cyclic-gmp and manumycin

Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in heme catabolism, which confers cytoprotection against oxidative injury and provides a vital function in maintaining tissue homeostasis. HMG-CoA reductase inhibitors (statins) possess several anti-inflammatory mechanisms and may be beneficial in the treatment of inflammatory diseases. Our previous study has shown that statins can inhibit iNOS gene expression in murine RAW264.7 macrophages. In this study, we showed that lovastatin, fluvastatin, atorvastatin, simvastatin, mevastatin and pravastatin are able to upregulate the mRNA expression of HO-1 gene. This effect of lovastatin was attenuated by farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), a protein kinase G (PKG) inhibitor (KT5823), a soluble guanylyl cyclase inhibitor (ODQ), a p38 MAPK inhibitor (SB203580), and MEK inhibitors (U0126 and PD98059), but not by inhibitors of protein kinase C (PKC), protein kinase A (PKA), c-jun N-terminal kinase (JNK) and Rho kinase. Consistent with this notion, our previous study has reported the ability of statins to activate ERK and p38 MAPK in RAW264.7 macrophages. Here we further found the participation of cyclic guanosine monophosphate (cGMP)/PKG pathway for ERK activation in cells stimulated with statin and the ability of statin to induce AP-1 activity, which is an essential transcription factor in the regulation of HO-1 gene expression. In addition, a Ras inhibitor (manumycin A) treatment also caused a marked induction of HO-1 mRNA followed by a corresponding increase in HO-1 protein; instead, inhibition of Rho activity by toxin B only led to a transient and weak induction of HO-1. The involvement of signal pathways in manumycin A-induced HO-1 gene expression was associated with p38 MAPK, JNK and ERK activation. Taken together, these results demonstrate for the first time that statins might activate PKG to elicit activations of ERK and p38 MAPK pathways and finally induce HO-1 gene expression, which provides a novel anti-inflammatory mechanism in the therapeutic validity.

Topics: Animals; Cell Line; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Enzymologic; Heme Oxygenase-1; Hydroxymethylglutaryl-CoA Reductase Inhibitors; JNK Mitogen-Activated Protein Kinases; Macrophages; Mice; p38 Mitogen-Activated Protein Kinases; Polyenes; Polyunsaturated Alkamides; ras Proteins; rho GTP-Binding Proteins; RNA, Messenger; Signal Transduction; Time Factors; Transcription Factor AP-1; Up-Regulation

We demonstrated previously that 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), an activator of soluble guanylate cyclase (sGC), induces cyclooxygenase-2 (COX-2) expression via cGMP- and p44/42 mitogen-activated protein kinase-dependent pathways in human pulmonary epithelial A549 cells. In this study, we explore the role of Ras, phosphoinositide-3-OH-kinase (PI3K), Akt, and transcription factor nuclear factor-kappaB (NF-kappaB) in YC-1-induced COX-2 expression in A549 cells. A Ras inhibitor (manumycin A), a PI3K inhibitor (wortmannin), an Akt inhibitor (1l-6-Hydroxymethyl-chiro-inositol2-[(R)-2-O-methyl-3-O-octadecylcarbonate]), and an NF-kappaB inhibitor [pyrrolidine dithiocarbamate (PDTC)] all reduced YC-1-induced COX-2 expression. The YC-1-induced increase in COX activity was also blocked by manumycin A, wortmannin, PDTC, and the dominant-negative mutants for Ras (RasN17), Akt (Akt DN), and IkappaBalpha (IkappaBalphaM). The YC-1-induced increase in Ras activity was inhibited by an sGC inhibitor [1H-(1,2,4)oxadiazolo[4,3-a]quinozalin-1-one (ODQ)], a protein kinase G (PKG) inhibitor [1-oxo-9.12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-I][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT-5823)], and manumycin A. YC-1-induced Akt activation was also inhibited by ODQ, KT-5823, manumycin A, and wortmannin. YC-1 caused the formation of an NF-kappaB-specific DNA-protein complex and an increase in kappaB-luciferase activity. YC-1-induced kappaB-luciferase activity was inhibited by ODQ, KT-5823, manumycin A, wortmannin, an Akt inhibitor, PDTC, RasN17, Akt DN, and IkappaBalphaM. Likewise, YC-1-induced IKKalpha/beta activation was inhibited by ODQ, KT-5823, manumycin A, wortmannin, and an Akt inhibitor. Furthermore, YC-1-induced COX-2 promoter activity was inhibited by manumycin A, RasN17, Akt DN, PDTC, and IkappaBalphaM. Taken together, these results indicate that YC-1 might activate the sGC/cGMP/PKG pathway to induce Ras and PI3K/Akt activation, which in turn initiates IKKalpha/beta and NF-kappaB activation and finally induces COX-2 expression in A549 cells.

Topics: Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclooxygenase 2; Dinoprostone; Enzyme Inhibitors; Epithelial Cells; Gene Expression; Humans; I-kappa B Kinase; I-kappa B Proteins; Indazoles; Interleukin-1; Isoenzymes; Lung; Membrane Proteins; Mutation; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphatidylinositol 3-Kinases; Polyenes; Polyunsaturated Alkamides; Proline; Prostaglandin-Endoperoxide Synthases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; ras Proteins; Thiocarbamates; Transcription Factors