Expression of signal transduction proteins during the differentiation of primary human erythroblasts

Abstract

The high number (>10(8-10)) of primary human pro-erythroblasts (CD36high/CD235alow) obtainable in HEMA culture (Migliaccio et al., 2002) is exploited here to analyse the expression of proteins implicated in erythropoietin (EPO)-signalling (STATs, PI-3K, and PLCs) during the process of erythroid maturation. Human pro-erythroblasts progressed in 4 days of culture with EPO into basophilic- (CD36high/CD235amedium, 24 h), polychromatic-(CD36high/CD235ahigh, 48 h), and, finally, orthochromatic-(CD36low/CD235ahigh, 72-96 h) erythroblasts. During this maturation, STAT-1 was expressed up to the orthochromatic stage, expression of STAT-5, as well as of its target proteins BclxL and IRF1, remained constant up to 48 h (polychromatic-erythroblasts) but decreased by 96 h (orthochromatic-erythroblasts), while that of STAT-3 decreased constantly from 24 h on and became undetectable by 96 h. Expression of PI-3K rapidly decreased with differentiation since only 50% of original protein levels were detected by 48 h. On the other hand, among the members of PLC families investigated, PLC beta4 was not expressed, PLC beta2, delta1, and gamma2 were expressed at constant levels throughout the maturation process, while expression of PLC beta3 and of PLC gamma1 decreased, as PI-3K, by 24 h and that of PLC beta1 was induced by 6 h and became undetectable by 24 h. In conclusion, these data depict the dynamic signalling scenario associated with the maturation of erythroid cells and provide the first indication that members of PLC families (PLC beta1, beta3, and gamma1) might be involved in the control of erythroid differentiation in humans.

Chronic IFN-γ production in mice induces anemia by reducing erythrocyte life span and inhibiting erythropoiesis through an IRF-1/PU.1 axis

“…43 A cooperative function between IRF-1 and PU.1 has been reported during all-trans retinoic acid-mediated granulopoiesis 44 and in IFN-␥-stimulated myeloid cells. 45 Furthermore, IRF-1 is naturally down-regulated toward the final stages of erythroid maturation, 46 which overall sustains the notion that suppression of the IRF-1-PU.1 axis is a prerequisite for normal erythropoiesis. Our findings provide the molecular mechanism by which IFN-␥ affects erythropoiesis, which implicates transcriptional regulation of hematopoietic-lineage differentiation in response to inflammation.…”

TRPC3 Is the Erythropoietin-regulated Calcium Channel in Human Erythroid Cells

“…Although the lack of complete inhibition of the rise in [Ca 2ϩ ] i by PLC␥-targeted siRNA probably resulted from incomplete suppression of PLC␥ expression (Fig. 5), we cannot eliminate the possibility that other PLC members also expressed on primary erythroid cells and inhibited by U-73122, such as PLC␤ family members (45), have a role. The lack of complete elimination of TRPC3 and PLC␥ binding with the TRPC3-F4 mutant is consistent with previous observations that other PLC␥ binding sites exist on TRPC3 (28).…”

“…In myoblasts, Epo stimulated expansion of the progenitor population during differentiation and an increase in [Ca 2ϩ ] i dependent on extracellular calcium influx (15). In neuronal cell lines, Epo stimulated an increase in cell viability and an increase in 45 Ca 2ϩ uptake (16,17). Determination of the mechanisms through which the erythropoietin receptor modulates Ca 2ϩ influx is important in understanding regulation of erythroid proliferation and differentiation as well as the role of Epo-R expression in nonerythroid tissues and is likely to be applicable to other cytokine receptor pathways.…”

Suppression of erythropoiesis in patients with chronic heart failure and anaemia of unknown origin: evidence of an immune basis

“…Burst-forming units-erythroid (BFU-E) constitute the earliest detectable erythroid lineage-restricted progenitors, and are identified in semi-solid media by their ability to form haemoglobinised progeny in a burst-like formation [7]. Subsequently, BFU-E differentiate into erythroid precursors (CD36 + /glycophorin A + cells [8]) and ultimately, reticulocytes. In the face of established anaemia, the inability to increase reticulocyte production (reticulocytosis) as marked by a normal or low absolute reticulocyte count or a reticulocyte production index (RPI) b2, is diagnostic of defective erythrocyte production [9].…”