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J. Biol. Chem., Vol. 277, Issue 22, 19448-19454, May 31, 2002

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Insulin-like Growth Factor-binding Protein-3 Activates a Phosphotyrosine Phosphatase

EFFECTS ON THE INSULIN-LIKE GROWTH FACTOR SIGNALING PATHWAY^*

Jean-Marc Ricort and Michel Binoux

From the Institut National de la Santé et de la Recherche Médicale, Unité 515, Assistance Publique-Hôpitaux de Paris, Université Paris VI, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75571 Paris CEDEX 12, France

Received for publication, January 15, 2002, and in revised form, March 19, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The proliferative action of insulin-like growth factors (IGF-I and -II) is mediated via the type I IGF receptor (IGF-IR) and is modulated by their association with high affinity binding proteins, IGFBP-1 to -6. We recently found that, in addition to its ability to bind IGFs, IGFBP-3 also inhibits IGF-IR activation independently of IGF binding and without interacting directly with IGF-IR. Here, we show that IGFBP-3 is capable of blocking the signal triggered by IGFs. Breast carcinoma-derived cells (MCF-7) were stimulated by des(1-3)IGF-I or [Gln³,Ala⁴,Tyr¹⁵,Leu¹⁶]IGF-I, two IGF analogues with intact affinity for IGF-IR, but with weak or virtually no affinity for IGFBPs, then incubated with IGFBP-3. The activated IGF-IR was desensitized through reversal of its autophosphorylation, following which both phosphatidylinositol 3-kinase and p42^MAPK activities were depressed. Direct measurement of phosphotyrosine phosphatase activity and reconstitution experiments using tyrosine-phosphorylated insulin receptor substrate-1 (IRS-1) indicated that IGFBP-3 activated a phosphotyrosine phosphatase (PTPase). This action appeared to be peculiar to IGFBP-3 among the IGFBPs, since neither IGFBP-1 nor IGFBP-5 (structurally the closest to IGFBP-3), had any such effect. Several cell lines derived from normal or tumor cells responsive to IGF-I were used to show that IGFBP-3-stimulated PTPase is cell type-specific. Although the precise nature of the phosphatase remains to be determined, the results of this study demonstrate that IGFBP-3 stimulates a phosphotyrosine phosphatase activity that down-regulates the IGF-I signaling pathway, suggesting a major role for IGFBP-3 in regulating cell proliferation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The insulin-like growth factors, IGF-I and -II,¹ are involved in regulating proliferation and/or differentiation in diverse cell types (for reviews, see Refs. 1 and 2). Their biological effects are transmitted via binding to and activation of their type I receptor. The stimulated tyrosine kinase activity of the receptor leads to tyrosine phosphorylation of cellular substrates like insulin receptor substrate 1 and 2 (IRS-1 and -2). This tyrosine phosphorylation allows binding to proteins containing SH2 domains that initiate activation of such pathways as the phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase (MAP kinase) signaling pathways. Since PI 3-kinase activity is essential for the mitogenic signaling of IGF-I in MCF-7 breast cancer cells (3), it probably plays a major role in IGF-I-responsive tumors. IGFs are potent determinants in cancer incidence (4, 5) and, at a cellular level, the tyrosine kinase activity of IGF-IR and the signaling pathways activated downstream of it constitute key elements in this mitogenic capacity. Understanding the molecular mechanisms regulating these activities is therefore of primary interest in elucidating IGF-dependent carcinogenesis.

In all biological fluids, IGFs are associated with high affinity binding proteins, the IGFBPs, six molecular species of which (IGFBP-1 to -6) have been characterized. These act as carriers in the bloodstream and also modulate IGF action mediated via the type I IGF receptor (IGF-IR) (1, 6, 7). Some IGFBPs are now known to possess intrinsic activities that are unrelated to their IGF binding (for review, see Ref. 8). For instance, IGFBP-3, which is present in most tissues (9), influences cell growth (10, 11) and induces apoptosis (12). We previously reported that preincubation of MCF-7 breast cancer cells with recombinant human (rh)-IGFBP-3 inhibits subsequent IGF-IR activation by IGF analogues with intact affinity for IGF-IR but weak or virtually no affinity for IGFBP-3 (13). However, the molecular mechanisms of this effect remained to be elucidated. In this study, we provide evidence that IGFBP-3 activates a tyrosine phosphatase capable of blocking the IGF-I signaling pathway.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Antibodies and Materials

Anti-phosphotyrosine antibodies used for immunoblotting were purchased from Upstate Biotechnology (Lake Placid, NY), antibodies to IRS-1 and to the p85 subunit of PI 3-kinase were a gift from J.-F. Tanti (INSERM, Nice, France), antibodies to p42^MAPK were purchased from Santa Cruz Inc. (Santa Cruz, CA), and rabbit anti-mouse immunoglobulin antibodies from ICN (Orsay, France). Non-glycosylated rh- IGFBP-3 (coli) was a gift from Celtrix Pharmaceuticals (Santa Clara, CA). Glycosylated IGFBP-3 obtained with the baculovirus/insect cell system was a gift from F. Godeau (INSERM U515, Paris, France). Amniotic fluid-derived IGFBP-1, rh-IGFBP-5, and rh-des(1-3)IGF-I (an IGF-I analogue with 80-100-fold reduced affinity for IGFBP-3) (14, 15) were provided by GroPep (Adelaide, Australia). [Gln³,Ala⁴,Tyr¹⁵,Leu¹⁶]IGF-I (an IGF-I analogue with 1000-fold reduced affinity for IGFBP-3) (15) was a gift from M. Cascieri (Merck Research Laboratories, Rahway, NJ), and rh-IGF-I was a gift from Ciba Geigy Ltd. (Basel, Switzerland). All other biochemicals were from Sigma (Saint-Quentin Fallavier, France) or ICN.

Cell Culture

The MCF-7, T47D, and MDA-MB 231 human breast cancer cell lines and CCL39 hamster lung fibroblast cells were grown to 85-90% confluence in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 100 units/ml penicillin and 100 µg/ml streptomycin. The CHO-IR cell line (16) was grown in Ham's-F-12 medium containing 400 µg/ml geneticin supplemented with 10% fetal calf serum and 100 units/ml penicillin and 100 µg/ml streptomycin. For 16-24 h before each experiment, cells were starved in their respective medium without serum.

Immunodetection of Phosphotyrosine-containing Proteins

First, cells were incubated without or stimulated with 3 nM IGF-I, des(1-3)IGF-I or [Gln³,Ala⁴,Tyr¹⁵,Leu¹⁶]IGF-I for periods up to 30 min. After the first 5 min of each period, cells were further incubated for the remaining times at 37 °C with or without different concentrations of IGFBP-3 or with 10 nM IGFBP-1 or IGFBP-5. 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 units/ml aprotinin) containing 1% Nonidet P-40, and the proteins were separated by SDS-PAGE and transferred to polyvylinidene difluoride sheets. These were incubated with anti-phosphotyrosine antibodies overnight at 4 °C and then washed three times (10 min each) in phosphate-buffered saline containing 1% Nonidet P-40. Thereafter, they were incubated for 1 h at room temperature with rabbit anti-mouse immunoglobulin G antibodies and washed as above before being incubated for 1 h at room temperature with ¹²⁵I-protein-A (5 × 10⁵ cpm/ml blocking buffer) and washed as above. Incorporated radioactivity was quantified using a Storm Imager (Amersham Biosciences).

Measurement of p42^MAPK Activity

Lysates obtained from cells treated as above were used to measure MAPK activity. Phosphorylated p42^MAPK was visualized by its characteristically reduced electrophoretic mobility (17).

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 to the PI 3-kinase 85-kDa subunit coupled to protein-A-Sepharose beads. Thereafter, immune pellets were washed twice with each of the three following buffers: (a) phosphate-buffered saline containing 1% Nonidet P-40 and 200 µM Na₃VO₄; (b) 100 mM Tris, pH 7.4, 0.5 M LiCl, 200 µM Na₃VO₄; and (c) 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 PI (18, 19). 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 PI 3-P.

Measurement of Phosphotyrosine Phosphatase Activity

MCF-7 cells were incubated with or without IGFBP-3 or -5 and PTPase activity measured using a kit purchased from Sigma. Briefly, cells were solubilized for 20 min at 4 °C in 50 mM Hepes buffer, pH 7.4, containing 0.5% Triton X-100, 10% glycerol, and protease inhibitors. Then, cell lysates were centrifuged for 10 min at 13,000 rpm and applied to a Sephadex G-25 column to remove phosphate salts. An aliquot of the eluate was used to measure PTPase activity by the dephosphorylation of provided tyrosine-phosphorylated peptides in a buffer comprising: 10 mM Tris, pH 7.4, 1 mM MgCl₂, 0.1% -mercaptoethanol, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride. The inorganic phosphate formed during the reaction complexes with Malachite Green/ammonium molybdate to generate a colored product quantified by spectrophotometry at 620 nm (using an enzyme-linked immunosorbent assay reader).

Reconstitution Experiments

First Step: Immunoprecipitation of Tyrosine-phosphorylated IRS-1-- MCF-7 cells in 100-mm culture dishes were stimulated for 10 min at 37 °C with 6.7 nM IGF-I. Cells were solubilized for 30 min at 4 °C in buffer A containing 1% Nonidet P-40. Lysates were centrifuged for 10 min at 12,000 × g and supernatants immunoprecipitated for 2 h at 4 °C with antibodies to IRS-1 coupled to protein-A-Sepharose beads. Immune pellets were washed twice with phosphatase buffer (10 mM Tris, pH 7.4, 1 mM MgCl₂, 0.1% -mercaptoethanol, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride).

Second Step: Preparation of Cell Lysates-- Cells were incubated with or without 10 nM IGFBP-3 or with IGFBP-1 or IGFBP-5 for 5 min at 37 °C. Cells were scraped into the phosphatase buffer described above and homogenized with a Dounce Potter homogenizer (20 strokes). Homogenates were centrifuged for 10 min at 12,000 × g and the supernatants incubated with immune pellets for 10 min at room temperature. In some samples, 1 mM ZnCl₂ was added to the incubation medium. To stop the reaction, immune pellets were washed three times with ice-cold buffer A containing 1% Nonidet P-40. Pellets were solubilized in Laemmli buffer and subjected to SDS-PAGE. Tyrosine-phosphorylated IRS-1 was quantified as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

IGFBP-3 Dose-dependently Reverses the Des(1-3)IGF-I- and [Gln³,Ala⁴,Tyr¹⁵,Leu¹⁶]IGF-I-stimulated Autophosphorylation and Tyrosine Kinase Activity of IGF-IR-- Having previously demonstrated that preincubation of MCF-7 breast cancer cells with IGFBP-3 dose-dependently inhibits subsequent IGF-IR activation by its ligand (13), we sought to determine whether or not IGFBP-3 could reverse this activation. MCF-7 cells were incubated with 3 nM des(1-3)IGF-I for 6 min, with or without increasing concentrations of IGFBP-3 for the last 3 min of incubation. After solubilization of the cells, proteins were separated by SDS-PAGE and immunodetected with anti-phosphotyrosine antibodies. As can be seen in Fig. 1, there was little phosphotyrosine in either IGF-IR or IRS-1 under basal conditions. Des(1-3)IGF-I stimulated IGF-IR autophosphorylation and its tyrosine kinase activity, as visualized by the tyrosine phosphorylation of IRS-1. Addition of IGFBP-3 to the culture medium dose-dependently decreased the des(1-3)IGF-I-induced tyrosine phosphorylation of both IGF-IR and IRS-1.

View larger version (41K): [in this window] [in a new window]   Fig. 1.   IGFBP-3 dose-dependently reduces the des(1-3)IGF-I-stimulated autophosphorylation and tyrosine kinase activity of IGF-IR. MCF-7 cells were incubated with or without 3 nM des(1-3)IGF-I for 6 min at 37 °C, with or without increasing concentrations of rh-IGFBP-3 for the last 3 min of stimulation, before being homogenized. Proteins were separated by SDS-PAGE, transferred to polyvylinidene difluoride sheets, and immunoblotted with anti-phosphotyrosine antibodies as described under "Materials and Methods." The levels of tyrosine phosphorylation are expressed as percentages of that measured in IGF-I-stimulated cells without rh-IGFBP-3. Results are the means ± S.E. for three to four separate experiments.

Time course experiments were performed to analyze the effects of IGFBP-3 in more detail. MCF-7 cells were incubated for varying periods with 3 nM des(1-3)IGF-I, in each case 10 nM rh-IGFBP-3 being added after 5 min. After solubilization of the cells, proteins were separated by SDS-PAGE and immunodetected with anti-phosphotyrosine antibodies. Des(1-3)IGF-I induced tyrosine phosphorylation of IGF-IR and IRS-1 within 2 min (Fig. 2). Stimulation of IRS-1 tyrosine phosphorylation peaked after 6 min and remained at a plateau up to 15 min, with a slight decrease after 30 min. The time course of des-1-3-IGF-I-induced IGF-IR tyrosine phosphorylation was slightly different, with a progressive increase up to 30 min of stimulation. When 10 nM IGFBP-3 was added to the culture media after 5 min of des(1-3)IGF-I stimulation, phosphotyrosine was rapidly dephosphorylated in both IGF-IR and IRS-1, the effect being significant after only 1 min of incubation with IGFBP-3.

View larger version (54K): [in this window] [in a new window]   Fig. 2.   IGFBP-3 reverses IGF-IR and IRS-1 phosphorylation induced by des(1-3)IGF-I. A, MCF-7 cells were stimulated for different time periods with 3 nM des(1-3)IGF-I and in each case treated with or without 10 nM IGFBP-3 after 5 min of stimulation, then treated as described in the legend to Fig. 1. Results are those of a typical experiment. B, the bands corresponding to the tyrosine-phosphorylated proteins, IGF-IR and IRS-1, were quantified using a Storm Imager (Amersham Biosciences). (, control cells; , IGFBP-3-treated cells). Results are the means ± S.E. for five separate experiments. *, p < 0.01; **, p < 0.001 compared with the 100% tyrosine phosphorylation at time 5 min.

With a view to demonstrating that the effect of IGFBP-3 was unrelated to its ability to bind des(1-3)IGF-I for which it has weak affinity, the same experiments were performed using [Gln³,Ala⁴,Tyr¹⁵,Leu¹⁶]IGF-I, which has virtually no affinity for IGFBP-3. As in the case of des(1-3)IGF-I, IGFBP-3 proved capable of reversing [Gln³,Ala⁴,Tyr¹⁵,Leu¹⁶]IGF-I-stimulated tyrosine phosphorylation of both IGF-IR and IRS-1 (Fig. 3).

View larger version (42K): [in this window] [in a new window]   Fig. 3.   IGFBP-3 reverses the phosphorylation of IGF-IR and IRS-1 induced by [Gln3,Ala4,Tyr15,Leu16]IGF-I. Same experiment as in Fig. 2. Cells were stimulated for different time periods with 3 nM [Gln3,Ala4,Tyr15,Leu16]IGF-I and in each case after 5 min of stimulation treated with or without 10 nM IGFBP-3. The results presented are those of a typical experiment.

IGFBP-3 Dose-dependently Reverses the Des(1-3)IGF-I-stimulated PI 3-Kinase and p42^MAPK Activities-- Since addition of IGFBP-3 decreased the IGF-IR autophosphorylation induced by IGF-I analogues, subsequent inhibition could be expected of the two major signaling pathways initiated by IGF-IR, i.e. the PI 3-kinase and MAP kinase signaling pathways. MCF-7 cells were incubated with des(1-3)IGF-I for different periods of time and, as described above, incubated with or without IGFBP-3. Des(1-3)IGF-I stimulated the phosphorylation of p42^MAPK as shown by its reduced electrophoretic mobility, and addition of 10 nM IGFBP-3 inhibited this phosphorylation (Fig. 4). Similarly, as shown by PI 3-kinase measurement in immune pellets obtained with antibodies to IRS-1 (Fig. 5A) or the PI 3-kinase p85 subunit (Fig. 5B), des(1-3)IGF-I stimulated both PI 3-kinase association with IRS-1 and PI 3-kinase activation, and addition of 10 nM IGFBP-3 induced significant inhibition of both activities.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   IGFBP-3 reverses des(1-3)IGF-I-induced p42MAPK phosphorylation. MCF-7 cells were treated as described in the legend to Fig. 2. Proteins from cell lysates were separated by SDS-PAGE, transferred to polyvylinidene difluoride, and immunodetected with anti p42MAPK antibodies as described under "Materials and Methods." Results are those of a typical experiment.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   IGFBP-3 reverses des(1-3)IGF-I-induced PI-3 kinase association with IRS-1 and PI 3-kinase activation. MCF-7 cells were treated as described in the legend to Fig. 2. Proteins from the cell lysates were immunoprecipitated with antibodies to IRS-1 (A) or to the PI 3-kinase p85 subunit (B) coupled to protein-A-Sepharose. PI 3-kinase activity was measured in immune pellets as described under "Materials and Methods." Results are the means of a typical experiment performed in triplicate.

De-activation of the IGF-I Signaling Pathway Is IGFBP-3-specific-- By way of checking that the action of IGFBP-3 was specific, IGFBP-1 and -5 were used in experiments similar to those illustrated in Figs 2 and 3. MCF-7 cells were stimulated with 3 nM des(1-3)IGF-I prior to being incubated with 10 nM IGFBP-1 or -5. Cells were solubilized and proteins separated and immunoblotted as described above. As shown in Table I, neither IGFBP-1 nor IGFBP-5 was capable of reversing the des(1-3)IGF-I-induced tyrosine phosphorylation of IGF-IR and IRS-1, whichever the incubation period with IGFBP.

IGFBP-3 Activates a Tyrosine Phosphatase in MCF-7 Cells-- The state of tyrosine phosphorylation of a protein depends upon the fine balance between tyrosine kinases and tyrosine phosphatases. We therefore sought to determine whether the dephosphorylation provoked by IGFBP-3 results from inhibition of a tyrosine kinase activity or stimulation of a tyrosine phosphatase activity. PTPase activity was measured as described under "Materials and Methods." MCF-7 cells were incubated at 37 °C with or without different concentrations of IGFBP-3 or 30 nM IGFBP-5 for different periods of time, and PTPase activity was measured in cell lysates. IGFBP-3 activated a PTPase capable of dephosphorylating synthetic tyrosine-phosphorylated peptides. PTPase activity was maximal with 10-20 nM IGFBP-3, although significant stimulation was achieved with 5 nM (Fig. 6A). As shown in Fig. 6B, IGFBP-3-induced PTPase activity increased rapidly to a maximum within 15 min (3.3-fold over basal), then plateaued for a further 15 min. Predictably on the basis of the previous experiments, IGFBP-5 had no effect, even used at 30 nM (the dosage of IGFBP-3 that induced the maximal response). Moreover, glycosylated IGFBP-3 had the same effect as the non-glycosylated form, indicating no effect of the state of IGFBP-3 glycosylation in stimulating this PTPase activity (data not shown).

View larger version (11K): [in this window] [in a new window]   Fig. 6.   IGFBP-3 activates a PTPase in MCF-7 cells. MCF-7 cells were either incubated with or without different concentrations of IGFBP-3 or 30 nM IGFBP-5 for 5 min at 37 °C (A) or incubated with or without 10 nM IGFBP-3 or -5 for different periods of time (B). Then cells were lysed and proteins tested for PTPase activity as described under "Materials and Methods." Results are the means ± S.E. for three separate experiments.

Reconstitution experiments were performed to assess the specificity of the PTPase activity to the IGF-I signaling pathway. Lysates from MCF-7 cells treated or not (control) with 10 nM IGFBP-3 or IGFBP-1 were incubated with immunopurified tyrosine-phosphorylated IRS-1 as described under "Materials and Methods." As shown in Fig. 7, neither phosphatase buffer (0) nor lysates from control cells dephosphorylated IRS-1, but lysates from IGFBP-3-treated MCF-7 cells did so, indicating that IGFBP-3 stimulated a PTPase activity that could be inhibited by ZnCl₂. The effect was specific to IGFBP-3, since IGFBP-1-treated cells failed to dephosphorylate tyrosine-phosphorylated IRS-1. Addition of IGFBP-3 directly to tyrosine-phosphorylated IRS-1 did not change its state of tyrosine phosphorylation, indicating that IGFBP-3 had no PTPase activity per se (data not shown).

View larger version (52K): [in this window] [in a new window]   Fig. 7.   IGFBP-3 activates a PTPase that is sensitive to ZnCl2. Tyrosine-phosphorylated IRS-1 immunopurified from IGF-I-stimulated cells was incubated for 10 min at room temperature with buffer (control) or lysates of cells incubated for 5 min at 37 °C with or without (0) 10 nM IGFBP-3 or IGFBP-1. In some samples, 1 mM ZnCl2 was added to the reaction medium. Immune pellets were treated as described under "Materials and Methods" and tyrosine-phosphorylated IRS-1 visualized by Western immunoblotting using anti-phosphotyrosine antibodies and revealed by autoradiography. Results are those of a typical experiment.

Cell Type-specific Activation of IGFBP-3-induced Tyrosine Phosphatase-- To assess the cell type specificity of IGFBP-3-stimulated PTPase activity, we measured PTPase activity in different cell lines that respond to IGF-I in terms of IGF-IR autophosphorylation and activation of its tyrosine kinase activity. As shown previously, IGFBP-3 stimulated PTPase activity in MCF-7 cells (MCF-7/A) and also in T47D human breast carcinoma and CHO-IR Chinese hamster ovary cells. The same stimulation was seen in another MCF-7 cell line that differs from the first at least in its ability endogenously to secrete low levels of IGFBP-3 (MCF-7/B) (Fig. 8). In contrast, no PTPase activity was detected in IGFBP-3-treated MDA-MB-231 human breast carcinoma cells or CCL39 Chinese hamster lung fibroblasts (Fig. 8). Reconstitution experiments confirmed these findings; lysates from IGFBP-3-treated MCF-7/B, T47D, and CHO-IR cells dephosphorylated IRS-1, whereas lysates from IGFBP-3-treated MDA-MB-231 cells did not (Fig. 9).

View larger version (64K): [in this window] [in a new window]   Fig. 8.   IGFBP-3 activates a PTPase in different cell lines. Cells were incubated with or without IGFBP-3 (10 nM) for 5 min at 37 °C. Cells were then lysed and proteins tested for PTPase activity as described under "Materials and Methods." Results are the means ± S.E. for three independent experiments, and values are expressed as -fold stimulation compared with IGFBP-3 untreated cells.

View larger version (36K): [in this window] [in a new window]   Fig. 9.   IGFBP-3 activates a PTPase in different cell lines. Tyrosine-phosphorylated IRS-1 immunopurified from IGF-I-stimulated MCF-7 cells was incubated for 10 min at room temperature with lysates of different cell lines incubated for 5 min at 37 °C with or without 10 nM IGFBP-3. Immune pellets were treated as described under "Materials and Methods" and tyrosine-phosphorylated IRS-1 visualized by Western immunoblotting using anti-phosphotyrosine antibodies and revealed by autoradiography. Results are those of a typical experiment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This study provides evidence indicating that IGFBP-3 is capable of blocking the signal initiated by IGF-I in MCF-7 cells, via a mechanism involving activation of a PTPase. Following activation of the IGF-I-IR by its ligand, which results in its autophosphorylation and tyrosine phosphorylation of IRS-1, addition of IGFBP-3 rapidly provoked dephosphorylation of the two proteins. The tyrosine kinase activity of IGF-IR and tyrosine phosphorylation of IRS-1 are crucial steps in activating downstream signaling pathways (20), and IGFBP-3 was shown also to inhibit the two major pathways induced by IGF-I, the PI 3-kinase and MAP kinase pathways. The regulatory mechanism induced by IGFBP-3 therefore has physiological implications, since it affects both the early and the later steps in the IGF-I signaling pathway. PI 3-kinase activity is crucial for the mitogenic signaling of IGF-I in MCF-7 cells (3), and it seems significant that IGFBP-3 inhibits IGF-I-stimulated PI 3-kinase activity in the same cells. Interestingly, in our MCF-7 cells, PI 3-kinase association with IRS-1 and activation of the enzyme (as measured in immunoprecipitation experiments using IRS-1 or p85 subunit antibodies, respectively) exhibited different kinetics. Association with IRS-1 was transient, whereas activation persisted for 30 min, indicating that PI 3-kinase remains active beyond its period of association with IRS-1.

Activation of the IGF-I signaling pathway was achieved using the IGF-I analogues, des(1-3)IGF-I and [Gln³,Ala⁴,Tyr¹⁵,Leu¹⁶]IGF-I, with 80-100 and 1000 times weaker affinity for IGFBP-3 than IGF-I itself (14, 15). This meant that the inhibitory effect of IGFBP-3 was unrelated to its IGF-binding capacity. In addition, the inhibition seemed to be specific to IGFBP-3, in terms of the IGFBPs, since neither IGFBP-5 (structurally the closest to IGFBP-3) nor IGFBP-1 had any such effect. It would therefore seem reasonable to hypothesize that the region of IGFBP-3 responsible for its stimulation of PTPase activity would be centrally situated in the domain of the protein that is the least conserved among the IGFBPs.

The extent of tyrosine phosphorylation of a protein reflects the fine equilibrium between the activities of tyrosine kinases and phosphotyrosine phosphatases. Several mechanisms could therefore account for the down-regulation of the IGF-I signaling pathway by IGFBP-3. In one, IGFBP-3 would block the tyrosine kinase activity of IGF-IR, after which endogenous tyrosine phosphatases could dephosphorylate IGF-IR and IRS-1. This seemed a likely mechanism, since we had recently shown IGFBP-3 to affect IGF-IR, but not the insulin receptor. Preincubation of MCF-7 cells with IGFBP-3 was found to inhibit IGF-IR activation independently of IGFBP-3's ability to bind IGFs (13) and possibly involving conformational changes (14). IGFBP-3 could also alter IRS-1 phosphorylation, as shown in other systems where serine/threonine phosphorylation inhibits tyrosine phosphorylation in response to insulin, hence down-regulating subsequent insulin signaling (21, 22). However, this appeared unlikely, since serine/threonine phosphorylation of IRS-1 results in an increase of its molecular mass, which is visible in SDS-PAGE, but which did not occur in our experiments. In another scenario, IGFBP-3 would stimulate phosphotyrosine phosphatase activity capable of down-regulating the early steps of the IGF-I signaling pathway by dephosphorylating IGF-IR and IRS-1. Direct measurement of PTPase activity and the reconstitution experiments showed that IGFBP-3 did indeed activate a PTPase in MCF-7 cells, which dephosphorylated synthetic tyrosine-phosphorylated peptides and the tyrosine-phosphorylated IRS-1 protein used as a substrate in vitro. The persistence of the activation of this PTPase for at least 30 min, which corresponds to the duration of activation by IGF-I of PI 3-kinase and the MAP kinases, suggests that IGFBP-3 is implicated in regulating the IGF-I signaling pathway in MCF-7 cells. This may account for the so-called IGF-independent effects of IGFBP-3, such as its induction of apoptosis (12), and would imply IGFBP-3 interaction with specific cell surface proteins, as identified in other cell systems (23-25). In view of the extremely rapid activation of PTPase in MCF-7 cells, it seems unlikely that the mechanism of action of IGFBP-3 necessitates an internalization step. It would therefore be plausible that IGFBP-3 may either bind to and activate cell surface receptors with tyrosine phosphatase activity or stimulate an intracellular tyrosine phosphatase that dephosphorylates the proteins required in the initial steps of the IGF-I signaling pathway. These include IRS-1, which was used as tyrosine-phosphorylated substrate in our reconstitution experiments. They would also include IGF-IR, the tyrosine phosphorylation of which is decreased after addition of IGFBP-3. IGF-IR tyrosine kinase activity is closely related to the extent of its tyrosine autophosphorylation, and it could be expected that such tyrosine dephosphorylation would lead to depressed tyrosine kinase activity of the IGF receptor. This phosphatase-related mechanism of action of IGFBP-3 would be complementary to the well established extracellular mechanism of IGF sequestration.

Our study also showed that tyrosine phosphatase activation by IGFBP-3 is not peculiar to MCF-7 cells, since T47D human breast carcinoma and CHO-IR Chinese hamster ovary cells exhibited the same activation. Nevertheless, it does appear to be cell type-specific, since it did not occur in MDA-MB-231 human breast carcinoma or CCL39 Chinese hamster lung fibroblast cells. It would therefore seem that expression of either the IGFBP-3 receptor or the proteins activated by IGFBP-3 (in the present case, the PTPase enzyme) is cell type-specific and that one or more are either not expressed or not functional in all cells. Nevertheless, earlier work on the IGF-independent effects of IGFBP-3 indicated IGFBP-3 binding to the cell surface of MDA-MB-231 cells (26, 27). This suggests that the IGFBP-3 receptor exists in this cell type, but that binding to an IGFBP-3-activated PTPase does not occur. The fact that human IGFBP-3 activated a PTPase in Chinese hamster ovary cells would mean that there may be inter-species cross-reactivity between human IGFBP-3 and the Chinese hamster IGFBP-3 receptor. This would suggest that the peptide sequence of IGFBP-3, which to our knowledge is still unknown in Chinese hamster, and its receptor must be very similar to the human forms. The observation that IGFBP-3 also activated PTPase activity in a MCF-7 cell line that endogenously secretes low levels of IGFBP-3 may seem surprising. In fact, long term contact between agonist and cell frequently elicits subsequent resistance to the agonist via a down-regulatory mechanism. The basal expression level of IGFBP-3 may therefore not be sufficient in this line to down-regulate the IGFBP-3 signaling pathway. In addition, in the course of our experiments there are numerous and repeated washing steps to reduce IGFBP-3 in the reaction medium and avoid such down-regulation.

The nature of the PTPase activated by IGFBP-3 in MCF-7 cells remains to be determined. A possible candidate would be PTPase 1B, shown to be a negative regulator of IGF-I-stimulated signaling via dephosphorylation of both IGF-IR and IRS-1 (28). We were nevertheless able to establish that the IGFBP-3-activated PTPase is inhibited by phenylarsine oxide (data not shown) and Zn²⁺, which act by targeting the active cysteine site. The activation of a PTPase by IGFBP-3 to inhibit the IGF signaling pathway is functionally significant. Some PTPases may be capable of dephosphorylating critical substrates involved in the transformation process, and they may represent a family of tumor-suppressor enzymes (29). It is possible that PTPases function as anti-oncogenes, in that the enhanced tyrosine phosphorylation seen in some transformed cells may result from inactivation of a PTPase rather than activation of a phosphotyrosine kinase. For instance, PTEN, whose sequence bears homology with protein tyrosine phosphatases and whose gene is mutated in a wide range of cancers, appears to be a tumor suppressor negatively regulating tyrosine kinase activities (30). Interestingly, vanadate treatment of NRK-1 cells enhances intracellular phosphotyrosine levels and leads to production of transformed morphology (31). PTPases could potentially be important targets for proteins like IGFBP-3 in regulating the phosphotyrosine kinase signaling pathways.

	ACKNOWLEDGEMENTS

We are grateful to C. Desbois-Mouthon and M. Caron for their gift of CHO-IR cells and particularly to Dr R.-A. Toillon for providing us with IGFBP-3-secreting MCF-7 cells and the T47D and MDA-MB 231 cell lines.

	FOOTNOTES

^* This work was supported by the Institut National de la Santé et de la Recherche Médicale, the University of Paris VI, and Beckman Coulter France S.A.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Fellow of the Association pour la Recherche contre le Cancer. To whom correspondence and reprint requests should be addressed: INSERM U.515, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75571 Paris CEDEX 12, France. Tel.: 33-1-4928-4631; Fax: 33-1-4343-1065; E-mail. ricort@ st-antoine.inserm.fr.

Published, JBC Papers in Press, April 8, 2002, DOI 10.1074/jbc.M200439200

	ABBREVIATIONS

The abbreviations used are: IGF, insulin-like growth factor; IGFBP, IGF-binding protein; IGF-IR, type I IGF receptor; IRS, insulin receptor substrate; PI 3-kinase, phosphatidylinositol 3-kinase; MAP, mitogen-activated protein; MAPK, MAP kinase; PTPase, phosphotyrosine phosphatase; rh, recombinant human.

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