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Endocrinology Vol. 141, No. 9 3098-3103
Copyright © 2000 by The Endocrine Society

ARTICLES

Insulin-Like Growth Factor (IGF) Binding Protein-3 Potentiation of IGF Action Is Mediated through the Phosphatidylinositol-3-Kinase Pathway and Is Associated with Alteration in Protein Kinase B/AKT Sensitivity1

C. A. Conover, L. K. Bale, S. K. Durham and D. R. Powell

Endocrine Research Unit (C.A.C., L.K.B.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905; and Department of Pediatrics (S.K.D., D.R.P.), Baylor College of Medicine, Texas Children’s Hospital, Houston, Texas 77030

Address all correspondence and requests for reprints to: Cheryl A. Conover, Ph.D., Mayo Clinic, 200 First Street SW, 5–194 Joseph, Rochester, Minnesota 55905. E-mail: conover.cheryl{at}mayo.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell-association and processing of insulin-like growth factor binding protein-3 (IGFBP-3) by cultured bovine fibroblasts results in markedly enhanced type I IGF receptor signaling at a step distal to ligand binding. The purpose of the present study was to determine the intracellular mediators of IGFBP-3’s potentiating effect. Preincubation of cultured bovine fibroblasts with 50 nM IGFBP-3 had no effect alone, but enhanced by 3- to 4-fold IGF-I-stimulated 3H-aminoisobutryric acid (AIB) uptake. IGFBP-3-induced potentiation was specifically prevented if an inhibitor of phosphatidylinositol 3 (PI3)-kinase activation (LY294002), but not an inhibitor of mitogen-activated protein kinase activation (PD98059), was present during the preincubation period. IGFBP-3 did not directly activate the downstream effector of PI3-kinase, protein kinase B (PKB)/Akt. However, the sensitivity of PKB/Akt to activation by IGF-I was increased by 2- to 4-fold with IGFBP-3 pretreatment. This increased sensitivity was accompanied by altered mobility of PKB/Akt on SDS-polyacrylamide gels, suggestive of a diminished phosphorylation state. Consistent with this, okadaic acid, a potent serine/threonine phosphatase inhibitor, was able to block the potentiation effect of IGFBP-3 and prevent the altered mobility of the PKB/Akt molecule in response to IGFBP-3 treatment. PKB/Akt immunoprecipitated from IGFBP-3-pretreated cells was no longer recognized by an antibody specific for phosphorylated threonine followed by proline. These data indicate that IGFBP-3 modulates type I IGF receptor signaling through an effect on PI-3-kinase pathway substrates and suggest a novel mechanism of dephosphorylation whereby PKB/Akt is transformed into a more sensitive substrate of type I IGF receptor signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor binding protein-3 (IGFBP-3) is one of the six known high affinity IGFBPs (1). IGFBP-3 is the most abundant IGFBP in adult serum, responsible for binding the majority of the IGFs in serum (2). However, IGFBP-3 is more than just a passive carrier of IGFs in the circulation. Indeed, IGFBP-3 has multiple growth regulatory functions at the cellular level (3, 4, 5).

IGFBP-3 can be a potent inhibitor of cell growth by virtue of its ability to bind IGF with high affinity, thereby preventing its interaction with membrane receptors (4, 6, 7, 8). The type I IGF receptor mediates the growth-stimulating effects of IGFs and related ligands (9, 10). Ligand binding to the {alpha}-subunit of the type I IGF receptor initiates a phosphorylation cascade impacting the receptor ß-subunit, immediate receptor substrates and two major intracellular signaling pathways involving phosphatidylinositol 3 (PI3) kinase and mitogen activated protein (MAP) kinase (9, 10, 11). IGFBP-3 can also function as a direct cell growth inhibitor, independent of its IGF binding activity (12). This inhibitory effect of IGFBP-3 has been demonstrated even in the absence of type I IGF receptors and appears to be mediated by distinctive cell membrane receptors, one of which is the type V transforming growth factor-ß (TGF-ß) receptor (13).

Furthermore, IGFBP-3 can potentiate IGF action in a number of cell systems (6, 8, 14, 15, 16, 17, 18, 19, 20, 21, 22). In previous studies using a bovine fibroblast model, we showed that this ability to stimulate cell growth required IGFBP-3 cell-association and subsequent modification of the cell-bound IGFBP-3 (8, 16, 23). It did not require IGFBP-3 binding to IGF, but it did require ligand activation of type I IGF receptor signaling. IGFBP-3 by itself was not stimulatory. The molecular mechanism underlying IGFBP-3 potentiation has not been elucidated as yet. However, the effect occurs at a step distal to IGF-I receptor binding, suggesting that cell-associated IGFBP-3 influences intracellular events.

The purpose of this study was to determine the elements of signal transduction involved in IGFBP-3 potentiation of IGF-I action in bovine fibroblasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human IGFBP-3 was expressed in baculovirus and purified as described (24). In some experiments, IGFBP-3 protein (ND variant), a kind gift from Celtrix Pharmaceuticals, Inc. (Santa Clara, CA), was used with equivalent results. Recombinant human IGF-I was purchased from R & D Systems, Inc. (Minneapolis, MN). PD98059, PKB/Akt antibody, phospho-specific (Ser473) PKB/Akt antibody, and phospho-specific Thr-Pro (p-Thr-Pro) antibody were obtained from New England Biolabs, Inc. (Beverly, MA). LY294002 was from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). Okadaic acid, FBS and RIA-grade BSA were from Sigma (St. Louis, MO). Protein G plus/protein A agarose was from Oncogene Science, Inc. (Uniondale, NY). Tissue culture media and supplements were obtained from Life Technologies, Inc. (Grand Island, NY).

Cell culture
Bovine dermal fibroblasts (GM06034) were purchased from the Human Genetic Mutant Cell Repository (Camden, NJ) and were cultured in DMEM supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 4 mM glutamine, and containing 10% FBS, as previously described (8, 16, 23). Fibroblasts were plated at approximately 30,000 cells/cm2 and grown to confluency (4–5 days) in 6- or 24-multiwell plates (Costar, Cambridge, MA). After this time in culture, IGFBP-3 and inhibitors were added directly to spent medium. Incubation was continued for 48 h.

Aminoisobutyric acid (AIB) uptake
[3H]AIB uptake was determined as previously described (8, 16, 23). Fibroblast cultures, with or without IGFBP-3 and inhibitor pretreatment, were washed three times with HBSS containing 1.75 g/l NaHCO3, 20 mM HEPES (pH 7.4), and 0.1% BSA (HBSS buffer). The medium was changed to HBSS buffer with or without 1 nM IGF-I, and the monolayers were incubated at 37 C for 6 h. [3H]AIB (0.5 µCi/ml; 8 µM) was added, and the incubation was continued for 12 min. Cultures were placed on ice and washed quickly four times with cold PBS. Monolayers were solubilized in 0.25 N NaOH, and aliquots were taken for liquid scintillation counting. Results are expressed as a percentage of the total counts in the incubation medium that were taken up by the cells.

Immunoblotting
Fibroblast cultures, with or without IGFBP-3 and inhibitor pretreatment, were washed three times and then stimulated without or with IGF-I for 10 min at 37 C. Cells were washed with ice-cold PBS, lysed, and equal amounts of lysate protein (~100 µg) were processed by SDS-PAGE under reducing conditions (100 mM dithiothreitol) using a 10% slab gel, and then transferred to nitrocellulose (11). Filters were blocked for 4–6 h, incubated overnight at 4 C with primary antibody, washed, and then incubated for 2 h at room temperature with horseradish peroxidase-conjugated secondary antibody. Antigen-antibody reactions were visualized using an enhanced chemiluminescent detection system (Amersham Pharmacia Biotech. Arlington Heights, IL).

PKB/Akt kinase assay
PKB/Akt kinase activity kit was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), and assays were performed according to manufacturer’s instructions. Briefly, bovine fibroblasts with and without IGFBP-3 pretreatment, were washed and then stimulated without or with IGF-I for 10 min at 37 C. Whole cell lysates were incubated with PKB/Akt antibody and protein A agarose. Washed immunoprecipitates were incubated for 20 min at 30 C in the presence of a PKB/Akt-specific substrate, {gamma}-32P-ATP (NEN Life Science Products, Boston, MA) and specific kinase inhibitors. Reactions were stopped with the addition of 40% trichloroacetic acid, and the reaction volume was spotted on phosphocellulose paper. Incorporated radioactivity was assayed using liquid scintillation counting.

Immunoprecipitation
Lysates from cells with and without IGFBP-3 pretreatment were incubated at 4 C overnight with PKB/Akt antibody (1:1000 final dilution) and protein G-plus/protein A agarose (11). Immunoprecipitated proteins were washed, run on SDS-PAGE, and immunoblotted with p-Thr-Pro antibody (1:5000 final dilution).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGFBP-3 potentiation of IGF action: PI3- and MAP-kinases
Because IGFBP-3 potentiation of IGF action in bovine fibroblasts required activation of type I IGF receptor signaling (8, 16, 23), initial experiments examined the effect of IGFBP-3 on the immediate effectors of type I IGF receptor activation in these cells: tyrosine phosphorylation of the receptor ß-subunit and insulin receptor substrate 2 (IRS-2) (11). However, in three separate experiments there was no apparent effect of IGFBP-3 on the basal protein tyrosine phosphorylation pattern or on IGF-I-stimulated tyrosine phosphorylation of receptor or IRS-2 as assessed by phospho-tyrosine immunoblot (data not shown). Therefore, we turned our attention to more downstream events in type I IGF receptor signaling. Two of the major intracellular signal transduction pathways used by many growth factors, including the IGFs, are phosphatidylinositol 3 (PI3)-kinase and mitogen-activated protein (MAP) kinase. To determine whether IGFBP-3 potentiation involved either of these pathways we used selective inhibitors. LY294002 is a specific inhibitor of PI3-kinase activity (25) and PD98059 is a specific inhibitor of MAP-kinase activation by upstream MAP kinase kinase (26).

Cultured bovine fibroblasts were preincubated for 48 h with 50 nM IGFBP-3 with and without inhibitors. Cells were washed and then stimulated with 1 nM IGF-I and 3H-AIB uptake measured (Fig. 1Go). This experimental design was previously determined to be optimal for these studies (8, 16, 23). Under Control conditions, IGF-I alone stimulated 3H-AIB uptake 2-fold. Preincubation with IGFBP-3 resulted in 4-fold enhancement of IGF-I bioeffectiveness. Preincubation with LY294002 by itself had no effect on basal or IGF-I-stimulated 3H-AIB uptake. However, LY294002 completely blocked the potentiating effect of IGFBP-3. On the other hand, preincubation with PD98059 did not prevent IGFBP-3 potentiation of IGF-I action. It is important to point out that both LY294002 and PD98059 effectively inhibited IGF-I signaling if added concomitantly with IGF-I (data not shown), but not when added 48 h before IGF-I as in these experiments. Thus, we were looking at selective effects of active inhibitors on IGFBP-3 action. These data suggest that IGFBP-3 potentiation involves the PI3-kinase pathway.



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  		Figure 1. IGFBP-3 potentiation of IGF-I action: effect of inhibitors of PI3kinase (LY294002) and MAP-kinase (PD98059). Bovine fibroblasts were preincubated for 48 h without (open bars) or with (solid bars) 50 nM IGFBP-3 and no inhibitor (Control), 50 µM LY294002, or 50 µM PD98059. Cells were washed and changed to serum-free medium ± IGF-I (1 nM) and [3H]AIB uptake was measured as described in Materials and Methods. Results are the mean ± SE of three experiments. *, Significant effect of IGFBP-3 preincubation, P < 0.05.

 
IGFBP-3 potentiation of IGF action: PKB/Akt
Activation of the PI3-kinase pathway generally results in selective phosphorylation of a downstream effector, protein kinase B (PKB) also known as Akt, the cellular homolog of retroviral oncogene v-akt (27, 28). Full activation requires specific phosphorylation of PKB/Akt on Ser473 and Thr308 (29). Figure 2Go shows immunoblots of PKB/Akt from lysates of bovine fibroblasts following a 48 h IGFBP-3 preincubation, and then with and without IGF-I stimulation for 10 min. In the upper panel, activated PKB/Akt was assessed using an antibody that specifically recognizes PKB/Akt phosphorylated on Ser473. Little PKB/Akt was phosphorylated on Ser473 in the basal state (lanes a and f), and IGFBP-3 preincubation by itself did not activate PKB/Akt (lane d). As expected, with the addition of IGF-I there was marked activation of PKB/Akt (lanes b, c, and e). Figure 2Go, lower panel, shows the same lysates probed with an antibody that detects phosphorylated and nonphosphorylated forms of PKB/Akt. Total amount of PKB/Akt protein was not altered by any of the treatments. However, changes in mobility of the PKB/Akt protein were observed. IGF-I treatment resulted in a slightly retarded mobility of PKB/Akt on SDS-PAGE (lanes b, c, and e). This electrophoretic mobility shift is indicative of differentially phosphorylated forms of PKB/Akt, i.e. phosphorylation on Ser473 and Thr308 induced by IGF-I (29, 30). Interestingly, IGFBP-3 treatment for 48 h appeared to produce a PKB/Akt form with increased mobility on SDS-PAGE compared with control, suggestive of a decreased phosphorylation state (lane d). IGFBP-3 may act to alter the basal PKB/Akt phosphorylation state making it more responsive to subsequent activating events. As assessed by phospho-specific PKB/Akt immunoblot (Fig. 3Go), preincubation with IGFBP-3 resulted in 2- to 4-fold enhanced activated PKB/Akt at every IGF-I concentration tested (0.2–2 nM). This enhanced sensitivity was also found in Fig. 2Go (compare lanes b and e, upper panel), but the film exposure makes it less apparent there than in Fig. 3Go. Assay of the in vitro kinase activity of PKB/Akt immunoprecipitates confirmed that preincubation with IGFBP-3 had no effect on basal PKB/Akt activity but enhanced IGF-I-stimulated PKB/Akt kinase activity (Table 1Go).



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  		Figure 2. PKB/Akt immunoblots. Bovine fibroblasts were preincubated for 48 h without (lanes a, b, c, and f) or with 50 nM IGFBP-3 (lanes d and e) as in Fig. 1Go. Cells were then washed and changed to serum-free medium ± IGF-I at 1 nM (lanes b and e) and 10 nM (lane c) for 10 min. Total cell lysates were separated by SDS-PAGE and immunoblotted with phospho-specific PKB/Akt antibody (upper panel) or total PKB/Akt antibody (lower panel).

 


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  		Figure 3. PKB/Akt activation by IGF-I: effect of IGFBP-3. Bovine fibroblasts were preincubated without (-) or with (+) 50 nM IGFBP-3 for 48 h, washed and stimulated with IGF-I at the indicated concentrations for 10 min. Total cell lysates were separated by SDS-PAGE and immunoblotted with phospho-specific PKB/Akt antibody.

 

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  		Table 1. PKB/Akt kinase activity

 
IGFBP-3 potentiation of IGF action: okadaic acid
In the basal state, PKB/Akt has no detectable tyrosine phosphorylation but has low level serine/threonine phosphorylation (29, 30). If IGFBP-3 pretreatment results in dephosphorylation of PKB/Akt on serine or threonine residues with subsequent increased sensitivity to activation, then an inhibitor of serine/threonine dephosphorylation, such as okadaic acid (OKA), should prevent IGFBP-3 potentiation (31). Bovine fibroblasts were preincubated with 50 nM IGFBP-3 without or with 20 nM OKA. Cells were washed and then stimulated with 1 nM IGF-I and 3H-AIB uptake measured as before. As shown in Fig. 4Go, OKA had little effect on IGF-I stimulation of 3H-AIB uptake. In contrast, OKA was able to significantly block IGFBP-3 potentiation of IGF-I action. Furthermore, OKA was also able to prevent the increased PKB/Akt mobility in response to IGFBP-3 treatment (Fig. 5Go).



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  		Figure 4. IGFBP-3 potentiation of IGF-I action: effect of okadaic acid. Bovine fibroblasts were preincubated for 48 h without (open bars) or with (solid bars) 50 nM IGFBP-3, and either no inhibitor (Control) or 20 nM OKA. Cells were washed and changed to serum-free medium ± 1 nM IGF-I and [3H]AIB uptake measured as in Fig. 1Go. Results are mean ± SE of three experiments. *, Significant effect of IGFBP-3 preincubation, P < 0.05.

 


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  		Figure 5. PKB/Akt immunoblot: effect of okadaic acid. Bovine fibroblasts were preincubated for 48 h without (C, control) or with 50 nM IGFBP-3 and either no inhibitor (C) or 20 nM OKA. Total cell lysates were separated by SDS-PAGE and immunoblotted with total PKB/Akt antibody as in Fig. 2Go.

 
PKB/Akt basal phosphorylation: effect of IGFBP-3
Studies of Alessi et al. (29) indicated that under nonstimulated conditions human PKB/Akt was phosphorylated predominantly on Ser124 and Thr450. Both of these residues are followed by proline in the PKB/Akt molecule (27, 29). Human and bovine PKB/Akt amino acid sequences show 94% identity (32). To probe whether IGFBP-3 treatment of bovine fibroblasts might be inducing dephosphorylation of PKB/Akt at analogous residues, we used a recently available antibody that specifically recognizes phosphorylated threonine when it is followed by proline. As shown in Fig. 6Go, upper panel, PKB/Akt from Control cells was recognized by the p-Thr-Pro antibody, whereas PKB/Akt from IGFBP-3-treated cells was not. Equal amounts of PKB/Akt are detected by total PKB/Akt antibody under both conditions, as shown in Fig. 6Go, lower panel.



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  		Figure 6. Basal phosphorylation of PKB/Akt: effect of IGFBP-3. PKB/Akt was immunoprecipitated from lysates of bovine fibroblast with or without IGFBP-3 pretreatment for 48 h, run out on SDS-PAGE, and immunoblotted with p-Thr-Pro antibody (upper panel), or total PKB/Akt antibody (lower panel).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies demonstrate that IGFBP-3 can directly modulate intracellular signaling through a mechanism involving the PI3-kinase pathway. Thus, a specific inhibitor of PI3-kinase activation, LY294002, could completely block IGFBP-3’s ability to potentiate subsequent IGF-I action in cultured bovine fibroblasts. Although LY294002 is known to inhibit other growth factor signaling through PI3-kinase, including IGF-I, the experimental design targeted IGFBP-3. IGFBP-3’s potentiating effect in this cell system requires a pretreatment period of more than 24 h (16, 23), and in these experiments we used 48 h. LY294002 added 48 h prior had no effect on basal or IGF-I stimulation.

IGFBP-3’s effect may involve PKB/Akt. PKB/Akt is an important downstream effector of PI3-kinase that mediates many of the growth-promoting effects of PI3-kinase activation (33), although a direct link between PI3-kinase activation and PKB/Akt was not established in this study. The phospholipid products of PI3-kinase regulate specific kinases that phosphorylate PKB/Akt on Ser473 and Thr308. These specific phosphorylation events are essential for full activation of the PKB/Akt molecule (29, 33). IGFBP-3 did not directly activate PKB/Akt, but IGFBP-3 treatment increased the sensitivity of PKB/Akt to activation by IGF-I 2- to 4-fold. Exactly how IGFBP-3 accomplishes this increased sensitivity of a key intracellular signaling molecule is unclear. However, the experiments in this study offer some clues. When PKB/Akt is activated by phosphorylation on Ser473 and Thr308 it exhibits reduced mobility on SDS-PAGE, as was seen in response to IGF-I stimulation. It has been shown in other studies, as well, that the different electrophoretic mobility forms reflect different phosphorylation states of PKB/Akt (30). Interestingly, IGFBP-3 treatment alone resulted in a PKB/Akt form with increased mobility on SDS-PAGE compared with control. Very little is known about the basal phosphorylation state of PKB/Akt. Alessi et al. (29) found low level serine/threonine phosphorylation of endogenous PKB/Akt in L6 myoblasts. Andjelkovic et al. (30) reported that PKB/Akt was phosphorylated predominantly on serine in unstimulated Swiss 3T3 cells; no tyrosine phosphorylation was evident. Thus, the physical change in PKB/Akt in response to IGFBP-3 treatment may be a decrease in basal serine/threonine phosphorylation. The results with OKA would be consistent with this hypothesis. OKA, at low concentrations, is a potent inhibitor of two of the major protein phosphatases in mammalian cells that dephosphorylate on serine and threonine residues (31). An OKA-induced block in the ability IGFBP-3 to dephosphorylate PKB/AKt was associated with altered PKB/Akt mobility on SDS-PAGE and loss in ability of IGFBP-3 to potentiate IGF-I action. Our data further suggest specific dephosphorylation of PKB/Akt on a threonine residue that is followed by proline. Alessi et al. (29) identified Thr450, one of three threonines followed by proline, as the predominate site of human PKB/Akt phosphorylation in the basal state. The availability of an antibody highly specific for the presence of phosphorylated threonine when followed by proline allowed us to test the hypothesis that IGFBP-3 induced dephosphorylation of PKB/Akt at one of three such threonines in the bovine PKB/Akt molecule (32). Under control conditions, p-Thr-Pro antibody recognized PKB/Akt in bovine fibroblast lysates, consistent with PKB/Akt being phosphorylated in the basal state. However, PKB/Akt in lysates from cells pretreated with IGFBP-3 was not readily detected by this antibody, even though equal amounts of PKB/Akt were present. Thus, IGFBP-3 pretreatment appears to result in a dephosphorylation of PKB/Akt at a threonine that is followed by proline, coincident with increased sensitivity to IGF activation. Thr450 is a likely candidate considering its role in basal phosphorylation of human PKB/Akt (29). This will need to be verified biochemically in our model.

The "receptor" mediating the enhancing effect of IGFBP-3 was not addressed in these studies. Clearly, it differs from that mediating the inhibitory effect of IGFBP-3 (12, 34) because the effect is not direct, i.e. IGFBP-3 has no stimulatory effect alone but rather enhances the stimulatory response mediated by activated type I IGF receptors. Furthermore, it is not mediated by IGFBP-3 binding to type V TGF-ß receptors (13). There were no detectable type V TGF-ß receptors in cultured bovine fibroblasts, preincubation with TGF-ß did not mimic IGFBP-3 potentiation of IGF-I action, IGFBP-3 not inhibit specific TGF-ß receptor binding and, conversely, TGF-ß did not alter IGFBP-3 cell-association (our unpublished data).

In fact, IGFBP-3 cell-association may not reflect a classic receptor. One possibility is that IGFBP-3 is interacting with integrins. Delcommenne et al. (35) recently identified integrin-linked kinase (ILK) activity as an upstream effector of PI3-kinase regulation of PKB/Akt. Mira et al. (36) postulated that membrane-bound matrix metalloprotease-9 acts as IGFBP-3 receptor enabling the enhancing mechanism. Either of these would fit with previous studies indicating that IGFBP-3 potentiation of IGF-I action in bovine fibroblasts requires heparin sulfate-independent cell association and processing of cell-bound IGFBP-3 to lower molecular weight forms (16, 23).

These studies provide further evidence of a complex role for IGFBP-3 in regulating cell growth and suggest a novel mechanism for heightened cell response to the stimulatory effects of IGF-I and related ligands through alterations in basal PKB/Akt phosphorylation. The significance of basal PKB/Akt phosphorylation as a determinant of signal transduction warrants further investigation.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Award RO1-DK-38773 (to D.R.P.) and the Mayo Foundation. Back

Received October 13, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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