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

Dig Dis Sci. 2015 Dec;60(12):3570-8. doi: 10.1007/s10620-015-3822-6. Epub 2015 Aug 2.

Abstract

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 Ca(2+) 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.

Keywords: ATP; GERD; Protease-activated receptor-2; Transient receptor potential vanilloid 4.

MeSH terms

  • Animals
  • Biopsy
  • Cells, Cultured
  • Esophagus / cytology*
  • Esophagus / metabolism
  • Gastroesophageal Reflux / metabolism
  • Gastroesophageal Reflux / pathology*
  • Humans
  • Keratinocytes / metabolism*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Receptor, PAR-2 / genetics
  • Receptor, PAR-2 / metabolism*
  • TRPV Cation Channels / genetics
  • TRPV Cation Channels / metabolism*
  • Up-Regulation

Substances

  • RNA, Messenger
  • Receptor, PAR-2
  • TRPV Cation Channels
  • Trpv4 protein, mouse