Protein tyrosine kinase, mitogen-activated protein kinase and protein kinase C are involved in the mitogenic signaling of platelet-activating factor (PAF) in HEC-1A cells

Abstract

We have recently demonstrated that the phospholipid platelet-activating factor (PAF) mediates an autocrine proliferative loop in the endometrial adenocarcinoma cell line HEC-1A. In the present study we investigated the signaling pathways involved in PAF-mediated increase of proliferation in these cells. In particular, we studied the effect of PAF on protein tyrosine phosphorylation and mitogen-activated protein kinase (MAPK) activity, as well as the effect of protein tyrosine kinase (PTK) and protein kinase C (PKC) inhibition on PAF-induced increase of c-fos gene expression and thymidine incorporation in HEC-1A cells. We found that PAF induced a rapid, time- and dose-dependent increase of tyrosine phosphorylation of a subset of proteins of 42, 44, 78, 99, and 150 kDa molecular weight. We also found that PAF increased tyrosine phosphorylation and activity of p42 MAPK, suggesting the involvement of this important intermediary enzyme in the proliferative effect of PAF. The effect of PAF on c-fos gene expression was not prevented by pre incubation with the PTK inhibitors genistein or methyl-2,5-dihydroxycinnamate, whereas was strongly affected by PKC down regulation after long term incubation with PMA or by PKC inhibition with sangivamycin. We also found that genistein and methyl 2,5-dihydroxycinnamate decreased both basal and PAF-stimulated [3H]thymidine uptake in these cells. Similar results were obtained with PD 098059, a specific inhibitor of MAP kinase cascade. PAF-stimulated [3H]thymidine uptake was also prevented by PKC down regulation after long term exposure to PMA and PKC inhibition with the two inhibitors sangivamycin and bis-indolylmaleimide. In conclusion, our results indicate that PAF-induced mitogenesis in HEC-1A cells is mediated by the activation of multiple signaling pathways, involving PTK, MAPK, and PKC activation.