adhesiveness (Fig. S3). The results suggest that acidosis/GPR4- induced HUVEC cell adhesion may not be linearly proportional to the overexpression of GPR4 per se but be related to downstream events triggered by GPR4 activation. To further delineate downstream pathways, we examined whether the Gs/adenylate cyclase/cAMP pathway is important for acidosis/GPR4-induced endothelial cell adhesion. In addition to acidosis-induced cAMP production (,7-fold) in HUVEC/GPR4 cells (Fig. 2A), a ,1.5-fold increase of cAMP upon acidic pH stimulation was detected in HUVEC/Vector cells (Fig. 3A). Furthermore, inhibition of adenylate cyclase by 29,59-dideoxyade- nosine (DDA) attenuated acidosis/GPR4-induced HUVEC adhe- sion (Fig. 3B). A similar extent of decrease in HUVEC adhesiveness was observed when cells were treated with another adenylate cyclase inhibitor SQ 22536 (Fig. 3C). Consistently, direct introduction of a cAMP analog, 8-bromo-cAMP, increased the adhesion of HUVECs at all tested pHs, recapitulating the effects of GPR4 activation (Fig. 3D). In line with this observation, treatment of HUVEC/GPR4 cells with pertussis toxin (PTX), which blocks the cAMP inhibitory G protein Gi, increased the adhesion of HUVECs (Fig. 3E). These results indicate that the Gs/cAMP pathway plays an important role in regulating acidosis/GPR4-mediated HUVEC adhesion. The cAMP effector, Epac, is involved in acidosis/GPR4- induced HUVEC cell adhesion Increase of intracellular cAMP can activate several down- stream effectors including protein kinase A (PKA) and Epac Acidosis/GPR4 and Endothelial Cell Adhesion [51]. To determine which effector(s) are involved in acidosis/ GPR4-induced HUVEC adhesion, we first examined the role of the best characterized cAMP target, PKA, in this process. Treating HUVEC/GPR4 cells with the PKA inhibitor H-89 did not affect cell adhesion induced by acidosis (Fig. 4A), suggesting that PKA is not important for this process. To verify that H-89 effectively inhibited the activity of PKA, we examined the phosphorylation status of the PKA substrate CREB (cAMP response element-binding) by Western blotting and showed that H-89 effectively blunted CREB phosphorylation induced by 8- bromo-cAMP in HUVECs (data not shown). Next, to elucidate the role of Epac in acidosis/GPR4-mediated HUVEC adhesion, a dominant-negative Epac mutant N-Epac or the pQCXIP control vector was expressed in HUVEC/Vector and HUVEC/ GPR4 cells. The N-Epac dominant-negative mutant lacks the kinase domain and blocks the activity of Epac by competitively binding with the substrates [39]. By Western blotting with a myc antibody, the exogenous expression of myc-N-Epac was confirmed in HUVEC/Vector and HUVEC/GPR4 cells (Fig. 4B). In the HUVEC-U937 adhesion assay, the expression of the N-Epac mutant inhibited acidosis/GPR4-induced HU- VEC cell adhesion (Fig. 4C). On the other hand, when HUVEC cells were directly treated with the Epac specific activator 8- CPT-2Me-cAMP [52], cell adhesion was enhanced at all pHs tested (Fig. 4D). These results suggest that Epac, but not PKA, is the downstream cAMP effector that mediates acidosis/GPR4- induced HUVEC adhesion. Figure 5. Real-time RT-PCR of adhesion molecules E-selectin, VCAM-1, and ICAM-1. HUVECs transduced with the control vector, GPR4 or GPR4 R115A mutant were treated with EGM-2/HEM media at pH 8.4, 7.4, or 6.4 for 5 h. Total RNAs were isolated and reverse transcribed. Real-time RT-PCR quantification of mRNA levels of E-selectin (A), VCAM-1 (B), and ICAM-1 (C) was performed in duplicate. Values were normalized to the housekeeping gene GAPDH. The expression level of the target gene in HUVEC/Vector cells at pH 8.4 was set as 1. The results are representative of at least two independent experiments. Error bars are the mean 6 SEM. doi:10.1371/journal.pone.0027586.g005 PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27586