Increased erythropoiesis of β‐thalassaemia/Hb E proerythroblasts is mediated by high basal levels of ERK1/2 activation

Abstract

Increased erythropoiesis of b-thalassaemia/Hb E proerythroblasts is mediated by high basal levels of ERK1/2 activationThe thalassaemias are a group of disorders characterized by the under production of globin chains as a consequence of a wide range of underlying genetic abnormalities (Weatherall & Clegg, 2001). The a and b chains of haemoglobin A are the most commonly affected genes and both a-and b-thalassaemias represent a worldwide clinical problem (Weatherall & Clegg, 2001). The underproduction of one chain leads to a resultant excess of the other chain in the red cell that is toxic to the cell and leads to a reduced red cell life span, increased haemolysis and ineffective erythropoiesis, which is believed to arise through accelerated cell death by apoptosis of the immature erythroid cell (Yuan et al, 1993), either as a direct or indirect consequence of the unpaired globin chain deposition within the cell (Mathias et al, 2000). In b-thalassaemia apoptosis is believed to occur primarily at the polychromatophilic normoblast stage (Mathias et al, 2000). The combination of ineffective erythropoiesis, increased haemolysis and reduced life span of the mature red cell lead to the anaemic condition, which stimulates the production of erythropoietin (EPO) leading to erythroid hyperplasia in the bone marrow (Chen et al, 1992;Centis et al, 2000;Mathias et al, 2000;Pootrakul et al, 2000;Kittikalayawong et al, 2005;Mai et al, 2007) and markedly increased levels of circulating EPO have been documented in thalassaemic patients (Nisli et al, 1997;Chaisiripoomkere et al, 1999;Paritpokee et al, 2002). However, despite the increase in erythroid precursors, the overall response to the anaemia is limited due to the ineffective erythropoiesis leading to the accelerated death of immature erythroid cells. In severe cases of Summary b-thalassaemia is one of the most common inherited anaemias, arising from a partial or complete loss of b-globin chain synthesis. In severe cases, marked bone marrow erythroid hyperplasia, believed to result from erythropoietin (EPO)-mediated feedback from the anaemic condition is common, however, as yet, no study has investigated EPO-mediated signal transduction in thalassaemic erythroid cells. Using proerythroblasts generated from peripheral blood circulating CD34 + haematopoietic progenitor cells, the activation of the mitogen-activated protein kinase/extracellular signalregulated kinases (MAPK/ERKs) pathway was examined under conditions of steady state growth, cytokine deprivation and post-EPO stimulation. Levels of cellular cyclic adenosine monophosphate (cAMP) and Ca 2+ were determined as was the degree of erythroid expansion. A significantly higher basal level of phosphorylation of ERK1/2 was observed in b-thalassaemia/Hb E proerythroblasts as compared to normal controls, which was coupled with significantly higher levels of both cAMP and Ca 2+ . Modulation of either cAMP or Ca 2+ or direct inhibition of MAPK/ERK kinase (MEK) reduced basal levels of ERK1/2 phosphorylation, as well as significantly...

High-Efficiency Serum-Free Feeder-Free Erythroid Differentiation of Human Pluripotent Stem Cells Using Small Molecules

“…The role of cAMP in HSC specification has recently been reported by Diaz et al [20], and increased cAMP has been shown to drive erythroid proliferation [21]. TNFα is a recognized inhibitor of hematopoietic progenitor development and of erythropoiesis [22].…”

Dengue virus infection of erythroid precursor cells is modulated by both thalassemia trait status and virus adaptation

“…Under these growth conditions more than 95% of cells show erythroblast morphology as established previously (Wannatung et al, 2009). Cell numbers were established by trypan blue exclusion assay and counting using a cell counting chamber.…”

“…CD34 þ hematopoietic stem cells (HSCs) were isolated from peripheral blood samples and cultured under conditions that produced differentiation in the erythroid lineage as described in previously (Lithanatudom et al, 2010;Wannatung et al, 2009) and see more detail in Supplemental materials and methods. Under these growth conditions more than 95% of cells show erythroblast morphology as established previously (Wannatung et al, 2009).…”

“…To investigate the susceptibility of erythroid committed precursor cells, CD34 þ HSCs were selected and cultured for 7 and 10 days under conditions that drive erythropoiesis (Leecharoenkiat et al, 2011;Lithanatudom et al, 2010;Wannatung et al, 2009) and infected with DENV at m.o.i. 50.…”

Emerging EPO and EPO receptor regulators and signal transducers

“…58 And in b-thalassemia proerythroblasts, phospho-C-Raf levels correlate with increased ERK activation. 59 Mek2 is dispensable for mouse development, 60 implicating prime roles for Mek1 in Erk1/2 signaling. In mice expressing a truncated EpoR-HM allele, pharmacologic inhibition of Mek reverses stage-specific EPC differentiation defects, 61 whereas in mice with somatic inactivation of Nf1, Mek1/2 inhibition decreases splenomegaly and enhances erythropoiesis.…”