Mortalin-2 (mthsp70/PBP74/Grp75) Mediates the Erythropoietin Signal To Stimulate Growth of Purified Human Erythroid Progenitor Cells.

Abstract Erythropoietin (EPO) stimulates erythroid growth by enhancing the proliferation, maturation and survival of late stage erythroid progenitor cells. Intracellular signaling molecules such as Janus kinase 2 (JAK2) and phosphoinositide-3 kinase (PI-3K)/Akt are considered mediators of the EPO signal; however, the entire process of EPO stimulation remains undetermined. Previously, we used siRNA to show that mortalin-2, which is a member of the heat shock protein 70 family of chaperones, mediates EPO signaling to stimulate the growth of human erythroid colony forming cells (ECFCs). In the present study, we examined the relationship between cell growth and mortalin-2 overexpressed in ECFCs, and analyzed the gene expression pattern of ECFCs treated with mortalin-2 siRNA using a DNA microarray, to further clarify the intracellular mechanism by which EPO and mortalin-2 interact. In the presence of different concentrations of EPO (0–1.0 U/ml), the effect of mortalin-2 overexpression on ECFC growth was determined by MTT assay. There was tendency to better viability of ECFCs treated with mortalin-2 expression vectors than control cells, especially in the EPO 0 U/ml group (p=0.08). BrdU ELISA, used to investigate the effects of mortalin-2 on the DNA synthesis of ECFCs, revealed that when ECFCs were treated with mortalin-2 expression vectors, the cells showed an increase in the amount of BrdU incorporation into DNA without EPO. Next we analyzed the gene expression pattern using mRNA obtained from ECFCs cultured with or without EPO after treatment with mortalin-2 siRNA or control siRNA. When ECFCs were cultured with EPO after treatment with mortalin-2 siRNA, the expression of 19 genes was suppressed to less than 0.6 fold, and these genes included those involved in cell growth, apoptosis or transport, such as interleukin 6 receptor, ATP-binding cassette, Mdm2, BCL2 interacting protein and interleukin 10 receptor alpha. Furthermore, the expression of 8 genes was upregulated to over 1.5 fold, and these genes included transcription or signal transmission related genes, such as ubiquitin A-52 residue ribosomal protein fusion product 1 and serum/glucocorticoid regulated kinase 2. There were also some genes whose expressions overlapped with genes obtained from ECFCs cultured without EPO after treatment with control siRNA. Our data suggests that mortalin-2 expression depends on various pathways, and that one of these pathways mediates EPO signaling to stimulate mortalin-2 expression, which is related to the growth of erythroid progenitor cells.

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Neuromedin U Peptide Activates STAT5 and S6 in a JAK-2 Dependent Manner and Promotes Erythroid Cell Growth in Primary Erythroid Progenitor Cells

Blood ◽

10.1182/blood.v120.21.1241.1241 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1241-1241

Author(s):

Rebecca Lenzo ◽

Martha Dua-Awereh ◽

Martin Carroll ◽

Susan E. Shetzline

Keyword(s):

Cell Growth ◽

Progenitor Cells ◽

Surrogate Marker ◽

Janus Kinase ◽

Erythroid Cell ◽

Dependent Manner ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Erythroid Progenitor Cells ◽

Pi3k Activation

Abstract Abstract 1241 Erythropoiesis is a multi-step process during which hematopoietic stem cells terminally differentiate into red blood cells (RBCs). Erythropoietin (EPO) is the only known cytokine regulator of terminal erythroid differentiation. Previously, we reported that the neuropeptide, neuromedin U (NmU), which interacts with NmU receptor type 1 (NMUR1), functions as a novel extracellular cofactor with EPO to promote the expansion of early erythroblasts, which are CD34−, CD71+, glycophorin A (GlyA)dim(Gambone et al, Blood. 2011). Here, we describe studies to understand the mechanism whereby NmU augments EPO effects on erythroid cell growth. EPO triggers Janus kinase (Jak)-2 dependent activation of signal transducer and activator of transcription (STAT) 5 and phosphatidylinositol 3-kinase (PI3K) to promote the proliferation and/or survival of erythroid progenitor cells. We hypothesized that NmU peptide would cooperate with EPO to promote the proliferation of early erythroblasts through STAT5 and/or PI3K activation. To address this hypothesis, we cultured primary human CD34+ cells in 2-stage liquid culture with IL-3, IL-6, and stem cell factor (SCF) from day 0 to day 6. On day 6, 2U/mL of EPO was added, and the cells were cultured for an additional 5 days to expand erythroid progenitors. On day 11, cells were briefly serum starved and then stimulated with EPO and/or NmU in the absence or presence of a Jak-1/2 inhibitor. Activation of STAT5 and S6, a surrogate marker for PI3K activation, were assessed by phospho-flow in ERY3 (CD34−, CD71+, GlyA+) and ERY4 (CD34−, CD71dim, GlyA+) cells. As expected, EPO alone activated STAT5 and S6 in ERY3 cells only, and the presence of a Jak-1/2 inhibitor diminished STAT5 activation. Interestingly, STAT5 and S6 were activated by NmU peptide alone in ERY3 and ERY4. Surprisingly, in the presence of a Jak-1/2 inhibitor, NmU peptide, which binds to NMUR1 a G-protein coupled receptor, did not activate STAT5 or S6 in ERY3 or 4 cells, suggesting that NmU functions through a JAK kinase in erythroid cells. No additive or synergistic activation of STAT5 and S6 is observed in the presence of both EPO and NmU peptide when EPO was used at a dose of 2 U/mL. The mechanism whereby NmU activates a JAK dependent signaling pathway is under investigation. Preliminary evidence suggests that EPO induces the physical association of NMUR1 with EPO receptor (EPOR). Taken together, we propose that NmU is a neuropeptide expressed in bone marrow cells that cooperates to regulate erythroid expansion during early erythropoiesis through the activation of cytokine receptor like signaling pathways and perhaps through direct interaction with EPOR. NmU may be useful in the clinical management of anemia in patients unresponsive to EPO or other erythroid-stimulating agents. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of β-globin gene expression by 3′-azido-3′-deoxythymidine in human erythroid progenitor cells

Antiviral Research ◽

10.1016/s0166-3542(99)00065-0 ◽

1999 ◽

Vol 44 (3) ◽

pp. 167-177 ◽

Cited By ~ 17

Author(s):

Maria-Grazia Spiga ◽

Douglas A Weidner ◽

Chantal Trentesaux ◽

Robert D LeBoeuf ◽

Jean-Pierre Sommadossi

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Globin Gene ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Globin Gene Expression

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Mortalin-2 (mthsp70/PBP74/Grp75), a Novel Mediator of Erythropoietin Signal.

Blood ◽

10.1182/blood.v106.11.3141.3141 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3141-3141

Author(s):

Rie Ohtsuka ◽

Yasunobu Abe ◽

Tomomi Fujii ◽

Eriko Nagasawa ◽

Hirotoshi Shimada ◽

...

Keyword(s):

Progenitor Cells ◽

Intracellular Signaling ◽

Antigen Processing ◽

Control Culture ◽

Janus Kinase ◽

Dependent Manner ◽

Transient Transfection Assay ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Peptide Mass

Abstract Erythropoietin (EPO) stimulates erythroid growth by enhancing the proliferation, maturation and survival of late stage erythroid progenitor cells. Intracellular signaling molecules such as Janus kinase 2 (JAK2) and phosphoinositide-3 kinase (PI-3K)/Akt are considered mediators of the EPO signal, however the entire process of EPO stimulation remains undetermined. To further clarify the intracellular mechanism by which EPO affects the growth of erythroid progenitor cells, we analyzed protein obtained from purified human erythroid colony forming cells (ECFCs) cultured with, or without EPO, by 2-dimensional gel electrophoresis. Five protein spots were apparently related to EPO stimuli, and these proteins were then examined by peptide mass fingerprinting. One of the 5 was identified as mortalin-2, which is a member of the heat shock protein 70 family of chaperones. Mortalin-2 is thought to perform multiple functions relevant to stress response, intracellular trafficking, antigen processing, control of cell proliferation and differentiation. When the expression of mortalin-2 was confirmed by Western blotting, the amount of mortalin-2 was found to be greater in ECFCs cultured with EPO. The amount of mortalin-2 mRNA was then analyzed by quantitative real time PCR. In the presence of EPO, the amount of mortalin-2 mRNA gradually increased during erythroid maturation, peaked on day 6, and then decreased in the terminal stage of maturation. When day 6 ECFCs were incubated with different concentrations of EPO, the amount of mortalin-2 mRNA increased in a dose-dependent manner. Next, we designed a small interfering RNA (siRNA) to RNA encoding mortalin-2, and used this in a transient transfection assay. When the siRNA was transfected into day 6 ECFCs, the expression of mortalin-2 mRNA was suppressed to 40–60% of that seen in the control culture. In the presence of different concentrations of EPO (0–1.0 u/ml), the effect of the siRNA on ECFC growth was determined by MTT assay. The siRNA significantly suppressed the growth of ECFCs in the presence of relatively low concentrations of EPO (0.5 u/ml). These data suggest that mortalin-2 mediates the EPO signal to stimulate the growth of erythroid progenitor cells.

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Chromatin-modifying agents promote the ex vivo production of functional human erythroid progenitor cells

Blood ◽

10.1182/blood-2010-10-314567 ◽

2011 ◽

Vol 117 (17) ◽

pp. 4632-4641 ◽

Cited By ~ 20

Author(s):

Pratima Chaurasia ◽

Dmitriy Berenzon ◽

Ronald Hoffman

Keyword(s):

Progenitor Cells ◽

Histone Deacetylase Inhibitors ◽

Ex Vivo ◽

Gene Expression Pattern ◽

Erythroid Progenitor ◽

Erythroid Precursor ◽

Differentiated Cells ◽

Erythroid Progenitor Cells ◽

H3 Acetylation

Abstract Presently, blood transfusion products (TPs) are composed of terminally differentiated cells with a finite life span. We have developed an ex vivo–generated TP composed of erythroid progenitor cells (EPCs) and precursors cells. Several histone deacetylase inhibitors (HDACIs) were used in vitro to promote the preferential differentiation of cord blood (CB) CD34+ cells to EPCs. A combination of cytokines and valproic acid (VPA): (1) promoted the greatest degree of EPC expansion, (2) led to the generation of EPCs which were capable of differentiating into the various stages of erythroid development, (3) led to epigenetic modifications (increased H3 acetylation) of promoters for erythroid-specific genes, which resulted in the acquisition of a gene expression pattern characteristic of primitive erythroid cells, and (4) promoted the generation of a TP that when infused into NOD/SCID mice produced mature RBCs containing both human adult and fetal globins as well Rh blood group Ag which persisted for 3 weeks and the retention of human EPCs and erythroid precursor cells within the BM of recipient mice. This ex vivo–generated EPC-TP likely represents a paradigm shift in transfusion medicine because of its potential to continue to generate additional RBCs after its infusion.

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Gene expression pattern in hepatic stem/progenitor cells during rat fetal development using complementary DNA microarrays

Hepatology ◽

10.1002/hep.20088 ◽

2004 ◽

Vol 39 (3) ◽

pp. 617-627 ◽

Cited By ~ 47

Author(s):

Petko M. Petkov ◽

Jiri Zavadil ◽

David Goetz ◽

Tearina Chu ◽

Robert Carver ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Expression Pattern ◽

Fetal Development ◽

Dna Microarrays ◽

Gene Expression Pattern ◽

Complementary Dna

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Stage-specific gene expression in erythroid progenitor cells (CFU-E)

Cell Differentiation ◽

10.1016/0045-6039(88)90017-6 ◽

1988 ◽

Vol 22 (3) ◽

pp. 259-265 ◽

Cited By ~ 4

Author(s):

Yuji Mishina ◽

Yasuhisa Matsui ◽

Masuo Obinata

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Specific Gene ◽

Erythroid Progenitor ◽

Specific Gene Expression ◽

Erythroid Progenitor Cells

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Differential gene expression analysis in early and late erythroid progenitor cells in β-thalassaemia

British Journal of Haematology ◽

10.1111/bjh.13432 ◽

2015 ◽

Vol 170 (2) ◽

pp. 257-267 ◽

Cited By ~ 7

Author(s):

Luke Forster ◽

John McCooke ◽

Matthew Bellgard ◽

David Joske ◽

Jill Finlayson ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Differential Gene Expression ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Erythroid Progenitor ◽

Differential Gene Expression Analysis ◽

Erythroid Progenitor Cells ◽

Differential Gene

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Developmental and Differential Changes of Gene Expression in Erythroid Progenitor Cells.

Blood ◽

10.1182/blood.v106.11.3648.3648 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3648-3648

Author(s):

Vladan P. Cokic ◽

Bhaskar Bhattacharya ◽

Raj K. Puri ◽

Alan N. Schechter

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

G Protein ◽

Peripheral Blood ◽

Globin Gene ◽

Globin Genes ◽

Erythroid Progenitor ◽

Gamma Globin ◽

Erythroid Progenitor Cells ◽

Globin Gene Expression

Abstract During erythropoiesis and human development different globin genes (α, β, γ, δ and ε) are expressed as a result of globin gene switching. We investigated globin gene expression in comparison to the expression of other genes in erythroid progenitor cells (EPC) during ontogenesis using in-house produced microarrays containing 16,659 oligonucleotides. Human primitive CD34+ cells were isolated from fetal liver (FL), cord blood (CB), adult bone marrow (BM), peripheral blood (PB) and mobilized peripheral blood (mPB), and developed into EPC in the presence of erythropoietin and other cytokines. The differentiation to EPC was confirmed by flow cytometry as 100% cells were CD71+. In microarray studies, a total of 2996 genes were highly expressed in FL, 2673 genes in CB, 2580 in mPB, 1465 in PB and 1259 in BM derived EPC. 661 of these genes were common for all type of cells. The high level of expression, beside globin genes, was observed for the following genes: transferrin receptor, proteoglycans, ALAS2, Charcot-Leyden crystal protein, nucleophosmin, eosinophil peroxidase, myeloperoxidase and ribonucleases. Most of the analyzed genes demonstrated down-regulation during ontogenesis (elastase 2, glutathione peroxidase 1, SERPINB1, nudix, mitochondrial proteins, ribosomal proteins, enthoprotin, serine proteinase inhibitor), but some showed up-regulation (hexokinase, superoxide dismutase 2, spectrin). Besides developmental changes of globin gene expression during ontogenesis, we also analyzed changes in their expression during erythropoiesis in these different tissues by quantitative PCR. Beta-globin gene expression reached the maximum levels in cells of adult blood origin: BM (176 fmol/μg) and PB (110 fmol/μg). Gamma-globin gene expression, of FL origin, had steady levels during erythroid differentiation (20 fmol/μg), whereas cord blood derived EPC demonstrated consistent up-regulation (60 fmol/μg) in contrast to cells originated from adult blood (3–15 fmol/μg at day 14th). G protein related genes and histone deacetylases were elevated in CB derived EPC, concomitant with increased gamma-globin gene expression. We also analyzed the gamma-globin induction by hydroxyurea, a well known inducer, and established which G protein-coupled receptors involved pathways are activated in PB derived EPC: dopamine receptors D1, D2 and D5, beta 2 adrenergic receptor, human DP prostanoid receptor and prostaglandin E receptor 1, as well as genes activated by cAMP/PKA, PI-3 kinase, MAP and NO/cGMP pathways. This study establishes concomitant changes in expression of globin genes and other known and/or previously unrecognized genes, which appear to be involved in erythropoiesis.

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