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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08DK075422-04
Application #
7629114
Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Program Officer
Bishop, Terry Rogers
Project Start
2006-07-01
Project End
2011-06-30
Budget Start
2009-07-01
Budget End
2010-06-30
Support Year
4
Fiscal Year
2009
Total Cost
$126,792
Indirect Cost
Name
University of Washington
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195