Mechanisms Of Phospholipase C-γ1 Survival Signaling In Oxidative Stress

Phospholipase C-γl (PLC-γl) is known to be activated in response to growth factor stimulation by a mechanism that relies on tyrosine phosphorylation and plays an important role in regulating cell proliferation and differentiation through the generation of the second messengers diacylglycerol and inositol 1,4,5-trisphosphate, leading to the activation of protein kinase C (PKC) and increased levels of intracellular calcium, respectively. Given the existing overlap between signaling pathways that are activated in response to oxidant injury and those involved in responding to proliferative stimuli, we investigated the role of PLC-yl and its downstream targets during the cellular response to oxidative stress by using mouse embryonic fibroblasts (MEF) genetically deficient in PLC-yl (Plcgl-/-) and its wild type (Plcgl+/+) as models.Both Plcgl-/- and Plcgl+/+ MEFs undergo rapid cell death when stimulated with high dose (5 mM) of H2O2. When treated with low doses (0.05-0.5 mM) of H2O2, however, Plcgl-/- MEF was much more sensitive (P<0.05) to H2O2 induced apoptosis than Plcgl+/+ MEF which PLC-yl was tyrosine phosphorylated in a dose- and time-dependent manner. The H2O2-induced PLC-yl tyrosine phosphorylation in Plcgl+/+ MEF could be blocked by herbimycin A, a protein tyrosine kinase (PTK)-specific inhibitor, but not by wortmannin, a phosphoinositide 3-kinase (PO-K)-specific inhibitor. It is suggested that PTK-mediated activation of PLC-yl enhances cell survival in oxidative stress.We further examined the downstream targets of PLC-yl activation in H2O2-induced oxidative stress. H2O2 stimulated extracellular regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and induced heat shock protein 70 (HSP70), p53 either in Plcgl'1' or in Plcgl*1* MEFs. Activation of PKC was also markedly increased in both cell lines treated with H2O2 (1-5 mM), but with low doses (50-200 |iM), PKC activation was-5-considerably decreased in Plcgl-/- cells (P<0.05). After treatment with H2O2, PKC-dependent phosphorylation of Bcl-2 and cell viability of Plcgl'1' cells decreased dramatically and caspase-3 activity increased significantly compared with that of the wild-type cells. Furthermore, pretreatment of Plcgl+/+ cells with PKC-specific inhibitor decreased levels of PKC dependent Bcl-2 phosphorylation, enhanced caspase-3 activity and their sensitivity to HaOi. On the contrary, treatment of Plcgl-/- cells with PKC-specific activator increased the Bcl-2 phosphorylation, decreased caspase-3 activity and improved their survival. In addition, stability of PKC and expression of Bag-1, a bcl-2-binding protein, were also significant decreased in Plcgl-/-MEF compared with that of wild-type cells after treatment by H2O2. These results suggested that Bag-1, PKC-dependent phosphorylation of Bcl-2 and inhibition of caspase-3 maybe the downstream of PLC-yl activation in oxidative stress. ERK, p38 MAPK, HSP70 and p53 are not involved in PLC-yl survival signaling.Taken together, we concluded that PLC-yl mediates survival signaling in oxidative-stress response by PKC-dependent phosphorylation of Bcl-2 and inhibition of caspase-3.