Study of Uncoupling Protein 2 (UCP2) Function during Erythroid Differentiation and Maturation.

Abstract

Mitochondrial oxidative stress is thought to play a key role in sideroblastic anemia and the myelodysplastic syndrome. Potential sources of reactive radicals reside in the heme biosynthetic pathway involving the import and production of pro-oxidant agents, such as ALA and iron and in the respiratory chain. Antioxidant mechanisms are, therefore, expected to be an integral function in erythroid differentiation and their impairment is expected to affect hemoglobinization and maturation. The mitochondrial uncoupling proteins have been shown to reduce oxidative stress through the generation of proton leak across the inner membrane of the mitochondria. They have been implicated in a wide range of physiological and pathological states, including obesity, diabetes, aging neurodegenerative, and immunological diseases.Here we report that UCP2 is induced during erythroid differentiation and that UCP2 deficient mice have a delayed recovery from anemia.We hypothesized that erythroid heme biosynthesis is accompanied by oxidative stress, which results in the induction of UCP2, and that UCP2 plays a role in erythroid maturation by preventing oxidative stress and damage.We found that UCP2 transcripts and protein are induced following the activation of GATA-1 in G1ER cells and during DMSO, butyrate and heme -induced differentiation of murine erythroleukemic (MEL) and K652 erythroid cell lines. Similarly, differentiation of primary mouse c-kit+ / Ter119− erythroid progenitors to Ter119+ is accompanied by induction of UCP2 transcripts. To test the functional significance of UCP2 in erythroid differentiation we studied a UCP2 null mouse. Peripheral blood analysis from UCP2 KO mice revealed a mild elevation of the reticulocyte index as compared to wild type (WT C57BL/6J) mice, which may be related to mild anemia. To test the role of UCP2 in recovery from anemia, we treated WT and UCP2 KO mice with phenylhydrazine for 3 days and studied erythropoiesis using FACS analysis of Ter119 and CD71 surface markers in cells isolated from bone marrow. Stimulation of erythropoiesis was more rapid in the WT mice as compared to the UCP2 KO. The delay in the mutant is more pronounced at the stage of the proerythroblast and is also reflected in the peripheral blood where a higher level of reticulocytes was transiently observed. By 9 days the UCP2 KO mice peripheral blood count was identical to the WT. Analysis of oxidative damage confirmed that UCP2 acts to reduce oxidative damage of mitochondrial proteins. The delayed reticulocytosis could not however be explained by cell death or by reduced hemoglobinization. The increased oxidative damage present in the UCP2 null cells during erythroid differentiation and maturation did not result in the stimulation of apoptosis as revealed by identical Annexin V staining profile of UCP2 KO and WT mice. Remarkably, iron incorporation and hemoglobin content assays ruled out a function of UCP2 in the process of heme biosynthesis per-se. We therefore conclude that UCP2 deficiency regulates maturation in the erythroid lineage independent of the heme biosynthetic pathway.