Epo-Induced Erythroid Maturation Is Dependent On Plcγ1 Signalling

Erythropoiesis is a multi-step process in which the development of red blood cells occurs through expansion and differentiation of hematopoietic stem cells (HSCs) into more committed progenitors and finally into erythrocytes. Erythropoietin (Epo) is strictly required for erythropoiesis as it promotes survival and late maturation. In vivo and in vitro studies have pointed out the major role of erythropoietin receptor (EpoR) signalling through JAK2 tyrosine-kinase and STAT5a/b as a central regulator of erythropoiesis. STAT5a/b is essential in regulating early erythroblast survival, however, with regard to differentiation of erythroid progenitors current data are not definitive in establishing a critical, non-redundant role. Phospholipase C gamma 1 (PLCγ1) is known to act as key mediator of calcium-signalling that can substitute for PI3K/AKT in oncogenic models. Interestingly, genetic deletion of murine PLCγ1 in embryonic development using a conventional knockout mouse model resulted in lethality at E9.0 due to generalized growth failure and there was absence of erythrogenesis and vasculogenesis.

Here, we revisited the role of Plcγ1 and investigated its function in signalling, differentiation and transcriptomic/epigenetic regulation of erythropoiesis: Upon Epo stimulation, we were able to demonstrate that Plcγ1 is a downstream target of EpoR/Jak2 signalling in lymphoid (Ba/F3) and myeloid (32D) progenitor cell lines (both transfected with EpoR and Jak2-WT) and in a erythroid progenitor (I/11) cell line. In order to specifically assess its role in erythroid development downstream of the EpoR-Jak2 axis, we focused on the murine pro-erythroblast cell line I/11 which is able to differentiate upon dexamethasone-/stem cell factor-withdrawal combined with erythropoietin stimulation. Interestingly, knockdown of Plcγ1 led to a dramatic delay (scr CD44^high 21% vs. Plcγ1 shRNA CD44^high 64%, p=0.02) in erythroid differentiation and accumulation of immature erythroid progenitors as assessed by flow cytometry technology. Knockdown of Plcγ1 did alter neither proliferation of cells nor the cell cycle distribution and activation of other EpoR downstream molecules as Stat5, Mek and Akt was not impaired. In addition, we analysed the colony-forming potential of Plcγ1-deficient I/11 and fetal liver cells (FLC) compared to controls. Colony formation was dramatically impaired in both - I/11 (scr 138 vs. Plcγ1 shRNA 32, p=0.03) and primary FLC (scr 107 vs. Plcγ1 shRNA 28, p<0.001) - when compared to control cells. Flow cytometry analysis of the colonies revealed a higher amount of immature populations (CD44^high, KIT⁺) in PLCγ1-deficient cells as compared to controls whereas the content of TER119⁺ cells, reflecting more mature erythroid cells, was higher in controls.

To elucidate on the mechanism of Plcγ1-mediated regulation of erythroid development, we performed global gene expression analysis in I/11 cells at various time points of differentiation after knockdown of Plcγ1. Several of the genes that change expression in the absence of Plcγ1 can be classified as transcription/co-transcription factors, epigenetic regulators, metabolic factors or adaptor molecules involved in intracellular signaling. Thus, Plcγ1-deficient cells showed up-regulation of the transcription factor RUNX1 and the adaptor molecule GRAP2 over time compared to controls whereas the epigenetic regulator H2AFY2 was significantly decreased. Stimulated by our observation that profound changes in global gene expression also included the epigenetic machinery (H2afy2), we speculated whether Plcγ1 signalling also modifies the global epigenetic landscape of I/11 pro-erythroblasts. Therefore, we performed genome-wide DNA methylome analysis in I/11 cells upon Plcγ1 knockdown using MCIP-seq (methyl-CpG immunoprecipitation combined with next-generation sequencing). The observed methylation changes were by far dominated by an apparent hypomethylation of differentially methylated regions (DMRs) in Plcγ1 knockdown cells as compared to control cells. In line with this, gene ontology analysis of DMRs revealed a highly significant enrichment of biological terms associated with developmental processes and cell differentiation.

Taken together, our findings provide evidence for an essential role of Plcγ1 in regulating erythroid differentiation through alteration of the transcriptomic and epigenetic landscape.