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

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 1α (HIF1α), 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 HIF1α. To test this hypothesis, bovine lung microvascular ECs (BLMECs) were transfected with HIF1α and BMK1 cDNAs, and stimulated by hypoxia. HIF1α activity was measured by a reporter gene assay in which luciferase expression was driven by HIF1α activation. Hypoxia (1% O 2 , 24 hours) stimulated HIF1α activity by 5.1±0.6 fold. In the presence of dominant negative (DN)-BMK1, which inhibited BMK1 activity, hypoxia induced HIF1α activity was enhanced significantly to 6.4±0.4 fold. BMK1 activation by constitutively active (CA)-MEK5 inhibited HIF1α activity by 46±4%, suggesting BMK1 functions as a negative regulator of HIF1α activation. Activation of BMK1 reduced HIF1α protein levels. Ubiquitination inhibitors (30 μmol/L ALLN, 2 μmol/L lactacystin, or 100 nmol/L MG132) reduced the BMK1-mediated effect on HIF1α expression by >80%, suggesting that BMK1 stimulated HIF1α proteolysis. The negative effect of BMK1 on HIF1α 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 HIF1α and angiogenesis by increasing HIF1α ubiquitination and inhibiting HIF1α activity in endothelial cells.