Accumulation of hypoxia-inducible factor-1α protein and its role in the differentiation of myeloid leukemic cells induced by all-trans retinoic acid

Background: The clinical activities of all-trans retinoic acid in the treatment of acute promyelocytic leukemia, a unique subtype of acute myeloid leukemia, have triggered extensive studies aimed at defining the mechanisms by which this compound induces differentiation of leukemic cells. Recent studies show that hypoxia-inducible factor-1α (HIF-1α) contributes to the differentiation of acute myeloid leukemia cells via transcriptional activity-independent mechanisms. We investigated whether all-trans retinoic acid affects HIF-1α protein and whether this has a role in all-trans retinoic acid-induced differentiation. Design and Methods: The acute myeloid leukemia cell lines NB4 and U937 were treated with all-trans retinoic acid, and HIF-1α/HIF-1β mRNA and proteins were measured respectively by real-time quantitative reverse transcriptase polymerase chain reaction and western blotting. To investigate the role of HIF-1α in all-trans retinoic acid-induced differentiation, NB4 cells, U937 cells, U937 cells in which HIF-1α was induced by the withdrawal of tetracycline and U937 cells with stable expression of specific short hairpin RNA against HIF-1α, Runx1, C/EBPα and PU.1, were treated with all-trans retinoic acid and/or the hypoxia-mimetic agent cobalt chloride (CoCl₂). Cellular differentiation was evaluated by morphological criteria and myeloid differentiation antigens. Results: all-trans retinoic acid rapidly increased endogenous and inducible expressed or CoCl₂-stabilized HIF-1α protein in leukemic cells under normoxia. Importantly, suppression of HIF-1α expression by specific short hairpin RNA partially but significantly inhibited all-trans retinoic acid-induced differentiation of the U937 cell line. Reciprocally, the differentiation induced by all-trans retinoic acid was significantly enhanced by conditional HIF-1α induction and HIF-1α-stabilizing CoCl₂ treatment. Furthermore, knock-down of PU.1, Runx1 and C/EBPα, three transcriptional factors crucial for normal hematopoiesis, greatly inhibited the differentiation cooperation of all-trans retinoic acid and HIF-1α induction. Conclusions: This work provides the first demonstration that HIF-1α, a protein rapidly responsive to all-trans retinoic acid, plays a role in all-trans retinoic acid-induced differentiation of leukemic cells. These observations shed new light on the molecular mechanisms underlying all-trans retinoic acid-induced differentiation of acute myeloid leukemia cells.