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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 4 1627-1633
Copyright © 2000 by The Endocrine Society

Original Studies

Testosterone and Insulin-like Growth Factor (IGF) I Interact in Controlling IGF-Binding Protein Production in Androgen-Responsive Foreskin Fibroblasts1

Atsuko Yoshizawa and David R. Clemmons

Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

Address correspondence and requests for reprints to: David R. Clemmons, Division of Endocrinology, The University of North Carolina at Chapel Hill, CB #7170, 6111 Thurston Bowles, Chapel Hill, North Carolina 27599-7170.


   Abstract
Top
 Abstract
Introduction
 Experimental Procedures
 Results
 Discussion
 References
 
The growth of the male external genitalia is primarily regulated by androgens. However, human genital fibroblast growth is also stimulated by insulin-like growth factor (IGF) I. In this study, we report that IGF-binding protein (IGFBP) production in human foreskin fibroblasts is regulated by androgens and IGF-I. Human foreskin fibroblasts secrete IGFBP-3, IGFBP-4, and IGFBP-5. IGF-I increased the abundance of both intact IGFBP-3 and -5 in the culture medium. Testosterone increased IGFBP-3, and the combination of IGF-I and testosterone had an additive effect. Following its secretion, IGFBP-5 was degraded, but the effect of IGF-I on IGFBP-5 peptide abundance in conditioned media did not seem to be due to inhibition of proteolysis. Testosterone had no effect on IGFBP-5 degradation. Intact IGFBP-4 was decreased by IGF-I, and the combination resulted in a similar reduction. The mechanism seemed to be decreased synthesis, since IGFBP-4 messenger RNA was also decreased. The increase in IGFBP-5 synthesis was associated with an increase in the abundance of intact IGFBP-5 in the extracellular matrix. The combination of testosterone and IGF-I resulted in a synergistic stimulation of total protein synthesis by the fibroblast cultures, suggesting that a maximum anabolic response requires both hormones. These observations suggest that combined exposure to androgen and IGF-I altered the abundance of some forms of IGFBPs and that the IGFBPs that are regulated may play a role in modulating the effects of IGF-I on the anabolic response.


   Introduction
Top
 Abstract
 Introduction
Experimental Procedures
 Results
 Discussion
 References
 
PEPTIDE growth factors have been shown to mediate some of the growth-promoting effects of androgens in androgen-responsive tissues. Insulin-like growth factor (IGF) I has been shown to be synthesized by androgen-responsive tissues and to stimulate locally regulated growth. In addition to their growth-promoting actions, both androgens and IGF-I have metabolic actions in gonadal cells. Similarly, they have been shown to interact in controlling testosterone biosynthesis by leydig cells. IGF-I has been shown to stimulate the testosterone production by testicular interstitial cells (1) and to enhance steroidogenesis by leydig cells in response to human CG (2, 3). Further evidence for an interaction between these trophic factors comes from the clinical observation that patients with congenital GH deficiency (4) or those with GH receptor mutations (5, 6, 7) develop micropenis despite normal androgen production. Patients with abnormalities in androgen production, such as congenital hypogonadism (8, 9) or androgen receptor mutations (10), may present with genitalia that show disordered growth regulation. These clinical observations suggest that interactions between IGFs and androgens may be required for normal genital growth.

In addition to production and responsiveness of IGF-I, androgen-sensitive tissues have also been shown to produce IGF-binding proteins (IGFBPs). IGFBP-2 to -4 messenger RNAs (mRNAs) have been shown to be expressed in normal rat testis (11). However, the regulation of IGFBP synthesis by gonadal tissues has not been studied in detail. Sertoli cells in culture have been shown to produce IGFBP-3, and this is regulated by IGF-I (12). Similarly, testosterone was reported to increase total IGFBP activity in media obtained from foreskin fibroblasts (13). Because the IGFBP bind the IGFs with high affinity, they have the potential to regulate the responsiveness of androgen-sensitive tissues to this growth factor. For this reason, we studied IGFBP production by androgen-sensitive fibroblasts and correlated these findings with changes in anabolic responsiveness. We chose to use androgen-sensitive fibroblasts as a model for studying androgen action because the responses of nonandrogen responsive fibroblasts to IGF-I had been characterized in detail.


   Experimental Procedures
Top
 Abstract
 Introduction
 Experimental Procedures
Results
 Discussion
 References
 
Cell culture

Normal human foreskin fibroblasts (GM8333A) were obtained from Coriell Institute (Camden, NJ) and grown in MEM without phenol red (Life Technologies, Inc., Grand Island, NY) supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), 2 mM L-glutamine, and 15% FBS (Sigma, St. Louis, MO). The cells were plated on 10-cm plates (Falcon #3003; Division of Becton-Dickinson, Plymouth, UK), 48-well plates (#3548; Costar, Cambridge, MA). The medium was changed every 3 days until confluency was attained (usually 7–10 days). At that time, the experiments were initiated without a period of serum deprivation. These fibroblasts have been extensively characterized (14, 15, 16). We have shown that they express androgen receptors and respond to dihydrotestosterone (Yoshizawa, A., F. S. French, and D. R. Clemmons, unpublished observations).

Western ligand blots and immunoblots

Conditioned medium was collected by adding 4.0 mL serum-free MEM to fibroblast cultures with or without the listed treatments. The treatments included IGF-I [50 ng/mL (a gift of Genentech, Inc., South San Francisco, CA) or testosterone 10 nM (Sigma)]. These concentrations were chosen because, after testing concentrations between 5–100 ng/mL IGF-I and 1–100 nM testosterone, they were shown to give the greatest response. After 24 h, the medium was removed and centrifuged at 5000 x g for 10 min, then stored at -20C until it was analyzed. In some experiments, 100 U/mL heparin (Sigma) was added to limit IGFBP-5 degradation. Each conditioned medium sample (100–150 µL) was lyophilized and reconstituted in 30 µL Laemmli sample buffer (17). Each sample and known molecular weight standards (Amersham Corp., Arlington Heights, IL) were electrophoresed through 12.5–15% SDS polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon; Millipore Corp., Bedford, MA). The membranes were probed with 125I-IGF-I (specific activity 150–250 µCi/µg) using 600,000 cpm/membrane, as described previously (18). Immunoblotting was performed using a 1:500 dilution of a polyclonal rabbit antihuman IGFBP-4 antiserum and a 1:1000 dilution of a guinea pig or a 1:2000 dilution of a rabbit antihuman IGFBP-5 antiserum. After an overnight incubation at room temperature, the immunoblots were developed by incubating for 3 h with goat antirabbit IgG alkaline phosphatase conjugate (Sigma) or sheep antiguinea pig IgG alkaline phosphatase conjugate (Chemicon, Temecula, CA). To visualize the bands, the Protoblot System immunoblotting reagents were used following the technique recommended by manufacturer (Promega Biotech, Madison, WI). The blots were analyzed by scanning densitometry (Hoeffer Scientific, San Francisco, CA). The signal intensity of each band was quantified using NIH Image.

IGFBP-4 degradation assay

The conditioned medium (50 µL) that had been collected after a 24-h exposure to confluent, quiescent foreskin fibroblasts, was incubated with 50 ng pure human IGFBP-4 for 14 h at 37C at 60 µL Tris (0.05 M) containing 50 mM NaCl and 2.0 mM CaCl2 (pH 7.4). The digestion products were analyzed by immunoblotting.

RNA isolation and Northern blot analysis

RNA was isolated from cells using TriReagent (Molecular Research Center, Inc., Cincinnati, OH). Fifteen micrograms of total RNA were loaded to a 1.0% agarose formaldehyde gel and transferred onto a nylon membrane (ICN Biochemical, Inc., Irvine, CA). The membranes were hybridized with a 1125-bp [32P]-dCTP-labeled human IGFBP-3 probe (19), a 600-bp IGFBP-4 probe (19), or a 627-bp IGFBP-5 complementary DNA probe (19). The amount of radiolabeled probe that was hybridized was determined by autoradiography. The signal intensity of each band was quantified using a Phospho Imager SF (Molecular Dynamics, Sunnyvale, CA). The abundance of each mRNA was normalized for changes in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. A complementary DNA probe for GAPDH was obtained from Ambion (Austin, TX).

Preparation of extracellular matrix (ECM)

The cells were rinsed twice with phosphate-buffered saline (PBS), and the cellular membranes were removed by incubating for 5 min in 0.5% Triton X-100 in PBS (pH 7.4). The adherent nuclei and cytoskeletal proteins were removed by incubating for 5 min in 25 mM ammonium acetate (pH 9.0). After washing twice with PBS, the ECM was scraped from the plates into Laemmli sample buffer. The extracts, 0.05 cc, were loaded onto SDS polyacrylamide gels (12.5%), transferred to polyvinylidene difluoride membranes, and analyzed by Western ligand blotting using 125I-IGF-I, as described previously. This method of ECM preparation has been shown to result in abundant fibronectin and other ECM components (20).

[35S]-methionine incorporation assay

The effects of the test substances on protein synthesis were determined by adding 20 µCi [35S]-methionine (specific activity 1218 Ci/mmol; ICN Pharmaceuticals, Irvine, CA) to 0.5 ml low methionine (10-6 M) medium and increasing concentrations of IGF-I or 50 ng/mL IGF-I plus increasing concentrations of testosterone for 6 h at 37C. The medium was aspirated, and wells were rinsed twice with PBS containing 0.1% BSA (Sigma). Cells were lysed, and the total intracellular protein precipitated in 5% TCA then centrifuged at 14,000 x g for 10 min. The pellets were resuspended in 0.1 M NaOH with 1% SDS and counted in a ß-scintillation counter.

Statistical analysis

Student’s t test was used to compare the difference between the control and test groups. Values represent mean ± SE.


   Results
Top
 Abstract
 Introduction
 Experimental Procedures
 Results
Discussion
 References
 
Western ligand blotting of fibroblast-conditioned medium obtained after exposure to either IGF-I (50 ng/mL), testosterone (10 nM), or IGF-I and testosterone showed that the cells released forms of IGFBPs with molecular weight estimates of 37–50 kDa, 30 kDa, and 24 kDa (Fig. 1Go). These molecular weight estimates corresponded to human IGFBP-3, -4, and -5. That the 30- and 24-kDa forms were IGFBP-5 and IGFBP-4, respectively, was confirmed by immunoblotting. The 37–50-kDa form has previously been shown by immunoblotting to be IGFBP-3 (19). Thus, the three forms of IGFBPs that are released are similar to those released by dermal fibroblasts obtained from other sites (19). Exposure to 10 nM testosterone resulted in an increase in IGFBP-3 but no change in IGFBP-4 or IGFBP-5. In contrast, exposure to IGF-I resulted in a major increase in IGFBP-5, a lesser increase in IGFBP-3, and a decrease in IGFBP-4. Scanning densitometry of autoradiographs from nine separate experiments (Table 1Go) showed that IGF-I increased IGFBP-3 by 212% of control (serum-free medium), whereas testosterone resulted in a 145% increase. In contrast, when dermal fibroblasts obtained from other sites (19) that have low androgen receptor number were analyzed, they did not show this testosterone response (data not shown). The combination of testosterone plus IGF-I resulted in a 336% increase over baseline, and this was significantly greater than the response to IGF-I or testosterone alone (P < 0.02). IGFBP-5 was not significantly increased by testosterone. IGF-I stimulated a 415% increase, but the response to testosterone + IGF-I was not significantly greater than IGF-I alone (TableGo I). Scanning densitometry of the band changes for IGFBP-4 showed very different results. Specifically, IGF-I reduced IGFBP-4 to 63% of control, and testosterone resulted in no significant change. The combination of testosterone plus IGF-I reduced it by 63%. IGFBP-1, -2, and -6 were not detected (data not shown).



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  		Figure 1. Ligand blot showing the effect of IGF-I and testosterone on IGFBP secretion. Conditioned media were collected after a 24-h incubation and analyzed by SDS PAGE with ligand blotting, as described in Experimental Procedures. The arrows denote the positions of IGFBP-3, -4, and -5. Lane 1, control; Lane 2, 10 nM testosterone; Lane 3, 50 ng/mL IGF-I; Lane 4, testosterone + IGF-I. This experiment was repeated nine times, yielding similar results.

 

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  		Table 1. Changes in IGFBP peptide abundance in fibroblast-conditioned medium

 
To further analyze the mechanisms that accounted for these changes, Northern blotting was performed for each of the three mRNA species (Fig. 2Go). As shown in Table 2Go, IGF-I had no effect on IGFBP-3 mRNA, whereas it decreased IGFBP-4 to 73 ± 6% of the control culture value and increased IGFBP-5 to 193 ± 33% above control cultures. Similar findings have been reported for dermal fibroblasts that do not possess androgen receptors (19, 21). Testosterone increased the abundance of IGFBP-5 mRNA and had no effect on IGFBP-3 and -4. The combination of IGF-I + testosterone also suppressed IGFBP-4 mRNA, but the degree of change was not different compared to IGF-I alone. In contrast, the combination of testosterone and IGF-I had an additive effect on IGFBP-5 mRNA (i.e. 333% stimulation). This response was significantly greater than the response to IGF-I or testosterone alone (P < 0.05). The combination of testosterone and IGF-I decreased IGFBP-3 mRNA significantly. Because IGFBP-3 fragments were not detected by immunoblotting (data not shown), the increase in IGFBP-3 in media seen with testosterone plus IGF-I is probably due to either enhanced secretion or release of IGFBP-3 from the cell surface.



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  		Figure 2. Northern blotting analysis of IGFBP abundance after testosterone IGF-I stimulation. Fibroblast cultures were stimulated with the treatments listed for 24 h, then total RNA was extracted and analyzed by Northern blotting, as described in Experimental Procedures. Lane 1, control; Lane 2, 50 ng/mL IGF-I; Lane 3, 10 nM testosterone; Lane 4, IGF-I + testosterone. The bands shown represent IGFBP-3, -4, and -5 mRNA, as noted. The abundance of GAPDH was also determined, and the results were used to normalize the level of each mRNA loaded. The experiment was repeated nine times with similar results.

 

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  		Table 2. Changes in IGFBP mRNA abundance in fibroblasts

 
To further analyze the changes that were occurring in IGFBP-4, two types of experiments were conducted (Fig. 3AGo). Exposure of the cultures to testosterone alone resulted in no change in the amount of intact IGFBP-4, and there was no change in the abundance of IGFBP-4 fragment as compared with control. In contrast, when IGF-I was incubated with the cultures, almost no intact protein was detected and two IGFBP-4 fragment bands were present. The combination of testosterone and IGF-I gave similar results. This experiment was repeated nine times, and the blots were analyzed by scanning densitometry. The results showed that there was a 67% ± 9% (P < 0.05) decrease in intact IGFBP-4 band intensity with IGF-I and a 39% ± 17% (P < 0.05) increase in the intensity of the IGFBP-4 fragment bands (Table 3Go). The combination of testosterone plus IGF-I also resulted in a decrease in intact IGFBP-4 and in an increase in the fragments, but these changes were not different from those induced by IGF-I alone.



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  		Figure 3. Change in IGFBP-4 secretion and degradation. A, Fibroblast cultures were exposed to serum-free medium for 24 h. Aliquots of conditioned medium were immunoblotted for IGFBP-4 using a specific antiserum, as described in Experimental Procedures. The arrows denote the positions of intact IGFBP-4 (top arrows) and the two major IGFBP-4 fragments. Lane 1, control; Lane 2, 10 nM testosterone; Lane 3, 50 ng/mL IGF-I; Lane 4, testosterone + IGF-I. Eight independent experiments were performed with similar results. B, Conditioned media were obtained after exposing the cultures to the treatments listed below for 24 h. At that time, 50 ng/mL IGFBP-4 was added to 50 µL conditioned medium, and the incubation continued for 14 h at 37C. The digestion products were analyzed by SDS-PAGE and immunoblotted with an anti-IGFBP-4 antiserum. Lanes 1, 3, 5, and 7 contain 50 ng/mL human IGFBP-4. Lanes 2, 4, 6, and 8 contain no added IGFBP-4. The media samples were obtained from the culture exposed to: Lanes 1 and 2, control, no additives; Lanes 3 and 4, 50 ng/mL IGF-I; Lanes 5 and 6, 10 nM testosterone; Lanes 7 and 8, testosterone + IGF-I. Three independent experiments were performed and showed the same results.

 

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  		Table 3. Changes in intact IGFBP-4 and fragments

 
To determine whether the appearance of IGFBP-4 fragments was due to a protease that was released into the medium or if degradation was occurring at an intracellular level or on the cell surface, 24-h conditioned medium was obtained, and the protease activity analyzed in vitro. It has been shown in several cell types that an IGFBP-4 protease that is released into medium is active in vitro, particularly if IGF-I or IGF-II is added to the incubation medium (22). However, when we analyzed degradation of IGFBP-4 by the conditioned medium obtained in the presence of IGF-I, there was no additional detectable degradation (Fig. 3BGo). Taken together, the results strongly suggest that degradation is occurring either intracellularly or on the cell surface.

To definitively identify the 30-kDa protein as IGFBP-5 and to further analyze the changes that occurred in IGFBP-5 peptide, conditioned medium was analyzed by immunoblotting. As shown in Figure 4AGo and Table 4Go, IGF-I induced a large increase in intact IGFBP-5, whereas testosterone had no effect, and the combination did not have a greater effect than IGF-I alone. To determine whether changes in IGFBP-5 degradation accounted for part of these changes, heparin was added to the culture medium to prevent IGFBP-5 degradation. IGF-I increased the abundance of intact IGFBP-5, whereas testosterone had no effect, and the combination of testosterone + IGF-I was no greater than the effect of IGF-I alone (Fig. 4BGo, Table 4Go). Although heparin was added at a concentration that effectively inhibits proteolysis (23), a 14-kDa fragment was still detected in the medium.



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  		Figure 4. Release of IGFBP-5 from fibroblast cultures. A, Cultures were exposed to various treatments. Following a 24-h incubation, as described in Experimental Procedures, the media were analyzed by immunoblotting for IGFBP-5 using a specific anti-IGFBP-5 antiserum. Lane 1, IGFBP-5 standard 40 ng; Lane 2, serum-free control; Lane 3, 50 ng/mL IGF-I; Lane 4, 10 nM testosterone; Lane 5, testosterone + IGF-I. The top band denotes the position of intact IGFBP-5, and the bottom band is the major proteolytic fragment. The experiment was repeated five times with similar results. B, Conditioned media were collected using the same treatment conditions listed in A, but heparin (100 µg/mL) was added to inhibit degradation. They were then analyzed by immunoblotting for IGFBP-5. Lane 1, control; Lane 2, 50 ng/mL IGF-I; Lane 3, 10 nM testosterone; Lane 4, testosterone + IGF-I. The top arrow corresponds to intact IGFBP-5, and the two bottom arrows to its two major fragments.

 

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  		Table 4. Changes in intact IGFBP-5 and fragments

 
Because IGFBP-5 is highly concentrated in the ECM of fibroblasts, and since matrix-associated IGFBP-5 can control the growth response of these cells to IGF-I (20), we also analyzed the effects of androgen and IGF-I treatment on the abundance of IGFBP-5 in the ECM. As can be seen in Fig. 5Go, under basal conditions after 24 h IGFBP-5 could easily be detected in the ECM. IGF-I treatment alone did not alter IGFBP-5 abundance at 24 h, but after a longer incubation period (e.g. 72 h), IGF-I or testosterone alone induced significant increase in the amount of IGFBP-5 that was detectable in the ECM compared to control, nonstimulated cultures. Testosterone alone and the combination of testosterone plus IGF-I induced significant increases over control cultures at 48 h (Table 5Go). Testosterone plus IGF-I progressively increased the amount of IGFBP-5 from 24 to 72 h. At the 72-h point, the effect of testosterone plus IGF-I was not greater than IGF-I alone.



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  		Figure 5. IGFBP-5 abundance in ECM analyzed by ligand blotting. ECM was prepared as described in Experimental Procedures after the three incubation times listed. The ECM extracts were then analyzed by ligand blotting. The arrow denotes the migratory position of IGFBP-5. Lane 1, control; Lane 2, 10 nM testosterone; Lane 3, 50 ng/mL IGF-I; Lane 4, testosterone + IGF-I. The experiment was repeated four times with similar results.

 

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  		Table 5. Changes in IGFBP-5 abundance in ECM

 
To determine the effects of testosterone and IGF-I on the cellular anabolic response, the cultures were exposed to increasing concentrations of IGF-I or to increasing concentrations of testosterone in the presence and absence of IGF-I, and the protein synthesis response was measured (Fig. 6Go). There was a dose-dependent increase in protein synthesis in response to increasing concentrations IGF-I between 25 and 100 ng/mL. The maximum increase was 34 ± 3%, P < 0.001, compared with control. In contrast, increasing concentrations of testosterone between 1 and 1000 nM resulted in no change in the absence of IGF-I. However, a response to testosterone was easily detected if 50 ng/mL of IGF-I was also present, and increasing concentrations of testosterone stimulated protein synthesis from 25–57% above the value obtained with testosterone alone. Comparison of the result obtained with concentrations of testosterone between 10–1000 nM plus IGF-I with the value obtained with 1 nM testosterone plus IGF-I showed that these concentrations induced significantly greater increases, P < 0.05.



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  		Figure 6. Protein synthesis response of cultures to IGF-I + testosterone. Fibroblast cultures were exposed to increasing concentrations of IGF-I alone (A). In the experiments represented in panel B, increasing concentrations of testosterone were added in the presence (•) or absence ({circ}) of IGF-I (50 ng/mL). After 6 h, the amount of 35S-methionine incorporated in the total protein was quantified. The results were expressed as percentage of control, with control being serum-free medium, with no additives. Each point represents a mean ± SEM of nine replicate determinations.

 

   Discussion
Top
 Abstract
 Introduction
 Experimental Procedures
 Results
 Discussion
References
 
This study clearly demonstrates that there is an interaction between androgens and IGF-I in controlling IGFBP-3 and IGFBP-5 synthesis or secretion and in stimulating total protein synthesis by fibroblasts. Specifically, the abundance of IGFBP-3, the most abundant form of IGFBP that is secreted by fibroblasts, in the conditioned medium was stimulated by both compounds, and they seemed to have an additive effect. The changes in IGFBP-3 seemed to be due to release from either the cell surface or an intracellular compartment, rather than increased synthesis, because neither IGF-I nor testosterone increased IGFBP-3 mRNA abundance.

The changes in IGFBP-4 were complex. Specifically, IGFBP-4 mRNA was suppressed by IGF-I, but the abundance of an IGFBP-4 fragment in the media was also increased. The fragment was probably generated by an intracellular or plasma membrane-associated protease, since the medium did not contain IGFBP-4 proteolytic activity. There was an increase in the amount of fragment after exposure of the cells to IGF-I, but there is no direct evidence that IGF-I stimulated protease activity. Testosterone had no effect on IGFBP-4 mRNA and resulted in no change in intact peptide. IGF-I plus testosterone also decreased both IGFBP-4 mRNA and peptide abundance, but the effect was not greater than IGF-I alone. These results suggest that the decrease in IGFBP-4 that is induced by exposure to IGF-I is due to both decreased synthesis and enhanced degradation.

IGFBP-5 was regulated differently than IGFBP-3 or -4. Both testosterone and IGF-I increased its mRNA abundance, and their effects were additive. When immunoblotting was used to detect intact peptide, an increase could be detected in the conditioned medium with IGF-I alone or testosterone plus IGF-I. Testosterone alone had no effect. Exposure to IGF-I probably resulted in stimulation of IGFBP-5 synthesis, since steady-state IGFBP-5 mRNA levels were increased, as were intact IGFBP-5 peptide levels. In contrast to IGF-I, the testosterone-induced increase in IGFBP-5 mRNA abundance did not result in a significant increase in intact peptide. This could have resulted from an increase in proteolytic cleavage of IGFBP-5, but there was no significant increase in the predominant fragment, making this an unlikely explanation. IGF-I exposure did not result in clear-cut inhibition of IGFBP-5 proteolysis, since the fragment abundance was increased 174% over control. However, this change was not significant, suggesting that inhibition of proteolysis may have contributed to the increase in peptide abundance. In other studies, IGF-I has been shown to inhibit IGFBP-5 proteolysis; therefore, this possibility cannot be definitively excluded (23). These findings suggest that cellular exposure to IGF-I or testosterone resulted in increased IGFBP-5 synthesis, but only IGF-I definitively increased peptide abundance in the medium. This is a result that has been reported previously for dermal fibroblasts that have low androgen receptor number (19, 23).

These changes in IGFBP-5 synthesis were also reflected by changes in IGFBP-5 content in the ECM. We have shown previously that IGFBP-5 preferentially adheres to fibroblast ECM (20) and that when IGFBP-5 is bound to ECM it is relatively resistant to proteolysis (24). In this study, both testosterone and IGF-I exposure resulted in substantially greater amounts of IGFBP-5 remaining in the ECM after 72 h compared with control cultures. The increase that occurred with exposure to testosterone or IGF-I alone for 24 or 48 h were of less magnitude, although the changes induced by testosterone at 48 h were significantly greater than control. IGFBP-5 in the medium undergoes extensive proteolysis. Our findings suggest that exposure to serum-free medium reduces the rate of IGFBP-5 synthesis to a level that is not adequate to maintain a stable level of intact peptide in the ECM. In contrast, cultures exposed to IGF-I, testosterone, or the combination are able to synthesize adequate amounts of IGFBP-5 to maintain stable levels of intact peptide in the ECM at 72 h. Therefore, both the rate of IGFBP-5 synthesis and the rate of proteolytic cleavage in the medium are major variables determining the concentration of IGFBP-5 in ECM.

The most interesting finding of this study was true synergism in the stimulation of protein synthesis by fibroblasts in response to testosterone/IGF-I. Increasing concentrations of testosterone up to 1000 nM had no effect on the protein synthesis response of these cells, but if IGF-I was added a testosterone concentration as low as 10 nM stimulated protein synthesis. Furthermore, the maximum effect that could be obtained with IGF-I was potentiated by testosterone, indicating a true synergism in the protein synthesis response. Dykstra et al. (25) had previously reported that IGF-I plus testosterone increased intracellular protein content in foreskin fibroblasts. However, they did not analyze the response to testosterone alone. Because the effects of testosterone and IGF-I on IGFBP-5 mRNA abundance and on IGFBP-3 peptide were additive, it is probable that these responses are related to the protein synthesis response of the cells. We have previously shown that enriching the fibroblast ECM in IGFBP-5 results in a potentiation of the fibroblast mitogenic response to IGF-I (20). Therefore, our finding of association between an enhancement of protein synthesis and the amount of IGFBP-5 in the ECM in these cells also suggests that it may have contributed to potentiation of the anabolic response.

The additive or synergistic responses between IGF-I and androgen suggests that there may be points in the IGF signal transduction pathway that may be influenced by activation of androgen receptor pathway. Specific genes whose transcription is activated in response to IGF-I include elastin (26, 27, 28), IGFBP-5 (29), crystallin (30), and a cholesterol sidechain cleavage enzyme (31). It will be important in future studies to determine whether increased IGFBP-5 synthesis in response to androgen is the result of a direct increase in transcription. Future studies should be directed toward determining whether the androgen/androgen receptor complex interacts with this 5' flanking regulatory sequence in the IGFBP-5 promoter and whether or not this accounts for the ability of androgens to potentiate IGF-I-stimulated synthesis of this protein. The extent to which androgen receptor activity and IGFBP/IGF receptor occupancy are required for maximum stimulation of the protein synthesis in this cell type is also worthy of further study, as is understanding the point at which the signal transduction pathways interact, and the genes that are coregulated by both stimuli that result in an enhanced protein synthesis response.


   Acknowledgments
 
We gratefully acknowledge the assistance of Mr. George Mosley in preparing this manuscript.


   Footnotes
 
1 Supported by NIH Grant AG02331. Back

Received September 5, 1997.

Revised June 2, 1999.

Accepted December 28, 1999.


   References
Top
 Abstract
 Introduction
 Experimental Procedures
 Results
 Discussion
 References
 

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