manumycin and prolinedithiocarbamate

Rapid recognition and ingestion of apoptotic cells by phagocytes are important for the prevention of toxic intracellular contents release, thereby attenuate inflammation and autoimmune diseases such as systemic lupus erythematosus (SLE). We have reported that oridonin isolated from Rabdosia rubescens enhanced phagocytosis of apoptotic U937 cells by macrophage-like U937 cells through TNFalpha and IL-1beta release. In this study, the molecular mechanisms involved in this phagocytic process are investigated. Inhibitors of Ras and Raf1 kinase significantly reduced oridonin-induced phagocytic stimulation as well as extracellular signal-regulated kinase (ERK) phosphorylation. Simultaneously, oridonin-enhanced engulfment was partially blocked by a nuclear factor (NF)-kappaB inhibitor PDTC or proteasome inhibitor MG132. Further studies revealed that oridonin induced IkappaBalpha degradation, which was prevented by Ras inhibitor manumycin A, ERK inhibitor PD98059, but not prevented by c-Jun N-terminal kinase (JNK) MAPK inhibitor SP600125, and up-regulated expression of IL-1beta precursor. These results demonstrate that Ras/Raf1/ERK signaling pathway-dependent IkappaBalpha degradation, resulting in NF-kappaB activation, participates in regulation of oridonin-enhanced phagocytosis, and one of its effector functions is to induce synthesis of IL-1beta, which partially contribute to phagocytic activity of oridonin.

Topics: Apoptosis; Blotting, Western; Diterpenes; Diterpenes, Kaurane; Extracellular Signal-Regulated MAP Kinases; Flow Cytometry; Genes, ras; Humans; I-kappa B Proteins; Interleukin-1; Leupeptins; Macrophages; Phagocytosis; Polyenes; Polyunsaturated Alkamides; Proline; Proto-Oncogene Proteins c-raf; ras Proteins; Thiocarbamates; U937 Cells

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