Tu1887 The Gene Encoding the G Protein-Coupled Receptor 68 (GPR68/ OGR1) is Regulated by TNF, Hypoxia, and Low pH in Human Monocytic Cells

Abstract

Background: The G protein-coupled receptors, ovarian cancer G-protein coupled receptor 1 (OGR1/GPR68), GPR4 and T-cell death associated gene 8 (TDAG8/GPR65) function as sensors for extracellular protons, leading to activation of adenylyl cyclase or phospholipase C. Inflammatory bowel disease (IBD) is associated with acidification of mucosal tissue, and subsequent proinflammatory cytokine production. The cytokine tumor necrosis factor (TNF) is of crucial importance in IBD pathogenesis, and many of its effects are mediated by the transcription factor nuclear factor-kB (NF-kB). Furthermore, tissue hypoxia, which stabilizes the transcription factor hypoxia-inducible factor-1a (HIF-1a), is a feature of active IBD. We studied whether TNF and hypoxia regulate the expression of OGR1 and TDAG8, which are expressed in both immune cells and colonic tissue. Methods/Results: Treatment of the human monocytic cell line MM6 cells with TNF, but none of the other cytokines (IFN-g, IL-1b, IL-6, TGF-b) tested, led to significant upregulation of OGR1 mRNA, in a dose-dependent manner, as determined by real-time PCR. Macrophagic differentiation of MM6 cells with phorbol myristate acetate (PMA) also led to a significant increase in OGR1 mRNA expression. TNF- and PMA-dependent induction of OGR1 mRNA was confirmed in primary human monocytes. Induction of OGR1 mRNA expression by TNF was reversed by simultaneous treatment of cells with specific NF-kB inhibitors. In agreement with the findings at the mRNA level, GPCR activity in MM6 cells was increased upon treatment with TNF or PMA, only at acidic pH, as quantified by intracellular calcium dyes and EPIC label-free assays. In hypoxia, both 0.2% and 2% oxygen conditions enhanced TNF- and PMA-induction of OGR1 mRNA expression. Consistent with the hypoxia effect in MM6 cells, OGR1 expression was elevated in colonic tumours in mice. Two alternative predicted promoter variants ~9 kpb apart, exist for the OGR1 gene in chromosome 14. In silico analysis using MatInspector software and visual inspection revealed several putative DNA-binding sites for NF-kB and HIF-1a within the proximal regions of the OGR1 promoter variants. In addition to TNF and hypoxia, OGR1 mRNA expression was elevated at low pH, suggesting positive feedforward regulation of OGR1 activity in acidic conditions. No induction of mRNA expression of the related GPCR, TDAG8, was observed upon treatment with TNF or PMA. Conclusion: OGR1 expression is induced in cells of human macrophage lineage and primary human monocytes by TNF and PMA. NF-kB inhibition reverses the induction of OGR1 mRNA expression by TNF. Interestingly, hypoxia, known to cross-talk with the NF-kB pathway, enhances the TNF-mediated induction of OGR1 expression. The stimulation of OGR1 expression by TNF and hypoxia, and subsequently pH-sensing activity, may play a role in IBD pathogenesis.