Journal
JOURNAL OF CELLULAR BIOCHEMISTRY
Volume 114, Issue 4, Pages 844-853Publisher
WILEY-BLACKWELL
DOI: 10.1002/jcb.24425
Keywords
EXENDIN-4 (EX-4); LPS; iNOS; NF-B; cAMP; PKA; Raw264; 7 MACROPHAGE
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Funding
- National Research Foundation of Korea [5-2010-A0154-00139]
- Basic Science Research Program through the National Research Foundation of Korea (NRF)
- Ministry of Education, Science and Technology [5-2010-A0154-00139]
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Glucagon-like peptide-1 (GLP-1) and its potent agonists have been widely studied in pancreatic islet -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-B to the iNOS promoter. Consistent with the result of EMSA, LPS-induced IB 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 PKA 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: 844853, 2013. (c) 2012 Wiley Periodicals, Inc.
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