title: Endothelial exposure to hypoxia induces Egr-1 expression involving PKCalpha-mediated Ras/Raf-1/ERK1/2 pathway.
abstract: Hypoxia induces endothelial dysfunction that results in a series of cardiovascular injuries. Early growth response-1 (Egr-1) has been indicated as a common theme in vascular injury. Here we demonstrates that in bovine aortic endothelial cells (ECs) subjected to hypoxia (PO(2) approximately 23 mmHg), rapidly increased Egr-1 mRNA expression which peaked within 30 min and decreased afterwards. Treatment of ECs with PD98059, a specific inhibitor to mitogen-activated protein kinase (MAPK/ERK), inhibited this hypoxia-induced Egr-1 expression. The involvement of ERK pathway was further substantiated by the inhibition of Egr-1 promoter activities when ECs were co-transfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf 301), or a catalytically inactive mutant of ERK2 (mERK). In addition, the hypoxia-induced transcriptional activity of Elk-1, an ERK substrate, was abolished by administration of PD98059. Addition of calphostin C, a protein kinase C (PKC) inhibitor, completely blocked the hypoxia-augmented Egr-1 expression. The likewise occurred while exposing ECs to D609 to inhibit phospholipase C and BAPTA/AM to chelate intracellular calcium. Hypoxia to ECs increased ERK phosphorylation within 10 min and which was abolished by administration of PD98095, calphostin C, and BAPTA/AM. Hypoxia triggered a transient translocation of PKCalpha from cytosol to membrane fraction concurrent with the association of PKCalpha to Raf-1. Involvement of PKCalpha in mediating ERK activation was further confirmed by the inhibition of ERK and the subsequent Egr-1 gene induction with antisense oligonucleotides to PKCalpha. These results indicate that ECs under hypoxia induce Egr-1 expression and this induction requires calcium, phospholipase C activation, and PKCalpha-mediated Ras/Raf-1/ERK1/2 signaling pathway. Our finding support the importance of specific PKC isozyme linked to MAPK pathway in the regulation of endothelial responses to hypoxia.
authors.affiliation: Departmant of Applied Chemistry, National Chi Nan University, Nantou, Taiwan, ROC.
authors.forename: L W
authors.initials: LW
authors.lastname: Lo
authors.forename: J J
authors.initials: JJ
authors.lastname: Cheng
authors.forename: J J
authors.initials: JJ
authors.lastname: Chiu
authors.forename: B S
authors.initials: BS
authors.lastname: Wung
authors.forename: Y C
authors.initials: YC
authors.lastname: Liu
authors.forename: D L
authors.initials: DL
authors.lastname: Wang
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D015687:Cell Hypoxia / Q000502:physiology*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D005799:Genes, Dominant; D017930:Genes, Reporter; D007527:Isoenzymes / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D015533:Transcriptional Activation; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D050807:ets-Domain Protein Elk-1; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D018631:ras Proteins; D002118:Calcium