Role of G proteins and ERK activation in hemin-induced erythroid differentiation of K562 cells.
ABSTRACT Heterotrimeric G proteins which couple extracellular signals to intracellular effectors play a central role in cell growth and differentiation. The pluripotent erythroleukemic cell line K562 that acquires the capability to synthesize hemoglobin in response to a variety of agents can be used as a model system for erythroid differentiation. Using Western blot analysis and RT-PCR, we studied alterations in G protein expression accompanying hemin-induced differentiation of K562 cells. We demonstrated the presence of G(alpha s), G(alpha i2) and G(alpha q) and the absence of G(alpha i1), G(alpha o) and G(alpha 16) in K562 cells. We observed the short form of G(alpha s) to be expressed predominantly in these cells. Treatment of K562 cells with hemin resulted in an increase in the levels of G(alpha s) and G(alpha q). On the other hand, the level of G(alpha i2) was found to increase on the third day after induction with hemin, followed by a decrease to levels lower of those of uninduced cells. The mitogen-activated protein kinase ERK1/2 pathway is crucial in the control of cell proliferation and differentiation. Both Gi- and Gq-coupled receptors stimulate MAPK activation. We therefore examined the phosphorylation of ERK1/2 during hemin-induced differentiation of K562 cells. Using anti-ERK1/2 antibodies, we observed that ERK2 was primarily phosphorylated in K562 cells. ERK2 phosphorylation increased gradually until 48 h and returned to basal values by 96 h following hemin treatment. Our results suggest that changes in G protein expression and ERK2 activity are involved in hemin-induced differentiation of K562 cells.
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ABSTRACT: Hydroxyurea (HU) is effectively used in the management of β-hemoglobinopathies by augmenting the production of fetal hemoglobin (HbF). However, the molecular mechanisms underlying HU-mediated HbF regulation remain unclear. We previously reported that overexpression of the HU-induced SAR1 gene closely mimics the known effects of HU on K562 and CD34(+) cells, including γ-globin induction and cell-cycle regulation. Here, we showed that HU stimulated nuclear factor-κB interaction with its cognate binding site on the SAR1 promoter to regulate transcriptional expression of SAR1 in K562 and CD34(+) cells. Silencing SAR1 expression not only significantly lowered both basal and HU-elicited HbF production in K562 and CD34(+) cells, but also significantly reduced HU-mediated S-phase cell-cycle arrest and apoptosis in K562 cells. Inhibition of JNK/Jun phosphorylation and silencing of Giα expression in SAR1-transfected K562 and CD34(+) cells reduced both γ-globin expression and HbF level, indicating that activation of Giα/JNK/Jun proteins is required for SAR1-mediated HbF induction. Furthermore, reciprocal coimmunoprecipitation assays revealed an association between forcibly expressed SAR1 and Giα2 or Giα3 proteins in both K562 and nonerythroid cells. These results indicate that HU induces SAR1, which in turn activates γ-globin expression, predominantly through the Giα/JNK/Jun pathway. Our findings identify SAR1 as an alternative therapeutic target for β-globin disorders.Blood 06/2014; · 9.78 Impact Factor
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ABSTRACT: BACKGROUND: The guanine nucleotide binding protein (G protein)-coupled receptors (GPCRs) regulate cell growth, proliferation and differentiation. G proteins are also implicated in erythroid differentiation, and some of them are expressed principally in hematopoietic cells. GPCRs-linked NO/cGMP and p38 MAPK signaling pathways already demonstrated potency for globin gene stimulation. By analyzing erythroid progenitors, derived from hematopoietic cells through in vitro ontogeny, our study intends to determine early markers and signaling pathways of globin gene regulation and their relation to GPCR expression. RESULTS: Human hematopoietic CD34+ progenitors are isolated from fetal liver (FL), cord blood (CB), adult bone marrow (BM), peripheral blood (PB) and G-CSF stimulated mobilized PB (mPB), and then differentiated in vitro into erythroid progenitors. We find that growth capacity is most abundant in FL- and CB-derived erythroid cells. The erythroid progenitor cells are sorted as 100% CD71+, but we did not find statistical significance in the variations of CD34, CD36 and GlyA antigens and that confirms similarity in maturation of studied ontogenic periods. During ontogeny, beta-globin gene expression reaches maximum levels in cells of adult blood origin (176 fmol/mug), while gamma-globin gene expression is consistently up-regulated in CB-derived cells (60 fmol/mug). During gamma-globin induction by hydroxycarbamide, we identify stimulated GPCRs (PTGDR, PTGER1) and GPCRs-coupled genes known to be activated via the cAMP/PKA (ADIPOQ), MAPK pathway (JUN) and NO/cGMP (PRPF18) signaling pathways. During ontogeny, GPR45 and ARRDC1 genes have the most prominent expression in FL-derived erythroid progenitor cells, GNL3 and GRP65 genes in CB-derived cells (high gamma-globin gene expression), GPR110 and GNG10 in BM-derived cells, GPR89C and GPR172A in PB-derived cells, and GPR44 and GNAQ genes in mPB-derived cells (high beta-globin gene expression). CONCLUSIONS: These results demonstrate the concomitant activity of GPCR-coupled genes and related signaling pathways during erythropoietic stimulation of globin genes. In accordance with previous reports, the stimulation of GPCRs supports the postulated connection between cAMP/PKA and NO/cGMP pathways in activation of gamma-globin expression, via JUN and p38 MAPK signaling.BMC Genomics 02/2013; 14(1):116. · 4.04 Impact Factor
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ABSTRACT: Background. β-thalassemia is caused by mutations in the β-globin locus resulting in loss of, or reduced, Hemoglobin A (Adult hemoglobin, HbA, α2β2) production. Hydroxyurea treatment increases fetal γ-globin (Fetal Hemoglobin, HbF, α2γ2) expression in postnatal life substituting for the missing adult β-globin, and is therefore an appealing therapeutic approach. Patients treated with Hydroxyurea fall into three categories: (i) Responders, who increase hemoglobin to therapeutic levels (ii) Moderate-responders, who increase hemoglobin levels but still need transfusions at longer intervals, and (iii) Non-responders, who do not reach adequate hemoglobin levels and remain transfusion-dependent. The mechanisms underlying these differential responses remain largely unclear. Design and Methods. We generated RNA expression profiles from erythroblast progenitors of 8 responder and 8 non-responder β-thalassemia patients. Results. These profiles revealed that Hydroxyurea treatment induced differential expression of many genes in cells from non-responders while it had little impact on cells from responders. Part of the gene program upregulated by Hydroxyurea in non-responders was already highly expressed in responders before Hydroxyurea treatment. Baseline HbF expression was low in non-responders, and Hydroxyurea treatment induced significant cell death. Conclusions. We conclude that cells from responders have adapted well to constitutive stress conditions and display a propensity to proceed to the erythroid differentiation program.Haematologica 10/2012; · 5.94 Impact Factor