Cell. Signal. Vol. 9, No. 1, pp. 109–116, 1997 ISSN 0898-6568/97 $17.00 Copyright  1997 Elsevier Science Inc. PII S0898-6568(96)00120-9 Evidence that Thrombin-stimulated DNA Synthesis in Pulmonary Arterial Fibroblasts Involves Phosphatidylinositol 3-kinase-dependent p70 Ribosomal S6 Kinase Activation Christopher M. Belham,*† Pamela H. Scott,† Dermot P. Twomey,† Gwyn W. Gould,‡ Roger M. Wadsworth† and Robin Plevin† †Department of Physiology & Pharmacology, University of Strathclyde, Royal College, 204 George Street, Glasgow G1 1XW, UK, and ‡Division of Biochemistry & Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, University Avenue, Glasgow G12 8QQ, UK ABSTRACT. We have examined the regulation of the 70 kDa ribosomal S6 kinase (p70 s6k ) by the G-protein- coupled receptor agonist -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 con- centration-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-stimu- lated 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)  and  iso- forms 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). Throm- bin also strongly stimulated (over 100 fold) the incorporation of [ 3 H]thymidine into growth arrested PA fibro- blasts 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 PI 3-kinase involved in signalling by PDGF, and PKC. 2) a p70 s6k -dependent pathway plays a role in mitogenic signalling by thrombin. Copyright  1997 Elsevier Sci- ence Inc. cell signal 9;1:109–116, 1997. KEY WORDS. p70 Ribosomal S6 Kinase, Thrombin, DNA synthesis, Pulmonary Arterial Fibroblast INTRODUCTION p85 s6k ) (for reviews see [4, 5]). Whereas a role for elements of the MAP kinase pathway in the mitogenic response to The serine protease -thrombin has been shown to be po- thrombin has been described [1, 2, 6], no studies to date tent mitogen for a number of cell types, notably fibroblasts have addressed the involvement of p70 s6k /85 s6k in thrombin- and vascular smooth muscle cells [1, 2, 3]. A number of sig- stimulated growth. nal transduction pathways have been implicated in regulat- Both p70 s6k /p85 s6k isoforms of the same kinase are en- ing cell growth and division in response to G-protein-cou- coded by a common gene and found in different subcellular pled receptor agonists, such as thrombin, and growth factors locations. The p70 s6k is found in the cytoplasm while the 23- which activate protein tyrosine kinase receptors. These in- amino acid amino terminus extension of p85 s6k contains a clude distinct cascades involving the serine-threonine ki- sequence that targets this isoform to the nucleus [7, 8]. The nase families mitogen-activated protein (MAP) kinase and p70 s6k has been shown to be obligatory for cell cycle progres- the 70 and 85 kDa 40S ribosomal protein S6 kinases (p70/ sion as serum-stimulated cells are arrested in G l phase when microinjected with antibodies to p70 s6k [9] or treated with * Author to whom correspondence should be addressed. the selective inhibitor of the p70 s6k pathway rapamycin [10]. Abbreviations: p70 s6k , p70 ribosomal protein S6 kinase; PA, pulmonary Phosphorylation of the S6 region of the 40S ribosomal sub- arterial; PI 3-kinase, phosphatidylinositol 3-kinase; PDGF, platelet-de- unit by p70 s6k [11] is believed to contribute to an increased rived growth factor; PTX, pertussis toxin; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; MAP kinase, mitogen-activated protein rate of protein synthesis during G l necessary for entry into kinase; RTK, receptor tyrosine kinase; DMEM, Dulbecco’s modified Ea- S phase, possibly by selectively upregulating translation of gle’s medium; NCS, newborn calf serum. Received 30 May 1996; and accepted 1 July 1996. a family of mRNAs essential for cell growth [12]. The func-