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HEMATOPOIESIS
From the Department of Cell Genetics, Sasaki Institute,
and the Department of Pathology, Juntendo University School of
Medicine, Tokyo, Japan.
PU.1 is an Ets family transcription factor essential for
myelomonocyte and B-cell development. We previously showed that
overexpression of PU.1 in murine erythroleukemia (MEL) cells inhibits
growth and erythroid differentiation and induces apoptosis of the
cells. In an effort to identify target genes of PU.1 concerning these phenomena by using a messenger RNA differential display strategy, we
found that some myeloid-specific and lymphoid-specific genes, such as
the osteopontin gene, are transcriptionally up-regulated in MEL cells
after overexpression of PU.1. We then found that expression of several
myelomonocyte-specific genes, including the CAAT-enhancer-binding
protein- Hematopoiesis is the process by which progenitor
cells acquire characteristics of certain types of hematopoietic cells.
It is believed that this process is mediated by hematopoietic
cell-specific transcription factors that confer cell specificity by
means of regulation of the expression of cell-type-specific
genes. Targeted disruption of such transcription-factor genes results
in impaired hematopoiesis or defects in the development of
particular types of hematopoietic cells.1-6
PU.1, a member of the Ets family of transcription factors, is expressed
in hematopoietic cells, predominantly in myelomonocytes and B cells.
PU.1 regulates cell-type-specific expression of genes such as the CD18
and granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR)
genes in myelomonocytes and the immunoglobulin (Ig) heavy- and
light-chain genes in B cells. Targeted disruption of the
PU.1 gene was found to cause severe defects in the
development process in both myeloid and lymphoid cells, characterized
by the absence of macrophages and B cells and the delayed appearance of
neutrophils.7,8 This finding indicates that PU.1 plays a
fundamental role in myelomonocyte and B-cell development.
In Friend virus-induced murine erythroleukemia (MEL), expression
of the PU.1 gene is deregulated by proviral integration of spleen focus-forming virus.9,10 This deregulated
expression of PU.1 in erythroblasts is thought to be one of the causes
of leukemogenesis.
In a previous study, which we conducted to elucidate the functional
role of PU.1 in the regulation of growth and differentiation of MEL
cells, we introduced an expression plasmid construct of the
PU.1 gene into MEL cells. We found that when PU.1 was
overexpressed in the cells in the presence of the
differentiation-inducing reagent dimethyl sulfoxide (DMSO), growth
inhibition and apoptosis were induced but erythroid differentiation was
not.11 These results suggest that although PU.1
contributes to the generation of erythroleukemia by inhibiting
differentiation of erythroid cells, it induces growth suppression and
cell death under special circumstances. Subsequently, we showed that
expression of the c-myc and bcl-2 genes is
down-regulated in apoptosis12 and DNA-binding activity of
GATA-1 transcription factor, which is important for the survival and
differentiation of erythroid cells,13,14 is markedly
reduced.15 However, the exact mechanism (or mechanisms) by
which PU.1 inhibits growth and erythroid differentiation and induces
apoptosis has not been determined. Because PU.1 is a transcription
factor, it is logical to assume that the cellular effects induced by
its overexpression are mediated by means of regulation of expression of
its target genes. Therefore, using a messenger RNA (mRNA) differential
display strategy, we attempted to isolate and identify the target genes of PU.1 that are transcriptionally up-regulated or down-regulated during induction of the effects of PU.1 in MEL cells.
We observed transcriptional up-regulation of some myeloid-specific and
lymphoid-specific genes in addition to up-regulation or down-regulation
of genes suggested to be involved in growth inhibition or apoptosis.
This finding indicated the possibility that PU.1 not only inhibits
erythroid differentiation of MEL cells but also induces expression of
myelomonocyte-specific and B-cell-specific genes in the cells. In the
current study, we addressed this possibility and found that PU.1
induces expression of several myelomonocyte-specific and
B-cell-specific genes as well as morphologic and functional changes in
MEL cells.
Cell culture
mRNA differential display
Northern blot analysis Twenty micrograms of total RNA samples was denatured with formamide and separated in an 1% agarose gel containing formaldehyde. The RNA was transferred to a nylon membrane and then hybridized with DNA probes labeled with phosphorus 32-deoxycytidine triphosphate. The hybridization process was done as described previously.16 The complementary DNA (cDNA) fragments used as probes for the osteopontin gene, the eosinophil cationic protein gene, and the B144 gene were obtained by using mRNA differential display. The cDNA fragments used as probes for the CAAT-enhancer-binding protein- (C/EBP-) and C/EBP- genes were amplified by RT-PCR from HL-60 human myelomonocytic leukemia cells by using the primers shown in Table
1. A BamHI fragment of mouse
 -actin cDNA was used as the internal control.
RT-PCR analysis Preparation of first-strand cDNA and PCR amplification were done as described previously.12 PCR amplification consisted of 1 minute at 94°C, 2 minutes at 55°C, and 3 minutes at 72°C for 25 to 35 cycles. PCR products were resolved in a 1% agarose gel containing ethidium bromide. The intensity of the bands was quantified by using the National Institutes of Health Image computer program. The primers used are listed in Table 1.Western blot analysis Fifty micrograms of total protein samples was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was then probed with an antimouse PU.1 antibody (T-21; Santa Cruz Biotechnology, Santa Cruz, CA) as described previously.11 Bound antibody was detected by using an electrogenerated chemiluminescence system (Amersham, United Kingdom).Flow cytometric analysis Flow cytometric analysis was done with a fluorescence-activated cell-sorter scanner (Becton Dickinson, Mountain View, CA), and data were processed with the Cell Quest program (Becton Dickinson). Cells (5 × 105) were incubated first with 2.4G2 monoclonal antibody (mAb) (Pharmingen, San Diego, CA) to block the Fc receptor and were then incubated with mAbs. Dead cells were stained with 0.05 µM YOYO-3 (Molecular Probes, Eugene, OR). The mAbs used were phycoerythrin-conjugated rat antimouse CD11b, biotin-conjugated rat antimouse F4/80 and B220, and fluorescein isothiocyanate-conjugated rat antimouse CD19 (Pharmingen).Immunohistochemical analysis Cells cytocentrifuged onto a slide were fixed in acetone and blocked with normal rabbit serum. The cells were incubated with rat antimouse CD3, CD4, CD5, CD8, B220, CD19, CD11b, Ter119 (Pharmingen), or F4/80 (Caltag, Burlingame, CA) mAb and then with a biotin-conjugated rabbit antirat Ig antibody (Dako, Denmark). Subsequently, the cells were incubated with streptavidin-horseradish peroxidase conjugate. Bound peroxidase was detected by using diaminobenzidine as a substrate.Nitroblue tetrazolium reduction assay Cells were incubated with 1 mg/mL nitroblue tetrazolium (NBT) and 100 ng/mL 12-O-tetradecanoyl phorbol 13-acetate for 1 hour at 37°C. After incubation, the proportion of formazan-positive cells was determined by examining the cells under a microscope.Measurement of phagocytic activity Cells were incubated with latex beads (diameter, 1.09 µm) for 2.5 hours at 37°C. After 4 washes with phosphate-buffered saline, the proportion of cells taking up the beads was determined by examining the cells under a microscope.
Identification of genes in MEL cells whose expression changed with PU.1-mediated growth inhibition, differentiation inhibition, and apoptosis Using an mRNA differential display strategy, we isolated 251 bands whose intensity was changed in one clone of MEL-B8/3 cells transfected with a zinc-inducible expression plasmid of the wild-type PU.1 gene (PU.1-1 cells) after overexpression of PU.1. Sequencing and a search of the Basic Local Alignment Search Tool database (National Center for Biotechnology Information) revealed that 100 of the 251 PCR products originated from known genes. Of these 100 genes, expression of 37 was up-regulated and that of 63 was down-regulated.Representative genes are listed in Table
2. The transcriptionally up-regulated
genes were, for instance, the CDC10 gene involved in
cytokinesis17 and hematopoietic cell-specific genes that are not normally expressed in the erythroid cell lineage. The transcriptionally down-regulated genes included, for instance, the
adenosine triphosphate synthase
Of particular interest was the up-regulation of expression of
hematopoietic cell-specific genes, including the osteopontin gene,
encoding for an extracellular adhesion protein thought to be involved
in monocyte maturation21,22; the eosinophil cationic protein gene, encoding for a toxic protein in the granules of eosinophils23; and the B144 gene, which is located in the
major histocompatibility complex class III region and abundantly
expressed in hematopoietic cells, macrophages, and B
cells.24,25 Northern blot analysis confirmed that
expression of these genes was induced in PU.1-1 cells treated with
ZnCl2 for 48 hours (Figure 1,
lane 11). In cells treated with both DMSO and ZnCl2, levels
of expression of these genes were lower than in cells treated with
ZnCl2 alone (Figure 1, lanes 11 and 12). This may have been
because of the reduction in cell viability resulting from apoptosis
induced under these conditions. Alternatively, it may be possible that
stimulation of DMSO, which triggers erythroid differentiation of MEL
cells, affected expression of these genes, although we previously
showed that erythroid differentiation of MEL cells induced by DMSO is inhibited by overexpression of PU.1.11 These results
suggested the interesting possibility that PU.1 could induce expression of genes specific for myeloid and lymphoid lineages in erythroid cells.
We therefore focused our study on assessing this possibility.
Induction of myelomonocyte-specific and B-cell-specific gene expression in MEL cells by overexpression of PU.1 Expression of several genes specific for myelomonocyte and B-cell lineages was examined by Northern blot and RT-PCR analyses in PU.1-1 cells treated with and without ZnCl2. As shown in Figure 2A, expression of the genes encoding for C/EBP-, C/EBP-, and C/EBP- transcription factors, which play
important roles in the development and maturation of myelomonocytes,
was up-regulated in the cells cultured for 48 hours with
ZnCl2 (lane 11). Similarly, levels of expression of the
genes encoding for receptors of myelomonocyte-specific growth factors
(the GM-CSFR, granulocyte colony-stimulating factor receptor
[G-CSFR], and macrophage colony-stimulating factor receptor [M-CSFR] genes), the myeloperoxidase (MPO) gene, and the CD18 gene
encoding for a chain of 2 family of integrins were higher in
PU.1-1 cells treated with ZnCl2 than in those of parental
MEL and mock cells treated with ZnCl2 (Figure 2A, lanes 3, 7, and 11).
The intensity of the bands obtained in the RT-PCR analysis was quantified, and the ratio of the value for PU.1-1 cells treated with ZnCl2 to the average value for parental and mock cells treated with ZnCl2 was calculated for each gene. According to the calculation, expression of the myelomonocyte-specific genes was up-regulated 1.7- to 22.4-fold in PU.1-1 cells compared with parental and mock cells (Figure 2B). The degree of expression of some genes was weaker in the cells cultured with DMSO and ZnCl2 (Figure 2A, lane 12) than in the cells cultured with ZnCl2 alone (Figure 2A, lane 11), possibly because of the reasons described above. The presence of detectable levels of expression of some genes in the parental and mock cells, as well as in the PU.1-1 cells cultured without ZnCl2, may have been due to the function of steady-state levels of PU.1 originally expressed in MEL cells. Changes in levels of expression of some genes in the parental and mock cells cultured in the presence of DMSO may have reflected erythroid differentiation of these cells. On the other hand, expression of the neutrophil elastase gene was not detected in the RT-PCR analysis (data not shown). Expression of the hematopoietic lineage switch 7 (HLS7) gene, which has been shown to be involved in cell-lineage conversion from erythroid to myeloid cells,26,27 and the mixed lineage leukemia (MLL, also known as acute lymphoblastic leukemia 1) gene, which is thought to be involved in monocyte differentiation,28 was not induced in PU.1-1 cells treated with ZnCl2 (lane 11). Expression of the gene encoding for the B-cell marker CD19 was
up-regulated in PU.1-1 cells cultured with ZnCl2 (Figure
2A, lane 11, and Figure 2B). Basal levels of expression of the
C/EBP- Induction of expression of myelomonocyte-specific and B-cell-specific genes is not peculiar to PU.1-1 cells because similar results were obtained in 2 additional independent clones of the transfectants, PU.1-2 and PU.1-3 cells, after overexpression of PU.1 (Figure 2C). Western blot analysis showed that the degree of induction of expression of the PU.1 protein was comparable in these clones after treatment with ZnCl2 (Figure 2D). Taken together, these results indicate that expression of a considerable number of myelomonocyte-specific and B-cell-specific genes is up-regulated in MEL cells after overexpression of PU.1. Induction of expression of myelomonocyte-specific proteins in MEL cells by overexpression of PU.1 The results described above prompted us to examine whether myelomonocyte-specific and B-cell-specific proteins are expressed in MEL cells after overexpression of PU.1. Flow cytometric analysis found that 70% to 80% of PU.1-1 cells treated with ZnCl2 expressed myelomonocyte-specific protein CD11b (Mac-1) on their surface. Moreover, 40% to 50% of CD11b-positive cells were also positive for another myelomonocyte-specific protein, F4/80 antigen (Figure 3, left). Similar results were obtained in studies using PU.1-2 cells; that is, about 50% of cells were positive for CD11b and 30% of CD11b-positive cells were also positive for F4/80 antigen (data not shown). These proteins were not detected on the surface of PU.1-1 and PU.1-2 cells cultured without ZnCl2 (data not shown) or mock cells cultured with or without ZnCl2 (Figure 3, right, and data not shown).
In contrast to the results regarding myelomonocyte-specific proteins, expression of the B-cell-specific proteins B220 and CD19 was not detected on the surface of PU.1-1 and PU.1-2 cells or mock cells cultured with or without ZnCl2 (Figure 3 and data not shown). Because expression of the CD19 gene was up-regulated in PU.1-1 cells after overexpression of PU.1, we used immunohistochemical analysis to assess whether B220 and CD19 proteins were present in the cytoplasm. Although CD11b and F4/80 antigen were detected in cells cultured with ZnCl2, neither of the B-cell-specific proteins was present (data not shown). These findings suggest that even though expression of B-cell-specific genes is induced by overexpression of PU.1 in MEL cells, this does not necessarily result in expression of the proteins. Morphologic and functional changes in MEL cells after overexpression of PU.1 We next examined the effects of overexpression of PU.1 on morphologic features and function of MEL cells by using PU.1-1 and PU.1-2 cells. These cells were originally spherical and grew in suspension. However, when treated with ZnCl2, some cells of both clones became spindle shaped and adherent by 48 hours (Figure 4A and data not shown). Such changes were not observed in the parental and mock cells treated with ZnCl2 (data not shown). Because expression levels of the genes encoding for some myelomonocyte-specific growth factor receptors were up-regulated in these cells treated with ZnCl2, we attempted to determine the effects of these factors on morphologic changes in the cells. As shown in Figure 4A, the number of adherent cells increased when granulocyte-macrophage colony-stimulating factor (GM-CSF; 1 ng/mL) was added to the culture along with ZnCl2. Similar results were obtained when macrophage colony-stimulating factor (M-CSF; 6 ng/mL) was added instead of GM-CSF (data not shown). These results suggest that the receptors are expressed on the cell surfaces and that they are functional.
Levels of NBT reduction activity were markedly increased in both PU.1-1 and PU.1-2 cells cultured with ZnCl2 for 48 hours compared with mock cells cultured under the same conditions (Figure 4B). Moreover, 11% to 14% of cells of both clones took up latex beads after treatment with ZnCl2 for 48 hours (Figure 4C and data not shown). No such activity was observed in cells cultured without ZnCl2 or mock cells cultured with or without ZnCl2 (data not shown). These results indicate that morphologic and functional changes are provoked in MEL cells as a result of the alteration in gene-expression profile after overexpression of PU.1. In summary, we found that PU.1 can reprogram MEL cells that have myelomonocytic characteristics. Activation domain of PU.1 is required for induction of myelomonocyte-specific and B-cell-specific gene expression in MEL cells In a previous study,11 we established a transfectant by introducing a zinc-inducible expression plasmid of a mutant version of PU.1 with a deletion of the glutamine-rich region of the activation domain (referred to as PU.1- A mutant) into MEL-B8/3 cells. Using this transfectant, we showed that induced expression of this mutant PU.1 in MEL cells inhibits erythroid differentiation of the cells, although it does not induce growth inhibition and
apoptosis.11 We therefore investigated whether expression
of myelomonocyte-specific and B-cell-specific genes is induced in the
transfectant when PU.1-A mutant is expressed.
As shown in Figure 5A and 5B, no
induction of expression was observed in any of the genes examined (the
osteopontin, eosinophil cationic protein, C/EBP-
In this study, we used an mRNA differential display strategy to identify PU.1 target genes involved in the inhibition of growth and erythroid differentiation and the induction of apoptosis in MEL cells. Some of the genes we identified are known to be involved in cell division and regulation of mitochondrial and ribosomal functions that might participate in growth inhibition or apoptosis. In addition, expression of 3 genes
PU.1 is known to bind to and activate the promoter of genes such as the
GM-CSFR,30 M-CSFR,31 G-CSFR,32
CD18,33 and CD11b.34 Ets-binding sites were
reported to be present in the promoter regions of the
osteopontin35 and C/EBP- It is remarkable that expression of the genes encoding for some C/EBP
transcription factors was up-regulated because C/EBP- The HLS7 gene was isolated from Raf-transformed and Myc-transformed erythroid cells that spontaneously acquired myeloid characteristics.26,27 The HLS7 gene was shown to be homologous to the human myeloid leukemia factor 1 gene and to inhibit differentiation of erythroid cells and induce differentiation of myeloid cells.27 The MLL gene is often rearranged by chromosomal translocations in human lymphocytic and myelomonocytic leukemias and is thought to play a role in the regulation of myelomonocyte differentiation.28 However, no increased expression was observed in these genes after overexpression of PU.1 in MEL cells. The results of this study suggest that the pathway through which PU.1 induces lineage switch in MEL cells is distinct from pathways used by HLS7 or MLL or that these 2 genes are not downstream from PU.1, even if they are in the same pathway as PU.1. Reflecting the induction of the gene-expression profile for myelomonocytes, morphologic and functional changes associated with macrophage-like phenotypes were produced in MEL cells. Induction of these changes appeared to depend on the levels of expression of PU.1, since no such changes were observed in PU.1 cell clones before induction of PU.1 overexpression or in mock cells, both of which originally expressed steady-state levels of PU.1 and detectable levels of some myelomonocyte-specific genes (Figure 2). Addition of GM-CSF or M-CSF to the culture medium increased the number of adherent cells. We also observed that addition of these growth factors at least partly, if not completely, rescued PU.1-1 cells from apoptosis induced by overexpression of PU.1 in the presence of DMSO (data not shown). These results suggest that the growth factor receptors induced to be expressed by overexpression of PU.1 are functional in MEL cells. In contrast to the morphologic findings, no apparent increase in NBT reduction activity or the proportion of phagocytic cells taking up latex beads was observed when GM-CSF or M-CSF was added (data not shown). These growth factors might not have any effects on these functions in MEL cells. In the flow cytometric and immunohistochemical analyses, induction of
expression of the myelomonocyte-specific proteins CD11b (Mac-1) and
F4/80 antigen was observed in PU.1 cell clones after overexpression of
PU.1, but there were no detectable levels of expression of
B-cell-specific B220 and CD19 proteins. Because induction of gene
expression was observed in the case of CD19, we suggest that
posttranscriptional modifications, including translation or
posttranslational modifications (or both) in these B-cell-specific proteins may not occur completely in the erythroid background. Expression of the Ig To address the possibility of whether loss of erythroid
characteristics was induced by the overexpression of PU.1 concomitant with the lineage switch in MEL cells, we determined levels of expression of the The experiments using PU.1- This study provides evidence that PU.1 induces a lineage switch in MEL cells, which have already been committed to the erythroid lineage, toward myelomonocytes. In line with this idea, Nerlov and Graf43 demonstrated that PU.1 induces myeloid lineage commitment in multipotent hematopoietic progenitor cells (MEPs) established by infecting chicken blastoderm cells with the Myb-Ets-encoding E26 leukemia virus. Taken together, these results indicate that PU.1 can impose myelomonocytic characteristics not only on hematopoietic progenitor cells but also on cells already committed to other lineages, thus suggesting that PU.1 acts as a master regulator to mediate lineage commitment of progenitors in hematopoiesis. Additionally, consistent with our observations, Nerlov and Graf43 also showed that the activation domain of PU.1 is required for myeloid lineage commitment of MEPs. Moreover, DeKoter and Singh44 found that a low concentration of PU.1 promotes B-cell differentiation, whereas a high concentration promotes macrophage differentiation in hematopoietic progenitors. Apart from PU.1, GATA-1 was shown to induce megakaryocyte and erythrocyte traits in M1 myeloid cells,45 and E2A was found to induce expression of B-cell-specific genes in macrophage-like cells.46 The findings of this study, along with the examples of lineage switch of hematopoietic cells induced by hematopoietic cell-specific transcription factors, provide evidence that transcription factors play fundamental roles in the commitment of hematopoietic cells and offer new insight into hematopoiesis in general.
We thank Dr Y. Hashimoto, the director of Sasaki Institute, for critical reading of the manuscript.
Submitted July 7, 2000; accepted December 21, 2000.
Supported by grants-in-aid for Scientific Research on Priority Areas and for Scientific Research (C) from the Ministry of Education, Science and Culture of Japan, and by the Uehara Memorial Foundation, Tokyo, Japan, and Obstetrics-Gynecology Akiyama Memorial Hospital, Hakodate, Japan.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Tsuneyuki Oikawa, Department of Cell Genetics, Sasaki Institute, 2-2, Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-0062, Japan; e-mail: oikawa{at}sasaki.or.jp.
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Top
Abstract
Introduction
), mock (
), and PU.1-1 (
)
cells treated with ZnCl2 are shown in the graphs. The
calculated ratio of the value for PU.1 cells to the average value for
parental and mock cells is shown in each graph. (C) Total RNA was
extracted from PU.1-2 cells (lanes 1 and 2) and PU.1-3 cells (lanes 3 and 4) cultured for 48 hours without reagents (lanes 1 and 3) or with
ZnCl2 (lanes 2 and 4). Expression of myelomonocyte-specific
and B-cell-specific genes was examined by RT-PCR analysis. (D) Total
protein was extracted from PU.1-1 cells (lanes 1 and 2), PU.1-2 cells
(lanes 3 and 4), and PU.1-3 cells (lanes 5 and 6) cultured for 8 hours
without reagents (lanes 1, 3, and 5) or with ZnCl2 (lanes
2, 4, and 6). Expression of PU.1 protein was examined by Western blot
analysis.
gene, which is highly expressed in early B
cells,
-chain and Igµ-chain genes was not detected in the RT-PCR
analysis in any cells cultured under any conditions (data not shown).
the osteopontin, eosinophil
cationic protein, and B144