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Endocrinology, doi:10.1210/en.2003-1377
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Endocrinology Vol. 145, No. 9 4213-4221
Copyright © 2004 by The Endocrine Society

Stimulation of Insulin-Like Growth Factor (IGF) Binding Protein-3 Synthesis by IGF-I and Transforming Growth Factor-{alpha} Is Mediated by Both Phosphatidylinositol-3 Kinase and Mitogen-Activated Protein Kinase Pathways in Mammary Epithelial Cells

Usha Sivaprasad, Jodie Fleming, Poonam S. Verma, Kelly A. Hogan, Gwenaëlle Desury and Wendie S. Cohick

Department of Animal Sciences, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901-8520

Address all correspondence and requests for reprints to: Wendie S. Cohick, Ph.D., Rutgers, The State University of New Jersey, 108 Foran Hall, 59 Dudley Road, New Brunswick, New Jersey 08901-8520. E-mail: cohick{at}aesop.rugters.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF binding protein (IGFBP)-3 is an important regulator of mammary epithelial cell (MEC) growth and can enhance the ability of both IGF-I and epidermal growth factor ligands such as TGF{alpha} to stimulate MEC proliferation. Here we investigate the role of the phosphatidylinositol-3 kinase (PI3K) and MAPK pathways in the regulation of IGFBP-3 expression by IGF-I and TGF{alpha} in bovine MECs. Both growth factors stimulated DNA synthesis, although IGF-I was the stronger mitogen. IGF-I and TGF{alpha} also stimulated IGFBP-3 mRNA and protein levels. TGF{alpha} stimulated rapid, transient activation of Akt that was maximal at 5 min and diminished by 15 min. In contrast, IGF-I-induced Akt activation was maximal between 15 and 90 min and was sustained for 6 h. Although ERK 1/2 was maximally stimulated by TGF{alpha} between 5 and 15 min, IGF-I did not stimulate discernible activation of ERK 1/2. In addition, TGF{alpha} but not IGF-I induced rapid phosphorylation of Shc, whereas only IGF-I activated insulin receptor substrate-1. Pretreatment with the PI3K inhibitor LY294002 or knockdown of p85 with small interfering RNA inhibited IGF-I or TGF{alpha}-stimulated IGFBP-3 expression. Similarly, MAPK kinase-1 inhibitors PD98059 and U0126 each abolished TGF{alpha}-stimulated increases in IGFBP-3 mRNA levels. In contrast to TGF{alpha}, IGF-I retained the ability to partially increase IGFBP-3 mRNA levels in the presence of MAPK kinase-1 inhibitors, indicating that IGF-I may activate alternative substrates of the PI3K pathway that are involved in IGFBP-3 regulation. In conclusion, stimulation of IGFBP-3 mRNA levels by mitogens is regulated through both the PI3K and MAPK pathways in bovine MECs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GROWTH OF THE mammary gland is regulated by an array of growth factors and hormones that interact through complex mechanisms to modulate cellular proliferation, differentiation, and apoptosis. In mice, genetic approaches have shown that the IGF and epidermal growth factor (EGF) families each play critical roles in the normal growth and development of the mammary gland (1, 2, 3, 4). In addition, overexpression of components of both families is implicated in the etiology of breast cancer (5, 6).

IGF-I and EGF ligands are mitogenic for both normal and tumorigenic mammary epithelial cells (MECs) in vitro. At the cellular level, these growth factors activate cell surface receptors [IGF receptors (IGFRs) and EGF receptors (EGFRs)] that belong to the superfamily of protein tyrosine kinase receptors (7). The majority of the effects of IGFs on cell proliferation are mediated through the type I IGFR (8). The EGF family comprises multiple ligands, including EGF, TGF{alpha}, heparin-binding EGF, amphiregulin, heregulin, and betacellulin (6). Of the four known EGF receptors (ErbB1 through ErbB4), ErbB1 (commonly referred to as the EGFR) is the primary receptor that binds EGF and TGF{alpha}. However, the four ErbB receptors undergo homo- and heterodimerization that results in an array of receptor dimers with varying affinities for the different EGF ligands (9). Transcripts for TGF{alpha} and EGF mRNA as well as EGFR have been detected in the bovine mammary gland (10, 11, 12, 13, 14); however, the existence of multiple EGF ligands and receptors is unexplored in this species. The IGFR and EGFRs can each activate common molecules in the Ras-Raf-MAPK and the phosphatidylinositol-3 kinase (PI3K) signaling pathways, and it is unclear how specificity ensues (15, 16). Recent studies indicate that cross-talk between these two receptor families may play a role in their ability to regulate cell proliferation in MECs (17, 18, 19).

IGF binding protein (IGFBP)-3 has been shown to regulate cell growth through a complex array of mechanisms (20). Whereas an inhibitory role for IGFBP-3 has been widely reported in vitro (21, 22), several studies have found that IGFBP-3 enhances the mitogenic effects of IGF-I or EGF in both normal and tumorigenic MECs (23, 24, 25, 26). Interestingly, breast tumors with a poor disease prognosis exhibit enhanced expression of IGFBP-3 (27, 28, 29). IGF-I and EGF have both been shown to stimulate IGFBP-3 expression (30, 31, 32). However, the signaling pathways that mediate these effects have not been determined. Because both IGF-I and EGF ligands activate the PI3K and MAPK pathways, our goal was to investigate whether these growth factors increase IGFBP-3 expression through these signaling pathways in normal MECs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Recombinant human IGF-I, TGF{alpha}, and EGF were obtained from GroPep (North Adelaide, Australia), Intergen Co. (Purchase, NY), and Sigma-Aldrich (St. Louis, MO), respectively. Antibodies against phosphorylated forms of Akt (Ser473 or Ser308) were purchased from Cell Signaling Technology (Beverly, MA). Antibodies that recognize phosphorylated ERK 1/2 or phosphorylated tyrosines (PY 99) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies that recognize phospho-Shc (Tyr317), Shc, insulin receptor substrate (IRS)-1, IRS-2, or the p85 subunit of PI3K were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Nonimmune rabbit IgG was from Sigma. The MAPK kinase (MEK)1 inhibitors PD98059 and U0126 and the PI3K inhibitor LY294002 were purchased from Calbiochem (La Jolla, CA), resuspended as 10 mM stock solutions in dimethyl sulfoxide, and stored at –20 C. Cell culture reagents were from Invitrogen Co. (Carlsbad, CA). Antisera against bovine IGFBP-2 and -3 were the generous gifts of Dr. David Clemmons (University of North Carolina, Chapel Hill, NC). A smartpool of double-stranded small interfering (si) RNA against the p85{alpha} regulatory subunit of PI3K (M-003020) as well as mutated p85 siRNA control were from Dharmacon Research Inc. (Lafayette, CO). All siRNAs were dissolved in Rnase-free buffer based on the manufacturer’s protocol to a concentration of 20 µM.

Cell culture experiments
The bovine MEC line MAC-T was established from primary bovine MECs by immortalization with the SV40 large-T antigen (33). Cells were routinely maintained in complete media consisting of DMEM supplemented with 4.5 g/liter D-glucose, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamicin, 10% fetal bovine serum (FBS), and 5 µg/ml bovine insulin at 37 C in a humidified atmosphere with 5% CO2. For experiments, cells were plated in complete media without insulin or phenol-red. For analysis of intracellular signaling molecules, cells were grown to confluence, washed twice with phenol-red-free serum-free (PRFSF) DMEM, and incubated in PRFSF DMEM with 0.2% BSA and 30 nM Na selenite for 24 h before exposure to treatments. Cell lysates were collected and handled as described previously (34). For RNA analysis, cells were handled similarly before treatment then lysed in Trizol (Invitrogen) and stored at –80 C until analysis. For analysis of secreted IGFBP, PRFSF DMEM without additives was used for the 24-h washout period. Media conditioned by MAC-T cells were collected, cleared by centrifugation, and stored at –20 C until analysis.

Small interfering RNA transfection
Cells were plated at 4 x 104 cells/cm2 in six-well tissue culture dishes in DMEM supplemented with 10% FBS without antibiotics. The following day cells were transfected in serum-free DMEM with 100 nM siRNA or a mutated siRNA control using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. After 6 h, media were aspirated and replaced with DMEM supplemented with 10% FBS. After an additional 42 h, media were aspirated, cells were washed, and media were replaced with DMEM supplemented with 0.2% BSA and 30 nM Na selenite. After 24 h, media were replaced with fresh DMEM without additives ± growth factors for the indicated times.

Immunoprecipitation and Western blotting
Cell lysates were centrifuged at 13,000 x g for 15 min at 4 C, and total protein content of the cytosolic fraction was determined using a protein assay (Bio-Rad Laboratories, Hercules, CA). For immunoprecipitation, 1000 µg total protein were preincubated with protein-A agarose beads (Upstate Biotechnology), collected by centrifugation, and then incubated overnight at 4 C with 4 µg anti-IRS-1. Immunocomplexes were captured by the addition of protein-A agarose beads, washed three times in cold lysis buffer, and centrifuged briefly at 13,000 x g after each wash. Before Western immunoblotting, samples were heated at 100 C for 5 min. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (0.2 µm; Bio-Rad Laboratories). Membranes were immunoblotted by following the manufacturer’s recommendations for each individual primary antibody and each horseradish peroxidase-linked secondary antibody. Peroxidase activity was detected by enhanced chemiluminescence (Amersham Pharmacia, Piscataway, NJ) followed by autoradiography. Membranes were stripped (100 mM 2-ß-mercaptoethanol, 2% sodium dodecyl sulfate, 62.5 mM Tris-HCl) and reprobed with antibodies recognizing the nonphosphorylated forms of the proteins.

In vitro kinase assay
Activity of ERK 1/2 in lysates from IGF-I and TGF{alpha}-treated cells was determined by assessing phosphorylation of Elk-1 (Ser383) using a nonradioactive kinase assay kit (Cell Signaling). After treatment, confluent monolayers were rinsed with ice-cold 1x PBS and lysed according to the manufacturer’s instructions. Phospho-ERK 1/2 (Thr202/Tyr204) were immunoprecipitated using phosphospecific antibodies immobilized on agarose hydrazide beads overnight at 4 C. Beads were resuspended in 50 µl kinase buffer supplemented with 2 µg recombinant Elk-1 and ATP to a final concentration of 200 µM. Kinase reactions were allowed to proceed for 30 min at 30 C. Samples were then analyzed by Western blotting using antibodies against phosphorylated Elk-1 (Ser383).

DNA synthesis
Cells were plated at 1 x 104 cells/cm2 in 96-well tissue culture plates in PRF complete media without insulin. Cells were incubated for 5 d without a media change and then washed twice and incubated overnight in PRFSF DMEM. The following day, spent media were replaced with PRFSF media containing varying concentrations of the growth factors and 1 µCi/well of [3H]-thymidine (ICN Pharmaceuticals, Irvine, CA) for 18 h. [3H]-thymidine incorporation into DNA was determined as previously reported (23).

Northern blotting
Total RNA was isolated and analyzed by Northern blotting as described previously (23). Briefly, denatured RNA (10–15 µg/lane) was separated by electrophoresis, transferred to nylon membranes (Biotrans, ICN), and then hybridized overnight with [32P]-dCTP-labeled cDNA probes for bovine IGFBP-2 and -3 or for 18S rRNA. Membranes were washed and then exposed to AR film (Kodak, Rochester, NY) with intensifying screens at –80 C. Differences in relative band intensities were determined by PhosphorImager analysis (Molecular Dynamics, Inc., Sunnyvale, CA).

Statistical analysis
Data from experiments with multiple treatment groups were analyzed by ANOVA followed by Student-Newman-Keuls post hoc test. All analyses were performed in SigmaStat (2.03) program for Windows (SPSS Inc., Chicago, IL). Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TGF{alpha} and IGF-I increase DNA synthesis in MAC-T cells
We previously reported that IGF-I stimulates DNA synthesis in MAC-T cells (23). Whereas EGF and TGF{alpha} have also been reported to be mitogens for MECs, interactions between IGF-I and EGF ligands have not been investigated in bovine MECs. As shown in Fig. 1Go, IGF-I and TGF{alpha} each significantly stimulated DNA synthesis with maximal increases of 3.9 ± 0.32- and 1.6 ± 0.18-fold, respectively. EGF evoked a similar response in DNA synthesis relative to TGF{alpha} (data not shown). TGF{alpha} maximally stimulated DNA synthesis at 5 ng/ml. In contrast, IGF-I induced maximal responses between 10 and 20 ng/ml. Each dose of IGF-I elicited a greater response, compared with similar doses of TGF{alpha}. At low doses (1.25–5 ng/ml), the combination of IGF-I and TGF{alpha} stimulated DNA synthesis in an additive manner.



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  		FIG. 1. IGF-I and TGF{alpha} stimulate DNA synthesis in MAC-T cells. Cells were plated and grown to confluence. Spent media were aspirated, replaced with serum-free media for 24 h, and then treated with fresh serum-free media with or without treatments and [3H]thymidine for 18 h. Data represent the mean ± SEM of five separate experiments with each experimental point performed in triplicate. *, Significantly different from serum-free control (P < 0.05); #, significantly different from IGF-I alone (P < 0.05); **, significantly different from serum-free control (P < 0.001).

 
TGF{alpha} and IGF-I induce IGFBP-3 mRNA and protein levels in an additive manner
Similar to our previous reports, IGF-I (200 ng/ml) increased IGFBP-3 mRNA by 10 ± 1.4-fold (n = 5; P < 0.001) within 8 h (Fig. 2AGo). TGF{alpha} alone (100 ng/ml) also increased IGFBP-3 mRNA levels, although to a smaller extent (2.2 ± 0.2-fold; n = 5; P < 0.05). The lowest concentration of IGF-I tested (i.e. 10 ng/ml) elicited a greater IGFBP-3 response relative to all doses of TGF{alpha} up to and including 100 ng/ml (data not shown). Together, IGF-I and TGF{alpha} increased IGFBP-3 mRNA and protein levels in an additive manner (n = 5; P < 0.05). This additive effect on IGFBP-3 message was also seen at lower concentrations of 10 ng/ml IGF-I and TGF{alpha} (data not shown). TGF{alpha} and IGF-I also increased IGFBP-3 protein secretion by MAC-T cells, indicating that the increases in IGFBP-3 mRNA translated into increased IGFBP-3 protein. In addition, IGFBP-3 secretion was further enhanced in the presence of both growth factors (Fig. 2BGo). These effects were specific for IGFBP-3 because mRNA and protein levels of IGFBP-2 were unchanged (Figs. 2BGo and 3AGo).



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  		FIG. 2. IGF-I and TGF{alpha} stimulate additive increases in IGFBP-3 mRNA and secreted protein levels in MAC-T cells. Confluent MAC-T cells were serum starved for 24 h before addition of IGF-I (200 ng/ml), TGF{alpha} (100 ng/ml), or IGF-I + TGF{alpha}. Cell lysates were collected after 8 h and total RNA (15 µg) was analyzed by Northern blotting. A, Densitometry analysis of IGFBP-3 mRNA corrected for 18S (mean ± SEM of five separate experiments). Significance is indicated as: a, P < 0.05, compared with serum-free control; b, P < 0.05, compared with IGF-I alone; c, P < 0.05, compared with TGF{alpha} alone. B, Representative immunoblot of two separate experiments. Media conditioned by confluent MAC-T cells were collected after 12 and 16 h. Lyophilized media (100 µl) were separated on 12.5% SDS-PAGE gels and Western blotted with antibodies recognizing bovine IGFBP-3 and -2.

 


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  		FIG. 3. Temporal induction of IGFBP-3 mRNA by IGF-I and TGF{alpha} occurs differentially. Confluent MAC-T cells were serum deprived for 24 h before addition of IGF-I (200 ng/ml), TGF{alpha} (100 ng/ml), or IGF-I + TGF{alpha}. Cell lysates were collected after 4, 8, 12, and 16 h and total RNA (15 µg) was analyzed by Northern blotting. A, Representative autoradiogram of two separate experiments. B, Densitometry analysis of IGFBP-3 mRNA levels (corrected for 18S) presented as mean ± SD of two separate experiments.

 
The time course of the additive induction of IGFBP-3 mRNA by IGF-I and TGF{alpha} is shown in Fig. 3Go. Maximal induction of IGFBP-3 mRNA by TGF{alpha} occurred between 4 and 8 h, whereas IGF-I maximally induced IGFBP-3 mRNA levels at 8 h. The additive effect of both growth factors on IGFBP-3 mRNA levels was seen at every time point starting from 4 until 16 h.

IGF-I and TGF{alpha} differentially activate Akt and ERK 1/2
To determine the intracellular signaling pathways that mediate the effect of these growth factors on IGFBP-3 expression, we first determined the signal transduction pathways activated by TGF{alpha} and IGF-I in MAC-T cells. As shown in Fig. 4Go, both IGF-I and TGF{alpha} caused a rapid phosphorylation of Akt (Ser473). The activation of Akt by IGF-I was maximal between 15 and 90 min and was sustained up to 6 h (data not shown). In contrast, maximal activation of Akt by TGF{alpha} was observed at 5 min and was relatively transient, diminishing within 90 min. Similar results were observed using an antibody that recognizes Thr308 of Akt (Fig. 4CGo). A similar time course of Akt activation was obtained with 50 ng/ml IGF-I and 5.0 ng/ml TGF{alpha} or EGF (data not shown).



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  		FIG. 4. Temporal induction of phosphorylated Akt by IGF-I and TGF{alpha} occurs differentially. Confluent MAC-T cells were serum starved for 24 h before treatment with IGF-I (200 ng/ml; A) or TGF{alpha} (100 ng/ml; B) for the indicated times. Cell lysates (20 µg total protein) were separated by SDS-PAGE and Western blotted with antibodies specific for phosphorylated Akt (Ser473 or Thr308). Membranes were then stripped and reprobed with an antibody recognizing total Akt. Representative immunoblots of five separate experiments are shown.

 
In contrast to Akt, basal phosphorylation of ERK 1/2 was readily detectable in untreated cells (Fig. 5Go). Surprisingly, concentrations of IGF-I (200 ng/ml) that induced maximal responses in IGFBP-3 mRNA had a negligible effect on phosphorylation of ERK 1/2. In contrast, TGF{alpha} (100 ng/ml) caused a strong activation of ERK 1/2 that was maximal between 5 and 15 min (Fig. 5AGo). To confirm that phosphorylation reflected activation, in vitro kinase assays were performed (Fig. 5BGo). TGF{alpha} alone caused a marked increase in ERK 1/2 kinase activity as evidenced by the increase in Elk-1 phosphorylation, whereas IGF-I had no effect. Kinase activity in response to IGF-I plus TGF{alpha} together was not different from that observed with TGF{alpha} alone. Lower concentrations of IGF-I (50 ng/ml) also elicited negligible activation of ERK 1/2 relative to 5 ng/ml of either TGF{alpha} or EGF (Fig. 5CGo). TGF{alpha} and EGF elicited similar increases in phosphorylation of ERK at both 5 and 100 ng/ml (Fig. 5DGo). Similar results were observed with concentrations from 2.5 to 20 ng/ml (data not shown).



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  		FIG. 5. ERK 1/2 is phosphorylated by TGF{alpha} and EGF but not IGF-I. MAC-T cells were grown to confluence and serum starved for 24 h before addition of EGF, TGF{alpha}, or IGF-I. A, C, and D, Cell lysates (20 µg total protein) were analyzed by Western blotting using an antibody specific for phosphorylated ERK 1/2. The membranes were then stripped and reprobed with an antibody recognizing total ERK 1/2. B, Phospho-p44/42 MAPK (Thr202/Tyr204) was immunoprecipitated from cell lysates collected 5 min post treatment and used for an in vitro kinase assay with an ELK-1 fusion protein as substrate. Samples were analyzed by Western immunoblotting using a P-ELK-1 (Ser383) antibody. Figures show representative immunoblots of three separate experiments.

 
TGF{alpha} and IGF-I activate different upstream signaling molecules
The findings that TGF{alpha} and IGF-I elicited different time courses of Akt activation and differentially regulated ERK suggested that they might each activate different upstream signaling molecules. Immunoprecipitation experiments indicated that IGF-I and EGF/TGF{alpha} each activated their respective receptors within 1 min (data not shown). The docking protein Shc is common to both the IGF and EGF receptor signaling pathways. Shc binds to Grb2/mammalian son of sevenless (mSOS), leading to activation of the Ras/Raf/MAPK pathway. As shown in Fig. 6Go, increased phosphorylation of Shc was observed as early as 1 min with activation persisting through 45 min after treatment with TGF{alpha} or EGF (5 or 100 ng/ml). In contrast, IGF-I had no effect on Shc activation at concentrations of 50 or 200 ng/ml.



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  		FIG. 6. Shc is phosphorylated by TGF{alpha} and EGF but not IGF-I. Confluent, 24-h, serum-starved cells were treated with IGF-I, TGF{alpha}, or EGF for the indicated times. Cell lysates (20 µg total protein) were analyzed by Western blotting using an antibody specific for phosphorylated Shc. The membrane was then stripped and reprobed with an antibody recognizing total Shc. Representative immunoblots of four separate experiments are shown.

 
IRS-1 is a major substrate of the IGFR and is activated by the EGFR in some cell types (35). Therefore, we determined whether differences in the activation of this molecule might play a role in the different time course of Akt activation. Immunoprecipitation experiments demonstrated that IRS-1 was phosphorylated as early as 1 min and was sustained through 15 min after exposure to IGF-I (Fig. 7Go). In contrast, TGF{alpha} had no effect on IRS-1 phosphorylation. Neither IGF-I nor TGF{alpha} stimulated IRS-2 phosphorylation, although total IRS-2 was readily detectable (data not shown).



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  		FIG. 7. IRS-1 is phosphorylated by IGF-I but not TGF{alpha}. Confluent MAC-T cells were serum deprived for 24 h before addition of IGF-I (200 ng/ml) or TGF{alpha} (100 ng/ml). Cell lysates (1000 µg) were immunoprecipitated with anti-IRS-1 or a nonimmune rabbit IgG. Protein was separated by SDS-PAGE and Western immunoblotted with an antibody recognizing phosphorylated tyrosines. Membranes were stripped and reprobed with an antibody recognizing total IRS-1. Figure shows a representative immunoblot of three separate experiments.

 
TGF{alpha} and IGF-I regulate IGFBP-3 expression via PI3K and MAPK
To determine whether the PI3K or MAPK pathways are involved in the IGFBP-3 mRNA response after treatment with IGF-I or TGF{alpha}, cells were pretreated with specific inhibitors of each pathway before addition of the growth factors (Fig. 8Go, A and B). Preincubation of MAC-T cells with an inhibitor of the PI3K pathway (LY294002) completely blocked the ability of IGF-I to increase IGFBP-3 mRNA levels. Similar results were obtained when protein levels of p85, the regulatory subunit of PI3K, were inhibited with siRNA (Fig. 9Go). Surprisingly, inhibition of ERK 1/2 with the MEK1 inhibitor PD98059 also decreased IGF-I-stimulated increases in IGFBP-3 message by 65%, even though a detectable increase in ERK 1/2 phosphorylation was not observed after exposure to IGF-I (Fig. 8Go). Similar results were obtained with a second MEK1 inhibitor, U0126, which acts through a different mechanism. Inhibitors of either the MAPK or PI3K pathways were each able to completely abolish the ability of TGF{alpha} to increase IGFBP-3 mRNA levels (Fig. 8Go). In addition, knockdown of p85 with siRNA also blocked the TGF{alpha}-induced increase in IGFBP-3 mRNA (Fig. 9Go). When both growth factors were present together, PD98059 or LY294002 each decreased the IGFBP-3 mRNA response by 50 and 90%, respectively. Pretreatment with both inhibitors together completely prevented any increase in IGFBP-3 mRNA levels by the growth factors.



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  		FIG. 8. IGF-I and TGF{alpha} regulate IGFBP-3 mRNA levels via the PI3K and MAPK pathways. Confluent, serum-starved cells were treated with PD98059 (10 µM), LY294002 (25 µM), or U0126 (5 µM) for 1 h before addition of IGF-I (200 ng/ml) and/or TGF{alpha} (100 ng/ml) for 8 h. Total RNA (10–15 µg) was analyzed by Northern blotting. A, Representative autoradiograms. B, Quantitation by PhosphorImager analysis of IGFBP-3 mRNA levels corrected for 18S. Data are the mean ± SEM of at least three separate experiments. Significance is indicated as: a, P < 0.001, compared with serum-free control; b, P < 0.001, compared with IGF-I alone; c, P < 0.05, compared with TGF{alpha} alone; d, P < 0.001, compared with IGF-I + TGF{alpha}.

 


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  		FIG. 9. Gene knockdown of p85 inhibits the ability of IGF-I and TGF{alpha} to regulate IGFBP-3 mRNA. Cells were transfected in serum-free DMEM with 100 nM siRNA against the p85 subunit of PI3K or with a mutated (scrambled) p85 siRNA control. Six hours after transfection, media were aspirated and replaced with DMEM supplemented with 10% FBS. After an additional 42 h, cells were washed, and media were replaced with DMEM supplemented with 0.2% BSA and 30 nM Na selenite. After 24 h, media were replaced with fresh DMEM without additives ± TGF{alpha} (100 ng/ml) or IGF-I (200 ng/ml). Cell lysates were collected after 5 min of treatment for analysis of p85 protein (A) or after 8 h of treatment for RNA analysis (B). Total ERK 1/2 served as a loading control for the p85 Western blot shown in A. B, Representative autoradiogram of three experiments.

 
PD98059 or LY297002 inhibits IGF-I-stimulated DNA synthesis in MAC-T cells
The ability of MEK1 inhibitors to attenuate the stimulation of IGFBP-3 mRNA levels by IGF-I was a surprising observation, given that IGF-I did not appear to activate ERK 1/2. Therefore, we next determined whether another physiological end point of IGF-I treatment (i.e. DNA synthesis) would also be disrupted by inhibition of the MAPK pathway. PD98059 inhibited IGF-I stimulation of DNA synthesis in a dose-dependent manner, with 10 µM PD98059 inhibiting DNA synthesis by approximately 50% (Fig. 10Go). Therefore, MEK1 inhibitors decrease the ability of IGF-I to stimulate both DNA synthesis as well as IGFBP-3 mRNA levels despite the absence of any perceptible activation of ERK 1/2. Inhibition of the PI3K pathway with LY294002 also attenuated IGF-I stimulated DNA synthesis, indicating that both the PI3K and MAPK pathways are involved in the ability of IGF-I to stimulate DNA synthesis.



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  		FIG. 10. PD98059 and LY294002 inhibit IGF-I-stimulated DNA synthesis in MAC-T cells. Cells were plated and grown to confluence. Spent media were aspirated and replaced with serum-free media for 24 h. Cells were pretreated with PD98059 (0.5–10 µM) or LY294002 (0.25–4 µM) for 30 min and then treated with serum-free media with or without IGF-I (200 ng/ml) and 1 µCi/well of [3H]thymidine for 18 h. Data represent the mean ± SEM of three separate experiments with each experimental point performed in triplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have demonstrated that IGF-I and TGF{alpha} each stimulate IGFBP-3 mRNA and protein levels in MAC-T cells through the PI3K and MAPK pathways. The ability of IGF-I to stimulate greater increases in IGFBP-3 synthesis relative to TGF{alpha} paralleled the effects of these mitogens on DNA synthesis. We previously reported that MAC-T cells transfected with an IGFBP-3 cDNA exhibit enhanced DNA synthesis and/or Akt activation in response to both IGF-I and TGF{alpha} (34). Firth et al. (36) reported that T47D cells transfected with IGFBP-3 were initially growth inhibited relative to control cells but exhibited a growth advantage after several passages in culture. Recently these findings have been extended to show that injection of late-passage IGFBP-3-expressing T47D cells into nude mice increased tumor growth rate and wet tumor weight relative to injection of control or early-passage IGFBP-3-transfected cells (26). This effect was mediated through up-regulation of the EGFR. Therefore, regulation of IGFBP-3 expression through the PI3K and MAPK pathways via the IGF and EGFR systems may represent an important component by which these growth factors regulate growth in both normal and tumorigenic MECs. Future studies should be directed at determining whether the regulation of IGFBP-3 expression in response to IGF-I and EGF ligands is altered during different stages of growth or as cells become more malignant.

IGF-I and TGF{alpha}/EGF activate both the PI3K and Ras-Raf-MAPK pathways in many cell types (37, 38). In general, activation of the PI3K pathway by IGF-I involves phosphorylation of IRS-1 or -2 by the IGFR and subsequent recruitment of the p85 regulatory subunit of PI3K. In contrast, p85 typically interacts directly with the EGFR without the involvement of docking intermediates. Activation of ERK occurs via complex formation between the EGFR or IGFR and Shc/Grb2/mSOS and subsequent activation of the Ras/Raf/MEK signaling pathway. IRS proteins can also bind the Grb2/mSOS complex, resulting in ERK activation (38, 39).

Others have also reported IGF-I to be a stronger mitogen for ruminant MECs, compared with EGF or TGF{alpha} (40, 41). However, this is the first report documenting the signaling molecules that are activated by these growth factors in ruminant MECs. Our studies found that IGF-I and TGF{alpha} each induced a rapid activation of Akt. However, a different time course of activation was observed for each. IGF-I induced a sustained activation of Akt, whereas activation by TGF{alpha} was relatively transient. When upstream signaling events were examined, we found that IGF-I induced rapid increases in IRS-1 phosphorylation, whereas TGF{alpha} had no effect. Similar differences in the time course of Akt activation and IRS-1 phosphorylation by IGF-I and TGF{alpha} have been reported in pancreatic ß-cells (42). The lack of a requirement for an IRS-1 intermediate after exposure to TGF{alpha} may explain the faster attainment of a maximal response in Akt. However, it is not clear what accounts for the difference in sustainability of the Akt response. In other systems such as nerve cells and hepatocytes, the differential response to growth factors has been proposed to relate to the duration of signaling, not activation of different pathways (43, 44). Therefore, it is interesting to speculate that the longer duration of Akt activation after IGF-I treatment may account for the greater response in DNA synthesis and IGFBP-3 mRNA expression.

In addition to their effects on Akt phosphorylation, TGF{alpha} and EGF each caused a rapid increase in the phosphorylation of Shc and ERK. In contrast, IGF-I had no detectable effect on Shc phosphorylation and subsequent downstream ERK activation. In a comparison of multiple breast cancer cell lines, a wide variation in ERK activation in response to IGF-I was observed (45). IGF-I induced the strongest ERK activation in those lines that overexpressed multiple components of the IGFR signaling system (i.e. MCF-7 and T47D). In MDA-231 cells, an estrogen receptor-negative cell line that constitutively expresses high basal ERK activity, IGF-I did not further increase the activity of ERK or Shc (18, 45, 46). There is increasing evidence to suggest that interactions between the EGF and IGF systems may be involved in the activation of ERK by IGF-I. In a recent study, the kinetics of ERK activation in a normal human MEC line was reported to differ between IGF-I and EGF, with IGF-I inducing a slower, weaker, and more transient activation (18). The authors found that the EGFR tyrosine kinase inhibitor ZD1839 prevented IGF-I induced ERK activation. In contrast, it had no effect on the ability of IGF to activate the IGFR or Akt. Interestingly, a dependence on the EGFR for ERK activation in response to IGF-I was not observed in MCF-7 cells, which overexpress IGFR and IRS-1. It is interesting to note that of six additional breast cancer cell lines examined, ERK was activated by IGF-I in only one (CAL-51) (18). Additional evidence that EGFR/IGFR interactions play a role in IGF-induced Erk activation comes from a study in which overexpressing the ErbB2 receptor in MCF-7 cells attenuated the ability of IGF-I to phosphorylate Shc or ERK but had no effect on Akt activation (47). Similarly, in COS-7 cells overexpressing the IGFR, an EGFR tyrosine kinase inhibitor attenuated IGF-I-induced ERK activation (19). Therefore, the level of expression of various components of the EGFR and IGFR systems may determine the extent to which IGF-I activates the Ras/Raf/MAPK pathway in a given cell system.

The ability of the Shc-Grb2 pathway and ensuing activation of Ras/Raf/MAPK to mediate the effect of IGF-I on DNA synthesis is also likely dependent on the balance between multiple components of interacting signaling cascades. For example, a dominant-negative mutant of Shc blocked cell cycle progression at G0-G1 and G2-M phases in breast cancer cells exhibiting high ErbB2 expression levels but had no effect in a normal breast cell line (48). Whereas IGF-I induced significant increases in DNA synthesis in MAC-T cells without apparent activation of this pathway, inhibiting ERK phosphorylation with the MEK1 inhibitor PD98059 inhibited IGF-I-stimulated DNA synthesis about 50%. One explanation for this may be that basal levels of phosphorylated Shc and ERK, which are detectable in untreated cells, may be required for IGF-I to activate DNA synthesis and IGFBP-3 mRNA. The ability of TGF{alpha} to further activate this pathway may contribute to the additive effects of IGF-I and TGF{alpha} on DNA synthesis and IGFBP-3 expression.

Whereas many studies have shown that IGF-I regulates IGFBP-3 synthesis in a variety of cell types (32, 49, 50), the signaling pathways mediating this effect have not been investigated before this report. However, in porcine vascular smooth muscle cells, activation of the PI3K pathway was sufficient for IGF-I to increase IGFBP-5 expression as shown by the ability of LY294002 or wortmannin to completely abolish this response (51). In contrast, PD98059 had no effect, even though it blocked IGF-stimulated ERK activation. This contrasts with the present study in which inhibiting either PI3K or ERK activation decreased the ability of IGF-I or TGF{alpha} to increase IGFBP-3 mRNA levels. Therefore, our data suggest that these two pathways converge at a downstream point. One possibility is that they both are required to activate a common signaling intermediate or transcription factor complex. Alternatively, they could each activate individual molecules that act in concert to regulate IGFBP-3 expression. The ability of IGF-I to induce vascular endothelial cell growth factor synthesis has been reported to exhibit a similar dependence on coordinate signaling from both pathways (52). Future studies will be directed at determining mechanisms by which the PI3K and MAPK pathways interact to regulate IGF-I-induced IGFBP-3 expression.

It is interesting that the ability of TGF{alpha} to increase IGFBP-3 was completely abrogated by inhibition of either pathway, whereas IGF-I retained the ability to partially increase IGFBP-3 mRNA levels in the presence of MEK-1 inhibitors. One possible explanation is that IGF-I stimulates a substrate(s) of the PI3K pathway that is not shared by TGF{alpha}. We investigated p70S6K as a possible candidate and found that IGF-I and TGF{alpha} each stimulated p70S6K phosphorylation. However, blocking this activation with the inhibitor rapamycin did not inhibit the ability of either growth factor to increase IGFBP-3 (data not shown). Other possible PI3K substrates are members of the protein kinase C (PKC) family including PKC-{zeta} and glycogen synthase kinase-3ß. Whereas neither IGF-I nor TGF{alpha} increased phosphorylation of PKC-{zeta} in MAC-T cells, the potential involvement of glycogen synthase kinase-3ß is presently under investigation.

In conclusion, we have found that IGFBP-3 expression is positively regulated via two intracellular signaling pathways that are critical regulators of cellular growth and survival. This raises the intriguing possibility that IGFBP-3 expression may be altered by changes that frequently occur in the IGF/EGF axis in pathological conditions such as breast cancer.


   Footnotes
 
This work was supported by the United States Department of Agriculture (awards 98-35206-6428 and 2003-35206-12811 to W.S.C.); Department of Defense (award to W.S.C.); New Jersey Agricultural Experiment Station; and Charles and Johanna Busch Memorial Fund at Rutgers, The State University of New Jersey.

Abbreviations: EGF, Epidermal growth factor; EGFR, EGF receptor; FBS, fetal bovine serum; IGFBP, IGF binding protein; IGFR, IGF receptor; IRS, insulin receptor substrate; MEC, mammary epithelial cell; MEK, MAPK kinase; mSOS, mammalian son of sevenless; PI3K, phosphatidylinositol-3 kinase; PKC, protein kinase C; PRFSF, phenol-red-free serum-free; si, small interfering.

Received October 14, 2003.

Accepted for publication June 4, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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