Published online before print May 5, 2005, doi: 10.1161/01.RES.0000168802.43528.e1
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Submitted on December 3, 2004
Revised on April 8, 2005
Accepted on April 27, 2005
BMK1/ERK5 Is a Novel Regulator of Angiogenesis by Destabilizing Hypoxia Inducible Factor 1
Xinchun Pi ;
From the Cardiovascular Research Institute and Departments of Medicine (X.P., G.G., Q.Z., H.W., J.A., C.Y., B.C.B.) and Pharmacology (L.X.), University of Rochester, NY
* To whom correspondence should be addressed. E-mail: Bradford_Berk{at}urmc.rochester.edu.
Big MAP kinase 1 (BMK1 or ERK5) is a key mediator of endothelial cell (EC) function as shown by impaired embryonic angiogenesis and vascular collapse in BMK1 knockout mice. Hypoxia inducible factor 1
(HIF1), a potent mediator of angiogenesis, is positively regulated by the MAP kinases, ERK1/2. Because BMK1 deficiency is associated with impaired angiogenesis we hypothesized that BMK1 might regulate HIF1. To test this hypothesis, bovine lung microvascular ECs (BLMECs) were transfected with HIF1 and BMK1 cDNAs, and stimulated by hypoxia. HIF1 activity was measured by a reporter gene assay in which luciferase expression was driven by HIF1 activation. Hypoxia (1% O2, 24 hours) stimulated HIF1 activity by 5.1±0.6 fold. In the presence of dominant negative (DN)-BMK1, which inhibited BMK1 activity, hypoxia induced HIF1 activity was enhanced significantly to 6.4±0.4 fold. BMK1 activation by constitutively active (CA)-MEK5 inhibited HIF1 activity by 46±4%, suggesting BMK1 functions as a negative regulator of HIF1 activation. Activation of BMK1 reduced HIF1 protein levels. Ubiquitination inhibitors (30µmol/L ALLN, 2µmol/L lactacystin, or 100 nmol/L MG132) reduced the BMK1-mediated effect on HIF1 expression by >80%, suggesting that BMK1 stimulated HIF1 proteolysis. The negative effect of BMK1 on HIF1 was functionally important because transfection with CA-MEK5 significantly decreased EC migration by 68±10%, and inhibited angiogenesis (in vitro Matrigel assay) by 76±7%. In summary, BMK1 is a novel negative regulator of HIF1 and angiogenesis by increasing HIF1 ubiquitination and inhibiting HIF1 activity in endothelial cells.
Key words: big MAP kinase 1 • hypoxia inducible factor 1
•
angiogenesis
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