BMK1/ERK5 Is a Novel Regulator of Angiogenesis by Destabilizing Hypoxia Inducible Factor 1α

Abstract

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 1alpha (HIF1alpha), 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 HIF1alpha. To test this hypothesis, bovine lung microvascular ECs (BLMECs) were transfected with HIF1alpha and BMK1 cDNAs, and stimulated by hypoxia. HIF1alpha activity was measured by a reporter gene assay in which luciferase expression was driven by HIF1alpha activation. Hypoxia (1% O2, 24 hours) stimulated HIF1alpha activity by 5.1+/-0.6 fold. In the presence of dominant negative (DN)-BMK1, which inhibited BMK1 activity, hypoxia induced HIF1alpha activity was enhanced significantly to 6.4+/-0.4 fold. BMK1 activation by constitutively active (CA)-MEK5 inhibited HIF1alpha activity by 46+/-4%, suggesting BMK1 functions as a negative regulator of HIF1alpha activation. Activation of BMK1 reduced HIF1alpha protein levels. Ubiquitination inhibitors (30 micromol/L ALLN, 2 micromol/L lactacystin, or 100 nmol/L MG132) reduced the BMK1-mediated effect on HIF1alpha expression by >80%, suggesting that BMK1 stimulated HIF1alpha proteolysis. The negative effect of BMK1 on HIF1alpha 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 HIF1alpha and angiogenesis by increasing HIF1alpha ubiquitination and inhibiting HIF1alpha activity in endothelial cells.