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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 1934-1941
Cytoplasmic Domains of the Leukemia Inhibitory Factor Receptor
Required for STAT3 Activation, Differentiation, and Growth
Arrest of Myeloid Leukemic Cells
By
Mikio Tomida,
Toshio Heike, and
Takashi Yokota
From the Saitama Cancer Center Research Institure, Ina, Saitama,
Japan; and the Department of Stem Cell Regulation, Institute of Medical
Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.
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ABSTRACT |
Leukemia inhibitory factor (LIF) induces growth arrest and
macrophage differentiation of mouse myeloid leukemic cells through the
functional LIF receptor (LIFR), which comprises a heterodimeric complex
of the LIFR subunit and gp130. To identify the regions within the
cytoplasmic domain of LIFR that generate the signals for growth arrest,
macrophage differentiation, and STAT3 activation independently of
gp130, we constructed chimeric receptors by linking the transmembrane
and intracellular regions of mouse LIFR to the extracellular domains of
the human granulocyte macrophage colony-stimulating factor receptor
(hGM-CSFR)  and  c chains. Using the full-length cytoplasmic
domain and mutants with progressive C-terminal truncations or point
mutations, we show that the two membrane-distal tyrosines with the YXXQ
motif of LIFR are critical not only for STAT3 activation, but also for
growth arrest and differentiation of WEHI-3B D+ cells. A
truncated STAT3, which acts in a dominant negative manner was
introduced into WEHI-3B D+ cells expressing
GM-CSFR-LIFR and GM-CSFRc-LIFR. These cells were not
induced to differentiate by hGM-CSF. The results indicate that STAT3
plays essential roles in the signals for growth arrest and
differentiation mediated through LIFR.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
LEUKEMIA INHIBITORY factor
(LIF)/differentiation-stimulating factor (D-factor) is a
multifunctional cytokine that was initially identified as a factor that
inhibits the proliferation and induces macrophage differentiation of
the murine myeloid leukemic cell line, M1.1-4 WEHI-3B
D+ leukemic cells transfected with LIF receptor (LIFR) cDNA
are also induced to differentiate by LIF.5 LIF maintains
embryonic stem cells in an undifferentiated, pluripotent state,
enhances the synthesis of acute phase proteins by hepatocytes,
regulates nerve differentiation, and induces cardiac myocyte
hypertrophy.6-11 It is a member of the interleukin (IL)-6
type cytokine family. Receptors for these cytokines are composed of
multisubunit complexes that share a common signaling subunit, gp130.
IL-6 and IL-11 induce the homodimerization of gp130, while LIF,
cardiotrophin (CT)-1, ciliary neurotrophic factor (CNTF), and human
oncostatinM(OSM) induce gp130 heterodimer formation with the LIFR
subunit.12 The essential role of gp130 in the signaling by
these receptors is demonstrated by the fact that antibodies to gp130
are capable of neutralizing the cell responses to all of these
cytokines.12 Targeted disruption of the LIFR gene causes
placental, skeletal, neural, and metabolic defects, and thereby results
in perinatal death.13,14 However, the functional
significance of the cytoplasmic domain of LIFR is not well understood.
Baumann et al8 constructed chimeric receptors consisting of
the extracellular domain of the granulocyte colony-stimulating factor
receptor (G-CSFR) and the cytoplasmic domain of LIFR and showed that
the cytoplasmic domain of LIFR was capable of inducing gene expression
in hepatic cells when induced to form a homodimer. It was not known
whether or not the cell differentiation and proliferation signals were
generated through the cytoplasmic region of LIFR alone. We fused the
transmembrane and intracellular regions of LIFR to the extracellular
parts of the human granulocyte-macrophage colony-stimulating factor
receptor (GM-CSFR)  and c chains, respectively. GM-CSFR is
composed of a specific GM-CSFR chain and a common c subunit that
is shared with the receptors for IL-3 and IL-5.15 Using
these chimeric receptors, we show that a homodimer of the cytoplasmic
domain of LIFR can generate the signals for growth arrest and
differentiation in mouse WEHI-3B D+ and M1 myeloid leukemic cells.
Dimerization of the hematopoietin receptors initiates intracellular
signaling by activating members of the receptor-associated tyrosine
kinase family, referred to as Jaks.16 The information is
next relayed by a family of transcription factors known as STATs
(signal transducers and activators of transcription).17 Tyrosine phosphorylation of the STAT proteins is necessary for the
formation of STAT homo- or heterodimers and subsequent DNA binding.
IL-6 type cytokines preferentially activate STAT-3,
STAT-1,18 and STAT-5.19 Tyrosine residues
within receptors mediate signal transduction through the recruitment of
molecules containing either Src homology 2 domains or phosphotyrosine
binding domains.18 There are six tyrosine residues within
the LIFR cytoplasmic domain.20,21 In this study,
mutational analysis of LIFR showed the tight correlation between STAT3
activation and induction of differentiation of WEHI-3B D+
cells. Overexpression of dominant negative STAT3 blocks the
induction of growth arrest and differentiation mediated through LIFR.
| |
MATERIALS AND METHODS |
Cells and cell culture.
WEHI-3B D+ leukemic cells (kindly provided by Dr Alan C. Sartorelli, Yale University School of Medicine, New Haven,
CT)) were cultured in McCoy's 5A modified medium
(GIBCO-BRL, Grand Island, NY) supplemented with 10% fetal calf serum
(FCS). M1 cells were cultured as described previously.1,2
Cytokines.
Recombinant human GM-CSF was purchased from Peprotech, London, England.
Recombinant human IL-6 was kindly provided by Ajinomoto Co (Kawasaki,
Japan). Recombinant human D-factor (LIF) was produced in Chinese
hamster ovary cells and purified to homogeneity as described
previously.4
Plasmid construction.
For the construction of pCAG-hGM-CSFR -mLIFR and pMKIT-hGM-CSFR
c-mLIFR, the cDNA of mouse LIFR was cloned from ES cell line A3.1 by
reverse transcription-polymerase chain reaction
(RT-PCR).21,39 The PCR products were inserted into pSP72
(Promega, Madison, WI) at the Pvu II site and the
nucleotide sequence was confirmed using a 373A DNA sequencing system
(Perkin Elmer, Foster City, CA). The hGM-CSFR (pCEV4-hGMR) and
hGM-CSFRc (pME18S-KH97) cDNAs were kindly provided by Dr A. Miyajima
(University of Tokyo, Tokyo, Japan). All of these cDNAs were subcloned
into pCAG22 at the Xho I site, which was provided
by Dr J. Miyazaki (Osaka University, Osaka, Japan). The
chimeric receptor constructions, which have the extracellular domain of
hGM-CSFR or c linked to the transmembrane and cytoplasmic regions
of mLIFR, were generated by overlap extension using PCR.23
In brief, complementary oligonucleotide primers and PCR were used to
generate two DNA fragments with overlapping ends. In the case of
hGM-CSFR-mLIFR, the following two components were first generated by
PCR; the extracellular domain fragment of hGM-CSFR with the first 9 bp of the transmembrane domain of mLIFR and the
transmembrane/intracellular domain of mLIFR with the last 9 bp of the
extracellular domain of hGM-CSFR. These fragments were combined in a
subsequent fusion reaction, in which the overlapping ends were
annealed, allowing the 3' overlap of each strand to serve as a
primer for the 3' extension of the complementary strand. The
resulting fusion product was further amplified by PCR. This PCR product
was inserted into pSP72 and its nucleotide sequence was confirmed using
the 373A DNA sequencing system. Subsequently, the following three
fragments were prepared: the first part of pCAG with the first part of
hGM-CSFR, the chimeric fragment from the latter part of hGM-CSFR
plus the first part of the transmembrane/intracellular mLIFR, and the
latter part of transmembrane/intracellular mLIFR plus the latter part
of pCAG. These three fragments were ligated, resulting in completion of
the chimeric receptor under control of the CAG promoter. For
constructing pMKIT-hGM-CSFRc-LIFR, the fragment of hGM-CSFRc-LIFR
with the CAG promoter was isolated and ligated at the Bam HI
and Not I sites of pMKIT neo, which was kindly provided by Dr
K. Maruyama (Tokyo Medical and Dental University, Tokyo, Japan).
Mutant receptors with progressive C-terminal truncations or cytoplasmic
domain tyrosine  phenylalanine substitutions were constructed
(see Fig 2). Chimeric cDNAs were subcloned into the Xho I site
of pGEM-7Zf (Promega). C-terminal truncations were generated by PCR
using a 5'oligonucleotide (MDR1F:
5'-CTGTAAGGCGCTACAGTTTCAGAA-3') located upstream of the
unique Bgl II site in LIFR cDNA, and 3' oligonucleotides
introducing termination codons and Bst EII sites (T112:
5'-ATGGTGACCTACTGCACATCGATGTACACC-3'; T129:
5'-ATGGTGACCTACTCTGCTTTGGCTTGCGGC-3'; and T149:
5'-ATGGTGACCTAGGGAAGGCGCATCTGTGG-3'). Receptor fragments with tyrosine phenylalanine substitutions were generated by recombinant PCR24 using MDR1F and MDR5R
(5'-GACAAAGGGTGACCTGGTTA-3', which includes the normal
termination codon and a Bst EII site) as external
primers. The following oligonucleotides were used as internal primers:
F5F, 5'-GTGGCAGGCTTTAAGCCACAG-3'; F5R,
5'-CTGTGGCTTAAAGCCTGCCAC-3'; F4F,
5'-CAGTCCATGTTTCAGCCGCAA-3'; F4R,
5'-TTGCGGCTGAAACATGGACTG-3'; F3F,
5'-CAGGTGGTGTTCATCGATGTG-3'; F3R,
5'-CACATCGATGAACACCACCTG-3'; F6F,
5'-ACCGCCGGTTTCAGACCTCAG-3'; and F6R,
5'-CTGAGGTCTGAAACCGGCGGT-3'.
Construct F3/4/5/6 was generated by recombination between F3 plasmid
DNA and a fragment of F4/5/6 digested with Cla I and Bst
EII. Similarly, F4/5/6 was generated by recombination between F4
and F5/6 at the Bam HI and Bst EII sites. F5/6 was
generated by PCR using F5 plasmid DNA as a template, and F6F and F6R as primers. The nucleotide sequences of the fragments derived on the PCR
were confirmed by dideoxy sequencing using an ALF DNA sequencer
(Pharmacia, Uppsala, Sweden). The construct of dominant negative
STAT325 was kindly provided by Dr Alice L.-F.Mui (DNAX Research Institute, Palo Alto, CA).
Transfection of DNA.
DNA was introduced into cells by electroporation using a Gene Pulser
(Bio-Rad, Richmond, CA). The cells (107) were
suspended in 0.8 mL of HEPES-buffered saline (50 mmol/L HEPES, 137 mmol/L NaCl, 6.8 mmol/L KCl, 0.28 mmol/L Na2
HPO4, and 0.1% dextrose, pH 7.1) containing 25 µg each
of the expression plasmids for chimeric GM-CSFR and c. The cells
were exposed to a 450 V (WEHI-3B D+) or 400 V (M1) pulse
with a capacitance of 960 µF. After 2 days culture, the cells were
transferred to 96-well plates and then cultured in medium containing
G-418 at the final concentration of 1.8 mg/mL. Surface expression
of the transfected chimeric receptor genes were analyzed by flow
cytometry with an Epics XL (Coulter Electronics, Luton, UK), using
anti-GM-CSFR (S-20; Santa Cruz Biotechnology, Santa Cruz, CA) and
anti-GM-CSFR c (S-16; Santa Cruz Biotechnology).
Transfectants expressing W238 receptors were further transfected with
30 µg of pME18S  STAT3B together with 1 µg of pPUR (Clontech, Palo Alto, CA), which carries the puromycin-resistant
gene. The cells were selected in a medium containing 2 µg/mL
puromycin and 1.8 mg/mL G418. The C-terminal truncated STAT3
protein was immunoprecipitated using anti-STAT3 antibody K15 (directed
against amino acids 626-640), which was obtained from Santa Cruz Biotechnology.
Properties of differentiated cells.
The differentiation of M1 cells was assayed by measuring the induction
of phagocytic activity in the cells. M1 cells (3 × 105 cells/mL) were incubated with cytokines for 2 days. The
cells were harvested, suspended in serum-free medium containing 0.2% of a suspension of polystyrene latex particles (average diameter, 0.944 µm, Seradyn, Indianapolis, IN), and then incubated for 4 hours at
37°C. The cells were then thoroughly washed three times with
phosphate-buffered saline (PBS). Cells containing more than 10 latex
particles were scored as phagocytic cells.
The ability of cells to reduce nitroblue tetrazolium (NBT) was used as
a functional marker of the differentiation of WEHI-3B D+
cells. The cells (104 cells/mL) were incubated with
cytokines for 4 days. The cells were harvested, and the cell numbers
were determined using a Model ZM Coulter Counter. The cells were
suspended in 1 mL of serum-free medium containing 1 mg/mL of NBT and
100 ng/mL of 12-O-tetra decanoylphorbol 13-acetate, and then incubated
for 1 hour at 37°C. The reaction was stopped by adding HCl to the
final concentration of 1 mol/L. The suspension was
centrifuged, and the formazan deposits were solubilized by adding
dimethyl sulfoxide. The absorption of the formazan solution at 560 nm
was measured with a spectrophotometer.
Agar cultures of WEHI-3B D+ cells were performed in 35-mm
Petri dishes, as described previously.26 Briefly, 200 cells
were cultured in 1 mL of 0.3% agar (DIFCO Laboratories,
Detroit, MI) in Dulbecco's modified Eagle's medium
containing 20% FCS and various concentrations of hGM-CSF or IL-6 (100 ng/mL) as a positive inducer of differentiation. Colony numbers and
morphology were scored after 7 days incubation at 37°C
under a fully humidified atmosphere of 5% CO2 in
air. Aggregates of more than 50 cells were scored as colonies. Colonies
with a halo of dispersed cells were scored as differentiated.
Immunoprecipitation and Western blot analysis.
WEHI-3B D+ cells (107) were incubated with or
without hGM-CSF (100 ng/mL) for 10 minutes at 37°C. The reaction
was stopped by adding ice-cold PBS. The cells were harvested, washed
once with ice-cold PBS, and then lysed in 400 µL of lysis buffer (1%
Triton X-100, 150 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 7.4, 2 mmol/L
EDTA, 1 mmol/L Na3VO4, 1 mmol/L
phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, 10 µg/mL
pepstatin, 1 µg/mL aprotinin, and 100 µg/mL Pefabloc) for 5 minutes
on ice. Cell lysates were centrifuged for 5 minutes at 4°C at
14,000 rpm, and the resulting supernatants were incubated with
antibodies to STAT3 (C-20), STAT5b (C-17), SHP-2 (Santa Cruz
Biotechnology), or Shc (Transduction Laboratories, Lexington, KY) for 2 hours at 4°C. Protein A-Sepharose was added to the reaction
mixture, and the incubation was continued overnight at 4°C. The
beads were pelleted by centrifugation and then washed three times
with cold PBS. The immune complexes were eluted with 20 µL of Laemmli
sample buffer, resolved by 10% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE), and then transferred to Immobilon P
(Millipore, Bedford, MA). The membrane was incubated with
antiphosphotyrosine antibodies (4G10; Upstate Biotechnology, Lake
Placid, NY) for 1 hour. After washing, the membrane was incubated with
horseradish peroxidase-conjugated second-antibodies for 1 hour. The
immune complex was detected using an enhanced chemiluminescence system
(ECL; Amersham Life Science, Little Chalfont, UK). Blots
were stripped with 2% SDS, 100 mmol/L 2-mercaptothanol, and 62.5 mmol/L Tris-HCl, reblocked, and then reprobed with antibodies to STAT3
(Transduction Laboratories).
| |
RESULTS |
Signal transduction through the chimeric receptor carrying the
full-length cytoplasmic domain of LIFR in M1 and WEHI-3B
D+ cells.
Stimulation of M1 cells with LIF or IL-6 induces macrophage
differentiation and growth arrest.1,2,27-29 Although
wild-type WEHI-3B D+ cells respond to IL-6, but not LIF,
cells transfected with LIFR cDNA respond to both LIF and
IL-6.5 M1 and WEHI-3B D+ cells were transfected
with both the hGM-CSFR-LIFR and hGM-CSFRc-LIFR constructs, and
expression of these receptors was determined by flow cytometric
analysis (Fig 1). The differentiation of M1
cells was assessed by measuring the induction of phagocytic activity in
the cells, as the induction of phagocytic activity in the cells was
associated with the induction of other phenotypic markers of cell
differentiation.1 M1 cells expressing both the and c
chimeric receptors responded to hGM-CSF, as well as LIF and IL-6
(Table 1). The differentiation of WEHI-3B
D+ cells was assessed first by measuring the
ability of the cells to reduce NBT, a differentiation marker.
The cells expressing both the and c chimeric receptors exhibited
enhanced ability to reduce NBT on treatment with hGM-CSF, as well as
IL-6 (see W238 in Fig 3). Cell migration and growth arrest of the
transfected cells were also induced by treatment with hGM-CSF in a soft
agar culture (see W238 in Fig 4). These results suggest that
homodimerization of the LIFR cytoplasmic domain is sufficient to induce
macrophage differentiation of M1 and WEHI-3B D+ cells
because hGM-CSF cannot bind to mouse GM-CSFR.
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| Fig 1.
Flow cytometric analysis of chimeric receptor expression
in M1 transformants. M1 cells were transfected with GM-CSFR-LIFR and
GM-CSFR c-LIFR cDNA. The cells were incubated with
anti-GM-CSFR (B) or anti-GM-CSFR c (C). Antibody binding
was detected by incubation with fluorescein-conjugated rabbit
antimouse immunoglobulin. Samples were analyzed using a flow cytometer.
(A) The flow cytometry profile stained with the fluorescein-conjugated
second antibody alone.
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Cytoplasmic domains that are required for inducing
differentiation and growth arrest of WEHI-3B D+ cells.
To determine the receptor domains required for growth arrest and
differentiation, LIFR mutants, as shown in
Fig 2, were constructed. and c
chimeric receptor cDNAs carrying these LIFR mutations were
transfected into WEHI-3B D+ cells. hGM-CSF could induce
differentiation and growth arrest of the transformants expressing
truncated forms of the LIFR cytoplasmic domain containing at least 149 amino acid residues (Figs 3 and 4). More extensively truncated forms of
the LIFR (T129 and T119) were unable to generate signals for
differentiation and cell growth arrest. A normal differentiation
response to IL-6 was observed in WEHI-3B D+ cells
expressing each of the various mutant receptors.
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| Fig 2.
Deletion and point mutations in LIFR. The cytoplasmic
regions of the LIFR mutants are schematically shown. The transmembrane
domain (TM), putative box 1 (B1), box 2 (B2), and box 3 (B3), and the
positions of tyrosine (Y) and phenylalanine (F) residues are
indicated.
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| Fig 3.
Differentiation of WEHI-3B D+ cells
expressing LIFR mutants. Cells expressing various chimeric receptors
were treated for 4 days with 10 ng/mL of hGM-CSF or 100 ng/mL of IL-6.
The NBT-reducing activity of the cells was determined by the
colorimetric assay. The data for representative clones are presented as
percentages of the values for untreated control cultures. The values
represent the averages of duplicate assays ± standard error.
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| Fig 4.
Soft agar colony assaying of WEHI-3B D+
cells expressing LIFR mutants. Cells expressing the various
chimeric receptors (W238, T149, T129, T119, F3, F4, F5, or
F3/4/5/6) were cultured in agar with increasing concentrations of
hGM-CSF or IL-6 (100 ng/mL). The colony numbers (left panels)
and the proportions of colonies containing differentiated
cells (right panels) were determined after 7 days. The means
of duplicate assays are shown.
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The phosphorylation of tyrosine residues is considered to be a crucial
process in the initiation of signal transduction from the receptors
after ligand binding. LIFR has six tyrosine residues in its cytoplasmic
domain (Fig 2). The third tyrosine residue from the membrane-proximal
region is located in a YXXV motif, a known docking site for
SHP-2. Three tyrosine residues, ie, the fourth, fifth, and sixth ones,
are located in a YXXQ motif, a STAT3 consensus binding
sequence.18 We introduced a mutation into either the
third (F3), fourth (F4), or fifth (F5) tyrosine residue, or into four
tyrosine residues, ie, the third, fourth, fifth, and sixth ones
(F3/4/5/6). Mutants, F3, F4, and F5, but not F3/4/5/6, could
induce growth arrest and macrophage differentiation (Figs 3 and 4).
STAT3 activation in relation to growth arrest and differentiation.
LIF and related cytokines activate a common set of signalling molecules
including components of the JAK-STAT pathway and of the Ras-MAP kinase
signalling cascade.9,11,12 To assess the ability of the
receptor mutants to activate Shc and SHP-2 in the Ras-MAP kinase
pathway, and STAT3 and STAT5, Western blot analysis was performed on
lysates of stimulated and unstimulated WEHI-3B D+ cells.
Shc, SHP-2, and STAT5 were already phosphorylated in all cell lines
cultured in medium containing FCS. Stimulation with hGM-CSF did not
cause an increase in the level of tyrosine phosphorylation of these
proteins (data not shown). On the contrary, phosphorylation of STAT3
was not detected in unstimulated cells. Stimulation with hGM-CSF
induced the tyrosine phosphorylation of STAT3 in the transformants of
W238 , T149, F3, F4, and F5, but not in the transformants of T129,
T119, and F3/4/5/6 (Fig 5). The results
show that the tight correlation between STAT3 activation and induction
of differentiation and growth arrest of the cells.
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| Fig 5.
Tyrosine phosphorylation of STAT3 through the chimeric
receptors. Cells expressing various chimeric receptors were either
stimulated (+) with hGM-CSF (10 ng/mL) for 10 minutes or left
unstimulated (-). STAT3 was immunoprecipitated from cell lysates using
anti-STAT3 antibodies and then probed with either antiphosphotyrosine
antibodies (upper panel) or anti-STAT3 antibodies (lower panel). The
immune complex was visualized by chemiluminescence.
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Effect of dominant-negative STAT3 on growth arrest and
differentiation.
To examine the role of STAT3, a truncated STAT3 (STAT3), which lacks
the transactivation domain and acts as a dominant negative manner was
generated.25 The construct was transfected with a puromycin-resistance selection marker into WEHI-3B D+ cells
expressing W238 receptors. Transfectants that were resistant to both
puromycin and neomycin were isolated. The responsiveness of the cells
to IL-6 was first examined because dominant negative forms of STAT3
were shown to block the IL-6-mediated growth arrest and
differentiation of M1 cells.28,29 Several clones exhibiting different sensitivities to IL-6 were selected, and the expression of
truncated STAT3 in these clones was examined by immunoblot analysis.
The expression of truncated STAT3 correlated with a decrease in
sensitivity to IL-6. To examine the activation of STAT3, cells
were stimulated with IL-6 or hGM-CSF. In the cells overexpressing truncated STAT3 (Fig
6B), the tyrosine-phosphorylation of endogenous STAT3 in response to
IL-6 was reduced and the response to hGM-CSF was almost completely
blocked (Fig 6A). We examined the effect of expression of the mutant
STAT3 protein on the induction of differentiation and growth arrest of
WEHI-3B D+ cells. As shown in
Figs 7 and 8,
in the cells expressing dominant negative STAT3, the induction of
differentiation and growth arrest by hGM-CSF or IL-6 was dramatically
reduced relative to the control. These results suggest that STAT3 plays
an essential role in the signals mediated by the LIFR homodimer.
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| Fig 6.
Inhibition of tyrosine phosphorylation of STAT3 by
dominant negative STAT3. (A) WEHI-3B D+ cells expressing
the chimeric LIFR (W238) and  STAT3 or the chimeric LIFR alone
(PurrNeor) were stimulated with hGM-CSF (10 ng/mL) or IL-6 (100 ng/mL) for 10 minutes. STAT3 was immunoprecipitated
from cell lysates using anti-STAT3 antibodies (C20; Santa Cruz
Biotechnology), and then probed with either antiphosphotyrosine
antibodies or anti-STAT3 antibodies (Transduction Laboratories). (B)
Expression of STAT3 in WEHI-3B D+ cells. STAT3 was
immunoprecipitated from cell lysates using anti-STAT3 antibodies (K-15;
Santa Cruz Biotechnology) and then probed with anti-STAT3 antibodies
(Transduction Laboratories).
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| Fig 7.
Inhibition of differentiation of WEHI-3B D+
cells expressing LIFR by dominant negative STAT3. Cells expressing LIFR
(W238) and STAT3, or LIFR alone (PurrNeor)
were treated for 4 days with 10 ng/mL of hGM-CSF or 100 ng/mL of IL-6.
The NBT-reducing activity of the cells was determined by the
colorimetric assay. The data for representative clones are presented as
percentages of the values for untreated control cultures. The values
represent the averages of duplicate assays ± standard error.
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| Fig 8.
Soft agar colony assaying of WEHI-3B D+
cells expressing LIFR and dominant negative STAT3. Cells expressing
LIFR (W238) and STAT3, or LIFR alone
(PurrNeor) were cultured in agar with
increasing concentrations of hGM-CSF or IL-6 (100 ng/mL). The colony
numbers (left panels) and the proportions of colonies containing
differentiated cells (right panels) were determined after 7 days. The
means of duplicate assays are shown.
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| |
DISCUSSION |
LIFR is a member of the hematopoietin receptor family.20 It
is structurally most similar to gp130, G-CSFR, IL-12 receptor, OSM
receptor, and leptin receptor.12,30,31 Among these
receptors, gp130 and G-CSFR have been extensively studied as to the
cytoplasmic domains required for cell proliferation and
differentiation.27-29,32,33 While these receptor subunits
function as homodimers, LIFR functions as a heterodimer with gp130. To
examine signal transduction by LIF, we fused the cytoplasmic domain of
mouse LIFR or gp130 to the extracellular domain of the human GM-CSFR
and c chains, respectively. By transfection of different
combinations of chimeric receptors into mouse cells, we analyzed the
signals of not only homodimers, but also heterodimers of LIFR and
gp130.39
In the present study, we transfected M1 and WEHI-3B D+
cells with GM-CSFR-LIFR and GM-CSFRc-LIFR cDNA and found that
hGM-CSF induced macrophage differentiation and growth arrest of these leukemic cells, suggesting that the cytoplasmic domain of LIFR is
capable of signal transduction when it is induced to form a homodimer.
Therefore, mutants of these chimeric receptors permit analysis of the
functional domain of the LIFR cytoplasmic domain in the absence of
gp130. We identified the first 149 amino acid residues of the
cytoplasmic domain of LIFR as the minimal region necessary for growth
arrest and differentiation of WEHI-3B D+ cells. The amino
acid 136-145 region, called box 3, is conserved among gp130, LIFR and
G-CSFR and contains the fifth tyrosine residue and a YXXQ motif. The
tyrosine residue in the motif was shown to play a role in activating
STAT3.18 The fourth and sixth tyrosine residues are also
contained in the YXXQ motif.21 However, the truncated
mutant, T129, containing the fourth tyrosine residue and a YXXQ motif,
neither activated STAT3 nor induced the differentiation of WEHI-3B
D+ cells. The mutant, T149, with the F5 mutation was also
inactive (data not shown). Therefore, the sixth tyrosine residue may
substitute for the fifth tyrosine residue in activating STAT3 and the
generation of the signals for growth arrest and differentiation because
the F5 mutation in the full-length cytoplasmic domain did not affect the function of the receptor (Figs 3 and 4).
Kuropatwinski et al19 and Stahl et al18 fused
the cytoplasmic domain of LIFR and the extracellular domains of G-CSFR
and the epidermal growth factor receptor, respectively. These chimeric receptors formed homodimers after binding to ligands. They showed that
similar constructs to our T129 mutant could activate STAT3 in COS-1 or
COS-7 cells. The difference between their and our present results seems
to originate from the difference between the lineages of the cells used.
Our present study showed that activation of STAT3 is tightly correlated
with the signals for growth arrest and macrophage differentiation, and
that a dominant negative form of STAT3 inhibited both LIFR-mediated
growth arrest and macrophage differentiation of the WEHI-3B
D+ transformants. These results are consistent with the
results obtained by Nakajima et al28 and Minami et
al.29 They showed that activation of STAT3 is essential for
gp130-mediated growth arrest and differentiation of M1 cells. On the
other hand, Alexander et al34 showed that activation of
STAT3 DNA binding was insufficient for c-Mpl (thrombopoietin
receptor)-mediated WEHI-3B D+ differentiation. They
suggested that activation of components of the Ras signalling cascade,
initiated by interaction of Shc with c-Mpl, plays a decisive role in
the differentiation signals. Therefore, we examined Shc phosphorylation
in our WEHI-3B D+ cells. In contrast to their results, Shc
was phosphorylated in the cells cultured in normal medium containing
FCS. Treatment of the cells with hGM-CSF did not cause a further
increase in phosphorylation. Nicholson et al33 analyzed
tyrosine residues in G-CSFR that mediate the induction of
differentiation of M1 cells. They showed that STAT3 was activated by
G-CSF in the cells expressing G-CSFR tyrosine mutants unable to mediate
G-CSF-induced differentiation. Therefore, activation of STAT3 is not
sufficient for G-CSF-mediated M1 differentiation. Tyrosine
phosphorylation of Shc was not required for the differentiation of M1
cells. All of these results, including ours, suggest that activation of
STAT3 is required, although not sufficient, for induction of the
differentiation of myeloid leukemic cells such as WEHI-3B
D+ and M1. Multiple signaling pathways through LIF in
various cells have been reported.35-37
Recently, Starr et al38 reported that homodimers of the
LIFR cytoplasmic domain were able to deliver the signal that blocks differentiation of ES cells, although they were not sufficient for
transducing a differentiation signal in M1 cells nor for induction of a
proliferation signal in Ba/F3 cells. We also examined the effects of
our chimeric receptors on ES and Ba/F3 cells.39 Homodimers of the LIFR cytoplasmic domain could not support the proliferation of
these cells. The reason for the difference between our results and
theirs is not known, but clonal variation of ES and M1 cells and
differences in the selection of transfectants may be possible explanations. In general, LIFR homodimers may be less efficient than
heterodimers of LIFR and gp130 in signaling biological responses. However, in the case of WEHI-3B D+ cells, LIFR homodimers
were equivalent in activity to heterodimers of LIFR and gp130. The
response to hGM-CSF of WEHI-3BD+ cells transfected with the
present chimeric receptors was very similar to the response to LIF of
WEHI-3B D+ cells transfected with the full-length LIFR,
which formed a heteromeric complex with endogenous gp130.5
We showed that the cytoplasmic domain of LIFR in the absence of gp130
could generate the signals for growth arrest and differentiation of
myeloid leukemic cells. It is possible that an as yet undiscovered cytokine could induce downstream signaling through homodimerization of
LIFR in some cell types. No signaling pathway specific to LIFR has been
identified. Our chimeric receptors will be useful for elucidating the
different signaling potentials of LIFR versus gp130.
| |
ACKNOWLEDGMENT |
We thank T. Higashihara for her technical assistance and Dr T. Matsuda
for the helpful discussions.
| |
FOOTNOTES |
Submitted July 8, 1998; accepted November 5, 1998.
Supported in part by a Grant-in-Aid for Scientific Research on Priority
Areas from the Ministry of Education, Science, Sports and Culture of Japan.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Mikio Tomida, PhD, Saitama Cancer Center
Research Institute, Ina, Saitama 362-0806, Japan.
| |
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ABSTRACT
INTRODUCTION