Protease-Activated Receptor-2 Up-Regulates Transient Receptor Potential Vanilloid 4 Function in Mouse Esophageal Keratinocyte

Background: The reflux of pancreatic–duodenal fluids is implicated in the pathophysiology of proton-pump inhibitor-resistant gastroesophageal reflux disease (GERD). Protease-activated receptor-2 (PAR-2) is activated by proteases, the pancreatic enzyme, trypsin, and the activated PAR-2 enhances transient receptor potential vanilloid 4 (TRPV4) function in neurons. TRPV4 stimulates ATP exocytosis in conjunction with the vesicular nucleotide transporter, which mediates mechano-transduction and vagal stimulation. The aim of the present study was to verify whether the activated PAR-2 up-regulates TRPV4 function in mouse esophageal keratinocytes, which may link to the pathophysiology in PPI-resistant GERD. Methods: TRPV4 and PAR-2 expressions were detected by RT-PCR, immunostaining, and western blotting in mouse esophageal keratinocytes. The functional response of TRPV4 to esophageal keratinocytes was analyzed using a Ca²⁺ imaging system. Cellular ATP release was examined by luciferase–luciferin reaction. TRPV4 phosphorylation was studied by immunoprecipitation and western blotting. Results: PAR-2 and TRPV4 mRNAs and proteins were expressed in esophageal keratinocytes. Pre-treatment with trypsin significantly increased the responses to TRPV4 activator in esophageal keratinocytes, probably via the phosphorylation of serine residue of TRPV4 by protein kinase C and resulted in cellular ATP release from the cells. Conclusions: Activated PAR-2 with trypsin exposure up-regulated TRPV4 function and increased ATP release in mouse esophageal keratinocytes. This mechanism might be related to the pathophysiology of GERD, especially non-erosive GERD.