Regulation and function of COX-2 gene expression in isolated gastric parietal cells

Abstract

We examined expression, function, and regulation of the cyclooxygenase (COX)-2 gene in gastric parietal cells. COX-2-specific mRNA was isolated from purified (>95%) canine gastric parietal cells in primary culture and measured by Northern blots using a human COX-2 cDNA probe. Carbachol was the most potent inducer of COX-2 gene expression. Gastrin and histamine exhibited minor stimulatory effects. Carbachol-stimulated expression was inhibited by intracellular Ca2+chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid-AM (90%), protein kinase C (PKC) inhibitor GF-109203X (48%), and p38 kinase inhibitor SB-203580 (48%). Nuclear factor (NF)-κB inhibitor 1-pyrrolidinecarbodithioic acid inhibited carbachol-stimulated expression by 80%. Similar results were observed in the presence of adenoviral vector Ad.dom.neg.IκB, which expresses a repressor of NF-κB. Addition of SB-203580 with Ad.dom.neg.IκB almost completely blocked carbachol stimulation of COX-2 gene expression. We examined the effect of carbachol on PGE2release by enzyme-linked immunoassay. Carbachol induced PGE2 release. Ad.dom.neg.IκB, alone or with SB-203580, produced, respectively, partial (70%) and almost complete (>80%) inhibition of carbachol-stimulated PGE2 production. Selective COX-2 inhibitor NS-398 blocked carbachol-stimulated PGE2 release without affecting basal PGE2production. In contrast, indomethacin inhibited both basal and carbachol-stimulated PGE2 release. Carbachol induces COX-2 gene expression in the parietal cells through signaling pathways that involve intracellular Ca2+, PKC, p38 kinase, and activation of NF-κB. The functional significance of these effects seems to be stimulation of PGE2 release.