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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.
J Cell Biochem. 2013 Apr; 114(4):844-53.JC

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+Show Affiliations

Department of Physiology, College of Medicine, The Catholic University, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

23097217

Citation

Chang, Seo-Yoon, et al. "Exendin-4 Inhibits iNOS Expression at the Protein Level in LPS-stimulated Raw264.7 Macrophage By the Activation of cAMP/PKA Pathway." Journal of Cellular Biochemistry, vol. 114, no. 4, 2013, pp. 844-53.
Chang SY, Kim DB, Ryu GR, et al. Exendin-4 inhibits iNOS expression at the protein level in LPS-stimulated Raw264.7 macrophage by the activation of cAMP/PKA pathway. J Cell Biochem. 2013;114(4):844-53.
Chang, S. Y., Kim, D. B., Ryu, G. R., Ko, S. H., Jeong, I. K., Ahn, Y. B., Jo, Y. H., & Kim, M. J. (2013). Exendin-4 inhibits iNOS expression at the protein level in LPS-stimulated Raw264.7 macrophage by the activation of cAMP/PKA pathway. Journal of Cellular Biochemistry, 114(4), 844-53. https://doi.org/10.1002/jcb.24425
Chang SY, et al. Exendin-4 Inhibits iNOS Expression at the Protein Level in LPS-stimulated Raw264.7 Macrophage By the Activation of cAMP/PKA Pathway. J Cell Biochem. 2013;114(4):844-53. PubMed PMID: 23097217.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Exendin-4 inhibits iNOS expression at the protein level in LPS-stimulated Raw264.7 macrophage by the activation of cAMP/PKA pathway. AU - Chang,Seo-Yoon, AU - Kim,Dong-Bin, AU - Ryu,Gyeong Ryul, AU - Ko,Seung-Hyun, AU - Jeong,In-Kyung, AU - Ahn,Yu-Bae, AU - Jo,Yang-Hyeok, AU - Kim,Myung-Jun, PY - 2012/08/29/received PY - 2012/10/08/accepted PY - 2012/10/26/entrez PY - 2012/10/26/pubmed PY - 2013/9/26/medline SP - 844 EP - 53 JF - Journal of cellular biochemistry JO - J. Cell. Biochem. VL - 114 IS - 4 N2 - 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. SN - 1097-4644 UR - https://www.unboundmedicine.com/medline/citation/23097217/Exendin_4_inhibits_iNOS_expression_at_the_protein_level_in_LPS_stimulated_Raw264_7_macrophage_by_the_activation_of_cAMP/PKA_pathway_ L2 - https://doi.org/10.1002/jcb.24425 DB - PRIME DP - Unbound Medicine ER -