Thus, ERK and mTORC1 are key components of the intra cellular signals regulating cell growth. Involvement of epidermal growth factor receptor transactivation in sPLA2 IIA enhanced microglial cell proliferation Next, we analyzed whether selleck chem Nutlin-3a sPLA2 IIA induced Inhibitors,Modulators,Libraries cell pro liferation involves EGFR signaling, since transactivation of this receptor is a crucial signaling mechanism for con trolling cell survival, migration and proliferation. Func tional expression of EGFR in microglial cells has been previously described, and a flow cytometry analysis revealed that resting BV 2 cells also constitutively express it. After that, we investigated whether sPLA2 IIA treatment caused tyrosine phosphor ylation of EGFR at Tyr 845, as well as at Tyr 1173, by using anti phospho specific antibodies and flow cytometry analysis.

As shown in Figure 2B. a, a rapid and sustained phos phorylation of EGFR at both Tyr 1173 and Tyr 845 was detected Inhibitors,Modulators,Libraries in BV 2 cells upon phospholipase Inhibitors,Modulators,Libraries stimulation. Phosphorylation of Tyr 845 is believed to stabilize the receptor activation loop and is required for the mito genic function of the receptor, whereas phosphorylation of Tyr 1173 is involved in MAPK activation. In addition, Inhibitors,Modulators,Libraries EGFR phosphorylation in response to sPLA2 IIA was similar in extent to that observed in response to EGF. Studies on primary micro glial cells also showed EGFR phospharylation at Tyr 1173 upon sPLA2 IIA treatment. These results indicate that sPLA2 IIA is able to cause transacti vation of EGFR in microglial cells.

Next, to determine whether EGFR transactivation is required for sPLA2 IIA induced mitogenic signals, we pre incubated primary and immortalized BV 2 cells in the presence of different doses of the selective EGFR tyrosine kinase Inhibitors,Modulators,Libraries inhibitor, AG1478. We found that the presence of the inhibitor diminished the proliferative response Imatinib Mesylate chemical structure induced by 24 h of phospholipase stimulation in a dose dependent manner. The activa tion and phosphorylation of the key signaling proteins ERK, P70S6K and rS6, as well as EGFR phospholylation at Tyr 1173 was fully abol ished in AG1478 pretreated BV 2 cells. The presence of AG1478 only partially suppressed phosphorylation of Tyr 845. These findings demonstrate that EGFR transactivation accounted for sPLA2 IIA promoted cell proliferation and intracellular signaling in microglial cells, and suggest that EGFR phosphor ylation initiated by sPLA2 IIA requires its intrinsic kin ase activity. Several lines of evidence have suggested that transacti vation of EGFR may be mediated via metalloproteinases by extracellular release of EGFR ligands, such as transforming growth factor, amphiregulin and heparin binding EGF like growth factor, from the cell membrane.