NAD(P)H oxidases regulate HIF-2alpha 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 (HIF-1alpha and HIF-2alpha) 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 p22phox, Nox4 protein levels and NADPH-dependent superoxide generation are increased. Reintroduction of VHL into the VHL-deficient cells down-regulates the expression of p22phox and NADPH-dependent superoxide generation. Inhibition of the 26 S proteasome in VHL-expressing cells increased p22phox protein levels, which correlated with an increase of NADPH-dependent superoxide generation. We also show that p22phox co-immunoprecipitates with VHL in vivo. Moreover, p22phox is a target of ubiquitination. Importantly, in VHL-deficient cells, diphenyleneiodonium chloride (DPI), an inhibitor of Nox oxidases, decreased the expression of HIF-2alpha. Down-regulation of Nox1, Nox4, and p22phox expression by small interfering RNA also decreased HIF-2alpha protein expression and inhibited Akt and 4E-BP1 phosphorylation, suggesting that a translational mechanism is involved in maintaining HIF-2alpha 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 p22phox-based Nox4/Nox1 NADPH oxidase-dependent reactive oxygen species generation.
Block
Karen
K
Division of Nephrology, Department of Medicine, George O'Brien Kidney Research Center, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA. block@uthscsa.edu
Gorin
Yves
Y
Hoover
Paul
P
Williams
Paul
P
Chelmicki
Tomasz
T
Clark
Robert A
RA
Yoneda
Toshiyuki
T
Abboud
Hanna E
HE
D051792:Basic Helix-Loop-Helix Proteins; D005786:Gene Expression Regulation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis*; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000378:metabolism; D000099162:Endothelial PAS Domain-Containing Protein 1
D051792:Basic Helix-Loop-Helix Proteins; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; D009861:Onium Compounds; D046988:Proteasome Endopeptidase Complex; D061988:Proteasome Inhibitors; D017382:Reactive Oxygen Species; D013481:Superoxides; D014157:Transcription Factors; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D000099162:Endothelial PAS Domain-Containing Protein 1; C007517:diphenyleneiodonium; C402799:NOX1 protein, human; C498910:NOX4 protein, human; C086986:CYBA protein, human; C061553:ATP dependent 26S protease; C497446:VHL protein, human