NAD(P)H Oxidases regulate HIF-2α protein expression

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.