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Exendin‐4 inhibits iNOS expression at the protein level in LPS‐stimulated Raw264.7 macrophage by the activation of cAMP/PKA pathway
Author(s) -
Chang SeoYoon,
Kim DongBin,
Ryu Gyeong Ryul,
Ko SeungHyun,
Jeong InKyung,
Ahn YuBae,
Jo YangHyeok,
Kim MyungJun
Publication year - 2013
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.24425
Subject(s) - microbiology and biotechnology , cycloheximide , electrophoretic mobility shift assay , chemistry , receptor , biology , gene expression , biochemistry , protein biosynthesis , gene
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. J. Cell. Biochem. 114: 844–853, 2013. © 2012 Wiley Periodicals, Inc.