Atiprimod Inhibits the Growth of JAK2 Tyrosine Kinase Over-Expressing Cells “In Vitro” and “Ex Vivo”.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4924-4924
Author(s):  
Roberto Nussenzveig ◽  
Taghi Manshouri ◽  
Hagop Kantarjian ◽  
Francis Giles ◽  
Jorge Cortes ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Annexin V ◽  
Dependent Manner ◽  
Wild Type ◽  
Erythroid Progenitor ◽  
Myeloid Metaplasia ◽  
Erythroid Progenitor Cells ◽  
Therapeutic Benefits

Abstract Background: A novel somatic single point non-synonymous mutation (1849 G to T) in the tyrosine kinase JAK2, rendering the enzyme constitutively active, has been reported in classical myeloproliferative disorders (MPD), specifically, in greater than 90% of patients with polycythemia vera (PV), and 50% of patients with essential thrombocythemia (ET) and myelofibrosis with myeloid metaplasia (MMM). Consequently, JAK2 tyrosine kinase has become a significant target for the development of specific inhibitors of its activity with possible therapeutic benefits for patients with classical MPD. We report here the results of a study using atiprimod, an anti-inflammatory and anti-cancer compound belonging to the azaspirane class of cationic amphiphilic drugs in JAK2 over-expressing cell lines and patient samples. In contrast to TKI inhibitors, this drug is causing downregulation of JAK2 protein by facilitating its degradation, possibly through the ubiquitin-proteosome pathway (Faderl, S. et al, Leukemia Research 2006 in press). Methods and Results: We have examined the growth inhibitory effect of atiprimod against two mouse FDCP cell lines transfected with erythropoietin receptor (Epo-R) and, either wild-type JAK2 (JAK2WT) or mutant JAK2 (JAK2V617F). Atiprimod growth inhibition, measured using 72 hours MTS assay, was significant against both mutant JAK2V617F cells (IC50 0.42μM) and wild-type JAK2WT cells (IC50 0.65μM). Atiprimod was found to induce apoptosis in cell lines as evidenced by an increase in the mitochondrial membrane potential and augmented annexin V positivity by flow cytometry. Mononuclear cells, isolated from peripheral blood of PV patients (JAK2V617F positive), were cultured in cytokine supplemented serum free media for expansion of erythroid progenitors. Expansion spanned a three week, three step sequential treatment of the erythroid progenitor cells with specific combinations of cytokines. Finally, treatment of expanded erythroid progenitor cells with atiprimod for 24 and 48 hours resulted in increased annexin V positive cells, in both, a dose and time dependent manner. Conclusion: Our preliminary pre-clinical results suggest that atiprimod might be a beneficial medication for MPD patients carrying the JAK2V617F mutation. Atiprimod is currently being tested in a phase I clinical study.

scholarly journals Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor.

1995 ◽  
Vol 15 (6) ◽  
pp. 3147-3153 ◽  
Author(s):  
G A Blobel ◽  
C A Sieff ◽  
S H Orkin
Keyword(s):  
Bone Marrow ◽  
Estrogen Receptor ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Erythroid Cell ◽  
High Dose ◽  
Dependent Manner ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. Interference with GATA-binding proteins may be one mechanism by which steroid hormones modulate cellular differentiation.

scholarly journals A constitutively activated erythropoietin receptor stimulates proliferation and contributes to transformation of multipotent, committed nonerythroid and erythroid progenitor cells.

1994 ◽  
Vol 14 (4) ◽  
pp. 2266-2277 ◽  
Author(s):  
G D Longmore ◽  
P N Pharr ◽  
H F Lodish
Keyword(s):  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Leukemia Virus ◽  
Active Form ◽  
Erythropoietin Receptor ◽  
Mutant Form ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

If the env gene of spleen focus-forming virus (SFFV) is replaced by a cDNA encoding a constitutively active form of the erythropoietin receptor, EPO-R(R129C), the resultant recombinant virus, SFFVcEPO-R, induces transient thrombocytosis and erythrocytosis in infected mice. Clonogenic progenitor cell assays of cells from the bone marrow and spleens of these infected mice suggest that EPO-R(R129C) can stimulate proliferation of committed megakaryocytic and erythroid progenitors as well as nonerythroid multipotent progenitors. From the spleens of SFFVcEPO-R-infected mice, eight multiphenotypic immortal cell lines were isolated and characterized. These included primitive erythroid, lymphoid, and monocytic cells. Some expressed proteins characteristic of more than one lineage. All cell lines resulting from SFFVcEPO-R infection contained a mutant form of the p53 gene. However, in contrast to infection by SFFV, activation of PU.1 gene expression, by retroviral integration, was not observed. One cell line had integrated a provirus upstream of the fli-1 gene, in a location typically seen in erythroleukemic cells generated by Friend murine leukemia virus infection. This event led to increased expression of fli-1 in this cell line. Thus, infection by SFFVcEPO-R can induce proliferation and lead to transformation of nonerythroid as well as very immature erythroid progenitor cells. The sites of proviral integration in clonal cell lines are distinct from those in SFFV-derived lines.

scholarly journals Ex Vivo-Generated CD36+ Erythroid Progenitors Are Highly Permissive to Human Parvovirus B19 Replication

2007 ◽  
Vol 82 (5) ◽  
pp. 2470-2476 ◽  
Author(s):  
Susan Wong ◽  
Ning Zhi ◽  
Claudia Filippone ◽  
Keyvan Keyvanfar ◽  
Sachiko Kajigaya ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Real Time ◽  
Cell Lines ◽  
Ex Vivo ◽  
Parvovirus B19 ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Human Virus ◽  
Erythroid Progenitor Cells ◽  
Large Numbers

ABSTRACT The pathogenic parvovirus B19 (B19V) has an extreme tropism for human erythroid progenitor cells. In vitro, only a few erythroid leukemic cell lines (JK-1 and KU812Ep6) or megakaryoblastoid cell lines (UT7/Epo and UT7/Epo-S1) with erythroid characteristics support B19V replication, but these cells are only semipermissive. By using recent advances in generating large numbers of human erythroid progenitor cells (EPCs) ex vivo from hematopoietic stem cells (HSCs), we produced a pure population of CD36+ EPCs expanded and differentiated from CD34+ HSCs and assessed the CD36+ EPCs for their permissiveness to B19V infection. Over more than 3 weeks, cells grown in serum-free medium expanded more than 800,000-fold, and 87 to 96% of the CD36+ EPCs were positive for globoside, the cellular receptor for B19V. Immunofluorescence (IF) staining showed that about 77% of the CD36+ EPCs were positive for B19V infection, while about 9% of UT7/Epo-S1 cells were B19V positive. Viral DNA detected by real-time PCR increased by more than 3 logs in CD36+ EPCs; the increase was 1 log in UT7/Epo-S1 cells. Due to the extensive permissivity of CD36+ EPCs, we significantly improved the sensitivity of detection of infectious B19V by real-time reverse transcription-PCR and IF staining 100- and 1,000-fold, respectively, which is greater than the sensitivity of UT7/Epo-S1 cell-based methods. This is the first description of an ex vivo method to produce large numbers of EPCs that are highly permissive to B19V infection and replication, offering a cellular system that mimics in vivo infection with this pathogenic human virus.

scholarly journals A role for calmodulin in the growth of human hematopoietic progenitor cells

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1446-1454 ◽  
Author(s):  
N Katayama ◽  
M Nishikawa ◽  
F Komada ◽  
N Minami ◽  
S Shirakawa
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Hematopoietic Progenitor Cells ◽  
Dependent Manner ◽  
Hematopoietic Progenitor ◽  
Erythroid Progenitor ◽  
Gm Csf ◽  
Erythroid Progenitor Cells ◽  
Dependent Inhibition ◽  
Dose Dependent

Abstract A possible role for calmodulin in the colony growth of human hematopoietic progenitor cells was investigated using pharmacologic approaches. We obtained evidence for a dose-dependent inhibition of colony formation of myeloid progenitor cells (CFU-C) stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) by three calmodulin antagonists, N- (6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N- (4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13), and trifluoperazine. Chlorine-deficient analogs of W-7 and W-13, with a lower affinity for calmodulin, did not inhibit the growth of CFU-C colonies. W-7, W-13, and trifluoperazine inhibited the colony formation of immature erythroid progenitor cells (BFU-E) stimulated by IL-3 plus erythropoietin (Ep) or GM-CSF plus Ep, in a dose-dependent manner, while they did not affect the colony formation of mature erythroid progenitor cells (CFU-E) induced by Ep. W-7, W-13, and trifluoperazine also led to a dose-dependent inhibition of GM-CSF-induced colony formation of KG-1 cells. Calmodulin-dependent kinase activity derived from the KG-1 cells was inhibited by these three calmodulin antagonists in a dose-dependent manner. These data suggest that calmodulin may play an important regulatory role via a common process in the growth of hematopoietic progenitor cells stimulated by IL-3, GM-CSF, and G-CSF. Mechanisms related to the growth signal of Ep apparently are not associated with calmodulin-mediated systems.

Tyrosine Phosphorylation Enhances ITIM-Dependent Internalization of CD33, the Target for the Anti-AML Immunoconjugate, Gemtuzumab Ozogamicin.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2581-2581
Author(s):  
Roland B. Walter ◽  
Brian W. Raden ◽  
Irwin D. Bernstein ◽  
Jonathan A. Cooper
Keyword(s):  
Cell Surface ◽  
Tyrosine Kinase ◽  
Gene Silencing ◽  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Myeloid Cell ◽  
Gemtuzumab Ozogamicin ◽  
Dependent Manner ◽  
Wild Type ◽  
Phosphorylation State

Abstract Background: CD33, the target for the anti-AML immunoconjugate, gemtuzumab ozogamicin (GO; Mylotarg™), contains two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). We have previously shown that these motifs control uptake of antibody-bound CD33 and GO-induced cytotoxicity. In this study, we determined which phosphorylation state favors uptake of antibody-bound CD33, identified proteins that bind to CD33 in an ITIM-dependent manner, and assessed their importance for CD33 internalization by siRNA-based gene silencing. Methods: Internalization of anti-CD33 antibodies was measured by flow cytometry in the presence or absence of the tyrosine phosphatase inhibitor, pervanadate, in human CD33+ AML cell lines (ML-1, HL-60, NB4, U937, TF-1) and CD33− Jurkat T cells infected with wild-type and mutant CD33. Pull-down experiments were performed with glutathione S-transferase (GST) proteins fused to phosphorylated cytoplasmic tails of CD33, using human myeloid cell lysates. Co-immunoprecipitations were performed with myeloid cell lines expressing HA-tagged wild-type CD33. Lentivirus-based siRNA constructs were generated for gene silencing, and expressed in human CD33+ AML cell lines. Results: Pervanadate significantly increased uptake of anti-CD33 antibodies in human AML cell lines; this effect was dependent upon the integrity of the ITIMs and was prevented by co-treatment with the Src tyrosine kinase inhibitor PP2, suggesting that Src family kinase-dependent phosphorylation of the ITIMs critically controls uptake of antibody-bound CD33, possibly by altering which proteins binds to CD33 or by facilitating binding of adaptor-proteins required for endocytosis. We identified several proteins, including the tyrosine phophatases, SHP-1 and SHP-2, and the non-receptor tyrosine kinase, Syk, which bound to phosphorylated wild-type and mutant CD33 in a manner that paralleled the endocytic properties of the corresponding CD33 protein. Since these three proteins have been implicated in endocytic processes of other cell surface proteins, we assessed their role in uptake of antibody-bound CD33 by siRNA-mediated gene silencing. Simultaneous depletion of SHP-1 and SHP-2, but not SHP-1 or SHP-2 alone, significantly increased internalization of antibody-bound CD33 in the two AML cell lines with the highest cell surface expression of CD33, whereas no effect was seen in two other cell lines with lower CD33 expression levels. In contrast, depletion of Syk, whose expression has previously been correlated to the inhibitory effect of anti-CD33 antibodies on AML cell growth, failed to affect antibody internalization in the cell lines assessed. Conclusion: These studies indicate that the phosphorylation status of the ITIMs controls uptake of antibody-bound CD33. In line with this model, SHP-1 and SHP-2, which have been shown to dephosphorylate CD33 in vitro, can affect this endocytic process. Thus, our data imply manipulation of the phosphorylation state of CD33, e.g. by activating Src family kinases or interfering with phosphatases as a novel means to increase uptake of anti-CD33 antibody-based therapeutics such as GO. Finally, the variable effect of SHP-1 and SHP-2 depletion suggests that there are significant cell-type specific differences in the response to anti-CD33 antibody ligation, for example differences in tyrosine phosphorylation levels and/or activation/recruitment or redundancies of tyrosine phosphatases.

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

Blood ◽  
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.

Phosphatidylinositol 3-Kinase Is Involved in the Protection of Primary Cultured Human Erythroid Precursor Cells From Apoptosis

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1568-1577 ◽  
Author(s):  
Yoshihito Haseyama ◽  
Ken-ichi Sawada ◽  
Atsushi Oda ◽  
Kazuki Koizumi ◽  
Hina Takano ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Maximal Effect ◽  
Erythroid Cells ◽  
Phosphatidylinositol 3 Kinase ◽  
Erythroid Progenitor ◽  
Erythroid Precursor ◽  
Erythroid Progenitor Cells ◽  
Dependent Cell ◽  
Phosphatidylinositol 3

Little is known about the physiologic role of phosphatidylinositol 3-kinase (PI-3K) in the development of erythrocytes. Previous studies have shown that the effects of the PI-3K inhibitor wortmannin on erythropoietin (EPO)-dependent cell lines differed depending on the cell type used. Wortmannin inhibited EPO-induced differentiation of some cell lines without affecting their proliferation; however, the EPO-induced proliferation of other cell lines was inhibited by wortmannin. In neither case were signs of apoptosis observed. We have previously reported that signaling in highly purified human colony forming units-erythroid (CFU-E), generated in vitro from CD34+ cells, differed from that in EPO-dependent cell lines. In the current study, we examined the effects of a more specific PI-3K inhibitor (LY294002) on human CFU-E. We found that LY294002 dose-dependently inhibits the proliferation of erythroid progenitor cells with a half-maximal effect at 10 μmol/L LY294002. LY294002 at similar concentrations also induces apoptosis of these cells, as evidenced by the appearance of annexin V–binding cells and DNA fragmentation. The steady-state phosphorylation of AKT at Ser-473 that occurs as a result of PI-3K activation was also inhibited by LY294002 at similar concentrations, suggesting that the effects of LY294002 are specific. Interestingly, the acceleration of apoptosis by LY294002 was observed in the presence or absence of EPO. Further, deprivation of EPO resulted in accelerated apoptosis irrespective of the presence of LY294002. Our study confirms and extends the finding that signaling in human primary cultured erythroid cells is significantly different from that in EPO-dependent cell lines. These data suggest that PI-3K has an antiapoptotic role in erythroid progenitor cells. In addition, 2 different pathways for the protection of primary erythroid cells from apoptosis likely exist: 1 independent of EPO that is LY294002-sensitive and one that is EPO-dependent and at least partly insensitive to LY294002.

scholarly journals A role for calmodulin in the growth of human hematopoietic progenitor cells

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1446-1454
Author(s):  
N Katayama ◽  
M Nishikawa ◽  
F Komada ◽  
N Minami ◽  
S Shirakawa
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Hematopoietic Progenitor Cells ◽  
Dependent Manner ◽  
Hematopoietic Progenitor ◽  
Erythroid Progenitor ◽  
Gm Csf ◽  
Erythroid Progenitor Cells ◽  
Dependent Inhibition ◽  
Dose Dependent

A possible role for calmodulin in the colony growth of human hematopoietic progenitor cells was investigated using pharmacologic approaches. We obtained evidence for a dose-dependent inhibition of colony formation of myeloid progenitor cells (CFU-C) stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) by three calmodulin antagonists, N- (6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N- (4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13), and trifluoperazine. Chlorine-deficient analogs of W-7 and W-13, with a lower affinity for calmodulin, did not inhibit the growth of CFU-C colonies. W-7, W-13, and trifluoperazine inhibited the colony formation of immature erythroid progenitor cells (BFU-E) stimulated by IL-3 plus erythropoietin (Ep) or GM-CSF plus Ep, in a dose-dependent manner, while they did not affect the colony formation of mature erythroid progenitor cells (CFU-E) induced by Ep. W-7, W-13, and trifluoperazine also led to a dose-dependent inhibition of GM-CSF-induced colony formation of KG-1 cells. Calmodulin-dependent kinase activity derived from the KG-1 cells was inhibited by these three calmodulin antagonists in a dose-dependent manner. These data suggest that calmodulin may play an important regulatory role via a common process in the growth of hematopoietic progenitor cells stimulated by IL-3, GM-CSF, and G-CSF. Mechanisms related to the growth signal of Ep apparently are not associated with calmodulin-mediated systems.

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

Blood ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document