H2S Signals through VEGFR-PI3K-Rac1-Cofilin Figure 2. H2S induces reorganization of the actin cytoskeleton in HUVECs. (A) The effects of H2S on the actin cytoskeleton and on paxillin- containing focal adhesions. Serum-starved HUVECs were stimulated or not by 50 mM NaHS for indicated times. Thereafter, immunofluorescence analysis was performed using DAPI (blue) to stain nucleus, rhodamine-conjugated phalloidin (red) to stain F-actin and a mouse anti-paxillin monoclonal antibody followed by Alexa Fluor-labeled secondary antibody (green) to stain paxillin-contained focal adhesions. An overlay of fluorescent signals, generating yellow color in areas of colocalization, is shown on the right column. The thin arrows indicate lamellipodia, arrowheads indicate cell protrusions and thick arrows show paxillin-containing focal adhesions. Scale bars, 10 mm. Representative blots (B) and statistical values (C) showing that NaHS (50 mM) treatment has no effect on the phosphorylation of paxillin. The results are representative of three independent experiments. Values are means 6 SE. NS, not significant. doi:10.1371/journal.pone.0044590.g002 phosphorylation levels of two other MAPKs members, p38 and JNK (Fig. 6A, B). Inhibiting VEGFR using SU5416 or PI3K (LY294002) completely abrogated the NaHS-induced phosphor- ylation of Akt (Fig. 6C, D), which demonstrates that H2S signals through the VEGFR-PI3K pathway in HUVECs. The observa- tion that both SU5416 and LY294002 have no effect on the NaHS-induced activation of ERK (Fig. 6C, D) signifies that ERK acts independently of the VEGFR-PI3K pathway. Both SU5416 and LY294002 could abolish the enhanced phosphorylation of cofilin by NaHS (Fig. 6C, D). Compared to NaHS treatment, VEGF induced a more pro- nounced augmentation of Rac1 activity (NaHS vs. VEGF: 140.9610.2% vs. 191.4618.3% Fig. 6E). Inhibition of Rac1 by NSC23766 significantly reduced NaHS-induced Rac1 activation, and the inhibition of VEGFR with SU5416 significantly reduced H2S-induced activation of Rac1 (Fig. 6E). Similar results were obtained using specific inhibitors of PI3K (LY294002) (Fig. 6E), whereas pretreating with the MEK inhibitor U0126 had no effect on Rac1 activation by H2S (Fig. 6E). These results suggest that H2S-induced Rac1 activation is regulated by the VEGFR-PI3K Figure 3. H2S selectively activates the Rho GTPase Rac1 in HUVECs but does not directly activate Rac1 in vitro. (A, B) Pull down assays were conducted to detect the activity of Rho GTPases. Serum-starved HUVECs were treated with 50 mM NaHS for indicated times. Cells were then lysed and incubated with GST-PAK1-PBD-bound or GST-Rhotekin-RBD-bound glutathione beads. GTP-loaded Rac1 and Cdc42 bound to GST-PAK1- PBD (A) and GTP-loaded RhoA bound to GST-Rhotekin RBD (B) were detected by western blotting with antibodies against Rac1, Cdc42 or RhoA (upper panel). GTPcS loaded as positive control. Total cell lysates were also probed with the same antibodies to demonstrate that equal amounts of total protein were used in individual assays (lower panel). The results are representative of three independent experiments. (C) The time course for Rac1 activation by H2S. ELISA-based GTPase activation assays (G-LISA) of Rac1 were performed at various times after treatment with 50 mM NaHS. (D) In vitro interaction of H2S with Rac1. Human recombinant Rac1 was allowed to react with different concentrations of NaHS, and G-LISA assays were performed to detect the level of GTP-bound Rac1. The data are presented as means 6 SE of three independent experiments each performed in triplicate. *P,0.05 vs control. NS, not significant. doi:10.1371/journal.pone.0044590.g003 PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e44590