title: Cyclooxygenase-2 expression in cultured cortical thick ascending limb of Henle increases in response to decreased extracellular ionic content by both transcriptional and post-transcriptional mechanisms. Role of p38-mediated pathways.
abstract: We showed previously that decreased extracellular salt or chloride up-regulates the cortical thick ascending limb of Henle (cTALH) COX-2 expression via a p38-dependent pathway. The present studies determined that low salt medium increased COX-2 mRNA expression 3.9-fold control by 6 h in cultured cTALH, which was blocked by actinomycin D pretreatment, suggesting transcriptional regulation. Luciferase activity (normalized to beta-galactosidase activity) of the full-length (-3400) COX-2 promoter in cTALH increased from 1.8 +/- 0.3 in control media to 5.8 +/- 0.7 in low salt (n = 9; p < 0.01). Low chloride medium had similar effects as low salt has on COX-2 promoter activity. Deletion constructs -815, -512, and -410 were similarly stimulated, but -385 could not be stimulated significantly by low salt (1.8 +/- 0.3 versus 2.4 +/- 0.5, n = 10). This suggested involvement of an NF-kappaB cis-element located in this region, which was confirmed by utilizing a construct with a point mutation of this NF-kappaB-binding site that was not stimulated by low salt medium. Co-incubation of the specific p38 inhibitor, SB203580 or PD169316, inhibited a low salt-induced increase in luciferase activity of the intact COX-2 promoter (5.8 +/- 0.7 versus 1.1 +/- 0.2, n = 8 and 1.4 +/- 0.4, n = 4 respectively, p < 0.01). Mobility shift assays indicated that the low salt medium stimulated NF-kappaB binding activity, and this stimulation was inhibited by p38 inhibitors. To test whether p38 also increased COX-2 expression by increasing mRNA stability, cTALH were incubated in low salt for 2 h, and actinomycin was then added with or without SB203580. p38 inhibition led to a decreased half-life of COX-2 mRNA (from 68 to 18 min, n = 4-7, p < 0.05). Therefore, these studies indicate that p38 stimulates COX-2 expression in cTALH and macula densa by transcriptional regulation predominantly via a NF-kappaB-dependent pathway and by post-transcriptional increases in mRNA stability.
authors.affiliation: George M. O'Brien Kidney and Urologic Diseases Center and Division of Nephrology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Hui-Fang
authors.initials: HF
authors.lastname: Cheng
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Harris
mesh_terms: D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D003609:Dactinomycin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D051550:I-kappa B Kinase; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D008138:Loop of Henle / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009154:Mutation; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D020871:RNA Stability; D011817:Rabbits; D012965:Sodium Chloride / Q000378:metabolism*; D014158:Transcription, Genetic; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D016328:NF-kappa B; D019384:Nucleic Acid Synthesis Inhibitors; D003609:Dactinomycin; D012965:Sodium Chloride; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases