title: Exendin-4 inhibits iNOS expression at the protein level in LPS-stimulated Raw264.7 macrophage by the activation of cAMP/PKA pathway.
abstract: Glucagon-like peptide-1 (GLP-1) and its potent agonists have been widely studied in pancreatic islet β-cells. However, GLP-1 receptors are present in many extrapancreatic tissues including macrophages, and thus GLP-1 may have diverse actions in these tissues and cells. Therefore, we examined the mechanism by which exendin-4 (EX-4), a potent GLP-1 receptor agonist, inhibits lipopolysaccharide (LPS)-induced iNOS expression in Raw264.7 macrophage cells. EX-4 significantly inhibited LPS-induced iNOS protein expression and nitrite production. However, Northern blot and promoter analyses demonstrated that EX-4 did not inhibit LPS-induced iNOS mRNA expression and iNOS promoter activity. Electrophoretic mobility shift assay (EMSA) showed that EX-4 did not alter the binding activity of NF-κB to the iNOS promoter. Consistent with the result of EMSA, LPS-induced IκBα phosphorylation and nuclear translocation of p65 were not inhibited by EX-4. Also, actinomycin D chase study and the promoter assay using the construct containing 3'-untranslated region of iNOS showed that EX-4 did not affect iNOS mRNA stability. Meanwhile, cycloheximide chase study demonstrated that EX-4 significantly accelerated iNOS protein degradation. The EX-4 inhibition of LPS-induced iNOS protein was significantly reversed by adenylate cyclase inhibitors (MDL-12330A and SQ 22536), a PKA inhibitor (H-89) and PKAα gene silencing. These findings suggest that EX-4 inhibited LPS-induced iNOS expression at protein level, but not at transcriptional mechanism of iNOS gene and this inhibitory effect of EX-4 was mainly dependent on cAMP/PKA system.
authors.affiliation: Department of Physiology, College of Medicine, The Catholic University, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea.
authors.forename: Seo-Yoon
authors.initials: SY
authors.lastname: Chang
authors.forename: Dong-Bin
authors.initials: DB
authors.lastname: Kim
authors.forename: Gyeong Ryul
authors.initials: GR
authors.lastname: Ryu
authors.forename: Seung-Hyun
authors.initials: SH
authors.lastname: Ko
authors.forename: In-Kyung
authors.initials: IK
authors.lastname: Jeong
authors.forename: Yu-Bae
authors.initials: YB
authors.lastname: Ahn
authors.forename: Yang-Hyeok
authors.initials: YH
authors.lastname: Jo
authors.forename: Myung-Jun
authors.initials: MJ
authors.lastname: Kim
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D000077270:Exenatide; D015971:Gene Expression Regulation, Enzymologic; D000067757:Glucagon-Like Peptide-1 Receptor; D007097:Imines / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D059748:Proteolysis; D020871:RNA Stability; D018027:Receptors, Glucagon / Q000819:agonists; D014688:Venoms / Q000494:pharmacology*
chemical_list: D020413:3' Untranslated Regions; D004791:Enzyme Inhibitors; C000593834:Glp1r protein, mouse; D000067757:Glucagon-Like Peptide-1 Receptor; D007097:Imines; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009573:Nitrites; D010455:Peptides; D018027:Receptors, Glucagon; D014688:Venoms; D003609:Dactinomycin; D009569:Nitric Oxide; C014925:RMI 12330A; D000077270:Exenatide; D000242:Cyclic AMP; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse