Endothelial exposure to hypoxia induces Egr‐1 expression involving PKCα‐mediated Ras/Raf‐1/ERK1/2 pathway

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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 (PO2 ≈ 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 PKCα from cytosol to membrane fraction concurrent with the association of PKCα to Raf‐1. Involvement of PKCα in mediating ERK activation was further confirmed by the inhibition of ERK and the subsequent Egr‐1 gene induction with antisense oligonucleotides to PKCα. These results indicate that ECs under hypoxia induce Egr‐1 expression and this induction requires calcium, phospholipase C activation, and PKCα‐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.