| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
Erythropoietin (Epo) gene expression is negatively regulated by GATA, which binds to the GATA site in the Epo promoter, and also positively regulated by hypoxia inducible factor-1 (HIF-1), which binds to the hypoxia responsive element (HRE) in the Epo enhancer. The disorders associated with anemia of chronic disease (ACD) are characterized by a deficiency of Epo, leading to Epo being considered as a treatment. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), which increase the binding activity of GATA and inhibit Epo promoter activity, are increased in patients with ACD. In a previous study, we demonstrated the ability of K-7174 (a GATA-specific inhibitor) to improve Epo production that had been inhibited by IL-1β or TNF-α treatment in Hep3B cells in vitro and in an in vivo mouse assay (FASEB J 17:1742; 2003). However, K-7174 was injected intraperitoneally into the mice. We examined the ability of K-11706, a novel GATA-specific inhibitor that has a 1,000-fold higher affinity
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than K-7174, to improve Epo production following inhibition by IL-1β or TNF-α in Hep3B cells in vitro. Oral administration of K-11706 reversed the decreases of hemoglobin, serum Epo, reticulocyte counts and CFU-E induced by IL-1β or TNF-α. These results raise the possibility of using orally administered K-11706 as a novel drug for treating ACD (Blood 104:4300; 2004).
In oxygenated cells, hypoxia inducible factor-1 (HIF-1) α 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 Epo and vascular endothelial growth factor (VEGF). The expression of Epo 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-1α protein level in Hep3B cells in hypoxic conditions. However, MG132 (the proteasome inhibitor) rescued the inhibition of HIF-1α 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. Less
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