H2S Signals through VEGFR-PI3K-Rac1-Cofilin Figure 8. Dominant negative Rac1 and Rac1 siRNA abolish the phosphorylation of cofilin initiated by H2S, but have no effect on that of ERK. Representative blots (A) and statistical analysis (B) showing the effects of dominant negative Rac1 on the phosphorylation of cofilin (Ser3) and ERK (Thr202/Tyr204). (C) Representative blots and statistical analysis (D) showing Rac1 siRNA on the phosphorylation of cofilin (Ser3), ERK (Thr202/Tyr204). Values represent means 6 SE; n = 3 in each group. *P,0.05. NS, not significant. doi:10.1371/journal.pone.0044590.g008 activated by H2S, whereas we could not find evidence of RhoA or Cdc42 activated by H2S in the experiment. In the present study, the finding that dominant negative plasmid of Rac1 decreased the formation of lamellipodia suggests that Rac1 mediates the H2S- induced reorganization of the actin cytoskeleton. Therefore, we propose that the activation of Rac1 is essential for cell lamellipodia formation provoked by H2S. A novel finding of the present study is the observation that Rac1 mediate the promoted cell migration effect induced by H2S in human endothelial cells, which is requisite for the angiogenesis. In the present study, suppression of Rac1 expression by RNA interference and inhibiting the endogenous activity of Rac1 by transfecting dominant negative plasmid both blunted the H2S- induced promoted migration effect of endothelial cells. Further- more, the enhanced tube formation effect of endothelial cells by H2S was also greatly decreased. Moreover, the lamellipodia formation induced by H2S was revoked by Rac1 activity inhibiting. These data provide us with the first piece of evidence that the promoted migration effect of H2S on human endothelial cells is dependent on Rac1 mediated actin cytoskeleton remodel- ing. H2S, a small, ubiquitous, gaseous, and diffusible molecule, can exert active properties in biological systems. Studies by Mustafa et al in the year of 2009 demonstrated that H2S can physiologically S-sulfhydrate proteins and that this sulfhydration modification of cysteine residues enhances the molecular activity of these proteins [28]. In our cell-free system, however, the observation that Rac1 was not directly activated by H2S gives us assured implication that Rac1 activation was potentially mediated by an upstream signaling molecule. To date, the mechanisms that regulate the activity of Rho GTPases during cell migration have not been studied extensively. Rac1 activation by receptor tyrosine kinases usually depends on PI3K activity, and inhibitors of PI3K can block Rac1 activation [29,30]. In the present study, pharmacologic inhibition of VEGFR and PI3K blunted the activated Rac1 and promoted cell migration induced by H2S, which gives us implication that VEGFR-PI3K pathway lay upstream of Rac1 and mediate the promoted cell migration effect of H2S. The observation that dominant negative Akt had no effect on the elevated activation of Rac1 induced by H2S indicates that H2S-induced Rac1 activation is independent of Akt. PI3Ks has a pivotal role in the regulation of cell growth, cell survival, and cell motility [31]. Class I PI3Ks have been classified into class IA and class IB. Class IA contains three catalytic isoforms, i.e. p110a, p110b, and p110d and class IB contains the p110c isoform [32]. Previous studies have shown that angiogenesis selectively requires the p110a isoform of PI3K to control PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e44590