HIF-1alpha-prolyl hydroxylase: molecular target of nitric oxide in the hypoxic signal transduction pathway.
We have investigated inhibitory mechanisms of hypoxic activation of HIF-1alpha by nitric oxide (NO). Using a Hep3B cell-derived cell line, HRE7 cells, we found that the inhibition of HIF-1alpha activity by NO requires a substantial amount of oxygen, albeit at a lower level. We further investigated the effect of NO on the binding activity of the von Hippel-Lindau tumor suppressor protein (pVHL) to the N-terminal activation domain (NAD) overlapping the oxygen-dependent degradation domain (ODD) of HIF-1alpha, because this reaction involves prolyl hydroxylation in NAD that requires oxygen. Although we could not detect any binding activity when NAD was incubated with whole cell extracts from cells treated with CoCl(2) or desferrioxamine, the binding capacity was manifested when Hep3B cells were treated together with NO. This activation was also observed when whole cell extracts from CoCl(2)-treated cells were incubated with NO. The prolyl hydroxylase from Hep3B cells treated with CoCl(2) was partially purified about 80-fold, and several enzymatic properties were examined. The enzyme required ferrous ion and 2-oxoglutaric acid. Strong activation of the prolyl hydroxylase by NO was observed without further addition of ferrous ion.
Wang
Feng
F
Department of Biomolecular Science, Graduate School of Life Sciences, Tohoku University, Aoba-ku Sendai 980-8578, Japan.
Sekine
Hiroki
H
Kikuchi
Yasuo
Y
Takasaki
Chikahisa
C
Miura
Chisa
C
Heiwa
Okuda
O
Shuin
Taro
T
Fujii-Kuriyama
Yoshiaki
Y
Sogawa
Kazuhiro
K
D015153:Blotting, Western; D002460:Cell Line; D003035:Cobalt / Q000378:metabolism; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007656:Ketoglutaric Acids / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism* / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012685:Sepharose / Q000737:chemistry; D015398:Signal Transduction; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D018076:DNA, Complementary; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007656:Ketoglutaric Acids; D014157:Transcription Factors; D009569:Nitric Oxide; D003035:Cobalt; D012965:Sodium Chloride; D012685:Sepharose; D008156:Luciferases; D011393:Procollagen-Proline Dioxygenase; C018021:cobaltous chloride; D010100:Oxygen