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Serotonin-Induced Growth of Pulmonary Artery Smooth Muscle Requires Activation of Phosphatidylinositol 3-Kinase/Serine-Threonine Protein Kinase B/Mammalian Target of Rapamycin/p70 Ribosomal S6 Kinase 1

Tufts–New England Medical Center, Pulmonary, Critical Care and Sleep Division, Tupper Research Institute, Boston, Massachusetts

Correspondence and requests for reprints should be addressed to Barry L. Fanburg, M.D., Pulmonary, Critical Care, and Sleep Division, Tufts–New England Medical Center, 750 Washington Street, #257, Boston, MA 02111. E-mail: bfanburg{at}tufts-nemc.org

We have previously found that both mitogen-activated protein kinase (MAPK)- and Rho kinase (ROCK)-related signaling pathways are necessary for the induction of pulmonary artery smooth muscle cell (SMC) proliferation by serotonin (5-hydroxytryptamine [5-HT]). In the present study, we investigated the possible additional participation of a phosphatidylinositol 3-kinase (PI3K)/serine-threonine protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (S6K1) pathway in this growth response. We found transient activation of Akt (Ser⁴⁷³) and more prolonged activation of S6K1 by 5-HT. Inhibition of PI3K with Wortmannin and LY294002 completely blocked these activations, but not that of MAPK or the ROCK substrate myosin phosphatase targeting subunit. Similarly, inhibition of MAPK and ROCK failed to block the Akt activation. Inhibition of Akt with NL-71–101 and downregulation of Akt expression with Akt small interfering RNA blocked 5-HT–induced S6K1 phosphorylation. Wortmannin, LY294002, and NL-71–101 dose-dependently inhibited 5-HT–induced SMC proliferation. 5-HT stimulated mTOR phosphorylation and the mTOR inhibitor, rapamycin, blocked activations of S6K1 and S6 ribosomal protein, and inhibited 5-HT–induced SMC proliferation. Akt phosphorylation and cell proliferation were also blocked by the antioxidants, N-acetyl-l-cysteine, Ginko biloba 501, and tiron, the reduced nicotinamide adenine dinucleotide phosphate oxidase inhibitor, diphenyleneiodonium, and the 5-HT2 receptor antagonists ketanserin and mianserin, but not by the 5-HT serotonin transporter or 5-HT 1B/1D receptor antagonists. We conclude from these studies that a parallel PI3K- and reactive oxygen species–dependent Akt/mTOR/S6K1 pathway participates independently from MAPK and Rho/ROCK in the mitogenic effect of 5-HT on pulmonary artery SMCs. From these and other studies, we postulate that independent signaling pathways leading to 5-HT–induced SMC proliferation are initiated through multiple 5-HT receptors and serotonin transporter at the cell surface.

Key Words: mammalian target of rapamycin • p70 ribosomal S6 kinase • phosphatidylinositol 3-kinase • pulmonary hypertension • serine/threonine protein kinase • serotonin • smooth muscle cells

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