I. Introduction
Recent advances in stem cell biology, together with
gene transfer technology, have led to the prospect of a new generation of cell
therapy. However, many obstacles must be overcome before this vision becomes a
reality. One major hurdle is to control transplanted cells in the recipientÕs
body, in particular, to expand the desired cell subsets so that they exhibit
therapeutic benefit. We have developed a novel system for selective expansion
of genetically modified cells to supplement current gene transfer vectors (Ito
et al, 1997; Kume et al, 2002). In this system, the target cells are harnessed
with a Ôselective amplifier gene (SAG)Õ which encodes a fusion protein
comprising the granulocyte colony-stimulating factor (G-CSF) receptor (GcR) and
the hormone binding domain (HBD) of the estrogen receptor (ER). The ER-HBD
works as a molecular switch so that the fusion protein generates a GcR-derived
growth signal upon binding to estrogen (Mattioni et al, 1994). Besides the
prototype SAG encoding a chimera of the full-length GcR and ER-HBD (GcRER), a
series of derivative fusion receptors were constructed to attain altered ligand
specificity and signal characteristics. The modifications include a deletion of
the G-CSF binding site (DGcR) (Ito et al, 1997), replacement of the ER with a mutant specific
for 4-hydroxytamoxifen (TmR) (Xu et al, 1999), and the substitution of
phenylalanine for the most proximal tyrosine residue in the GcR cytoplasmic
domain (Y703FGcR) (Matsuda et al, 1999a).
The Y703F mutant is of
particular interest because this amino acid substitution apparently led to a
differentiation block in myeloid progenitor 32D cells (Matsuda et al, 1999a). To
explore the mechanisms of granulocyte differentiation in 32D cells, we examined
JAK-STAT pathways involved in GcR signaling, and identified reduced STAT3
phosphorylation associated with the Y703F mutation.
II.
Materials and methods
A. Plasmids and cells
Bicistronic vector plasmids
were constructed with the pMX retrovirus backbone and the encephalomyocarditis
virus (EMCV)-derived internal ribosome entry site (IRES; nucleotides 259-833 of
EMCV-R genome) (Duke et al, 1992; Onishi et al, 1996). pMX/DGcRER-IRES-CD8a encodes a fusion protein of DGcR and ER-HBD, and murine
CD8a as a selectable marker (Fukunaga et al, 1991; Koike et al, 1987; Nakauchi
et al, 1985). The Y703F mutation in the GcR part was introduced into this
plasmid as previously described (pMX/DY703FGcRER-IRES-CD8a)
(Matsuda et al, 1999a). The recombinant DNA experiments were carried out
following the National Institutes of Health guidelines and approved by the
Jichi Medical School Recombinant DNA Research Advisory Board.
The murine myeloid
progenitor line 32D and its derivatives were maintained in RPMI-1640 medium
(Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum
(Bioserum, Victoria, Australia) and 0.5% conditioned medium of C3H10T1/2 cells
transfected with a murine IL-3 expression plasmid pBMG-hph-IL-3 (Valtieri et
al, 1987; Matsuda et al, 1999a; Xu et al, 1999).
B. Immunoprecipitation and western blotting
32D cells were deprived of serum and IL-3 for 3 hours
at a density of 5 x 105 cells/ml, and incubated in RPMI medium
containing 1 mM Na3OV4 for an additional 1 hour at 1 x 107
cells/ml. After starvation, cells were stimulated with either 10-7 M
E2 (Sigma, St. Louis, MO) or 10-9 M recombinant human
G-CSF (provided by Chugai Pharmaceuticals, Tokyo, Japan) for given periods,
then washed with ice-cold phosphate-buffered saline (PBS) containing 100 mM Na3OV4. Subsequently, cells
were solubilized in lysis buffer (1% NP-40, 20 mM Tris-HCl [pH 7.4], 137 mM
NaCl, 1 mM phenylmethylsulfonyl fluoride, 50 mg/ml aprotinin and 2 mM Na3OV4)
on ice for 30 minutes, and centrifuged for 10 minutes. The soluble proteins
were measured by Protein Assay (Bio-Rad, Hercules, CA).
For immunoprecipitation, the cell lysate containing 1 mg of protein was incubated with one of the following antibodies for 8 hours at 4¡C: anti-JAK1 (Upstate Biotechnology, Lake Placid, NY), anti-JAK2 (Upstate Biotechnology), anti-STAT3 (C-20; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-STAT5 (C-17; Santa Cruz Biotechnology). The immune complexes were absorbed by protein G-Sepharose beads (Sigma) for 2 hours at 4¡C. The beads were washed with the lysis buffer and boiled in sample buffer (60 mM Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate [SDS], 10% glycerol and 5% 2-mercaptoethanol) for 3 minutes. After centrifugation, the supernatants were subjected to SDS-7.5% polyacrylamide gel electrophoresis and blotted onto polyvinylidene fluoride membranes (Immobilon-P; Millipore, Yonezawa, Japan). After blocking treatment with 5% bovine serum albumin (Fraction V; Roche Diagnostics, Mannheim, Germany), the membranes were incubated with an anti-phosphotyrosine antibody (4G10; Upstate Biotechnology) for 1 hour at room temperature. Immunoreactive proteins were visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Little Chalfont, UK). In some instances, membranes were stripped by incubation in denaturing buffer (62.5 mM Tris-HCl [pH 6.7], 2% SDS and 100 mM 2-mercaptoethanol) for 30 minutes at 50¡C and reprobed with another antibody.
III. Results
A. Construction of
conditionally activated G-CSF receptors
Structures of the chimeric receptors used in this
study are shown in Figure 1. The
fusion protein system is based on the fact that ER-HBD functions as an
estrogen-specific molecular switch to control heterologous effector proteins,
in our case, GcR (Mattioni et al, 1994). GcR belongs to the type I cytokine
receptor superfamily, and its cytoplasmic domain comprises functionally
distinct subdomains: the membrane-proximal region is sufficient for mitogenic
signaling, and the membrane-distal portion is essential for granulocyte
maturation (Dong et al, 1993; Fukunaga et al, 1993; Avalos, 1996; Koay and
Sartorelli, 1999). All of the four
conserved tyrosine residues in the
cytoplasmic domain of GcR (at positions 703, 728, 743 and 763 in the murine
GcR) are in the membrane-distal region and phosphorylated upon G-CSF
stimulation. Among these, the tyrosine at position 703 (Y703) was most
prominently phosphorylated and involved in granulocyte differentiation
(Yoshikawa et al, 1995). However, previous
studies on functional domains of GcR were carried out with ectopically
expressed wild-type and mutant molecules in receptor-negative cells. It may be more informative if mutant
receptors are analyzed in the natural intracellular environment where the
endogenous molecule functions. From this viewpoint, the ER-HBD fusion system
provides a valuable experimental tool. Estrogen specifically activates the
introduced GcRER (and its derivatives) without influencing the endogenous GcR
in the same cell, and the downstream events can be studied independently.
Figure
1. Structures of the chimeric
receptors involved in this study.
GcRER is a fusion of the full-length murine granulocyte
colony-stimulating factor (G-CSF) receptor (GcR) and the hormone binding domain
(HBD) of rat estrogen receptor (ER).
DGcRER
is a derivative of GcRER deleted of the G-CSF binding site (amino acids 5-195).
DY703FGcRER carries a
substitution of phenylalanine for a cytoplasmic tyrosine at position 703
(Y703F) in GcR. Ext, extracellular
domain; G, G-CSF binding site; TM, transmembrane domain; Cyt, cytoplasmic
domain; TA, transactivation domain; DNA, DNA binding domain; YYYY, conserved
tyrosine residues in GcR cytoplasmic domain; FYYY, Y703F mutation in GcR.
In our previous report, the biological response to the
DGcRER- and DY703FGcRER-mediated signal was evaluated in murine
myeloid progenitor 32D cells (D designates a deletion of amino acids 5-195 required
for G-CSF binding; Matsuda et al, 1999a). Parental 32D cells are dependent on
interleukin-3 (IL-3) for continuous growth, and switching from IL-3 to G-CSF
makes the cells differentiate into morphologically mature neutrophils (Valtieri
et al, 1987). By retrovirus-mediated gene transfer, stable clones expressing DGcRER (32D/DGcRER) or DY703FGcRER (32D/DY703FGcRER) were established and stimulated by
estrogen. While estrogen-treated 32D/DGcRER cells underwent granulocyte differentiation
indistinguishable from that seen in G-CSF-treated cells, 32D/DY703FGcRER cells showed a distinct phenotype. Estrogen
supported a long-term proliferation of 32D/DY703FGcRER with myeloblastic appearance, indicating
that the Y703F mutation abrogated the differentiation signal (Matsuda et al,
1999a). This observation prompted us to characterize signaling molecules
downstream of GcR in more detail.
Following ligand-induced homodimerization, GcR induces a wide array of intracellular signaling events (Avalos, 1996). Like many other cytokine receptors, GcR has no intrinsic kinase activity; instead, it recruits and activates other cytoplasmic kinases such as Janus kinases (JAKs), signal transducer and activation of transcription (STAT) proteins, Src family kinases and components of the mitogen-activated protein kinase pathway. The activation of JAKs is one of the earliest events in the GcR signaling cascade, followed by the tyrosine phosphorylation of STATs and GcR itself (Nicholson et al, 1994; Dong et al, 1995). Since the signal transduction for granulocyte differentiation has been ascribed to the JAK-STAT pathway, we focused on these molecules in DGcRER and DY703FGcRER cells.
B. Estrogen-induced
phosphorylation of JAK1 and JAK2 via fusion receptors
First, we examined the tyrosine phosphorylation of
JAK1 and JAK2. As shown in Figure 2,
these kinases were not tyrosine-phosphorylated in resting 32D/DGcRER and 32D/DY703FGcRER cells. Addition of G-CSF rapidly induced
phosphorylation of JAK1 and JAK2; this event was induced by dimerization of the
endogenous GcR, and maximal activation was observed within 10 minutes (data not
shown). Similarly, 10-7 M 17b-estradiol (E2) induced tyrosine
phosphorylation of JAK1 and JAK2 in these cells (Figure 2). The
estrogen-induced activation of JAK1 and JAK2 was mediated by chimeric
receptors, at a slower rate than the activation mediated by the endogenous GcR; the maximal phosphorylation was observed 60
minutes after E2 addition (time course not shown). The
difference in kinetics of JAK1/JAK2 phosphorylation may be due to different
mechanisms of receptor activation. While G-CSF directly crosslinks GcR at the extracellular domain, the
activation of ER-HBD fusion receptors is a ligand-induced derepression that
involves other proteins such as HSP90 (Mattioni et al, 1994). Nevertheless, the
levels of JAK1/JAK2 phosphorylation were comparable whether the cells were
stimulated with G-CSF or estrogen. As shown in Figure 2, the levels of estrogen-induced JAK1/JAK2
phosphorylation in 32D/DY703FGcRER cells were comparable to those seen in 32D/DGcRER cells. Reprobing of the blots with anti-JAK1 and
anti-JAK2 antibodies showed that approximately equal amounts of the kinases
were loaded on these lanes (not shown). Thus, we concluded that the Y703F
mutation had little, if any, effect on the tyrosine phosphorylation of JAK1 and
JAK2. Considering that JAK1 and JAK2 are constitutively associated with the
membrane-proximal region of GcR which is sufficient to activate them (Nicholson et al, 1994; Dong et al,
1995; Avalos, 1996), it is conceivable that the kinases were not affected by
the GcR mutation in the membrane-distal region.
C. Comparable STAT5 phosphorylation following fusion receptor activation
Next, we investigated the activation of STAT proteins
in 32D/DGcRER
and 32D/DY703FGcRER
cells. It was shown that G-CSF-induced
signaling involves STAT1, STAT3 and STAT5 (Tian et al, 1994; de Koning et al,
1996; Tian et al, 1996; Shimozaki et al, 1997; Dong et al, 1998; Chakraborty et
al, 1999; Ward et al, 1999). Since the membrane-distal cytoplasmic region of
GcR was not required for STAT1 activation (de Koning et al., 1996), we
addressed whether the phosphorylation of STAT5 and STAT3 is affected by the
Y703F mutation. Figure 3 shows the time course of STAT5 activation in 32D/DGcRER and 32D/DY703FGcRER cells (upper panel). STAT5 was not
tyrosine-phosphorylated in unstimulated 32D cells, and addition of 10-9
M G-CSF induced a rapid phosphorylation of this molecule through crosslinking
of the endogenous GcR. On the other hand, 10-7 M of E2
induced a slower and less extensive phosphorylation of STAT5.
Figure
2. Tyrosine phosphorylation of JAK1
and JAK2. Serum- and
cytokine-starved 32D/DGcRER and 32D/DY703FGcRER cells were harvested before (0Õ) and after 60 minutes (60Õ)
of incubation with 10-7 M of estradiol (E2). Lysates from
32D/DGcRER and 32D/DY703FGcRER cells were immunoprecipitated (IP) with
either an anti-JAK1 (aJAK1; upper panel) or an anti-JAK2 (aJAK2; lower panel) antibody. Immunoblotting (IB) was carried out with an
anti-phosphotyrosine antibody (aPY).
The
estrogen-induced STAT5 activation was comparable in 32D/DGcRER and 32D/DY703FGcRER cells at 60 minutes after stimulation, and
reprobing of the blot with an anti-STAT5 antibody showed that approximately
equal amounts of STAT5 were loaded (Figure 3, lower panel). The delay in STAT5 phosphorylation may
be associated with a slower JAK1/JAK2 activation through estrogen-induced
dimerization of the chimeric receptors. The reason for the reduced STAT5
phosphorylation in the E2-stimulated cells is currently unknown; we
speculate that the linking of ER-HBD to the C-terminal of GcR might hinder STAT
proteins from freely accessing the membrane-distal region of the receptor. In
any case, STAT5 appeared to be phosphorylated to the same extent in 32D/DGcRER and 32D/DY703FGcRER cells. Others demonstrated that STAT5 was activated even when the membrane-distal region of GcR was deleted or the receptor
tyrosine phosphorylation was abrogated (Shimozaki et al, 1997; Tian et al,
1996). Taken together with our observation that JAK1 and JAK2 were
activated in both 32D/DGcRER and 32D/DY703FGcRER cells (Figure 2),
we concluded that the Y703F mutation did not affect the tyrosine
phosphorylation of STAT5.
D. Decrease in STAT3 Activation
by Y703F G-CSF Receptor Mutant
Finally, we addressed
whether the Y703F mutation in GcR affects tyrosine phosphorylation of STAT3. After
cytokine starvation, 32D/DGcRER and 32D/DY703FGcRER clones were incubated with 10-7 M of E2 for
60 minutes. While estrogen induced a significant tyrosine phosphorylation of
STAT3 in 32D/DGcRER, only a slight
activation of STAT3 was detected in 32D/DY703FGcRER clones (Figure
4, upper panel, arrow). Reprobing of the
membrane with an anti-STAT3 antibody revealed an even loading of STAT3 in these
lanes (Figure 4, lower panel).
Figure 3. Tyrosine
phosphorylation of STAT5. Starved
32D/DGcRER and 32D/DY703FGcRER cells were harvested before (0Õ) and after
10, 30, and 60 minutes (10Õ, 30Õ, 60Õ) of incubation with 10-9 M of
G-CSF or 10-7 M of estradiol (E2). Lysates were immunoprecipitated (IP)
with an anti-STAT5 antibody (aSTAT5) and immunoblotted (IB) with an
anti-phosphotyrosine antibody (aPY; upper panel).
The blot was reprobed with the anti-STAT5 antibody to confirm the equal loading
of STAT5 (lower panel).
Repeated experiments constantly
demonstrated a decreased STAT3 phosphorylation in 32D/DY703FGcRER.
Consistent with this observation, Tian et al showed that the G-CSF-induced
STAT3 activation was greatly abrogated in UT-7epo cell transfectants by
deleting a membrane-distal part including Y703 from GcR (Tian et al, 1996). We
therefore concluded that Y703 in GcR was involved in STAT3 activation, and that
the event is crucial to granulocyte differentiation in 32D cells.
IV. Discussion
The
phosphotyrosine residues in GcR create potential docking sites for the
recruitment of signaling molecules such as STATs that contain a Src homology 2
(SH2) domain. STAT3 is recruited via the interaction of its SH2 domain with
receptor tyrosine residues that are present in a tyrosine-X-X-glutamine (YXXQ)
sequence (Stahl et al, 1995). Among four conserved tyrosine residues in the
cytoplasmic region of GcR, only Y703 provides a YXXQ motif, accounting for the
reduced STAT3 activation by the Y703F mutant. However, there was a residual
level of STAT3 activation in DY703FGcRER
and other GcR mutants devoid of this motif, which suggested the presence of
another STAT3 binding site in GcR or some bridging molecule (Avalos, 1996;
Chakraborty et al, 1999). We observed a few additional phosphorylated proteins
coimmunoprecipitated with STAT3 including a 130 kDa species (Figure 4, upper panel, arrowheads). These proteins are yet to be
identified; at least they did not react with an antibody against GcR in a
subsequent reprobing (data not shown).
Figure
4. Tyrosine phosphorylation of STAT3.
Starved 32D/DGcRER
and 32D/DY703FGcRER (clone 1 and clone 2) cells were
harvested before (0Õ) and after 60 minutes (60Õ) of incubation with 10-7
M of estradiol (E2). Lysates were immunoprecipitated (IP) with an
anti-STAT3 antibody (aSTAT3) and immunoblotted (IB) with an anti-phosphotyrosine antibody (aPY; upper panel). The blot was reprobed with the
anti-STAT3 antibody to confirm the equal loading of STAT3 (lower panel). Besides STAT3 (92 kDa,
arrow), several phosphoproteins including a 130 kDa species
(arrowheads) were coimmunoprecipitated.
A consensus has been reached that tyrosine
phosphorylation of GcR and activation of STAT3 is crucial to granulocyte
differentiation, but there remains some controversy over the relative
contribution of each tyrosine residue depending on the cells used (Tian et al, 1994, 1996; de
Koning et al, 1996; Shimozaki et al, 1997; Chakraborty et al, 1999; Ward et al,
1999). Previous reports employed either GcR-negative cells to
examine the function of the receptor and associated molecules, or overexpression
of dominant-negative forms of GcR to elucidate the mechanisms for growth and
differentiation. By using ER-HBD fusion proteins to bypass endogenous GcR, we
herein provided additional data suggesting the major involvement of Y703 in
STAT3 activation. It is of particular note that the cells retained the
expression of wild-type GcR and downstream signaling molecules, thereby rapidly
undergoing granulocyte differentiation in response to G-CSF, indistinguishable
from the parent 32D cells (Matsuda et al, 1999a).
Contrary to its promoting function in myeloid cell
differentiation, STAT3 was shown to play a central role in the maintenance of
the pluripotent phenotype of embryonic stem cells (Matsuda et al, 1999b; Niwa
et al, 1998). STAT3 appears to dictate widely divergent instructions such as
differentiation and proliferation depending on the cell type. Thus, it is
crucial to set up an appropriate venue to study the physiological molecular
interaction involving a promiscuous molecule such as STAT3. The HBD fusion system
provides a powerful tool to examine the behavior of mutated proteins controlled
by specific ligands, in the exact milieu where the wild-type molecules coexist
but remain unstimulated.
Acknowledgments
We are grateful to
Chugai Pharmaceuticals for providing G-CSF. This work was supported by grants
from the Ministry of Education, Culture, Sports, Science and Technology, and
the Ministry of Health, Labor and Welfare, Japan
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Dr. Akihiro
Kume