TGF‐β3 inhibits chondrogenesis of cultured chick leg bud mesenchymal cells via downregulation of connexin 43 and integrin β4

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Abstract
Transforming growth factor β (TGF‐β) 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‐β3 suggests its role in chick limb development, it is not well characterized how TGF‐β3 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‐β3 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 β4 protein, in a manner similar to that observed when these cells were exposed to TGF‐β3. Similarly, exposure of cultured leg chondroblasts to a functional blocking antibody against integrin‐β4 induced an increase in apoptosis. Treatment of cells with TGF‐β3 decreased the membrane translocation of PKC‐α, leading to activation of ERK. The increase in apoptotic cell death triggered by TGF‐β3 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‐β3 treatment downregulates Cx43 and induces apoptotic cell death via downregulation of integrin β4, activation of ERK and suppression of PKC‐α activation. J. Cell. Physiol. 214: 345–353, 2008.