(Received for publication, April 4, 1997, and in revised form, May 15, 1997)
From the Endocrine Research Unit, Mayo Clinic and Mayo Foundation, 5-164 W. Joseph, Rochester, Minnesota 55905
Insulin-induced desensitization to insulin-like
growth factor-I (IGF-I) stimulated mitogenesis in bovine fibroblasts
involves steps distal to IGF-I binding to its tyrosine kinase receptor. When quiescent cultures of bovine fibroblasts were stimulated with 10 nM IGF-I and total cell lysates immunoblotted with
anti-phosphotyrosine antibody, we observed a band at ~97 kDa,
representing the -subunit of the IGF-I receptor, and a predominant
tyrosyl-phosphorylated species migrating as a broad band between 170 and 190 kDa. The majority of proteins in this latter band were
immunoprecipitated by antibodies against insulin receptor substrate
(IRS)-2 and not by antibodies against IRS-1. Pretreatment of bovine
fibroblasts with 10 nM insulin for 48 h blocked
subsequent IGF-I-stimulated DNA synthesis and the IGF-I-induced
increase in tyrosyl-phosphorylated IRS-2. Insulin pretreatment did not
alter IRS-1 or IRS-2 expression by these cells, as assessed by
metabolic labeling and direct immunoblotting with IRS antibodies. The
interleukin-4 (IL-4) cytokine receptor also has IRS-2 as its major
substrate for tyrosine phosphorylation. Although 10 nM IL-4
was as effective as 10 nM IGF-I in stimulating IRS-2
phosphorylation, 10 nM IL-4 did not have comparable
mitogenic power in these cells. Nonetheless, pretreatment of bovine
fibroblasts with IL-4 inhibited IGF-I-stimulated DNA synthesis by
50-60%, concomitant with a decrease in IGF-I-induced IRS-2
phosphorylation. Insulin-induced desensitization could be prevented if
a specific inhibitor of phosphatidylinositol 3-kinase (LY294002), but
not an inhibitor of mitogen-activated protein kinase (PD98059), was present during the preincubation period. LY294002 also prevented the
shift in IRS-2 molecular mass in response to prolonged incubation of
cells with insulin. These data indicate that, in a nontransformed cell
system, IRS-2 plays a key role in cellular desensitization to
IGF-I-stimulated mitogenesis most likely through a feedback mechanism
in the phosphatidylinositol 3-kinase pathway. Furthermore, they suggest
that signaling through IRS-2 may provide an important point of
integration for hormone, growth factor, and cytokine receptor systems
that regulate critical cellular growth responses.
Peptide growth factors bind to specific transmembrane receptors to initiate intracellular events that must be highly coordinated and controlled to elicit appropriate changes in nuclear gene expression and consequent cell replication. This coordination must include important molecular mechanisms that operate to restrain mitogenic signaling as well, since constitutive activation of growth factor pathways would be dysfunctional. Thus, negative feedback mechanisms and internal checks are implicit, if not always explicit, in every important regulatory system involved in cell growth. Furthermore, cross-talk and overlap in receptor intracellular signaling pathways, especially as they apply to heterologous interactions, are a powerful means of processing and coordinating external signals to set cell context and generate an appropriate response to the environment. Little is known about the molecular basis of these interactions, however.
Insulin-like growth factor-I (IGF-I)1 is an essential growth-promoting peptide that shares structural and functional features with insulin (1). We have previously shown that IGF-I is a potent mitogen for normal bovine fibroblasts in culture (2, 3). However, pretreatment with physiologic insulin concentrations rendered these cells refractory to subsequent IGF-I stimulation of DNA synthesis (3). This insulin-induced desensitization to IGF-I is selective (i.e. no loss of responsiveness to serum), is mediated by specific insulin receptors on bovine fibroblast, and involves events proximal to induction of the nuclear proto-oncogene, c-myc,2 and distal to IGF-I receptor binding and activation (3). These data suggested that insulin controlled cell response to IGF-I at an intracellular step along a mitogenic pathway shared by insulin and IGF-I.
The biological effects of insulin and IGF-I are mediated by specific
cell surface receptors. These receptors are structurally homologous
2
2 heterotetramers belonging to the
family of ligand-activated receptor tyrosine kinases (4-7).
Extracellular binding of ligand to receptor
-subunits induces
conformational changes and phosphorylation of receptor
-subunits on
tyrosines leading to autoactivation of the receptor tyrosine kinase
toward specific intracellular proteins. The major receptor substrates
include IRS (insulin receptor substrate) and Shc (Src-homology
2/
-collagen) proteins. In most cell systems studied, IRS-1 (~170
kDa) is the predominant substrate phosphorylated in response to
stimulation by insulin or IGF-I (8, 9); and several observations
suggest a key role for IRS-1 in control of cell proliferation by
insulin and IGF-I (10-13). Phosphorylation of IRS-1 on multiple
specific tyrosines has the potential for divergent and amplified
signaling. Phosphorylated IRS-1 stimulates engagement and activation of
phosphatidylinositol 3-kinase (PI 3-kinase) and its cell growth
signaling pathway (14-16). Recent data suggest that feedback from
secondary signals may attenuate the initial signaling potential of
IRS-1 through activation of protein-tyrosine phosphatases and/or
serine/threonine kinases (17, 18). Phosphorylated IRS-1 can also
activate the mitogen-activated protein (MAP) kinase/extracellular
signal-related kinase pathway (8, 19). Shc phosphorylation is
stimulated strongly by mitogens and growth factors, and appears to act
primarily through MAP kinase activation (20). Indeed, MAP kinase has
been suggested to be a point of integration for multiple intracellular
signals transmitted by various mitogens, and to serve as the
cytoplasmic connection between plasma membrane and nuclear events
(21).
IRS-2 was discovered as an alternative insulin receptor substrate in
IRS-1 knock-out mice (22, 23). IRS-2 is slightly larger (~190 kDa)
and immunologically distinct from IRS-1, but in most in
vitro systems IRS-2 appears to function as does IRS-1, i.e. IRS-2 has the potential to link insulin and IGF-I
signaling to both PI 3-kinase and MAP kinase activation (22-25). A
physiologic role for IRS-2 has not been documented as yet, although two
phosphotyrosine binding elements in IRS-2, which are not found in
IRS-1, suggest a distinct functional role in vivo (26).
Furthermore, Peraldi et al. (27) recently reported a
difference between IRS-1 and IRS-2 in their sensitivity to tumor
necrosis factor -induced inhibition in myeloid 32D cells. In
addition, IRS-2 was found to be identical to 4PS, the major
tyrosine-phosphorylated substrate of the interleukin-4 (IL-4) receptor
(23). Unlike the insulin and IGF-I receptors, the IL-4 receptor is not
a tyrosine kinase itself, but upon activation it stimulates an
associated Janus kinase, which phosphorylates tyrosine residues on the
IL-4 receptor (23, 25, 28, 29). 4PS has also been shown to engage and activate PI 3-kinase (23).
The purpose of this study was to define the molecular events underlying insulin-induced desensitization to IGF-I using the nontransformed bovine fibroblast model. Herein we report a novel role for IRS-2, an intracellular signaling molecule shared by IGF-I, insulin, and IL-4 receptors, in regulating the cells' mitogenic response to IGF-I.
Recombinant human IGF-I and interleukin-6 (IL-6)
were purchased from R & D Systems, Inc. (Minneapolis, MN). Crystalline
bovine insulin was kindly provided by Lilly (Indianapolis, IN). IL-4, fetal bovine serum and radioimmunoassay grade bovine serum albumin were
from Sigma. Tissue culture media and supplements were obtained from
Life Technologies, Inc. Antibodies against phosphotyrosine (PY20)
were purchased from Transduction Laboratories (Lexington, KY), and
antibodies against IRS-1 (
IRS-1) and IRS-2 (
IRS-2) were from
Upstate Biotechnology Inc. (Lake Placid, NY). A monoclonal IGF-I
receptor antibody generated against a synthetic peptide corresponding
to sequences in the C terminus of the
-subunit was generously
provided by Dr. R. J. Smith (Boston, MA). PD98059 and LY294002 were
obtained from New England Biolabs, Inc. (Beverly, MA) and Biomol
Research Laboratories, Inc. (Plymouth Meeting, PA), respectively.
Bovine dermal fibroblasts (GM06034) were purchased from the Human Genetic Mutant Cell Repository (Camden, NJ). Fibroblasts were cultured in Dulbecco's modified Eagle's medium supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and 4 mM glutamine, and containing 10% fetal bovine serum. Cultures were used between passages 7 and 14. For all experiments, confluent cells were washed and incubated for 48 h in serum-free Waymouth's medium:Dulbecco's modified Eagle's medium plus 0.1% bovine serum albumin (SFM) with or without the indicated additions (3). After this preincubation period, cells were treated as described for the different methodologies.
DNA SynthesisCells were washed three times and the medium changed to SFM with or without peptide stimulus. [3H]Thymidine (0.5 µCi/ml; 1.5 µM) was added at 22 h, and the cells were harvested at 26 h. Details of this method have been reported previously (2, 3). Results are expressed as the percent of total counts in the incubation medium incorporated into acid-precipitable material.
Cell LysatesCells were acutely treated with peptide stimulus, washed with ice-cold phosphate-buffered saline containing 2 mM Na3VO4, and then lysed with 20 mM Tris, pH 7.6, containing 137 mM NaCl, 1 mM MgCl2, 2 mM Na3VO4, 1% Nonidet P-40, 10% glycerol, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, leupeptin (10 µg/ml), and aprotonin (10 µg/ml). Cell lysates were sonicated and centrifuged at 12,000 rpm for 10 min at 4 °C. Protein content of the lysate was determined using a BCA protein assay (Pierce Chemical Co.).
ImmunoblotEqual amounts of lysate protein (~100 µg) were processed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (100 mM dithiothreitol) using a 5-15% linear gradient, and then transferred to nitrocellulose. Filters were blocked for 4-6 h, incubated overnight at 4 °C with primary antibody or nonspecific IgG, washed, and then incubated for 2 h at room temperature with horseradish peroxidase-conjugated secondary antibody. Antigen-antibody reactions were visualized using an enhanced chemiluminescent detection system (Amersham Life Sciences).
ImmunoprecipitationCell lysates were incubated for 4-6 h at 4 °C with 4 µg of primary antibody/mg of total protein. Protein A-agarose beads (Oncogene Science Inc., Uniondale, NY) were added and incubation continued overnight as described previously (30). Immunoprecipitated proteins were washed and analyzed by immunoblot.
Metabolic LabelingConfluent bovine fibroblasts were incubated in SFM for 24 h with and without additions and then changed to methionine- and cysteine-free Dulbecco's modified Eagle's medium with 0.1% bovine serum albumin and Tran35S-label (50 µCi/ml, ICN, Costa Mesa, CA) with and without the same additions for 24 h. Cell lysates were precleared with protein A-agarose beads for 2 h at 4 °C, and the supernatants subjected to immunoprecipitation with the appropriate antibodies. Immunoprecipitated proteins were separated by 5-15% SDS-PAGE under reducing conditions. The resultant gel was soaked in Enlightening reagent (NEN Life Sciences Products Inc.) and analyzed by autoradiography.
MAP Kinase ActivityMAP kinase activity was determined
using an in-gel renaturation assay modified from Gotoh et
al. (31). Cells were treated without and with IGF-I or insulin for
5 and 90 min at 37 °C. Total cell lysates (25 µg of protein) were
resolved on a 10% SDS-polyacrylamide gel containing 0.2 mg/ml myelin
basic protein. The gel was rinsed four times for 20 min with 20%
isopropyl alcohol in 50 mM Tris (pH 8.0), and then
denatured by two 30-min washes with 50 mM Tris (pH 8.0)
containing 6 M guanidine HCl and 5 mM
mercaptoethanol. Renaturation proceeded overnight with four changes of
5 mM mercaptoethanol and 0.04% Tween 40 in 50 mM Tris (pH 8.0) buffer. The following morning the gel was
incubated in assay buffer (40 mM HEPES, pH 8.0, containing
0.1 mM EGTA, 5 mM magnesium acetate, and 2 mM dithiothreitol) for 30 min at 30 °C. The in-gel
kinase reaction was then initiated with the addition of 20 µM [-32P]ATP (90 µCi), and allowed to
proceed for 1 h at room temperature. The reaction was terminated
by washing the gel 6 times for 20 min in 5% trichloroacetic acid with
1% sodium pyrophosphate. After these washes the gel was vacuum-dried
and subjected to autoradiography.
Bovine
fibroblasts possess abundant IGF-I receptors and respond to exogenous
IGF-I with marked increases in nuclear proto-oncogene expression,2 DNA synthesis, and cell replication (2, 3). To
investigate the initial signaling events in IGF receptor activation,
quiescent cultures of bovine fibroblasts were stimulated with 10 nM IGF-I for 10 min and total cell lysates were prepared.
Lysate proteins were separated by SDS-PAGE, transferred to
nitrocellulose filters, and immunoblotted with anti-phosphotyrosine
antibody (PY20). With IGF-I treatment, we observed a band at ~97
kDa, and a predominant tyrosyl-phosphorylated species migrating as a
broad band between 170 and 190 kDa (Fig. 1). Using a
specific IGF-I receptor antibody with broad species recognition, the
97-kDa protein was documented to represent the
-subunit of the IGF-I
receptor (data not shown). The 170-190-kDa band approximated the size
of IRS protein, and we initially presumed it represented IRS-1, the
major insulin and IGF-I receptor substrate. However, the majority of
tyrosyl-phosphorylated protein in this 170-190-kDa band was
immunoprecipitated by antibodies against IRS-2 and not by antibodies
against IRS-1 (Fig. 2). The protein immunoprecipitated
by
IRS-2 appeared to represent the higher molecular weight component
of the 170-190-kDa band. Nonspecific IgG did not precipitate any
PY20 reactivity.
Both IRS-1 and IRS-2 proteins could be identified by metabolic labeling
of cultured bovine fibroblasts with Tran35S-label, followed
by immunoprecipitation (Fig. 3). The antibodies used to
identify IRS-1 and IRS-2 were specific for each of the molecules, since
the two proteins could be distinguished by migration on SDS-PAGE. The
labeled protein species immunoprecipitated with IRS-1 migrated at
~170 kDa; the species immunoprecipitated with
IRS-2 migrated at
~190 kDa. No cross-reactivity of antibodies and antigens was
evident.
Insulin-induced Desensitization
As shown in Fig.
4, preincubation but not co-incubation with low
concentrations of insulin desensitized bovine fibroblasts to the
mitogenic effects of IGF-I. Bovine fibroblasts were washed and changed
to serum-free medium for 48 h without or with 10 nM insulin. Cells were washed extensively and then 10 nM
IGF-I, 10 nM insulin, or 10 nM IGF-I plus 10 nM insulin added, and [3H]thymidine
incorporation determined. IGF-I stimulated [3H]thymidine
incorporation 45-fold. Insulin alone produced a much smaller but
significant increase in [3H]thymidine incorporation. The
co-addition of insulin had no significant effect on IGF-I-stimulated
DNA synthesis. However, pretreatment with the same amount of insulin
inhibited IGF-I-stimulated [3H]thymidine incorporation by
85%. This insulin-induced cellular desensitization has been
characterized previously and cannot be accounted for by changes in
IGF-I receptor binding (3).
Investigating where the insulin-induced desensitization occurs along
the IGF-I receptor signaling pathway, bovine fibroblasts were treated
as in Fig. 4 and total cell lysates immunoblotted for
tyrosine-phosphorylated proteins (Fig. 5). IGF-I
stimulation was associated with the appearance of bands on PY20
immunoblots at 97 and 170-190 kDa, as in Fig. 1. Pretreatment with 10 nM insulin for 48 h alone had no visible effect on
tyrosine phosphorylation of proteins at 170-190 kDa, but completely
prevented the ability of IGF-I to induce phosphorylation of the higher
molecular weight bands associated with IRS-2. The lower molecular
weight bands in this complex, presumably including IRS-1, did not
appear to be affected by insulin pretreatment. A specific decrease in
tyrosine-phosphorylated IRS-2 was verified by immunoprecipitation (data
not shown). Prolonged insulin treatment also diminished
IGF-I-stimulated phosphorylation of the 97-kDa band associated with
IGF-I receptor
-subunit, even though some IGF-I receptor-mediated
responses are not impaired under these conditions (Ref. 3, and see Fig.
8).
Treatment with 10 nM insulin for 48 h had little or no effect on IRS-1 or IRS-2 protein content as assessed by direct immunoblotting with IRS antibodies (not shown) or by metabolic labeling (Fig. 3). The level of IRS-1 and IRS-2 protein present with and without insulin treatment was nearly equal, as determined by measuring the radioactivity in each band by scintillation counting (Table I). However, following insulin treatment, IRS-2 migrated slower and as a broader band on SDS-PAGE. This decrease in mobility, despite the decrease in tyrosine phosphorylation, is often associated with increased serine/threonine protein phosphorylation (10).
|
Since the IL-4
cytokine receptor also has IRS-2 as its major substrate for tyrosine
phosphorylation, we assessed the influence of IL-4 in our cell system.
As shown in Fig. 6, 10 nM IGF-I stimulated [3H]thymidine incorporation 30-fold, whereas IL-4 showed
no significant dose-dependent stimulation. However,
pretreatment of bovine fibroblasts with 10 nM IL-4
inhibited IGF-I-stimulated DNA synthesis by 50-60% (p < 0.05) in four separate experiments. Pretreatment with two other
cytokines, IL-1 and IL-6, had no effect on IGF-I action in these
experiments. In parallel experiments, 10 nM IL-4 was as
effective as 10 nM IGF-I in stimulating IRS-2 tyrosyl
phosphorylation (Fig. 7A), even though, as
noted in Fig. 6, this did not translate into a comparable mitogenic
effect. Nonetheless, pretreatment with 10 nM IL-4 inhibited
IGF-I-induced IRS-2 phosphorylation. This was not due to
down-regulation of IRS-2 since IL-4 had no effect on IRS-2 protein
content (Fig. 7B). Pretreatment with IL-1
or IL-6 had no
effect on IRS-2 phosphorylation or expression in these cells (data not
shown).
Desensitization in the Receptor Signaling Network
IRS-2 has the potential to link IGF-I signaling to both MAP kinase and PI 3-kinase activation. MAP kinase is thought to be the more important pathway for directing IGF-I receptor signaling toward mitogenesis (20); therefore, MAP kinase activity in bovine fibroblasts was measured using a functional in-gel assay. In preliminary time course experiments (not shown), we found that 10 nM insulin and 10 nM IGF-I were equivalent in increasing by 3-5-fold the activity of the p42 and p44 isoforms of MAP kinase. Activation was transient with peak activity occurring within 5 min and returning to near baseline by 90 min. No MAP kinase activity was detectable 24 h after stimulation. This pattern of acute MAP kinase activation is characteristic, and differences in duration of response may influence cell signaling decisions (32, 33). As shown in Fig. 8, IGF-I increased p42/p44 MAP kinase activity 5-fold, and neither the magnitude nor the duration of this stimulation was affected by pretreatment with insulin for 48 h. Similarly, we saw no effect of IL-4 pretreatment on the ability of IGF-I to activate the MAP kinase pathway (data not shown). Therefore, despite activation of the MAP kinase signaling pathway, IGF-I-stimulated mitogenesis did not occur if preceded by insulin or IL-4 receptor activation. That insulin-induced cellular desensitization does not involve the MAP kinase signaling pathway was supported by experiments using PD98059, a specific inhibitor of MAP kinase activation by upstream MAP kinase kinase (34). Although we could show that PD98059 was an effective inhibitor of insulin-stimulated MAP kinase activity in our system, the presence of PD98059 during insulin pretreatment did not prevent cellular desensitization to subsequent IGF-I stimulation (Table II). PD98059 did not have an independent inhibitory effect since pretreatment for 48 h with PD98059 alone did not interfere with IGF-I stimulation.
|
On the other hand, insulin-induced desensitization to IGF-I could be
prevented if LY294002, a specific inhibitor of PI 3-kinase activation
(35), was present during the preincubation period (Fig.
9). In three separate experiments, IGF-I stimulation
following preincubation with insulin and LY294002 was 94-106% of
maximum versus 21-25% of maximum following preincubation
with insulin alone. LY294002 pretreatment in the absence of insulin
produced a decrease in IGF-I-stimulated [3H]thymidine
incorporation, which appeared to reflect a residual effect of the
inhibitor on IGF-I receptor signaling (data not shown). Retention of
IRS-2 tyrosine phosphorylation was concomitant with the
IGF-I-stimulated DNA synthesis following preincubation with insulin and
LY294002 (data not shown). Interestingly, LY294002 also prevented the
decrease in mobility and broadening of the IRS-2 band on SDS gels in
response to prolonged insulin treatment (Fig. 10).
The control of cell growth is complex, involving coordinated integration of signals arising from a variety of activated receptors. The vast majority of studies use transfected or immortalized cells overexpressing a particular receptor to delineate its signal transduction pathway for mitogenesis. This approach has been crucial to our present understanding of cell growth. Nevertheless, there is a growing appreciation that feedback loops and intracellular cross-talk between signaling pathways may underlie natural mechanisms of receptor activity regulation, and that "normal" cell models may be more relevant to these types of investigations. Cultured bovine fibroblasts, naturally expressing a high number of IGF-I receptors, have been a useful cell model for studying various aspects of IGF physiology (2, 3, 36, 37). In the present study we identified signaling cascades involved in IGF-I-induced mitogenesis in these cells, characterized the interplay among IGF-I, insulin, and cytokine receptor signaling, and produced evidence of a distinct role for IRS-2 in cell growth regulation.
IGF-I, Insulin, and Cytokine Receptor Signaling in Normal Bovine FibroblastsCultured bovine fibroblasts possess classic IGF-I
tyrosine kinase receptors with mitogenic end points similar to those
reported for insulin tyrosine kinase receptors, proto-oncogene
expression, DNA synthesis, and cell replication (2, 3). We found the initial signaling events to be similar as well, i.e.
autophosphorylation of the receptor -subunit on tyrosines and
subsequent tyrosine phosphorylation of specific intracellular
substrates, primarily IRS. Interestingly, the major IRS protein
tyrosine-phosphorylated in response to IGF-I in bovine fibroblasts was
IRS-2, rather than IRS-1. IRS-1 is expressed by these cells, however,
as determined by metabolic labeling. No other IGF-responsive
tyrosine-phosphorylated proteins were evident under our conditions.
Phosphorylation of Shc protein represents an IGF-I-stimulated signaling
event alternative to IRS (8, 9, 20). However, there was no specific
increase in tyrosine-phosphorylated proteins of 46, 52, and 66 kDa that could represent Shc proteins and no specific protein immunoprecipitated with
Shc antibodies (data not shown), suggesting low abundance of
Shc in these cells.
Insulin receptors also mediate proto-oncogene expression and DNA synthesis in bovine fibroblasts. However, pre-exposure to low concentrations of insulin that do not interfere with IGF-I receptor binding renders the cells refractory to subsequent IGF-I-stimulated DNA synthesis (3). Under these conditions, preincubation with insulin completely blocked the IGF-I-induced increase in tyrosine-phosphorylated 190-kDa IRS-2. The tyrosyl-phosphorylated band at 170 kDa, presumably IRS-1, was relatively unaffected by insulin. These data implicate IRS-2 in insulin/IGF-I post-receptor interplay, and suggest that IRS-2, and not IRS-1, is involved in insulin-induced desensitization to IGF-I in this model. Giorgino and Smith (38) similarly concluded that IRS-1 is not involved in dexamethasone-induced potentiation of IGF-I receptor signaling in muscle cells.
IL-4, a pluripotent cytokine, was equivalent to IGF-I in rapid tyrosine
phosphorylation of IRS-2 in bovine fibroblasts. This effect was not
seen with the other interleukins tested (IL-1 and IL-6). Unlike
IGF-I and insulin receptors, the IL-4 receptor does not possess
intrinsic tyrosine kinase activity and presumably gains this function
through association with Janus kinases (23, 25, 28, 29). IL-4-induced
IRS-2 phosphorylation was not associated with a mitogenic response in
these cells. Nonetheless, IL-4 pretreatment inhibited both
IGF-I-stimulated DNA synthesis and IGF-I-induced IRS-2 tyrosine
phosphorylation, suggesting that IRS-2 is not sufficient for mitogenic
signaling in this system but that it may be necessary for mitogenic
signaling by other growth factors. Alternatively, IGF-I and IL-4
tyrosine phosphorylation of IRS-2 may be qualitatively different (39).
Furthermore, these data indicate that preactivation of a mitogenic
signaling pathway is not essential for desensitization, since IL-4 was
able to induce cellular resistance without affecting DNA synthesis.
Although the IL-4 receptor is structurally and functionally distinct
from the insulin and IGF-I receptors, all three share a common motif in
their juxtamembrane region that may directly or indirectly interact
with IRS protein (40). It will be important to determine whether this
domain is essential for heterologous receptor desensitization. There
are other recent examples where cytokines have been shown to influence
cell growth response in vivo and in vitro.
Leptin, an adipocyte-derived cytokine, attenuated insulin-induced
tyrosine phosphorylation of IRS-1 and stimulation of gluconeogenesis in HepG2 cells (41). Tumor necrosis factor
is an important
mediator of insulin resistance in muscle and fat tissue (42).
Collectively, these data indicate significant interplay among IGF-I,
insulin, and cytokine receptor signaling, which may represent part of
normal physiologic cell growth regulation.
Our
data suggest that IGF-I, insulin, and IL-4 receptor signaling pathways
converge at a step where regulation of IRS-2 activity takes place, and
that the phosphorylation status of IRS-2 determines cellular
sensitivity. Our findings of unaltered IGF-I-stimulated amino acid
transport and glucose uptake (3) and of IGF-I-stimulated MAP kinase
activation following prolonged pre-exposure to low concentrations of
insulin or IL-4, suggest a functional IGF-I receptor and primary check
point beyond receptor autophosphorylation. However, decreased tyrosine
phosphorylation of the 97-kDa -subunit of the IGF-I receptor may
indicate possible upstream effects of receptor autophosphorylation that
will need to be addressed. Furthermore, unlike models of chronic
stimulation with 10-100-fold higher concentrations of insulin (19,
42-44), down-regulation of IRS-2 (or IRS-1) expression did not occur.
In our system, desensitization is also unlikely to be the result of
simple competition for a limiting substrate because co-treatment as
opposed to pretreatment with insulin or IL-4 had no effect on IGF-I
stimulation of DNA synthesis or IRS-2 phosphorylation on tyrosines,
although both insulin and IL-4 alone stimulate IRS-2 tyrosine
phosphorylation. Thus, time appears to be an important dimension of
signal transduction by virtue of its impact on intracellular response
patterns which may permit the defined order of the cell cycle to
proceed.
The decrease in tyrosine phosphorylation of IRS-2 appears to be central
to cellular desensitization to IGF-I. This could be accomplished by
dephosphorylation of phosphotyrosines on IRS-2 or, alternatively, by
phosphorylation on serine/threonines. Phosphorylation of signal
elements by activated downstream serine/threonine kinases has been
shown to inhibit subsequent tyrosine phosphorylation of the substrate
(18, 45). What was striking in our metabolic labeling studies was that
insulin-induced cellular desensitization was invariably accompanied by
an increase in IRS-2 apparent molecular mass on SDS gels. Sun et
al. (10) demonstrated that a similar molecular mass shift in IRS-1
after prolonged insulin treatment of Chinese hamster ovary cells
transfected with human IRS-1 reflected an increase in degree of serine
phosphorylation of the protein. Phosphorylation of IRS-1 on serine and
threonine residues interfered with the subsequent tyrosine
phosphorylation of IRS-1 by insulin receptors in this system. Our model
may be analogous in that activation of insulin receptor or IL-4
receptor signaling may cause increased expression or activation of a
downstream serine kinase which phosphorylates IRS-2, thereby preventing
subsequent tyrosine phosphorylation by IGF-I receptors. LY294002, an
inhibitor of dual specificity PI 3-kinase that possesses both lipid and
serine kinase activities (46), was able to prevent this IRS-2 mobility
shift and the insulin-induced desensitization to IGF-I, consistent with
a role for serine phosphorylation of IRS-2 in cellular desensitization. Interestingly, tumor necrosis factor has been shown to induce serine phosphorylation of IRS-1 and thereby convert IRS-1 into an
inhibitor of insulin receptor tyrosine kinase activity (18). Studies
are in progress to determine whether IRS-2 can directly inhibit IGF-I
receptor kinase activity in our system. A role for insulin and/or
IL-4-induced protein-tyrosine phosphatase activity has not been ruled
out, however. It was of particular interest to us that protein-tyrosine
phosphatase 1B was shown to be increased during prolonged treatment of
rat L6 muscle cells with low dose insulin, and that protein-tyrosine
phosphatase 1B acted as a negative regulator of insulin and IGF-I
signaling (17). However, our preliminary experiments (not shown) failed
to reveal any change in protein-tyrosine phosphatase 1B with insulin
treatment of bovine fibroblasts as assessed by immunoblot.
As noted above, inhibition of PI 3-kinase activation by LY294002 blocked the ability of insulin to induce desensitization to IGF-I, implicating the PI 3-kinase signal pathway in cellular desensitization to IGF-I-stimulated mitogenesis. Although MAP kinase is considered a key mitogenic signaling pathway (9, 20, 21), MAP kinase activation was not sufficient to propagate IGF-I-stimulated mitosis in bovine fibroblasts. Furthermore, given the lack of effect of insulin pretreatment on IGF-I-stimulated MAP kinase activity, it seems unlikely that desensitization occurs within the cascade leading to activation of MAP kinase. The latter was supported by the finding that a potent inhibitor of MAP kinase activation, PD98059, did not prevent desensitization during pretreatment with insulin.
ConclusionsThe impaired response of bovine fibroblasts to IGF-I as a consequence of pretreatment of cells with insulin or IL-4 is associated with post-receptor signaling alterations at the level of IRS-2. The molecular mechanism for the decrease in IRS-2 tyrosine phosphorylation in response to IGF-I under these conditions is not clear, but the data suggest a feedback mechanism mediated by a preactivated receptor tyrosine kinase pathway (i.e. by insulin or IL-4) that induces serine phosphorylation of IRS-2 which, in turn, interferes with IGF-I receptor-stimulated signal transduction through IRS-2 in these cells. IRS-2 is expressed in many cell types, and is not peculiar to bovine fibroblasts, suggesting a role in normal insulin, IGF-I, and IL-4 signaling (39, 47). Bovine fibroblasts appear to be a particularly useful model for further studies in this regard. Better understanding IRS-2 will be of critical importance to our understanding of integrated cell signaling and post-receptor desensitization, and perhaps be of relevance to growth-resistant states produced by insulin and cytokine receptor activation.