receptor activation [16,17]. GPR4, together with OGR1, TDAG8 and G2A, comprise a novel proton-sensing GPCR family [15,16,17,18,19,20,21]. We have recently shown that activation of GPR4 by acidosis stimulates the Gs/cyclic adenosine mono- phosphate (cAMP) signaling in endothelial cells and regulates microvessel growth [15]. GPR4-null neonatal mice exhibit a partially penetrant phenotype of spontaneous hemorrhaging with small blood vessel defects [15]. Recent analysis of GPR4-null mice has revealed that GPR4 is involved in acid-base homeostasis in the kidney [31]. These data suggest that GPR4 is a functional proton sensor in cells. GPR4 can be stimulated by extracellular acidic pH to transduce downstream signals through Gs/cAMP, Gq/phospholipase C (PLC), and G13/Rho pathways [15,16,17,20]. cAMP is a ubiquitous second messenger that controls a wide range of cellular processes mainly through the downstream effector protein kinase A (PKA). More recently, Epac (exchange protein directly activated by cAMP) has been identified as a new effector of cAMP and is involved in many important cellular processes including cell adhesion [32,33]. The cAMP/Epac/Rap1 signaling pathway has been reported to regulate a number of PKA-independent processes, such as b2-adrenergic receptor-mediated ovarian carcinoma cell adhesion to fibronectin [34], monocyte adhesion and chemotaxis [35], and adenosine receptor-stimulated activation of ERK1/2 in HUVEC cells [36]. Here we have identified a novel function of GPR4 in response to acidosis. Activation of GPR4 by acidic pH increases endothelial cell adhesion with leukocytes and this biological effect is mediated through the cAMP/Epac pathway. Acidosis/GPR4 signaling also up-regulates the expression of several adhesion molecules in endothelial cells. These findings suggest that the acidosis/GPR4 signaling may represent a new pathway in vascular inflammation responding to acidic microenvironments in many pathological conditions. Materials and Methods Reagents and chemicals 29,59-dideoxyadenosine (DDA), SQ 22536, 8-bromoadenosine 39,59-cyclic monophosphate (8-Br-cAMP), 8-(p-chlorophenylthio)- 29-O-methyladenosine-39,59-cyclic monophosphate (8-CPT-2Me- cAMP), H-89, and pertussis toxin (PTX) were purchased from Calbiochem/EMD4Biosciences (La Jolla, CA). 3-isobutyl-1-meth- ylxanthine (IBMX), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), N-(2-hydroxyethyl)-piperazine-N9-3-propanesulfo- nic acid (EPPS), 2-(4-morpholino)-ethanesulfonic acid (MES), and protease inhibitor cocktail were from Sigma-Aldrich (St Louis, MO) and Fisher Scientific (Fair Lawn, NJ). Anti-c-myc mouse monoclonal antibody (clone 9E10) was purchased from Roche Diagnostics (Indianapolis, IN). E-selectin (clone BBIG-E4), VCAM-1 (clone BBIG-V1), inter-cellular adhesion molecule-1 (ICAM-1) (clone BBIG-I1) and P-selectin (clone 9E1) monoclonal antibodies were from R&D Systems (Minneapolis, MN). Plasmid constructs The open reading frame of human GPR4 (GenBank accession number NM 005282) was amplified from human cDNAs by polymerase chain reaction (PCR) with the high-fidelity pfu-turbo DNA polymerase (Stratagene, La Jolla, CA). The PCR primer pair was: sense strand 59- ATAAGAATGCGGCCGCACCA- TGGGCAACCACACGTG -39 and antisense strand 59- ATAA- GAATGCGGCCGCTCATTGTGCTGGCGGCAGCAT -39. A NotI site was introduced at each end of the PCR fragment for molecular cloning and the Kozak consensus sequence was added Acidosis/GPR4 and Endothelial Cell Adhesion before the translation start codon ATG for efficient translation. The amplified PCR fragment was digested with NotI and cloned into the retroviral expression vector MSCV-IRES-GFP as previously described [37,38]. The coding region of the resultant construct, designated as MSCV-huGPR4-IRES-GFP, was verified by DNA sequencing from both strands. To generate the dominant-negative construct of Epac1 [39], a cDNA fragment encoding the N-terminal 420 amino acids of Epac1 was amplified by PCR from the HUVEC cDNA. The nucleotide sequence of the myc tag was designed to be added to the 59 of the Epac1 sequence. A NotI site at the 59 end and an EcoRI site at the 39 end were designed for cloning into expression vectors. The PCR primers for amplifying the dominant-negative Epac1 fragment were: sense strand 59- AGAGGGCGGCCG- CACCATGGAGCAAAAGCTCATTTCTGAAGAGGACTTG- GTGTTGAGAAGGATGCACCGGCCC -39 and antisense strand 59-GAGAGGAATTCCTACCTCTTGTTGCAGACGT- AGGTGCT-39. The amplified Epac fragment was digested with NotI and EcoRI and subcloned into the pQCXIP vector (Clontech, CA). The construct was sequenced to verify the accuracy of the coding sequence. The dominant-negative Epac construct was designated as pQCXIP-N-Epac. Site-directed mutagenesis The DRY motif of GPCRs is important for G protein coupling and signaling [40]. A pair of primers was designed to generate an arginine to alanine mutation at the residue 115 (namely R115A) of the DRY motif of GPR4. The sequences of the primer pair were: sense strand 59- TGCATCTCGGTGGACGCCTACCTGGCT- GTG -39 and antisense strand 59- CACAGCCAGGTAGG- CGTCCACCGAGATGCA -39. The MSCV-huGPR4-IRES- GFP plasmid construct was used as the template and the R115A mutation was generated using the QuikChange site-directed mutagenesis kit (Stratagene, Inc., CA). The mutagenesis procedure was performed according to the manufacturer’s instruction. The R115A mutation was confirmed by DNA sequencing and no other mutations were detected. RNAi knockdown The microRNA-based short-hairpin RNA interference (miRNA) vector system (Invitrogen, Carlsbad, CA) was used for the RNAi knockdown of human GPR4 gene expression in HUVEC cells. Perfectly matched miRNA molecules mainly cause the degradation of target mRNAs. Several sets of miRNAs against human GPR4 were designed according to the manufacturer’s instructions (www.invitrogen.com/rnai). The core nucleotide sequences of two effective human GPR4 miRNA molecules were: miRNA #1: (sense) 59-ATCCCTCTACATCTTTGTCAT-39 and (antisense) 59-ATGACAAAGATGTAGAGGGAT-39, and miRNA #2: (sense) 59-CAAGAGGAACAGCACAGCCAA-39 and (antisense) 59-TTGGCTGTGCTGTTCCTCTTG-39. A negative control miRNA that does not have sequence homology with any known genes was provided by Invitrogen. The core sequence of the negative control miRNA was: (sense) 59-GT- CTCCACGCGCAGTACATTT-39 and (antisense) 59-AAATG- TACTGCGCGTGGAGAC-39. The pre-miRNA oligos were cloned into the pcDNA 6.2-GW/RFP-miR vector and transferred to the lentiviral vector FCW attR1-attR2 using the BP/LR recombination reaction system (Invitrogen) as previously described [41]. All miRNA constructs were verified by DNA sequencing. The miRNA lentiviral vector was co-transfected with the packaging vectors pRSV-Rev, pCMV-VSV-G, and pMDL into HEK 293T cells (ATCC, Manassas, VA). Viral particles were PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27586