Evidence that thrombin-stimulated DNA synthesis in pulmonary arterial fibroblasts involves phosphatidylinositol 3-kinase-dependent p70 ribosomal S6 kinase activation

We have examined the regulation of the 79 kDa ribosomal S6 kinase (p70(s6k)) by the G protein-coupled receptor agonist alpha-thrombin and the role of this signalling molecule in the mitogenic effect of thrombin in cultured bovine pulmonary arterial (PA) fibroblasts. Thrombin stimulated p70(s6k) activity in a time and concentration-dependent manner which was abolished by the macrolide rapamycin. The phosphatidylinositol (PI) 3-kinase inhibitor wortmannin also completely blocked p70(s6k) activity in response to thrombin but did not affect p70(s6k) activity evoked by platelet-derived growth factor (PDGF) at a concentration that abrogated PDGF-stimulated PI 3-kinase activity. Activation of p70(s6k) by thrombin, but not PDGF, was also inhibited (by 48.3+/-5.4%) by pre-incubation of cells with pertussis toxin (PTX). Downregulation of protein kinase C (PKC) alpha and epsilon isoforms by pretreatment of fibroblasts for 48 h with phorbol 12-myristate 13-acetate (PMA), markedly attenuated both thrombin and PDGF-stimulated p70(s6k) activation (by 74.8+/-4.4% and 82.3+/-7.9% respectively). Thrombin also strongly stimulated (over 100 fold) the incorporation of [H-3]thymidine into growth arrested PA fibroblasts which was inhibited by rapamycin (by 33.6+/-2.0%). From these results we propose that in PA fibroblasts which was inhibited by rapamycin (by 33.6+/-2.0%). From these results we propose that in PA fibroblasts: 1) thrombin stimulates the activation of p70(s6k) in a manner consistent with an involvement of a heterotrimeric G protein of the G(i) family, a PI 3-kinase other than the PI3-kinase other than the PI3-kinase involved in signalling by PDGF, and PKC. 2) a p70(s6k)-dependent pathways plays a role in mitogenic signalling by thrombin. Copyright (C) 1997 Elsevier Science Inc.