This application describes a five-year mentored training program to develop an independent clinician- scientist whose career in academic medicine will focus on furthering our understanding of human red blood cell development. Cats naturally or experimentally infected with feline leukemia virus, subgroup C (FeLV-C) develop profound pure red cell aplasia (PRCA) or on rare occasion ineffective erythropoiesis. Our lab previously demonstrated that the cell surface receptor for feline leukemia virus, subgroup C (FLVCR) exports cytoplasmic heme, and is required for CFU-E/proerythroblast survival or differentiation. My goal in these studies is to determine if these observations connect, and more broadly, to use FLVCR as a tool to study early erythroid differentiation. I plan three specific aims to evaluate this: 1) to determine if intracellular free heme increases when FLVCR expression or function is downregulated in erythroid cells; 2) to determine if erythroid differentiation is blocked or apoptosis is induced when FLVCR function or expression is downregulated in primary human cells and in vivo in mice; 3) to determine if reducing intracellular free heme in cells where FLVCR is downregulated (restoring heme balance) will allow erythroid differentiation. These studies will test my hypothesis that FLVCR acts as a mandatory safety (overflow) valve that protects early erythroid cells from heme toxicity, will provide insight into the physiology responsible for PRCA in cats viremic with FeLV-C, and will further our understanding of normal and disordered erythropoiesis. I will accomplish these specific aims by the following methods: 1) design a dual luciferase assay based on the transcription factor Bach1 to directly measure intracellular free heme during the erythroid differentiation of K562 and primary human hematopoietic cells and determine the effects of downregulation of FLVCR function (inhibitory antibody) or expression (siRNA) using this assay system; 2) characterize erythroid differentiation in primary human hematopoietic cells when FLVCR is downregulated by antibody and breed a viable Flvcr(null/null) mouse and characterize its erythropoiesis; 3) attempt to restore intracellular free heme balance when FLVCR is downregulated (by either blocking heme synthesis or increasing its catabolism) and thereby permit erythroid differentiation of human and murine cells. These studies focus on basic red blood cell physiology and may lend insight into human diseases where red blood cell development is disturbed.
| Keel, Sioban B; Phelps, Susan; Sabo, Kathleen M et al. (2012) Establishing Rps6 hemizygous mice as a model for studying how ribosomal protein haploinsufficiency impairs erythropoiesis. Exp Hematol 40:290-4 |
