Phospholipase D signaling in serotonin-induced mitogenesis of pulmonary artery smooth muscle cells.
We have previously reported the participation of mitogen-activated protein, Rho, and phosphoinositide-3 (PI3) kinases in separate pathways in serotonin (5-HT)-induced proliferation of pulmonary artery smooth muscle cells (SMCs). In this study, we investigated the possible participation of phospholipase D (PLD) and phosphatidic acid (PA) in this growth process. 5-HT stimulated a time-dependent increase in [(3)H]phosphatidylbutanol and PA generation. Exposure of SMCs to 1-butanol or overexpression of an inactive mutant of human PLD1R898R blocked 5-HT-induced proliferation. Furthermore, 1-butanol inhibited 5-HT activation of S6K1 and S6 protein, downstream effectors of mammalian target of rapamycin (mTOR), by 80 and 72%, respectively, and partially blocked activation of extracellular signal-regulated kinase (ERK) by 30% but had no effect on other associated signaling pathways. Exogenous PA caused cellular proliferation and revitalized cyclin D1 expression by 5-HT of the 1-butanol-treated cells. PA also reproduced activations by 5-HT of mTOR, S6K1, and ERK. Transfection with inactive human PLD1 reduced 5-HT-induced activation of S6K1 by approximately 50%. Inhibition of 5-HT receptor 2A (R 2A) with ketaserin blocked PLD activation by 5-HT. Inhibition with PI3-kinase inhibitor failed to block either activation of PLD by 5-HT or PA-dependent S6K1 phosphorylation. Taken together, these results indicate that ligation of the 5-HTR 2A by 5-HT initiates PLD activation in SMCs, and that its product, PA, is an early signaling molecule in 5-HT-induced pulmonary artery SMC proliferation. Signaling by PA produces its downstream effects primarily through the mTOR/S6K1 pathway and to a lesser extent through the ERK pathway. Hydrolysis of cell membrane lipid may be important in vascular effects of 5-HT.
Liu
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Tufts Medical Center, 750 Washington St., Boston, MA 02111, USA.
Fanburg
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BL
D000818:Animals; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008954:Models, Biological; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000378:metabolism / Q000494:pharmacology; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D044402:Receptor, Serotonin, 5-HT2A / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects
D010712:Phosphatidic Acids; D044402:Receptor, Serotonin, 5-HT2A; D011994:Recombinant Proteins; D012701:Serotonin; D010739:Phospholipase D; C106801:phospholipase D1