EGF receptor transactivation and MAP kinase mediate proteinase-activated receptor-2-induced chloride secretion in intestinal epithelial cells

Abstract

We examined the stimulus-secretion pathways whereby proteinase-activated receptor 2 (PAR-2) stimulates Cl⁻ secretion in intestinal epithelial cells. SCBN and T84 epithelial monolayers grown on Snapwell supports and mounted in modified Ussing chambers were activated by the PAR-2-activating peptides SLIGRL-NH₂ and 2-furoyl-LIGRLO-NH₂. Short-circuit current ( I_sc) was used as a measure of net electrogenic ion transport. Basolateral, but not apical, application of SLIGRL-NH₂ or 2-furoyl-LIGRLO-NH₂ caused a concentration-dependent change in I_sc that was significantly reduced in Cl⁻-free buffer and by the intracellular Ca²⁺ blockers thapsigargin and BAPTA-AM, but not by the Ca²⁺ channel blocker verapamil. Inhibitors of PKA (H-89) and CFTR (glibenclamide) also significantly reduced PAR-2-stimulated Cl⁻ transport. PAR-2 activation was associated with increases in cAMP and intracellular Ca²⁺. Immunoblot analysis revealed increases in phosphorylation of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase, Src, Pyk2, cRaf, and ERK1/2 in response to PAR-2 activation. Pretreatment with inhibitors of cyclooxygenases (indomethacin), tyrosine kinases (genistein), EGFR (PD-153035), MEK (PD-98059 or U-0126), and Src (PP1) inhibited SLIGRL-NH₂-induced increases in I_sc. Inhibition of Src, but not matrix metalloproteinases, reduced EGFR phosphorylation. Reduced EGFR phosphorylation paralleled the reduction in PAR-2-stimulated I_sc. We conclude that activation of basolateral, but not apical, PAR-2 induces epithelial Cl⁻ secretion via cAMP- and Ca²⁺-dependent mechanisms. The secretory effect involves EGFR transactivation by Src, leading to subsequent ERK1/2 activation and increased cyclooxygenase activity.