Kit transduced signals counteract erythroid maturation by MAPK-dependent modulation of erythropoietin signaling and apoptosis induction in mouse fetal liver

Abstract

Signaling by the stem cell factor receptor Kit in hematopoietic stem and progenitor cells is functionally associated with the regulation of cellular proliferation, differentiation and survival. Expression of the receptor is downregulated upon terminal differentiation in most lineages, including red blood cell terminal maturation, suggesting that omission of Kit transduced signals is a prerequisite for the differentiation process to occur. However, the molecular mechanisms by which Kit signaling preserves the undifferentiated state of progenitor cells are not yet characterized in detail. In this study, we generated a mouse model for inducible expression of a Kit receptor carrying an activating mutation and studied its effects on fetal liver hematopoiesis. We found that sustained Kit signaling leads to expansion of erythroid precursors and interferes with terminal maturation beyond the erythroblast stage. Primary KIT D816V erythroblasts stimulated to differentiate fail to exit cell cycle and show elevated rates of apoptosis because of insufficient induction of survival factors. They further retain expression of progenitor cell associated factors c-Myc, c-Myb and GATA-2 and inefficiently upregulate erythroid transcription factors GATA-1, Klf1 and Tal1. In KIT D816V erythroblasts we found constitutive activation of the mitogen-activated protein kinase (MAPK) pathway, elevated expression of the src kinase family member Lyn and impaired Akt activation in response to erythropoietin. We demonstrate that the block in differentiation is partially rescued by MAPK inhibition, and completely rescued by the multikinase inhibitor Dasatinib. These results show that a crosstalk between Kit and erythropoietin receptor signaling cascades exists and that continuous Kit signaling, partly mediated by the MAPK pathway, interferes with this crosstalk. Erythroid cell proliferation, differentiation and survival are tightly regulated to ensure supply of the organism with sufficient numbers of red blood cells. Regulation of these processes is governed by two major signaling receptors, the stem cell factor (SCF) receptor Kit and the erythropoietin receptor (EpoR). Kit is expressed in hematopoietic stem and progenitor cells and becomes downregulated upon differentiation of colony-forming unit erythroid cells.1 EpoR gets upregulated after erythroid commitment and is expressed until later erythroblast stages. Both receptors are essential for erythroid development, as Kit or EpoR-deficient mice die in utero because of impaired fetal liver erythropoiesis.3,4 The roles of Kit and EpoR in erythropoiesis are partially overlapping and signal integration after co-stimulation results in proliferative synergy and enhanced survival. [5][6][7] However, Kit has been attributed a primary role in proliferation, 8,9 whereas EpoR has its main role in mediating differentiation and survival.

Characterization, regulation, and targeting of erythroid progenitors in normal and disordered human erythropoiesis

“…SCF/c-kit mutant and EPO-R deficient mice both exhibit severe anemia with varying erythroid progenitor defects, whereas erythropoiesis remains unaffected in the absence of IL-3R (31–33). Activating mutations in the c-kit receptor prevent erythroblast maturation, and expand progenitors (34*). Further, EPO acts directly on CFU-Es to promote survival as opposed to proliferation (35).…”

Integrative proteomics reveals principles of dynamic phospho-signaling networks in human erythropoiesis

“…Combining phosphoproteomics and a functional screen, our data highlights a general decrease in kinase activity across the erythroid proteome during terminal maturation and in particular a critical role for downregulation of Ras/MAP kinases activity is suggested. Previous studies have demonstrated defective terminal maturation in systems expressing either constitutively active forms of c-Kit or Ras proteins (Haas et al, 2015;Matsuzaki et al, 2000). Our systems-wide analysis of erythropoiesis derived from primary healthy donor human CD34 + cells suggests c-Kit drives expansion of early erythroid precursors and inhibits terminal maturation via Ras/MAPK signaling whereas EPOR drives signaling predominantly via the JAK-STAT5 pathway to foster later stages of terminal maturation.…”

“…Our erythroid culture system contained both SCF (c-Kit Ligand) and EPO in the first and second phase (day0-7) and EPO only in the third phase (day12-14) ( Figure 1A). The rationale for this culture system is based on findings that persistently elevated SCF-c-Kit signaling inhibits terminal erythroid maturation (Haas et al, 2015;Muta et al, 1995). The levels of c-Kit and EPOR/JAK2 proteins decreased during differentiation but with differing kinetics (Figures 6D).…”

Dissecting Regulatory Mechanisms Using Mouse Fetal Liver-Derived Erythroid Cells

“…A variety of mechanisms are in place to control the erythroid cell response to cytokines, and it would not be surprising if major additional mechanisms remain to be discovered. As cellular responses to cytokines can depend on the maturation status of the cells [27–29], this protocol provides a sensitive assay to quantitate cellular responses to cytokine signals in specific cell types delineated by their ensemble of cell surface proteins detected by flow cytometry.…”