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.
Jin
Eun-Jung
EJ
Department of Biology, College of Natural Sciences (BK21), Kyungpook National University, Daegu, Korea.
Lee
Sun-Young
SY
Jung
Jae-Chang
JC
Bang
Ok-Sun
OS
Kang
Shin-Sung
SS
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002458:Cell Fractionation; D002478:Cells, Cultured; D002642:Chick Embryo; D020219:Chondrogenesis / Q000187:drug effects*; D018031:Connexin 43 / Q000378:metabolism*; D015536:Down-Regulation; D018274:Electroporation; D005121:Extremities / Q000196:embryology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D039663:Integrin beta4 / Q000378:metabolism*; D008648:Mesoderm / Q000166:cytology / Q000187:drug effects*; D012333:RNA, Messenger / Q000378:metabolism; D053782:Transforming Growth Factor beta3 / Q000494:pharmacology*; D014921:Wings, Animal / Q000166:cytology
D018031:Connexin 43; D039663:Integrin beta4; D012333:RNA, Messenger; D053782:Transforming Growth Factor beta3