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

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-NH(2) and 2-furoyl-LIGRLO-NH(2). Short-circuit current (I(sc)) was used as a measure of net electrogenic ion transport. Basolateral, but not apical, application of SLIGRL-NH(2) or 2-furoyl-LIGRLO-NH(2) caused a concentration-dependent change in I(sc) that was significantly reduced in Cl(-)-free buffer and by the intracellular Ca(2+) blockers thapsigargin and BAPTA-AM, but not by the Ca(2+) 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 Ca(2+). 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-NH(2)-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 Ca(2+)-dependent mechanisms. The secretory effect involves EGFR transactivation by Src, leading to subsequent ERK1/2 activation and increased cyclooxygenase activity.

van der Merwe JQ ，Hollenberg MD ，MacNaughton WK 《AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY》

Protease-activated receptor-2 stimulates intestinal epithelial chloride transport through activation of PLC and selective PKC isoforms.

Serine proteases play important physiological roles through their activity at G protein-coupled protease-activated receptors (PARs). We examined the roles that specific phospholipase (PL) C and protein kinase (PK) C (PKC) isoforms play in the regulation of PAR(2)-stimulated chloride secretion in intestinal epithelial cells. Confluent SCBN epithelial monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short-circuit current (I(sc)) responses to basolateral application of the selective PAR(2) activating peptide, SLIGRL-NH(2), were monitored as a measure of net electrogenic ion transport caused by PAR(2) activation. SLIGRL-NH(2) induced a transient I(sc) response that was significantly reduced by inhibitors of PLC (U73122), phosphoinositol-PLC (ET-18), phosphatidylcholine-PLC (D609), and phosphatidylinositol 3-kinase (PI3K; LY294002). Immunoblot analysis revealed the phosphorylation of both PLCbeta and PLCgamma following PAR(2) activation. Pretreatment of the cells with inhibitors of PKC (GF 109203X), PKCalpha/betaI (Gö6976), and PKCdelta (rottlerin), but not PKCzeta (selective pseudosubstrate inhibitor), also attenuated this response. Cellular fractionation and immunoblot analysis, as well as confocal immunocytochemistry, revealed increases of PKCbetaI, PKCdelta, and PKCepsilon, but not PKCalpha or PKCzeta, in membrane fractions following PAR(2) activation. Pretreatment of the cells with U73122, ET-18, or D609 inhibited PKC activation. Inhibition of PI3K activity only prevented PKCdelta translocation. Immunoblots revealed that PAR(2) activation induced phosphorylation of both cRaf and ERK1/2 via PKCdelta. Inhibition of PKCbetaI and PI3K had only a partial effect on this response. We conclude that basolateral PAR(2)-induced chloride secretion involves activation of PKCbetaI and PKCdelta via a PLC-dependent mechanism resulting in the stimulation of cRaf and ERK1/2 signaling.

van der Merwe JQ ，Moreau F ，MacNaughton WK 《AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY》

Signal transduction for proteinase-activated receptor-2-triggered prostaglandin E2 formation in human lung epithelial cells.

Kawao N ，Nagataki M ，Nagasawa K ，Kubo S ，Cushing K ，Wada T ，Sekiguchi F ，Ichida S ，Hollenberg MD ，MacNaughton WK ，Nishikawa H ，Kawabata A ... -  《JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS》

Prostaglandin E2 derived from cyclooxygenases 1 and 2 mediates intestinal epithelial ion transport stimulated by the activation of protease-activated receptor 2.

Proteinase-activated receptor (PAR)(2) is activated by trypsin-like serine proteinases and has been implicated in intestinal inflammation. However, its role in the regulation of intestinal mucosal function remains unclear. Using the intestinal epithelial cell line, SCBN, we have studied the stimulus-secretion coupling mechanisms of PAR(2)-induced epithelial chloride transport, focusing on cyclooxygenase (COX)-1 and COX-2 activities and prostaglandin (PG) E(2) secretion. SCBN monolayers were grown on Snapwell supports, mounted in modified Ussing chambers, and exposed to the activating peptide, SLIGRL-NH(2) (50 microM), to activate PAR(2). Pretreatment with inhibitors of cytosolic PLA(2) (cPLA(2)) (AACOCF3, arachidonyltrifluoromethyl ketone), COX-1 [SC560, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole], and COX-2 (celecoxib) resulted in a significant concentration-dependent attenuation of PAR(2)-induced changes in short-circuit current. Immunoblot analysis showed a PAR(2)-induced increase in cPLA(2) phosphorylation that was blocked by the mitogen-activated protein kinase kinase inhibitor, PD98059 [2-(2-amino-3methoxyphenyl)-4H-1benzopyran-4-one, C(16)H(13)NO(3)], and the pan-protein kinase C inhibitor, GFX (bisindolylmaleimide). PAR(2) stimulation also resulted in a large increase in the production of PGE(2) as determined by enzyme-linked immunosorbent assay and was also blocked by PD98059 and GFX. Immunofluorescence and immunoblot analysis determined that EP2 and EP4 are expressed at the basolateral membrane of SCBN cells. Through the use of selective inhibitors (EP2, AH6809 [6-isopropoxy-9-oxoxanthene-2-carboxylic acid]; EP4, GW627368X [N-[2[4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl] acetyl]benzene sulphonamide]), it was found that both EP2 and EP4 were involved in mediating the PAR(2)-induced chloride secretory response. We conclude that basolateral PAR(2) activation induces epithelial chloride secretion that is mediated by cPLA(2), COX-1, COX-2, and the subsequent release of PGE(2). The production of PGE(2) results in an autocrine secretory response that is dependent on basolateral EP2 and EP4 receptors.

van der Merwe JQ ，Ohland CL ，Hirota CL ，MacNaughton WK ... -  《-》

Epidermal growth factor receptor transactivation is required for proteinase-activated receptor-2-induced COX-2 expression in intestinal epithelial cells.

Hirota CL ，Moreau F ，Iablokov V ，Dicay M ，Renaux B ，Hollenberg MD ，MacNaughton WK ... -  《-》