Erythropoiesis is a process by which hematopoietic cells become red blood cells (RBC). Mice that do not express the proto-oncogene c-myb, a transcription factor expressed predominantly in hematopoietic cells and functions to regulate their proliferation, survival, and differentiation, die at embryonic day 15 of severe anemia, strongly suggesting that c-Myb through the transactivation of its target genes is essential during erythropoiesis. Using a microarray approach to identify Myb-gene targets, we observed the greatest change in the expression of the neuropeptide neuromedin U (NmU) in hematopoietic cells which also express NmU's cognate receptor type-1 (NMUR1). Transient inhibition of NmU gene expression by mature siRNA in primary human CD34+ cells impaired the formation of erythroid progenitors even when erythropoietin (EPO) was present in the culture. Co-culturing primary human CD34+ in which NmU was transiently inhibited with EPO and exogenous NmU restored the frequency of erythroid progenitor generation to that observed with control siRNA treated cells. It is important to note that NmU alone could not induce eyrthroid progenitor formation in primary human CD34+ cells. But, without NmU expression in primary human CD34+ cells, EPO and stem cell factor (SCF), two indispensable factors that support erythropoiesis, could not effectively induce erythroid progenitor formation. Combined, these data strongly indicate that during erythropoiesis NmU is critical for erythroid progenitor formation from primary human CD34+ cells. Based on our novel findings, we hypothesize that sustained inhibition of NmU in primary CD34+ cells will impair erythropoiesis in vivo. To test this hypothesis, we will study NmU's in vivo function and gene expression regulation with the following specific aims:
Aim #1 - Determine the effect of sustained NmU inhibition in primary human CD34+ cells on their ability to provide full human hematopoietic reconstitution in NOD-SCID-gcnull (NSG) mice. Once we have achieved sustained inhibition of NmU in primary human CD34+ cells, we will transplant them into the NSG mice and evaluate the impact of NmU inhibition on the generation of erythroid progenitor cells.
Aim #2 - Investigate the transcriptional regulation of NmU in hematopoietic and erythroid progenitor cells. We will identify the components of the c-Myb transcription factor complex that induce NmU expression and determine the ability of Elk-1 to activate NmU transcription.
Aim #3 - Determine the regulation of NmU protein synthesis by microRNA (miRNA) in hematopoietic and erythroid progenitor cells. We will identify miRNA molecules that interact with NmU's 3'-UTR and determine the ability of c-Myb to regulate these miRNA molecules through a regulatory loop. The results from these proposed studies may provide a new therapeutic avenue for anemia.
Anemia is a condition marked by a deficiency in red blood cell number or of hemoglobin expressed in red blood cells. The neuropeptide neuromedin U has emerged as a novel factor that functions during erythropoiesis which is the process by which immature blood cells differentiate into red blood cells. Understanding neuromedin U's function during erythropoiesis may offer a new therapeutic avenue for anemia.
| Gambone, Julia E; Dusaban, Stephanie S; Loperena, Roxana et al. (2011) The c-Myb target gene neuromedin U functions as a novel cofactor during the early stages of erythropoiesis. Blood 117:5733-43 |
