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Endocrinology Vol. 138, No. 2 741-750
Copyright © 1997 by The Endocrine Society

Articles

Roles of Insulin Receptor Substrate-1 and Shc on Insulin-Like Growth Factor I Receptor Signaling in Early Passages of Cultured Human Fibroblasts

Yoshihiko Takahashi, Kazuyuki Tobe, Hiroko Kadowaki, Daisuke Katsumata, Yoshimitsu Fukushima, Yoshio Yazaki, Yasuo Akanuma and Takashi Kadowaki

Institute for Diabetes Care and Research (Y.T., H.K., Y.A.), Asahi Life Foundation, Tokyo 100, Japan; Third Department of Internal Medicine (Y.T., K.T., Y.Y., T.K.), Faculty of Medicine, University of Tokyo, Tokyo 113, Japan; and Saitama Children’s Medical Center (D.K., Y.F.), Iwatsuki 339, Japan

Address all correspondence and requests for reprints to: Takashi Kadowaki, Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-like growth factor-I (IGF-I) improves glucose metabolism and growth in patients with leprechaunism. We investigated signal transduction through IGF-I receptor in comparison with epidermal growth factor (EGF) receptor in early passages of cultured skin fibroblasts from a normal subject and a patient with leprechaunism whose insulin receptor tyrosine kinase was almost nonexistent. Insulin receptor substrate-1 (IRS-1) became tyrosine-phosphorylated and bound growth factor receptor-bound protein 2 (GRB2) quickly by IGF-I. The association of Shc with GRB2 by IGF-I was detected by immunoblot with anti-Shc antibody but was hardly visible with antiphosphotyrosine antibody, which was in marked contrast to efficient tyrosine phosphorylation of Shc by EGF. However, the potency of IGF-I for DNA synthesis was far stronger than EGF, which was not parallel with the potency of these growth factors to activate Shc or MAP kinase. Rather, phosphatidylinositol (PI) 3-kinase activity, which was activated by IGF-I about 5- to 10-fold more strongly than EGF, appeared to correlate with mitogenesis. Signal transduction pathways following IGF-I receptor or EGF receptor activation were indistinguishable between the normal subject and the patient. Our results strongly suggest that in human skin fibroblasts, which represent a more physiological cell culture: 1) IRS-1, rather than Shc, is the major tyrosine-phosphorylated protein binding GRB2 in initial phase of IGF-I signaling; 2) mitogenic potency of receptor tyrosine kinases such as IGF-I receptor and EGF receptor may not be determined solely by the amount of Shc-GRB2 complex or the activity of MAP kinase; and 3) in contrast to previous reports, IGF-I and EGF receptor signalings are not defective in leprechaunism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE GROWTH FACTOR I (IGF-I) is known to have metabolic and growth-promoting effects and can compensate for some insulin action in patients with mutations in the insulin receptor gene (1). Its receptor (IGF-I receptor) has tyrosine kinase activity, phosphorylates insulin receptor substrate-1 (IRS-1), and Shc directly, which leads to activation of MAP kinase cascade, PI 3-kinase, and so on in various established cell lines and murine tissues such as liver, muscle, and adipose tissue (2, 3). However, its postreceptor signaling in primary culture of human cells has not been well documented. Here, we characterize IGF-I receptor (IGF-I R) signaling in early passages of cultured human skin fibroblasts in order to investigate how IGF-I R mediates its signals through insulin receptor substrate-1 (IRS-1) (4) and Shc (5) in the activation of mitogen-activated protein kinase (MAP kinase) pathway in a human cell system with no overexpression of signaling molecules. There is still controversy over the importance of IRS-1 and Shc in insulin and IGF-I R signaling (4, 6, 7, 8, 9). To assess the contribution of Shc in IGF-I R signaling in mitogenesis, we compared it with signaling of epidermal growth factor receptor (EGF R), taking advantage of the coexpression of EGF R together with insulin receptor (IR) and IGF-I R in human skin fibroblasts.

Studies using human skin fibroblasts for insulin- or IGF-I-mediated signaling have always been hampered by the fact that there is ligand cross-reactivity between IGF-I R and IR. Because the similarity and the difference in postreceptor signaling between the two remain to be learned, we cannot rule out the possibility that a small effect of such cross-reactivity may affect interpretation of the results. To minimize these problems, we used cells from both a normal subject and a patient with leprechaunism who lacked almost completely the tyrosine kinase activity of IR due to mutations in both alleles of IR gene (10, and this study).

Another purpose for using the patient’s cells was to examine postreceptor signalings of IGF-I receptor and EGF receptor in leprechaunism, which always has severe growth retardation. We also found that, although there are several reports on functional defects of multiple receptor tyrosine kinases (RTKs) in leprechaunism (11, 12, 13, 14), our patient had normal signalings for IGF-I and EGF as described below.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Primary cultures of skin fibroblasts were obtained from the normal subject and the patient with leprechaunism by a standard method. Cells were cultured in DMEM containing 10% FCS and antibiotics and were maintained at 37 C in a CO2 incubator. Two days after reaching confluence, cells were washed once with PBS and serum-starved in DMEM containing 0.05% BSA for 20 h and subjected to the experiments, except for 3H-thymidine incorporation study. Cells used for the experiments were from fifth to tenth passage because further culture would diminish their proliferation and viability.

Detection of mutations
PCR-direct sequencing was performed as described by Kadowaki et al. (15). To confirm the mutation and analyze the expression of the mutant allele, RT-PCR and allele-specific oligonucleotide hybridization was performed. In brief, total RNA was extracted from EB virus-transformed lymphocytes by the acid-guanidine phenol-chloroform method, and complementary DNA (cDNA) was generated by the standard method using a specific antisense primer located in exon 22 of the IR gene (5'-GGA AGG ATT GGA CCG AGG C-3') (16). RT-PCR was performed with an upstream primer located in exon 18 (5'-AGT CAG CCA GTC TCC GAG AG-3') and a downstream primer located in exon 20 (5'-GCC ATC CAC CGT ACA GGG AG-3') under the following conditions: 94 C, 1 min; 55 C, 1 min; 30 sec; 72 C, 1 min, 30 sec x35 cycles. 32P-labeled oligonucleotide (wild-type: 5'-GAC TTT GGA ATG ACC AGA G-3' or mutant type: 5'-GAC TTT GGA ACG ACC AGA G-3') was probed with RT-PCR products on a nitrocellulose sheet at 37 C for 12 h. The sheet was washed at 60 C for 10 min and subjected to autoradiography (17).

Insulin binding and tyrosine kinase activity of patient’s insulin receptor
Insulin binding in EB virus-transformed lymphocytes, and the following Scatchard analysis was performed as previously described. As for immune-complex receptor kinase assay, EB virus-transformed lymphocytes were solubilized, and the lysates were immunoprecipitated with monoclonal {alpha}IR-1 antibody, which is known to be specific for human IR, and antimouse IgG preadsorbed to protein A-sepharose. Immunoprecipitates were extensively washed, and the content of IR was normalized by insulin binding. Tyrosine kinase activity was assessed by 32P incorporation into histone H2B (18), and the amount of IR was confirmed by immunoblot analysis with an antibody raised against the C-terminal domain of IR (19).

Immunoprecipitation and blotting
Antiphosphotyrosine antibody for immunoprecipitation was prepared from hybridoma cells (PY20), and polyclonal rabbit antiphosphotyrosine antibody was used for blotting. Rabbit polyclonal anti-GRB2, antihuman Sos-1, anti-c-raf-1 antibodies, and antiextracellular signal-regulated kinase (ERK)1 antibody, which recognizes mainly p44-MAP kinase (ERK1), were purchased from Santa-Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal anti-Shc antibody and anti-p85 of PI-3 kinase were from Transduction Laboratories (Lexington, KY). Polyclonal anti-IRS-1 antibody, which is raised against rat IRS-1 and reacts with human IRS-1 as manufacturer’s protocol, was from Upstate Biotechnology Inc. (Lake Placid, NY). Cells on 10-cm dishes (approximately 5 x 106 cells/dish at confluence) were stimulated with indicated growth factors for various times at 37 C and quickly frozen in liquid nitrogen. They were then lysed with lysis buffer A [1% Triton X-100, 50 mM HEPES, pH 7.4, 10 mM EDTA, 10 mM NaF, 10 mM Na4P2O7, 10 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF) and aprotinin]; 1-3 µg of each antibody was added to the lysates and they were incubated for 1-2 h on ice. Then, protein G or protein A-sepharose was added and the samples were further incubated at 4 C for 1 h. The immunoprecipitates were washed three times with lysis buffer A containing 0.15 M NaCl and boiled with Laemmli’s sample buffer containing 2-mercaptoethanol for 6 min. They were subjected to SDS-PAGE followed by electrical protein transfer to polyvinylidene difluoride membrane (Millipore). Immunoblot analyses were performed by a standard protocol. Detection of the proteins was done by 125I-protein A followed by autoradiography in most of the experiments, and the alkaline phosphatase detection system was used to identify migratory changes in some experiments. For suitable detection of various proteins, 7.5 or 8.5% SDS-PAGE was used for 60-kDa–200-kDa proteins, and 10 or 12% SDS-PAGE was used for less than 60-kDa proteins.

Activity of MAP kinase in polyacrylamide gels
The assay for mitogen-activated protein kinase (MAP kinase) was performed as described by Tobe et al. (20). Cells on 3.5-cm dishes were treated with IGF-I or EGF for indicated time and lysed with 150 µl of buffer B (25 mM Tris-HCl, pH 7.4, 25 mM NaCl, 10 mM NaF, 10 mM sodium pyrophosphate, 1 mM Na3VO4, 0.5 mM EGTA, and 1 mM PMSF). The lysates were electrophoresed on an SDS-PAGE containing 0.5 mg/ml of myelin basic protein (MBP). SDS was removed from the gel by washing the gel with two changes of 20% 2-propanol in 50 mM Tris-HCl, pH 8.0, for 1 h and then 250 ml of 50 mM Tris-HCl, pH 8.0, containing 5 mM 2-mercaptoethanol for 1 h at room temperature. The enzyme was denatured by treating the gel with two changes of 100 ml of 6 M guanidine HCl at room temperature for 1 h and then renatured with five changes of 250 ml each of 50 mM Tris-HCl, pH 8.0, containing 0.04% Triton X-100 and 5 mM 2-mercaptoethanol for 8–10 h. After renaturation, the gel was preincubated at room temperature for 1 h in 5 ml of 50 mM Tris-HCl, pH 8.0, 2 mM dithiothreitol, 0.5 mM EGTA, 10 mM MgCl2, and 2 µM protein kinase inhibitor (rabbit sequence; Sigma Chemical Co., St. Louis, MO). Forty micromoles/liter of unlabeled ATP and 50 µCi of [{gamma}-32P] ATP were then added and the gel was further incubated for 1 h. After phosphorylation reaction, the gel was washed frequently with 5% trichloroacetate (TCA) until the background was negligible. The washed gel was dried and subjected to autoradiography. The activities of bands excised from the gels were quantitated by Cerenkov counting. In addition, the rest of total cell lysates were subjected to immunoblot analysis for detection of changes in the mobility of p44- and p42-MAP kinase. For blotting, polyclonal antibody for both p44- and p42-MAP kinase ({alpha}Y91) was prepared as described (20).

MAP kinase activity in immune-complex
In brief, cells from 60-mm dishes were lysed with buffer B containing 1% Triton X-100, and the lysates were immunoprecipitated with anti-p44 MAP kinase antibody (anti-ERK1) preadsorbed to protein A-sepharose. The immunoprecipitates were washed three times with buffer B containing 0.1% Triton X-100, and the following immune-complex kinase assay was performed as described (20).

3H-thymidine incorporation
Cells at subconfluence on 12-well plates were washed and starved in DMEM 0.4% FCS for 36 h, then stimulated with various concentrations of the indicated growth factor for 16 h. Two microcuries of 3H-thymidine were added to each well, and the cells were further incubated for 4 h, washed with PBS twice, and treated with 10% TCA for 1 h on ice. Finally, they were washed with 5% TCA three times and lysed. 3H-thymidine incorporation was counted in a liquid-scintillation counter.

PI 3-kinase activity in immune-complex
Confluent monolayers on 60-mm dishes were starved, stimulated with 100 nM IGF-I or 10 nM EGF for 1 min and quickly frozen into liquid nitrogen. They were solubilized with lysis buffer A, and the lysates were incubated with anti-PY or anti-GRB2 preadsorbed to protein G-sepharose for 2 h. Then, the immunoprecipitates were washed and PI 3-kinase activity was measured as previously described (21, 22).

For statistical analysis on 3H-thymidine incorporation between control cells and the patient’s cells, Student’s t test was used and P value of less than 0.05 was considered as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Detection of a mutation in the insulin receptor gene
Direct sequencing of amplified genomic DNA revealed that the patient was a heterozygote for a missense mutation at codon 1153 (Met->Thr) that was located in the tyrosine kinase domain of the receptor (Fig. 1AGo). A substitution of Ile for Met at codon 1153 has previously been reported (23), and the mutation Met1153->Ile causes a severe defect in tyrosine kinase activity of the receptor without affecting insulin binding affinity. In fact, insulin binding in EB virus-transformed lymphocytes from a control subject was 15.8% and that from the patient was 11.4%, both of which were within our lower normal range. IR expressed in our patient’s cells had normal insulin binding affinity [Kd value: control 0.45 nM vs. patient 0.36 nM in a representative experiment (Fig. 1CGo)], but had almost no tyrosine kinase (Fig. 1DGo). Allele-specific oligonucleotide hybridization study revealed that the opposite allele of the patient was hardly expressed (Fig. 1BGo). Therefore, the patient was a compound heterozygote with a missense mutation (Met1153->Thr) in the tyrosine kinase domain in one allele that would severely decrease tyrosine kinase activity, and with an unknown mutation in the other that would decrease messenger RNA (mRNA) level in a cis-dominant manner. Direct sequencing analysis spanning exon 16-21 and cDNA subcloning over exon 4-7 and exon 12-14 revealed no other mutation in the patient (data not shown).



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  		Figure 1. Detection of a mutation in the insulin receptor gene in the patient with leprechaunism. A, Direct sequencing of amplified genomic DNA spanning exon 20 of the insulin receptor gene. B, Allele-specific oligonucleotide hybridization. C, Scatchard analysis of insulin binding in lymphocytes. D, Tyrosine kinase activity of the insulin receptor in EB virus-transformed lymphocytes of the patient. A, Patient was a heterozygote for the mutation (Met1153(ATG)->Thr(ACG)), which was confirmed by subcloning of the PCR product (data not shown). The substitution of C for T is indicated by an arrow. Panel B, a) and b), show the expression level of the wild type allele and the mutant allele, respectively (C, control; Pt, patient). B, a) shows that the opposite (wild type) allele was hardly expressed in the patient. B, b), shows the mutant allele was definitely expressed in the patient. C, Insulin binding affinity of insulin receptor expressed in control and patient’s lymphocytes. Insulin binding was 15.8% in the control subject and 11.4% in the patient (not shown), and the Kd values estimated from the plot was 0.45 nM in the control subject and 0.36 nM in the patient, both of which were within our normal range. D, Tyrosine kinase activity in {alpha}IR-1 immune-complexes measured by 32P-incorporation into histone H2B. The immune-complexes were incubated with (+) or without (-) 100 nM insulin. The content of the receptor was normalized by insulin binding and immunoblotting (data not shown).

 
IGF-I binding of the skin fibroblasts subjected to the experiments
We characterized the expression level of IGF-I R and tyrosine-phosphorylation of IRS-1 in control cells and the patient’s cells. 125I-IGF-I binding in intact cells was 8.2% and 5.1%, respectively, in a fifth passage of culture (Fig. 2Go). In further culture, 125I-IGF-I binding in the control cells and the patient’s cells gradually decreased in parallel (for example, control 4.9% and patient 3.2% in the sixth passage). Thus, the ratio of the expression of IGF-I R in the experiments below is considered as control: patient = 3:2. Insulin binding in control and patient’s skin fibroblasts was 1.8% and 1.0% in the sixth passage, respectively. We failed to obtain reliable Scatchard analysis on IGF-I and insulin receptors in skin fibroblasts, but ED50 on IGF-I receptor (Fig. 2Go) suggested that IGF-I binding affinity was not significantly different in control and patient’s fibroblasts. Phosphorylation of IRS-1 as well as the amount of p85 subunit of PI-3 kinase in antiphosphotyrosine immunoprecipitates was approximately in parallel with the IGF-I binding in these cells (data not shown) (21).



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  		Figure 2. IGF-I binding of the intact skin fibroblasts. The figure shows 125I-IGF-I binding in intact cells. The maximal binding of 125I-IGF-I was 8.2% in the control cells, and 5.1% in the patient’s cells. ED50 was approximately 4 nM in both cells (45).

 
Detection of proteins phosphorylated on tyrosines by growth factors
We further investigated RTK signaling in skin fibroblasts. Immunoblotting for detection of tyrosine-phosphorylated protein is indicated in Fig. 3Go. When the cells were stimulated by 100 nM IGF-I (Fig. 3AGo) tyrosine phosphorylation of IRS-1 was clearly detected and the response was maximal within 1 min, but the change in tyrosine phosphorylation of Shc was not detected at least up to 3 min. Tyrosine phosphorylation of EGF receptor and Shc was maximal at 1 min with 10 nM EGF stimulation (Fig. 3BGo). Other EGF-dependent tyrosine-phosphorylated proteins (110 kDa, 100 kDa, and 60-kDa) were detected, but their identity is not known at present. When cells were treated with 10 ng/ml basic fibroblast growth factor (bFGF) without exogenous heparin, tyrosine-phosphorylated protein with an Mr of approximately 140K was observed at 2, 5, and 10 min (Fig. 3CGo), probably representing the autophosphorylation of FGF receptor. Tyrosine phosphorylation of Shc or coimmunoprecipitation of Sck (23) in anti-Shc immunoprecipitates with bFGF stimulation was not detected in our cell system (right side of panel C). Blotting of the same sheet with anti-Shc antibody revealed that the amounts of 66 kDa and 52 kDa Shc in PY20 immunoprecipitates did not change within 10 min of bFGF stimulation (data not shown). Thus, tyrosine phosphorylation of Shc was apparently distinct among these growth factor stimulations.



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  		Figure 3. Detection of tyrosine-phosphorylated (p-Tyr) proteins by growth factors. A–C, Polyclonal anti-phosphotyrosine immunoblottings following the immunoprecipitations by indicated antibodies. A, Time-course of p-Tyr proteins at 100 nM IGF-I. Phosphorylation of IRS-1 was maximal within 1 min, and IGF-I-dependent phosphorylation of Shc or IGF-I receptor itself was not detected at least within 3 min. B, p-Tyr proteins with various concentrations of EGF at 1 min. Activation of EGF receptor and phosphorylation of 52-kDa Shc were maximal at 10 nM EGF. Also, approximately 110-kDa-, 100-kDa-, and 60-kDa proteins (indicated by arrows) were tyrosine-phosphorylated. C, p-Tyr proteins with 10 ng/ml of bFGF. Activation of 140-kDa p-Tyr protein was observed in PY20 immunoprecipitates, but the phosphorylation of p66- or p52-Shc was barely detected (B). Molecular size markers (kDa) are indicated on the left side, i.p., immunoprecipitation.

 
Tyrosine phosphorylation of Shc and its association with GRB2 following EGF stimulation
We tested anti-GRB2 and anti-Shc antibodies used for experiments by analyzing EGF-stimulated cells. Activated EGF receptor binds GRB2 and Shc, and phosphorylates Shc on a tyrosine residue (24). Antiphosphotyrosine antibody (PY20) was able to immunoprecipitate activated EGF receptor (Fig. 4AGo), Shc (Fig. 4BGo) and coimmunoprecipitate GRB2 (Fig. 4CGo) in a time-dependent manner. Similarly, anti-GRB2 antibody could coimmunoprecipitate activated EGF receptor (Fig. 4AGo) and Shc (Fig. 4Go, A and B). Anti-Shc antibody could coimmunoprecipitate GRB2 in a time-dependent manner (Fig. 4CGo), which was consistent with tyrosine phosphorylation of Shc (Fig. 4AGo). Thus, these antibodies worked efficiently enough for the detection of each complex of signaling molecules. The maximal responses of these associations occurred at 1 min after the EGF stimulation. Although 46-kDa Shc phosphorylation appeared to be weak in 7.5% SDS-PAGE analysis in Fig. 4AGo, the amount of the three species of Shc associated with GRB2 was almost equal in 12% SDS-PAGE as shown in the middle of Fig. 4BGo.



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  		Figure 4. Tyrosine phosphorylation of Shc and its association with GRB2 following EGF stimulation. Cell lysates treated with 10 nM EGF were immunoprecipitated by PY20, anti-GRB2 or anti-Shc antibody. A–C, Immunoblotting with {alpha}PY, anti-Shc and anti-GRB2, respectively. PY20 could immunoprecipitate activated EGF receptor, Shc and also coimmunoprecipitate GRB2 binding to tyrosine-phosphorylated proteins. Anti-GRB2 could co-immunoprecipitate EGF receptor, Shc (A and B), and 120 kDa protein (A). The identity of this 120-kDa tyrosine-phosphorylated protein has not yet been determined. Anti-Shc could coimmunoprecipitate EGF receptor and GRB2. Molecular size markers (kDa) are indicated on the left.

 
Tyrosine phosphorylation of IRS-1 or Shc and their association with GRB2 following IGF-I stimulation
The association of IRS-1 and Shc with GRB2 was thought to be a critical event for the activation of p21 ras, and we studied the time-course of this association in physiological cells. As indicated in Fig. 5AGo, IRS-1 became associated with GRB2 (22) quickly after IGF-I stimulation in intact cells, and its time course was similar in the control and the patient’s cells. In contrast, association of Shc with GRB2 was slower and its maximum was seen 3–5 min after the stimulation by means of anti-Shc blotting (Fig. 5BGo), which was difficult to be detected by {alpha}PY (Fig. 5AGo). Thus, the sensitivity of detection was different with the use of different antibodies. Next, we compared the Shc-GRB2 complex formation induced by IGF-I and EGF because its formation induced by IGF-I appeared so slight, and the complex induced by EGF was a good control for signal strength. As expected, the amount of Shc-GRB2 complex formed via IGF-I R was increased, but it was far less than the complex formation induced via EGF R (Fig. 5CGo). EGF R induced the association of GRB2 with 66, 52, and 46-kDa Shc to approximately similar extents (Figs. 4BGo and 5CGo), but IGF-I promoted the association of GRB2 mainly with 52 kDa-Shc (Fig. 5Go, B and C).



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  		Figure 5. Tyrosine phosphorylation of IRS-I and Shc and their association with GRB2 following IGF-I stimulation. A, Tyrosine-phosphorylated proteins in anti-GRB2 immuno-precipitates. B and C, Detection of Shc in anti-GRB2 immunoprecipitates. Cells were stimulated with 100 nM IGF-I, and immunoblotting was performed with the probe of {alpha}PY (A) or anti-Shc antibody (B and C). The 46-kDa Shc was overlapped by the immunoglobulin heavy chain. A, Tyrosine-phosphorylated 52-kDa Shc was so weakly observed at 3 or 5 min after 100 nM IGF-I stimulation that it is hardly visible in the photograph. Blotting with anti-Shc was more sensitive (B), and the amounts of Shc associated with GRB2 by 100 nM IGF-I and 10 nM EGF stimulation were compared (C). i.p., Immunoprecipitation.

 
We further investigated the amount of IRS-1-GRB2 complex and Shc-GRB2 complex by using anti-IRS-1 antibody and anti-Shc antibody. In the presented result, the maximal IRS-1-GRB2 and Shc-GRB2 complexes in control cells at the time corresponding to maximal association of GRB2 with each protein were compared (Fig. 6Go). The amount of GRB2 with IRS-1 maximal at 1 min was almost equal to that of Shc-GRB2 complex maximal at 5 min. It should be noted that the efficiency of immunoprecipitation of anti-IRS-1 antibody was less than that of anti-Shc, and tyrosine-phosphorylated IRS-1 precipitated by the anti-IRS-1 used was approx. half of the IRS-1 precipitated by anti-PY (data not shown). The maximal amount of GRB2 precipitated by anti-Shc on IGF-I stimulation was about 2-fold over basal (Fig. 6Go), which was consistent with the experiment in Fig. 5Go where Shc was precipitated by anti-GRB2. Taking together the results indicated in Figs. 5Go and 6Go, IRS-1-GRB2 complexes appears to predominate over Shc-GRB2 complex in at least initial phase (within 1 min) of IGF-I signaling, although the definite quantification of these GRB2 complexes should be determined by more sensitive immunoprecipitation system.



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  		Figure 6. Detection of GRB2 associated with IRS-1 and Shc on IGF-I stimulation. Based upon the time course of IRS-1-GRB2 and Shc-GRB2 association in Fig. 5Go, we compared the maximal amounts of the two complexes by immunoprecipitation with anti-IRS-1 and anti-Shc antibodies. The maximal IRS-1-GRB2 complex appeared to be equal to the maximal Shc-GRB2 complex. However, it should be noted that this anti-IRS-1 antibody has lower efficiency of immunoprecipitation than anti-Shc, thus total IRS-1-GRB2 may be more abundant than indicated.

 
Detection of hSos-1 in anti-Shc immunoprecipitates
In most of the transformed cells, GRB2 is thought to constitutively bind son of sevenless (Sos) (26), and this was also true in our cells, that is, hSos-1 was detectable in anti-GRB2 precipitates before growth factor stimulation (Fig. 7AGo, left). Interestingly, the amount of hSos-1 precipitated by anti-GRB2 significantly increased at 1–3 min with EGF treatment (Fig. 7AGo, left). When cells were treated with EGF for 1 min, human Sos-1 (hSos-1) was clearly detected in anti-Shc immunoprecipitates (Fig. 7AGo, right, and B), indicating that tyrosine-phosphorylated Shc efficiently recruited GRB2-Sos complex with the stimulation of EGF. However, hSos-1 was scarcely detected in anti-Shc immunoprecipitates at least within 5 min of IGF-I treatment (Fig. 7BGo). This was consistent with the fact that the amount of Shc-GRB2 complex formation induced via IGF-I R was far less than that via EGF R as described above. The experiment described was of the patient’s cells, and similar results were obtained from control cells (data not shown).



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  		Figure 7. Detection of hSos-1 in anti-Shc immunoprecipitates. A, Detection of hSos-1 in anti-GRB2 and in anti-Shc with EGF treatment; B, representative blot for the detection of hSos-1 in anti-Shc immunoprecipitates with the treatment of 100 nM IGF-I in the patient’s cells. GRB2-hSos-1 complex was present before EGF stimulation, but interestingly the amount of the complex increased with EGF stimulation (A, left). Also, hSos-1 was coprecipitated by anti-Shc in EGF-dependent manner (A, right). Based on this finding, we further investigated Shc-GRB2-Sos complex formation by IGF-I. As indicated in B, hSos-1 was undetectable in anti-Shc precipitates up to 5 min of IGF-I treatment. The far left lane indicates hSos-1 in anti-hSos-1 immunoprecipitate from the untreated whole lysate, and the far right lane indicates hSos-1 in anti-Shc immunoprecipitate from the EGF-treated cell lysate, both of which are presented as controls for anti-hSos-1 blotting.

 
Activation of MAP kinase
IGF-I is thought to be more potent in mitogenesis than insulin, and the activation of MAP kinase is critical for mitogenesis by receptor tyrosine kinases. Therefore, we performed gel-shift assay to evaluate the activation of the p44- and p42-MAP kinases by the treatment of IGF-I and EGF (28). As indicated in Fig. 8Go, A and B, p42-MAP kinase was completely shifted 3 min after EGF stimulation. With the IGF-I treatment, it was more slowly but clearly shifted, and complete shift was observed after 10 min in both the control and the patient’s cells. Thus, activation of p42-MAP kinase was far more efficient with EGF stimulation than IGF-I in human skin fibroblasts. The mobility shift of p44-MAP kinase was unremarkable as compared with that of p42-MAP kinase. The activities of MAP kinase on EGF- and IGF-I stimulation assessed by both in-gel phosphorylation assay and immune-complex kinase assay were parallel with the gel-shift, and far stronger activation by EGF (~4-fold over basal) than by IGF-I (~2-fold over basal) was observed (data not shown). Also, hyperphosphorylation of c-raf-1 (27), which lies upstream from MAP kinase and is directly activated by p21ras, was assessed by changes in the mobility on SDS-PAGE. The change in the mobility was initiated 3 min after EGF stimulation, and clearly observed 10 min afterward, whereas the change began more slowly on IGF-I stimulation and was detectable 10–15 min after IGF-I stimulation (data not shown). These observations indicate that MAP kinase cascade was activated by both IGF-I and EGF, although the degree of activation was significantly larger in EGF treatment in human skin fibroblasts.



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  		Figure 8. Fig. 8. Activation of MAP kinase. Activity of MAP kinase induced by the treatment of 10 nM EGF or 100 nM IGF-I was analyzed by immunoblot analysis to detect changes in the mobilities of p42- and p44- MAP kinase, which reflect the phosphorylation state of MAP kinases and their activities. p42 MAP kinase was apparently shifted during both of these growth factor treatments, whereas the shift of p44-MAP kinase was not remarkable as compared to p42-MAP kinase.

 
Mitogenic potency of IGF-I, EGF, and bFGF
The maximal 3H-thymidine incorporation into DNA with the treatment of IGF-I and bFGF was obtained at the concentrations of 100 nM IGF-I and 2 ng/ml of bFGF, respectively, and the maximal responses for IGF-I and bFGF are presented in Fig. 9Go. The dose-response for EGF is also indicated in this figure, and the maximal response for EGF was obtained at the concentration of 0.1 nM, which was consistent with a report on rat-1 fibroblasts (29). Interestingly, IGF-I was the strongest in mitogenic potency and promoted DNA synthesis about 2-fold over basal (Fig. 9Go). bFGF promoted DNA synthesis about 1.4-fold. Unexpectedly, EGF promoted DNA synthesis only about 1.2–1.3 fold (Fig. 9Go), despite the fact that, in many of the cell types, EGF is more potent in mitogenesis than insulin or IGF-I. Higher concentrations of EGF tended to be suppressive in DNA synthesis (29).



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  		Figure 9. Fig. 9. 3H-thymidine incorporation assay. Cells were treated with IGF-I, EGF, or bFGF under the same culture conditions and DNA synthesis was assessed by 3H-thymidine incorporation. The result is indicated as -fold over basal incorporation, which is an average of three independent experiments. Standard deviations are indicated as bars on top of the columns. P-values from Student’s t test (control vs. patient) are as follows: 100 nM IGF-I:0.592, 0.1 nM EGF:0.497, 1 nM EGF:0.086, 10 nM EGF:0.433, bFGF:0.264. Therefore, DNA synthesis induced by these growth factors over basal level was not significantly different in control and the patient. In both of the control and the patient, IGF-I-induced DNA synthesis was significantly larger than EGF-induced one (overall p < 0.005).

 
PI 3-kinase activation by IGF-I and EGF
PI 3-kinase is thought to play a crucial role in mitogenesis by PDGF or other cytokines; therefore, it may be implicated in the difference in mitogenesis by IGF-I and EGF as described above. As indicated in Fig. 10Go, PI 3-kinase activity in both of anti-PY and anti-GRB2 precipitates were significantly greater with IGF-I stimulation than with EGF. Besides, the amount of p85 subunit of PI 3-kinase coprecipitated by anti-PY or anti-GRB2 with IGF-I or EGF treatment was parallel with the activity (data not shown). PI 3-kinase activation by EGF appears to depend on cell types, and in our cells it was really activated by EGF, although the strength was far less than that of IGF-I-treated cells.



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  		Figure 10. Fig. 10. PI 3-kinase activity by IGF-I and EGF. IGF-I- and EGF-stimulated PI 3-kinase activities in anti-PY and anti-GRB2 immunoprecipitates were compared. PI 3-kinase activity in anti-PY precipitate was approximately 10-fold larger in IGF-I treatment than in EGF treatment. Also, PI 3-kinase activity in anti-GRB2 was 4-fold larger in IGF-I. Quantitation of the activity was performed by Fuji BAS-2000 image analyzer.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IRS-1 and Shc are both phosphorylated on tyrosine residue(s) by IR and IGF-I R(2, 3, 4, 7, 30). IR directly binds IRS-1 and Shc at tyrosine residue 960 when it is phosphorylated (31, 32). IGF-I R interacts directly with IRS-1 and Shc at tyrosine 950 (33, 34), additionally with IRS-1 at tyrosine 1131, 1135, and 1136 (major autophosphorylation sites of IGF-I R) (33), but it remains to be well defined what role IRS-1 and Shc play in mitogenic and metabolic actions of IGF-I R. Recent studies have suggested that Shc has an important function in mitogenesis induced by receptor tyrosine kinases including IR and IGF-I R in several fibroblast cell lines (7). Furthermore, Pruett et al. have reported that Shc-GRB2 complex is dominant in the activation of MAP kinase in L6 myoblasts from comparison of the signalings of native IR and overexpressed IL-4 receptor, both of which can phosphorylate IRS-1 (9). However, IRS-1 knockout mice established by two laboratories have shown growth retardation and mild insulin resistance, suggesting that IRS-1 plays a role in growth and metabolism in vivo (35, 36).

We have focused on the physiological role of IRS-1 and Shc in human cells and therefore investigated human skin fibroblasts in early passages from primary culture. In the culture, IGF-I, EGF, and FGF-stimulated DNA synthesis was observed due to physiologic expression of these receptors, and IGF-I-dependent glucose transport and glycogen synthesis could also be observed (Ref. 11, 37, and data not shown). We also checked the dose-dependent or time-dependent changes of immunoprecipitates in most of the experiments to confirm the adequacy of immunoprecipitation by the antibodies used.

In both the patient’s cells and the control cells, IRS-1 became associated with GRB2 immediately after IGF-I stimulation, but p66- and p52-Shc were hardly detected in anti-GRB2 immunoprecipitates by immunoblot with antiphosphotyrosine antibody (Fig. 5Go). Thus, IRS-1, rather than Shc, is the major tyrosine-phosphorylated protein associated with GRB2 by IGF-I stimulation especially at initial response in our more physiological cells. This is apparently in contrast to EGF R signaling, where EGF R itself and Shc were strongly tyrosine-phosphorylated and associated with GRB2 (Fig. 4AGo). These observations differ from a recent report on rat-1 fibroblasts (7), where p52-Shc is the major tyrosine-phosphorylated protein with the stimulation of insulin or IGF-I. In our cell system, the association of GRB2 with p52-Shc and, to a much lesser extent, with p66-Shc was detected by anti-Shc blotting, but its association was slower in IGF-I stimulation (Fig. 5BGo). Another finding was that EGF receptor used three isoforms (p66, p52, and p46) of Shc proteins equally in our cells, but IGF-I R appeared to primarily use p52-Shc (Fig. 5BGo), which was in part consistent with a previous report on Chinese hamster ovary cells overexpressing both IR and EGF R (38).

EGF receptor binds Shc and also GRB2 directly and rapidly after the ligand binding (25, 39, 40). Our data suggest that the activated EGF receptor binds GRB2 directly rather than Shc (Fig. 4AGo). Thus, EGF receptor will activate p21ras very strongly through EGF receptor-GRB2-Sos complex and Shc-GRB2-Sos complex, and we detected the latter complex at 1 min after EGF stimulation. In contrast, Shc-GRB2-Sos complex was hardly observable at least within 5 min after IGF-I stimulation, which was reasonable in view of the much lower Shc-GRB2 complex formation (Fig. 5CGo). Therefore, activation of MAP kinase would occur very slowly and weakly if Shc were the main component for p21ras activation through IGF-I R. However, as a matter of fact, activation of MAP kinase began 1–3 min after the stimulation (Fig. 8Go), and the half maximal response was obtained at 5 min as observed in MBP-containing gel assay (data not shown). Additionally, hyperphosphorylation of c-raf-1 was noted within 10 min after IGF-I stimulation. In the context of IRS-1 as a common substrate for IR and IGF-I R, this observation differed from an in vivo study, which reported that IR could not induce hyperphosphorylation of c-raf-1 in rat liver (41). Taking the weak tyrosine phosphorylation of Shc and the undetectable recruitment of Shc-GRB2-Sos via IGF-I R into account, it remains to be determined whether Shc is a major component in the early phase IGF-I R signaling toward MAP kinase cascade. Rather, IRS-1, the major tyrosine-phosphorylated protein via IGF-I R, appears to contribute to the early activation of MAP kinase cascade in human skin fibroblasts.

Because Shc is thought to play an important role in mitogenesis, we compared the mitogenic potency of IGF-I, EGF, and bFGF, which apparently differ in the early activation of the Shc-GRB2 pathway. In general, EGF is believed to be a more potent growth factor than IGF-I, and in some cell types, IGF-I treatment is reported to be more effective in combination with EGF cotreatment (42, 43). Unexpectedly, the most potent for DNA synthesis assessed by 3H-thymidine incorporation was IGF-I; EGF was even less potent than bFGF, which hardly affected tyrosine phosphorylation of Shc in human fibroblasts. This suggests that, although many RTKs can phosphorylate Shc directly or indirectly, their mitogenic potency may not be determined solely by the signal strength of the Shc-GRB2 pathway.

We have also shown that the maximal activity of MAP kinase was not parallel with the mitogenic potency of IGF-I and EGF. However, we have to consider the duration of MAP kinase activation as well as the maximal activity, because timing effect of MAP kinase activation on differentiation of PC12 cells by nerve growth factor, which is mimicked by overexpression of EGF receptor, has been reported (44). In that report, sustained activation of MAP kinase by nerve growth factor causes differentiation of PC12 cells, which can be mimicked by overexpression of EGF receptor, which results in longer activation of MAP kinase, although native EGF receptor by itself activates MAP kinase transiently and cannot induce the differentiation. Therefore the possibility that EGF treatment causes differentiation or other effects rather than DNA synthesis should be ruled out. In our cells, EGF-induced MAP kinase activation and the corresponding mobility shift of both p44- and p42-MAP kinases were transient, peaked within 10 min, quickly fell, and returned to the basal level within 120 min (data not shown). IGF-I-induced MAP kinase activity, as well as bFGF-induced MAP kinase activity, returns to its basal level within 60 min (data not shown). Thus, these three growth factors activate MAP kinase transiently in human skin fibroblasts and do not appear to resemble the effect of nerve growth factor on PC12 cells, although we need further investigation, for instance, on nuclear translocation of MAP kinase induced by its sustained activation (44).

Rather, the potency for DNA synthesis in human skin fibroblasts appears to correlate with the activation of PI 3-kinase (Fig. 10Go). Recent reports on IR signaling show the pivotal roles of PI 3-kinase in metabolic action, but many other growth factors or cytokines require PI 3-kinase activation for mitogenesis. Thus, it is possible that PI 3-kinase is engaged in mitogenesis by IGF-I R and that IRS-1 is a main component to mediate its signal. To address this issue, we have now been investigating the effect of various concentrations of wortmannin, a commonly used inhibitor for pI 3-kinase, on DNA synthesis. In our preliminary experiments, 100 nM wortmannin, which can inhibit PI 3-kinase activity, coprecipitated by anti-PY antibody by 70% in vitro and does not affect IGF-I- and EGF-induced mobility shift of p42 MAP kinase, inhibited both IGF-I- and EGF-induced increase in thymidine incorporation by only 20–30% when it was administered 30 min before growth factor stimulation (data not shown). This result suggests the requirement of PI 3-kinase in DNA synthesis independently of MAP kinase, and at the same time indicates the contribution of some other signals. We need further experiments, however, to state definitely the effect of wortmannin on cultured skin fibroblasts because the degree of the inhibitory effect appears to differ not only by the dose of the drug, but also by the culture condition and incubation time of wortmannin prior to stimulation.

In any case, our study suggests that the tyrosine phosphorylation of Shc may be insufficient, even if it is needed as the common component, for cell proliferation in early passages of cultured human skin fibroblasts. The activation of MAP kinase may be insufficient as well, although the requirement of its activation is well established. Other signaling pathways besides the strength of Shc and/or MAP kinase pathway such as PI 3-kinase may be important in mitogenesis mediated by growth factor receptor tyrosine kinases.

Another implication in our report has to do with the features of leprechaunism, a genetic syndrome of insulin resistance. Several reports suggested that leprechaunism had a primary or secondary defect in IGF-I R (11, 12), EGF R (13) or both (14), raising the possibility that the abnormality in fetal growth and differentiation in leprechaunism also involves defects of other growth factor receptors in addition to IR. However, our patient’s cells with IR defect showed no apparent impairment in IGF-I or EGF signal transduction in MAP kinase activation and mitogenesis by these growth factors; also, bFGF-induced mitogenesis was normal. Thus, our results demonstrate that receptor signalings for IGF-I, EGF, and bFGF are not responsible for our patient’s clinical symptoms: marked hyperinsulinemia (fasting plasma insulin: 2847 µU/ml, 17082 pM), diabetes mellitus (fasting plasma glucose: 251 mg/dl, 13.9 mM), intrauterine growth retardation (body weight: 1296 g at birth), abnormal segmentation of the lung, and so on. Although characterization of skin fibroblasts from other patients with leprechaunism is needed for confirmation, these data strongly suggest that defects in multiple growth factor receptors other than insulin receptor are not essential for the features of leprechaunism and that insulin receptor per se may play a direct role in fetal growth and differentiation.

Received July 15, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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