Transcriptional cross-talk between Smad, ERK1/2, and p38 mitogen-activatedprotein kinase pathways regulates transforming growth factor-beta-induced aggrecangene expression in chondrogenic ATDC5 cells

In chondrogenesis, members of the transforming growth factor-beta (TGF-beta ) superfamily play critical roles by inducing gene expression of cartilage- specific molecules. By using a chondrogenic cell line, ATDC5, we investigat ed the TGF-beta -mediated signaling pathways involved in expression of the aggrecan gene (Agc). At confluency, TGF-beta induced Age expression within 3 h, and cycloheximide blocked this induction, indicating that de novo prot ein synthesis is essential for this response. At this stage, TGF-beta induc ed rapid, transient phosphorylation of Smad2, extracellular signal-activate d kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (MAPK). Inh ibition of the Smad pathways by transfection with a dominant negative Smad4 construct significantly reduced TGF-beta -induced Age expression, indicati ng that Smad signaling is essential for this response. Furthermore, an inhi bitor of the ERK1/2 pathway, U0126, or inhibitors of the p38 MAPK pathway, SB203580 and SKF86002, repressed TGF-beta -induced Age expression in a dose -dependent manner, indicating that ERK1/2 or p38 MAPK activation is also re quired for TGF-beta -induced Agc expression in confluent ATDC5 cells. In di fferentiated ATDC5 cells, persistently high basal levels of ERK1/2 and p38 MAPK phosphorylation correlated with elevated basal Age expression, which w as inhibited by incubation with inhibitors of these pathways. Whereas Smad2 was rapidly phosphorylated by TGF-beta and involved in the initial activat ion of Agc expression in confluent cells, Smad2 activation was not required for maintaining the high level of Age expression. Taken together, these re sults suggest an important role for transcriptional cross-talk between Smad and MAPK pathways in expression of early chondrocytic phenotypes and ident ify important changes in the regulation of Age expression following chondro cyte differentiation.