title: Nitric oxide and hypoxia stimulate erythropoietin receptor via MAPK kinase in endothelial cells.
abstract: Erythropoietin receptor (EPOR) expression level determines the extent of erythropoietin (EPO) response. Previously we showed that EPOR expression in endothelial cells is increased at low oxygen tension and that EPO stimulation of endothelial cells during hypoxia can increase endothelial nitric oxide (NO) synthase (eNOS) expression and activation as well as NO production. We now observe that while EPO can stimulate NO production, NO in turn can regulate EPOR expression. Human umbilical vein endothelial cells (HUVEC) treated with 10-50 μM of NO donor diethylenetriamine NONOate (DETANO) for 24h showed significant induction of EPOR gene expression at 5% and 2% of oxygen. Also human bone marrow microvascular endothelial cell line (TrHBMEC) cultured at 21 and 2% oxygen with 50 μM DETANO demonstrated a time and oxygen dependent induction of EPOR mRNA expression after 24 and 48 h, particularly at low oxygen tension. EPOR protein was also induced by DETANO at 2% oxygen in TrHBMEC and HUVEC. The activation of signaling pathways by NO donor stimulation appeared to be distinct from EPO stimulation. In reporter gene assays, DETANO treatment of HeLa cells at 2% oxygen increased EPOR promoter activity indicated by a 48% increase in luciferase activity with a 2 kb EPOR promoter fragment and a 71% increase in activity with a minimal EPOR promoter fragment containing 0.2 kb 5'. We found that DETANO activated MAPK kinase in TrHBMEC both in normoxia and hypoxia, while MAPK kinase inhibition showed significant reduction of EPOR mRNA gene expression at low oxygen tension, suggesting MAPK involvement in NO mediated induction of EPOR. Furthermore, DETANO stimulated Akt anti-apoptotic activity after 30 min in normoxia, whereas it inhibited Akt phosphorylation in hypoxia. In contrast, EPO did not significantly increase MAPK activity while EPO stimulated Akt phosphorylation in TrHBMEC in normoxia and hypoxia. These observations provide a new effect of NO on EPOR expression to enhance EPO response in endothelial cells, particularly at low oxygen tensions.
authors: Clinic for Endocrinology, Diabetes and Metabolic Diseases, Genetic Laboratory, Clinical Center of Serbia, Belgrade, Serbia. Electronic address: miljuc@yahoo.com.
authors: Bojana B Beleslin
authors: BB
authors: Cokic
authors: Laboratory of Experimental Hematology, Institute for Medical Research, University of Belgrade, Belgrade, Serbia;
authors: Vladan P
authors: VP
authors: Cokic
authors: Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
authors: Sukanya
authors: S
authors: Suresh
authors: Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
authors: Stacey
authors: S
authors: Wirt
authors: Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
authors: Constance Tom
authors: CT
authors: Noguchi
mesh_terms: D015687:Cell Hypoxia / Q000502:physiology*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004921:Erythropoietin / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D009603:Nitroso Compounds / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D012333:RNA, Messenger; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; C094210:2,2'-(hydroxynitrosohydrazono)bis-ethanamine; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt; D020929:Mitogen-Activated Protein Kinase Kinases