In this study, we analyzed in rat myometrial cells the signaling pathways involved in the endothelin (ET)-1-induced extracellular signal-regulated kinase (ERK) activation required for the induction of DNA synthesis. We found that inhibition of protein kinase C (PKC) by Ro-31-8220 abolished ERK activation. Inhibition of phospholipase C (PLC) by U-73122 or of phosphoinositide (PI) 3-kinase by wortmannin partially reduced ERK activation. A similar partial inhibition was observed after treatment with pertussis toxin or PKC downregulation by phorbol ester treatment. The effect of wortmannin was additive with that produced by PKC downregulation but not with that due to pertussis toxin. These results suggest that both diacylglycerol-sensitive PKC, activated by PLC products, and diacylglycerol-insensitive PKC, possibly activated by a G(i)-PI 3-kinase-dependent process, are involved in ET-1-induced ERK activation. These two pathways were found to be activated mainly through the ET(A) receptor subtype. ET-1 and phorbol ester stimulated Src activity in a PKC-dependent manner, both responses being abolished in the presence of Ro-31-8220. Inhibition of Src kinases by PP1 abrogated phorbol ester- and ET-1-induced ERK activation. Finally, ET-1 activated Ras in a PP1- and Ro-31-8220-sensitive manner. Altogether, our results indicate that ET-1 induces ERK activation in rat myometrial cells through the sequential stimulation of PKC, Src, and Ras.

Class I PI3Ks, through the formation of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), are thought of as essential elements of the neutrophil response to chemotactic factors. Moreover, the recent development of PI3K-deficient mice and isoform-specific inhibitors enabled examinations of the contribution of the distinct PI3K isoforms in neutrophil activation. However, the results of these various studies are conflicting, and the exact role of the different PI3K isoforms is not yet clearly established, particularly in human cells. In the present study, we used a different approach to assess the role of the distinct PI3K isoforms in response to the chemotactic agent fMLP. We inhibited PI3K activities by the transient expression following nucleofection of dominant negative mutants of either p85α or p110γ in the human myeloid cell line PLB-985, which can be induced to express a neutrophil-like phenotype. The data obtained with this approach showed that the production of PI(3,4,5)P3 triggered by fMLP is biphasic, with a peak of production observed in a short time period that entirely depends on p110γ activity, and a delayed phase that is mediated by class IA PI3K. We also provide evidence that the PI3K-dependent functional responses (i.e., superoxide production and chemotaxis) induced by the chemotactic factor mainly involve PI3K IA and, by implication, the delayed phase of PI(3,4,5)P3 production, whereas p110γ and the early peak of PI(3,4,5)P3 do not play major roles in the initiation or the control of these responses.

Endothelin-1 (ET-1), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) stimulated thymidine incorporation with different efficiency (PDGF >> EGF = ET-1) in rat myometrial cells. They also stimulated ERK activation, which culminated at 5 min and then declined to reach a plateau (at 45 min: EGF > 90%, PDGF = 50%, and ET-1 < 10% of maximum). Inhibition and downregulation of PKC demonstrated that ERK activation at 5 min involved PKC delta and -zeta for ET-1 and PKC alpha plus another PKC isoform for PDGF. By contrast, the EGF response did not involve PKC. Stimulation of Ras was more important with EGF than with PDGF, with ET-1 being the weakest activator. The simultaneous incubation of the cells with EGF and ET-1 potentiated the ERK activation at 5 min and mimicked the plateau phase obtained with PDGF. Under these conditions thymidine incorporation was comparable to that induced by PDGF. Taken together, our results indicated that the kinetic profile of ERK activation and its impact on cell proliferation can be modulated by the differential involvement of PKC isoforms and the amplitude of Ras activation.