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 70 kDa ribosomal S6 kinase (p70s6k) 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 p70s6k 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 p70s6k activity in response to thrombin but did not affect p70s6k activity evoked by platelet-derived growth factor (PDGF) at a concentration that abrogated PDGF-stimulated PI 3-kinase activity. Activation of p70s6k 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 p70s6k activation (by 74.8 +/- 4.4% and 82.3 +/- 7.9% respectively). Thrombin also strongly stimulated (over 100 fold) the incorporation of [3H]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: 1) thrombin stimulates the activation of p70s6k in a manner consistent with an involvement of a heterotrimeric G protein of the G(i) family, a PI 3-kinase other than the PI 3-kinase involved in signalling by PDGF, and PKC. 2) a p70s6k-dependent pathway plays a role in mitogenic signalling by thrombin.
Belham
C M
CM
Department of Physiology & Pharmacology, University of Strathclyde, Royal College, Glasgow, UK.
Scott
P H
PH
Twomey
D P
DP
Gould
G W
GW
Wadsworth
R M
RM
Plevin
R
R
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D004247:DNA / Q000096:biosynthesis*; D004789:Enzyme Activation; D005260:Female; D005347:Fibroblasts; D008934:Mitogens / Q000494:pharmacology*; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011651:Pulmonary Artery; D019893:Ribosomal Protein S6 Kinases; D013917:Thrombin / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D004247:DNA; D008934:Mitogens; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D010982:Platelet-Derived Growth Factor; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases; D013917:Thrombin; D010566:Virulence Factors, Bordetella; D000077191:Wortmannin