Abstract
We have confirmed that the NO donor (±)-S-nitroso-N-acetylpenicillamine (SNAP) stabilizes the transactive form of hypoxia-inducible factor-1α (HIF-1α), leading to the induction of HIF-1α target genes such as vascular endothelial growth factor and carbonic anhydrase 9. Activation of HIF-1α should require inhibition of the dual system that keeps it inactive. One is ubiquitination, which is triggered by hydroxylation of HIF-1α-proline and the subsequent binding of E3 ubiquitin ligase, the von Hippel Lindau (VHL) protein. The other is hydroxylation of HIF-1α-asparagine, which reduces the affinity of HIF-1α for its coactivator, cAMP responsive element binding protein/p300. We examined the effects of the NO donor SNAP on proline and asparagine hydroxylation of HIF-1α peptides by measuring the activities of the corresponding enzymes, HIF-1α-specific proline hydroxylase 2 (PHD2) and the HIF-1α-specific asparagine hydroxylase, designated factor inhibiting HIF-1α (FIH-1), respectively. We found that the SNAP did not prevent PHD2 from hydroxylating the proline of HIF-1α. Instead, it blocked the interaction between VHL and the proline-hydroxylated HIF-1α, but only when the reducing agents Fe(II) and vitamin C were limiting. The fact that the absence of cysteine 520 of HIF-1α abolishes its responsiveness to SNAP suggests that this residue mediates the inhibition by SNAP of the interaction between VHL and HIF-1α, presumably by S-nitrosylation of HIF-1α. Un-like PHD2, asparagine hydroxylation by FIH-1 was directly inhibited by SNAP, but again only when reducing agents were limiting. Substitution of cysteine 800 of HIF-1α with alanine failed to reverse the inhibitory effects of SNAP on asparagine hydroxylation, implying that FIH-1, not its substrate HIF-1α, is inhibited by SNAP.
Footnotes
-
This study was supported by grant 2004-01969 from the Neurobiology Research Program, grant R01-2007-000-11672-0 from the Korea Science and Engineering Foundation, by grant CBM1-B113-001-1-0-0 from the center for Biological Modulators of the 21st Century Frontier R&D Program of the Ministry of Science and Technology (to H.P.), and by grant from the Korea Institute of Science and Technology (to E.G.Y.). Y.-K.P. was supported by a Brain Korea 21 Research Fellowship from the Ministry of Education and Human Resources Development and by a Seoul Science Fellowship.
-
All experiments reported in this article were repeated with similar results.
-
ABBREVIATIONS: HIF-1α, hypoxia-inducible factor-1α; α-KG, α-ketoglutarate; CA9, carbonic anhydrase 9; CBP, cAMP-responsive element binding protein; FIH-1, factor inhibiting hypoxia-inducible factor-1α; HRE, hypoxia-responsive element; ODD, oxygen-dependent degradation domain of hypoxia-inducible factor-1α; PHD, hypoxia-inducible factor-1α-specific prolyl hydroxylase; ROS, reactive oxygen species; SNAP, (±)-S-nitroso-N-acetylpenicillamine; spermine NONOate, N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine; TPEN, N,N, N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine; VHL, von Hippel-Lindau; VEGF, vascular endothelial growth factor; HSP70, 70-kDa heat shock protein; FITC, fluorescein isothiocyanate; PAGE, polyacrylamide gel electrophoresis; NOC18, 2,2′-(hydroxynitrosohydrazino) bis-ethanamine; GSNO, S-nitrosoglutathione; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase polymerase chain reaction; HA, hemagglutinin; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal.
-
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received January 14, 2008.
- Accepted April 21, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|