Epolones induce erythropoietin expression via hypoxia-inducible factor-1 alpha activation

Induction of erythropoietin (Epo) expression under hypoxic conditions is me diated by the heterodimeric hypoxia-inducible factor (HIF)-1. Following bin ding to the 3' hypoxia-response element (HRE) of the Epo gene, HIF-1 marked ly enhances Epo transcription. To facilitate the search for HIF-1 (ant)agon ists, a hypoxia-reporter cell line (termed HRCHO5) was constructed containi ng a stably integrated luciferase gene under the control of triplicated het erologous HREs, Among various agents tested, we identified a class of subst ances called epolones, which induced HRE-dependent reporter gene activity i n HRCHO5 cells. Epolones are fungal products known to induce Epo expression in hepatoma cells. We found that epolones (optimal concentration 4-8 mu mo l/L) potently induce HIF-1 alpha protein accumulation and nuclear transloca tion as well as HIF-1 DNA binding and reporter gene transactivation. Intere stingly, the activity of a compound related to the fungal epolones, ciclopi rox olamine (CPX), was blocked after addition of ferrous iron. This suggest s that CPX might interfere with the putative heme oxygen sensor, as has bee n proposed for the Iron chelator deferoxamine mesylate (DFX). However, abou t 10-fold higher concentrations of DFX (50-100 mu mol/L) than CPX were requ ired to maximally induce reporter gene activity in HRCHO5 cells, Moreover, structural, functional, and spectrophotometric data imply a chelator:iron s toichiometry of 1:1 for DFX but 3:1 for CPX. Because the iron concentration in the cell culture medium was determined to be 16 mu mol/L, DFX but not C PX function can be explained by complete chelation of medium iron. These re sults suggest that the lipophilic epolones might induce HIF-1 alpha by intr acellular iron chelation, (Blood, 2000; 96:1558-1565) (C) 2000 by The Ameri can Society of Hematology.