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

We have recently demonstrated that the phospholipid platelet-activatin g factor (PAF) mediates an autocrine proliferative loop in the endomet rial adenocarcinoma cell line HEC-1A. In the present study we investig ated the signaling pathways involved in PAF-mediated increase of proli feration in these cells. In particular, we studied the effect of PAF o n protein tyrosine phosphorylation and mitogen-activated protein kinas e (MAPK) activity, as well as the effect of protein tyrosine kinase (P TK) and protein kinase C (PKC) inhibition on PAF-induced increase of c -fos gene expression and thymidine incorporation in HEC-1A cells. We f ound that PAF induced a rapid, time- and dose-dependent increase of ty rosine phosphorylation of a subset of proteins of 42, 44, 78, 99, and 150 kDa molecular weight. We also found that PAF increased tyrosine ph osphorylation 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 i ncubation with the PTK inhibitors genistein or methyl-2,5-dihydroxycin namate, whereas was strongly affected by PKC down regulation after lon g 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 [H-3]thymidine uptake in these cells. Si milar results were obtained with PD 098059, a specific inhibitor of MA P kinase cascade. PAF-stimulated [H-3]thymidine uptake was also preven ted by PKC down regulation after long term exposure to PMA and PKC inh ibition with the two inhibitors sangivamycin and bis-indolylmaleimide. In conclusion, our results indicate that PAF-induced mitogenesis in H EC-1A cells is mediated by the activation of multiple signaling pathwa ys, involving PTK, MAPK, and PKC activation.