Principal Investigator Harvey Lodish
In situations of severe loss of red blood cells mammals and birds respond by a process known as stress erythropoiesis (SE), in which there is increased formation of erythroid progenitors. Johan Flygare hypothesizes that if the molecular pathways that induce SE are understood it will be possible to develop erythropoiesis stimulating agents that will complement or replace Epo treatment in anemic patients. Glucocorticoids (GCs) are known to be very potent enhancers of SE. This stimulatory effect of GCs on SE is utilized in the therapeutic regimen of Diamond-Blackfan Anemia (DBA), an erythropoietin-resistant congenital red cell aplasia. While an Epo-dependent balance of late red cell precursor survival normally maintains red cell homeostasis, Johan’s findings indicate that the physiology of SE involves a stimulation of earlier erythroid progenitors, which when activated are able to rescue red cell production in conditions such as DBA, where erythropoietin has little effect.
Flygare showed that glucocorticoids stimulate self-renewal of early Epo-independent progenitor cells (burst-forming units erythroid or BFU-Es), over time increase production of colony-forming units erythroid (CFU-E) erythroid progenitors from the BFU-E cells, and enhance terminal erythroid differentiation. He first established two FACS-based methods to separate and purify BFU-E and CFU-E cells from mouse fetal liver. He demonstrated that GCs induce self-renewal of BFU-E cells, and not of CFU-E cells or erythroblasts. GCs thereby protect BFU-E cells from exhaustion, and in parallel increase the number of CFU-E cells formed from each BFU-E >10-fold. He further demonstrated that GCs do not inhibit erythropoietin-dependent terminal differentiation of freshly isolated erythroid CFU-E progenitors.
In mRNA-seq experiments, he found that glucocorticoids induced expression of ~86 genes more than 2- fold in BFU-E cells. Computational analyses indicated that, of all transcription factors, binding sites for hypoxia-induced factor 1 alpha (HIF1α) were most enriched in the promoter regions of these genes, suggesting that activation of HIF1α may enhance or replace the effect of glucocorticoids on BFU-E self-renewal. Indeed, HIF1 alpha activation by the prolyl hydroxylase inhibitor (PHI) DMOG synergized with glucocorticoids and enhanced production of CFU-Es and later erythroblasts over 170-fold. PHI-induced stimulation of BFU-E progenitors thus represents a conceptually new therapeutic window for treating Epo-resistant anemia.
Flygare proposes a physiological model of stress erythropoiesis where increased levels of GCs and reduced oxygen help maintain the earliest erythroid progenitors, increase CFU-E output, and at the same time stimulate terminal differentiation, thus promoting both a rapid and long-lasting increase in red blood cell production.
Since the main action of the activated GCR is to interact with chromatin and regulate transcription Flygare and his technical assistant together with a graduate student, hope to answer many questions by mapping exactly where in the chromatin the activated GCR binds by ChIP-Seq, which binding partners it has, and how transcription is repressed and/or activated at these sites in BFU-E cells.
Further insight into the mechanism of GC stimulation of SE will come from ongoing work by an MIT undergraduate student Evelyn Wang and research associate Gregory Hyde, who are determining exactly which functional domains of the glucocorticoid receptor are necessary to stimulate SE; to this end they are expressing specific mutant forms of the GR in BFU-Es purified from GR knock-out mice and studying their subsequent proliferation and differentiation in culture.
Flygare is currently establishing his own group at the Lund Stem Cell Center in Sweden. In Sweden he will continue to collaborate with David Root at the Broad institute in a search for genes, molecular pathways and compounds that modify the red cell progenitor defect in Diamond Blackfan anemia. The aim of this work is to develop novel treatments for this disorder.