Dexamethasone Accelerates the Transition of Human BFU-E to CFU-E and Enhances CFU-E Proliferation through Cell Cycle Regulation

Abstract Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is frequently managed with glucocorticoids to increase red cell mass. Despite sustained effective clinical use for many decades in DBA and other anemias, we still do not fully understand the mechanistic basis for the regulation of human erythropoiesis by glucocorticoids. In order to improve the clinical management of patients with DBA, we studied the mechanisms of action of dexamethasone (Dex) on erythroid progenitors. To study the effects of Dex on erythroid progenitor biology, we employed a serum-free erythroid culture system to differentiate primary human CD34+ cells isolated from the peripheral blood of adults or DBA patients as well as cord blood samples. We found that Dex increases the total proliferation of CD34+ cells from adult peripheral blood but not in cord blood over 14 days in culture. Dex treatment also led to an acceleration of the BFU-E to CFU-E transition in peripheral blood with a minimal effect in cord blood. In methylcellulose colony forming assays, peripheral blood derived CFU-E treated with Dex for 24hrs produced more colonies or larger colonies than the untreated controls while a lesser effect was seen in cord blood derived CFU-E. Both assays revealed that Dex treatment enhanced self-renewal of peripheral blood derived CFU-E. We also noted that BFU-E and CFU-E differentially express the glucocorticoid receptor (GR) isoforms, GRα and GRβ. BFU-E expressed comparable levels of GRα and the dominant-negative GRβ, whereas only the GRα transcript was identified in CFU-E. We observed similar expression patterns for GR between peripheral and cord blood derived progenitors. Importantly, following Dex treatment, GRα was shown to translocate to the nucleus of erythroid progenitors derived from peripheral blood derived CD34+ cells but not in the cord blood derived progenitors. When we examined the cell cycle progression of sorted human erythroid progenitors, CFU-E and BFU-E, we observed a G0/G1 arrest of peripheral blood CFU-E treated with Dex compared to untreated CFU-E. In marked contrast, no such change was noted for either Dex treated BFU-E from peripheral blood or for CFU-E or BFU-E from cord blood. In addition, we found an increase in the expression of cell cycle regulators p57Kip2 and p27Kip1 in Dex treated peripheral blood CFU-E but no such increased expression was seen in cord blood CFU-E. When peripheral blood derived erythroid progenitors were treated with olomoucine (Olo), a small molecule CDK inhibitor, we observed an acceleration of the BFU-E to CFU-E transition similar to that induced by Dex. Upon co-treatment with Dex and Olo, there was a synergistic effect in the accelerated formation of CFU-E population from BFU-Es. To validate this finding of the dependence of CFU-E on cell cycle regulators, we transduced peripheral blood derived CD34+ cells with a lentiviral shRNA construct to knockdown p57 expression. Following successful knockdown of the expression levels of p57, the numbers of CFU-E indeed decreased implying a role for cell cycle in defective transition of BFU-E to CFU-E. Erythroid progenitors derived from cultures of primary CD34+ cells from DBA patients were also studied. Importantly, we noted increased baseline expression of p57Kip2 and p27Kip1 in erythroid progenitors from steroid resistant DBA patients and in contrast to controls, their expression did not increase following Dex treatment. Our findings provide novel mechanistic insights into the regulation of human erythropoiesis by Dex. We have shown that alterations in cell cycle regulation of erythroid progenitors plays a key role in the effects of glucocorticoids on red blood cells. Furthermore, this work has direct relevance for the development of improved therapeutic treatment strategies for patients with bone marrow failure syndromes such as DBA. Disclosures No relevant conflicts of interest to declare.