Glucocorticoids (GCs) are hormonal regulators of stress. They accelerate red blood cell production rate, an effect that is well established by mouse genetics, by in vitro erythropoiesis systems, and by human disease syndromes in which GCs are dysregulated. The use of GCs in the treatment of anemia is complicated, however, by their severe side effects, and is therefore limited to conditions where Erythropoietin (Epo) treatment is refractory or contraindicated, including Diamond Blackfan Anemia and other bone-marrow failure syndromes. The translational importance of GCs is apparent from their use in systems currently under development for the in-vitro generation of red blood cells for transfusion. Understanding the molecular action of GCs in erythroid progenitors could facilitate the development of novel erythropoiesis-stimulating agents that have fewer side-effects than GCs, and that improve the efficiency of generating red blood cells in vitro. Functionally, GCs increase erythropoietic rate by delaying the switch from self-renewal to differentiation in erythroid progenitors. The molecular mechanisms underlying this action are largely unknown. Based on our recently published work and on preliminary data, we propose a novel hypothesis of GC action that implicates the cell cycle S phase and DNA methylation as novel regulatory targets. We recently showed that both fetal and adult erythropoiesis entail genome-wide DNA demethylation, a unique global epigenetic modification in somatic cells (Shearstone et al., Science 2011). Global demethylation is tightly correlated with demethylation at erythroid gene promoters, and is a rate-limiting for their transcriptional activation. Further, global demethylatin is dependent on a marked change in S phase of the cell cycle, which becomes shorter and 50% faster with the switch from self-renewal to differentiation. The cyclin-dependent kinase inhibitor (CDKI) p57KIP2 is a key negative regulator of this switch. p57KIP2 is also a direct transcriptional target of GCs. Our preliminary data show that, in the presence of GCs, erythroid progenitors fail to downregulate p57KIP2, fail to accelerate S phase, and fail to undergo DNA demethylation, thereby delaying erythroid gene transcription. In this proposal, we investigate the hypothesis that high levels of GCs during erythropoietic stress inhibit the switch from self-renewal to differentiation by inducing p57KIP2, thereby inhibiting S phase acceleration, global DNA demethylation and erythroid gene induction. We will test this hypothesis in vivo using mouse models of erythropoietic stress and mice deleted or mutated for either p57KIP2, the GC receptor, or DNA methyl transferase 1 (Dnmt1), with the following three aims: 1) Determine the role of p57KIP2 in the GC-mediated erythropoietic stress response 2) Determine whether GCs prolong S phase in erythroid progenitors during stress 3) Determine whether GCs delay the onset of global DNA demethylation during stress. This work focuses on a unique epigenetic modification and has the potential to identify conceptually novel regulatory mechanisms, with translational implications for therapy of Epo-resistant anemia.
In health, approximately 1% of the body's red blood cells are replaced every day. The production of red blood cells increases dramatically, by as much as ten-fold, in response to anemia and other clinical settings that limit oxygen supply to tissues, such as respiratory/cardiovascular disease or bone-marrow transplantation. The hormone erythropoietin (Epo) is a successful therapy for many types of anemia, but there are types of anemia that are refractory to Epo treatment, such as those associated with myelodysplastic syndrome, Blackfan Diamond anemia and other bone-marrow failure syndromes. In addition, Epo treatment is often contra- indicated in cancer and in post-chemotherapy anemia, due to its off-target effects that may promote tumor relapse, and due to the risk of generating antibodies against Epo itself. It is therefore critical to identify new erythropoiesis-stimulating drugs that ill aid in the treatment of these anemias. The work in this proposal investigates the mechanism of action of glucocorticoids in red cell production. Glucocorticoids stimulate red cell production in cooperation with Epo. They are used to treat some types of anemia that are refractory to Epo, such as the bone-marrow failure syndrome Blackfan Diamond anemia. Their mechanism of action is in not well understood, however, and treatment with these drugs is limited by their multiple and severe side effects, including loss of bone density and diabetes. The proposed work in this application focuses on new discoveries from our laboratory that suggest new ways in which glucocorticoids may stimulate red cell production, specifically, by altering epigenetic marks on DNA and by affecting the manner in which cells synthesize DNA. This work may elucidate glucocorticoids'mechanism of action and help to develop new treatments that mimic its positive effects on blood production but do not have its deleterious effects;this will improve the body's ability to rapidly replenish its red blood cells and provide alternatives to Epo.