Abstract
Targeting the oxygen-sensing mechanisms of the hypoxiainducible factor (HIF) pathway provides pharmacological ways of manipulating the HIF response. Because HIF-1α-specific prolyl-4 hydroxylases (PHDs) prime degradation of HIF-1α, we have made an effort to find a small molecule capable of up-regulating the HIF pathway by inhibiting prolyl hydroxylation. Through an in vitro high-throughput screen, we have discovered a PHD2 inhibitor baicalein, which is also found to abrogate asparaginyl hydroxylation of HIF-1α. Such inhibitory effects are reversed by the addition of excess 2-oxoglutarate and iron(II), suggesting the involvement of baicalein's binding at the enzyme active sites, which has also been corroborated by spectroscopic binding assays between baicalein and enzyme. In addition, baicalein suppresses ubiquitination of HIF-1α, which works in concert with the inhibition of the HIF-specific hydroxylases to increase the HIF-1α content, leading to induction of HIF-1-mediated reporter gene activity and target gene transcription in tissue culture cells, whereas it induces HIF-independent activation of other genes. Furthermore, in vivo organ models based on the chick chorioallantoic membrane assay demonstrate that baicalein promotes new blood vessel formation. Together, our results indicate that baicalein possesses a proangiogenic potential and thus might have the therapeutic utility in the treatment of ischemic diseases.
Footnotes
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This work was supported by the InterFrontier Project from the Functional Proteomics Research Center of the 21st Century Frontier Research Program funded by the Korean Ministry of Science and Technology and from the Intelligent Microsystem Center of the 21st Century Frontier R&D Program sponsored by the Korean Ministry of Commerce, Industry and Energy, a Korean Institute of Science and Technology grant, and a grant (2004-01969) from the Neurobiology Research Program of the Ministry of Science and Technology (to H.P.).
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H.C. and H.-Y.L. contributed equally to this work.
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ABBREVIATIONS: HIF, hypoxia-inducible factor; VHL, von Hippel-Lindau tumor suppressor protein; VBC, von Hippel-Lindau protein-Elongin B-Elongin C; PHD, hypoxia-inducible factor-1α-specific prolyl-4 hydroxylase; 2-OG, 2-oxoglutarate; FIH-1, factor-inhibiting hypoxia-inducible factor-1; HRE, hypoxia-response element; VEGF, vascular endothelial growth factor; FP, fluorescence polarization; GST, glutathione transferase; DMSO, dimethyl sulfoxide; B-P564, biotin-DLDLEALAPYIPADDDFQLR; MALDI, matrix-assisted laser desorption ionization; TOF, time of flight; RT, reverse transcriptase; PCR, polymerase chain reaction; CAM, chorioallantoic membrane; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; CQ, clioquinol; shRNA, short hairpin RNA; bFGF, basic fibroblast growth factor; B, baicalein; H, hypoxia.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received July 19, 2007.
- Accepted April 21, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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