Pi Xinchun, Garin Gwenaele, Xie Liang, Zheng Qinlei, Wei Heng, Abe Jun-ichi, Yan Chen, Berk Bradford C
Cardiovascular Research Institute, Department of Medicine, University of Rochester, NY, USA.
Circ Res. 2005 Jun 10;96(11):1145-51. doi: 10.1161/01.RES.0000168802.43528.e1. Epub 2005 May 5.
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.
大丝裂原活化蛋白激酶1（BMK1或ERK5）是内皮细胞（EC）功能的关键调节因子，BMK1基因敲除小鼠的胚胎血管生成受损和血管塌陷就证明了这一点。缺氧诱导因子1α（HIF1α）是血管生成的一种有效调节因子，受丝裂原活化蛋白激酶ERK1/2的正向调控。由于BMK1缺陷与血管生成受损有关，我们推测BMK1可能调节HIF1α。为了验证这一假设，将HIF1α和BMK1的cDNA转染到牛肺微血管内皮细胞（BLMECs）中，并进行缺氧刺激。通过报告基因检测来测量HIF1α的活性，其中荧光素酶表达由HIF1α激活驱动。缺氧（1% O2，24小时）使HIF1α活性提高了5.1±0.6倍。在存在抑制BMK1活性的显性负性（DN）-BMK1的情况下，缺氧诱导的HIF1α活性显著增强至6.4±0.4倍。组成型活性（CA）-MEK5激活BMK1可使HIF1α活性降低46±4%，表明BMK1作为HIF1α激活的负调节因子发挥作用。BMK1的激活降低了HIF1α蛋白水平。泛素化抑制剂（30 μmol/L ALLN、2 μmol/L乳胞素或100 nmol/L MG132）使BMK1对HIF1α表达的影响降低了80%以上，表明BMK1刺激了HIF1α的蛋白水解。BMK1对HIF1α的负性作用在功能上很重要，因为转染CA-MEK5可使内皮细胞迁移显著降低68±10%，并在体外基质胶试验中抑制血管生成76±7%。总之，BMK1是一种新型的HIF1α和血管生成的负调节因子，它通过增加内皮细胞中HIF1α的泛素化并抑制HIF1α活性来实现。
