Sphingosine-1-phosphate–induced oxygen free radical generation in smooth muscle cell migration requires Gα12/13 protein-mediated phospholipase C activation

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Sphingosine-1-phosphate–induced oxygen free radical generation in smooth muscle cell migration requires Gα12/13 protein-mediated phospholipase C activation Eliza Roztocil, BS, Suzanne M. Nicholl, PhD, Mark G. Davies, MD, PhD Journal of Vascular Surgery Volume 46, Issue 6, Pages 1253-1259.e3 (December 2007) DOI: 10.1016/j.jvs.2007.08.013 Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 1 Activation of phospholipase C-β (PLC-β). A, Sphingosine-1-phosphate (S-1-P, 0.1μM)–induced time-dependent activation of PLC-β, PLC-γ1, or PLC-γ2, as measured by Western blotting. Maximal activation was at 5 minutes. PLC-β activation was blocked by U7 (10nM) but not by InactiveU7 (10nM). B, A representative Western blot for PLC-β is shown. C, Bar chart shows quantification of S-1-P–induced time-dependent activation of PLC-β, PLC-γ1, or PLC-γ2, in the presence and absence of U7 and InactiveU7. Values are the mean ± SEM of the ratio of the phosphorylation of the respective protein relative to the total unphosphorylated protein (n = 6). P < .05 compared with the control. DMEM, Dulbecco minimal essential medium. Journal of Vascular Surgery 2007 46, 1253-1259.e3DOI: (10.1016/j.jvs.2007.08.013) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 2 G-proteins and phospholipase C-β (PLC-β). A, Activation of PLC-β by sphingosine-1-phosphate (0.1μM) was Gαi-independent; that is, it was not blocked by pertussis toxin (PTx), a Gαi inhibitor, or Gαi2 and Gαi3 small interfering RNA (siRNA); and it was Gαq-independent (not blocked by GP-2A, a Gαq inhibitor, or Gαq siRNA). Small interfering RNA to Gα12/13 blocked PLC-β activation. Transfection with adenoviral COOH-terminus β-adrenergic receptor kinase (β-ΑRKCT) also blocked PLC-β activation. Representative Western blots are shown with α-actin as a loading control. B, The bar chart shows the quantification of the experiments representative in Fig 1, A and also shows additional controls. Scrambled siRNA and empty adenovirus did not significantly alter the response compared with control values. Values are the mean ± SEM of the ratio of the phosphorylation of PLC-β (p-PLC-β) relative to the total unphosphorylated PLC-β (t-PLC-β; n = 6). *P < .05 compared with control. DMEM, Dulbecco minimal essential medium; GP2A, glycoprotein 2A. Journal of Vascular Surgery 2007 46, 1253-1259.e3DOI: (10.1016/j.jvs.2007.08.013) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 3 Phospholipase C-β (PLC-β) and cell migration. A, Vascular smooth muscle cell migration in response to sphingosine-1-phosphate (S-1-P; 0.1μM) in both the wound assay and in (B) the Boyden chamber assays of migration was inhibited by increasing concentrations of the PLC-β inhibitor U7 (0.1 to 1000nM, preincubated with the cells for 1 hour before each assay) but not the inactive analogue, InactiveU7 (0.1 to 1000nM, preincubated with the cells for 1 hour before each assay). C, Migration in response to S-1-P (0.1μM) in the Boyden chamber assay was inhibited by small interfering RNA (siRNA) to Gα12/13 and adenoviral COOH-terminus β-adrenergic receptor kinase (adeno β-ΑRKCT) transfection. The transfection of scrambled siRNA or empty adenoviral had no effect. D, Cell migration in response to S-1-P (0.1μM) in the Boyden chamber was also inhibited by the nicotinamide dinucleotide phosphate hydrogen (NAD[P]H) oxidase inhibitor, diphenyleneiodonium chloride (DPI). Values are the mean ± SEM (n = 6). *P < .05; **P < .01 compared with control. Journal of Vascular Surgery 2007 46, 1253-1259.e3DOI: (10.1016/j.jvs.2007.08.013) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig E1 (online only) Western blots of small interfering RNA (si RNA) for G-proteins. We examined the expression of the G-proteins in the presence of specific siRNA to Gαi2, Gαi3, Gαq, and Gα12/13 after performing concentration-dependent suppression experiments. Western blots of Gαi2, Gαi3, Gαq, and Gα12/13, are shown in the presence of each siRNA used and the scrambled siRNA control. There is effective downregulation of each G-protein with the respective G-protein siRNA without affecting other G-proteins. We have also added Gαs and α-actin as loading controls. Journal of Vascular Surgery 2007 46, 1253-1259.e3DOI: (10.1016/j.jvs.2007.08.013) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig E2 (online only) Oxygen free radical production: sphingosine-1-phosphate (S-1-P; 0.1μM)–induced oxygen free radical production as measured by quantitative dihydroethidium staining was significantly inhibited by U7 (10nM) but not by InactiveU7 (10nM). Representative photographs of the staining are shown. A, Dulbecco minimal essential medium (DMEM); (B) S-1-P; (C) S-1-P with InactiveU7; and (D) S-1-P with U7). E, The bar chart shows the quantification of the experiment A to D. F, The bar chart shows oxygen free radical production in response to angiotensin II and S-1-P in the presence and absence of absence of the nicotinamide dinucleotide phosphate hydrogen oxidase inhibitor, diphenyleneiodonium chloride (DPI; 10μM), as measured by quantitative dihydroethidium staining. Values are the mean ± SEM (n = 6). *P < .05; **P < 0.01 compared with control. Journal of Vascular Surgery 2007 46, 1253-1259.e3DOI: (10.1016/j.jvs.2007.08.013) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig E3 (online only) Mitogen-activated protein kinase (MAPK) and phospholipase C-β (PLC-β). A, The presence of U7 (10nM) attenuated sphingosine-1-phosphate (S-1-P; 0.1μM)–mediated (ERK1/2) and (B) p38MAPK activation but (C) had no effect on akt activation as measured by Western blotting. The presence of InactiveU7 (10nM) had no effect, and the results could be superimposed on the control curves. D, A representative Western blot of the maximum response for each kinase is shown. Both activated and total protein expression is shown. Values are the mean ± SEM of the ratio of the phosphorylation of the respective protein relative to the total unphosphorylated protein (n = 6). *P < .05 compared with control. DMEM, Dulbecco minimal essential medium. Journal of Vascular Surgery 2007 46, 1253-1259.e3DOI: (10.1016/j.jvs.2007.08.013) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions