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Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1264-1273
HEMATOPOIESIS
From the Departments of Hematology/Oncology, Internal Medicine II,
Molecular Oncology, Biomedical Research Center, Osaka University
Medical School, Osaka, Japan; the Department of Immunology, Osaka City
University Medical School, Japan; and the Center for TARA and Institute
of Basic Medical Institute, University of Tsukuba, Tsukuba, Japan.
Cytokines exert pleiotropic effects on target cells in a manner
dependent on the cell type or stage of differentiation. To determine
how instinctive cell properties affect biological effects of cytokine,
we introduced an erythroid/megakaryocyte lineage-specific transcription
factor, GATA-1, into a murine myeloid cell line M1, which is known to
undergo macrophage differentiation in response to interleukin 6 (IL-6).
Overexpression of GATA-1 changed the phenotype of M1 cells from myeloid
to megakaryocytic lineage. Furthermore, GATA-1 blocked both
IL-6-induced macrophage differentiation and apoptosis of M1 cells.
Although STAT3 is essential for IL-6-induced macrophage differentiation
of M1 cells, GATA-1 had little or no effect on tyrosine
phosphorylation, DNA binding, and transcriptional activities of STAT3
in Western blot analysis, electropholic mobility shift
assay (EMSA), and luciferase assays. During IL-6-induced macrophage
differentiation of M1 cells, IL-6 down-regulated cyclin D1 expression
and induced p19INK4D expression, leading to reduction in
cdk4 activities. In contrast, sustained expression of cyclin D1 and a
significantly lesser amount of p19INK4D induction were
observed in IL-6-treated M1 cells overexpressing GATA-1. Furthermore,
although bcl-2 expression was severely reduced by IL-6 in M1 cells, it
was sustained in GATA-1-introduced M1 cells during the culture with
IL-6. Both IL-6-induced macrophage differentiation and apoptosis were
significantly abrogated by coexpression of cyclin D1 and bcl-2, whereas
overexpressions of cyclin D1 or bcl-2 inhibited only differentiation or
apoptosis, respectively. These results suggested that GATA-1 may not
only reprogram the lineage phenotype of M1 cells but also disrupt the biologic effects of IL-6 through the sustained expression of cyclin D1
and bcl-2.
(Blood. 2000;95:1264-1273)
Growth, differentiation, and survival of hematopoietic
cells are regulated by a number of cytokines and growth factors that activate multiple signal transduction pathways through binding to their
cognate receptors. Among various hematopoietic growth factors,
interleukin 6 (IL-6) exerts pleiotropic effects on hematopoietic cells
(for reviews, see 1 and 2). IL-6 has been
shown, for example, to augment growth of multipotential hematopoietic
progenitor cells and plasmacytoma/myeloma cells.3-5 IL-6
has also been implicated in differentiation of various cell types,
including normal B cells, megakaryocytes, and myeloid cell lines M1,
Y6, and 1A9-M.6-10
The IL-6 receptor is a heterodimeric complex, consisting of an IL-6
specific ligand-binding subunit, It has become increasing apparent that transcription factors play a key
role in hematopoiesis. Transcription factors of the GATA-family are
composed of six members and are essential for the development and
subsequent growth and differentiation of diverse cell types (for a
review, see 17). The first cloned member of this family,
GATA-1, was identified as an erythroid nuclear protein that binds to
consensus GATA motifs, (A/T)GATA(A/G), in globin gene promoters,
enhancers, and locus control regions (LCRs).18,19 It has
been shown that GATA-1 is expressed at high levels in erythroid cells,
megakaryocytes, and mast cells and at lower levels in hematopoietic progenitor cells and Sertoli cells of testis.20-22 In
contrast, GATA-2 is ubiquitously expressed, and GATA-3 is exclusively
expressed in T lymphocytes.23 The functional roles of
GATA-1 in the hematopoietic system have been elucidated by
gene-targeting experiments. In chimeric mice generated by mutant
embryonic stem (ES) cells lacking GATA-1, the mutant cells did not
contribute to erythropoiesis.24 In addition, GATA-1-null ES
cells were unable to differentiate into mature erythroid cells in
vitro.25,26 Thus, GATA-1 has been implicated in regulating
terminal differentiation of erythroid progenitor cells. Recently,
Shivdasani et al27 reported that lineage-selective GATA-1
knock out mice exhibited striking thrombocytopenia as well as severe
anemia because of the decreased proliferation and impaired cytoplasmic
maturation of megakaryocytes. Furthermore, Takahashi et
al28 demonstrated that heterozygous mutant mice chimeric
for GATA-1 gene expression displayed marked splenomegaly, anemia, and
thrombocytopenia because of progenitor proliferation in the spleen and
consequently differentiation arrest of immature erythroid and
megakaryocytic cells. Those results suggest that GATA-1 plays
essential roles not only in erythropoiesis but also in
megakaryopoiesis or thrombopoiesis.
Previous studies have also shown that GATA-1 is unique in its ability
to influence the lineage phenotype of hematopoietic cells. Enforced
expression of GATA-1 reprograms transformed chicken myeloblasts into
erythroblasts, thromboblasts, or eosinophiles.29 Moreover,
overexpression of GATA-1 in the early myeloid 416B cells provoked
megakaryocytic differentiation accompanied by a marked decrease in
myeloid surface phenotype.30 Recently, Yamaguchi et
al31 reported that forced GATA-1 expression in the murine myeloid M1 cells, which differentiate into macrophage and undergo apoptosis in response to IL-6, led to megakaryocytic or erythroid differentiation. Those findings led us to speculate that GATA-1 might
also affect cytokine-mediated signaling pathways that are involved in
gene transcription necessary for growth, differentiation, and survival
of hematopoietic stem or progenitor cells. In this study, therefore, we
introduced GATA-1 into murine myeloid M1 cells and examined the effect
of GATA-1 on IL-6-induced macrophage differentiation and
apoptosis of the cells. Over-expression of GATA-1 was found to
block IL-6-induced macrophage differentiation and apoptosis of M1 cells
without affecting the STAT3 pathway. The effects of GATA-1 were
mediated, at least partially, through the sustained expression of
cyclin D1 and bcl-2. Thus, we here provide evidence that GATA-1 is
capable of modulating biological activities of IL-6 through regulating
specific members of cell cycle and apoptosis regulating molecules.
Reagents and antibodies
Plasmid construct and complementary DNAs
Cell lines and cultures M1, a murine myeloid leukemia cell line originally established by Ichikawa32 was cultured in RPMI (Nakarai Tesq, Kyoto, Japan) supplemented with 10% fetal calf serum (FCS) (Flow, North Ryde, Australia). HepG2 and NIH3T3 cells were cultured in DMEM supplemented with 10% FCS.Morphological analysis The morphological characteristics of cultured cells were determined by staining the cytospin preparations (Shandon, Pittsburgh, PA) with May-Grünwald-Giemsa.Flow cytometry The surface phenotypes of cells were examined with the indirect immunofluorescent method by using rabbit anti-c-Mpl antiserum, anti-CD61, anti-CD32, or anti-F4/80 mAb as previously reported.33 The cell cycle analysis of cultured cells was performed by staining with propidium iodide as previously described.34Northern blot analysis The isolation of total cellular RNA and the method for Northern blot were described previously.35Immunoprecipitation and immunoblotting The isolation of total cellular lysates, immunoprecipitation, gel electrophoresis, and immunoblotting were performed according to the methods described previously.36 Immunoreactive proteins were visualized with the enhanced chemiluminescence detection system (DuPont NEN, Boston, MA).Metabolic labeling and measurement of protein turnover To examine the half-lives of cyclin D1 and bcl-2 proteins, 1 × 107 cells for each sample were radiolabeled with 200 µCi of 35S]methionine in 1 mL of methionine-free DMEM for 30 minutes or 6 hours. The cells were then washed and resuspended in DMEM containing 2 mmol/L of unlabeled methionine. After the culture with or without IL-6, total cellular lysates were prepared at the time indicated. Cyclin D1 and bcl-2 were immunoprecipitated from the lysates and subjected to SDS-PAGE, respectively. The gels were dried and subjected to the autoradiography. The radioactivities of the bands corresponding to cyclin D1 or bcl-2 protein were measured by a densitometric analysis.Preparation of M1 clones expressing GATA-1, cyclin D1, bcl-2, or a combination M1 cells were transfected with 30 µg of pcDNA3-GATA-1, pcDNA3-cyclin D1, pcDNA3-bcl-2, or an empty pcDNA3 by electroporation (250 V, 960 µFD) (Bio-Lad Laboratory, Richmond, CA). The transfected cells were screened by the culture with 1.2 mg/mL of G418 (Sigma). Of several G418-resistant clones, expression levels of each transgene were examined by Northern blot and Western blot analyses. To prepare a stable transformant designated M1-W, in which both cyclin D1 and bcl-2 are overexpressed, cyclin D1-transfected M1 (M1-D1) was further cotransfected with 50 µg of pcDNA3-bcl-2 and 5 µg of pSV2bsr, an expression vector of blasticidin S deaminase (Kaken Pharmaceutical Co, Tokyo, Japan) and screened by the culture with 30 µg/mL of blasticidin S hydrochloride (Funakoshi, Tokyo, Japan).Luciferase assays Luciferase assays were performed with a reporter gene for STAT3, named 4 × APRE-Luc.13 NIH3T3 or HepG2 cells were transfected with various amounts of an effector gene of pcDNA3-GATA-1 or pCAGGS-neo-HA-STAT3D along with 1 µg of a 4 × APRE-Luc reporter gene and 10 ng of pRL-CMV-Rluc, an expression vector of renilla luciferase, by calcium phosphate coprecipitation method. Total amounts of DNA for each transfection were equalized by the addition of an empty pcDNA3 or pCAGGS. After 12 hours of culture, the cells were washed, serum starved for 24 hours, and then stimulated with 20 ng/mL of rhIL-6 for 5 hours. Luciferase assays were performed by using the Dual-Luciferase Reporter System (Promega, Madison, WI) in which relative firefly luciferase activities were calculated by normalizing transfection efficiency according to the renilla luciferase activities.cdk4-associated GST-Rb kinase assay In vitro cdk4-associated GST-Rb (Rb: retinoblastoma protein) kinase assay was performed as previously described.37 Briefly, cdk4 was immunoprecipitated from equal amounts of cell lysates prepared from cultured cells. Immune complex kinase assay was performed in kinase buffer containing 5 µg of GST-Rb fusion protein (Santa Cruz) and 20 µCi of -32P]ATP for 30 minutes at
30°C. After the addition of protein loading buffer,
samples were boiled and subjected to SDS-PAGE. The gels were stained
with Coomassie blue to confirm the amounts of immunoprecipitates, then
destained, dried, and subjected to autoradiography.
Electropholic mobility shift assay (EMSA) The isolation of nuclear extracts was performed as previously described.37 A double-stranded oligonucleotide containing STAT3-binding sequence (APRE) was synthesized and used as a probe or a competitor (5'-AGCTTCCTTCTGGGAATTCCT-3', APRE sequence is underlined).38 Also, one more double-stranded oligonucleotide, which contains mutated APRE sequence, was used as a competitor (5'-AGCTTCCTGCTGGGACTTCCT-3', mutated recognition site is underlined). Nuclear extract (15 µg of each sample) was incubated in 20 µL of binding buffer containing 2 µg of poly (dI-dC) (Pharmacia) and labeled probe (30 000 cpm) for 20 minutes at 4°C. The reaction mixture was loaded onto 4% polyacrylamide gel, electrophoresed, dried, and subjected to autoradiography.
Preparation and characterization of GATA-1introduced M1 cells We initially introduced an expression vector of GATA-1 into M1 cells. After the selection with G418, expression of GATA-1 transgene was examined by Northern blot analysis. As shown in Figure 1A, GATA-1 mRNA was hardly detected in a control clone (M1-V1) transfected with an empty vector. In contrast, expression of GATA-1 mRNA was observed in GATA-1-transfectants (M1-G1, M1-G2, M1-G3, and M1-G4) at high levels almost similar to that in a human erythroleukemia cell line (HEL), whereas GATA-1 transgene showed more slowly migrating bands. In addition, Western blot analysis showed that comparable amounts of GATA-1 proteins were expressed in M1-G1, M1-G2, M1-G3, M1-G4, and HEL cells, but not in M1-V1 cells (Figure 1A). Because the ectopic overexpression of GATA-1 was reported to induce erythroid or megakaryocytic differentiation, or both,29-31 we examined the effects of GATA-1 on surface expression of two megakaryocytic lineage markers, TPO receptor (c-Mpl) and CD61 (GPIIb/IIIa). As shown in Figure 1B, although c-Mpl and CD61 were scarcely expressed on M1-V1, a weak but easily detectable level of c-Mpl and CD61 expression was observed on M1-G1 and M1-G2. These results suggested that ectopic overexpression of GATA-1 reprogrammed M1 cells, to some degree, toward megakaryocytic lineage.
Effect of GATA-1 on IL-6-induced growth arrest and subsequent macrophage differentiation of M1 cells Next, we examined the growth and differentiation of M1-V1 and M1-G1 in the presence or absence of rhIL-6 (Figure 2). Without the addition of rhIL-6, no significant difference was seen between growth curves of M1-G1 and M1-V1 (Figure 2A). However, when cultured with rhIL-6, M1-V1 ceased to grow after 3 days in the same way as parental M1, whereas M1-G1 showed continuous growth during the 4-day culture. Similar results were observed in other GATA-1-transfected clones, M1-G2, M1-G3, and M1-G4 (data not shown).
Effect of GATA-1 on IL-6 signaling Because IL-6 failed to exert biologic effects on M1-G1 and M1-G2, we examined as to which signaling pathway(s) of IL-6 was perturbed by GATA-1. To first determine whether IL-6 signaling could be transduced into the nucleus, we investigated the induction of IL-6-responsive immediate early genes, TIS11 and jun B,39 in M1-V1, M1-G1, and M1-G2. As shown in Figure 3A, the treatment with rhIL-6 resulted in rapid induction of Tis11 and Jun B mRNA in M1-V1, M1-G1, and M1-G2 at a comparable level, suggesting that IL-6 signaling to the nucleus was not disrupted in GATA-1 transfectants. We next examined tyrosine phosphorylation of STAT3 before and after treatment with rhIL-6, because STAT3 was reported to be essential for IL-6-induced macrophage differentiation of M1.12-14 As shown in Figure 3B, rhIL-6 was capable of inducing tyrosine phosphorylation of STAT3 in M1-G1, M1-G2, and M1-V1 in a similar fashion.
Cell cycle analysis on M1-V1 and M1-G1 cells during the culture
with rhIL-6
Expression of cell cycle regulatory and apoptosis-related molecules in M1-V1 and M1-G1 cells during the culture with IL-6 To clarify the mechanism underlying the differential biological responses of M1-V1 and M1-G1 to rhIL-6, we examined the expression of cell cycle regulatory molecules, including cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors, and apoptosis-related genes during 72-hour culture with rhIL-6. As shown in Figure 5A, Northern blot analysis showed that expression of cyclin D1 mRNA decreased gradually to an undetectable level after 24 hours in M1-V1, whereas it was up-regulated at 4 hours and retained at a detectable level throughout the culture in M1-G1. The expression of p19INK4D mRNA was up-regulated by rhIL-6 in M1-V1 and, to a lesser extent, in M1-G1 from 4 hours to 24 hours. By contrast, the expression levels of cyclin D2, cyclin D3, p18INK4C, and p27Kip1 mRNA did not show apparent difference between M1-V1 and M1-G1. Expression of p21WAF1 was not detectable in both clones during the test period. With reference to apoptosis-related genes, expression of bcl-2 mRNA was down-regulated markedly in M1-V1 after 12 hours, whereas it was detected constantly for up to 72 hours in M1-G1. However, an apparent difference was not observed in expression levels of bax, bak, or bcl-xL mRNA in M1-V1 and M1-G1. In addition to mRNA levels, Western blot analysis demonstrated that cyclin D1 and bcl-2 proteins were expressed in M1-G1 during 72-hour culture with rhIL-6, whereas their expression was severely reduced in M1-V1 at 72 hours (Figure 5B upper panel and Figure 5C). Because D-type cyclins and the INK4 family of cyclin-dependent kinase inhibitors have been reported to complex with cdk4 and to regulate its activities, we examined the changes in cdk4 activities with an immune complex kinase assay by using GST-Rb as a substrate (Figure 5B, lower panel). Although expression of cyclin-dependent D2 and cyclin D3 was retained in M1-V1 during 72-hour culture with rhIL-6, cdk4 activities were gradually reduced in M1-V1, possibly owing to the decreased expression of cyclin D1 and to the induction of p19INK4D. By contrast, cdk4 activities were sustained at an almost constant level for up to 72 hours in M1-G1. These results raised the possibility that GATA-1 might protect M1 cells from IL-6-induced cell cycle arrest and apoptosis through the sustained expression of cyclin D1 and bcl-2.
Sustained expression of cyclin D1 and bcl-2 proteins during IL-6 treatment in M1-G1 predominantly regulated at the transcriptional level We next investigated the mechanisms by which expression of cyclin D1 and bcl-2 was regulated in M1-V1 and M1-G1. At first, we examined changes in expression levels of cyclin D1 and bcl-2 mRNA in the presence of RNA synthesis inhibitor, actinomycin D. M1-V1 and M1-G1 cells were pretreated with 10 µg/mL of actinomycin D for 2 hours and then subjected to the cultures with or without rhIL-6 in the presence of actinomycin D. As shown in Figure 6A, the kinetics of cyclin D1 and bcl-2 mRNA disappearance was not affected by rhIL-6 in M1-V1 and M1-G1, and a significant difference was not detected between M1-V1 and M1-G1 (Figure 6A). These results suggested that the stability of cyclin D1 and bcl-2 mRNA was almost the same in M1-V1 and M1-G1 and that rhIL-6 treatment shows little or no effect on their stability. Therefore, we speculated that sustained expression of cyclin D1 and bcl-2 mRNA in M1-G1 during rhIL-6 treatment may result from the continued transcription of these genes but not from the stabilization of these mRNAs. Next, we assessed the half-life of cyclin D1 and bcl-2 proteins in M1-V1 and M1-G1 during the culture with or without rhIL-6. M1-V1 and M1-G1 cells were pulsed with 35S-methionin, and changes in expression levels of 35S-labeled cyclin D1 and bcl-2 proteins were examined. Degradation of cyclin D1 was very rapid and was not affected by rhIL-6 in M1-V1 and M1-G1, and an apparent difference in the kinetics was not observed between M1-V1 and M1-G1 (Figure 6B). Also, bcl-2 protein was degraded in M1-V1 and M1-G1 in a similar time course regardless of the treatment with rhIL-6 (Figure 6C). These results suggested that the stability of cyclin D1 and bcl-2 proteins was almost identical in M1-V1 and M1-G1, respectively, and that rhIL-6 treatment hardly affects their stability in these clones. Thus, it was assumed that the continued expression of cyclin D1 and bcl-2 proteins was due to the sustained expression of their mRNA but not their stabilization.
Effects of cyclin D1 or bcl-2 overexpression or both on IL-6-induced macrophage differentiation and apoptosis To examine the roles of cyclin D1 and bcl-2 in rhIL-6-induced differentiation and apoptosis, we introduced expression vectors of cyclin D1, bcl-2, or both into M1; M1 clones expressing cyclin D1, bcl-2, and both vectors were designated M1-D1, M1-bcl-2, and M1-W, respectively. As shown by Northern blot analysis (Figure 7A), the expression of cyclin D1, bcl-2, or both transgenes was more abundant than that of endogenous genes in each transfectant. Furthermore, Western blot analysis showed more abundant expression of the transgene products in each transfectant (data not shown). After culture with or without rhIL-6, growth potential of each clone was assessed by counting viable cells (Figure 7B). In the absence of rhIL-6, all clones showed consistent growth, although M1-D1 and M1-W grew slightly faster than M1-V1 and M1-bcl-2 (Figure 7B, left panel). When rhIL-6 was added to the culture medium, however, viable cell number of M1-V1 or M1-bcl-2 did not increase and that of M1-D1 decreased significantly after 1-2 days (Figure 7B, right panel). By contrast, M1-W showed continuous cell growth for 4 days in the presence of rhIL-6, although its growth was rather slow as compared with that of M1-G1 (Figure 2A right panel vs Figure 7B right panel).
During the past decade, a number of growth factors have been
identified, and their roles in hematopoiesis have been elucidated. In
those studies, a single cytokine was shown to reveal pleiotropic effects dependently on the target cell types. However, it is not well
understood as to which instinctive cell properties are associated with
biological effects of each cytokine. By using a murine M1 myeloid cell
line that is a suitable model for elucidating cytokine-induced cell
differentiation accompanied by growth arrest, we investigated the
effects of GATA-1 on IL-6-induced macrophage differentiation and apoptosis.
Submitted June 1, 1999; accepted October 7, 1999.
Supported in part by grants from the Japanese Ministry of
Education, Science, Sports and Culture; the Japanese Ministry of Health
and Welfare; Senri Life Science Foundation; Uehara Memorial Foundation;
Naito Foundation; and the Japan Medical Association.
Reprints: Itaru Matsumura, Department of Hematology and
Oncology, Osaka University Medical School, 2-2, Yamada-oka, Suita,
Osaka 565, Japan; e-mail: matumura{at}bldon.med.osaka-u.ac.jp.
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.
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Abstract
Introduction
chain, and a signal-transducing subunit, gp130. Structural analysis has revealed that both subunits belong to the cytokine receptor superfamily, and gp130 is shared by the
receptors for ciliary neurotrophic factor, leukemia inhibitory factor,
oncostatin M, and cardiotropin 1. The binding of IL-6 to 
C 21), and anti-cdk4 (C-22-G)
polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc.
(Santa Cruz, CA). Anti-cyclin D1 mAb was purchased from Medical
Biological Laboratories (MBL) Company Ltd (Nagoya, Japan).
) with a reference to rabbit preimmune serum or control antibody of
the same isotype (---).
nwald-Giemsa
(magnification × 100). (C) Flow cytometric analyses on
expression of F4/80 and CD32 before and after 72-hour culture with
rhIL-6. Expression of F4/80 and CD32 in the cultured cells was examined
by staining with anti-F4/80 or anti-CD32 monoclonal antibody (
2 (IFN
