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Activation of specific MEK-ERK cascade is necessary for TGFβ signaling and crosstalk with PKA and PKC pathways in cultured rat articular chondrocytes

2000; Elsevier BV; Volume: 8; Issue: 4 Linguagem: Inglês

10.1053/joca.1999.0297

1522-9653

Yasuo Hirota, T Tsukazaki, Akihiko Yonekura, Yoshitsugu Miyazaki, Makoto Osaki, Hiroyuki Shindo, Shunichi Yamashita,

Inflammatory mediators and NSAID effects

TGFbeta is a potent stimulator of cell growth in cultured rat articular chondrocytes (CRAC). The stimulatory effect is mediated through the immediate induction of c-fos gene by activating ERK of MAPK. The present study was undertaken to investigate the upstream regulators involved in TGFbeta-induced ERK activation in CRAC and to compare the results with the events in HepG2 cells.In vitro kinase and trans-reporting assays showed that TGFbeta preferentially activated ERK and JNK pathways in CRAC and HepG2, respectively. ERK activation in CRAC was selectively inhibited by PD98059, a MEK inhibitor. Overexpression of wild or active forms of MEKK1, the upstream activator of ERK and JNK, decreased the TGFbeta-induced 3TP-luciferase activity in CRAC. In contrast, in HepG2 dominant negative form of MEKK1 or SEK1 ligand-dependent reporter activity was diminished. Transfection of TAK1, another MAPKKK, also positively and negatively regulated 3TP transcriptional activity of HepG2 and CRAC, respectively. Activation of PKA by 8-bromo-cyclic AMP or forskolin, and inhibition of PKC by calphostin C, resulted in a significant decrease in 3TP activity as well as in vitro ERK kinase activity in CRAC.The results indicate that TGFbeta transduces a predominant signal pathway through MEK-ERK-Elk1, independent of MEKK1 or TAK1 pathway in CRAC. However, in HepG2, activation of MEKK1 and TAK1 is essential for TGFbeta-induced signal transmission. The results also demonstrated that in CRAC, MEK-ERK pathway activated by TGFbeta is negatively regulated by PKA cascade but transactivated by PKC.