Differential phosphorylation of p38 induced by apoptotic and anti-apoptotic stimuli in murine hepatocytes

Abstract

To investigate the differential phosphorylation and activation of p38 in hepatocytes by pro-apoptotic Transforming Growth Factor-beta1 (TGF-beta1), pro-survival factors Epidermal Growth Factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) and the potential mechanisms. The phosphorylation and activation of p38 were determined by immunoblotting. Apoptosis was analyzed by morphological staining and observation, FACS analysis of sub-G1 content and DNA fragmentation assay. To quantitatively determine caspase activation, caspase activity assay was performed in vitro. TGF-beta1-induced apoptosis was associated with the phosphorylation of p38, and SB202190, a specific inhibitor of p38, which was able to inhibit TGF-beta1-induced caspase activation and apoptosis. TPA and EGF also blocked apoptosis induced by TGF-beta1. Both of them induced the phosphorylation of p38. The results showed SB202190 had no effect on TGF-beta1-induced phosphorylation of p38, but effectively inhibited both EGF and TPA-induced phosphorylation of p38. Pro-apoptotic TGF-beta1, anti-apoptotic TPA and EGF induce the phosphorylation of p38 through different mechanisms that can be distinguished by SB202190. The data suggest that TPA and EGF-induced p38 phosphorylation is through an autophosphorylation-dependent mechanism. Since p38 phosphorylation induced by TGF-beta1 plays an important role in caspase activation and apoptosis, TPA and EGF-induced p38 phosphorylation may not be requisite for their anti-apoptotic function.