TGF-beta3 inhibits chondrogenesis of cultured chick leg bud mesenchymal cells via downregulation of connexin 43 and integrin beta4.

Transforming growth factor beta (TGF-beta) is a multifunctional cytokine that regulates a number of biological responses including chemotaxis, cell cycle progression, differentiation, and apoptosis of cells. Even though temporal and spatial expression of TGF-beta3 suggests its role in chick limb development, it is not well characterized how TGF-beta3 regulates chondrogenic differentiation of limb bud mesenchymal cells. In this study, differential display polymerase chain reaction (DD-PCR) screening and reverse transcription PCR analysis revealed that the mRNA expression of the gap junction protein, connexin 43 (Cx43), was significantly decreased during the first treatment of TGF-beta3 for 24 h in cultured chick leg bud mesenchymal cells. Treatment of these cells with lindane, a general gap junction blocker, or expression of dominant negative Cx43 increased apoptotic cell death and decreased the level of integrin beta4 protein, in a manner similar to that observed when these cells were exposed to TGF-beta3. Similarly, exposure of cultured leg chondroblasts to a functional blocking antibody against integrin-beta4 induced an increase in apoptosis. Treatment of cells with TGF-beta3 decreased the membrane translocation of PKC-alpha, leading to activation of ERK. The increase in apoptotic cell death triggered by TGF-beta3 and dominant negative Cx43 was blocked by inhibition of ERK but increased by inhibition of PKC. Collectively, these data indicate that, in cultured chick leg bud mesenchyme cells, TGF-beta3 treatment downregulates Cx43 and induces apoptotic cell death via downregulation of integrin beta4, activation of ERK and suppression of PKC-alpha activation.