calyculin-a and cobaltous-chloride

During hypoxia, hypoxia-inducible factor-1alpha (HIF-1alpha) is required for induction of a variety of genes including erythropoietin and vascular endothelial growth factor. Hypoxia increases mitochondrial reactive oxygen species (ROS) generation at Complex III, which causes accumulation of HIF-1alpha protein responsible for initiating expression of a luciferase reporter construct under the control of a hypoxic response element. This response is lost in cells depleted of mitochondrial DNA (rho(0) cells). Overexpression of catalase abolishes hypoxic response element-luciferase expression during hypoxia. Exogenous H(2)O(2) stabilizes HIF-1alpha protein during normoxia and activates luciferase expression in wild-type and rho(0) cells. Isolated mitochondria increase ROS generation during hypoxia, as does the bacterium Paracoccus denitrificans. These findings reveal that mitochondria-derived ROS are both required and sufficient to initiate HIF-1alpha stabilization during hypoxia.

Topics: Androstadienes; Animals; Cell Line; Cell Nucleus; Chelating Agents; Cobalt; Cytosol; Deferoxamine; DNA-Binding Proteins; Dose-Response Relationship, Drug; Electron Transport Complex III; Electron Transport Complex IV; Enzyme Inhibitors; Genes, Reporter; Humans; Hydrogen Peroxide; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoblotting; Marine Toxins; Mitochondria; Mitochondria, Liver; Models, Biological; Nuclear Proteins; Oxazoles; Oxidation-Reduction; Oxygen; Paracoccus denitrificans; Rats; Reactive Oxygen Species; Time Factors; Transcription Factors; Transfection; Tumor Cells, Cultured; Wortmannin

In response to hypoxia, sickle red blood cells (SS RBC) and leukocytes exhibit increased adherence to the vascular endothelium, while diapedesis of leukocytes through the blood vessel increases. However, the cellular signaling pathway(s) caused by hypoxia is poorly understood. We utilized CoCl2 as a mimetic molecule for hypoxia to study cellular signaling pathways. We found that in human umbilical vein endothelial cells (HUVEC), CoCl2 at 2 mM concentration induced the surface expression of a subset of CAMs (VCAM-1) and activation of transcription factor NF-kappaB in the nuclear extracts of HUVEC. Furthermore, CoCl2 also caused time-dependent tyrosine phosphorylation of mitogen-activated protein (MAP) kinase isoform ERK2 without significantly affecting ERK1, indicating ERK2 is the preferred substrate for upstream kinase of the MAPK pathway. Inhibitors of MAP kinase (PD98059) or platelet-activating factor (PAF)- receptor antagonist (CV3988) inhibited the CoCl2-induced NF-kappaB activation and VCAM-1 expression. Augmented expression of VCAM-1 led to increased SS RBC adhesion, inhibitable by a VCAM-1 antibody. Additionally, CoCl2 caused a two- to threefold increase in the rate of transendothelial migration of monocyte-like HL-60 cells and a twentyfold increase in phosphorylation of platelet endothelial cell adhesion molecules (PECAM-1). The transendothelial migration of monocytes was inhibited by an antibody to PECAM-1. Both phosphorylation of PECAM-1 and transendothelial migration of monocytes in response to CoCl2 were inhibited by protein kinase inhibitor (GF109203X) and augmented by protein phosphatase inhibitor (Calyculin A). Our data suggests that CoCl2-induced cellular signals directing increased expression of VCAM-1 in HUVEC involve downstream activation of MAP kinase and NF-kappaB, while the phosphorylation of PECAM-1 occurs as a result of activation of PKC. We conclude that PAF-receptor antagonist inhibits the CoCl2- or hypoxia-induced increase in the adhesion of SS RBC, PECAM-1 phosphorylation, and the concomitant transendothelial migration of monocytes.

Topics: Adult; Anemia, Sickle Cell; Base Sequence; Calcium-Calmodulin-Dependent Protein Kinases; Cell Adhesion; Cell Movement; Cells, Cultured; Cobalt; E-Selectin; Endothelium, Vascular; Enzyme Inhibitors; Erythrocytes, Abnormal; Flavonoids; HL-60 Cells; Humans; Hypoxia; Indoles; Intercellular Adhesion Molecule-1; Maleimides; Marine Toxins; Mitogen-Activated Protein Kinase 1; Molecular Sequence Data; NF-kappa B; Oxazoles; Phospholipid Ethers; Phosphoprotein Phosphatases; Phosphorylation; Platelet Endothelial Cell Adhesion Molecule-1; Platelet Membrane Glycoproteins; Protein Processing, Post-Translational; Receptors, Cell Surface; Receptors, G-Protein-Coupled; RNA, Messenger; Signal Transduction; Transcription Factor AP-1; Umbilical Veins; Vascular Cell Adhesion Molecule-1