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Endocrinology Vol. 144, No. 11 4811-4819
Copyright © 2003 by The Endocrine Society

Growth Factor-Specific Regulation of Insulin Receptor Substrate-1 Expression in MCF-7 Breast Carcinoma Cells: Effects on the Insulin-Like Growth Factor Signaling Pathway

Claudine Lassarre and Jean-Marc Ricort

Institut National de la Santé et de la Recherche Médicale (C.L., J.-M.R.), Unité 515, Hôpital Saint-Antoine, 75012 Paris, France; and Centre National de la Recherche Scientifique (J.-M.R.), Unité Mixte de Recherche 8113, Ecole Normale Supérieure de Cachan, 94235 Cachan Cedex, France

Address all correspondence and requests for reprints to: Jean-Marc Ricort, Ecole Normale Supérieure de Cachan, Laboratoire de Biotechnologies et de Pharmacologie Génétique Appliquée, Bâtiment d’Alembert, 61 avenue du Président Wilson, 94235 Cachan Cedex, France. E-mail: ricort{at}lbpa.ens-cachan.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGFs are potent mitogens that play a crucial role in cell proliferation and/or differentiation and tumorigenesis. Insulin receptor substrate-1 (IRS-1) is a key protein in the IGF signaling pathway in the estrogen-dependent MCF-7 breast carcinoma cell line. In this study, three growth factors [fibroblast growth factor (FGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF)] were tested for their ability to modulate IRS-1 protein expression and the IGF-I signaling pathway. FGF and, to a lesser extent, EGF were found to increase IRS-1 protein, whereas PDGF had no effect. This indicates that growth factors can specifically modulate IRS-1 protein content. The increases provoked by EGF and FGF were dependent on the MAPK signaling pathway but independent of phosphatidylinositol 3-kinase (PI 3-kinase) signaling and required de novo protein synthesis. We noted that the kinetics of MAPK activation was continuous in response to FGF but transient in response to EGF. In addition, transfection of cells with a constitutively active form of MAPK kinase, which results in continuous MAPK activity, increased IRS-1 expression. Taken together, these results suggest that stimulation of IRS-1 expression was therefore stronger when MAPK activity was sustained. Pretreatment of cells with EGF, FGF, or PDGF for 24 h reduced IGF-I-induced tyrosine phosphorylation per molecule of IRS-1. However, IGF-I-induced PI 3-kinase activity was decreased by 24 h of pretreatment with EGF or PDGF but not with FGF. Our results therefore demonstrate that different growth factors are capable of specifically modulating the IGF-I signaling via IRS-1. They further suggest that the FGF-induced increase in IRS-1 counterbalances the inhibition of IRS-1 tyrosine phosphorylation to allow normal stimulation of IGF-I-induced PI 3-kinase activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF-I AND IGF-II are potent polypeptide mitogens implicated in cell proliferation, differentiation, and survival (for review, see Ref. 1). They are produced by most tissues and their action may be endocrine, paracrine, or autocrine. Their biological effects are transmitted via the type I IGF receptor (IGF-IR), knockout of which is lethal (2). IGF-IR is a heterotetrameric transmembrane glycoprotein composed of two extracellular {alpha}-subunits and two transmembrane ß-subunits, and it shares strong homology with the insulin receptor (3). Binding of IGFs to the {alpha}-subunits activates the tyrosine kinase activity of the ß-subunits. The activated IGF-IR associates with intracellular targets, such as Shc and Grb2 proteins and insulin receptor substrate-1 (IRS-1) (4).

IRS-1 was originally identified downstream of the insulin receptor and is a member of a family comprising at least four related proteins, IRS-1 to -4 (4, 5, 6, 7, 8, 9). It possesses 18 potential tyrosine phosphorylation sites after binding of IGFs to IGF-IR and activation of the receptor. Once phosphorylated, these sites serve to anchor IRS-1 to Src homology 2 domain-containing proteins like Shc, Nck, Grb2, Grb10, and the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), (for review, see Ref. 10). It is via these protein-protein interactions that coordinated activation of different signaling pathways is achieved, resulting in the specific biological activities of the IGFs.

As a protein pivot in the IGF signaling pathway, IRS-1 regulates the efficiency of IGF signal transmission, specifically depending on its expression levels [overexpression of IRS-1 induces cell transformation (11)] and the extent of its tyrosine phosphorylation. Several mechanisms appear to be involved in inhibiting IGF-stimulated tyrosine phosphorylation of IRS-1, including proteasome-mediated degradation (12), phosphatase-mediated dephosphorylation (13, 14), and serine/threonine phosphorylation mediated by kinases like MAPK (15), protein kinase C (16), protein kinase B (17), casein kinase II (18), and PI 3-kinase (19, 20, 21). For example, phosphorylation of IRS-1 on serine 307 appears to be an important event in inhibiting further IGF-induced tyrosine phosphorylation (22).

Understanding the regulation of IRS-1 production and its susceptibility to appropriate tyrosine phosphorylation in response to IGFs will therefore contribute toward elucidating the mechanisms that control IGF signaling. With this in view, we investigated the ability of three growth factors [epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF)] to modulate IRS-1 protein levels and subsequent steps in the IGF-I signaling pathway. An estrogen-dependent breast carcinoma cell line, MCF-7, was used, in which IRS-1 is a crucial IGF-I-activated signaling molecule that leads to the mitogenic response (23).

We demonstrated that IRS-1 protein levels are increased after 24 h of treatment with EGF or FGF but are unaffected by PDGF. Calculated per molecule of IRS-1, subsequent IGF-I-induced IRS-1 tyrosine phosphorylation is inhibited by all three growth factors tested, whereas IGF-I-stimulated PI 3-kinase activity is inhibited by EGF and PGDF but is unaffected by FGF. In conclusion, different growth factors are shown to be capable of specifically modulating IGF-I signaling via IRS-1, which may represent an element of fine control in the activity and efficiency of the IGF-I signal transduction pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies and materials
Antiphosphotyrosine (clone 4G10), anti-IRS-1, anti-p85, and anti ERK1/2 antibodies used for immunoblotting were purchased from Upstate Biotechnology (Lake Placid, NY); antibodies to IRS-1 used for the PI3-kinase assay were a gift from J.-F. Tanti (Institut National de la Santé et de la Recherche Médicale, Nice, France); antibodies to ERK2, IGF-IR, and p110 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies to phosphorylated-ERK1/2 were from New England Biolabs (Beverly, MA); and rabbit antimouse Ig antibodies were purchased from ICN (Orsay, France). Plasmid coding for the constitutively active form of MAPK kinase was a generous gift from J. Pouysségur (Centre National de la Recherche Scientifique, Nice, France). Recombinant human PDGF-BB was obtained from Tebu (Le Perray-en-Yvelines, France), basic FGF was from R&D Systems (Minneapolis, MN), EGF was from PeproTech (Rocky Hill, NJ), and recombinant human des(1–3)IGF-I was provided by GroPep (Adelaide, Australia). All other biochemicals were from Sigma (Saint-Quentin Fallavier, France) or ICN.

Cell culture
The MCF-7 cell line was grown to 85–90% confluence in DMEM supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. For 16–24 h before each experiment, cells were starved in DMEM without serum.

Western immunoblotting
MCF-7 cells were cultured as described above and treated for different periods of time with or without FGF, EGF or PDGF. The cells were then solubilized in buffer A [20 mM Tris (pH 7.4), 137 mM NaCl, 100 mM NaF, 10 mM EDTA, 2 mM Na3VO4, 10 mM pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 100 U/ml aprotinin] containing 1% Nonidet P-40 and the proteins (50 µg as quantified by DC protein assay, Bio-Rad Laboratories, Hercules, CA) separated by SDS-PAGE and transferred to polyvylinidene difluoride (PVDF) sheets. These were incubated with the specific antibodies for 1 h at room temperature and revealed by enhanced chemiluminescence (Amersham, Orsay, France).

Measurement of ERK activity
Lysates obtained from cells treated as above were used to measure ERK1/2 activities. Phosphorylated ERK1/2 (i.e. activated) were identified via their characteristically reduced electrophoretic mobility (24) or using specific antibodies directed against phosphorylated forms of ERK1/2.

Determination of PI 3-kinase activity
Lysates from cells treated as above were incubated for 2 h at 4 C with antibodies to IRS-1, or p85, coupled to protein-A-Sepharose beads. Thereafter, immune pellets were washed twice with each of the following three buffers: 1) PBS containing 1% Nonidet P-40 and 200 µM Na3VO4; 2) 100 mM Tris (pH 7.4), 0.5 M LiCl, 200 µM Na3VO4; and 3) 10 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EDTA, 200 µM Na3VO4. Immunoprecipitated PI 3-kinase activity was measured in the immune pellets by in vitro phosphorylation of phosphatidylinositol (25, 26). The reaction products were separated by thin-layer chromatography on silica plates in methanol/chloroform/ammoniac buffer. After autoradiography, PI 3-kinase activity was quantified by Cerenkov analysis of the spots corresponding to phosphatidylinositol 3-phosphate.

Transient transfection of MCF-7 cells
Twenty-four hours after trypsinization, cells were transiently transfected as follows: 3 µg of the expression vector (pECE vector encoding the constitutively active form of p45-MAPK kinase) was diluted in 100 µl serum-free OPTI-MEM for further transfection in dishes and 6 µl PLUS reagent added. Lipofectamine was diluted (1/25) in serum-free OPTI-MEM (100 µl), mixed with the plasmid/OPTI-MEM/PLUS mixture, and incubated for 15 min at room temperature. Thereafter, the mixture was carefully added to the cells preincubated in 800 µl OPTI-MEM. Cells were then analyzed 24 h after transfection.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EGF and FGF stimulate IRS-1 expression in MCF-7 cells
To test the ability of growth factors to modulate IRS-1 expression, MCF-7 cells were treated with different concentrations of EGF, FGF, or PDGF for 24 h, then lysed, and equal amounts of protein separated by SDS-PAGE before being transferred to PVDF sheets. IRS-1 protein content was determined by Western immunoblotting. EGF dose-dependently increased IRS-1 (Fig. 1AGo), with maximal effects (2- to 2.4-fold increases) between 10 and 40 ng/ml EGF. FGF also dose-dependently increased IRS-1, but the dose-response curve was biphasic. A significant increase was observed with as little as 0.5 ng/ml FGF (2.1 ± 0.15-fold increase), the maximal effect was obtained with 5 ng/ml (6.3 ± 0.2-fold increase), and higher dosages yielded smaller IRS-1 increases (Fig. 1BGo). PDGF had no effect on IRS-1, whichever the concentration used (Fig. 1CGo). The response to EGF and FGF appeared to be relatively specific to IRS-1 because none of the three growth factors tested modulated protein content of IGF-IR (see Fig. 7DGo), the p85 and p110 subunits of PI 3-kinase (see Fig. 8Go, inset) or ERK2 (data not shown).



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  		FIG. 1. EGF and FGF, but not PDGF, stimulate IRS-1 expression in MCF-7 cells. Top, Cells were incubated for 24 h with different concentrations of EGF (A), FGF (B), or PDGF (C). Bottom, Cells were incubated for different periods of time with or without EGF (40 ng/ml) or FGF (5 ng/ml). Proteins from cell lysates were separated by SDS-PAGE, transferred to PVDF sheets and immunoblotted with anti-IRS-1 antibodies as described in Materials and Methods. The autoradiograms presented are those of typical experiments. The graphs represent quantitative analysis of the relative IRS-1 content at each point, corrected for background and corresponding to the mean ± SEM for three to eight separate experiments.

 


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  		FIG. 7. EGF, FGF, and PDGF inhibit des(1–3)IGF-I-induced tyrosine phosphorylation of IRS-1. Cells were incubated with or without EGF (40 ng/ml), FGF (5 ng/ml), or PDGF (40 ng/ml) for 1 h (blot A) or 24 h (blot B) and then stimulated for 5 min with des(1–3)IGF-I (40 ng/ml). Proteins from lysed cells were separated by SDS-PAGE, transferred to PVDF sheets, and immunoblotted with antiphosphotyrosine antibodies as described in Materials and Methods. The results presented are those of typical experiments. Bars represent quantitative analysis of total phosphotyrosylated IRS-1 under each set of conditions, corrected for background, and expressed as percentages of phosphotyrosylated IRS-1 measured after 5 min of des(1–3)IGF-I stimulation without pretreatment with EGF, FGF, or PDGF (control). Results are the means ± SEM for four separate experiments (*, P < 0.05; **, P < 0.01). C, Cells were stimulated with or without IGF-I (40 ng/ml) for 7 min. Cells were lysed and proteins from cell lysates (LYSATES) or anti-IRS-1-immunoprecipitated proteins (IP anti IRS-1) were separated by SDS-PAGE and immunodetected with antiphosphotyrosine antibodies. The results presented are those of typical experiments. D, Lysates from cells incubated with or without EGF (40 ng/ml), FGF (5 ng/ml), or PDGF (40 ng/ml) for 24 h were separated by SDS-PAGE and immunodetected with anti-IGF-IR antibodies. The results presented are those of typical experiments.

 


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  		FIG. 8. Effects of EGF, FGF, and PDGF pretreatment on des(1–3)IGF-I-induced PI 3-kinase activity. A, Cells were incubated with or without EGF (40 ng/ml), FGF (5 ng/ml), or PDGF (40 ng/ml) for different periods of time and then stimulated for 7 min with or without des(1–3)IGF-I (40 ng/ml). Proteins from cell lysates were immunoprecipitated with antibodies to IRS-1 coupled to protein-A-Sepharose. PI 3-kinase activity was measured in immune pellets as described in Materials and Methods. Results are the means ± SEM for four independent experiments. Results are expressed as percentages of the activity measured after 7 min of des(1–3)IGF-I stimulation without pretreatment with EGF, FGF, or PDGF. Insets, Immunodetection of p85 and p110 subunits of PI 3-kinase in cell lysates from cells treated for 24 h with or without EGF (40 ng/ml), FGF (5 ng/ml), or PDGF (40 ng/ml). B, Cells were incubated with or without EGF (40 ng/ml), FGF (5 ng/ml), or PDGF (40 ng/ml) for different periods of time. Proteins from cell lysates were immunoprecipitated with antibodies to p85 coupled to protein-A-Sepharose. PI 3-kinase activity was measured in immune pellets as described in Materials and Methods. The results presented are those of typical experiments.

 
Time course of the effects of EGF on IRS-1 expression differs from that of FGF in MCF-7 cells
The time course of IRS-1 production in response to EGF and FGF was investigated, using the concentrations that in Fig. 1Go, A and B, elicited maximal stimulation (40 and 5 ng/ml, respectively). EGF slightly but significantly increased IRS-1 (2-fold) after 20 h of treatment (Fig. 1DGo). The FGF-induced increase in IRS-1 was more rapid: a significant 1.6-fold rise was observed after 4–6 h, with a progressive increase up to 6-fold after 24 h (Fig. 1DGo).

FGF, EGF, and PDGF fail to prevent des(1–3)IGF-I-induced down-regulation of IRS-1
A previous study demonstrated that EGF is capable of blocking the IGF-induced down-regulation of IRS-1 in an epithelial prostate cell line (27). Because EGF and FGF increased IRS-1 protein content in MCF-7 cells, we tested their ability to counterbalance the effects of IGF-I on IRS-1. Des(1–3)IGF-I (an IGF-I analog with reduced affinity for IGF-binding protein (IGFBP) but intact affinity for IGF-IR) (28, 29) was used to avoid interference from binding between IGF-I and endogenously secreted IGFBPs. MCF-7 cells were treated with des(1–3)IGF-I in combination with or without EGF, FGF, or PDGF. Cells lysates were then analyzed for IRS-1 protein content. As previously described for IGF-I (12), des(1–3)IGF-I decreased IRS-1 levels in MCF-7 cells (Fig. 2Go). EGF, FGF, and PDGF all failed to inhibit des(1–3)IGF-I-induced down-regulation of IRS-1 (Fig. 2Go), despite their own effects on IRS-1 (Figs. 1Go and 2Go).



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  		FIG. 2. FGF, EGF, and PDGF fail to prevent des(1–3)IGF-I-induced IRS-1 down-regulation. Cells were incubated with des(1–3)IGF-I (40 ng/ml) in the presence or absence of EGF (40 ng/ml), FGF (5 ng/ml), or PDGF (40 ng/ml) for 24 h. Proteins from cell lysates were separated by SDS-PAGE, transferred to PVDF sheets, and immunoblotted with anti-IRS-1 antibodies as described in Materials and Methods. The results presented are representative of eight independent experiments.

 
FGF and EGF stimulation of IRS-1 expression requires de novo protein synthesis
To test the requirement for ongoing protein synthesis, MCF-7 cells were treated with the translation inhibitor, cycloheximide, before addition of EGF or FGF. Cycloheximide treatment virtually abolished both EGF- and FGF-induced increase in IRS-1, confirming the need for de novo protein synthesis (Fig. 3Go).



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  		FIG. 3. FGF-induced IRS-1 expression requires de novo protein synthesis. Cells were incubated for 2 h with or without cycloheximide (C) (10 µg/ml) and then for 24 h with or without FGF (F) (5 ng/ml) or EGF (E) (40 ng/ml). Proteins from lysed cells were separated by SDS-PAGE, transferred to PVDF sheets, and immunoblotted with anti-IRS-1 antibodies as described in Materials and Methods. The results presented are representative of three independent experiments.

 
FGF-induced increase in IRS-1 is blocked by MAPK inhibitor
In view of the marked influence of FGF on IRS-1 levels, we inhibited two well-characterized pathways activated by FGF, the MAPK and PI 3-kinase pathways, to determine which may account for the effect. MCF-7 cells were preincubated or not with wortmannin (a PI 3-kinase inhibitor) or PD98059 (a MAPK kinase inhibitor), after which they were or were not incubated with FGF for 24 h. PI 3-kinase and ERK2 activities were checked to confirm that they were inhibited during wortmannin or PD98059 incubation, respectively (data not shown). In the absence of inhibitor, FGF increased IRS-1 protein content (Fig. 4AGo). Wortmannin failed to alter IRS-1 content in either untreated or FGF-treated cells, indicating that FGF stimulation of IRS-1 synthesis does not involve a PI 3-kinase-dependent signaling pathway. PD98059 treatment did not affect basal IRS-1 expression but totally blocked the FGF stimulation. This indicates that a MAPK-dependent signaling pathway is required for FGF induction of IRS-1 production in MCF-7 cells (Fig. 4BGo). Similar results were obtained with EGF (data not shown).



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  		FIG. 4. Inhibition of MAPK blocks the FGF-induced increase in IRS-1. Cells were incubated with or without 100 nM wortmannin (Wort) for 20 min (A) or 100 nM PD98059 (P) for 1 h (B) before being incubated for 24 h with or without FGF (5 ng/ml). Proteins from lysed cells were separated by SDS-PAGE, transferred to PVDF sheets, and immunoblotted with anti-IRS-1 antibodies as described in Materials and Methods. The results presented are representative of seven independent experiments.

 
EGF and FGF differentially stimulate MAPK activity in MCF-7 cells
Because EGF and FGF (but not PDGF) stimulated MAPK activity (Fig. 5AGo), which seemed necessary for IRS-1 stimulation (Fig. 4BGo), we considered whether differences in the time course of MAPK activation may account for the different levels of IRS-1 stimulation. Cells were treated for different periods of time with EGF or FGF and ERK activity visualized as described in Materials and Methods. EGF transiently stimulated the activities of ERK1/2 (Fig. 5BGo), with no phosphorylated proteins detectable beyond 2 h of EGF incubation. In contrast, FGF stimulation of ERK1/2 persisted over 24 h of treatment (Fig. 5CGo).



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  		FIG. 5. EGF and FGF differentially stimulate MAPK activity in MCF-7 cells. A, Cells were incubated for 7 min with or without EGF (40 ng/ml), FGF (5 ng/ml), or PDGF (40 ng/ml). B and C, Cells were incubated for different periods of time with or without EGF (40 ng/ml) (B) or FGF (5 ng/ml) (C). Proteins from lysed cells were separated by SDS-PAGE, transferred to PVDF sheets, and immunoblotted with anti-ERK2 (A) or anti-phospho-ERK (B and C) antibodies as described in Materials and Methods. The results presented are representative of three to six independent experiments.

 
A constitutively active form of p45 MAPK kinase mimics FGF stimulation of IRS-1 synthesis in MCF-7 cells
Because EGF and FGF differentially activated MAPK, we hypothesized that prolonged activation of ERK2 by FGF may account for its stronger stimulation of IRS-1 synthesis, as compared with EGF. To test this hypothesis, we mimicked the continuous activation of ERK by FGF by transiently transfecting the cells with a constitutively activated form of p45 MAPK kinase. Whereas transient transfection with an empty vector (MOCK) had no effect, expression of p45 MAPK kinase in transfected cells resulted in persistent and similar activation of both ERK1 and ERK2 (Fig. 6Go, top, A and B) and a 3.6-fold increase in IRS-1 levels (Fig. 6Go, middle and bottom). These results strongly suggest that prolonged MAPK activation correlates with increased stimulation of IRS-1 production in MCF-7 cells.



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  		FIG. 6. A constitutively active form of p45-MAPK kinase mimics FGF stimulation of IRS-1 synthesis in MCF-7 cells. MCF-7 cells were transfected or not with either an empty vector (MOCK) or a vector encoding a constitutively active form of p45-MAPK kinase (MAPKK+) as described in Materials and Methods. Thereafter, cells were treated for 24 h with or without 5 ng/ml FGF. Proteins from lysed cells were separated by SDS-PAGE, transferred to PVDF sheets, and immunoblotted with either anti-ERK2 antibodies (top, A) or anti-ERK1/2 (top, B) or anti-IRS-1 antibodies (middle) as described in Materials and Methods. The results presented are those of typical experiments. Bottom, Bars represent quantitative analysis of the relative IRS-1 protein content under each set of conditions, corrected for background, and correspond to the means ± SEM for four independent experiments.

 
EGF, FGF, and PDGF inhibit des(1–3)IGF-I-induced tyrosine phosphorylation of IRS-1
The next step was to establish whether, in addition to modulating IRS-1 protein expression, growth factors are capable of modulating steps in the IGF-I signaling pathway. Cells were pretreated with EGF, FGF, or PDGF for 1 or 24 h and then acutely stimulated for 5 min with des(1–3)IGF-I. Induction of tyrosine phosphorylation of IRS-1 by des(1–3)IGF-I was then analyzed as described in Materials and Methods. First, we checked that the phosphotyrosylated band observed in the total lysates corresponded to tyrosine phosphorylated IRS-1. Proteins in the total lysate and proteins previously immunoprecipitated using anti-IRS-1 antibody were immunodetected using antiphosphotyrosine antibody. (Fig. 7CGo). The identical migration of the bands confirmed that the phosphotyrosylated band found in total lysates in response to des(1–3)IGF-I treatment was indeed IRS-1. No tyrosine phosphorylation of IRS-1 was induced by EGF, FGF, or PDGF alone (data not shown), and none was detectable under control conditions (Fig. 7Go). After 5 min of incubation with des(1–3)IGF-I in the absence of EGF, FGF, or PDGF, IRS-1 tyrosine phosphorylation was strongly stimulated (control). PDGF pretreatment for 1 h reduced des(1–3)IGF-I-induced tyrosine phosphorylation of IRS-1 by 40%, and the level of inhibition remained virtually the same after 24 h. In contrast, although 1 h of pretreatment with EGF and FGF also inhibited des(1–3)IGF-I-induced IRS-1 tyrosine phosphorylation by almost 40%, after 24 h of pretreatment, inhibition was reduced to 20% in the case of EGF and totally reversed in the case of FGF. Finally, we checked that at each time point, treatment with EGF, FGF, or PDGF had no effect on type 1 IGF receptor levels (Fig. 7DGo).

Effects of EGF, FGF, and PDGF on the IRS-1-associated PI 3-kinase activity induced by des(1–3)IGF-I
PI 3-kinase is pivotal for IGF-I signaling (30) and the role of IGF-I in antiapoptosis (31, 32) in MCF-7 cells. Its activity depends directly on the tyrosine phosphorylation state of IRS-1 (33, 34) and the quantities of IRS-1 protein available, both of which were affected differently by EGF, FGF, and PDGF (Figs. 1Go and 7Go). We investigated the effects of these growth factors on des(1–3)IGF-I-stimulated PI 3-kinase activity by pretreating cells with or without EGF, FGF, or PDGF for different periods of time (1, 6, 15, 20, or 24 h), followed by stimulation for 7 min with or without des(1–3)IGF-I. After 1 h of pretreatment with EGF, FGF, and PDGF, des(1–3)IGF-I-stimulated PI 3-kinase activity was inhibited by 53%, 33%, and 32%, respectively (Fig. 8AGo). In the case of PDGF, the level of inhibition remained virtually constant whichever the duration of pretreatment. With EGF, longer periods of pretreatment (20 and 24 h) resulted in decreased inhibition, and with FGF the effect was more rapid (within 15 h), inhibition having totally disappeared after 24 h. We checked that at each time point, growth factor treatment did not alter expression levels of the p85 and p110 subunits of PI 3-kinase (Fig. 8Go, insets). PI 3-kinase activity under the influence of EGF, FGF, or PDGF was also checked at each time point. No detectable PI 3-kinase activation was measured with EGF or FGF between 1 and 24 h (Fig. 8BGo). With PDGF, there was slight activation after 1 h, but this totally disappeared after 2.5 h.

Effects of EGF, FGF, and PDGF on ERK activity
Having shown that pretreatment with EGF, FGF, or PDGF affected IGF-I-induced IRS-1 tyrosine phosphorylation and IRS-1-associated PI 3-kinase activity, we investigated the possibility that other IGF-I-activated signaling pathways may be similarly modulated. Cells were pretreated for 24 h with EGF, FGF, or PDGF and then treated for 7 min with or without des(1–3)IGF-I and tested for ERK2 activity. Seven minutes of des(1–3)IGF-I treatment alone stimulated ERK2 activity. This stimulation was unaffected by 24 h of pretreatment with either EGF or PDGF (Fig. 9Go, A and C), but in the case of FGF, it was impossible to draw a conclusion because FGF-induced ERK2 activity (as seen in Fig. 5Go) was persistently strong (Fig. 9BGo).



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  		FIG. 9. Effects of EGF, FGF, and PDGF pretreatment on des(1–3)IGF-I-induced ERK2 activity. Cells were incubated with or without EGF (40 ng/ml) (A), FGF (5 ng/ml) (B), or PDGF (40 ng/ml) (C) for 24 h and then stimulated for 7 min with or without des(1–3)IGF-I (40 ng/ml). Proteins from cell lysates were separated by SDS-PAGE, transferred to PVDF sheets, and immunoblotted with anti-ERK2 antibodies as described in Materials and Methods. The results presented are representative of four independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, three growth factors have been shown to exert differential effects in regulating IRS-1 expression and modulating IGF signaling in MCF-7 cells. EGF and, to a much greater extent, FGF provoked dose- and time-dependent increases in IRS-1 levels owing to de novo synthesis, whereas PDGF had no effect. The cellular response to each growth factor therefore appeared to be specific, indicating that either distinct signaling pathways are activated or the same signaling pathways are differently affected in MCF-7 cells. Investigation of two major signaling pathways in these cells revealed PI 3-kinase activation by all three growth factors (data not shown) but in the case of MAPK, specifically a lack of activation by PDGF. The regulation of IRS-1 expression by EGF and FGF therefore probably involved a MAPK-dependent signaling pathway, which was confirmed by the experiments using specific protein inhibitors. Possibly this reflects the ability of MAPKs to modulate gene expression by directly phosphorylating transcription factors and/or by activating other protein kinases (e.g. Rsk, Mnk) that then phosphorylate proteins involved in gene expression (for review, see Ref. 35). FGF more potently increased IRS-1 protein than EGF, and because MAPK was evidently important in this regulation, we considered that differences in MAPK activation might account for the differential effects of FGF and EGF. Both EGF and FGF equally and potently stimulated ERK1/2 activities during the shorter periods of stimulation, but differences in the kinetics of activation appeared thereafter. The effect of EGF was transient (ERK1/2 activities persisted for about 1 h), whereas that of FGF was sustained and robust for at least 24 h. These differences in ERK activation kinetics may then account for the differences in potency in stimulating IRS-1 expression. Similar results have been described in PC12 cells, in which the kinetics of MAPK activation are transient with EGF and prolonged with nerve growth factor, resulting in cell proliferation in the former case and differentiation in the latter (36, 37, 38). MAPK activation kinetics represent the balance between a turn-on signal and a turn-off signal generated by MAPK phosphatases (MKPs) (for review, see Ref. 39), which determines the duration of activation. It would therefore appear that in MCF-7 cells, EGF and FGF differently regulate deactivation of MAPKs. Possible explanations may be growth factor-specific regulation of the activities of MKP isoforms and/or subcellular localization of MAPKs to sites close to MKP isoforms. On the suspicion that growth factor-induced IRS-1 expression might correlate with sustained MAPK activation (as with FGF), we mimicked continuous MAPK activation by transfecting MCF-7 cells with a constitutively active form of MAPK kinase. The result was continuous stimulation of ERK1/2 activities and a 3.6-fold increase in IRS-1 protein. The effects appeared to be stronger than those elicited by EGF (partially confirming our suspicion) and weaker than those with FGF. However, quantification of the efficiency of the transient transfection method showed that only about 20–30% of MCF-7 cells were transfected (data not shown). Relatively, therefore, our findings indicate that when MAPK activity is sustained, increases in IRS-1 protein are more pronounced. These results strongly support the hypothesis that MAPK activation accounts for the stimulation of IRS-1 synthesis by growth factors in MCF-7 cells.

Although EGF and FGF increased IRS-1 in MCF-7 cells, none of the three growth factors studied counteracted IGF-I-induced down-regulation of IRS-1. This would appear to contradict the results reported for EGF in a model of prostate epithelial cells (27). The discrepancy may reside in specificity of intracellular response to EGF and/or FGF and PDGF that would not inhibit the proteasome-mediated IGF-I-dependent degradation of IRS-1 in MCF-7 cells, in which ubiquitination of IRS-1 is a key determinant (40, 41). Another explanation may involve expression of secreted IGFBPs. EGF modulates IGFBP expression in several cell types (42, 43, 44) and, if EGF increases IGFBP expression in this prostate epithelial cell line, the IGF-I response would be inhibited owing to uptake and sequestration of IGF-I by IGFBPs in the conditioned medium.

IRS-1 overexpression provokes transformation (11) and IRS-1 is a major mediator of IGF-I signaling in MCF-7 cells (23). Because EGF and FGF stimulated IRS-1 expression, we investigated the possibility that increased IRS-1 modulates IGF-I signaling. Although IGFBP secretion was unaffected by EGF, FGF, and PDGF in MCF-7 cells (data not shown), we elected to use des(1–3)IGF-I to elicit IGF-I signaling to preclude binding to secreted IGFBPs. After 1 h of pretreatment with EGF, FGF, or PDGF, des(1–3)IGF-I-induced tyrosine phosphorylation of IRS-1 was significantly reduced (by about 40%). The inhibitory effect of PDGF was sustained over 24 h, whereas that of EGF decreased over time. In the case of FGF, inhibition was abolished after 24 h. Nevertheless, EGF increased IRS-1 protein 1.8-fold and FGF 6-fold, which means that, calculated per molecule of IRS-1, tyrosine phosphorylation was in fact strongly inhibited (by about 60%). These values are based on the assumption that constitutively synthesized and growth factor-stimulated IRS-1 proteins are equally phosphorylable following subsequent des(1–3)IGF-I stimulation. Several possibilities may account for this inhibition. One is that these growth factors may provoke translocation of IRS-1 proteins to a subcellular compartment in which they are less accessible to IGF-IR tyrosine kinase activity. Such IRS-1 movement has been described in 3T3-L1 adipocytes in which insulin provokes translocation of IRS-1 from low-density microsomes to the cytosol (45, 46). However, this translocation is sensitive to insulin but not to PDGF in adipocytes (46, 47), and investigation of the other growth factors will be necessary to clarify this point. Another possibility involves serine/threonine phosphorylation of IRS-1. Although protein kinase B-induced phosphorylation up-regulates IRS-1 (17), serine/threonine phosphorylation of IRS-1 more often prevents further tyrosine phosphorylation. The serine/threonine phosphatase inhibitor, okadaic acid (48, 49), and PDGF (50) increase serine phosphorylation of IRS-1 and inhibit insulin action because the serine/threonine-phosphorylated form of IRS-1 has diminished ability to interact with the insulin receptor (49). This is achieved by numerous kinases, such as MAPK (15), protein kinase C (16), protein kinase B (17), casein kinase II (18), and PI 3-kinase (19, 20, 21), all of which can be activated by EGF, FGF, and PDGF.

Our study therefore reveals at least two regulatory mechanisms affecting IRS-1 in response to growth factors. One, modulating its expression, is apparently MAPK dependent, and the other, mediating its IGF-I-induced phosphotyrosylation, which in view of the differential effects of EGF, FGF, and PDGF, is not MAPK dependent in MCF-7 cells. Studies are in progress to determine which signaling pathway(s) may be implicated in such regulation, although, as demonstrated in 3T3-L1 adipocytes (50), PI 3-kinase activity would be a good candidate.

Because EGF, FGF, and PDGF affect IGF-I-induced tyrosine phosphorylation of IRS-1, we investigated IRS-1-associated PI 3-kinase activity, which is determined by the extent of IRS-1 tyrosine phosphorylation (33, 34). Having shown that EGF, FGF, and PDGF themselves fail to stimulate PI 3-kinase activity at the time points studied (1–24 h of pretreatment), we could exclude the possibility that their effects on IGF-I-induced PI 3-kinase could reflect interference between the PI 3-kinase activity involved in the EGF, FGF, and PDGF pathways and that involved in the IGF-I pathway. PDGF pretreatment of 3T3-L1 adipocytes reduces insulin-induced IRS-1 tyrosine phosphorylation and results in inhibition of subsequent insulin-induced PI 3-kinase activity (50). We therefore explored the effects of the three growth factors on IGF-I-stimulated PI 3-kinase activity, which after 1 h of pretreatment with EGF, FGF, or PDGF, was inhibited by 30–50%. With PDGF, the inhibitory effect was sustained over at least 24 h. However, with EGF and FGF, a novel mechanism emerged: inhibition was suppressed over time, following kinetics that mirrored those of the IRS-1 increase that they provoke. These increases could therefore compensate, either totally or partially, for the reduced tyrosine phosphorylation per molecule of IRS-1. In essence then, these results suggest that activation of PI 3-kinase by IGF-I could effectively remain slightly or totally unaltered by exposure of the cells to EGF and FGF. PI 3-kinase is pivotal for IGF-I signaling (30) and the role of IGF-I in antiapoptosis (31, 32) in MCF-7 cells. The enlarged IRS-1 protein pool would represent a mechanism to counterbalance inhibition of IGF-I-stimulated IRS-1 tyrosine phosphorylation induced by growth factors like EGF and FGF. Thus, the IGF signal and consequently cell metabolism can remain unrestrained and the proliferative action of powerful mitogens like FGF maximally efficacious.

We also noted another signaling pathway in MCF-7 cells that was activated by IGF-I and led to ERK2 activation but was unaffected by 24 h of pretreatment with EGF and PDGF. A certain specificity therefore appears to exist with regard to inhibition of PI 3-kinase signaling in response to these growth factors. There could be several explanations for this. MAPK signaling could be activated independently of IRS-1 via another docking protein such as Shc in these cells. It could also be presupposed that the inhibition of IRS-1 phosphotyrosylation does not affect the phosphotyrosine residues involved in binding of the Src homology 2 domain of Grb2, the adaptor protein responsible for activation of the MAPK signaling pathway.

In summary, our results demonstrate that modulation of IRS-1 expression in MCF-7 cells can be growth factor specific, affecting the IGF-I signaling pathway at least with regard to IRS-1 tyrosine phosphorylation and PI 3-kinase activity. It was not possible in this study to provide direct evidence of effects on downstream biological activities like cell proliferation because all three growth factors are potent mitogens in MCF-7 cells, and any further stimulation by IGF-I was impossible to measure. Studies are under way on a different cell line, in which differentiation is differently modulated by IGF-I, EGF, FGF, and PDGF, in which growth factor pretreatment provokes differences in IRS-1 tyrosine phosphorylation and PI 3-kinase activity as well as the process of differentiation. Nevertheless, the present findings provide insights into negative or cooperative cross-talks that exist among the EGF, FGF, and PDGF signaling pathways, on the one hand, and the IGF-I signaling pathway on the other. It is pertinent that these mechanisms may be of importance in tumors harboring activating or inhibiting mutations of the different growth factor receptors.


   Acknowledgments
 
We are grateful to M. Binoux for critical reading of the manuscript.


   Footnotes
 
This work was supported by the Institut National de la Santé et de la Recherche Médicale, the University of Paris VI, the Centre National de la Recherche Scientifique, and Ecole Normale Supérieure de Cachan. J.-M.R. was a fellow of the Association Française de lutte contre la Myopathie and of Fondation Singer-Polignac.

Abbreviations: EGF, Epidermal growth factor; FGF, fibroblast growth factor; IGFBP, IGF-binding protein; IGF-IR, type 1 IGF receptor; IRS, insulin receptor substrate; MKP, MAPK phosphatase; PDGF, platelet-derived growth factor; PI 3-kinase, phosphatidylinositol 3-kinase; PVDF, polyvylinidene difluoride.

Received December 30, 2002.

Accepted for publication July 15, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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