sphingosine-1-phosphate and diphenyleneiodonium

Sphingosine-1-phosphate (S-1-P) is a bioactive sphingolipid that stimulates the migration of vascular smooth muscle cell (VSMC) through G-protein coupled receptors; it has been shown to activate reduced nicotinamide dinucleotide phosphate hydrogen (NAD[P]H) oxidase. The role of phospholipase C (PLC) in oxygen free radical generation, and the regulation of VSMC migration in response to S-1-P, are poorly understood.. Rat arterial VSMC were cultured in vitro. Oxygen free radical generation was measured by fluorescent redox indicator assays in response to S-1-P (0.1microM) in the presence and absence of the active PLC inhibitor (U73122; U7, 10nM) or its inactive analog U73343 (InactiveU7, 10nM). Activation of PLC was assessed by immunoprecipitation and Western blotting for the phosphorylated isozymes (beta and gamma). Small interfering (si) RNA to the G-proteins Galphai, Galphaq, and Galpha12/13 was used to downregulate specific proteins. Statistics were by one-way analysis of variance (n = 6).. S-1-P induced time-dependent activation of PLC-beta and PLC-gamma; PLC-beta but not PLC-gamma activation was blocked by U7 but not by InactiveU7. PLC-beta activation was Galphai-independent (not blocked by pertussis toxin, a Galphai inhibitor, or Galphai2 and Galphai3 siRNA) and Galphaq-independent (not blocked by glycoprotein [GP] 2A, a Galphaq inhibitor, or Galphaq siRNA). PLC-beta activation and cell migration was blocked by siRNA to Galpha12/13. Oxygen free radical generation induced by S-1-P, as measured by dihydroethidium staining, was significantly inhibited by U7 but not by InactiveU7. Inhibition of oxygen free radicals with the inhibitor diphenyleneiodonium resulted in decreased cell migration to S-1-P. VSMC mitogen-activated protein kinase activation and VSMC migration in response to S-1-P was inhibited by PLC- inhibition.. S-1-P induces oxygen free radical generation through a Galpha12/13, PLC-beta-mediated mechanism that facilitates VSMC migration. To our knowledge, this is the first description of PLC-mediated oxygen free radical generation as a mediator of S-1-P VSMC migration and illustrates the need for the definition of cell signaling to allow targeted strategies in molecular therapeutics for restenosis.

Topics: Animals; Cell Movement; Cells, Cultured; Enzyme Activation; Estrenes; GTP-Binding Protein alpha Subunits, G12-G13; Lysophospholipids; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NADPH Oxidases; Onium Compounds; p38 Mitogen-Activated Protein Kinases; Pertussis Toxin; Phosphodiesterase Inhibitors; Phospholipase C beta; Phospholipase C gamma; Phosphorylation; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Signal Transduction; Sphingosine; Time Factors

A growing amount of evidence suggests that reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, regulate intracellular signalling and have a role in cell proliferation. In the present study, we show that platelets increase the mitogenic rate in human fibroblasts and that this effect was inhibited by the intracellular antioxidant N-acetyl-L-cysteine (NAC) and the NADPH-oxidase inhibitor diphenyleneiodonium chloride (DPI). The mitogenic effects of platelets were mimicked by the platelet factors platelet-derived growth factor BB-isoform (PDGF-BB), transforming growth factor beta1 (TGF-beta1) and sphingosine-1-phosphate (S1P). The sphingosine kinase inhibitor DL-threo-dihydrosphingosine (DL-dihydro) abrogated the platelet-induced growth, while antibodies directed against PDGF or TGF-beta had modest effects. Exposure of fibroblasts to platelets, PDGF-BB, TGF-beta1 or S1P caused an extensive intracellular ROS production, measured as changes in dichlorofluorescein fluorescence. This ROS production was totally inhibited by NAC, pyrrolidinethiocarbamate (PDTC), DPI and apocynin. In conclusion, the results presented are indicative of a crucial role of ROS in the platelet-mediated regulation of fibroblast proliferation.

Topics: Acetophenones; Acetylcysteine; Becaplermin; Blood Platelets; Cell Division; Cells, Cultured; Enzyme Inhibitors; Fibroblasts; Fluoresceins; Humans; Lysophospholipids; NADPH Oxidases; Onium Compounds; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Reactive Oxygen Species; Skin; Sphingosine; Thiocarbamates; Transforming Growth Factor beta; Transforming Growth Factor beta1