From the Kimmel Cancer Center, Thomas Jefferson
University, Philadelphia, Pennsylvania 19107 and § Instituto
Regina Elena, Centro Ricerca Sperimentale, Laboratorio Oncogenesi
Molecolare, 00158 Rome, Italy
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ABSTRACT |
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The type 1 insulin-like growth factor receptor
(IGF-IR) plays an important role in the growth of cells both in
vivo and in vitro. The IGF-IR is also capable of
inducing differentiation in a number of cell types, raising the
question of how the same receptor can send two seemingly contradictory
signals, one for growth and one for differentiation. Using 32D cells,
which are murine hemopoietic cells, we show that the activated IGF-IR
can induce differentiation along the granulocytic pathway in a manner similar to the granulocyte colony-stimulating factor. We find that one
of the major substrates of the IGF-IR, the insulin receptor substrate-1
inhibits IGF-I-mediated differentiation of 32D cells. In the absence of
insulin receptor substrate-1, functional impairment of another major
substrate of the IGF-IR, the Shc proteins, is associated with a
decrease in the extent of differentiation. Although the end points of
the respective pathways remain to be defined, these results show for
the first time that IGF-I-mediated growth or differentiation of
hemopoietic cells may depend on a balance between two of its substrates.
The type 1 insulin-like growth factor receptor
(IGF-IR),1 activated by its
ligands plays an important role in the growth of cells in at least
three different ways: it is mitogenic, both in vivo and
in vitro, it is quasiobligatory for transformation, and it
can protect cells from a variety of apoptotic injuries (1). The IGF-IR
can also induce differentiation in certain types of cells, notably
myoblasts (2, 3), adipocytes (4), osteoblasts (5), and cells of the
central nervous system (6-8). These apparently contradictory actions
of the IGF-IR, and indeed of other growth factor receptors (growth
promotion on one side and induction of differentiation on the other)
are usually dismissed as due to the "cell context," a somewhat
unsatisfactory answer. It is self-evident that, at some point, the
mitogenic and differentiation signals originating from the IGF-IR will
diverge. The question we have asked in this investigation is whether
these signals can already be separated at the level of the receptor
itself or its immediate substrates. We have used as a model 32D cells
(9), which are well characterized diploid murine hemopoietic cells. 32D
cells have an absolute requirement for interleukin-3 (IL-3), and
undergo apoptosis when IL-3 is withdrawn (10, 11). IGF-I or
overexpression of the IGF-IR prevent or markedly decrease apoptosis caused by IL-3 withdrawal (12-16). Indeed, an overexpressed IGF-IR causes 32D cells to grow in the absence of IL-3 at least for several days (14, 16). Another interesting characteristic of 32D cells is that
they are completely devoid of insulin receptor substrate-1 (IRS-1) and
IRS-2 (12, 17, 18). While the IGF-IR, by itself, protects 32D cells
from apoptosis, overexpression of the insulin receptor (IR) is not
sufficient for the growth of these cells in the absence of IL-3 (17).
However, the combined overexpression of the IR and IRS-1 renders 32D
cells IL-3-independent (17).
We show here that 1) the IGF-IR can also induce differentiation of 32D
cells, along the granulocytic pathway; 2) IRS-1, one of the major
substrates of the IGF-IR, inhibits IGF-I-mediated differentiation; 3)
mutations of tyrosine 950 and of tyrosines 1250/1251 in the C terminus
of the IGF-IR, in the absence of IRS-1, cause a decrease in the extent
of IGF-I-mediated differentiation; 4) when these three tyrosine
residues are mutated, the phosphorylation of Shc, another major
substrate of the IGF-IR, is impaired; 5) overexpression of Shc promotes
differentiation of 32D cells, while a dominant negative mutant of the
Shc protein partially inhibits differentiation; and 6) the inhibitory
effect of IRS-1 on the differentiation of 32D cells is associated with
changes in the Akt/p70 S6 kinase pathway. We suggest that, at least in
the case of 32D cells overexpressing the IGF-IR, the outcome
(i.e. growth or differentiation) depends on a balance
between the signaling pathways originating from two of its substrates.
Retroviral Transduction--
32D clone 3 cells were transduced
with a murine leukemia virus-based retroviral vector system (19) to
express the wild type IGF-IR or its various mutants or its substrates,
IRS-1 and Shc. These constructs have been described in previous papers
from one of our laboratories (16, 20-22). The retroviral vector stocks were generated with a transient expression system (23) and used to
transduce 32D cells as described previously (24). The cDNAs were
inserted either into retroviral transfer vector MSCV.neoEB (carrying
the neomycin resistance gene) or MSCV.hph, (carrying the hygromycin
resistance gene), which were kindly provided by Dr. R. G. Hawley
(University of Toronto, Canada) and are described elsewhere (25).
The Shc-SH2 cDNA was constructed as described (26). Briefly, the 5'
portion of Shc cDNA, corresponding to amino acid residues 1-377,
was deleted and substituted with a 15-base pair sequence containing an eukaryotic initiation site for translation
(TAAAGCACTATGGGC). This construction was carried out by polymerase
chain reaction (PCR), using a sense primer containing the above
mentioned initiation translation site and a XhoI restriction
site in its overhang and an antisense primer containing the TAG stop
codon and an EcoRI restriction site in its overhang. The
sequence of the sense primer was 5'-GGTCAACCCTATATTCCTctcgagTAA
AGCACTATGGGCTGGTTCCATGGGAAGCTGAGCCGGC-3' (the
XhoI restriction site is indicated in lowercase letters, and
the translated region sequence of 5' Shc-SH2 cDNA is underlined), whereas the sequence of the antisense primer was 5'-GGTCAACCTTA ACCGCAGCACCGGTTAATCgaattcGTCCATGCTACTCCCAGCTCTGACACAAGGC-3'
(the EcoRI restriction site is indicated in lowercase
letters, and the stop codon and the translated region sequence of 3'
Shc-SH2 cDNA are underlined). The PCR conditions were as follows: a
denaturation step of the template at 94 °C for 1 min, followed by a
1-min interval at 52 °C to allow the annealing of the primers to the
template and a 3-min incubation at 72 °C for polymerase elongation
of the primers. This cycle was repeated 35 times. At the end of these 35 cycles, an additional incubation at 72 °C for 15 min was included to allow completion of the amplification. The PCR product was electrophoresed on a 1% agarose gel. The correct size product was
excised from the gel and purified with a gel extraction kit (Qiagen,
Santa Clarita, CA), following the manufacturer's recommendations. The
end PCR product was in 50 µl of water and digested with
XhoI and EcoRI. After the digestion, the enzymes
were heat-inactivated at 68 °C for 15 min, and the PCR fragment was
gel-purified as already described. The digested and gel-purified PCR
fragment was then ligated into the XhoI-EcoRI
cloning site of MSCV.pac retroviral vector, carrying the puromycin
resistance gene (25). The Shc-SH2 cDNA was then sequenced to
monitor for mutations.
Survival and Differentiation Analysis--
32D cells and the
derived mixed populations obtained by retroviral transduction were
grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal
bovine serum (Life Technologies, Inc.), 10% WEHI cell conditioned
medium as a source of IL-3 and the required antibiotics for the
selection (G418, 1 mg/ml (Life Technologies) or 1 mg/ml hygromycin
(Calbiochem)). For survival and granulocytic differentiation
experiments, 32D cells and their derivatives were collected, washed
three times, and seeded at a density of 5 × 104
cells/ml in the medium specified for the different experiments. At each
time point, viable cells were counted by trypan blue exclusion (Life
Technologies), and Wright-Giemsa-stained cytospins were evaluated for
differentiation. Bands and polymorphonuclear cells were considered as
differentiated cells.
Myeloperoxidase Staining--
Myeloperoxidase expression was
detected in cell cytospins using a leukocyte myeloperoxidase kit,
following the instructions of the manufacturer (Sigma).
Immunoprecipitation and Immunoblots--
Cells were incubated in
serum-free medium supplemented with 0.1% bovine serum albumin for
3 h before stimulation with the indicated growth factors, 20 ng/ml
IGF-I (Life Technologies), 100 units/ml granulocyte colony-stimulating
factor (G-CSF; Life Technologies) or 10% fetal bovine serum. Cell
lysates were resolved directly or after immunoprecipitation by
SDS-polyacrylamide gel electrophoresis and transferred to a
nitrocellulose filter. Immunoprecipitation and immunoblotting
procedures are described by Valentinis et al. (27). The
phosphotyrosine blots were performed with an antiphosphotyrosine horseradish peroxidase-conjugated antibody (PY20; Transduction Laboratories). The IGF-I receptor was immunoblotted with an
anti-
Phospho-Akt and Akt protein were detected using the PhosphoPlus Akt
(Ser473) antibody kit (New England Biolabs), following the
manufacturer's instructions. Phospho-p70 S6 kinase
(Thr421/Ser424) and p70 S6 kinase protein were
visualized with a PhosphoPlus p70 S6 kinase antibody kit, also from New
England Biolabs, and also following the manufacturer's instructions.
The present observations stem from our previous finding that, at
variance with the IR, an overexpressed IGF-IR protects 32D cells from
apoptosis although these cells do not have IRS-1 or IRS-2 (12, 17, 18).
As mentioned above, the IR requires the combined overexpression of
IRS-1 to promote growth of 32D cells in the absence of IL-3 (17). When
32D cells overexpress the IGF-IR, they not only survive but actually
grow in the complete absence of IL-3 (12, 14). We had noticed, however,
that, after a period of rapid growth, the 32D IGF-IR cells stop growing
and, after 4-5 days, begin to decrease in number, especially when the culture is not supplemented daily with IGF-I (see below). The period of
reduced growth coincided with the appearance of differentiated 32D
cells. We were intrigued by the finding that the IGF-IR can protect 32D
cells from apoptosis, while at the same time inducing terminal
differentiation, which usually results in cell death (28). We therefore
decided to examine the effects of the IGF-IR on 32D cells in more
detail, using transduction with appropriate retroviral vectors.
The Activated IGF-I Receptor Induces Differentiation of 32D
Cells--
The use of retroviral vectors (see "Experimental
Procedures") gives a good efficiency of transduction of 32D cells
(notoriously resistant to transfection by plasmids) and generates mixed
populations, expressing, in the case of the IGF-IR, approximately
100,000 to 200,000 receptors/cell (see below). The use of mixed
populations obviates the problem of clonal variation and, since the
expression levels are high, also compensates for slight variations in
individual cells. The levels of expression are also reasonably uniform
in the mixed population, as one would expect from retroviral
transduction (24). By flow cytometric analysis of cells stained with an
antibody to the IGF-IR, the width of the peak is essentially the same
as the width of the peak in a cloned cell line (data not
shown). The mixed population expressing
the wild type human IGF-IR is here designated as 32D IGF-IR. Fig.
1 shows that these cells survive after
IL-3 withdrawal and, with the addition of IGF-I, can actually grow, as
already reported for 32D cells transfected with an IGF-IR plasmid (12,
14). 32D IGF-IR cells differentiate all the way to granulocytes,
reaching a level of 50% differentiated cells by day 6 after IL-3
withdrawal and IGF-I addition (Fig. 1). Omission of IGF-I results in
cell death, despite the high levels of receptor expression. It should
be noted, however, that in these experiments, we have used
heat-inactivated serum, a procedure that can destroy many growth
factors, including the IGFs. For clarity, Fig. 1 gives only the data on
days 0, 4, and 6, but both survival and differentiation were determined
also at other intervals. The differentiation is morphologically
detectable (Giemsa staining), and can be confirmed by staining for
myeloperoxidase (Fig. 1).
Fig. 1 also shows G-CSF-induced differentiation of 32D IGF-IR cells.
G-CSF has been known to induce differentiation of parental 32D cells
(29), confirmed here, whether using the parental 32D cells or the 32D
IGF-IR cells. The data in Fig. 1 are not intended as a comparison
between IGF-I and G-CSF, since conditions are different. It is given
here simply to show that the extent of IGF-I-mediated differentiation
is not trivial.
Mutational Analysis of IGF-I-induced Differentiation of 32D
Cells--
We investigated several mutants of the IGF-IR to determine
the specific residues that are necessary for the induction of
differentiation. These mutants have been described in previous papers
from one of our laboratories (20-22) and are listed in Fig.
2a, which gives the levels of
expression of the IGF-I receptors in mixed populations of transduced
32D cells. For simplicity, we are showing the results obtained with an
antibody to the
Fig. 3a gives the survival at
4 days after IL-3 withdrawal of three of these mixed populations. The
overexpressed mutant receptors, 32D 4 basic aa, 32D 6 serine, and 32D
1293-94 (Fig. 3a), are as effective as the wild type
receptor in protecting 32D cells from apoptosis induced by IL-3
withdrawal, and all of them induce differentiation upon IGF-I addition
(Fig. 3b). Other mutant receptors (32D 3YF, 32D d1245, 32D
Y950, and 32D Y1250-51) were not capable of fully protecting 32D cells
from apoptosis after the first 48 h. Since differentiation, even
with the wild type receptor, does not become clearly visible until day
4, it was necessary to prolong the survival of these cell lines by the
addition of 0.1% WEHI medium (hereafter designated as IL-3). This
procedure partially decreases the extent of differentiation in 32D
cells expressing the wild type receptor to about 18% of viable cells
(Fig. 3d). However, the cells still differentiate. Using the
same conditions, we can see that the three mutant receptors, Y950F,
Y1250F/Y1251F, and d1245, have lost the ability to induce
differentiation (Fig. 3d). The level of differentiation is
roughly the same as that of parental 32D cells growing in IL-3 (see
Fig. 1). It may be argued that these mutant receptors are nonfunctional
receptors, incapable of transmitting an IGF-I-mediated signal. The
addition of 0.1% IL-3 was not sufficient to induce survival in the
cell line described in Fig. 3c (not shown). The addition of
IGF-I made these cell lines grow with two exceptions: the parental cell
line and the 32D 3YF cell line (Fig. 3c). The 3YF mutant
receptor has been known to be an inactive receptor (31). The dependence
on IGF-I for survival and growth of the 32D Y950, 32D Y1250-51, and
32D d1245 cell lines indicates that these mutant receptors are
responsive to stimulation by IGF-I, as indeed they are when transfected
into other cell lines (20-22). We can therefore say that these mutant
receptors can still transmit a mitogenic signal but have lost the
ability to transmit a differentiation signal.
It could be objected that 32D Y950, 32D Y1250-51, and 32D d1245 cell
lines may have lost the ability to differentiate. We therefore treated
these same cell lines with G-CSF (100 units/ml). The wild type receptor
cells gave (day 4) 19.5% differentiated cells, 20.8% Y950, 16.3%
Y1250-51, and 14.1% Role of IRS-1 in IGF-I-induced Differentiation of 32D
Cells--
Since 32D cells are devoid of IRS-1 and IRS-2 (17), we
inquired whether the absence of these proteins played a role in
IGF-I-mediated differentiation of 32D cells. For this purpose, we
examined other cell lines, all derived from the parental 32D cells: 1)
a cell line in which we introduced, through a retroviral vector, the IGF-IR into a 32D cell line overexpressing IRS-1 (32D IRS/IGF-IR) (the
original 32D IRS-1 cell line was a kind gift from Dr. Morris White and
was described previously also from our laboratory (16)); 2) a cell line
in which a retroviral vector expressing IRS-1 was introduced into 32D
IGF-IR cells (in order to avoid confusion, these cells are designated
as 32D GR15/IRS cells); and 3) a cell line overexpressing the IGF-IR
and transduced with the empty retroviral vector used to deliver IRS-1
(32D IGF-IR/hph). Fig. 2b shows that IRS-1 is not detectable
in the parental 32D cells (lane 1), in the 32D
IGF-IR cells (lane 2), and in 32D IGF-IR/hph
(lane 3). It is modestly expressed in 32D
GR15/IRS cells (lane 4) and strongly overexpressed in the 32D IRS/IGF-IR (lane 5).
This blot was purposely overexposed in order to demonstrate again the
absence of IRS-1 from the parental 32D cells. The levels of the
expression of the IGF-IR in these cell lines are also given in Fig.
2b. Notice the comparable level of the overexpressed
receptor in all four 32D-derived cell lines and the very low level of
expression of the IGF-IR in parental 32D cells.
Table I shows a representative experiment
on the survival and differentiation of these cell lines, in the absence
of IL-3 but with the addition of IGF-I. The 32D IGF-IR cells and the
same cells transduced with an empty retroviral vector for IRS-1 (32D IGF-IR/hph) survive after IL-3 withdrawal, grow, and differentiate (40-45% differentiated cells). The combined overexpression of IRS-1
and the IGF-IR (32D GR15/IRS and 32D IRS/IGF-IR) results in cell lines
that also survive and grow extremely well in the absence of IL-3. These
last two cell lines, however, do not differentiate as well as the cells
expressing only the IGF-IR (Table I). The mixed population of 32D cells
expressing the IGF-IR and moderate amounts of IRS-1 (32D GR15/IRS) had
8.9% differentiated cells; the one expressing high amounts of IRS-1
(32D IRS/IGF-IR) had 0.3%. The difference between these two cell lines
expressing both the IGF-IR and different levels of IRS-1 will be
discussed further below. Table I shows the extent of differentiation at
day 4, but we examined these cell lines also at day 6, and the cell
lines that did not differentiate remained undifferentiated even at day 6, when 50% of the 32D IGF-IR cells are already differentiated. It
seems therefore, that IRS-1 inhibits IGF-I-induced differentiation of
32D cells. We have confirmed the observation that overexpressed IR, by
itself, does not protect 32D cells from apoptosis but that the combined
overexpression of the IR and IRS-1 makes these cells grow in the
absence of IL-3 (17, 32). Interestingly, the 32D IR/IRS1 cells do not
differentiate (data not shown).
Role of Shc Proteins in Differentiation of 32D Cells--
The Shc
proteins (33) are a second major substrate of the IGF-IR. The 46- and
52-kDa isoforms are strongly expressed in 32D cells, roughly 10 times
the levels of expression in 3T3 cells (data not
shown). The p66 isoform is not expressed
in these cells (34). We transduced 32D cells with a retroviral vector
carrying the Shc protein cDNA that codes for the 46- and 52-kDa
forms (a kind gift of Dr. Joseph Schlessinger). Even with retroviral
vectors, it was not easy to obtain 32D clones overexpressing Shc
proteins, because the cells seemed to differentiate spontaneously even
in the presence of IL-3. We finally obtained two clones that expressed levels of Shc roughly twice the endogenous levels (Fig.
4a) and that grew in 10% IL-3
at least for 2-3 months. The increase in expression is modest but was
confirmed by densitometric measurements and may be due to the fact that
Shc proteins are already strongly expressed in 32D cells. The 32D cells
overexpressing Shc proteins differentiate rapidly even in the presence
of 5% IL-3 (Table II), while under the
same conditions the parental 32D cells have a percentage of
differentiated cells of 1.3%.
Since it has been reported that dominant negative mutants of Shc
proteins (26) can inhibit differentiation of other cell types (35, 36),
we transduced in 32D IGF-IR cells a retroviral vector expressing the
SH2 domain of the Shc proteins (see "Experimental Procedures").
This construct is poorly expressed, and previous authors (26) could
only detect it after immunoprecipitation. We were able to detect its
expression on Western blots, but it was necessary to expose the blot
longer than the time required for detection of the endogenous Shc
proteins (Fig. 4b). Note that the endogenous Shc proteins
are at the same levels in all three cell lines (Fig. 4b).
Despite the modest expression, the Shc dominant negative mutant
partially inhibits IGF-I-mediated differentiation (Table II). The
experiments were repeated, and the cells expressing the SH domain of
Shc consistently had a decreased extent of cell differentiation, which,
however, was never abrogated.
Tyrosyl Phosphorylation of Shc Proteins--
We have examined Shc
phosphorylation in the cell lines expressing the mutant receptors that
fail to induce differentiation of 32D cells (32D Y950 and 32D
Y1250-51). As expected, 32D IGF-IR cells, stimulated with IGF-I (Fig.
5a), show a clear increase in
Shc phosphorylation. As previously reported for the IR (37), the 52-kDa
isoform is the one that is preferentially phosphorylated (lane 5); serum is less effective than IGF-I in
inducing tyrosyl phosphorylation of Shc (lane 6).
A co-precipitation of Grb2 with Shc in these two lysates is clearly
visible, especially prominent in immunoprecipitates of 32D IGF-IR cells
stimulated with IGF-I. However, the Y950F mutant, which fails to induce
differentiation, also fails to increase Shc phosphorylation and to
co-precipitate Grb2. The results are not as clear cut with the
1250/1251 mutant; as Fig. 5a shows, there is a slight
increase in Shc phosphorylation and a slight increase in Grb2
co-precipitation when IGF-I is added to the cells expressing the
1250/1251 mutant, although substantially less than with the wild type
receptor. This experiment was repeated several times, with exactly the
same results.
Since both IRS-1 and Shc are known to bind to the Tyr950
residue of the IGF-IR (38), we attempted to explore further the
mechanism(s) by which IRS-1 inhibits and Shc proteins promote
differentiation of 32D cells by IGF-I. One important question is
whether the effect of IRS-1 on differentiation may be due to its
ability to compete with Shc for a common receptor binding site and to
sequester substrates common to both IRS-1 and Shc, for instance Grb2 as
suggested some years ago by Yamauchi and Pessin (39). For this purpose,
we tested the two cell lines described above, both of them
overexpressing the IGF-IR and IRS-1 but where one of the cell lines
expresses IRS-1 in much larger amounts than the other. The results
(Fig. 5b, right) show that Shc is
tyrosyl-phosphorylated in both the 32D IGF-IR and the 32D GR15/IRS
(comparatively low levels of IRS-1). In the cell line strongly
overexpressing IRS-1, Shc phosphorylation is not increased by
stimulation with IGF-I (lanes 5 and
6). Moreover, Grb2 co-precipitation with Shc is detectable
only in 32D IGF-IR/hph cells (no IRS-1) and in the cells expressing
lower levels of IRS-1 (32D GR15/IRS). A marked reduction in Grb2
co-precipitation is visible in anti-Shc precipitates of 32D IRS/IGF-IR
cells (high levels of IRS-1). Finally, and in agreement with the
results of Yamauchi and Pessin (39), when IRS-1 is immunoprecipitated
from the same lysates, Grb2 co-precipitates with the phosphorylated IRS-1 in larger amounts in 32D IRS/IGF-IR cells than in 32D GR15/IRS cells, as expected (Fig. 5b, left). The amounts
of IRS-1 and Shc that were immunoprecipitated by the respective
antibodies were monitored by blotting the membranes after stripping and
were similar (not shown).
IRS-1 Signaling in Transduced 32D Cells--
The results with Shc
were intriguing, but inconclusive, especially in consideration of the
fact that a role of Shc in differentiation was detectable only in the
absence of IRS-1. We therefore asked whether other signaling may be
different in 32D IGF-IR and in 32D IGF-IR/IRS-1 cells. One of the known
pathways activated by the IGF-IR is the mitogen-activated protein
kinase pathway, but in preliminary experiments we found that
mitogen-activated protein kinase was activated similarly in both 32D
IGF-IR cells and in 32D IGF-IR cells expressing IRS-1. There was a
sustained activation of mitogen-activated protein kinase, for at least
2 h, and the signals were very strong in either
case.2 Another pathway
activated by the IGF-IR is through phosphatidylinositol 3-kinase, Akt,
p70 S6 kinase (40). The 32D IGF-R cells and the two cell lines
overexpressing both the IGF-IR and IRS-1 were incubated for 3 h in
serum-free medium and subsequently stimulated with IGF-I; lysates were
made at the indicated times, and the phosphorylation of either Akt or
p70 S6 kinase was determined with the appropriate antibodies. Fig.
6a shows that Akt is strongly
phosphorylated when the IGF-IR and IRS-1 are both expressed in 32D
cells, whereas in the cells expressing only the IGF-IR, Akt is less
phosphorylated (the protein amounts are shown in Fig. 6b).
Even clearer are the results with p70 S6 kinase phosphorylation: the
wild type IGF-IR, by itself, cannot phosphorylate p70 S6 kinase, while
the combination of the receptor and IRS-1 causes a marked increase in
phosphorylation (Fig. 6c, with protein amounts in Fig.
6d).
There are several novel findings in the experiments reported above
that we think are of interest. 1) We show for the first time that an
activated IGF-IR can induce differentiation of hemopoietic cells along
the granulocytic lineage. 2) The absence of IRS-1 (which is missing in
parental 32D cells) is crucial for differentiation. When IRS-1 is
reintroduced into 32D IGF-IR cells, differentiation is inhibited, and
the extent of inhibition is dependent on the levels of IRS-1
expression. 3) IGF-I-induced differentiation (in the absence of IRS-1)
requires the C terminus of the receptor. 4) At least three residues are
important for differentiation, the tyrosine residue at 950 and the two
tyrosines at 1250/1251. 5) Shc phosphorylation is decreased in cells
expressing the two mutant receptors that fail to induce differentiation
(Y950F and Y1250F/Y1251F). 6) Overexpression of Shc proteins favors
differentiation, while a dominant negative mutant of Shc partially
inhibits IGF-I-mediated differentiation. 7) While the IGF-IR, by
itself, can promote temporary growth of 32D cells, the cells eventually
differentiate, whereas the 32D IGF-IR cells expressing IRS-1 can grow
indefinitely in the absence of IL-3 and IGF-I addition. Interestingly,
the IGF-IR, by itself, seems to be defective in the activation of the
Akt/p70 S6 kinase pathway, in respect to cells also expressing
IRS-1.
Although we acknowledge that the end points of these two processes
remain to be defined, our results show for the first time that the
stimulation of the same receptor can lead to different biological
outcomes (growth or differentiation), depending on a balance between
substrate levels (the "cell context").
The finding that an activated IGF-IR can induce differentiation of
hemopoietic cells along the granulocytic lineage is a novel finding,
although Merchav et al. (41) did report that injections of
IGF-I in animals increased granulopoiesis. The induction of differentiation is not trivial, according to the literature about on
the same level as differentiation induced by G-CSF (29), and, as in the
case of G-CSF (11), eventually results in a decrease in cell number.
Thus, although we started out to resolve an ambiguity of the IGF-IR
(cell proliferation versus differentiation), we find
ourselves with another ambiguity. The IGF-IR promotes cell survival in
IL-3-dependent cell lines (12-16, 22) and yet, at the same
time, induces a differentiation program that eventually leads to cell
death. Incidentally, IGF-I-mediated survival in the absence of IRS-1
and decreased Akt phosphorylation is due to the ability of the IGF-IR
to use two alternative pathways for BAD phosphorylation; one pathway
goes through the activation of mitogen-activated protein kinase, and a
third one depends on the integrity of serine 1283, which results in the
mitochondrial translocation of
Raf-1.3
The number of receptors is reasonably uniform in our cell lines, both
from cell line to cell line and, within a cell line, from cell to cell.
By fluorescence-activated cell sorting analysis of cell lines stained
with an antibody to the IGF-IR, we cannot find any difference in the
width of the peak between mixed populations and clonal populations
(24). The seemingly random differentiation induced by the IGF-IR is not
different from the differentiation induced in 32D cells by other
agents, such as G-CSF (29) or TPA (42).
The most interesting findings, however, are those related to IRS-1 and
the Shc proteins, two major substrates of both the IR and the IGF-IR.
It is clear that IRS-1 can inhibit the IGF-I-mediated differentiation
process of 32D. Incidentally, the reason we decided to examine two cell
lines overexpressing both the IGF-IR and IRS-1 is that the original 32D
IRS-1 clone expresses large amounts of IRS-1 (see Fig. 2b,
lane 5), while our mixed population of 32D GR15
cells transduced with IRS-1 expresses a much lower amount (Fig.
2b, lane 4). In both cell lines,
overexpression of IRS-1 in 32D cells with IGF-IR inhibited
differentiation, and the extent of inhibition was dependent on the
level of expression of IRS-1. IRS-1 has been repeatedly shown to be
important for mitogenesis induced by either insulin or IGF-I (43-45),
and therefore, it makes sense that, when overexpressed, it will
stimulate growth. However, this is the first time that IRS-1 is shown
unequivocally to inhibit cell differentiation. As mentioned above, the
combined expression of the IR and IRS-1 has already been reported by
Wang et al. (17) as inducing growth of 32D cells in the
absence of IL-3, while the IR, by itself, is insufficient. We have
confirmed their findings; although 32D cells overexpressing both the IR
and IRS-1 survive now in the absence of IL-3, they do not differentiate
(data not shown).
The IGF-IR protects 32D cells from IL-3 withdrawal, as previously
reported (12, 14). Indeed, the 32D IGF-IR cells grow for at least 2-3
days before they begin to differentiate. On the other hand, 32D
IGF-IR/IRS cells grow indefinitely, and, in fact, we can passage them
continuously in the absence of IL-3 and IGF-I addition. An explanation
of this difference may be found in the findings of Fig. 6; the Akt/p70
S6 kinase pathway is strongly stimulated in 32D IGF-IR cells expressing
IRS-1 but not in cells expressing only the IGF-IR. The Akt/p70 S6
kinase pathway (46) is the generally accepted pathway for
IGF-I-mediated protection from apoptosis, and it is probably the main
antiapoptotic pathway (47). The activation of Akt is generally
dependent on the activation of phosphatidylinositol 3-kinase (48, 49),
which is strongly activated by IRS-1 (50-52). Akt, in turn, activates
p70 S6 kinase (53). However, we have previously reported that the
IGF-IR protects cells from apoptosis in the absence of IRS-1 (12, 14)
and, furthermore, that it protects cells from apoptosis even in the presence of inhibitors of phosphatidylinositol 3-kinase, such as
wortmannin and LY294002 (54, 55). It is reasonable to conclude that, in
the absence of IRS-1, the IGF-IR may use an alternative antiapoptotic
pathway, which is Akt-independent and is not shared by the IR (55). The
existence of an alternative, secondary pathway has already been
suggested by several investigators (12, 32, 47). However, for
continuous stimulation of growth, at least in 32D cells, IRS-1 and the
activation of the Akt/p70 S6 kinase pathway are required.
Interestingly, Yao and Cooper (56) had also observed that p70 S6 kinase
activation was not necessary for growth factor-dependent survival.
Although the IGF-IR, by itself, can protect 32D cells from apoptosis
and induce even a temporary period of growth, the cells eventually
undergo differentiation. A legitimate question is whether the
inhibition of IGF-I-mediated differentiation by IRS-1 is simply the
effect of the stimulation of cell proliferation. In other words, it is
possible that differentiation may occur by default, when 32D cells are
stimulated to proliferate, but the mitogenic stimulus is not sustained.
In favor of this interpretation are the findings of Fig. 6, discussed
above. However, we believe that the situation is more complicated, and
that a positive signal may also be required for the induction of differentiation.
There are several reasons for this belief. In the first place,
IGF-I-induced differentiation of 32D cells is dependent on the presence
of specific residues of the IGF-IR; these include Tyr950
and the tyrosine residues at 1250/1251 (see also below). These mutant
receptors are functional, since they can transmit an IGF-I-mediated mitogenic signal (Fig. 3c), as we have already demonstrated
in mouse fibroblasts (21, 57, 58). In fibroblasts, we also have shown
that these mutant receptors protect from anoikis (58), an assay that,
unfortunately, cannot be carried out in 32D cells. Their inability to
induce differentiation is independent of their mitogenic capacity.
Second, Shc proteins seem to favor differentiation. True, it has been
difficult to obtain strong expression of Shc proteins in 32D cells,
which already express high levels of the 46- and 52-kDa isoforms. But
even a modest overexpression seems to increase the tendency of these
cells to differentiate. In addition, a dominant negative mutant of Shc
partially inhibited IGF-I-mediated differentiation. A role of Shc in
differentiation has also been suggested in PC12 cells induced by NGF
(35) and in 32D cells induced to differentiate by thrombopoietin (36).
We did not detect the 66-kDa isoform of Shc in 32D cells, confirming
previous results in the literature (34).
There are other findings that relate the Shc proteins to
differentiation by IGF-I. As mentioned above, Tyr950 and
Tyr1250/Tyr1251 seem to be necessary for the
differentiation process. The 1250/1251 residues of the IGF-IR have been
reported to bind a protein (59), although the relevance of this protein
to the differentiation process remains to be established. More
important is perhaps the fact that a mutation at Tyr1250
markedly decreases, but does not abrogate, the internalization of the
IGF-IR (57), and recent reports (37, 60) have shown that the IGF-I and
insulin receptors have to be internalized to phosphorylate Shc. This
could explain why Shc phosphorylation by the 1250/1251 mutant IGF-IR is
only decreased instead of being completely abolished, as in the case of
the Tyr950 mutant. If this interpretation is correct, the
requirement for 1250/1251 residues would be secondary to its effect on
internalization, and the really crucial residue for differentiation
would then be Tyr950 and its ability to phosphorylate Shc.
Indeed, the 32D cells expressing the Y950F mutant receptor completely
fail to phosphorylate Shc. Admittedly, the evidence for Shc inducing or
favoring differentiation is circumstantial; the results are not clear
cut, since we have partial inhibition by a dominant negative and
partial inhibition of Shc phosphorylation by the 1250/1251 mutant.
Perhaps another event is necessary that we have not yet identified.
Most important, however, is the notion that any differentiating effect
of Shc depends on the absence of IRS-1, whose overexpression simply
overwhelms any tendency to differentiation.
The Shc proteins have also been thought to regulate the mitogenic
process initiated by either insulin or IGF-I (61, 62). We can speculate
upon the relative proportions of IRS-1 and Shc proteins in determining
mitogenesis or differentiation, a hypothesis that was indirectly
suggested by the work of Yamauchi and Pessin (39). In agreement with
them (39), as already mentioned, the amount of Grb2 that
co-precipitates with either IRS-1 or Shc depends on the levels and
phosphorylation status of these substrates.
It will be an interesting pursuit to determine where the IRS-1 and Shc
pathways diverge and converge. A clue may be given by reports that the
interactions of the IGF-IR with IRS-1 and Shc, although both centered
at Tyr950, differ in the requirements for the surrounding
amino acids (50, 63) and that IRS-1 may also bind directly to the
tyrosine kinase domain (50). Another intriguing aspect of this
investigation is the relationship between survival and terminal
differentiation, which is known to result in cell death.
In conclusion, using 32D cells, we have established that the IGF-IR
induces granulocytic differentiation of murine hemopoietic cells and
that the outcome (differentiation versus proliferation) may
depend on a balance between two of its signaling pathways, IRS-1
inhibiting differentiation, and Shc favoring it. It seems reasonable to
conclude that, in the case at least of the IGF-IR, "cell context"
in determining growth or differentiation may depend on specific domains
of the receptor and the availability of different substrates.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit IGF-I receptor polyclonal antibody (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA). IRS-1 was immunoprecipitated and
immunoblotted with polyclonal anti-IRS-1 antibody (Upstate
Biotechnology, Inc., Lake Placid, NY). Shc proteins were
immunoprecipitated or immunoblotted with a polyclonal (Transduction
Laboratories) or a monoclonal (Santa Cruz Biotechnology) anti-Shc
antibody, respectively. Grb2 was immunoblotted with a monoclonal
anti-Grb2 antibody (Transduction Laboratories).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Overexpression of the IGF-I receptor induces
differentiation of 32D cells. 32D cells overexpressing the human
wild type IGF-IR (32D IGF-IR), like the parental 32D cells, grow well
in IL-3 and show no evidence of differentiation. After removal of IL-3,
32D IGF-IR cells were incubated in 10% serum supplemented with 50 ng/ml IGF-I or with G-CSF (100 units/ml). The number of surviving
(surv) and differentiating (diff) cells was
determined at various times thereafter. On the right is the
number of surviving cells and the percentage of differentiated 32D
cells at the times indicated after IL-3 withdrawal (range indicated to
the right of each value). On the left is the
morphology of these cells, either by Giemsa staining or after staining
for myeloperoxidase. For comparison, we are also showing the levels of
survival and G-CSF-induced differentiation in the parental 32D cells.
Survival is expressed as the fraction of cells recovered in relation to
the number of cells seeded. Differentiation is expressed as percentage
of band and polymorphonuclear cells in the surviving cells. These
experiments were repeated 3-5 times for each condition.
-subunit of the IGF-IR, but the same results were
obtained with an antibody to the
-subunit (not shown). The reason we
chose to present the
-subunit results is that the antibody to the
-subunit does not recognize the receptor truncated at residue 1245. Notice that the
-subunit of
1245 is of normal size, but the
proreceptor is a little shorter, because of the truncated
-subunit.
We have compared levels of expression with those of cell lines with a
known number of IGF-IRs (30). We can say that all populations express
at least 50,000 receptors/cell, which is more than it is needed for
other functions of the IGF-IR (30).
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Fig. 2.
Levels of expression of the transduced IGF-IR
or IRS-1 in 32D cells. a, Western blot of the various
mixed populations of 32D cells transduced with either a wild type or
different mutants of the IGF-IR, using an antibody to the -subunit
of the IGF-IR. The upper band is the proreceptor; notice that the
proreceptor is shorter in
1245, in which the last 92 amino acids of
the
-subunit are missing. Mutant receptors (20-22) are labeled as
follows. Y950, mutation to Phe at tyrosine 950;
3Y, tyrosines 1131, 1135, and 1136 mutated to Phe;
Y1250-1251, tyrosines 1250 and 1251 mutated to Phe; 6 serine, serines 1272, 1278, and 1280-1283 mutated to alanine;
4 basic aa, R1289F,H1290L,H1293F,K1294L;
1293-1294, H1293F,K1294L; 1245, wild type IGF-IR
truncated at residue 1245. b, IRS-1 and IGF-IR expression
levels in 32D cells and derivative cell lines. Western blot using the
antibody to IRS-1 or IGF-IR was as indicated under "Experimental
Procedures." See "Results" for explanation of cell lines.
Molecular masses (in kilodaltons) of marker proteins are
indicated.
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Fig. 3.
Survival and differentiation of 32D cells
expressing various mutants of the IGF-IR. Both survival and
differentiation were determined at day 4 after IL-3 withdrawal.
Survival is expressed as -fold increase (or decrease) over number of
cells plated. A 1-fold increase means that the number of cells has
doubled. Differentiation is expressed as in Fig. 1. a,
survival of the indicated cell lines in 10% serum, no IL-3, plus IGF-I
(50 ng/ml). b, differentiation of the same cell lines under
the same conditions. c, survival of the indicated cell lines
in 10% serum plus 0.1% IL-3 and plus IGF-I (50 ng/ml). d,
differentiation under the same conditions.
1245. Therefore, these cell lines can
differentiate when induced by G-CSF but not when induced by IGF-I.
Survival and differentiation of 32D-derived cell lines
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Fig. 4.
Expression of Shc and of a dominant negative
mutant of Shc in 32D cells. a, 32D cells were
transduced with a retroviral vector carrying the cDNA for the 46- and 52-kDa Shc proteins (a kind gift of Dr. Joseph Schlessinger). A
Western blot is shown of Shc proteins in parental 32D cells and in 32D
cells transduced with the Shc proteins cDNA. b, 32D
cells transduced with a retroviral vector expressing the SH2 domain of
the Shc proteins (see "Experimental Procedures"). A Western blot
shows both the endogenous Shc proteins and, below, the SH2 domain. 32D
IGF-IR/pac are 32D cells transduced with the empty retroviral vector
used for the SH2 domain mutant. Please note that the blot for the SH2
domain was exposed longer than the blot for the endogenous Shc
proteins.
Differentiation of 32D cells overexpressing Shc or the SH2 domain of
Shc
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Fig. 5.
Tyrosyl phosphorylation of IRS-1 and Shc
proteins under various conditions. a, Shc was
immunoprecipitated (IP) from lysates of the indicated cell
lines, and the blot was stained with an antiphosphotyrosine antibody
(P-tyr; first row) or with an
anti-Grb2 antibody (third row). After stripping,
the membrane was immunoblotted with a monoclonal anti-Shc antibody to
detect the amount of protein immunoprecipitated (second
row). Plus and minus signs
indicate that the cells were stimulated or not with IGFI (20 mg/ml) for
10 min. S, stimulation with 10% fetal bovine serum for 10 min. b, tyrosyl phosphorylation of IRS-1 or Shc and
co-precipitation of Grb2 with IRS-1 or Shc. The antibodies used are
described under "Experimental Procedures." On the left,
cell lysates were immunoprecipitated with an antibody to IRS-1, and the
membrane was blotted with an anti-phosphotyrosine antibody
(upper row) or with an antibody anti-Grb2
(lower row). On the right are the same
lysates but immunoprecipitated with an anti-Shc antibody. Molecular
masses (in kilodaltons) of marker proteins are indicated.
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Fig. 6.
Expression of IRS-1 increases the activation
of the Akt/p70 S6 kinase pathway. The cell lines 32D IGF-IR, 32D
GR15/IRS, and 32D IRS/IGF-IR were incubated in serum-free medium for
3 h and were then stimulated with IGF-I (20 ng/ml) for the
indicated times. Western blots are shown of lysates using the
antibodies described under "Experimental Procedures." a
and b, phosphorylation of Akt and levels of Akt proteins.
c and d, phosphorylation and protein levels of
p70 S6 kinase. Protein levels were determined on the same blots after
membrane stripping.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. R. G. Hawley for providing the murine stem cell virus vectors and to Dr. A. J. Kingsman for providing the pHIT vectors; to J. Verdone for skilled technical support; and to B. Vega for secretarial assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM 33694 and CA 53484.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Kimmel Cancer Center, Thomas Jefferson University, 233 S. 10th St., 624 BLSB, Philadelphia, PA 19107. Tel.: 215-503-4507; Fax: 215-923-0249; E-mail: r_baserga{at}lac.jci.tju.edu.
2 M. Dews and R. Baserga, manuscript in preparation.
3 F. Peruzzi, M. Prisco, M. Dews, P. Salomoni, E. Grassilli, B. Calabretta, G. Romano, and R. Baserga, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: IGF-IR, type I insulin-like growth factor receptor; IGF-I, insulin-like growth factor I; IR, insulin receptor; IRS, insulin receptor substrate; IL-3, interleukin-3; G-CSF, granulocyte-colony stimulating factor; PCR, polymerase chain reaction.
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