Figure 4. Dominant negative Rac1 inhibits the H2S-induced reorganization of the actin cytoskeleton in HUVECs. (A) HUVECs were transfected with plasmids encoding EGFP only (EGFP) or dominant-negative Rac1 (EGFP-Rac1-T17N). Western blot analysis showed manifest levels of the EGFP-Rac1 fusion protein in HUVECs transfected with EGFP-Rac1-T17N vector but not in the cells transfected with EGFP control vector. (B) Transfected cells were starved and then treated with or without 50 mM NaHS for 12 min. Cells were then fixed and stained with rhodamine-labeled phalloidin for F-actin and anti- EGFP antibody for EGFP-tagged Rac1. Representative images are shown. Thin arrows point to the formed lamellipodia. Thick arrows indicate the fragments of disrupted F-actin around the nucleus. Scale H2S Signals through VEGFR-PI3K-Rac1-Cofilin bars, 10 mm. (C, D) Rac1 siRNA decreased the expression of Rac1 both on the mRNA levels (C) and protein levels (D). (C) Electroporation was used to transfect HUVECs with siRNA. 48 h after transfection, mRNA transcripts of Rac1 and GAPDH as measured by real-time PCR were significantly reduced by siRNA separately. (D) Representative blots were shown on the protein levels after 72 h transfection. Transfection of HUVECs with Rac1 siRNA and GAPDH siRNA specifically knocked down the expression of their respective target genes without affecting each other’s targets. Values represent the means 6 SE, n = 9. *P,0.05. doi:10.1371/journal.pone.0044590.g004 pathway and that the activation of Rac1 triggered by H2S is independent of the MEK-ERK pathway. Moreover, the cell migration rate promoted by NaHS (50 mM) was also significantly blunted by the VEGFR inhibitor SU5416 and the PI3K inhibitor LY294002 (Fig. 6F). Interestingly, the MEK inhibitor U0126 also has an attenuating effect on cell migration induced by H2S (Fig. 6F). The PI3K p110a isoform is required for H2S-induced Rac1 activation and endothelial cell migration-H2S-induced Rac1 activation is dependent on the VEGFR-PI3K pathway. As the direct downstream effector of PI3K, Akt can be phosphorylated on residue Ser473 by H2S treatment (Fig. 6A). As shown in Fig. 7G, the observation that dominant negative Akt has no effect on the H2S-induced elevated activity of Rac1 indicates that H2S-induced Rac1 activation is independent of Akt. The successful transfection of dominant negative Akt was confirmed by western blot analysis using antibodies to the HA-tag (Fig. 7H). Knockdown of the individual PI3K isoforms did not affect the expression levels of the other isoforms (Fig. 7A, B). Knockdown of p110a, p110b, and p110c significantly reduced Akt phosphory- lation at Ser473 (Fig. 7A, B). The strongest reduction was observed after p110a depletion, whereas p110d siRNA had only a small effect on Akt phosphorylation. As shown in Fig. 7C, depletion of the p110a isoform using p110a siRNA significantly blunted the H2S-induced activation of Rac1, whereas p110b siRNA, p110c siRNA and p110d siRNA had little effect on the activated Rac1. Moreover, in the transwell boyden chamber assay, only the deletion of p110a markedly prevented H2S-induced endothelial cell migration (Fig. 7D, E). Cofilin, but not ERK, acts as the downstream effector of Rac1-HUVECs exposed to 50 mM NaHS exhibited a marked increase in cofilin phosphorylation in a time-dependent manner (Fig. 6A). Dominant negative Rac1 prevented the augmentation of cofilin phosphorylation at the site of Ser3 induced by 50 mM NaHS, but it failed to blunt the activation of ERK triggered by NaHS (Fig. 8A, B). Similarly, RNA interference of Rac1 significantly decreased the elevated phosphorylation level of cofilin that was induced by 50 mM NaHS, whereas it had no effect on NaHS-induced elevation of phosphorylated ERK (Thr202/Tyr204) (Fig. 8C, D). Discussion The process of endothelial cell migration is the main characteristic associated with blood vessel formation. Our previous study has shown that H2S is a proangiogenic factor both in vivo and in vitro [18,19]. Cell migration is driven by the mechanical force provided by dynamic remodeling of the actin cytoskeleton which is regulated by several signaling pathways including members of the Rho GTPases family [23]. Here, we demonstrate that in human endothelial cells, H2S selectively activates the Rho GTPase Rac1, which in turn inactivates its effector molecule cofilin, inducing lamellipodia formation. The presented data also support that VEGFR-PI3K activates the Rac1-cofilin pathway PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e44590