Abstract
Background: A single layer of epithelial cells creates a physical barrier between the external lumen and the internal milieu of the gastrointestinal tract. During inflammation, which is typically associated with a decrease in local pH, the intestinal barrier can be disrupted. Defects in barrier integrity have been implicated in the pathophysiology of inflammatory bowel disease (IBD). However, the molecular mechanisms mediating the responses to changes in pH are not well understood. G-protein-coupled receptor 68 (GPR68), also known as ovarian cancer GPR1 (OGR1), is a proton-sensing receptor, which upon acidification stimulates second messenger signaling, such as intracellular Ca2+ flux, inositol phosphate (IP) formation, and extracellular signal-regulated kinase (ERK) phosphorylation. We have investigated OGR1-mediated signaling pathways in response to lowered pH in intestinal epithelialcells. The colon carcinoma-derived-Caco2 cell monolayers can be used as In Vitro cell culture models of the human intestinal epithelium. Methods/Results: The OGR1 cDNA fragment was generated by PCR, and cloned into the pcDNA3.1 vector carrying a neomycin resistance gene. Clonal selection of stably transfected Caco2s was by limiting dilution or cloning cylinders, using the selection agent G418 (400 mg/ml). OGR1-overexpressing clones were selected by positive mRNA/protein expression and immunofluorescence, and those exhibiting strong IP formation upon acidification were chosen for further studies. The selected Caco2-OGR1 clones showed a typical pH-dependent GPCR response, in both intracellular Ca2+ signaling and label-free assays (EPIC). In addition, they exhibited weak but significant formation of cAMP upon extracellular acidification. When compared to the Caco2 vector control clone, the Caco2-OGR1 clones showed ERK phosphorylation after a pH shift from pH 7.9 to pH 6.8 for 5 minutes, by Western blotting. No pH-dependent phosphorylation remained present after 30 minutes. The Caco2-OGR1 clones exhibited significant serum response factor (SRF) activity at acidic pH but not at alkaline or neutral pH, in dual luciferase assays using SRE-dependent promoter constructs. Consistent with this, mRNA expression of a SRF target gene c-FOS was elevated in parallel. Electric Cell-Substrate Impedance Sensing (ECIS) assays showed tightening of the barrier in the Caco2-OGR1 monolayers at acidic pH, but not at neutral or alkaline pH. Conclusion: We have created Caco2 cell clones stably overexpressing OGR1, and have shown that these serve as valid tools to study OGR1 function and OGR1-mediated signaling. OGR1 overexpression in intestinal epithelial cells enhanced ERK signaling, led to increased tight junction formation, and increased SRF activity. Unravelling OGR1-dependent signaling may aid our understanding of the pathophysiology of IBD.
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