RESUMO
Late-phase and sustained activation of p44/42(MAPK) has been reported to be a critical factor in cell mitogenesis. We therefore hypothesized that p44/42(MAPK) is involved in mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth-muscle cells (ASMC). Treatment of adherent ASMC with beta-hexosaminidase A (Hex A, 50 nM), an endogenous mannosyl-rich glycoprotein, resulted in a late-onset (30-min) activation of p44/42(MAPK) that lasted for 4 h. Activation of p44/42(MAPK) induced by Hex A was inhibited by an 18-mer phosphorothioate-derivatized antisense oligonucleotide (1-5 microM) directed to human p44(MAPK); the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059 (5 microM); the p42(MAPK) inhibitor Tyrphostin AG-126 (0.2 microM); the farnesyl transferase inhibitors SCH-56582 (10 microM) and FPT III (10 miroM), which inhibit p21Ras activation; and Calphostin C (0.2 microM), an inhibitor of protein kinase C. These agents also inhibited Hex A-induced cell proliferation in bovine ASMC. These data suggest that Hex A activates p44/42(MAPK) in a p21Ras- and PKC-dependent manner and that this activation mediates Hex A- induced mitogenesis in bovine ASMC.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Músculo Liso/efeitos dos fármacos , Proteína Quinase C/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Traqueia/efeitos dos fármacos , beta-N-Acetil-Hexosaminidases/farmacologia , Alquil e Aril Transferases/antagonistas & inibidores , Animais , Bovinos , Divisão Celular , Células Cultivadas , Ativação Enzimática , Hexosaminidase A , MAP Quinase Quinase 1 , Proteína Quinase 3 Ativada por Mitógeno , Músculo Liso/citologia , Oligonucleotídeos Antissenso/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Fatores de Tempo , Traqueia/enzimologiaRESUMO
beta-Hexosaminidases (Hex) A and B promote mitogenesis via airway smooth muscle (ASM) mannose receptor. The objective of this study was to elucidate the contribution of protein kinase C (PKC) in Hex-induced mitogenesis in ASM cells (ASMC). Exposure of ASMC to Hex caused increases in both the calcium-dependent and the calcium-independent PKC activities. Both downregulation of PKC and PKC inhibitors staurosporine and calphostin C diminished Hex-induced DNA synthesis and cell number. Hex-induced DNA synthesis was enhanced by a diacylglycerol kinase inhibitor, R-59022, which was blocked by calphostin C. These data suggest that activation of PKC in part mediates Hex-induced mitogenesis in ASMC.