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Endocrinology Vol. 141, No. 9 3104-3110
Copyright © 2000 by The Endocrine Society

ARTICLES

Differential Regulation of Insulin-Like Growth Factor-Binding Protein-3 Protease Activity in MCF-7 Breast Cancer Cells by Estrogen and Transforming Growth Factor-ß11

Houta Salahifar, Robert C. Baxter and Janet L. Martin

Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia

Address all correspondence and requests for reprints to: Dr. Janet Martin, Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, 2065 New South Wales, Australia. E-mail: janetlm{at}med.usyd.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have examined the regulation of an insulin-like growth factor-binding protein-3 (IGFBP-3) protease secreted by MCF-7 human breast cancer cells using a ligand-binding assay that relies on the decrease in affinity for des(1–3)IGF-I that occurs when IGFBP-3 becomes proteolyzed. IGFBP-3 protease activity was not altered by treatment of MCF-7 cells with all-trans-retinoic acid, vitamin D, epidermal growth factor, platelet-derived growth factor, insulin, or forskolin. However, estradiol was a potent stimulator of IGFBP-3 protease activity, with a significant and maximal effect at 1 nM. This was prevented by cotreatment with tamoxifen, which had no significant effect in the absence of estradiol. By contrast, TGFß1 dose dependently inhibited the amount of protease activity secreted by MCF-7 cells, with complete reversal of IGFBP-3 degradation apparent in response to 10 ng/ml TGFß1. This study has demonstrated that estrogens and TGFß1, factors that are stimulatory and inhibitory, respectively, for MCF-7 cell growth, also stimulate and inhibit the production of an enzyme capable of proteolyzing the growth inhibitory protein IGFBP-3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor I (IGF-I) and IGF-II are potent mitogens for human breast cancer cells in vitro (1), and the recent observation that a high circulating level of IGF-I in premenopausal women correlates with increased risk of the development of breast cancer (2) may have a link with a role for this growth factor in the development of malignant breast disease.

The growth-promoting effects of IGFs are regulated by their high affinity interaction with members of the family of IGF-binding proteins (IGFBPs) (3). IGFBP-3, the predominant IGFBP in the circulation, is also secreted by many breast cancer cells (4, 5) where it has the potential to act as an autocrine/paracrine factor to inhibit (5) or enhance (6) the proliferative effects of IGFs. Thus, the IGFs and their regulatory IGFBPs may be significant in the development and progression of breast cancer, where IGFs act as mitogens and promote the growth of breast tumor cells, and IGFBPs regulate the impact of IGFs on tumor growth.

IGFBP-3 is a glycosylated protein of 43–45 kDa when intact; however, proteolysis of IGFBP-3 in serum has been demonstrated in pregnancy and a variety of pathological conditions, including postsurgical and severely ill patients (7, 8), patients with malignancies (9), and adults with noninsulin-dependent diabetes (10). The secretion of IGFBP-degrading proteases by cells in vitro is also well documented, with cathepsin D (11, 12), plasmin (13), and matrix metalloproteases (14) each capable of causing IGFBP-3 proteolysis. The fragments generated by enzymatic cleavage of IGFBP-3 frequently have reduced affinity for IGFs (15), and this is thought to be the explanation for altered responsiveness to IGFs in cells where IGFBP-3 proteolysis is modified (13). However, IGFBP-3 fragments may also have bioactivity independent of their modulation of type 1 IGF receptor activation (16, 17). IGFBP proteases therefore have the potential to both increase the free IGF concentration and generate IGFBP-3 fragments with intrinsic growth-promoting or -inhibiting activities.

We have previously shown that MCF-7 cells secrete an IGFBP-3 protease with biochemical characteristics different from those of other known IGFBP-3 proteases (18). In this study we describe a simple assay developed for the quantitative measurement of IGFBP-3 proteolysis and use it to examine the regulation of this protease activity in MCF-7 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
BSA, protein A, media for cell culture, glutamine, antibiotics, bovine insulin, estradiol, tamoxifen, forskolin, insulin, 1,25-dihydroxyvitamin D3, and all-trans-retinoic acid were purchased from Sigma (St. Louis. MO). Trypsin-EDTA solution was obtained from Flow Laboratories (North Ryde, Australia), and FCS was purchased from Trace Biosciences (North Ryde, Australia). Epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-ß1 (TGFß1) were purchased from Austral Biologicals (San Ramon, CA). Des(1, 2, 3)IGF-I was purchased from GroPep Pty. Ltd. (Adelaide, South Australia). Nonidet P-40 was a product of Fluka Chemical Co. (Basel, Switzerland). Recombinant human IGF-I was donated by Kabi Peptide Hormones (Stockholm, Sweden) and Genentech, Inc. (South San Francisco, CA). [Methyl-3H]thymidine was obtained from ICN Biomedical Corp. (Seven Hills, Australia). IGF-I, des(1, 2, 3)IGF-I, and protein A were radioiodinated with Na125I using chloramine-T. Molecular weight markers and electrophoresis equipment and materials were purchased from Pharmacia Biotech (Uppsala, Sweden) and Bio-Rad Laboratories, Inc. (Australia), and Hyperfilm-MP autoradiography film was purchased from Amersham Pharmacia Biotech (Aylesbury, U.K.). All other reagents were of analytical grade.

Cell culture and stimulation experiments
MCF-7 breast adenocarcinoma cells were obtained from American Type Culture Collection (Manassas, VA) and were maintained in RPMI buffered at pH 7.4 with 15 mM HEPES and containing 10% FCS, 4 mM glutamine, and 10 mg/liter bovine insulin. For stimulation experiments, cells were seeded into 24-well plates or 25-cm2 flasks at approximately 5 x 104 cells/cm2 in growth medium and grown to confluence. Media were replaced by phenol red-free RPMI containing 1 g/liter BSA for 24 h before the addition of test substances. Peptides, steroids, and growth factors were added to cells in phenol red- and serum-free medium (0.5 ml/well or 10 ml/flask), cultures were incubated for a further 48 h, then media were collected and prepared for analysis of IGFBP-3 protease activity. Cell numbers were determined on trypsin-dispersed cultures using a hemocytometer.

Preparation of conditioned media and IGFBP-3 digests
Conditioned media were equilibrated with sodium acetate buffer at pH 5.5 using a Centricon 10 ultrafiltration unit (Amicon, Danvers, MA) as follows. Medium (2–5 ml) was concentrated 50-fold by centrifugation in the Centricon 10, then flushed with 5 vol 0.1 M sodium acetate buffer, pH 5.5. The final concentrate was readjusted to starting volume in this buffer. Protease activity in these samples was assessed by incubating plasma-derived IGFBP-3 (100 ng) with 100 µl prepared medium at 37 C for 24 h at pH 5.5. Proteolysis of IGFBP-3 was monitored by immunoblot after SDS-PAGE or by ligand binding assay as described below.

Ligand binding assay
A decrease in binding of IGFBP-3 to [125I]des(1, 2, 3)IGF-I was used to assay IGFBP-3 proteolysis. This truncated IGF-I tracer was used because it retains considerable affinity for IGFBP-3, but not for other IGFBPs (19). IGFBP-3 digests prepared as described above were serially diluted in binding assay buffer (50 mM sodium phosphate, 0.25% BSA, and 0.02% sodium azide, pH 6.5) to give final IGFBP-3 concentrations of 0.1–20 ng/50 µl, then incubated with [125I]des(1, 2, 3)IGF-I (10,000 cpm) for 16 h at 22 C in a final assay volume of 300 µl. IGFBP-3-bound tracer was precipitated by the addition of anti-IGFBP-3 antibody (R100, 0.5 µl) in 25 µl binding buffer containing 0.1 M sodium phosphate, 0.2 g/liter sodium azide, and 2.5 g/liter BSA. After 1 h at 22 C, complexes were precipitated by the addition of goat antirabbit {gamma}-globulin (2.5 µl) and 1 ml cold 60 g/liter polyethylene glycol 6000 in 0.15 M NaCl and centrifugation for 20 min at 4,000 rpm. Supernatants were decanted, and pellets were counted for 2 min.

SDS-PAGE and Western blotting
Electrophoresis and Western blotting of proteolyzed IGFBP-3 were carried out as previously described (5), using 12% SDS-polyacrylamide gels. Separated proteins were transferred to a Hybond C membrane, then membranes were blocked in Tris-buffered saline (TBS; 10 mM Tris and 50 mM sodium chloride, pH 7.4) containing 10 g/liter BSA, 0.2 g/liter sodium azide, and 0.5 ml/liter Nonidet P-40 (blocking buffer) for 2–3 h at 22 C. For immunoblotting, blocked membranes were incubated overnight at 4 C with IGFBP-3 antiserum (R30) at a 1:5,000 final dilution. Membranes were then rinsed briefly in TBS containing 0.5 ml/liter Nonidet P-40, and incubated for an additional 2 h with [125I]protein A (1 x 106 cpm/50 ml in blocking buffer). Blots were washed three times for 10 min each time in TBS containing 0.5 ml/liter Nonidet P-40, then air-dried and autoradiographed for 4 days at -70 C.

Data analysis
Data analysis was performed using the StatView statistical package (SAS Institute, Inc., Cary, NC), and statistical significance, taken as P < 0.05, was determined by ANOVA followed by Fisher’s protected least significant differences (Fisher’s PLSD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We initially examined the effect of hormones and growth factors that have been implicated in regulating breast cancer cell proliferation on the activity of the MCF-7 cell-derived IGFBP-3 protease. Cells were treated for 48 h with effectors, then conditioned media were used to digest IGFBP-3 at pH 5.5, as described in Materials and Methods. Samples were analyzed by immunoblot after SDS-PAGE.

As previously reported (18), proteolysis of IGFBP-3 by untreated (control) conditioned medium was characterized by a marked decrease in the 43- to 45-kDa IGFBP-3 doublet and the appearance of an immunoreactive species of 21 kDa (Fig. 1Go, lane 2). An increase in the amount of protease activity was apparent in medium conditioned in the presence of estradiol (10 nM), with the 43- to 45-kDa IGFBP-3 doublet undetectable in these samples (Fig. 1Go, lane 12). By contrast, IGFBP-3 protease activity in medium from cells treated with TGFß1 (10 ng/ml) was markedly decreased compared with that in controls, with the intact 43- to 45-kDa IGFBP-3 predominating and the 21-kDa band reduced in intensity. Of the remaining test agents, vitamin D, the antiestrogen tamoxifen, and insulin caused a modest reduction in protease activity (Fig. 1Go).



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  		Figure 1. IGFBP-3 protease activity in MCF-7-cell conditioned medium. MCF-7 cells were treated for 48 h with no addition (lane 2), 100 nM all-trans-retinoic acid (lane 3), EGF (10 ng/ml) plus PDGF (10 ng/ml; lane 4), 10 nM 1,25-dihydroxyvitamin D (lane 5), 10 ng/ml TGFß1 (lane 6), 10 µg/ml bovine insulin (lane 7), 100 nM tamoxifen (lane 8), 0.5 µM forskolin (lane 9), 10 ng/ml EGF (lane 10), 10 ng/ml PDGF (lane 11), or 10 nM estradiol (lane 12). Conditioned media (100 µl) were used to digest IGFBP-3 (100 ng) as described in Materials and Methods, then IGFBP-3 proteolysis was analyzed by SDS-PAGE and immunoblot using IGFBP-3 antiserum. IGFBP-3 incubated with unconditioned medium is shown in lane 1. The migration position of molecular mass markers is indicated on the left of the figure.

 
To examine the regulation of MCF-7-derived protease in detail, we considered it necessary to develop a more quantitative measure of IGFBP-3 proteolysis. We had previously shown that the 21-kDa fragment generated by the action of this protease did not bind IGFs by ligand blot (18); therefore, we investigated whether an assay that measured a change in ligand binding in solution could be developed to quantify proteolysis of IGFBP-3.

IGFBP-3 was digested with conditioned media at pH 5.5, then ligand binding was carried out on serially diluted samples as described in Materials and Methods. These experiments were initially carried out using [125I]IGF-I as ligand; however, cell-derived IGFBPs present in the media were found to interfere in the binding assay by competing for ligand (data not shown). We therefore proceeded to optimize the assay using [125I]des(1, 2, 3)IGF-I as ligand. Binding of this analog to IGFBP-3 occurs with approximately one third to one fifth the activity of full-length IGF-I, whereas binding to other IGFBPs is much more markedly reduced (19).

To compare the abilities of intact and proteolyzed IGFBP-3 to bind des(1, 2, 3)IGF-I tracer, IGFBP-3 was incubated with 0, 25, or 50 µl MCF-7 conditioned medium for 16 h at pH 5.5, then [125I]des(1, 2, 3)IGF-I binding to samples was analyzed. Under our normal ligand binding assay incubation conditions of 2 h at 22 C, the 5-fold decrease in affinity of IGFBP-3 for des(1, 2, 3)IGF-I caused a reduction in binary complex formation to almost undetectable levels (data not shown); however, this was overcome by increasing the time of incubation to 16 h. Under these conditions, dose-dependent binding of tracer was observed when IGFBP-3 was incubated in the absence of conditioned medium, with approximately 50% of tracer bound at 10 ng/tube IGFBP-3 (Fig. 2AGo). IGFBP-3 incubated with 25 µl MCF-7 conditioned medium showed that binding activity decreased by two thirds, whereas binding was completely abolished after incubation with 50 µl conditioned medium.



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  		Figure 2. Binding of proteolyzed IGFBP-3 to [125I]des(1 2 3 )IGF-I. A, IGFBP-3 (100 ng) was incubated without medium ({blacksquare}) or with 25 µl ({circ}) or 50 µl ({triangleup}) MCF-7 conditioned medium at pH 5.5 for 16 h at 37 C. Samples were diluted to give the indicated concentrations of IGFBP-3, then binding of [125I]des(1 2 3 )IGF-I was carried out over 16 h at 22 C, as described in Materials and Methods. Results are the mean of duplicates from one of two experiments and show the percentage bound of the total added [125I]des(1 2 3 )IGF-I. Nonspecific binding was 6% of the total. B, IGFBP-3 (100 ng) was incubated with the indicated volume of unconditioned medium ({blacksquare}) or MCF-7 conditioned medium at pH 7.0 ({triangleup}) or at pH 5.5 in the absence ({circ}) or presence of 250 µM leupeptin ({blacktriangleup}) or 25 mM EDTA (•) for 16 h at 37 C. Binding of [125I]des(1 2 3 )IGF-I to 5 ng IGFBP-3 in each sample was determined. Results are the mean of duplicates from one of two experiments and show the percentage bound of total added [125I]des(1 2 3 )IGF-I. Nonspecific binding was 6% of the total. C, Western ligand blot of IGFBP-3 (50 ng) incubated with the indicated volume of MCF-7-conditioned medium at pH 5.5 for 16 h at 37 C, then separated by SDS-PAGE. The blot was probed with [125I]IGF-I as described in Materials and Methods. Intact IGFBP-3 of 43–45 kDa only is shown.

 
We had previously shown by immunoblotting that IGFBP-3 protease activity was inhibited by the protease inhibitors EDTA and leupeptin (18). To confirm this in the solution assay, we measured binding of des(1, 2, 3)IGF-I to 5 ng IGFBP-3 in samples incubated with conditioned medium in the presence of either leupeptin (250 mM) or EDTA (25 mM). As shown in Fig. 2BGo, [125I]des(1, 2, 3)IGF-I tracer binding to IGFBP-3 decreased with increasing volumes of MCF-7 conditioned medium in the absence of inhibitors, reflecting proteolysis of IGFBP-3. Inclusion of EDTA or leupeptin during IGFBP-3 digestion prevented this loss of binding activity, with the binding curves superimposable on those in which IGFBP-3 was incubated with either unconditioned medium or conditioned medium at pH 7 (Fig. 2BGo). The samples analyzed by ligand binding assay shown in Fig. 2AGo were reanalyzed by ligand blot. Figure 2CGo shows decreased 43- to 45-kDa IGFBP-3 after incubation with 25 and 50 µl medium, indicating that a decrease in the amount of intact IGFBP-3 in these samples parallels a decrease in [125I]des(1, 2, 3)IGF-I binding.

Using this assay we reassessed IGFBP-3 protease activity in the samples originally analyzed by immunoblotting shown in Fig. 1Go. With the exception of those from estradiol- or TGFß1-treated cells, none had significantly altered IGFBP-3 protease activity compared with untreated samples when examined by ligand binding assay (data not shown). However, treatment with 10 nM estradiol resulted in stimulation of proteolysis so that [125I]des(1, 2, 3)IGF-I binding to IGFBP-3 was reduced from 12.5% in the untreated medium sample to about 6% by medium from estradiol-treated cells (Fig. 3Go). By contrast with this, medium from cells treated with 5 ng/ml TGFß1 showed a 70% inhibition of the loss of des(1, 2, 3)IGF-I binding activity compared with control medium (Fig. 3Go), confirming the earlier finding of reduced protease activity obtained by immunoblot. In view of the effects of estradiol and TGFß1 on IGFBP-3 protease activity, we then focused on further characterization of its regulation by these agents.



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  		Figure 3. IGFBP-3 protease activity in MCF-7-cell conditioned medium measured by binding of [125I]des(1 2 3 )IGF-I. The untreated (ctl), estradiol-treated (E2), and TGFß1-treated (TGFß1) samples analyzed by immunoblot in Fig. 1Go were reassayed for [125I]des(1 2 3 )IGF-I binding using 5 ng IGFBP-3 from each digest. Binding of unproteolyzed IGFBP-3 is indicated in lane 1. Results are expressed as the mean ± SD of pooled data from two experiments, with each sample analyzed in duplicate.

 
Regulation of IGFBP-3 protease activity by estradiol
When IGFBP-3 was digested with 5 µl medium from 10 nM estradiol-treated cells, a more than 50% reduction in [125I]des(1, 2, 3)IGF-I binding was apparent compared with that using medium from untreated cells, reflecting an increase in IGFBP-3 proteolysis (Fig. 4AGo). When cells were treated with graded concentrations of estradiol, a significant increase in protease activity was apparent with 1 nM estradiol, and 10 nM estradiol reduced [125I]des(1, 2, 3)IGF-I binding to approximately 50% of the control value (P < 0.001). There was no significant difference in [125I]des(1, 2, 3)IGF-I binding between the 1 and 10 nM samples. To confirm that the observed increase in protease activity did not reflect increased cell number over the 48-h incubation, cell numbers were determined at the end of the conditioning period. There was no significant difference in cell number between control and estradiol-treated cultures (29.9 ± 3.7 x 105 cells/flask for control and 32.1 ± 2.5 x 105 cells/flask for 10 nM estradiol, mean ± SE of three flasks for each; P = 0.56).



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  		Figure 4. Stimulation of IGFBP-3 protease activity by estradiol. A, Intact IGFBP-3 (50 ng) was incubated with unconditioned medium ({blacksquare}) or with 5 µl medium from untreated cells (•) or cells treated with 10 nM estradiol ({circ}). The indicated concentrations of IGFBP-3 from each sample were then assayed for [125I]des(1 2 3 )IGF-I binding. Results are shown as the mean of pooled data from two experiments conducted in duplicate and represent specific binding of tracer. B, MCF-7 cells were treated for 48 h with the indicated concentration of estradiol. Medium (100 µl of each) was used to proteolyze 100 ng IGFBP-3, then 5 ng IGFBP-3 from each digest were analyzed by [125I]des(1 2 3 )IGF-I binding. Results shown are the mean ± SEM of pooled data from six experiments carried out in duplicate. Statistical significance, determined by ANOVA and Fisher’s PLSD, is shown (**, P < 0.001 compared with untreated).

 
Our original study describing this protease indicated that its activity was inhibited by [LR3]IGF-I (18), an IGF-I analog with extremely low affinity for IGFBP-3. To confirm that the estradiol-stimulated IGFBP-3 protease activity represented the same protease, we tested the effect of [LR3]IGF-I on estradiol-stimulated IGFBP-3 protease activity. Conditioned medium from estradiol-stimulated cells was incubated with 50 ng IGFBP-3 in the presence or absence of [LR3]IGF-I at 37 C and pH 5.5 for 16 h. SDS-PAGE and immunoblot analysis of these digests confirmed that [LR3]IGF-I blocked the proteolysis of IGFBP-3 by estradiol-treated conditioned medium (data not shown).

We then examined the effects of the antiestrogenic compound tamoxifen. Protease activity in medium conditioned by MCF-7 cells treated with estradiol, tamoxifen, or a combination of the two agents was determined by immunoblot and ligand binding assay. As shown by the IGFBP-3 immunoblot in Fig. 5AGo, the dose-dependent disappearance of the 43- to 45-kDa IGFBP-3 doublet was accompanied by the appearance of the 21-kDa fragment for both the control (untreated) and estradiol-treated samples. Almost complete disappearance of the 43- to 45-kDa IGFBP-3 was observed with 50 µl untreated medium, but only 10 µl estradiol-treated medium were required for the same degree of proteolysis. This suggested an approximately 5-fold increase in protease activity in medium from estradiol-treated cells and confirmed the results obtained by ligand binding assay. Treatment with both tamoxifen and estradiol produced the same pattern of proteolysis as that for control conditioned medium, indicating greatly reduced IGFBP-3 protease activity and inhibition of the stimulatory effect of estradiol (Fig. 5AGo).



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  		Figure 5. Effects of tamoxifen on estradiol-stimulated protease activity. A, IGFBP-3 (50 ng) was proteolyzed by the indicated volume of conditioned medium from untreated cells, cells treated with 10 nM estradiol, or cells treated with 10 nM estradiol plus 100 nM tamoxifen. IGFBP-3 proteolysis was assessed by SDS-PAGE and IGFBP-3 immunoblot, as described in Materials and Methods. The migration positions of molecular mass markers are indicated on the left. B, [125I]des(1 2 3 )IGF-I binding to IGFBP-3 (2.5 ng) digested by medium from untreated cells or those treated as indicated was determined. Binding of intact IGFBP-3 (i.e. IGFBP-3 incubated with unconditioned medium) is indicated as 100% (Ctl). Results are expressed as the mean ± SEM of pooled data from three experiments carried out in duplicate. Statistical significance, determined by ANOVA and Fisher’s PLSD, is shown (**, P < 0.005 compared with untreated).

 
When these samples were analyzed by ligand binding assay, tamoxifen (100 nM) did not significantly affect IGFBP-3 protease activity in the absence of estradiol (Fig. 5BGo). However, coincubation of cells with tamoxifen (100 nM) and estradiol (10 nM) led to inhibition of the stimulatory effect of estradiol on IGFBP-3 protease activity (P < 0.005 compared with estradiol alone).

Regulation of IGFBP-3 protease activity by TGFß1
We next examined the regulation of the protease activity by TGFß1. As shown in Fig. 6AGo, when IGFBP-3 was incubated with medium from cells treated with TGFß1, the loss of IGF-binding activity was markedly reduced compared with that using medium from untreated cells, indicating a decrease in protease activity in this medium. Immunoblot analysis, as illustrated in Fig. 6BGo, indicated that the disappearance of the 43- to 45-kDa IGFBP-3 doublet was inhibited up to the highest volume of medium from TGFß1-treated cells, indicating very little residual protease activity in these samples (compare with Fig. 5AGo, left panel). The presence of some 21-kDa material was still apparent. IGFBP-3 proteolysis decreased in a dose-dependent manner with increasing concentrations of TGFß1, and complete inhibition of protease activity was apparent in medium from cells treated with 10 ng/ml TGFß1 (Fig. 7Go). Over the 48-h incubation period, there was no significant change in cell number in response to TGFß1 (39.5 ± 4.3 x 105 cells/flask for control and 33.9 ± 1.4 x 105 cells/flask for 10 ng/ml TGFß1; mean ± SE of three flasks for each; P = 0.16). Proteolysis of IGFBP-3 by medium from untreated MCF-7 cells was unchanged by the inclusion of 5 ng/ml TGFß1 in the protease incubation mixture (data not shown), suggesting that the mechanism of action of TGFß1 involved inhibition of protease production at the cellular level, rather than direct interaction with either IGFBP-3 or the protease.



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  		Figure 6. Inhibition of IGFBP-3 protease activity by TGFß1. A, Intact IGFBP-3 (50 ng) was incubated with unconditioned medium ({blacksquare}) or with 50 µl medium from untreated cells (•) or cells treated with 10 ng/ml TGFß1 (•). The indicated concentrations of IGFBP-3 from each sample were then assayed for [125I]des(1 2 3 )IGF-I binding. Results are shown as the mean of pooled data from two experiments conducted in duplicate and represent specific binding of tracer. B, IGFBP-3 (50 ng) was digested with the indicated volume of medium from cells treated with 5 ng/ml TGFß1, and IGFBP-3 proteolysis was analyzed by the IGFBP-3 immunoblot. The migration positions of molecular mass markers are indicated on the left.

 


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  		Figure 7. Dose-response curve of TGFß1 inhibition of IGFBP-3 protease activity. MCF-7 cells were treated with TGFß1 at the indicated concentrations, then 50 ng IGFBP-3 were digested with 50 µl of each conditioned medium. [125I]Des(1 2 3 )IGF-I binding was measured using 5 ng IGFBP-3 from each digest. Results are shown as the mean ± SEM of pooled data from three determinations carried out in duplicate. The dotted line indicates the equivalent binding of intact IGFBP-3. **, P < 0.005 compared with untreated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously reported that proteolytic degradation of IGFBP-3 by an enzyme in MCF-7 conditioned medium results in the generation of fragments that fail to bind IGFs on ligand blots (18). In this study we have described a simple assay for the quantitation of IGFBP-3 proteolysis based on the decrease in IGFBP-3’s affinity for IGF-I that occurs when it is proteolyzed; increased protease activity results in a decreased proportion of intact IGFBP-3, and therefore decreased ligand binding. The level of IGFBP-3 protease activity in a given volume of sample is inversely related to the percentage of IGF tracer bound. Thus, if IGFBP-3 proteolysis is inhibited, for example by EDTA or leupeptin (18), the decrease in IGF tracer binding is reduced or abolished.

We were unable to use radiolabeled IGF-I in this assay due to competition by other IGFBPs in the cell-conditioned medium for ligand. In medium from MCF-7 cells, IGFBP-2 probably has the largest influence (4), because although IGFBP-6 is also secreted by this cell line (5), it binds IGF-I with lower affinity than either IGFBP-2 or IGFBP-3 (15) and would be less likely to have a significant effect. By using [125I]des (1, 2, 3)IGF-I in the ligand binding assay, this interference was overcome. The three amino-terminal residues of IGF-I are crucial for IGF-I binding to IGFBP-1 and IGFBP-2, but are of relatively minor importance in binding to IGFBP-3; therefore the affinity of this IGF analog for IGFBP-3 is reduced only about 5-fold (19).

This ligand binding assay was used in combination with the more conventional technique involving SDS-PAGE and immunoblot to examine regulation of IGFBP-3 protease activity secreted by MCF-7 breast cancer cells. A range of known growth regulators of breast cancer cells were investigated, including all-trans-retinoic acid, vitamin D, steroids, and peptide growth factors such as insulin, EGF, and PDGF. Of those investigated, two well recognized modulators of cancer cell proliferation (estradiol and TGFß) were found to modify the activity of this protease in opposite ways.

Estradiol had a significant and maximal stimulatory effect on protease activity when used in cells at 1–10 nM, concentrations previously shown to stimulate the synthesis of many proteins including IGFBPs (5, 20) and the proliferation of MCF-7 breast cancer cells (21, 22, 23, 24). However, increased protease activity was apparent within 48 h of addition of steroid, and the cell-proliferative effects of estradiol are not apparent until 4–6 days after its addition (21, 22), suggesting that the observed increase in IGFBP-3-protease activity was not a function of increased cell number.

Estradiol also stimulates the activity of cathepsin D, a lysosomal enzyme overexpressed in primary breast tumors and associated with increased risk of metastasis (25). Cathepsin D secreted by MCF-7 cells also shows protease activity against IGFBP-3 (11); however, its activity is optimum at pH 3, whereas the protease under investigation in the present study shows maximal activity at pH 5.5 (18). This suggests that estradiol may modulate the actions of a number of IGFBP proteases secreted by breast cancer cells.

Tamoxifen blocked the stimulatory effect of estradiol on IGFBP-3 protease activity in cell-conditioned medium. In the absence of estrogenic stimulation, antiestrogens may attenuate the actions of a number of growth factors, including IGF-I (26), suggesting that these agents can actively reduce growth inhibitory signals, rather than merely block estrogen-stimulated proliferation. However, we found no change in IGFBP-3 protease activity after treatment of cells with tamoxifen alone, indicating that its inhibition of the stimulatory effect of estradiol was via blocking estradiol action only.

Although a role for TGFß1 has been described in carcinogenesis and stimulation or inhibition of malignant cell proliferation (27, 28), the effect of TGFß1 on regulation of proteolysis has not been addressed extensively. The concentration of TGFß1 required for inhibition of IGFBP-3 protease activity (1–10 ng/ml) is similar to that required for inhibition of MCF-7 cell growth (29, 30). As with estradiol, however, the effects on cell number and protease activity were temporally discordant, with the decrease in protease activity in response to TGFß1 occurring before a decrease in cell number was apparent. Therefore, reduced protease activity cannot be explained by decreased cell number.

The mechanism by which TGFß1 inhibits IGFBP-3 protease activity is not yet known, and although a direct effect on the expression of the protease and/or its regulatory proteins (such as activators or inhibitors) is likely, other possibilities should also be considered. We have previously shown that IGF-I inhibits the MCF-7-derived IGFBP-3 protease activity in a cell-free system, suggesting a nonreceptor-mediated effect (18). This was found not to be the case for TGFß1, however, as it did not inhibit protease activity when added to IGFBP-3 digests containing medium from untreated cells. It is also possible that because TGFß1 stimulates IGFBP-3 production in some cells (31, 32), saturation of protease by endogenous substrate could result in an apparent loss of protease activity. However, the amount of IGFBP-3 secreted by MCF-7 cells is low (5), and the magnitude of the increase in response to TGFß1 is insufficient to affect protease availability.

The IGFBP-3 protease described in the present study has a pH optimum in vitro of 5.5 (18), raising the question of its activity in vivo where such acidic microenvironments are generally found only in endosomes and lysosomes. However, a pH of 5.5 has been demonstrated in some human tumors (33), implying that an environment conducive to the activation of this IGFBP-3 protease is possible in vivo. Although it is still not clear how such a low pH may be achieved, it has been suggested that localized acidic pH at the cell surface may be due to increased release of H+ from cell membrane proteins or H+ adenosine triphosphatase (34, 35), or an increase in the sialic acid content of oligosaccharide chains of cancer cells (36). Activation of cathepsin D to enable it to degrade the extracellular matrix of MCF-7 cells has been suggested to involve such acidic microenvironments (37).

In breast cancer, a significant increase in the level of secreted proteases has been correlated with increased disease recurrence, more frequent metastasis, and increased mortality in breast cancer patients (38, 39). This study has demonstrated that estrogens and TGFß1, factors that are stimulatory and inhibitory, respectively, for MCF-7 cell growth, also stimulate or inhibit the production of an enzyme capable of proteolyzing the growth inhibitory protein IGFBP-3. An important and ongoing study to identify this protease will provide further insight into both its significance in breast cancer cell biology, and the role of IGFBP-3 in the development and progression of malignant breast disease.


   Footnotes
 
1 This work was supported by the National Health and Medical Research Council of Australia (Grant 950199) and the University of Sydney Medical Foundation. Back

Received February 3, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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