not well defined. Our results suggest that GPR4 is a novel sensor for vascular endothelial cells to respond to acidic tissue microenvironments and elicit inflammatory responses. Data presented here reveal that acidosis activates GPR4 to increase endothelial cell adhesiveness through multiple adhesion molecules such as E-selectin, VCAM-1, and ICAM-1, which then facilitate the binding of leukocytes. We have also shown that hypercapnic acidosis, similar as isocapnic acidosis, can activate GPR4 to increase the expression of adhesion molecules and the adhesive capacity of HUVECs. In the literature, there are seemingly conflicted reports regarding the effects of hypercapnia on endothelial cell adhesion. Takeshita et al. showed that hypercapnia decreased the lipopolysaccharide (LPS)- induced upregulation of ICAM-1 in human pulmonary artery endothelial cells [69]. However, Liu et al. recently demonstrated that hypercapnia increased the LPS-induced expression of inflammatory molecules such as ICAM-1, VCAM-1 and E- selectin in human pulmonary microvascular endothelial cells and in a LPS-induced lung injury animal model [70]. Our own results using HUVECs as a model system are in line with the observations by Liu et al. [70] but in contrast to the reports by Takeshita et al. [69]. Although the reasons for the paradoxical findings are not exactly clear, the use of endothelial cells from different vessel types has been proposed as a potential explanation [70]. Indeed, endothelial cells isolated from different types of blood vessels have been shown to exhibit different phenotypes, gene expression profiles and responses to stimuli [71,72]. It should also be noted that hypercapnia leads to the increase of not only protons but also carbon dioxide and bicarbonate, all of which can trigger downstream signaling pathways through different mechanisms [11]. Thus, hypercapnia may elicit more complicated biological effects than hypercapnic acidosis alone. In the HUVEC model system, our results strongly suggest that hypercapnic acidosis can activate the proton-sensing receptor GPR4 in a gene dose- dependent manner. This observation may have implications in certain hypercapnia-associated clinical problems. For instance, the low-tidal-volume mechanical ventilation, used for the treatment of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), commonly causes hypercapnia [73]. While some studies indicate that hypercapnia is potentially beneficial in attenuating lung inflammation during ventilation, other studies suggest the opposite [9,69,70,74,75]. These observations underscore the complex biological effects of hypercapnia and the importance of further research to better understand the manifold pro- and anti- inflammatory signaling pathways related to hypercapnia. In this regard, GPR4 signaling may represent a novel pathway that senses hypercapnic acidosis and modulates inflammatory responses. Moreover, we have further demonstrated that G protein signaling and the Gs/adenylate cyclase/cAMP/Epac pathway are important for acidosis/GPR4-induced HUVEC adhesion. This conclusion is supported by several lines of evidence. First, the R115A DRY motif mutant of GPR4, which is deficient in G protein signaling, fails to increase HUVEC adhesiveness in response to acidosis. Second, inhibition of adenylate cyclase or Epac substantially blunts the acidosis/GPR4-induced HUVEC adhesion. Third, treatment with the cAMP analog 8-bromo- cAMP or the Epac specific activator 8-CPT-2Me-cAMP recapit- ulates the HUVEC adhesive phenotype induced by acidosis/ GPR4 signaling. Our observations are in line with several previous studies. Rangarajan et al. demonstrate that upon the stimulation of b2-adrenergic receptor, cAMP induces the adhesion of Ovcar3 ovarian cancer cells through the Epac/Rap1 pathway [34]. Fukuhara et al. show that the cAMP/Epac pathway increases VE-cadherin-mediated cell-cell contact and promotes endothelial Acidosis/GPR4 and Endothelial Cell Adhesion barrier function [76]. Our results indicate that the Gs/cAMP/ Epac pathway is also involved in acidosis/GPR4-induced endothelial cell adhesion, illustrating a recurrent theme in signaling transduction that diverse stimuli and receptor activation may converge into common downstream signaling pathways. It should also be pointed out that activation of GPR4 by acidosis has been shown to stimulate multiple G protein pathways including Gs, Gq and G13, of which the physiological role of Gq and G13 pathways in endothelial cell acidosis responses is not clear and needs to be further studied. Emerging evidence suggests that the GPR4 family of proton- sensing GPCRs, including GPR4, TDAG8, OGR1 and G2A, plays a role in inflammation. Previous studies have demonstrated that acidosis activates TDAG8 to produce cAMP in thymocytes and splenocytes [19,77]. Stimulation of TDAG8 by acidosis regulates the viability of eosinophils and the production of cytokines [78,79]. Our results show that activation of GPR4 by acidosis increases the adhesiveness of endothelial cells. While we have not studied the effects of acidosis/GPCR signaling on leukocyte adhesiveness, this aspect warrants further investigation because the reciprocal interaction between endothelial cells and leukocytes is critical for inflammation. Current data indicate that the proton-sensing GPCRs, such as GPR4 and TDAG8, potentially serve as the pH sensors for vascular cells and blood cells to perceive the acidic microenvironment at inflammatory sites and transduce signaling pathways to regulate the function of blood vessels and leukocytes. As acidosis is closely associated with inflammation, ischemia, cancer, and other diseases, the GPR4 proton-sensing receptor family may play a role in these pathological conditions. Supporting Information Figure S1 Acidosis/GPR4-induced HUVEC adhesion using different types of culture media or leukocytes. (A) HUVEC/Vector and HUVEC/GPR4 cells were treated with EGM-2/HEM media at indicated pHs for 5 to 15 h. RPMI medium supplemented with 10% FBS was used to replace DMEM medium for growing U937 cells and washing the plate to remove non-adhered U937 cells. The cell adhesion assay was performed as described in the ‘‘Materials and Methods’’. **, P,0.01; compared with the pH 8.4 group. (B) HUVEC/Vector and HUVEC/GPR4 cells were treated with EGM-2/HEM media at different pHs as indicated for 5 to 15 h. Afterwards, 66104 cells/well of HL-60 promyelocytic cells were added to adhere with HUVECs for 1 h at pH 7.4, and the cell adhesion assay was performed. **, P,0.01; ***, P,0.001; compared with the pH 8.4 group. The results are representative of two or more independent experiments. Error bars are the mean 6 SEM. (TIF) Figure S2 Endogenous mRNA level of GPR4 in HUVECs is decreased during acidic pH treatment. HUVEC/Vector cells were treated with EGM-2/HEM media at pH 8.4, 7.4 or 6.4 pH for 5 h. Total RNAs were isolated and the level of GPR4 mRNA was determined by real-time RT-PCR. Values were normalized to the housekeeping gene GAPDH. The expression level of GPR4 at pH 8.4 was set as 1. Error bars are the mean 6 SEM. ***, P,0.001; compared with pH 8.4. (TIF) Figure S3 Correlation between the intensity of GFP signal and the adhesiveness of HUVEC cells. In HUVEC/ GPR4 cells, GPR4 was co-expressed with a bicistronic GFP marker, which can serve as an indicator of GPR4 expression. PLoS ONE | www.plosone.org 12 November 2011 | Volume 6 | Issue 11 | e27586