From the Departments of Clinical Oncology and
§ Molecular and Developmental Biology, Institute of Medical
Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo
108-8639, Japan and the ¶ Department of Hematology and Oncology,
Nagano Children's Hospital, 3100 Toyoshina, Toyoshina-cho,
Minamiazumi-gun, Nagano 399-82, Japan
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ABSTRACT |
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HML/SE is a cytokine-dependent cell line established from childhood acute megakaryoblastic leukemia. Granulocyte-macrophage colony-stimulating factor or stem cell factor (SCF) alone could stimulate proliferation of HML/SE cells, however interleukin-3, interleukin-6, granulocyte colony-stimulating factor and thrombopoietin could not. Although erythropoietin (EPO) alone stimulated neither proliferation nor differentiation of HML/SE cells, it did stimulate proliferation of HML/SE cells and production of hemoglobin in the presence of SCF. SCF activated the human EPO receptor promoter and induced EPO receptor gene expression. Given these results, we speculate that HML/SE cells acquired responsiveness to EPO via the EPO receptor induced by SCF. Mutation analysis of putative transcription factor binding sites in the human EPO receptor promoter suggested that Sp1, rather than the GATA-1 binding site, contributed to the induction of the hEPOR gene. Although it is well documented that hematopoietic stem cells and primitive progenitors require both an early-acting cytokine and a lineage-specific cytokine to differentiate to a certain lineage, related mechanisms are not well understood. HML/SE may serve as an excellent model system to analyze functions of early-acting cytokine SCF and lineage-specific cytokine EPO related to proliferation and differentiation of hematopoietic stem cells.
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INTRODUCTION |
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Proliferation and differentiation of hematopoietic stem/progenitor cells are modulated by cytokines such as interleukin-3 (IL-3),1 granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO) (1, 2), and thrombopoietin (TPO) (3). Hematopoietic cytokines can be classified into lineage-nonspecific early-acting cytokines and lineage-specific late-acting cytokines by biological activities and target cells (1). Cytokines such as SCF, IL-3, and GM-CSF belong to the former group and EPO, G-CSF, and TPO belong to the latter group. Several studies indicated that hematopoietic stem cells required both groups of these cytokines to differentiate and maturate to the certain lineages in vitro. Early-acting cytokines can effectively interact with late-acting cytokines in the production of more mature cells (4). In both humans and mice, hematopoietic progenitors lose their responsiveness to IL-3 as they differentiate (4, 5). In erythropoiesis, erythroid progenitors lose their responsiveness to SCF and acquire responsiveness to EPO during differentiation (6, 7). The expression patterns of cytokine receptors throughout hematopoiesis are not abundantly documented. The down-regulation of early-acting cytokine receptors and up-regulation of lineage-specific late-acting cytokine receptors may possibly occur as hematopoietic stem/progenitor cells differentiate.
Attempts to clarify the mechanism of these processes are hindered as the numbers of hematopoietic stem/progenitor cells in various sources such as peripheral blood, bone marrow and cord blood are limited. Although several human cell lines had been established, one that requires both early-acting and lineage-specific late-acting cytokines and is suitable to elucidate these problems has apparently not been documented.
Here, we demonstrate that the early-acting cytokine SCF induces the receptor gene of the lineage-specific cytokine EPO and consequent acquisition of responsiveness to EPO. We used a human hematopoietic stem cell-like line, HML/SE, established from childhood acute megakaryoblastic leukemia.
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MATERIALS AND METHODS |
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Cytokines and Antibodies-- Recombinant human (h) EPO, hGM-CSF, hIL-3, and hTPO were provided by Kirin Brewery (Tokyo, Japan), recombinant hSCF and hG-CSF were from Amgen Inc. (Thousand Oaks, CA), and recombinant hIL-6 was from Tosoh Co. (Kanagawa, Japan).
Mouse monoclonal antibodies (mAbs) for hCD2, hCD3, hCD4, hCD13, hCD14, hCD19, hCD33, hCD34, hCD38, and hCD45RO conjugated with phycoerythrin and anti-hCD8 and hCD45RA mAbs conjugated with fluorescein isothiocyanate were from Becton Dickinson (San Jose, CA). Fluorescein isothiocyanate-conjugated anti-hCD41b and HLA-DR mAbs were from Nichirei, (Tokyo, Japan). Anti-hCD117 mAb conjugated with phycoerythrin was from Immunotech (Marseille, France). Purified mAbs for hCD130 and h-glycophorin A were from PharMingen (San Diego, CA) and Immunotech, respectively. The preparation of anti-hCD126 mAb has been described elsewhere (8). Anti-h-hemoglobinEstablishment and Culture of HML/SE--
HML was originally
established from mononuclear cells in peripheral blood obtained from a
patient with childhood acute megakaryoblastic leukemia and 21-trisomy
(9). HML/SE is one of the subclones of HML. The cells were cloned as
follows; cells were cultured in semisolid culture containing 100 units/ml hIL-3, 10 ng/ml hGM-CSF, and 2 units/ml hEPO. On day 14, individual colonies were transferred to liquid cultures in the
-medium (Flow Laboratories, Rockville, MD) containing 10% fetal
bovine serum (HyClone, Logan, UT), 1% bovine serum albumin (Sigma),
with hIL-3, hGM-CSF, and hEPO at the same concentration as semisolid
cultures. After 1 month, the clones were expanded in the presence of 10 ng/ml hGM-CSF alone. Here, we used one of these subclones of HML,
designated HML/SE, which was maintained stably in the presence of
hGM-CSF for over 1 year.
Cell Proliferation Assay-- Cell proliferation was estimated by a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma) as described (10, 11). For long term growth assay, the number of live cells were counted by trypan blue dye exclusion test.
Flow Cytometric Analysis and Immunohistochemical
Staining--
Flow cytometric analysis was done as described (11).
Immunostaining with the alkaline phosphatase/anti-alkaline
phosphatase method, using anti-h-hemoglobin mAb was done as
described (12, 13).
Northern Blotting Analysis--
Messenger RNAs (mRNAs) were
isolated from HML/SE cells cultured under various conditions using a
QuickPrep Micro mRNA purification kit (Amersham Pharmacia Biotech).
The same amount (1 µg) of each mRNA was electrophoresed on
agarose formaldehyde gels and transferred to nylon membranes
(Boehringer Mannheim). Hybridizations were done in Quick Hyb solution
(Stratagene, La Jolla, CA) using cDNA fragment of h-globin, hEPO
receptor (generous gift from Dr. H. Nakauchi, University of Tsukuba,
Japan), hGATA-1 and -2 (generous gifts from Dr. M. Yamamoto, University
of Tsukuba, Japan), or G3PDH (CLONTECH, Palo Alto,
CA) as probes. The probe for h
-globin was cloned by RT-PCR (31-471)
from normal human erythroid cells, and the sequence was verified using
an ABI PRISM 310 genetic analyzer (Perkin-Elmer, Foster City, CA). The
membranes were visualized using a BAS2000 image analyzer (Fuji Film,
Tokyo, Japan).
Construction of Plasmid for Luciferase Assay--
Human EPO
receptor (hEPOR) 5' flanking sequence (197 to
1) containing both
GATA and Sp1 binding sites was cloned by PCR from human genomic DNA
(14, 15). The PCR products were subcloned into pCR2.1 TA-cloning vector
(Invitrogen, San Diego, CA), and the sequence was verified. The plasmid
for the luciferase assay, pEPOR-W, was constructed using XhoI and
HindIII restriction enzyme sites within both pCR2.1 and pGL3-Basic
luciferase vector (Promega, Madison, WI). Mutants of pEPOR-W containing
a mutation of the GATA site (pEPOR-
G), the Sp1 binding site
(pEPOR-
S), and mutations of both GATA and Sp1 sites (pEPOR-
G
S)
were constructed by PCR mutagenesis using pEPOR-W in which AGATAA was
replaced with AGTTAA (for GATA mutation) and GGGCGG with GGGAAA (for
Sp1 mutation), respectively (16, 17).
Transfection and Luciferase Assay--
HML/SE cells (3 × 106) were transfected by electroporation with 15 µg of
either pGL3-Basic as a control or pEPOR-W, -G, -
S, or -
G
S,
as described (18) but with minor modifications. Briefly, cells were
electroshocked using 975 microfarads at 200 V by a Gene Pulser
electroporation apparatus (Bio-Rad), divided into two aliquots, and
cultured with 10 ng/ml hGM-CSF and 100 ng/ml hSCF, respectively. For
luminescence assay, the substrate was automatically injected into the
sample in the luminometer (TD-20/20, Turner Designs, Sunnyvale, CA).
Luciferase activities were normalized by protein concentration, which
was estimated using the BCA protein assay reagent (Pierce).
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RESULTS |
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Expression of Cell Surface Antigens-- We first examined by flow cytometric analysis the phenotype of HML/SE cells maintained in the presence of hGM-CSF (Table I). Most HML/SE cells highly expressed myeloid markers such as CD13 and a megakaryocyte-specific antigen CD41b, often the basis of a diagnosis of acute megakaryoblastic leukemia. They also weakly expressed an erythroid-specific marker, glycophorin A, on their surface. These results are taken to mean that HML/SE is a multi-potent cell line that has the potential to differentiate toward three lineages: myeloid, erythroid, and megakaryocytic.
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Effects of Cytokines on Proliferation of HML/SE Cells--
We next
analyzed proliferation of HML/SE cells in the presence of various
cytokines. Cells maintained in the presence of hGM-CSF were washed
three times with factor-free -medium, and started to culture at a
density of 1 × 104/ml in the presence of indicated
cytokines. Examination of viable cells was made by trypan blue dye
exclusion test every 24 h. As shown in Fig.
1A, HML/SE cells proliferated
in the presence of hGM-CSF and hSCF alone stimulated proliferation,
albeit to a lesser extent than seen with hGM-CSF. When these cytokines
were removed from the culture, all the HML/SE cells died within 72 h. Neither hEPO nor hIL-6 alone stimulated proliferation of HML/SE
within 72 h. Proliferation of HML/SE in the presence of hIL-3,
hG-CSF, and hTPO was not observed within 72 h (data not shown).
When we continued to examine proliferation of HML/SE cells until day 10 (Fig. 1B), the cells continued to proliferate in the presence of either
hGM-CSF or hSCF, but not in the presence of hIL-6, hEPO, hG-CSF, and
hTPO (data not shown).
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Synergistic Action of Early-acting Cytokines and Lineage-specific
Cytokines on Proliferation of HML/SE Cells--
It is well documented
that hematopoietic stem/progenitor cells require stimulation of the
combination of two or more cytokines to proliferate and differentiate.
We next investigated the synergistic action of early-acting cytokines
SCF and GM-CSF, which can stimulate proliferation of this cell line
alone, and lineage-specific cytokines EPO, G-CSF, and TPO on
proliferation of HML/SE cells. The cells maintained in the presence of
hGM-CSF were washed three times with factor-free -medium, and
started to culture at a density of 1 × 104/ml in the
presence of 10 ng/ml hGM-CSF or 100 ng/ml hSCF, with or without 2 units/ml hEPO. The culture was continued to day 10, as described under
"Materials and Methods." In combination with hEPO and hGM-CSF, the
cell number increased at almost the same rate as seen in the presence
of hGM-CSF alone (Fig. 2A). In
contrast, in combination with hEPO and hSCF, the cell number increased
more rapidly than in the presence of SCF alone (Fig. 2A). We
further analyzed effects of various concentrations of hEPO on HML/SE
cells, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Addition of either hGM-CSF or hSCF alone stimulated proliferation of HML/SE, in a dose-dependent manner, but
hEPO alone failed to do so, at any concentration used (Fig.
2B). When SCF was present in the medium, EPO stimulated
proliferation of HML/SE cells, in a dose-dependent manner
(Fig. 2C). We also examined the proliferation response of
HML/SE cells in combination with hGM-CSF or hSCF and either one of
hG-CSF or hTPO. No additional effect of hG-CSF and hTPO was observed in
the presence of hGM-CSF or hSCF (data not shown).
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Synergistic Action of SCF and EPO on Differentiation of HML/SE
Cells--
Because a synergistic action between SCF and EPO on
proliferation of HML/SE cells occurred, we further examined synergistic actions of early-acting cytokines and lineage-specific cytokines on
differentiation of HML/SE cells. In combination with hGM-CSF and hEPO,
expression of an erythroid-specific surface antigen glycophorin A did
not increase (Fig. 3A). In
contrast, with 10 days of culture in combination with hSCF and hEPO,
more than 50% of HML/SE cells markedly expressed glycophorin A on
their surface (Fig. 3A), and -globin mRNA markedly
increased in comparison with hGM-CSF plus hEPO or hSCF alone (Fig.
3B). As induction of
-globin mRNA was detected in
case of a combination of hSCF and hEPO, we examined the production of
hemoglobin, at the protein level. Hemoglobin production was evident in
cells cultured with hSCF plus hEPO, as determined immunohistochemically
(Fig. 3C, d), but was not detectable in cells
cultured with hGM-CSF (Fig. 3C, b), hGM-CSF plus
hEPO, or hSCF alone (data not shown). In addition, the cells had
erythroid features such as condensed nucleus and a basophilic cytoplasm
(Fig. 3C, b) compared with cells maintained in
the presence of hGM-CSF (Fig. 3C, a) and the cell
pellet was reddish (data not shown), suggesting that EPO signaling led
to erythroid differentiation in the presence of SCF.
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Induction of EPO Receptor mRNA by SCF-- In the presence of SCF, HML/SE cells seemed to acquire responsiveness to EPO; therefore, we investigated whether the expression of the EPOR had been changed by SCF. In general, it is difficult to detect EPOR protein due to the low expression of endogenous EPOR on the cell surface. We then examined changes of hEPOR mRNA expression. The mRNA expression of hEPOR was analyzed by Northern blotting using 1 µg of purified mRNAs extracted from HML/SE cells cultured for 10 days in the presence of hGM-CSF, hSCF, hSCF plus hEPO, and hGM-CSF plus hEPO, respectively. When HML/SE cells were maintained in the presence of hGM-CSF alone, the expression of hEPOR mRNA was hardly detectable (Fig. 4A). With addition of hEPO, the amount of hEPOR mRNA did not increase (Fig. 4A). In the presence of hSCF alone, the expression of hEPOR mRNA increased (Fig. 4A), and in combination with hEPO, the hEPOR mRNA level was dramatically increased (Fig. 4A). As it appeared that SCF transcriptionally activated the EPOR gene, we examined the time course of hEPOR mRNA changes; hEPOR mRNA in HML/SE cells increased time-dependently in the presence of hSCF (Fig. 4B).
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SCF Activated EPOR Promoter--
As described above, SCF induced
EPOR mRNA. To clarify the mechanism of EPOR gene induction by SCF,
we investigated whether SCF activated the EPOR promoter by transient
system. We cloned hEPOR promoter region containing both GATA and Sp1
binding sites from human genomic DNA, and the cloned fragment was fused
with luciferase coding region as described under "Materials and
Methods." HML/SE cells (3 × 106) were transfected with
15 µg of either pGL3-Basic or pEPOR-W, divided into two aliquots and
cultured with 10 ng/ml hGM-CSF and 100 ng/ml hSCF, respectively. After
72 h, the cells were lysed and luminescence was measured. GM-CSF
did not activate the hEPOR promoter; however, SCF did activate it
10-fold more compared with the sample transfected with the luciferase
vector lacking the hEPOR promoter (Fig.
5A). Because the EPOR promoter
we used contains putative GATA and Sp1 binding sites, we next
investigated whether these sites contributed to the activation of the
hEPOR promoter by SCF using pEPOR-W mutants lacking the GATA and/or Sp1
binding site. Luciferase activity of the hEPOR promoter containing a
mutation of the GATA binding site (pEPOR-G) induced by SCF was
higher than that observed with the intact promoter. On the other hand, when the cells were transfected with pEPOR-
S, containing a mutation of the Sp1 binding site, luciferase activity was dramatically reduced.
As expected, only background luciferase activity was observed with
pEPOR-
G
S (both GATA and Sp1 mutated) (Fig. 5A). We
also examined both GATA-1 and GATA-2 mRNA expression levels of the
HML/SE cultured in the presence of either GM-CSF or SCF by Northern
blotting analysis. As shown in Fig. 5B, neither the level of
GATA-1 nor GATA-2 was changed by the addition of SCF. These results
support the idea that Sp1 rather than GATA plays a role in the
induction of the hEPOR promoter by SCF.
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DISCUSSION |
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We obtained evidence of induction of the receptor gene of the lineage-specific cytokine EPO by the early-acting cytokine SCF and the consequent acquisition of responsiveness to EPO in HML/SE, a human leukemic cell line. Modulation of the process of hematopoietic differentiation by a series of early-acting hematopoietic cytokines, including SCF, IL-3, GM-CSF, and lineage-specific hematopoietic cytokines such as EPO, G-CSF, and TPO has been proposed (2). Multi-potent progenitor cells must interact with an appropriate combination of both groups of these hematopoietic cytokines in order to proliferate and to progress to the next stage of differentiation. In erythropoiesis, both the early-acting cytokines and the lineage-specific cytokine EPO are required (19). The importance of SCF in erythroid cell development is demonstrated by the severe macrocytic anemia in Sl/Sl and W/W mice, which are mutated at the loci encoding SCF and its receptor c-Kit, respectively (20). Reduced levels of CFU-E in these mice (21) means that generation of CFU-E from pluri-potent stem cell is largely dependent on SCF. Studies on knock-out of EPO or EPOR showed that generation of committed erythroid BFU-E and CFU-E progenitors did not require EPO or EPOR (22). It was also reported that most purified BFU-E were insensitive to EPO and had a very low number of EPOR on their surface, but the number of EPOR did increase and acquired responsiveness to EPO when differentiated to CFU-E (6). EPO alone could stimulate neither proliferation nor differentiation to erythroid, and both SCF and EPO were indispensable for erythroid proliferation and differentiation of HML/SE cells. These results suggest that HML/SE cells consist of very early stage progenitors that have not acquired responsiveness to EPO as the number of EPOR is scanty. Other cell lines reported to proliferate and differentiate to erythroid in the presence of EPO alone such as F36, UT-7, MTAT, and TF-1, seem to have already expressed EPOR and consequently acquired responsiveness to EPO (23-26). Thus, HML/SE cells seem to have a unique feature corresponding to normal progenitors that are more immature than CFU-E. It may be that early-acting cytokines SCF up-regulate EPOR of BFU-E level progenitors and consequently give rise to CFU-E with acquired responsiveness to EPO. Lack of this mechanism may result in reduced levels of CFU-E in Sl/Sl and W/W mice. The physical interaction between c-Kit and EPOR has been reported (27). The binding of SCF to its receptor c-Kit may cause trans-phosphorylation of EPOR and result in transduction of differentiation signal. We propose another mechanism to explain the co-operation between SCF and EPO.
Because EPOR mRNA was much more increased by SCF plus EPO than by SCF alone, EPO signaling seems to lead to further erythroid differentiation by positive feedback regulation. It was reported that the EPOR gene was trans-activated by transcription factor GATA-1 (15, 17, 28), and that GATA-1 expression was stimulated by EPO (28-30). GATA-1 has been also reported to trans-activate other erythroid-specific genes such as globin and GATA-1 itself (31). In the presence of SCF and EPO, as well as EPO in normal erythroid, erythroid-specific genes such as globin and EPOR were induced in HML/SE cells. In contrast, in the presence of SCF alone, only induction of the EPOR gene was observed. We found that SCF activated the hEPOR promoter, determined using a luciferase vector inserted the hEPOR promoter containing the GATA binding site. However, activation of the hEPOR promoter by SCF was not affected by a GATA binding site mutation and SCF did not increase mRNA expression levels of either GATA-1 or GATA-2, which is assumed to be involved in the induction of EPOR mRNA (32). Taken together, these results suggest that neither GATA-1 nor GATA-2 participate in the induction of the hEPOR gene by SCF. Abrogation of hEPOR promoter activation by SCF through a mutation of the Sp1 binding site suggests a role for Sp1 in hEPOR promoter activation by SCF. Because other erythroid-related genes are reported to be regulated by GATA-1, it can be speculated that specific induction of the EPOR gene by SCF is achieved by Sp1.
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ACKNOWLEDGEMENTS |
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We thank I. Suyama for technical support and M. Ohara for comments on the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
81-3-5449-5694; Fax: 81-3-5449-5428; E-mail:
nakahata{at}ims.u-tokyo.ac.jp.
1 The abbreviations used are: IL, interleukin; EPO, erythropoietin; EPOR, EPO receptor; h, human; PCR, polymerase chain reaction; mAb, monoclonal antibody; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; SCF, stem cell factor; TPO, thrombopoietin; GM-CSF, granulocyte-macrophage colony-stimulating factor; G-CSF, granulocyte colony-stimulating factor; CFU-E, colony-forming unit erythroid; BFU-E, burst-forming unit erythroid.
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REFERENCES |
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