Stimulation of the Proliferation of Mouse and Human Definitive Erythroid Progenitors By Activation of the Vitamin D Receptor Transcription Factor

Abstract How the proliferation of erythroid progenitors is regulated is still now well understood. We found, using a computational analysis, that the vitamin D receptor (Vdr) nuclear hormone receptor transcription factor gene is expressed in the fetal and adult (definitive) but not the embryonic (primitive) stage of mouse erythroid ontogeny. Vdr is transcribed in definitive erythroid (EryD) progenitors and was downregulated during their maturation. VDR transcription factor activation by its ligand vitamin D3 (1,25(OH)2D3) results in conformational changes that stabilize the protein and induce its translocation into the nucleus, where it recruits co-regulatory complexes. The VDR signaling pathway has been studied primarily in the biology of bone but has been largely unexplored in erythropoiesis, where the limited published studies were performed almost exclusively in leukemic cell lines and not in normal primary cells. Activation of Vdr signaling by the vitamin D3 agonist calcitriol increased the outgrowth of EryD colonies from fetal liver and adult bone marrow, maintained progenitor potential, and delayed terminal erythroid maturation, as revealed by clonogenic assays, suspension culture studies, cell surface phenotype, and gene expression analyses. The stimulation in growth of erythroid progenitors resulted in a large increase in the numbers of mature red blood cells. The early (cKit+CD71lo/neg) but not the late (cKit+CD71hi) EryD progenitor subset of Linneg cKit+ cells was responsive both to calcitriol and to calcipotriol (which is 1-to-200 fold less potent in its calcemic effects than calcitriol). Therefore, the increase in progenitor numbers in response to calcitriol is mediated through activation of VDR rather than by effects on calcium flux. In preliminary studies of human Lineageneg cells, we find that the vitamin D agonists calcitriol and calcipotriol increase the numbers of CFU-E colony numbers from peripheral blood or BM. These results are similar to our findings for mouse. The glucocorticoid receptor (Gr), like Vdr, is a member of the nuclear hormone receptor transcription factor family and has been shown to stimulate the proliferation of cKit+CD71lo/neg cells. To determine whether the Vdr and Gr signaling pathways can cooperate to modulate erythroid progenitor growth, cKit+CD71lo/negcells were cultured with or without calcitriol, dexamethasone, or the two ligands in combination. Culture of cKit+CD71lo/neg progenitors in the presence of both calcitriol and dexamethasone resulted in an increase in proliferation that was at least additive, compared to either ligand alone, suggesting a role in stress erythropoiesis. This possibility is supported by our recent finding that an erythroid specific deletion in Vdr that interferes with DNA binding results in a reticulocytosis that occurs earlier and is more pronounced than in control animals, in response to phenylhydrazine (PHZ)-induced anemia. In addition, this deletion in Vdr blocked the increase in early erythroid progenitors from fetal liver seen for wild type mice. Lentivirus shRNA-mediated knockdown of Vdr expression abrogated the stimulation of early erythroid progenitor growth by calcitriol. These findings suggest that Vdr has a cell-intrinsic function in early erythroid progenitors. Activation of Vdr by calcitriol blocked the up regulation of the erythroid transcription factor genes Gata1, Fog1 and Klf1. In contrast, expression of genes known to regulate erythroid progenitors (Gata2, Zfp36l2, Bmi1, and Hopx) was not affected by Vdr signaling. Therefore, other genes must be involved in the Vdr signaling pathway in erythroid progenitors. Targeting of downstream components of the VDR signaling pathway could lead to new approaches for expansion of erythroid progenitors ex vivo. (This work was supported by grants to MHB from the NIH, R01 DK102945 and HL62248.) Disclosures No relevant conflicts of interest to declare.