Key points are not available for this paper at this time.
In chondrogenesis, members of the transforming growth factor-β (TGF-β) superfamily play critical roles by inducing gene expression of cartilage-specific molecules. By using a chondrogenic cell line, ATDC5, we investigated the TGF-β-mediated signaling pathways involved in expression of the aggrecan gene (Agc). At confluency, TGF-β induced Agcexpression within 3 h, and cycloheximide blocked this induction, indicating that de novo protein synthesis is essential for this response. At this stage, TGF-β induced rapid, transient phosphorylation of Smad2, extracellular signal-activated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (MAPK). Inhibition of the Smad pathways by transfection with a dominant negative Smad4 construct significantly reduced TGF-β-induced Agcexpression, indicating that Smad signaling is essential for this response. Furthermore, an inhibitor of the ERK1/2 pathway, U0126, or inhibitors of the p38 MAPK pathway, SB203580 and SKF86002, repressed TGF-β-induced Agc expression in a dose-dependent manner, indicating that ERK1/2 or p38 MAPK activation is also required for TGF-β-induced Agcexpression in confluent ATDC5 cells. In differentiated ATDC5 cells, persistently high basal levels of ERK1/2 and p38 MAPK phosphorylation correlated with elevated basal Agc expression, which was inhibited by incubation with inhibitors of these pathways. Whereas Smad2 was rapidly phosphorylated by TGF-β and involved in the initial activation of Agc expression in confluent cells, Smad2 activation was not required for maintaining the high level ofAgc expression. Taken together, these results suggest an important role for transcriptional cross-talk between Smad and MAPK pathways in expression of early chondrocytic phenotypes and identify important changes in the regulation of Agc expression following chondrocyte differentiation. In chondrogenesis, members of the transforming growth factor-β (TGF-β) superfamily play critical roles by inducing gene expression of cartilage-specific molecules. By using a chondrogenic cell line, ATDC5, we investigated the TGF-β-mediated signaling pathways involved in expression of the aggrecan gene (Agc). At confluency, TGF-β induced Agcexpression within 3 h, and cycloheximide blocked this induction, indicating that de novo protein synthesis is essential for this response. At this stage, TGF-β induced rapid, transient phosphorylation of Smad2, extracellular signal-activated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (MAPK). Inhibition of the Smad pathways by transfection with a dominant negative Smad4 construct significantly reduced TGF-β-induced Agcexpression, indicating that Smad signaling is essential for this response. Furthermore, an inhibitor of the ERK1/2 pathway, U0126, or inhibitors of the p38 MAPK pathway, SB203580 and SKF86002, repressed TGF-β-induced Agc expression in a dose-dependent manner, indicating that ERK1/2 or p38 MAPK activation is also required for TGF-β-induced Agcexpression in confluent ATDC5 cells. In differentiated ATDC5 cells, persistently high basal levels of ERK1/2 and p38 MAPK phosphorylation correlated with elevated basal Agc expression, which was inhibited by incubation with inhibitors of these pathways. Whereas Smad2 was rapidly phosphorylated by TGF-β and involved in the initial activation of Agc expression in confluent cells, Smad2 activation was not required for maintaining the high level ofAgc expression. Taken together, these results suggest an important role for transcriptional cross-talk between Smad and MAPK pathways in expression of early chondrocytic phenotypes and identify important changes in the regulation of Agc expression following chondrocyte differentiation. During development, cartilage serves as a template for most bones. Cartilage formation is initiated with condensation of mesenchymal cells, followed by progression of chondrocyte differentiation toward proliferation, prehypertrophy, and hypertrophy (1Cancedda R. Descalzi Cancedda F. Castagnola P. Int. Rev. Cytol. 1995; 159: 265-358Crossref PubMed Scopus (351) Google Scholar, 2Wallis G.A. Curr. Biol. 1996; 6: 1577-1580Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). A number of growth factors, such as fibroblast growth factors (3Karsenty G. Dev. Genet. 1998; 22: 301-313Crossref PubMed Scopus (85) Google Scholar, 4Niswander L. Martin G.R. 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TGF-β phosphorylation of ERK1/2 that of the basal phosphorylation as early as the at and was until TGF-β 3 between phosphorylation and activation of ERK1/2 was by an ERK1/2 using as a at using basal levels of and TGF-β phosphorylated p38 with ERK1/2 phosphorylation of p38 MAPK was 3 was not phosphorylated or by indicating that this is not involved in TGF-β signaling in these cells not we used a number of inhibitors of the MAPK pathways to the of p38 MAPK and ERK1/2 activation expression. the ERK1/2 pathway, we used U0126, a inhibitor of which is an that ERK1/2 M.F. F. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the p38 MAPK pathway, SB203580 S. D. S.M. R.S. G.A. S. 1997; PubMed Scopus Google Scholar) and J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. D. D. J.E. R. S.M. 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Full Text Full Text PDF PubMed Scopus Google Scholar), with a gene the of and a construct of inhibitors the basal in of U0126, and inhibited TGF-β-induced transcriptional of both and Whereas and SB203580 inhibited basal activation by and TGF-β-induced activation was inhibited by and Furthermore, little the basal indicating that the of by these inhibitors is used as an to the of these of induced of the gene of or SB203580 in of by not results that these inhibitors the p38 MAPK and ERK1/2 Taken together, these results that TGF-β-induced activation of p38 MAPK or ERK1/2 is essential for transcriptional activation of Smad2 and Smad4 and that this is for activation of a transcriptional in ATDC5 cells. this of the Smad and MAPK pathways was restricted to confluent ATDC5 cells or was a at in chondrocyte differentiation, we performed a their to TGF-β at of differentiation. we levels of Agcexpression, using in basal Agc expression of ATDC5 cells differentiation. levels to and at and confluency, the level at is chondrocyte differentiation in this Although TGF-β Agc expression differentiation, the by TGF-β to at as with in confluent of Agc in ATDC5 levels of Agc by the levels of in a levels of Agc by the levels of we phosphorylation of Smad2, and p38 by TGF-β following differentiation in ATDC5 cells. TGF-β phosphorylated Smad2 at confluency, and indicating that for TGF-β and their phosphorylation of Smad2 differentiation Furthermore, low levels of Smad2 phosphorylation in the of TGF-β that is TGF-β in these cells. In the of p38 MAPK and ERK1/2 to TGF-β was differentiation. At the phosphorylation level of ERK1/2 was by by TGF-β the high basal phosphorylation of ERK1/2 and p38 levels of both ERK1/2 and p38 MAPK with elevated basal Agcexpression in differentiated ATDC5 cells, that activation of both ERK1/2 and p38 MAPK required for Agcexpression in differentiated cells. Therefore, we the of inhibitors Agc expression in differentiated ATDC5 cells. in both and SB203580 expression in differentiated cells. the role of ERK1/2 and p38 MAPK expression and that in as to ATDC5 cells, Agc expression is activation of the Smad of Agc expression in differentiated ATDC5 cells by inhibitors of ERK1/2 and p38 MAPK pathways. cells for and or SB203580 was to the for cells with TGF-β for was and was and SB203580 Agc levels by results in In these we have that both and the p38 MAPK and ERK1/2 pathways are rapidly and following with TGF-β in a mouse chondrogenic cell line, ATDC5, and that the activation of of these pathways is required for the transcriptional activation of Furthermore, we that the TGF-β-induced transcriptional activation of a is the activation of p38 MAPK and ERK1/2 pathways in these cells. that an important level of transcriptional cross-talk an between these Smad proteins and of p38 MAPK and ERK1/2 in these cells. studies have that TGF-β of ATDC5 cells the expression of chondrocyte differentiation type II C. 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Furthermore, the that p38 MAPK and ERK1/2 activation following with TGF-β and that both pathways are required for TGF-β-induced activation ofAgc expression that or of of these pathways that is involved in Agc expression. is of these MAPK pathways are involved in this with these in cells, we that differentiated ATDC5 cells a of following with Although is of Smad2 phosphorylation following with TGF-β in these cells, indicating that is an TGF-β signaling pathway, the persistently high basal phosphorylation of p38 MAPK and ERK1/2 is inhibited following with Furthermore, the confluent ATDC5 cells, the differentiated cells have a high basal level of Agcexpression which p38 MAPK and ERK1/2 activation activation of the Smad2, indicating that Agc expression regulation by role of proteins in this is as these not to essential to the high basal levels of Agcexpression in differentiated ATDC5 cells. Although TGF-β basal Agc expression, to a in these cells, the for the protein to the of p38 MAPK and ERK1/2 In the of persistently high levels of p38 MAPK and ERK1/2 activation in the differentiated cells, activation of is at low transient levels of p38 MAPK and ERK1/2 activation in the confluent cells, the is In differentiated members of the TGF-β superfamily also in high levels of and are induced in ATDC5 cells. been to p38 MAPK and ERK1/2 in ATDC5 cells, and with of or chondrocyte differentiation K. T. S. S. K. F. Exp. Cell Res. 1999; PubMed Scopus Google Scholar). of these proteins for the basal MAPK activation and Agcexpression following differentiation in ATDC5 cells. have also that TGF-β-induced transcriptional activation of in confluent ATDC5 cells is the activation of ERK1/2 and p38 MAPK pathways by TGF-β in transcriptional Although this is an that not the between these pathways in the of a with Agc expression, it suggest that levels of cross-talk between these pathways involved in these for this cross-talk is that of these pathways in the transcriptional we not which are of the p38 MAPK and ERK1/2 pathways in confluent ATDC5 cells, we that Smad4 is to with a of factors, which is a of p38 MAPK H. J. M. J. H. K. E. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). not for the cross-talk between and MAPK pathways is that the are the in these cells. from the phosphorylation of MAPK in Smad2 this phosphorylation is required for its transcriptional its to with is that we not in differentiated cells in the of a high basal levels of MAPK it is that the a not these phosphorylated in of the is with that growth and growth activation of the ERK1/2 Smad2 of the and the transcriptional of P. Dev. 1998; PubMed Scopus Google Scholar). Furthermore, the role of Jr., J.N. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) have that transient activation of these MAPK pathways phosphorylation and transcriptional activation of Smad2 and with with the that which is with persistently high levels of MAPK activation of Smad2 and M. J. J. 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TGF-β also not expression in ATDC5 cells. M. P. de and Y. is in ATDC5 cells, and its expression level is not with TGF-β Thus, and not to involved in TGF-β-mediated of aggrecan expression in ATDC5 cells. Taken together, these that are transcriptional cross-talk mechanisms between p38 and Smad signaling pathways that are involved in Agcexpression at of chondrocyte differentiation, and these the mechanisms chondrocytic to the of the cartilage-specific transcriptional Agc expression. J. for expression and J. and for and for transforming growth factor-β mitogen-activated protein kinase extracellular kinase bone morphogenetic proteins extracellular matrix kinase dominant negative cycloheximide protein
Watanabe et al. (Sun,) studied this question.