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[CANCER RESEARCH 60, 2263–2272, April 15, 2000]
Insulin-like Growth Factor I Receptor Signaling in Differentiation of Neuronal
H19-7 Cells1
Andrea Morrione, Gaetano Romano, Magali Navarro, Krzysztof Reiss, Barbara Valentinis, Michael Dews, Eva Eves,
Marsha Rich Rosner, and Renato Baserga2
Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [A. M., G. R., M. N., K. R., B. V., M. D., R. B.], and Ben May Institute for Cancer
Research, University of Chicago, Chicago, Illinois 60637 [E. E., M. R. R.]
ABSTRACT
The type I insulin-like growth factor receptor (IGF-IR) is known to
send two seemingly contradictory signals inducing either cell proliferation
or cell differentiation, depending on cell type and/or conditions. H19-7
cells are rat hippocampal neuronal cells immortalized by a temperaturesensitive SV40 large T antigen that grow at 34°C in epidermal growth
factor or serum but differentiate at 39°C when induced by basic fibroblast
growth factor. At 39°C, expression of the human IGF-IR in H19-7 cells
induces an insulin-like growth factor (IGF) I-dependent differentiation.
We have investigated the domains of the IGF-IR required for differentiation of H19-7 cells. The tyrosine 950 residue and serines 1280 –1283 in the
COOH terminus of the receptor are required for IGF-I-induced differentiation at 39°C, although they are dispensable for IGF-I-mediated growth
at 34°C. Both domains have to be mutated to inactivate the differentiating
function. The inability of these mutant receptors to induce differentiation
correlates with mitogen-activated protein kinase activation. In contrast,
inhibitors of phosphatidylinositol 3ⴕ-kinase have no effect on IGF-Imediated differentiation of H19-7 cells, although they do inhibit the
mitogenic response.
INTRODUCTION
The role of the IGF-IR3 in cell growth, transformation, and protection from apoptosis has been well established in our laboratory and
several other laboratories (reviewed in Refs. 1 and 2). The IGF-IR
also promotes differentiation in different cell types, such as myoblasts, osteoblasts, hemopoietic cells (reviewed in Ref. 3), and macrophages (4). There is also substantial evidence that the IGF-IR plays a
role in neural differentiation, beginning with the previous observations on oligodendrocytes (5) and neuronal cells, where the IGFs
promoted neurite outgrowth and tubulin mRNA production (6 – 8).
Additional references on the participation of the IGF-IR in neuronal
differentiation can be found in two recent reviews by Leventhal et al.
(9) and Anlar et al. (10). Even more interesting is a recent study by
Arsenijevic and Weiss (11), in which the authors state that IGF-I is a
differentiation factor for central nervous system stem cell-derived
neuronal precursors. These findings should not be construed as meaning that the IGF-IR is the sole or even the most important receptor in
neuronal differentiation. However, they indicate a role of the IGF-IR
in this process, in association with other growth factor receptors.
Given a role of the IGF-IR in neuronal differentiation, it is reasonable to ask how the IGF-IR participates in the differentiation of
neuronal cells. One approach is to determine the domains of the
IGF-IR required for neuronal differentiation because the identification
of these domains could give important clues on the mechanisms
involved. For this purpose, we infected H19-7 cells with retroviral
vectors expressing a WT or several mutants of the human IGF-IR.
H19-7 cells are rat hippocampal cells that have been conditionally
immortalized by transducing a retroviral vector expressing a temperature-sensitive SV40 large T antigen (12). This cell line proliferates at
the permissive temperature (34°C) in response to epidermal growth
factor or serum and differentiates to a neuronal phenotype in N2
medium supplemented with bFGF at the nonpermissive temperature
(39°C; Refs. 12 and 13). Differentiated H19-7 cells do not respond to
serum, extend neurites, and express neuronal markers, such as neurofilament proteins and brain type II sodium channels, and display
action potentials (12–14). Cells similarly immortalized by a temperature-sensitive SV40 large T antigen show region-specific neuronal
differentiation on transplantation into rat brains (15, 16).
In the present experiments, we wished first to establish whether the
activated IGF-IR could induce differentiation of H19-7 cells at 39°C
and then to determine the domains in the IGF-IR required for the
induction of differentiation. As a control, we examined the WT and
mutant IGF-IRs for their ability to respond to IGF-I with mitogenesis
at 34°C. We show here that tyrosine 950 and the serines 1280 –1283
in the COOH terminus of the IGF-IR are necessary for differentiation
of H19-7 cells but are dispensable for IGF-I-mediated mitogenesis.
This finding clearly separates the mitogenicity of the IGF-IR from its
ability to modulate differentiation in neuronal cells. We also carried
out preliminary experiments on IGF-IR signaling in these cells at the
two temperatures. The inability of certain mutant receptors to promote
differentiation correlates with their inability to give a sustained activation of ERK1/2, and differentiation is inhibited by MEK inhibitors,
thus confirming previous results by other investigators on the role of
MAPK in the differentiation of neuroblastoma cells (8, 17, 18).
However, inhibitors of the PI3K pathway have no effect on the
differentiation of H19-7/IGF-IR cells, although they completely inhibit the mitogenic response at 34°C.
MATERIALS AND METHODS
Cell Lines. H19-7 are rat hippocampal cells immortalized by transduction
of a retroviral vector expressing a temperature-sensitive SV40 large T antigen
(12). H19-7 cells are maintained in DMEM supplemented with 10% fetal
bovine serum and 200 ␮g/ml G418 (selection for the T antigen plasmid) in
Received 11/17/99; accepted 2/17/00.
flasks coated with 15 ␮g/ml poly-L-lysine (Sigma).
The costs of publication of this article were defrayed in part by the payment of page
R12, R508, R600, and p6 cells are mouse embryo fibroblasts expressing
charges. This article must therefore be hereby marked advertisement in accordance with
different numbers of human IGF receptor as described previously (19, 20).
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by NIH Grants CA 56309 and AG 16291 (to R. B.) and NS 33858 (to
Plasmids and Retroviral Vectors. pHIT60 and pHIT123 were kindly
M. R. R.).
provided by Dr. Alan Kingsman (Oxford University, Oxford, United King2
To whom requests for reprints should be addressed, at Kimmel Cancer Center,
dom) and are described elsewhere (21). pHIT60 contains the murine leukemia
Thomas Jefferson University, 233 South 10th Street, 624 BSLB, Philadelphia, PA 19107.
virus gag/pol cassette under the control of the human cytomegalovirus immePhone: (215) 503-4507; Fax: (215) 923-0249; E-mail: [email protected].
3
The abbreviations used are: IGF-IR, type I insulin-like growth factor receptor;
diate early promoter, whereas pHIT123 contains the human cytomegalovirus,
MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3⬘-kinase; IGF,
i.e., driven murine leukemia virus ecotropic envelope. pMSCV vectors were
insulin-like growth factor; bFGF, basic fibroblast growth factor; ERK, extracellular
kindly provided by Dr. Robert G. Hawley (University of Toronto, Toronto,
signal-regulated kinase; MEK, mitogen-activated protein/ERK kinase; SFM, serum-free
Canada). The human IGF-IR receptor cDNA was excised from the CVN
medium; NF68, neurofilament protein 68; BrdUrd, 5-bromo-2⬘-deoxyuridine; WT, wildplasmid (20, 22) by HindIII and HpaI digestion, filled in with Klenow
type; FGF, fibroblast growth factor; IRS-1, IR substrate 1; IR, insulin receptor.
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IGF-IR AND DIFFERENTIATION OF H19-7 NEURONAL CELLS
fragment, and inserted in the HpaI restriction site of pMSCVpac to generate
the plasmid pMSCVpac-IGF-IR. Therefore, this retroviral vector also carries
the gene for puromycin resistance (23). All of the mutants of the IGF-IR in
retroviral vectors were generated by the same strategy and have been described
previously (21). The Y950F-4S IGF-IR mutant was obtained by inserting the
HindIII/BamHI fragment from the SK-IGF-IR-4S (24) into the HindIII/BamHI
sites of the SK-IGF-IR-Y950F plasmid (25). The SK-Y950F-4S plasmid was
cut with SalI/BamHI, and the insert containing the full-length IGF-IR double
mutant was cloned into the XhoI/BglII sites of the pMSCVpac retroviral vector.
Transduction and Selection of H19-7 Cell Lines. Viral transduction for
the establishment of the various H19-7 cell lines was performed as described
in detail by Romano et al. (21). Mixed populations of transduced cells were
selected in medium containing puromycin (2 ␮g/ml).
Analysis of Differentiation. For differentiation experiments, the transduced H19-7 cell lines were plated at a density of 105 cells/35-mm plate in
medium containing serum at the permissive temperature. After 18 h, the cells
were washed extensively and shifted to the nonpermissive temperature in N2
SFM (DMEM-high glucose medium supplemented with 0.11 mg/ml sodium
pyruvate, 2 mM glutamine, 0.1 mg/ml transferrin, 20 nM progesterone, 0.1 mM
putrescine, and 30 nM sodium selenite) supplemented or not supplemented with
50 ng/ml IGF-I (Life Technologies, Inc.) or 5 ␮g/ml insulin. As a positive
control, cells were also plated in N2 SFM with IGF-I/insulin and 10 ng/ml
bFGF, a combination that is known to induce differentiation of parental H19-7
cells. After 48 h, the cells were analyzed, scored for neurite formation (processes longer than the cell body were considered neurites) with a Zeiss
microscope at a ⫻1000 magnification, and photographed with a 35 mm Nikon
camera (8, 26, 27).
Immunofluorescent Staining of NF68. On differentiation, H19-7 cells
show an increased expression of neurofilaments detectable by immunofluorescence cytochemistry using an antibody against NF68, and this increase in
expression of neurofilaments can be used as a marker of neuronal differentiation (27). The immunofluorescent staining of NF68 was performed as described by Kuo et al. (27), with some modifications. Cells were plated at a
density of 105 cells/35-mm, poly-L-lysine (Sigma)-coated plate in medium
containing serum at the permissive temperature; after 18 h, the cells were
washed extensively and shifted to 39°C in N2 SFM supplemented with 50
ng/ml IGF-I. As a positive control, cells were also plated in N2 SFM with
IGF-I and 10 ng/ml bFGF. After 48 h, the cells were fixed in 10% formalin
(methanol free) for 10 min at room temperature and permeabilized in 2%
formalin/0.2% Triton X-100 for 5 min at room temperature, and after washing
with PBS, the cells were blocked in 1% goat serum (in PBS) for 30 min at
37°C. After washing with PBS, the cells were than incubated for 1 h at 37°C
with a monoclonal antibody against NF68 (Sigma; clone NR4) used at a 1:200
dilution. The cells were washed extensively with PBS, and a secondary
antibody, fluorescein-conjugated antimouse IgG (Boehringer), was added at a
1:50 dilution and incubated for 30 min at 37°C. After washing with PBS, the
cells were covered with Vectashield (Vector Laboratories, Inc.), analyzed with
an immunofluorescence microscope, and photographed with a Nikon 35 mm
camera.
Analysis of Cell Growth. For cell growth experiments, the transduced
H19-7 cell lines were plated at a density of 5 ⫻ 104 cells/35-mm plate in
medium containing serum at the permissive temperature; after 18 h, the cells
were washed extensively and shifted to N2 SFM supplemented either with 50
ng/ml IGF-I (Life Technologies, Inc.) or 5 ␮g/ml insulin (Sigma). Cells were
counted after 3 days in a hemocytometer.
DNA Synthesis. This parameter was determined by using the BrdUrd
Detection and Labeling Kit (Boehringer Mannheim, Indianapolis, IN) as
described in detail by Reiss et al. (28).
Immunoprecipitation and Immunoblots. The expression of the IGF-IR
in the various cell lines was determined by Western blot on cells growing at
34°C as described previously (29 –31), using a polyclonal antibody against
either the ␣ or ␤ subunit of the IGF-IR (Santa Cruz Biotechnology). The
antibody against the ␣ subunit also recognizes the truncated forms of the
IGF-IR (␦1245 mutant). For detection of phosphorylated IGF-IR, IRS-1, or
Shc, the cells were serum-starved in N2 SFM for 24 h either at the permissive
or nonpermissive temperature and then stimulated with 50 ng/ml IGF-I for 10
min. Immunoprecipitations were performed as described previously (29 –31)
using a monoclonal antibody against the ␣ subunit of the IGF-IR (Oncogene
Sciences) or a polyclonal antibody against IRS-1 (Upstate Biotechnology) or a
polyclonal antibody against Shc (Transduction Laboratories). Phosphotyrosine
blots were performed with an anti-phosphotyrosine horseradish peroxidaseconjugated antibody (PY20; Transduction Laboratories). Endogenous Shc
proteins were detected using an anti-Shc monoclonal antibody from Santa Cruz
Biotechnology.
Phosphorylated ERK1 and ERK2 were detected using antibodies from
Promega. Akt (Ser-473), was detected using PhosphoPlus kits (New England
Biolabs) according to the manufacturer’s instructions. The level of endogenous
ERK1/2 was detected using polyclonal antibodies from Santa Cruz Biotechnology, whereas Akt was detected with antibodies also included in the PhosphoPlus kits.
Kinase Inhibitors. The PI3K inhibitor LY294002 (Biomol Research Laboratories) was dissolved in DMSO. For cell growth and differentiation experiments, LY294002 was added to the cells at the time of stimulation at a
concentration of 10 or 30 ␮M. For Akt phosphorylation, cells were preincubated for 15 min with 30 ␮M LY294002 before stimulating with IGF-I. For
inhibition of MAPK, we used the MEK inhibitor PD98059 (New England
Biolabs). For cell growth and differentiation experiments, PD98059 was added
to the cells at the time of stimulation at a concentration of 25 or 50 ␮M. For
ERK1/2 phosphorylation, cells were preincubated for 30 min with 50 ␮M
PD98059 before stimulation with IGF-I. U0126 (Calbiochem) was used at a
concentration of 1, 2.5, 5, and 10 ␮M at 39°C and was added at the time of
stimulation.
RESULTS
Establishment of H19-7/IGF-IR Cells. H19-7 rat hippocampal
cells proliferate at the permissive temperature (34°C) in response to
epidermal growth factor or serum and differentiate to a neuronal
phenotype in N2 medium supplemented with bFGF at the nonpermissive temperature of 39°C (12, 13). We infected H19-7 cells with a
retroviral vector expressing the human IGF-IR, and after selection, the
mixed population was tested by Western blot for the expression of the
receptor. Fig. 1C shows the levels of expression in the parental (Lane
1) and transduced cell lines, either with an empty vector (Lane 2) or
with the WT IGF-IR vector (Lane 3). This last cell line expresses a
considerable amount of the IGF-IR protein (arrow) compared with
parental or vector-transduced H19-7 cells. The other lanes in Fig. 1C
are lysates from different cell lines (mouse embryo fibroblasts) with a
known number of IGF-IRs (19, 20). For instance, R600 cells (Lane 6)
are known to express 3 ⫻ 104 receptors/cell, whereas p6 cells (Lane
7) express roughly 5 ⫻ 105 receptors/cell. On this basis, the level of
IGF-IR in transduced H19-7 cells can be estimated to be about
100,000 receptors/cell.
The IGF-IR Promotes Differentiation of H19-7 Cells. H19-7/
IGF-IR cells were tested for differentiation at 39°C or growth at 34°C
in NT2 SFM alone or supplemented with either IGF-I or insulin (see
“Materials and Methods”). The extent of differentiation was determined by the number of cells with neurite formation (see “Materials
and Methods” and the text below), although other markers were
occasionally used for confirmation. Fig. 1A shows that H19-7/IGFIR
cells differentiate in the presence of IGF-I or insulin at supraphysiological concentration. This concentration of insulin is known to
activate the IGF-IR (32). In NT2 SFM medium, there is a small
background level of differentiation, which is only slightly above the
level of differentiation of the parental cell line (or H19-7 cells transduced with the empty vector). Fig. 1B shows that all cell lines fail to
grow and actually decrease in number in N2 SFM. Only H19-7/
IGF-IR cells grow at 34°C, when the medium contains either IGF-I or
insulin. The other two cell lines still die under these conditions.
Interestingly, insulin at 5 ␮g/ml is as good as IGF-I in inducing
differentiation but is less effective in inducing cell growth. We did not
pursue this observation, which may be caused by differences in insulin
binding to the IGF-IR at the two temperatures. Another interesting
feature of Fig. 1 is that the parental H19-7 cells are incapable of
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IGF-IR AND DIFFERENTIATION OF H19-7 NEURONAL CELLS
signal, we transduced H19-7 cells with several mutants of the IGF-IR.
These mutants are listed in Table 1 and have been described previously (21, 24, 25, 33–36). After selection, mixed populations were
analyzed for the expression of the IGF-IR protein, using an antibody
Fig. 1. The IGF-IR induces differentiation of H19-7 cells. Three different cell lines
were used: the parental H19-7 cells and the same cells stably infected with an empty
retroviral vector (H19-7/V) or with a retroviral vector expressing the WT human IGF-IR.
The last two cell lines were mixed populations selected with puromycin. Three conditions
were used: N2 SFM only or N2 SFM supplemented with either insulin (5 ␮g/ml) or IGF-I
(50 ng/ml). A, differentiation at 39°C as determined by counting cells expressing neurites
after 48 h. B, cell proliferation, expressed as the percentage increase in cell number over
the number of cells plated after 72 h. C, Western blot of the IGF-IR in lysates from various
cell lines, using an antibody to the ␤ subunit. Lane 1, H19-7 parental cells; Lane 2,
H19-7/V cells; Lane 3, H19/7/IGFIR cells; Lane 4, R12 cells; Lane 5, R508 cells; Lane
6, R600 cells; Lane 7, p6 cells (See “Materials and Methods”).
growing at 34°C in IGF-I, despite the presence of an active SV40 T
antigen and a modest number of IGF-IRs.
H19-7/IGFIR cells at 39°C show a clear concentration dependence
on IGF-I for differentiation (Fig. 2A). The cells extend neurites (Fig.
2B, right) and show increased expression of NF68 (Fig. 2C, right),
which was used as a marker of neuronal differentiation (27). H19-7/V
cells were used as a control (Fig. 2, B and C, left). These experiments
indicate that increased expression of the human IGF-IR induces either
IGF-I-mediated growth at 34°C or IGF-I-mediated differentiation at
39°C.
Mutational Analysis of the IGF-IR. To identify the specific domains or residues in the receptor required for the differentiation
Fig. 2. IGF-I-mediated differentiation of H19-7/IGFIR cells. A, extent of differentiation of H19-7/IGFIR cells as a function of IGF-I concentration. B, neurite formation in
H19-7/IGFIR cells 48 h after IGF-I stimulation (right). H19-7/V cells were used as a
control (left). C, increased expression of NF68 in differentiating H19-7/IGFIR cells
(right). H19-7/V cells were used as a control (left).
Table 1 IGF-IR mutants used in these experiments
The generation of these mutants has been described in several studies from one of our
laboratories (21, 24, 25, 33–35).
H19-7 code
␦1245
Y950F-␦1245
Y950F
1289, 90, 93, and 94A
Y1316F
Y1250 and 51F
4S
6S
Y950F-4S
Y950F-3YF
IGF-IR mutants
Truncation at residue 1245
Tyrosine 950 to phenylalanine and truncation at
residue 1245
Tyrosine 950 to phenylalanine
Residues 1289, 90, 93, and 94 to alanine
Tyrosine 1316 to phenylalanine
Tyrosines 1250 and 1251 to phenylalanine
Serines 1280–1283 to alanine
Serines 1272, 1278, and 1280–1283 to alanine
Tyrosine 950 to phenylalanine and serines 1280–1283
to alanine
Tyrosines 950, 1131, 1135, and 1136 to
phenylalanine
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IGF-IR AND DIFFERENTIATION OF H19-7 NEURONAL CELLS
Fig. 3. Mutational analysis of the IGF-IR in
neuronal cell differentiation. The cell lines expressing the various IGF-IRs are indicated on the abscissa, and the mutations are summarized in Table
1. A, differentiation at 39°C in N2 SFM supplemented with IGF-I (50 ng/ml). B, growth at 34°C in
N2 SFM supplemented solely with IGF-I. C, Western blots of the IGF-IR from lysates of the various
cell lines (indicated above each lane). The lysates
were blotted with an antibody to the ␣ subunit of the
IGF-IR. The upper band is the proreceptor. For
convenience of comparison, the vector and the WT
receptor (WT) are repeated.
to the ␣ subunit. Fig. 3C shows that all mutant receptors were
expressed at a substantial level. Note that the proreceptors of the
␦1245 and Y950F-␦1245 cells are, as expected, slightly shorter than
the other proreceptors. Although there is some variability, all mutant
receptors are clearly overexpressed and much in excess of the 3 ⫻ 104
receptors/cell that are sufficient for mitogenicity and transforming
activity of the IGF-IR (Ref. 19; see also Fig. 1). These cell lines were
tested at 39°C for differentiation. As shown in Fig. 3A, all but four
mutant receptors induce differentiation at a level that is not significantly different from that of the WT receptor. The four exceptions are:
(a) the Y950F-␦1245 IGF-IR mutant; (b) the Y950-4S mutant; and (c)
the Y950F-3YF mutant, which have completely lost the ability to
induce differentiation; and (d) the ␦1245 mutant, which is defective in
inducing differentiation. The difference from the WT receptor is
statistically significant by the Student’s t test, where values of
P ⬍ 0.05 were considered to be significant.
The loss of the differentiating function could simply be due to the
fact that a mutant receptor is a disabled receptor, incapable of transmitting an IGF-I-mediated signal. The same cells were then tested for
their ability to grow at 34°C in N2 SFM supplemented solely with
IGF-I. The results are shown in Fig. 3B. All mutant receptors can
induce the growth of the transduced H19-7 cells, with the exception of
the Y950F-3YF mutant. This receptor is indeed a disabled receptor, as
also shown in mouse embryo fibroblasts (21). However, the other
three mutant receptors that were defective in differentiation are fully
capable of responding to IGF-I at 34°C with a growth response. For
simplicity, we have omitted the values of cell growth in N2 SFM only.
As shown in Fig. 1B, even the cell line with the WT receptor is not
proliferating without IGF-I. We can therefore say that a functional
IGF-IR is an absolute requirement for either mitogenesis or differentiation of H19-7 cells in N2 SFM supplemented solely with IGF-I. A
mutation at tyrosine 950 in combination with a deletion of the COOH
terminus or a mutation at serines 1280 –1283 results in receptors that
are normal for IGF-I-mediated mitogenesis (21, 35, 37) but incapable
of inducing differentiation.
It could be objected that the H19-7/Y950F-␦1245 and H19-7/
Y950 – 4S cell lines may have simply lost the capacity to differentiate.
They were therefore tested at 39°C for differentiation induced by a
combination of IGF-I and bFGF, a combination that is known to
promote differentiation of H19-7 parental cells (12, 13). Under these
conditions, these cells were fully differentiating, ruling out the possibility that they might have lost the ability to differentiate (data not
shown).
Fate of Differentiating Cells. Two important questions at this
point concern the ability of parental cells to respond to IGF-I and the
possibility that the lack of differentiation by some of the mutant
receptors may be due to their inability to sustain survival. To answer
the first question, we tested the levels of DNA synthesis at 34°C by
BrdUrd incorporation on selected cell lines. All of the cell lines tested
showed a clear increase in DNA synthesis after stimulation with IGF-I
(data not shown). The most relevant experiments are summarized in
Table 2. Interestingly, even the vector-transduced H19-7 cells used as
a control showed increased BrdUrd incorporation. This suggests that
the level of endogenous IGF-IR in H19-7 cells is sufficient to sustain
DNA synthesis (Table 2) but is not sufficient to induce cell division
(Fig. 1B; Fig. 3B). The dissociation between DNA synthesis and
mitosis is not surprising, and it has been reported for the IGF-IR in
previous studies (23, 28, 38, 39). However, this result shows that even
the parental cell line is sensitive to the action of IGF-I.
Table 2 DNA synthesis at 34°C and survival at 39°C of various H19-7 cell lines
For DNA synthesis, 105 cells were starved for 24 h in N2 SFM at 34°C and then
stimulated with IGF-I (50 ng/ml). BrdUrd was added 2 h after stimulation, and the cells
were fixed after 24 h. For survival, 105 cells were plated at 34°C in serum-containing
medium. After 24 h, the cells were shifted to 39°C in N2 SFM supplemented with IGF-I
(50 ng/ml; T ⫽ 0) and counted after 24 h. The number of cells is expressed in 104 and
represents averages from two separate experiments. V, vector; WT, wild type receptor;
Y950F-␦1245, double mutant.
% BrdUrdpositive cells
(34°C)
No. of cells (39°C)
H19-7 cell lines
SFM
IGF-I
T⫽0
T ⫽ 24 h
% decrease
V
WT
Y950F-␦1245
13.7
20.4
18.7
42
60
44.2
12
13
13
8.6
6
8.7
30
55
30
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IGF-IR AND DIFFERENTIATION OF H19-7 NEURONAL CELLS
To answer the second question, we determined the number of viable
cells for various cell lines at 39°C. There were no significant differences in the number of surviving cells after induction of differentiation between differentiating and nondifferentiating cell lines. If anything, the nondifferentiating cells survived slightly better (Table 2).
We also tried to correlate differentiation with DNA synthesis by
counting the cells that incorporated BrdUrd and displayed neurite
outgrowth. We could not find any correlation between these two
processes (data not shown).
Autophosphorylation of the IGF-IR. We have compared the
autophosphorylation of the WT IGF-IR with selected mutant receptors: (a) the Y950F-␦1245 mutant, which is mitogenic but does not
induce differentiation; and (b) the ␦1245 single mutant, which is
defective in differentiation. Fig. 4A shows that all receptors were
autophosphorylated on IGF-I stimulation at either temperature (compare with vector-transduced cells). The decrease in receptor autophosphorylation in the double mutant at 39°C is due in part to a slight
decrease in the amount of the IGF-IR protein immunoprecipitated
(Fig. 4B). However, the truncated receptors are expected to show a
decreased level of phosphorylation because they lack three tyrosine
residues (tyrosine 1250, tyrosine 1251, and tyrosine 1316) that are
known to be phosphorylated on IGF-I stimulation. As expected, the ␤
subunit of the IGF-IR is detected as a faster migrating band by
phosphotyrosine antibodies in the two cell lines expressing the truncated receptors (Fig. 4A). This difference is not evident in the total
protein blot (Fig. 4B), where we used antibodies against the ␣ subunit
of the IGF-IR.
The endogenous IGF-IR in H19-7/V cells is not detectable (Fig. 4B)
under these conditions; increasing the exposure time can visualize it,
although the overexpressed receptors would be grossly overexposed.
Phosphorylation of IRS-1 and Shc. Although we could not detect
significant differences in the autophosphorylation of the IGF-IR, it is
reasonable to ascertain whether these receptors may differ in the
activation of IRS-1 (40) and Shc proteins (41), the two major substrates of the IGF-IR. We determined tyrosyl phosphorylation of these
two substrates at both temperatures. The results for Shc are shown in
Fig. 4C (tyrosyl phosphorylation) and Fig. 4D (amounts of Shc
protein immunoprecipitated), and the results for IRS-1 are shown in
Fig. 4E (tyrosyl phosphorylation). The most important comparison is
between the cells with the WT receptor and the cells with the double
mutant Y950F-␦1245 receptor.
The Mr 52,000 Shc was tyrosyl phosphorylated in cells with either
the WT receptor or the ␦1245 receptor, but not in cells with the Y950F
mutation or the double mutant Y950F-␦1245 (Fig. 4C). This was
expected because tyrosine 950 is a major binding site for Shc proteins
(42) and is required for their activation. However, the results were the
same at both temperatures. When we tested the level of phosphorylation of IRS-1, we could not detect any significant difference in
tyrosyl phosphorylation of IRS-1 between the cells expressing the WT
IGF-IR and the double mutant on stimulation with IGF-I at either
temperature (Fig. 4E). In fact, IRS-1 is phosphorylated in all four cell
lines tested. We also determined the level of tyrosyl phosphorylation
of IRS-2 (43) at 39°C, and we did not detect any difference in tyrosyl
phosphorylation of IRS-2 between cells expressing the WT and cells
expressing the Y950F-␦1245 mutant receptor (data not shown).
These results show that these receptors, whether capable of inducing differentiation or not, are signaling to one or the other of their
major substrates. The results with Shc proteins will be discussed
below.
The PI3K Pathway Is Dispensable for Differentiation of H197/IGF-IR Cells. According to a number of investigators (44), there
are two major signaling pathways for the IGF-IR. The first is through
IRS-1, the activation of PI3K (45) and Akt/PKB kinase (46 –50),
Fig. 4. Phosphorylation of the IGF-IR and its major substrates. Lysates were made
from cells stimulated for 10 min with IGF-I or from cells left unstimulated. The respective
proteins were immunoprecipitated with the appropriate antibody, and the blots were then
stained with an anti-phosphotyrosine antibody (A, C, and E) and, after stripping, reblotted
with a specific antibody to determine the amount of protein that was immunoprecipitated
(B and D). Cell lines are indicated above the lanes, antibodies are indicated on the left, and
temperatures are indicated at the bottom (see “Materials and Methods” for details).
whereas the second major pathway is through MAPK (51, 52). We
investigated the role of PI3K in differentiation of H19-7/IGFIR cells
by incubating them with an inhibitor of PI3K, LY294002. Fig. 5A
shows that the inhibitor LY294002 does not inhibit IGF-I-mediated
differentiation of H19-7/IGF-IR cells, even at a concentration of 30
␮M. The inhibitor is effective on these cells because when the experiment is conducted at 34°C, it markedly inhibits IGF-I-mediated
growth (Fig. 5B). Thus, the activation of PI3K is necessary for the
mitogenic response of H19-7/IGFIR cells, but not for their differentiation. This was confirmed by examining Akt/PKB activation in these
same cells. Fig. 5C shows that Akt is activated by IGF-I in H19-7/
IGFIR cells (Lanes 2 and 5). The addition of LY294002 causes a
complete inhibition of Akt/PKB activation at both 34°C and 39°C
(Lanes 3 and 6). However, despite the complete inhibition of Akt/
PKB activation, H19-7/IGFIR cells still differentiate at 39°C. Incidentally, this last experiment also rules out the possibility that
LY294002 may be inactivated at 39°C because its effect on Akt/PKB
activation is just as dramatic as it was at 34°C.
Because the PI3K Akt/PKB pathway activates p70s6k (44, 53), we
investigated the activation of p70s6k in the cell lines mentioned above
(Fig. 4, C–E) at either temperature. p70s6k was activated in all four
cell lines at both temperatures (data not shown). The activation of
p70s6k confirms that IGF-I activates this pathway in H19-7/IGF-IR
cells, although the pathway is not crucial for differentiation.
MAPK Activation. In these experiments, we examined activation
of ERK1 and ERK2 at various times (up to 2 h) after stimulation with
IGF-I. For convenience, we show only five cell lines (those expressing the WT receptor, the ␦1245 receptor, the Y950F receptor, and the
two double mutants). The other receptors listed in Table 1 are already
known to activate MAPK (21, 32). A representative experiment is
shown in Fig. 6, but these experiments were repeated several times
with essentially the same results. In all cell lines, stimulation with
IGF-I at 34°C causes a strong and prolonged activation of ERK1 and
ERK2, as illustrated for two of the cell lines tested in Fig. 6G.
However, at 39°C (Fig. 6, A, C, and E), the cells with the double
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Effect of a MAPK Inhibitor. To confirm the importance of
MAPK signaling in differentiation of H19-7/IGFIR cells, we incubated these cells with the MEK inhibitor PD98059. Fig. 7A shows that
PD98059 inhibits the differentiation of H19-7/IGFIR cells in a concentration-dependent manner. The inhibition is not complete; nevertheless, it is both significant and reproducible. Fig. 7B shows that
PD98059 markedly inhibits the activation of MAPK at a concentration of 50 ␮M. The MEK inhibitor was also tested at 34°C, and Fig.
7D shows that it also inhibits IGF-I-mediated mitogenesis. We also
tried another inhibitor of MEK, UO126 (54), which has a higher
affinity for MEK than PD98059. At 2.5 ␮M, UO126 inhibited IGF-Imediated differentiation in H19-7/IGF-IR cells by 50%, and the
inhibition was complete at 10 ␮M (data not shown). Therefore, using
the inhibitors, one can say that in these cells MAPK activation is
required for both mitogenesis and differentiation, whereas the PI3K
pathway is required only for mitogenesis.
DISCUSSION
The primary conclusions of these experiments are summarized as
follows. (a) The expression of a WT human IGF-IR promotes IGFI-mediated differentiation of H19-7 rat hippocampal cells at 39°C.
The ability of the IGF-IR to induce differentiation of H19-7 cells is
dependent on the concentration of IGF-I. (b) The WT IGF-IR is also
mitogenic at 34°C. A functional IGF-IR is necessary for IGF-Imediated mitogenesis at 34°C or differentiation at 39°C. (c) We have
identified two domains in the IGF-IR that are necessary to induce
differentiation of H19-7 cells. The first domain is tyrosine 950, and
the second domain localizes in the COOH terminus at a serine quartet
in residues 1280 –1283. Both domains have to be mutated for differentiation to be inhibited. The double mutant receptors that fail to
induce differentiation are still capable of responding to IGF-I at 34°C
with mitogenesis. Secondary conclusions of these experiments include: (a) sustained activation of MAPKs correlates with the ability of
the various receptors to induce differentiation of H19-7 cells. Inhibitors of the MAPK pathway inhibit both mitogenesis and differentiation; and (b) in contrast, the PI3K pathway is necessary for IGF-Iinduced mitogenesis but not for differentiation of H19-7/IGF-IR cells.
These points will be discussed separately.
Fig. 5. PI3K activation is not required for differentiation of H19-7/IGFIR cells. A,
differentiation of H19-7/IGFIR cells in the presence of LY294002 at 39°C. B, growth of
H19-7/IGFIR cells at 34°C in the presence of LY294002. C, activation of Akt/PKB by
IGF-I in the presence or absence of LY294002. D, amounts of Akt/PKB protein in the
lysates. C and D: Lanes 1, unstimulated cells at 34°C; Lanes 2, IGF-I stimulation; Lanes
3, IGF-I plus LY294002 (30 ␮M); Lanes 4 – 6, same treatments as Lanes 1–3 at 39°C. The
methods are described in “Materials and Methods.”
mutant receptors show an activation that is not sustained but returns to
basal levels after 10 min. In the other cell lines (WT receptor and
Y950F), activation of ERK1 and ERK2 is sustained for at least 2 h,
even at 39°C. This result indicates a correlation between sustained
MAPK activation and IGF-I-mediated differentiation of H19-7/IGFIR
cells. Because the cells with the ␦1245 receptor are slightly defective
in differentiation, one would have expected a stronger impairment in
MAPK activation in these cells. Although there is a clear decrease
between 10 and 30 min, the level of MAPK activation in the ␦1245
mutant is still above the basal level at 2 h (Fig. 6A). We will return to
this observation in the “Discussion.”
Fig. 6. MAPK activation in H19-7-derived cell lines. ERK1/2 activation was determined as described in “Materials and Methods.” The cells were either quiescent or
stimulated with IGF-I for the indicated times (in min). A, C, and E show ERK1/2
activation in the cell lines tested at 39°C, whereas G shows the activation of ERK1/2 at
34°C in cells expressing the WT receptor and one of the double mutants. The amount of
ERK1/2 proteins is shown in B, D, F, and H.
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Fig. 7. Inhibitors of MAPK activation decrease IGF-I-mediated differentiation. The
cells chosen for this experiment were H19-7/IGFIR cells, which differentiate on IGF-I
stimulation. A, effect of the MEK inhibitor PD98059 on differentiation as determined after
48 h in N2 SFM supplemented with IGF-I at 39°C. B, ERK1/2 activation in the absence
or presence of PD98059 (50 ␮M). C, Western blot of total ERK1/2 proteins. D, effect of
MAPK inhibitors on growth of cells at 34°C, as determined at 72 h.
The role of the IGFs and the IGF-IR in the central nervous system
has been well established (see “Introduction” and Refs. 55 and 56).
This study therefore deals not with the role of the IGF-IR in neuronal
differentiation but with the mechanism(s) by which this receptor
participates in the differentiation process. The domains of the IGF-IR
necessary for mitogenesis, transformation, and survival have been
well characterized (for reviews, see Refs. 2 and 57). There is one
report on the domains of the IGF-IR required for the granulocytic
differentiation of murine hemopoietic cells (58), but no data are
available at the moment on the domain(s) of the IGF-IR necessary for
the differentiation signal in neuronal cells.
Several mutants of the IGF-IR have been examined. A mutant
receptor truncated at residue 1245 (therefore lacking the last 92 amino
acids) was defective in differentiation. A double mutant (mutation at
tyrosine 950 in combination with a truncation at the COOH terminus,
Y950F-␦1245 mutant) completely lost the ability to induce differentiation while maintaining its ability to induce growth at 34°C. Another
double mutant, Y950F-serines 1280 –1283A (Y950F-45), has also lost
the ability to differentiate H19-7 cells while still being capable of
giving a mitogenic response. We can therefore say that we have
identified two domains in the IGF-IR required for IGF-I-induced
differentiation of H19-7 rat hippocampal cells: (a) tyrosine 950
(which is located in the juxta-membrane region); and (b) the serines
1280 –1283 in the COOH terminus. The receptor with these two
mutations is not a disabled receptor that cannot transmit an IGF-Iinduced signal because it can induce mitogenesis in H19-7 cells at
34°C. Interestingly, similar results have been reported recently for
FGF receptor 1, where both the juxtamembrane and the COOHterminal regions of the receptor were identified as necessary for
induction of FGF-stimulated neurite outgrowth of PC12 cells (59).
It is important to establish that the receptors defective in differentiating ability are not disabled receptors. Using the double mutant
receptors, we have shown that these receptors are autophosphorylated
at 39°C and can activate some of the transducing molecules downstream of the IGF-IR, including tyrosyl phosphorylation of IRS-1 and
IRS-2 and activation of the p70s6k protein. The tyrosine 950 mutants
are defective in Shc phosphorylation, and this may be related to the
defect in MAPK activation that will be discussed below. A truly
disabled IGF-IR is the Y950F-3YF mutant. H19-7 cells expressing
this mutant do not differentiate, but they are also totally insensitive to
IGF-I-mediated mitogenesis. This receptor has also been found be
incapable of stimulating growth in mouse embryo fibroblasts, where
it cannot induce tyrosyl phosphorylation of either IRS-1 or Shc (21).
Of the two domains we have identified, the tyrosine 950 residue is
not required for mitogenesis but is required for transformation of
mouse embryo fibroblasts (21). Its importance in apoptosis is ambiguous because it provides only partial protection (21, 60). A single
mutation at tyrosine 950 does not seem to affect IGF-I-mediated
differentiation of H19-7 cells. This is in sharp contrast to the results
in the differentiation of hemopoietic 32D cells, where a single mutation at tyrosine 950 did inhibit IGF-I-mediated differentiation (58).
The second domain we have identified in these experiments is
constituted by the serine quartet at 1280 –1283. We have previously
shown in one of our laboratories that an IGF-IR with mutations at
serines 1280 –1283 in the COOH terminus is defective in transformation and protection from apoptosis but is perfectly normal for monolayer growth induced by IGF-I (21, 24). The fact that these two
mutations do not affect the mitogenicity of the IGF-IR (Refs. 21 and
24 and this study) but do affect differentiation clearly separates these
two functions of the IGF-IR to different domains.
The next question is how these mutations may influence IGF-IR
signaling. Tyrosine 950 is the main binding site for one of the major
substrates of the IGF-IR, the Shc proteins (42). Because Shc proteins
seem to play a role in IGF-I-mediated differentiation of 32D cells
(58), we examined the behavior of Shc proteins during the differentiation of H19-7 cells. The phosphorylation of the Mr 52,000 Shc
protein (61) was severely impaired in cells expressing the Y950F␦1245 receptor. Unfortunately, it is even less phosphorylated in cells
expressing the Y950F mutant, which differentiate normally. Kim et al.
(17) reported that a dominant negative mutant of Shc inhibited differentiation of neuroblastoma cells. We tested a dominant negative
mutant of Shc in H19-7/IGF-IR cells, but it had no effect on differ-
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entiation (data not shown). The same mutant was quite effective in
one of our laboratories in inhibiting IGF-I-mediated differentiation of
32D cells (58). We also overexpressed Shc p46 and p52 proteins by
retroviral infection in parental H19-7 cells, but we could not detect
any differentiation induced by IGF-I in H19-7/Shc cells at 39°C (data
not shown). It seems that in these cells, the role of Shc proteins in
differentiation is ambiguous. They may play a role, but only in
combination with other signal(s).
As to the other domain, the serines at 1280 –1283 are a binding site
for 14-3-3 adapter proteins (62, 63), and this interaction is valid only
for the IGF-IR and is not shared by the IR (62, 63). 14-3-3 proteins
have been shown to stabilize and activate Raf kinases (64 – 68). A
mutation at serine 1280 –1283 has also been shown to interfere with
the mitochondrial translocation of Raf, which occurs when cells
expressing the WT receptor are stimulated with IGF-I (69). The
interaction of 14-3-3 proteins with the IGF-IR at the serines in the
COOH terminus could therefore serve as an alternative pathway to
activate Raf kinases in a Ras-independent way, a pathway that would
not be shared by the IR. Indeed, although it may be coincidental, we
have shown that the IR, even when overexpressed, cannot induce
differentiation of H19-7 cells (70).
It could be argued that the COOH terminus signal may be more
important than the tyrosine 950 signal because there is a small but
significant decrease in differentiation with the ␦1245 receptor but not
with the Y950F receptor. It seems that an intact tyrosine 950 can
partially replace the need for a COOH terminus signal. On the other
hand, the presence of a COOH terminus signal seems to completely
obviate the need for tyrosine 950. It is difficult, at this point, to explain
this difference. One could speculate that the COOH terminus sends
two separate signals, one that is specific to the COOH terminus (the
four serines?) and one that is redundant with the signal from tyrosine
950 (Shc proteins?). In an attempt to gain some information on this
point, we have explored IGF-IR signaling in these cells.
One pathway of the IGF-IR that does not seem to be required for
differentiation of H19-7/IGFIR cells is the class I PI3K pathway (53).
This statement is supported by the following findings: (a) inhibitors of
PI3K activity have no effect on IGF-I-mediated differentiation, although they inhibit mitogenesis; (b) these inhibitors completely inhibit Akt/PKB activation at both temperatures, but only mitogenesis is
affected; and (c) the p70s6k kinase (53) is normally activated in WT
and relevant mutant receptors at both temperatures.
In contrast, it is clear that MAPK activation is required for differentiation of H19-7/IGF-IR cells. The importance of MAPKs in differentiation of neuronal cells has already been reported in different
cellular models, and, in this respect, our experiments simply confirm
and extend the results previously reported from other laboratories with
different cells of neuronal origin. Sustained activation of MAPKs
(71–73) has been shown to promote either FGF- or nerve growth
factor-mediated differentiation of PC12 cells (74, 75). A role of
MAPKs in differentiation of PC12 cells has been also reported by
Nguyen et al. (76). Activation of MAPKs is also necessary for
IGF-I-mediated neurite outgrowth of SH-SY5Y neuroblastoma cells
(8, 17, 18), but the signaling leading to MAPK activation is still
controversial. One report shows a role of the Shc-Grb2 complex in
mediating ERK activation (17), whereas in another report, PI3K
seems to be required for ERK activation and differentiation (18).
Another important difference between SH-SY5Y neuroblastoma cells
and H19-7 cells is that the former cells do not have IRS-1 (18). IRS-1
is known to have a profound effect on the differentiation of murine
hemopoietic cells (58), and the presence of IRS-1 in H19-7/IGF-IR
cells could also explain the lack of effect on differentiation, as we
mentioned previously, of a dominant negative of Shc. The difference
at the two temperatures in the duration of MAPK activation in the
double mutant receptors can be explained. The SV40 T antigen, active
at 34°C is sending an additional signal to activate MAPK, a signal that
is lost at 39°C, where the T antigen is inactive. We have shown
previously that the interaction between the T antigen and IRS-1
promotes transformation of mouse embryo fibroblast (77) and protects
32D cells from apoptosis (78). The activation of IRS-1 by the SV40
T antigen is sending a strong mitogenic signal that is prevailing over
the differentiation program at 34°C, a signal that is missing at 39°C,
where the differentiation program prevails.
As mentioned above, PI3K activation seems dispensable for differentiation of H19-7/IGF-IR cells. Clearly, IGF-IR signaling and functions vary from one cell type to another, and this variability has been
vigorously demonstrated in a recent review by Petley et al. (3). The
variability in signals probably depends on the availability of substrates
and transducing molecules, as demonstrated in the granulocytic differentiation of 32D cells (58).
In conclusion, our experiments have identified two domains in the
IGF-IR required for IGF-I-mediated differentiation of H19-7 neuronal
cells. When both domains are mutated, the IGF-IR no longer induces
H19-7 cell differentiation. Our experiments also point out how little
certain mutations of the IGF-IR affect its mitogenicity. Unless the
receptor is simply inactivated (mutations at the ATP-binding site or at
the tyrosine kinase domain), other mutations have little effect on the
ability of the IGF-IR to transmit a mitogenic signal, as we had
observed repeatedly in other cell lines (2, 57). The two domains we
have identified send signals that apparently converge on the activation
of ERKs. Additional studies will be necessary to prove our hypothesis
that one of these signals is ras dependent (through Shc signaling),
whereas the other one is ras independent, perhaps by the activation of
Raf kinases through their interaction with the 14-3-3 proteins.
ACKNOWLEDGMENTS
We thank J. Verdone for skilled technical support and B. Vega for secretarial assistance.
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Insulin-like Growth Factor I Receptor Signaling in
Differentiation of Neuronal H19-7 Cells
Andrea Morrione, Gaetano Romano, Magali Navarro, et al.
Cancer Res 2000;60:2263-2272.
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