Hypoxic up-regulation of erythroid 5-aminolevulinate synthase

The erythrold-specific isoform of 5-amino-levulinate synthase (ALAS2) catalyzes the rate-limiting step in heme blosynthesis. The hypoxia-inducible factor-1 (HIF-1) transcriptionally up-regulates erythropoletin, transferrin, and transferrin receptor, leading to increased erythropoiesis and hematopoietic Iron supply. To test the hypothesis that ALAS2 expression might be regulated by a similar mechanism, we exposed murine erythroleukemia cells to hypoxia (1% O-2) and found an up to 3-fold up-regulation of ALAS2 mRNA levels and an Increase In cellular heme content. A fragment of the ALAS2 promoter ranging from -716 to +1 conveyed hypoxia responsiveness to a heterologous luciferase reporter gene construct In transiently transfected HeLa cells. In contrast, iron depletion, known to induce HIF-1 activity but Inhibit ALAS2 translation, did not increase ALAS2 promoter activity. Mutation of a previously predicted HIF-1-binding site (-323/-318) within this promoter fragment and DNA-binding assays revealed that hypoxic up-regulation is Independent of this putative HIF-1 DNA-binding site. (C) 2003 by The American Society of Hematology.