endothelial cell migration [33]. Researchers have also demon- strated that the activation of Rac1 is selectively dependent on the p110a isoform of PI3K [34]. In our study, the depletion of p110a using siRNA significantly inhibited the activation of Rac1 by H2S, whereas p110b siRNA, p110c siRNA and p110d siRNA had no effect on the activation of Rac1. Moreover, only depletion of p110a significantly inhibited H2S-induced cell migration. Taken together, these results suggest that H2S-induced Rac1 activation and cell migration are specifically dependent on the p110a isoform of PI3K. With respect to mitogen-activated protein kinases (MAPKs), ERK can also regulate the activity of Rac1, allowing the formation of cellular protrusions and thereby contributing to tumor cell motility and invasion [35]. Studies have also demonstrated that ERK mediates the action of EGF by activating Rac1 to regulate cell migration [36]. But our results that inhibiting the activation of ERK has no effect on the activated Rac1 induced by H2S strongly suggest that Rac1 activation by H2S is independent of ERK. Rac1 activates PAK which subsequently stimulates LIM-kinase (LIMK). Activated LIMK can inactivate cofilin by inducing phosphorylation of cofilin, while non-phosphorylated cofilin can induce actin depolymerization [37]. In this context, Rac1 has the ability to inhibit cofilin-induced actin depolymerization. There is also strong evidence that cofilin is required for and promotes lamellipodia extension and cell migration [38]. In our study, the phosphorylation level of cofilin, which was stated as inactive form, elevated in a time-dependent manner after H2S treatment. Besides, suppression of Rac1 expression by RNA interference and inhibiting the endogenous activity of Rac1 by transfecting dominant negative plasmid both abolished the augmented phosphorylation level of cofilin. Moreover, pharmacologic inhibition of the VEGFR-PI3K pathway significantly attenuated the level of phosphorylated cofilin. Based on these observations, we validate that cofilin acts as a downstream effector of Rac1 and may take its effect to modulate actin dynamics triggered by H2S. Whether there exist other downstream effectors still needs further investigation. It is well established that MAPKs, involving p38, ERK and Jun N-terminus kinase (JNK), have pivotal roles in the regulation of cell migration via various signaling pathways. P38 regulate cell migration by inducing phosphorylation of MAPK-activated pro- tein kinase 2/3 (MK 2/3) and subsequent phosphorylation of HSP27 which is involved in directional migration of the cell [39,40]. ERK has been shown to regulate cell motility by inducing phosphorylation of myosin light chain kinase (MLCK) [41], paxillin [42] or FAK [43]. While JNK regulates cell migration by inducing phosphorylation of paxillin [44]. In our present study, we observed transient phosphorylation level elevations on ERK. Neither JNK nor p38 were under phosphorylated changements after NaHS treatment. And the finding that inhibition of MEK- ERK markedly reduced the migrated cell number that was augmented by H2S indicates that MEK-ERK pathway partially mediates the promotion of cell migration caused by H2S. ERK has also been found to be a downstream regulator of Rac1 in the regulation of cell motility [45]. But our evidence that the ERK phosphorylation caused by H2S was not inhibited by Rac1 suppression, indicating that the action of ERK on cell migration is independent of the Rac1 pathway. Previous data have shown that ERK activation play an important role in the regulation of actin- myosin mediated contraction [46]. MLCK (MLC kinase) can be directly activated by ERK and thereby leading to the phosphor- ylation of myosin light chains, which is important for the key processes in cell migration including the formation of adhesion complexes and focal adhesions [47]. Accordingly, we speculate that ERK may affect endothelial cell motility by its substrates (eg. H2S Signals through VEGFR-PI3K-Rac1-Cofilin Figure 9. Model illustrating the underlying signal transduction pathways of H2S that affect the migration of endothelial cells. doi:10.1371/journal.pone.0044590.g009 MLCK ) to modulate focal adhesion dynamics. On the other hand, our data demonstrate the pathway of VEGFR/PI3K/ Rac1/cofilin mediate the H2S-induced endothelial cell migration through actin cytoskeleton remodeling. Taken together, we conclude that the endothelial cell migration induced by H2S seems to be independently mediated through the pathway of VEGFR/PI3K/Rac1/cofilin and MEK-ERK, which suggest their different roles in the action of H2S. However, the exact role of the two respective signaling pathway in H2S-promoted endothelial cell migration should be further investigated. In conclusion, H2S signals through the VEGFR-PI3K pathway to activate Rac1 and thus inactivates the downstream effector cofilin, which in turn influences actin dynamics by triggering the formation of lamellipodia. In addition, our data support that H2S-induced Rac1 activation is specifically dependent on the p110a isoform of PI3K. These observations demonstrate that H2S activates Rac1 in a PI3K p110a-dependent fashion and ascribes a novel function to H2S in the regulation of the actin cytoskeleton and cell migration. The induction of the VEGFR/PI3K/Rac1/cofilin pathway by H2S contributes to the regulation of endothelial cell motility via actin cytoskeleton reorganization. Moreover, the MEK-ERK pathway is also involved in the effect of H2S on endothelial cell migration but is independent of Rac1. MEK-ERK pathway may play its effect through focal adhesion dynamics (Fig. 9). Author Contributions Conceived and designed the experiments: LJZ YCZ. Performed the experiments: LJZ BBT MJW. Analyzed the data: LJZ HMJ YCZ. Wrote the paper: LJZ YCZ. PLOS ONE | www.plosone.org 12 September 2012 | Volume 7 | Issue 9 | e44590