NAD(P)H Oxidases regulate HIF-2α protein expression
NAD(P)H Oxidases regulate HIF-2α protein expression
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DOI：
10.1074/jbc.m611569200
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发表时间：
2007-03-16
影响因子：
4.8
通讯作者：
Abboud, Hanna E.
中科院分区：
文献类型：
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作者：
Block, Karen;Gorin, Yves;Abboud, Hanna E.
von Hippel-Lindau肿瘤抑制基因（VHL）的双等位基因失活与遗传性和散发性肾细胞癌（RCC）的发生有关。在VHL不存在的情况下，异源二聚体低氧诱导转录因子（HIT-1 α和HIF-2 α）的α亚基是稳定的。由NAD（P）H氧化酶产生的活性氧参与恶性生长的信号级联。我们发现，在VHL缺陷细胞p22（phox），Nox 4蛋白水平和NADPH依赖的超氧化物生成增加。将VHL重新引入VHL缺陷细胞下调p22（phox）的表达和NADPH依赖的超氧化物生成。VHL表达细胞中26 S蛋白酶体的抑制增加了p22（phox）蛋白水平，这与NADPH依赖性超氧化物生成的增加相关。我们还表明，p22（phox）与VHL在体内免疫共沉淀。此外，p22（phox）是泛素化的靶标。重要的是，在VHL缺陷细胞中，氯化二苯碘铵（DPI），一种Nox氧化酶的抑制剂，降低了HIF-2a的表达。通过小干扰RNA下调Nox 1、Nox 4和p22（phox）表达也降低了HIF-2 α蛋白的表达，并抑制了Akt和4 E-BP 1磷酸化，这表明在VHL缺陷细胞中维持HIF-2 α的翻译机制。DPI能明显抑制RCC 786-O在软琼脂上的集落形成。此外，DPI显着抑制RCC 786-O裸鼠肿瘤形成。总的来说，这些数据表明，VHL蛋白发挥其肿瘤抑制作用，至少部分，通过抑制p22（phox）为基础的Nox 4/Nox 1 NADPH氧化酶依赖性活性氧的产生。
Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is linked to the development of hereditary and sporadic renal cell carcinoma (RCC). In the absence of VHL, the alpha subunits of heterodimeric hypoxia-inducible transcription factors (HIT-1 alpha and HIF-2 alpha) are stabilized. Reactive oxygen species, generated by NAD(P)H oxidases, are involved in signaling cascades of malignant growth. We show that in VHL-deficient cells p22(phox), Nox4 protein levels and NADPH-dependent superoxide generation are increased. Reintroduction of VHL into the VHL-deficient cells down-regulates the expression of p22(phox) and NADPH-dependent superoxide generation. Inhibition of the 26 S proteasome in VHL-expressing cells increased p22(phox) protein levels, which correlated with an increase of NADPH-dependent superoxide generation. We also show that p22(phox) co-immunoprecipitates with VHL in vivo. Moreover, p22(phox) is a target of ubiquitination. Importantly, in VHL-deficient cells, diphenyleneiodonium chloride (DPI), an inhibitor of Nox oxidases, decreased the expression of HIF-2a. Down-regulation of Nox1, Nox4, and p22(phox) expression by small interfering RNA also decreased HIF-2 alpha protein expression and inhibited Akt and 4E-BP1 phosphorylation, suggesting that a translational mechanism is involved in maintaining HIF-2 alpha in VHL-deficient cells. Colony formation by RCC 786-O in soft agar was markedly inhibited by DPI. Moreover, DPI significantly inhibited RCC 786-O tumor formation in athymic mice. Collectively, the data demonstrate that VHL protein exerts its tumor suppressor action, at least partially, via inhibition of p22(phox)-based Nox4/Nox1 NADPH oxidase-dependent reactive oxygen species generation.