2-Oxoglutarate downregulates expression of vascular endothelial growth factor and erythropoietin through decreasing hypoxia-inducible factor-1alpha and inhibits angiogenesis.
In oxygenated cells, hypoxia-inducible factor-1 (HIF-1) alpha subunits are rapidly degraded by a mechanism that involves ubiquitination by the von Hippel-Lindau tumor suppressor E3 ligase complex using 2-oxoglutarate as a substrate. We examined the effect of 2-oxoglutarate on the production of erythropoietin and vascular endothelial growth factor (VEGF). The expression of erythropoietin and VEGF protein were dose-dependently downregulated in Hep3B cells by the addition of 2-oxoglutarate. The promoter activity of VEGF-luciferase was dose-dependently downregulated by the addition of 2-oxoglutarate. Gel mobility shift assays revealed that the addition of 2-oxoglutarate dose-dependently inhibited HIF-1 binding activity, but did not affect GATA binding activity. Western blot analysis revealed that 2-oxoglutarate dose-dependently inhibited the HIF-1alpha protein level in Hep3B cells in hypoxic conditions. However, MG132 (the proteasome inhibitor) rescued the inhibition of HIF-1alpha protein expression by 2-oxoglutarate. Furthermore, under hypoxic conditions, 2-oxoglutarate dose-dependently inhibited tube formation in in vitro angiogenesis assays. These results indicate that 2-oxoglutarate treatment may be useful for the inhibition of angiogenesis.
Matsumoto
Ken
K
Department of Clinical and Experimental Hematology, Doctoral Program in Advanced Biomedical Applications, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Imagawa
Shigehiko
S
Obara
Naoshi
N
Suzuki
Norio
N
Takahashi
Satoru
S
Nagasawa
Toshiro
T
Yamamoto
Masayuki
M
D015687:Cell Hypoxia / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects*; D004921:Erythropoietin / Q000378:metabolism*; D050980:GATA Transcription Factors / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000494:pharmacology*; D050505:Mutant Proteins / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
D050980:GATA Transcription Factors; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007656:Ketoglutaric Acids; D050505:Mutant Proteins; D042461:Vascular Endothelial Growth Factor A; D004921:Erythropoietin