Activation of GPR4 by Acidosis Increases Endothelial Cell Adhesion through the cAMP/Epac Pathway Aishe Chen1., Lixue Dong1., Nancy R. Leffler1, Adam S. Asch1,3, Owen N. Witte4, Li V. Yang1,2,3* 1 Department of Internal Medicine, Brody School of Medicine, East Carolina University, Greenville, North Carolina, United States of America, 2 Department of Anatomy and Cell Biology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, United States of America, 3 UNC Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina, United States of America, 4 Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles, California, United States of America Abstract Endothelium-leukocyte interaction is critical for inflammatory responses. Whereas the tissue microenvironments are often acidic at inflammatory sites, the mechanisms by which cells respond to acidosis are not well understood. Using molecular, cellular and biochemical approaches, we demonstrate that activation of GPR4, a proton-sensing G protein-coupled receptor, by isocapnic acidosis increases the adhesiveness of human umbilical vein endothelial cells (HUVECs) that express GPR4 endogenously. Acidosis in combination with GPR4 overexpression further augments HUVEC adhesion with U937 monocytes. In contrast, overexpression of a G protein signaling-defective DRY motif mutant (R115A) of GPR4 does not elicit any increase of HUVEC adhesion, indicating the requirement of G protein signaling. Downregulation of GPR4 expression by RNA interference reduces the acidosis-induced HUVEC adhesion. To delineate downstream pathways, we show that inhibition of adenylate cyclase by inhibitors, 29,59-dideoxyadenosine (DDA) or SQ 22536, attenuates acidosis/GPR4-induced HUVEC adhesion. Consistently, treatment with a cAMP analog or a Gi signaling inhibitor increases HUVEC adhesiveness, suggesting a role of the Gs/cAMP signaling in this process. We further show that the cAMP downstream effector Epac is important for acidosis/GPR4-induced cell adhesion. Moreover, activation of GPR4 by acidosis increases the expression of vascular adhesion molecules E-selectin, VCAM-1 and ICAM-1, which are functionally involved in acidosis/GPR4-mediated HUVEC adhesion. Similarly, hypercapnic acidosis can also activate GPR4 to stimulate HUVEC adhesion molecule expression and adhesiveness. These results suggest that acidosis/GPR4 signaling regulates endothelial cell adhesion mainly through the Gs/cAMP/Epac pathway and may play a role in the inflammatory response of vascular endothelial cells. Citation: Chen A, Dong L, Leffler NR, Asch AS, Witte ON, et al. (2011) Activation of GPR4 by Acidosis Increases Endothelial Cell Adhesion through the cAMP/Epac Pathway. PLoS ONE 6(11): e27586. doi:10.1371/journal.pone.0027586 Editor: Niels Olsen Saraiva Caˆmara, Universidade de Sao Paulo, Brazil Received April 13, 2011; Accepted October 20, 2011; Published November 16, 2011 Copyright: 2011 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by research grants from North Carolina Biotechnology Center, Brody Brothers Endowment Fund, Golfers Against Cancer, the American Heart Association, and the Startup Fund from the Department of Internal Medicine, East Carolina University (to L.V.Y.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Li V. Yang is the applicant for a patent which includes the utilization of GPR4 in the treatment of inflammation, related to the data described in this manuscript. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: yangl@ecu.edu . These authors contributed equally to this work. Introduction Local or systemic acidosis is associated with a variety of pathological conditions such as inflammation, ischemia, tumor, diabetic ketoacidosis, and lung and renal diseases due to defective blood flow, hypoxia, and glycolytic metabolism [1,2,3,4,5,6]. For instance, interstitial pH in ischemic organs often decreases to 7.0 - 6.0 and sometimes even below 6.0 [5,7,8]. There are two major types of acidosis: isocapnic acidosis, such as metabolic acidosis caused by excessive metabolic acids, and hypercapnic acidosis, such as respiratory acidosis caused by carbon dioxide accumula- tion [9,10,11]. Acidosis has profound effects on blood vessels, immune cells, inflammatory responses, and tissue injury [4,8,12,13,14,15], but the molecular mechanisms by which acidosis regulates vascular function, endothelium-leukocyte inter- action and inflammation are not well known. The GPR4 family of proton-sensing G protein-coupled receptors (GPCRs) has recently been identified as novel pH sensors [15,16,17,18,19,20,21]. GPR4, originally cloned as an orphan GPCR, is expressed in a wide range of tissues such as the lung, kidney, heart, and liver [22,23,24]. GPR4 is highly conserved during evolution, with more than 90% amino acid sequence homology among mammalian orthologs and more than 70% homology between human and zebrafish orthologs. Howev- er, the biological function of GPR4 is not clearly defined. GPR4 was previously reported as a receptor for sphingosylphosphor- ylcholine (SPC) and lysophosphatidylcholine (LPC) [25], but this observation has not always been confirmed [20,26,27] and the original publication has been withdrawn [25]. Several studies indicated that GPR4 mediates the SPC-induced endothelial tube formation, LPC-induced impairment of endothelial barrier function, and LPC-induced vascular cell adhesion molecule-1 (VCAM-1) expression [28,29,30]. Since the ligand-receptor relationship between SPC, LPC and GPR4 is not validated, it is unclear whether GPR4 directly or indirectly mediates the biological effects of SPC and LPC. More recent studies from several research groups demonstrated that GPR4 predominantly functions as a proton sensor activated by extracellular acidic pH [15,16,17,20]. Protonation of several extracellular histidine residues of GPR4 is important for the PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27586