Transforming Growth Factor-β Stimulates Cyclin D1 Expression through Activation of β-Catenin Signaling in Chondrocytes

Transforming growth factor-β (TGF-β) plays an essential role in chondrocyte maturation. It stimulates chondrocyte proliferation but inhibits chondrocyte differentiation. In this study, we found that TGF-β rapidly induced β-catenin protein levels and signaling in murine neonatal sternal primary chondrocytes. TGF-β-increased β-catenin induction was reproduced by overexpression of SMAD3 and was absent in Smad3-/- chondrocytes treated with TGF-β. SMAD3 inhibited β-transducin repeat-containing protein-mediated degradation of β-catenin and immunoprecipitated with β-catenin following TGF-β treatment. Both SMAD3 and β-catenin co-localized to the nucleus after TGF-β treatment. Although both TGF-β and β-catenin stimulated cyclin D1 expression in chondrocytes, the effect of TGF-β was inhibited with β-catenin gene deletion or SMAD3 loss of function. These results demonstrate that TGF-β stimulates cyclin D1 expression at least in part through activation of β-catenin signaling. Transforming growth factor-β (TGF-β) plays an essential role in chondrocyte maturation. It stimulates chondrocyte proliferation but inhibits chondrocyte differentiation. In this study, we found that TGF-β rapidly induced β-catenin protein levels and signaling in murine neonatal sternal primary chondrocytes. TGF-β-increased β-catenin induction was reproduced by overexpression of SMAD3 and was absent in Smad3-/- chondrocytes treated with TGF-β. SMAD3 inhibited β-transducin repeat-containing protein-mediated degradation of β-catenin and immunoprecipitated with β-catenin following TGF-β treatment. Both SMAD3 and β-catenin co-localized to the nucleus after TGF-β treatment. Although both TGF-β and β-catenin stimulated cyclin D1 expression in chondrocytes, the effect of TGF-β was inhibited with β-catenin gene deletion or SMAD3 loss of function. These results demonstrate that TGF-β stimulates cyclin D1 expression at least in part through activation of β-catenin signaling. Endochondral bone formation involves condensation and differentiation of mesenchymal cells into chondrocytes, followed by chondrocyte proliferation, maturation, hypertrophic differentiation, and apoptosis. Eventually, the calcified cartilage tissue formed in the growth plate is replaced by bone tissue (1Kronenberg H.M. Nature. 2003; 423: 332-336Crossref PubMed Scopus (2177) Google Scholar). Each step of the endochondral bone formation process is precisely regulated by local growth factors. Among these factors, transforming growth factor-β (TGF-β) 3The abbreviations used are: TGF-β, transforming growth factor-β; PBS, phosphate-buffered saline; FBS, fetal bovine serum; TCF, T cell factor; Ad, adenovirus; CMV, cytomegalovirus; GFP, green fluorescent protein; ICAT, inhibitor of β-catenin and TCF; β-TrCP, β-transducin repeat-containing protein; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; BrdUrd, 5-bromo-2′-deoxyuridine; Cdks, cyclin-dependent kinases; Rb, retinoblastoma. plays important roles in chondrocyte proliferation and hypertrophy. TGF-β promotes chondrocyte proliferation but inhibits chondrocyte differentiation and hypertrophy (2Beier F. Ali Z. Mok D. Taylor A.C. Leask T. Albanese C. Pestell R.G. LuValle P. Mol. Biol. Cell. 2001; 12: 3852-3863Crossref PubMed Scopus (117) Google Scholar, 3Ferguson C.M. Schwarz E.M. Reynolds P.R. Puzas J.E. Rosier R.N. O'Keefe R.J. Endocrinology. 2000; 141: 4728-4735Crossref PubMed Scopus (115) Google Scholar, 4Pateder D.B. Rosier R.N. Schwarz E.M. Reynolds P.R. Puzas J.E. D'Souza M. O'Keefe R.J. Exp. Cell Res. 2000; 256: 555-562Crossref PubMed Scopus (50) Google Scholar, 5Ionescu A.M. Schwarz E.M. Zuscik M.J. Drissi H. Puzas J.E. Rosier R.N. O'Keefe R.J. Exp. Cell Res. 2003; 288: 198-207Crossref PubMed Scopus (36) Google Scholar, 6Dong Y. Drissi H. Chen M. Chen D. Zuscik M.J. Schwarz E.M. O'Keefe R.J. J. Cell. Biochem. 2005; 95: 1057-1068Crossref PubMed Scopus (65) Google Scholar). The mechanism of TGF-β-induced chondrocyte proliferation remains undefined. In this study, we investigated the interaction between TGF-β and β-catenin signaling in chondrocytes. We found that TGF-β activates β-catenin signaling through SMAD3. SMAD3 interacted with β-catenin and increased β-catenin nuclear translocation and signaling. Although TGF-β stimulated cyclin D1 expression in chondrocytes, this effect was abolished by inhibition of β-catenin signaling. These results demonstrate for the first time that TGF-β stimulates cyclin D1 expression in chondrocytes at least in part through activation of β-catenin signaling. These findings provide novel insight regarding the mechanism through which TGF-β regulates chondrocyte proliferation. Cell Culture—Smad3-/- mice derived from a C57/B6 lineage, in which exon 8 of the Smad3 gene is deleted (a kind gift from Dr. C. X. Deng, National Institutes of Health, Bethesda, MD) (7Yang X. Chen L. Xu X. Li C. Huang C. Deng C.X. J. Cell Biol. 2001; 153: 35-46Crossref PubMed Scopus (536) Google Scholar), were bred using heterozygote pairs. 3-day-old neonatal mice were killed and genotyped using tail tissues obtained at the time of death. The anterior rib cage and sternum were harvested en bloc, washed with sterile phosphate-buffered saline (PBS), and then digested with Pronase (Roche Applied Science) dissolved in PBS (2 mg/ml) in a 37 °C water bath with continuous shaking for 60 min. This was followed by incubation in a solution of collagenase D (3 mg/ml dissolved in serum-free Dulbecco's modified Eagle's medium; Roche Applied Science) for 90 min at 37 °C. The soft tissue debris was thoroughly removed. The remaining sterna and costosternal junctions were further digested in fresh collagenase D solution in Petri dishes in a 37 °C incubator for 5 h with intermittent shaking. This step allows remnant fibroblasts to attach to the Petri dish while the chondrocytes remain afloat in the medium. The digestion solution was filtered through Swinex to remove all residual bone fragments. The solution was centrifuged, and the cells were resuspended in complete medium (Dulbecco's modified Eagle's medium with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 100 mml-glutamine, and 50 μg/ml ascorbic acid, pH 7.1). The cells were counted and plated at the appropriate density. To remove any remaining fibroblasts, 24-h cultures were treated with 0.05% trypsin for 1 min to lift the fibroblasts from the culture dish while allowing the chondrocytes to remain attached. A similar procedure was used for chondrocyte isolation from β-cateninflox/flox mice (8Brault V. Moore R. Kutsch S. Ishibashi M. Rowitch D.H. McMahon A.P. Sommer L. Boussadia O. Kemler R. Development (Camb.). 2001; 128: 1253-1264PubMed Google Scholar) and β-catenin reporter TOPGAL transgenic mice, in which a β-galactosidase gene is under the control of a lymphoid enhancer factor/T cell factor (TCF)- and β-catenin-inducible promoter. In these transgenic mice, TOPGAL expression is directly stimulated by a stabilized form of β-catenin (9DasGupta R. Fuchs E. Development (Camb.). 1999; 126: 4557-4568Crossref PubMed Google Scholar). The chondrocyte cell line RCJ3.1C5.18 (C5.18) was cultured in α-minimal essential medium containing 10% FBS. In cyclin D1 reporter and Western blot assays, all cells were synchronized for 3 days in serum-free medium prior to different treatments. Adenovirus Production and Infection—The full-length mouse β-catenin cDNA was cloned into the TOPO entry vector using the Gateway system (Invitrogen). By LR reaction, a β-catenin insert was subcloned into the ViraPower™ adenoviral expression vector. The plasmid was linearized using PacI and transiently transfected into 293A cells. After several cycles of amplification, the adenovirus was purified using the CsCl binding method. Ad5-CMV-Cre-GFP and Ad5-CMV-enhanced GFP were purchased from the Baylor College of Medicine. Infection (multiplicity of infection of 10) lasted for 24 h, and cells were allowed to recover for 48 h prior to treatments. β-Cateninflox/flox chondrocytes were infected with Ad5-CMV-Cre-GFP for 24 h and recovered in full medium for 48 h. Ad5-CMV-enhanced GFP was used in the same way as a control. β-Galactosidase Activity Assay—TOPGAL chondrocytes were plated for 24 h. TGF-β (1 ng/ml) was added to cultures in serum-free medium for 24 h. β-Galactosidase activity was measured with a luminescent β-galactosidase detection kit (BD Biosciences) on a luminometer (Opticom 1, MGM Instruments, Inc., Hamden, CT). Each cell preparation was tested in triplicate, and the values were standardized by protein concentrations. The results are presented as the means ± S.E. Transfections and Luciferase Assay—Transient transfection was performed using a Targefect F-2 reagent kit (Targeting Systems, Santee, CA). To increase the transfection efficiency, Virofect provided in the kit was added to all reactions. The transfection complex was formed after incubation at 37 °C for 20 min. At the same time, chondrocytes were pretreated with hyaluronidase (200 ng/ml; Sigma) at 37 °C for 30 min. The transfection complex was then added to cell culture dishes containing Dulbecco's modified Eagle's medium with 10% FBS. The following plasmids were transfected into chondrocytes for 12 h before treatments: TOPflash and FOPflash reporter plasmids (a gift from Dr. Jennifer Westendorf, Mayo Clinic College of Medicine, Rochester, MN), the cyclin D1 promoter (-1745CD1-Luc, a gift from Dr. Phyllis LuValle, University of Florida, Gainesville, FL), a gift from Dr. The University MD) V. H. PubMed Scopus Google Scholar), (a gift from Dr. University of Rochester, Rochester, and inhibitor of β-catenin and a gift from Dr. University of T. M. J. S. Y. S. S. Y. H. T. 2000; Google Scholar, D. Xu Mol. Cell. PubMed Scopus Google Scholar). was with the to results for transfection Luciferase activity in the cell was using a luminometer (Opticom β-transducin repeat-containing protein cDNA was by using from cells and then cloned into the vector The was by the was performed using a and kit cell primary (1 and were added to of acid, pH and in the provided at °C for 1 h with After the protein were using of and at °C was used in the Western were in with inhibitor (Roche Applied 1 1 1 and 1 The protein was using a protein kit The protein were using (Invitrogen). After to a and with the were with the following mouse at β-catenin and D1 at a of was used to protein were then to the and for 60 min. The were using detection of different in the same we used solution Inc., CA). and cells J.E. Endocrinology. PubMed Scopus Google Scholar) were plated in Rochester, for 24 h and then treated with TGF-β (1 ng/ml) for h. After with PBS, the cells were with at °C for 30 min. binding was by incubation with PBS containing 10% serum at for 1 h. After serum was mouse and to in PBS containing 10% serum and Inc., were to and at °C. After with PBS, the were in the for 1 h with fluorescein and tetramethylrhodamine Inc., in PBS containing and 10% The were then with water for 30 min and with medium CA). was using a with different after were through a on a or through a on a with with of and to and FITC, The were using the was from cultures using an kit Inc., CA). 1 of was using an kit (BD was performed using the system and the fluorescent to Applied CA). The used in this were as and and cyclin and The a step at °C for followed by cycles of at °C for 20 for 20 and at °C for 30 of the fluorescent was at the of the °C were to a and the were and with the and were used to remove Each was tested at least in and for cell In and solution was into the of neonatal mice (1 of were killed h and at were harvested and tissue were using a kit with and with chondrocytes in the growth plate were and at least from were In and was added to cultures containing primary chondrocytes and for h. The cells were washed with PBS, and with at °C for 24 h. the of the cells were washed with PBS and with solution for 30 min. After and the cells were resuspended with 1 of PBS containing and the cells were with 1 of PBS containing at for 30 min. (Roche Applied Science) was added to the solution and for 60 min in the After cells were treated with A (1 mg/ml) at for 30 min. cells were with μg/ml in for min at filtered through and a (BD were between using of or as values were and are in the TGF-β β-catenin signaling in cells and in primary chondrocytes from the sterna of TOPGAL transgenic of the TOPflash reporter with the β-catenin expression plasmid in cells in of reporter infection of TOPGAL chondrocytes with in increased β-galactosidase activity 1, a and To TGF-β activates β-catenin signaling in chondrocytes, we the effect of TGF-β (1 ng/ml) on β-galactosidase activity in primary chondrocytes from TOPGAL transgenic mice and found that TGF-β stimulated β-galactosidase activity in a To TGF-β β-catenin protein levels in chondrocytes, we treated cells with TGF-β (1 ng/ml) for different of time and in β-catenin protein levels by Western blot TGF-β increased the protein levels of the and of β-catenin in a TGF-β increased β-catenin protein levels and effect was at the time after TGF-β In TGF-β effect on β-catenin expression in these cells These results that TGF-β activates β-catenin signaling by β-catenin protein levels in chondrocytes. TGF-β through β-catenin protein levels are by we the protein levels of β-catenin in Smad3 chondrocytes. Cell were from sternal chondrocytes derived from and Smad3 mice, and in β-catenin protein levels were by Western using The results that both and β-catenin levels were in chondrocytes with chondrocytes In the cyclin D1 protein was in Smad3-/- chondrocytes To further TGF-β activates β-catenin signaling through we transfected the TOPflash reporter into chondrocytes from or Smad3 mice and treated these cells with TGF-β (1 TGF-β stimulated TOPflash reporter activity in but Smad3 chondrocytes To further the role of SMAD3 in β-catenin the SMAD3 expression plasmid was with the β-catenin reporter into cells. Although transfection of SMAD3 increased β-catenin reporter of SMAD3 with TOPflash reporter activity that overexpression of β-catenin a of reporter These results demonstrate that TGF-β activates β-catenin signaling through SMAD3 in chondrocytes. SMAD3 with in SMAD3 directly with β-catenin in chondrocytes, we performed chondrocytes were cultured in the or of TGF-β for 30 and cell were and to using or control followed by Western blot using A interaction between SMAD3 and β-catenin was in the of TGF-β. In a interaction between SMAD3 and β-catenin was in the of TGF-β In with β-catenin in cells These results that β-catenin with TGF-β in chondrocytes. TGF-β TGF-β β-catenin nuclear we performed using chondrocytes were and then treated with TGF-β (1 ng/ml) for h. and that with TGF-β induced the nuclear of both SMAD3 and β-catenin in cells The that β-catenin with SMAD3 in the nucleus of cells that these in chondrocytes. SMAD3 is a in the protein complex that β-catenin degradation in cell H. J. Kemler R. J. PubMed Scopus Google Scholar, M. S. M. M. J. 1999; PubMed Scopus Google Scholar, E. M. 1999; PubMed Scopus Google Scholar, P. P. 1999; PubMed Scopus Google Scholar, Xu Mol. Cell. 2003; PubMed Scopus Google Scholar). To β-catenin degradation in chondrocytes, we transfected the expression plasmid into cells and the effect of on the protein levels of of the β-catenin protein in cells. of SMAD3 the of β-catenin and β-catenin degradation in cells These results that SMAD3 β-catenin D1 in then the effect of β-catenin on cyclin D1 gene and expression in chondrocytes. chondrocytes were with the plasmid and the cyclin D1 promoter. of stimulated cyclin D1 promoter activity in chondrocytes D1 protein expression was increased chondrocytes were infected with In chondrocytes with the cyclin D1 promoter and cyclin D1 promoter activity To further the role of β-catenin in cyclin D1 expression in chondrocytes, we primary chondrocytes from mice (8Brault V. Moore R. Kutsch S. Ishibashi M. Rowitch D.H. McMahon A.P. Sommer L. Boussadia O. Kemler R. Development (Camb.). 2001; 128: 1253-1264PubMed Google Scholar) and infected these cells with infected with were used as a control. The results in chondrocytes, cyclin D1 expression was with cells these results demonstrate that β-catenin plays an important role in cyclin D1 expression in chondrocytes. TGF-β D1 and Cell through the role of TGF-β in chondrocyte cell we in cell proliferation in Smad3 mice and found that chondrocytes in the of the growth plate were by This was by an in using primary chondrocytes from Smad3 mice and The results that cells were in Smad3 chondrocytes, of serum In Smad3 chondrocytes, expression of cyclin D1 and protein was and these results that TGF-β stimulates cyclin D1 expression and cell growth through SMAD3 in chondrocytes. TGF-β-induced D1 by both TGF-β and β-catenin cyclin D1 expression and TGF-β activates β-catenin signaling in chondrocytes, we that TGF-β cyclin D1 expression through activation of β-catenin signaling. To this primary chondrocytes were from the mice, infected with or and treated with TGF-β. TGF-β stimulated cyclin D1 expression in but chondrocytes with these transfection of the TGF-β R. J. J. PubMed Scopus Google Scholar) increased cyclin D1 which was inhibited by of is an protein that with the binding of β-catenin to and inhibits of T. M. J. S. Y. S. S. Y. H. T. 2000; Google Scholar, D. Xu Mol. Cell. PubMed Scopus Google Scholar). These results that TGF-β regulates cyclin D1 expression in a TGF-β to cyclin D1 expression and chondrocyte proliferation (2Beier F. Ali Z. Mok D. Taylor A.C. Leask T. Albanese C. Pestell R.G. LuValle P. Mol. Biol. Cell. 2001; 12: 3852-3863Crossref PubMed Scopus (117) Google Scholar, X. Chen L. Xu X. Li C. Huang C. Deng C.X. J. Cell Biol. 2001; 153: 35-46Crossref PubMed Scopus (536) Google Scholar, F. R.J. Taylor A.C. Pestell R.G. LuValle P. S. 1999; PubMed Scopus Google Scholar). A that β-catenin chondrocyte proliferation and differentiation H. Y. X. R. Z. Deng T. PubMed Scopus Google Scholar). The interaction of TGF-β and β-catenin signaling in chondrocytes In this study, we that TGF-β β-catenin signaling through SMAD3. SMAD3 directly interacted with TGF-β increase β-catenin protein levels by β-catenin degradation TGF-β increased β-catenin protein levels but effect on β-catenin TGF-β to cyclin D1 expression the β-catenin gene was deleted in chondrocytes, that TGF-β-induced cyclin D1 expression is by β-catenin in chondrocytes. These findings provide novel into the interaction between TGF-β and β-catenin signaling and the mechanism of chondrocyte proliferation. TGF-β stimulates cell proliferation through induction of cyclin D1 F. R.J. Taylor A.C. Pestell R.G. LuValle P. S. 1999; PubMed Scopus Google Scholar, F. Ali Z. Mok D. Taylor A.C. Leask T. Albanese C. Pestell R.G. LuValle P. Mol. Biol. 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Cell. 2005; PubMed Scopus Google Scholar), promotes chondrocyte in growth plate chondrocytes by chondrocyte proliferation and chondrocyte gene the β-catenin gene is deleted in chondrocytes, chondrocyte proliferation is and hypertrophic chondrocyte differentiation is H. Y. X. R. Z. Deng T. PubMed Scopus Google Scholar). The effect of β-catenin on chondrocyte is regulated by factor the of β-catenin and inhibits the interaction of β-catenin with H. Y. X. R. Z. Deng T. PubMed Scopus Google Scholar). findings demonstrate that stimulates expression through activation of bone protein signaling in chondrocytes M. Y. Y. Drissi H. Chen D. O'Keefe R.J. J. Res. 2005; Scholar). These demonstrate that β-catenin with or signaling chondrocyte maturation. Although the mechanism of TGF-β β-catenin degradation and β-catenin nuclear translocation further findings demonstrate that TGF-β regulates cyclin D1 expression through activation of β-catenin signaling. We Y. Drissi H. Chen M. Chen D. Zuscik M.J. 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In and hypertrophic chondrocytes to maturation, β-catenin differentiation Y. Drissi H. Chen M. Chen D. Zuscik M.J. Schwarz E.M. O'Keefe R.J. J. Cell. Biochem. 2005; 95: 1057-1068Crossref PubMed Scopus (65) Google Scholar, M. J. E. Y. C. T. H. T. T. V. T. M. M. Biol. PubMed Scopus Google Scholar). The findings in and chondrocytes, β-catenin of TGF-β and to chondrocyte proliferation, with an effect on proliferation in cell H. Z. Chen J. C. PubMed Scopus Google Scholar, Y. Li C.M. J. 2005; PubMed Scopus Google Scholar, P. C. J. PubMed Scopus Google Scholar). a of the role of TGF-β signaling in signaling cell growth differentiation is that SMAD3 β-catenin signaling cell proliferation but inhibits signaling in chondrocytes maturation. at a β-catenin to differentiation, the in the from proliferation to differentiation and the role of β-catenin in these remain the is that with the TGF-β and bone protein signaling are We Dr. for the Dr. for the Dr. Jennifer for the TOPflash and FOPflash reporter Dr. Phyllis LuValle for the cyclin D1 Dr. for the and Dr. of and of Medicine, University of for the RCJ3.1C5.18 cells.

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