou J.C. Tonegawa S. Nature. 1997; 385: 434-439Crossref PubMed Scopus (428) Google Scholar, 6Hilton M. Middleton G. Davies A.M. Curr. Biol. 1997; 7: 798-800Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Bcl-2 is also necessary for the early maturational change in embryonic sensory neurons (10Middleton G. Pinon L.G. Wyatt S. Davies A.M. J. Neurosci. 1998; 18: 3344-3350Crossref PubMed Google Scholar) and dictates the differentiation of hematopoietic progenitor cells (5Haughn L. Hawley R.G. Morrison D.K. von Boehmer H. Hockenbery D.M. J. Biol. Chem. 2003; 278: 25158-25165Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 9Matsuzaki Y. Nakayama K. Nakayama K. Tomita T. Isoda M. Loh D.Y. Nakauchi H. Blood. 1997; 89: 853-862Crossref PubMed Google Scholar). Furthermore, the terminal differentiation of keratinocytes is inhibited by Bcl-2 (4Harada H. Mitsuyasu T. Seta Y. Maruoka Y. Toyoshima K. Yasumoto S. J. Oral Pathol. Med. 1998; 27: 11-17Crossref PubMed Scopus (48) Google Scholar), and up-regulation of urokinase plasminogen activator receptor expression is induced by Bcl-2 through an extracellular signal-regulated kinase (ERK) 1The abbreviations used are: ERK, extracellular signal-regulated kinase; PKC, protein kinase C; FBS, fetal bovine serum; IRC, Immortalized rat chondrocyte; m.o.i., multiplicity of infection; MEK, mitogen-activated protein kinase/ERK kinase; Z-VAD, benzyloxycarbonyl-VAD; siRNA, small interference RNA. 1The abbreviations used are: ERK, extracellular signal-regulated kinase; PKC, protein kinase C; FBS, fetal bovine serum; IRC, Immortalized rat chondrocyte; m.o.i., multiplicity of infection; MEK, mitogen-activated protein kinase/ERK kinase; Z-VAD, benzyloxycarbonyl-VAD; siRNA, small interference RNA. signaling pathway that activates Sp1 DNA binding in cancer cells (11Trisciuoglio D. Iervolino A. Candiloro A. Fibbi G. Fanciulli M. Zangemeister-Wittke U. Zupi G. Del Bufalo D. J. Biol. Chem. 2004; 279: 6737-6745Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Bcl-2 overexpression in melanoma cells under hypoxia conditions modulates vascular endothelial growth factor expression (2Biroccio A. Candiloro A. Mottolese M. Sapora O. Albini A. Zupi G. Del Bufalo D. FASEB J. 2000; 14: 652-660Crossref PubMed Scopus (118) Google Scholar, 7Iervolino A. Trisciuoglio D. Ribatti D. Candiloro A. Biroccio A. Zupi G. Del Bufalo D. FASEB J. 2002; 16: 1453-1455Crossref PubMed Scopus (113) Google Scholar). Finally, the activation of the c-Jun N-terminal kinase pathway by interleukin-1-β is inhibited by Bcl-2 in an apoptosis-independent manner in fibroblasts (8Lee L. Irani K. Finkel T. Mol. Genet. Metab. 1998; 64: 19-24Crossref PubMed Scopus (21) Google Scholar). Although there is a growing consensus that the above functions of Bcl-2 are not entirely a consequence of the anti-apoptotic process, the specific signaling pathways underlying these processes have not been fully identified. Chondrocytes, the only resident cells in cartilage, synthesize matrix proteins, including aggrecan, collagen type II, and link protein that are important for tissue function (12Yamada Y. Horton W. Miyashita T. Savagner P. Hassell J. Doege K. J. Craniofac. Genet. Dev. Biol. 1991; 11: 350-356PubMed Google Scholar). The transcription factor Sox9 binds to regulatory regions of genes coding for these matrix proteins as well as collagen type XI and cartilage derived retinoic acid-sensitive protein (13Bridgewater L.C. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 14Lefebvre V. Huang W. Harley V.R. Goodfellow P.N. de Crombrugghe B. Mol. Cell. Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar, 15Sekiya I. Tsuji K. Koopman P. Watanabe H. Yamada Y. Shinomiya K. Nifuji A. Noda M. J. Biol. Chem. 2000; 275: 10738-10744Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 16Xie W.F. Zhang X. Sakano S. Lefebvre V. Sandell L.J. J. Bone Miner Res. 1999; 14: 757-763Crossref PubMed Scopus (108) Google Scholar). Sox9 is also required for chondrocyte differentiation and chondrocyte proliferation (17Akiyama H. Chaboissier M.C. Martin J.F. Schedl A. de Crombrugghe B. Genes Dev. 2002; 16: 2813-2828Crossref PubMed Scopus (1354) Google Scholar, 18Bi W. Deng J.M. Zhang Z. Behringer R.R. de Crombrugghe B. Nat. Genet. 1999; 22: 85-89Crossref PubMed Scopus (1389) Google Scholar). During development, chondrocytes differentiate from mesenchymal cells and are responsible for longitudinal growth of long bones (19Yoon B.S. Lyons K.M. J. Cell. Biochem. 2004; 93: 93-103Crossref PubMed Scopus (249) Google Scholar). Chondrocytes in the prehypertrophic zone of the growth plate express Bcl-2 along with cartilage matrix proteins and Sox9 (19Yoon B.S. Lyons K.M. J. Cell. Biochem. 2004; 93: 93-103Crossref PubMed Scopus (249) Google Scholar, 20Amling M. Neff L. Tanaka S. Inoue D. Kuida K. Weir E. Philbrick W.M. Broadus A.E. Baron R. J. Cell Biol. 1997; 136: 205-213Crossref PubMed Scopus (272) Google Scholar, 21Vornehm S.I. Dudhia J. Von der M.K. Aigner T. Matrix Biol. 1996; 15: 91-98Crossref PubMed Scopus (58) Google Scholar, 22Wang Y. Toury R. Hauchecorne M. Balmain N. Histochem. Cell Biol. 1997; 108: 45-55Crossref PubMed Scopus (37) Google Scholar, 23de Crombrugghe B. Lefebvre V. Behringer R.R. Bi W. Murakami S. Huang W. Matrix Biol. 2000; 19: 389-394Crossref PubMed Scopus (401) Google Scholar). However, in the hypertrophic zone where chondrocytes undergo terminal differentiation and start to express collagen type X and bone related matrix proteins, there is decreased expression of Bcl-2 and cartilage matrix proteins as well as loss of Sox9 expression (20Amling M. Neff L. Tanaka S. Inoue D. Kuida K. Weir E. Philbrick W.M. Broadus A.E. Baron R. J. Cell Biol. 1997; 136: 205-213Crossref PubMed Scopus (272) Google Scholar, 25Nerlich A.G. Kirsch T. Wiest I. Betz P. Von der M.K. Histochemistry. 1992; 98: 275-281Crossref PubMed Scopus (42) Google Scholar, 26Ng L.J. Wheatley S. Muscat G.E. Conway-Campbell J. Bowles J. Wright E. Bell D.M. Tam P.P. Cheah K.S. Koopman P. Dev. Biol. 1997; 183: 108-121Crossref PubMed Scopus (560) Google Scholar, 27Zhao Q. Eberspaecher H. Lefebvre V. de Crombrugghe B. Dev. Dyn. 1997; 209: 377-386Crossref PubMed Scopus (435) Google Scholar). In vivo, suppression of Bcl-2 expression leads to accelerated terminal differentiation of chondrocytes and shortened long bones and Bcl-2 is downstream of parathyroid hormone related peptide (20Amling M. Neff L. Tanaka S. Inoue D. Kuida K. Weir E. Philbrick W.M. Broadus A.E. Baron R. J. Cell Biol. 1997; 136: 205-213Crossref PubMed Scopus (272) Google Scholar). Previously we reported that Bcl-2 functions as a regulator of cartilage matrix protein expression in chondrocytes (29Feng L. Precht P. Balakir R. Horton Jr., W.E. J. Cell. Biochem. 1998; 71: 302-309Crossref PubMed Scopus (43) Google Scholar, 30Feng L. Balakir R. Precht P. Horton Jr., W.E. J. Cell. Biochem. 1999; 74: 576-586Crossref PubMed Scopus (37) Google Scholar, 31Kinkel M.D. Horton Jr., W.E. J. Cell. Biochem. 2003; 88: 941-953Crossref PubMed Scopus (28) Google Scholar). The suppression of Bcl-2 in chondrocytes results in decreased aggrecan expression even when the apoptotic pathway is blocked by inhibition of caspase activity (30Feng L. Balakir R. Precht P. Horton Jr., W.E. J. Cell. Biochem. 1999; 74: 576-586Crossref PubMed Scopus (37) Google Scholar). Additionally, aggrecan, collagen type II, link protein, and Sox9 mRNA expression is significantly decreased in chondrocytes expressing a low level of Bcl-2 (30Feng L. Balakir R. Precht P. Horton Jr., W.E. J. Cell. Biochem. 1999; 74: 576-586Crossref PubMed Scopus (37) Google Scholar, 31Kinkel M.D. Horton Jr., W.E. J. Cell. Biochem. 2003; 88: 941-953Crossref PubMed Scopus (28) Google Scholar). However, collagen type I mRNA expression is increased suggesting that Bcl-2 may be involved in regulating the phenotype of chondrocytes (31Kinkel M.D. Horton Jr., W.E. J. Cell. Biochem. 2003; 88: 941-953Crossref PubMed Scopus (28) Google Scholar). Furthermore, chondrocytes constitutively expressing Bcl-2 are protected from down-regulation of aggrecan, collagen type II, and Sox9 following serum withdrawal (30Feng L. Balakir R. Precht P. Horton Jr., W.E. J. Cell. Biochem. 1999; 74: 576-586Crossref PubMed Scopus (37) Google Scholar, 31Kinkel M.D. Horton Jr., W.E. J. Cell. Biochem. 2003; 88: 941-953Crossref PubMed Scopus (28) Google Scholar). From these studies we hypothesize that Bcl-2 may function to regulate cartilage matrix expression in addition to or independent of its role in regulating apoptosis. However, the precise mechanism by which this occurs is still unknown. Here we tested three possible pathways that might be involved in the regulation of matrix gene expression by Bcl-2: PKCα, NFκB, and ERK1/2. PKCα positively regulates expression of collagen type II during chondrocyte differentiation and is down-regulated during dedifferentiation (32Chang S.H. Oh C.D. Yang M.S. Kang S.S. Lee Y.S. Sonn J.K. Chun J.S. J. Biol. Chem. 1998; 273: 19213-19219Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 33Yang M.S. Chang S.H. Sonn J.K. Lee Y.S. Kang S.S. Park T.K. Chun J.S. Mol. Cells. 1998; 8: 266-271PubMed Google Scholar, 34Yoon Y.M. Oh C.D. Kim D.Y. Lee Y.S. Park J.W. Huh T.L. Kang S.S. Chun J.S. J. Biol. Chem. 2000; 275: 12353-12359Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). NFκB inhibits Sox9 activity, destabilizes Sox9 mRNA, and decreases collagen type II expression with tumor necrosis factor-α treatment (35Murakami S. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 2000; 275: 3687-3692Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 36Sitcheran R. Cogswell P.C. Baldwin Jr., A.S. Genes Dev. 2003; 17: 2368-2373Crossref PubMed Scopus (103) Google Scholar). The ERK1/2 signaling pathway is known to negatively regulate collagen type II, aggrecan, and Sox9 mRNA in mesenchymal cells prior to chondrocyte differentiation (37Bobick B.E. Kulyk W.M. J. Biol. Chem. 2004; 279: 4588-4595Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 38Seghatoleslami M.R. Roman-Blas J.A. Rainville A.M. Modaressi R. Danielson K.G. Tuan R.S. J. Cell. Biochem. 2003; 88: 1129-1144Crossref PubMed Scopus (29) Google Scholar). Conversely, the ERK pathway has been shown to positively regulate Sox9 mRNA and activity during chondrogenesis induced by fibroblast growth factor (39Murakami S. Kan M. McKeehan W.L. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1113-1118Crossref PubMed Scopus (311) Google Scholar). In general, the pattern of expression of active PKCα is directly correlated with differentiation and collagen type II expression, whereas ERK1/2 expression is inversely correlated, even though PKCα and ERK appear to regulate chondrogenesis independently (40Yoon Y.M. Kim S.J. Oh C.D. Ju J.W. Song W.K. Yoo Y.J. Huh T.L. Chun J.S. J. Biol. Chem. 2002; 277: 8412-8420Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The findings presented here support a role for Bcl-2 in regulating the differentiated phenotype of chondrocytes. In addition, the data support a model whereby Bcl-2 suppresses an inhibitory action of the MEK-ERK1/2 signaling pathway on Sox9-dependent regulation of chondrocyte gene expression downstream of Bcl-2. Cell Culture—Primary chondrocytes were isolated from femoral condyles of 6-day-old Sprague-Dawley rats (Charles River). Cartilage was digested with 0.4% (w/v) collagenase (Worthington Biochemical Corp.) in a shaking incubator at 37 °C for 1 h. The cells were recovered by centrifugation at 4 °C. The cell pellet was washed with phosphate-buffered saline two times, and cells were seeded on dishes in Ham's F-12 medium (Invitrogen) with 10% FBS. Immortalized rat chondrocytes (IRC) were cultured in Ham's F-12 medium with 10% FBS. The IRC cells are known to have a differentiated phenotype similar to articular chondrocytes (41Horton Jr., W.E. Cleveland J. Rapp U. Nemuth G. Bolander M. Doege K. Yamada Y. Hassell J.R. Exp. Cell Res. 1988; 178: 457-468Crossref PubMed Scopus (70) Google Scholar). Cells were transfected with plasmids containing sense or antisense Bcl-2 coding sequences and selected in G418 as described previously (29Feng L. Precht P. Balakir R. Horton Jr., W.E. J. Cell. Biochem. 1998; 71: 302-309Crossref PubMed Scopus (43) Google Scholar, 30Feng L. Balakir R. Precht P. Horton Jr., W.E. J. Cell. Biochem. 1999; 74: 576-586Crossref PubMed Scopus (37) Google Scholar). The cells were plated at a density of 1 × 106 in T-25 flasks for protein isolation or 3.85 × 105 in 6-well pates for RNA extraction and were cultured at 37 °C with 5% CO2 in Ham's F-12 medium. Adenoviral Vectors and Infection in Chondrocytes—The adenovirus vectors were generated with the Adeno-X expression system (BD Clontech). The recombinant adenovirus vector expressing rat Bcl-2 cDNA was constructed using the identical sequence coding for Bcl-2 as previously described (29Feng L. Precht P. Balakir R. Horton Jr., W.E. J. Cell. Biochem. 1998; 71: 302-309Crossref PubMed Scopus (43) Google Scholar). Adenoviral vectors containing the luciferase cDNA (Adeno-x-LacZ, BD Clontech) was used as control vector. The purified recombinant adenoviral DNA containing the coding region in the sense orientation for Bcl-2 or LacZ was transfected into HEK293 cells, and a recombinant adenovirus was produced. The viral stocks were concentrated, and the purified stock was titered using the Adeno-X Rapid Titer Kit (BD Clontech). The infection efficiency of adenovirus, as determined by β-galactosidase staining (Invitrogen), was 100% with 30 m.o.i. in IRC cells and 45 m.o.i. in primary chondrocytes. Primary chondrocytes or IRC cells were plated at a density of 6 × 105 in 6-well plates. 45 m.o.i. or 30 m.o.i. recombinant virus diluted with 500 μl of Opti-MEM (Invitrogen) was added to cells, and the mixture was incubated for 90 min. Next, 1 ml of complete medium was added to each well, and, following incubation for an additional 6 h, the virus-containing medium was replaced by complete culture medium. Antibodies and Other Reagents—Antibodies recognizing total ERK1/2 and phosphorylated ERK1/2 were purchased from Cell Signaling. Antibodies to phosphorylated PKCα and β-actin were obtained from Santa Cruz Biotechnologies. Anti-Bcl-2 was obtained from BD Transduction Laboratories. For immunofluorescence staining, the primary Bcl-2 antibody and anti-rabbit fluorescein isothiocyanate secondary antibody were purchased from Santa Cruz Biotechnologies. The MEK1/2 inhibitor, U0126 (Cell Signaling), was used at concentrations of 20 or 25 μm depending on experiments. The PKCα inhibitors, Go6983 (42Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marme D. Schachtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar) and GF109203X (43Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar) (Sigma), were used at concentrations of 1 μm and 5 μm, which have been shown to suppress PKCα activity (40Yoon Y.M. Kim S.J. Oh C.D. Ju J.W. Song W.K. Yoo Y.J. Huh T.L. Chun J.S. J. Biol. Chem. 2002; 277: 8412-8420Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 44Kim S.J. Kim H.G. Oh C.D. Hwang S.G. Song W.K. Yoo Y.J. Kang S.S. Chun J.S. J. Biol. Chem. 2002; 277: 30375-30381Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). 50 μm Z-VAD (Alexis) was used to inhibit general caspase activities. Western Blotting—Cells were lysed and scraped in radioimmune precipitation assay buffer consisting of 20 mm Tris (pH 7.9), 140 mm NaCl, 5 mm EDTA, 1 mm EGTA, 10 mm NaF, 1% Nonidet P-40, 1% Triton X-100, 10% glycerol, 1 μg/ml aprotinin, 10 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, and phosphatase inhibitor mixture I (Sigma). The cell lysate was incubated on ice and centrifuged at 14,000 × g for 15 min at 4 °C. The protein concentration was determined by BCA protein assay (Pierce). 15-40 μg of total protein was mixed with Tris-glycine SDS sample buffer and sample reducing agent (Invitrogen) and loaded on a 7.5% or 10% SDS-PAGE gel. Following electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes (Invitrogen). The membranes were blocked in Tris-buffered saline with 0.1% Tween and 5% nonfat milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibody. After washing with TBS-T, membranes were incubated with secondary antibody for 1 h at room temperature. The membranes were developed with chemiluminescence reagents (Pierce). The expression level of positive bands of proteins were quantified using Image Station 440CF (Kodak) and normalized to the level of expression of β-actin. Transient Transfection, β-Galactosidase, and Luciferase Assay—Cells were plated at 3 × 105 in 12-well plates 24 h prior to transfection. A construct containing four repeats of a Sox9 binding site with the collagen type II promoter driving the luciferase reporter gene (14Lefebvre V. Huang W. Harley V.R. Goodfellow P.N. de Crombrugghe B. Mol. Cell. Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar, 45Lefebvre V. Zhou G. Mukhopadhyay K. Smith C.N. Zhang Z. Eberspaecher H. Zhou X. Sinha S. Maity S.N. de Crombrugghe B. Mol. Cell. Biol. 1996; 16: 4512-4523Crossref PubMed Google Scholar) (generous gift from Dr. V. Lefebvre, Cleveland Clinic Foundation) or an NFκB reporter construct (Stratagene) was co-transfected with a β-galactosidase reporter vector as an internal control of transfection efficiency. The dominant negative IκB expression vector (Stratagene), or Bcl-2 or control siRNA vector (IMGENEX) were also co-transfected with the Sox9 construct and β-galactosidase reporter vector. All transfections were performed with Lipofectamine2000 (Invitrogen) mixed with Opti-MEM (Invitrogen). Cells were exposed to DNA-Lipofectamine complex in 10% FBS Ham's F-12 without antibiotics for 4 h and then cultured overnight in normal growth media containing 10% FBS. After recovery, the cells were exposed to inhibitors or serum withdrawal as indicated. The cells were then lysed with Reporter Lysis Buffer (Promega). β-Galactosidase activities were determined by measuring absorbance at 420 nm, and Luciferase activities were determined by the Luciferase Assay System (Promega) using Lumat LB 9501/16 luminometer (Berthold). Immunofluorescence Staining—Cells were seeded at 1.4 × 105/well in 4 well chamber slides and transfected with Bcl-2 or control siRNA expression vectors (IMGNEX). 48 h after transfection, cells were fixed with 5% formalin for 30 min, incubated with 0.2% Triton X, and then blocked with 2% goat serum. Next, cells were incubated overnight with the Bcl-2 primary antibody at 4 °C and then incubated with fluorescein isothiocyanate secondary antibody. 4′,6-Diamidino-2-phenylindole mounting medium (Vector) was applied, the slides were covered with coverslips, and fluorescence was observed with an immunofluorescence microscope (BX60, Olympus). The Analysis of Sulfated Cartilage Matrix Proteoglycan—After treatments as indicated, cells were fixed and stained with 0.5% Alcian blue 8GX, pH 1.0, as described previously (46Hassell J.R. Horigan E.A. Teratog. Carcinog. Mutagen. 1982; 2: 325-331Crossref PubMed Scopus (110) Google Scholar). The Alcian blue bound to matrix proteoglycan was extracted with 4 m guanidine hydrochloride and measured at 600 nm (32Chang S.H. Oh C.D. Yang M.S. Kang S.S. Lee Y.S. Sonn J.K. Chun J.S. J. Biol. Chem. 1998; 273: 19213-19219Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Quantitative Real-time PCR—Total RNA was isolated using TRIzol reagent (Invitrogen) and treated with DNase (Invitrogen) to remove genomic DNA. Next, 1 μg of total RNA was reverse transcribed to cDNA using TaqMan reverse transcription reagents (Applied Biosystems) following the manufacturer's protocol. The aggrecan, collagen type II, Sox9, and 18 S primers were designed and examined for primer efficiency as described previously (31Kinkel M.D. Horton Jr., W.E. J. Cell. Biochem. 2003; 88: 941-953Crossref PubMed Scopus (28) Google Scholar). Briefly, primers were designed using Primer Express software (Applied Biosystems), and primer efficiency was performed with a standard curve of 60, 6, and 0.6 ng of cDNA with each primer compared with 18 S. The specificity of amplified products was confirmed by dissociation curve analysis (Applied Biosystems). The quantitation of mRNA expression was performed using the Applied Biosystems ABI Prism 7700 sequence detection system (Applied Biosystems). The PCR reactions were performed using 50 ng of cDNA and SYBR Green PCR core reagents (Applied Biosystems) in 96-well plates following the manufacturer's protocol. The data were analyzed using Sequence Detector version 1.7 software (Applied Biosystems). Relative expression was calculated using the Comparative CT Method (User Bulletin no. 2, Applied Biosystems, and Ref. 31Kinkel M.D. Horton Jr., W.E. J. Cell. Biochem. 2003; 88: 941-953Crossref PubMed Scopus (28) Google Scholar). According to accepted standards, relative differences of 2-fold or greater are considered as biologically significant (Applied Biosystems). The Level of Bcl-2 Protein Modulates Sox9-dependent Chondrocyte Matrix Gene Expression—We previously demonstrated that suppression of Bcl-2 protein level with integrated antisense constructs resulted in down-regulation of expression of mRNA coding for cartilage matrix proteins even in the presence of caspase inhibitors that blocked full apoptosis (29Feng L. Precht P. Balakir R. Horton Jr., W.E. J. Cell. Biochem. 1998; 71: 302-309Crossref PubMed Scopus (43) Google Scholar, 30Feng L. Balakir R. Precht P. Horton Jr., W.E. J. Cell. Biochem. 1999; 74: 576-586Crossref PubMed Scopus (37) Google Scholar, 31Kinkel M.D. Horton Jr., W.E. J. Cell. Biochem. 2003; 88: 941-953Crossref PubMed Scopus (28) Google Scholar). Here we examine the effects of direct down-regulation of Bcl-2 using siRNA on Sox9-dependent collagen type II promoter activity. IRC cells were transfected with a plasmid vector expressing siRNA for Bcl-2 along with the Sox9 reporter construct. Immunocytochemistry was used to demonstrate no change in the Bcl-2 level in IRC chondrocytes transfected with control siRNA containing random DNA sequences (Fig. 1A), whereas there was an obvious decrease in the percentage of chondrocytes expressing Bcl-2 following transfection with the Bcl-2 siRNA (Fig. 1B). The Sox9-dependent reporter construct contains four copies of the Sox9 binding site upstream from a minimal collagen II promoter (14Lefebvre V. Huang W. Harley V.R. Goodfellow P.N. de Crombrugghe B. Mol. Cell. Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar, 45Lefebvre V. Zhou G. Mukhopadhyay K. Smith C.N. Zhang Z. Eberspaecher H. Zhou X. Sinha S. Maity S.N. de Crombrugghe B. Mol. Cell. Biol. 1996; 16: 4512-4523Crossref PubMed Google Scholar). This reporter construct is dependent on transcriptional activation by Sox9 in IRC chondrocytes, because mutations in the Sox9 binding sites eliminate promoter activity (data not shown). The Sox9-dependent reporter activity was decreased by 70% in chondrocytes with suppressed Bcl-2 expression compared with cells transfected with control siRNA (Fig. 2A). This suppression of Sox9 activity was observed even in the presence of 50 μm Z-VAD, a general caspase inhibitor that blocks full apoptosis in IRC cells (30Feng L. Balakir R. Precht P. Horton Jr., W.E. J. Cell. Biochem. 1999; 74: 576-586Crossref PubMed Scopus (37) Google Scholar) compared with control siRNA-transfected cells treated with 50 μm Z-VAD (Fig. 2B). These results confirm that Sox9-dependent promoter activity is dependent on Bcl-2 in chondrocytes. A specific role for Bcl-2 was further established by increasing basal expression of Bcl-2 using an adenoviral construct prior to exposure of the chondrocytes to Bcl-2 siRNA. Cells were first infected with either sense Bcl-2 or LacZ adenovirus, and then co-transfected with either Bcl-2 or control siRNA along with the Sox9 activity reporter construct in the presence of 10% FBS. Cells infected with LacZ virus and transfected with Bcl-2 siRNA showed a 50% down-regulation of Sox9-dependent promoter activity compared with control siRNA (Fig. 3). However, if the cells were first treated with Bcl-2 adenovirus to increase the basal level of Bcl-2, there was no decrease in Sox9-dependent promoter activity following transfection with Bcl-2 siRNA (Fig. 3). Previously we demonstrated that constitutive expression of Bcl-2 protected IRC chondrocytes against serum withdrawal-induced down-regulation of mRNA transcripts coding for several cartilage matrix proteins and Sox9 (31Kinkel M.D. Horton Jr., W.E. J. Cell. Biochem. 2003; 88: 941-953Crossref PubMed Scopus (28) Google Scholar). Here we extend these studies to look directly at Sox9-dependent promo
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