title: Activation of a cyclic amp-guanine exchange factor in hepatocytes decreases nitric oxide synthase expression.
abstract: Adenosine 3',5'-cyclic adenosine monophosphate (cAMP) activates intracellular signaling by regulating protein kinase A, calcium influx, and cAMP-binging guanine nucleotide exchange factors (Epac [exchange protein directly activated by cAMP] or cAMP-GEF). Cyclic adenosine monophosphate inhibits cytokine-induced expression of inducible nitric oxide synthase (iNOS) in hepatocytes by a protein kinase A-independent mechanism. We hypothesized that Epac mediates this effect. A cyclic AMP analog that specifically activates Epac, 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (OPTmecAMP), and overexpression of liver-specific Epac2 both inhibited interleukin 1β/interferon γ-induced iNOS expression and nitrite production. OPTmecAMP inactivated Raf1/MEK/ERK signaling, but ERK had no effect on iNOS expression. OPTmecAMP induced a persistent Akt phosphorylation in hepatocytes that lasted up to 8 h. Overexpression of a dominant-negative Akt blocked the inhibitory effect of OPTmecAMP on iNOS production. A specific PI3K inhibitor, LY294002, attenuated the inhibition of nitrite production and iNOS expression produced by overexpressing a liver-specific Epac2 (LEpac2). OPTmecAMP also induced c-Jun N-terminal kinase (JNK) phosphorylation in hepatocytes. Overexpression of dominant-negative JNK enhanced cytokine-induced iNOS expression and nitrite production and reversed the inhibitory effects of LEpac2 on nitrite production and iNOS expression. We conclude that Epac regulates hepatocyte iNOS expression through an Akt- and JNK-mediated signaling mechanism.
authors.affiliation: Department of Surgery and Price Institute of Surgical Research, University of Louisville, Louisville, KY, USA. b.zhang@louisville.edu
authors.forename: Baochun
authors.initials: B
authors.lastname: Zhang
authors.forename: Ikenna
authors.initials: I
authors.lastname: Nweze
authors.forename: Jaganathan
authors.initials: J
authors.lastname: Lakshmanan
authors.forename: Brian G
authors.initials: BG
authors.lastname: Harbrecht
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D022781:Hepatocytes / Q000276:immunology / Q000378:metabolism*; D007371:Interferon-gamma / Q000276:immunology; D053583:Interleukin-1beta / Q000276:immunology; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D008297:Male; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D009573:Nitrites / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D053583:Interleukin-1beta; D009573:Nitrites; C531571:Rapgef4 protein, rat; D007371:Interferon-gamma; D000242:Cyclic AMP; D019001:Nitric Oxide Synthase; D051057:Proto-Oncogene Proteins c-akt; D048670:MAP Kinase Kinase 4