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12 PAR-2-INDUCED EPITHELIAL CHLORIDE SECRETION INVOLVES EGF RECEPTOR TRANSACTIVATION BY A SRC-DEPENDENT MECHANISM JQ van der Merwe, WK MacNaughton
BACKGROUND: Proteinase-activated receptor (PAR)-2 is activated by trypsin-like serine proteinases and selective synthetic peptides. It has been implicated in intestinal inflammation, but its role in the regulation of intestinal mucosal function remains unclear. We studied the stimulus-secretion coupling mechanisms of PAR-2 activation on epithelial chloride transport in a cell culture model.
METHODS: 1. RT-PCR and immunocytochemistry were used to confirm PAR-2 expression in SCBN intestinal epithelial cells. 2. SCBN monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short circuit current (Isc) was monitored as a measure of net electrogenic ion transport. The roles of PKC, MAP kinases (specifically ERK1/2), Src, matrix metallo-proteinases (MMPs) and the EGF receptor (EGFr) in the response to PAR-2 activation by SLIGRL-NH2 were determined using specific inhibitors (GFX, PD98059, PP1, GM6001, PD153035). 3. Western blot analysis was conducted for ERK1/2, Src and EGFr phosphorylation in response to PAR-2 activation by SLIGRL-NH2. Western blots for total amounts of each protein were conducted as controls.
RESULTS: 1. RT-PCR and immunocytochemistry showed PAR-2 expression in SCBN cells. 2. Pretreatment of the cells with GFX resulted in a significant reduction in the response to PAR-2 activation by SLIGRL-NH2 (P<0.001), thereby suggesting a role for PKC in PAR-2 mediated chloride secretion. PD98059 pretreatment significantly reduced responses to PAR-2 activation (P<0.001) as did pretreatment with PD153035 (P<0.001), implicating EGF receptor tyrosine kinase and MAP kinase pathways, and more specifically ERK1/2. Inhibition of Src, but not MMPs, resulted in a significant decrease in response to PAR-2 activation by SLIGRL-NH2, therefore suggesting a role for Src in EGF receptor-dependent pathways. 3. PAR-2 activation resulted in EGFr, Src and ERK1/2 phosphorylation with no change in total levels of each protein. Inhibiton of Src, but not MMPs, resulted in significantly reduced phosphorylated EGFr following activation of PAR-2 with SLIGRL-NH2, indicating that Src is required for EGFr transactivation. Furthermore, inhibition of Src, EGFr or PKC prevented ERK1/2 phosphorylation, suggesting that ERK1/2 phosphorylation is dependent upon EGFr transactivation and PKC-dependent pathways.
CONCLUSIONS: PAR-2 activation induces epithelial chloride secretion that is mediated by Src-dependent EGF receptor transactivation, subsequent ERK1/2 activation and PKC-mediated signalling.