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Molecular Regulation of the IGF-Binding Protein-4 Protease System in Human Fibroblasts: Identification of a Novel Inducible Inhibitor

Endocrine Research Unit, Division of Endocrinology, Metabolism, and Nutrition, Mayo Clinic and Mayo Foundation (B.-K.C., L.K.B., Z.T.R., C.A.C.), Rochester, Minnesota 55905; University of Aarhus, Department of Molecular and Structural Biology (M.T.O., C.O.), DK-8000 Aarhus C, Denmark; and Statens Serum Institute (M.C.), 2300 Copenhagen S, Denmark

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

	Abstract

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The IGF-binding protein-4 (IGFBP-4) protease system is an important regulator of local IGF bioavailability and cell growth. Recently, the IGF-dependent IGFBP-4 protease secreted by cultured human fibroblasts was identified as pregnancy-associated plasma protein A (PAPP-A). In pregnancy serum, PAPP-A circulates as a disulfide-bound complex with the precursor form of major basic protein (pro-MBP), and in this complex PAPP-A’s proteolytic activity is not evident. In this study we analyzed the IGFBP-4 protease system in normal human fibroblasts to determine regulation outside of pregnancy. Treatment with the phorbol ester tumor promoter, ß-phorbol 12,13-didecanoate (ß-PDD), resulted in time-dependent inhibition of the IGF-dependent IGFBP-4 protease activity in cell-conditioned medium, which was evident at 6 h and complete by 24 h. PAPP-A mRNA was constitutively expressed in control cells, and levels were decreased only after 24 h of ß-PDD treatment. Secretion of PAPP-A protein into conditioned medium did not change with ß-PDD treatment. On the other hand, pro-MBP mRNA was undetectable in control human fibroblasts, and treatment with ß-PDD induced pro-MBP mRNA and protein expression within 6 h. ß-PDD-induced pro-MBP mRNA expression and protease inhibition were blocked with an inhibitor of RNA synthesis, actinomycin D. Actinomycin D had no effect on PAPP-A mRNA levels in the absence or presence of ß-PDD. Similarly, transformation of human fibroblasts with simian virus 40 large T antigen resulted in the synthesis of pro-MBP mRNA and protein and inhibition of IGFBP-4 protease activity. Coculture of fibroblasts with cells transfected with pro-MBP cDNA resulted in inhibition of IGFBP-4 proteolytic activity without having any effect on PAPP-A synthesis. In summary, phorbol ester tumor promoters and simian virus 40 transformation regulate IGFBP-4 proteolysis in human fibroblasts through induction of a novel inhibitor of PAPP-A, pro-MBP. These findings expand our understanding of the IGFBP-4 protease system and suggest an additional level of local cell growth control.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE IMPORTANCE OF limited proteolysis in directing IGF-binding protein (IGFBP) function in vitro and in vivo is well established (reviewed in Refs. 1 and 2). In particular, the IGFBP-4/IGFBP-4 protease system has been implicated in control of the local growth-promoting effects of IGF-I. Intact IGFBP-4 binds and inhibits IGF-I action in a variety of cell systems (1, 2, 3, 4, 5). Cleavage of IGFBP-4 results in increased IGF-I available for receptor-mediated stimulation of cell growth (6, 7, 8, 9). A specific IGFBP-4 protease has been described in medium conditioned by normal human fibroblasts, osteoblasts, vascular smooth muscle cells, and granulosa cells in culture (5, 6, 7, 8, 10, 11, 12, 13). IGFBP-4 proteolysis in these systems is characterized by dependence on IGFs, in particular IGF-II, for functional activity. The IGF-dependent IGFBP-4 protease is postulated to amplify local IGF-I activity in wound healing, vascular repair, bone remodeling, and development of the dominant follicle and was recently identified in these systems as pregnancy-associated plasma protein A (PAPP-A) (14, 15, 16).

To better understand the physiology and pathophysiology of local IGF-regulated cell growth it will be important to determine the molecular regulation of IGFBP-4 protease/PAPP-A expression in the various systems. To date, all studies of this enzyme have been based on activity assays. We previously reported that treatment of normal human fibroblasts and osteoblasts in culture with phorbol ester tumor promoters resulted in decreased IGF-dependent IGFBP-4 protease activity in the medium (17, 18). Furthermore, conditioned medium from these treated cells could suppress the proteolysis of both endogenous and exogenous IGFBP-4. As this inhibition of IGFBP-4 proteolysis could be blocked by inhibitors of RNA and protein synthesis, we postulated that phorbol ester tumor promoters induced the expression of an IGFBP-4 protease inhibitory protein, but were unable to verify this directly.

In the present study we demonstrate quantitative measure of PAPP-A gene and protein expression and determine that the inducible inhibitor is the pro form of eosinophil major basic protein (pro-MBP).

	Materials and Methods

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Materials
Recombinant human IGFBP-4 was provided by Dr. S. Mohan (Loma Linda, CA), and IGF-II was purchased from Bachem (Torrance, CA). ß-Phorbol 12,13-didecanoate (ß-PDD), -PDD, phorbol 12-myristate 13-acetate (PMA), forskolin, actinomycin D, and BSA (RIA grade) were purchased from Sigma (St. Louis, MO). Reagents for SDS-PAGE were purchased from Bio-Rad Laboratories, Inc. (Richmond, CA).

Cell culture and cell-conditioned medium
Human dermal fibroblasts from normal male adult donors (GM03440 and GM00037) and simian virus 40 (SV40)-transformed human fibroblasts (GM00637) were purchased from the Human Genetic Mutant Cell Repository (Coriell Institute, Camden, NJ). Fibroblasts were cultured in DMEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 4 mM glutamine and containing 10% Cosmic calf serum (HyClone Laboratories, Inc., Logan, UT).

At confluence fibroblasts were washed twice and preincubated in a 1:1 (vol/vol) mixture of Waymouth’s medium to DMEM plus 0.1% BSA serum-free medium (SFM) for 24 h. The cells were then washed, and the medium was changed to SFM with or without experimental additions for the indicated times. At the end of the incubation period, the cell-conditioned medium was collected, centrifuged at 2,000 x g at 4 C for 30 min, and stored frozen at -70 C.

IGFBP-4 protease assay
Fifty microliters of fibroblast-conditioned medium were incubated in a microfuge tube with [¹²⁵I]IGFBP-4 without or with 5 nM IGF-II at 37 C for 6 h as previously described (8, 10, 14, 15, 16). Reaction products were separated by SDS-PAGE on a 7.5–15% linear gradient. Gels were dried and exposed to film or analyzed using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

RNA isolation and cDNA synthesis
Total RNA was extracted from human fibroblasts using RNeasy Mini Kit (QIAGEN, Valencia, CA) and treated with deoxyribonuclease (DNA-free, Ambion, Inc., Austin, TX). Four hundred nanograms of RNA were reverse transcribed using TaqMan RT reagents (PE Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. The absence of genomic DNA contamination was confirmed by PCR (see below) using isolated RNA without RT as template.

Real-time PCR
Real-time quantitative PCR analyses were performed using the ABI PRISM 7700 Sequence Detection System and software (PE Applied Biosystems). All reagents were from PE Applied Biosystems. Primer and probe sequences for specific amplification and detection of the targets (PAPP-A and pro-MBP) as well as the reference gene (nuclear DNA-encoded gene 28S rRNA) were selected using Primer Express software from the published sequences of each gene (Table 1). Using these primers, PCR products of the expected size were obtained (data not shown). The target probes were labeled at the 5'-end with a reporter dye (6'-carboxyfluorescein) and at the 3'-end with a quencher dye (6'-carboxytetramethyl-rhodamine) and were phosphate-blocked at the 3'-end to prevent extension. The 28S probe was constructed identically, except that the 5'-end of the probe was labeled with a different fluorescent dye. The theoretical basis of the method has been detailed previously (19). All samples were run in triplicate and quantitated by normalizing the target signal with the 28S signal. C_T is the number of amplification cycles required by each gene to reach a fixed threshold of signal intensity. The higher the abundance, the lower the C_T. The practical working range is within C_T values of 15–35. The nontemplate control C_T is 40. Relative abundance is calculated as 2^CT, where C_T = control C_T - treatment C_T.

View larger version (17K): [in this window] [in a new window]   Figure 1. Colinearity of dilution and assay range for PAPP-A, pro-MBP, and 28S RNA. Human fibroblast RNA was reverse transcribed, serially diluted, and PCR-amplified using the PAPP-A (), pro-MBP (), and 28S () primers and probes listed in Table 1. Dilution values of the samples are plotted vs. amplification cycle threshold (CT). All points represent the mean ± SEM of triplicate PCR amplifications.

Plasmid generation
Human pro-MBP cDNA (GenBank accession no. Y0089), cloned into the NheI/XhoI sites of pcDNA3.1⁺ (Invitrogen, Carlsbad, CA), was excised with NheI and XhoI and subcloned into pDC315 (Microbix Systems, Inc., Toronto, Canada) to generate pDC315/pro-MBP. This construct was verified by restriction site mapping. Plasmids were propagated in the DH5 strain of Escherichia coli (Invitrogen, San Diego, CA) and purified by CsCl₂ banding.

Transfection studies
An ovarian cancer cell line (OV177) established in our laboratory (22) has no detectable expression of PAPP-A or pro-MBP (our unpublished data). OV177 cells were recovered by trypsinization, washed with PBS, and resuspended in Cytomix (120 mM KCl, 0.15 mM CaCl₂, 10 mM mono- and dibasic KH₂PO₄, 25 mM HEPES, 2.0 mM EGTA, and 5.0 mM MgCl₂, pH 7.6) at a concentration of 5.0 x 10⁶ cells/ml. Optimization of electroporation volume, cell number, and plasmid DNA concentration were determined for OV177 cells using the pEGFP-N₁ plasmid (provided by Dr. Larry Karnitz, Mayo Clinic, Rochester, MN) and flow cytometric analysis of the green fluorescent protein. Cell suspension (0.4 ml) was added to a 0.4-cm disposable cuvette (Invitrogen), and plasmid DNA was added at a concentration of 10 µg/1.0 x 10⁶ cells. Electroporation was conducted at 325 V at a pulse frequency of 2 x 5.0 msec. Vector control (pDC315) or pDC315/pro-MBP-transfected OV177 cells were seeded at 1.5 x 10⁶ cells on approximately 60% confluent human fibroblasts cultured in six-well plates. The OV177 cells were allowed to attach for 16 h, at which time the cultures were washed twice, and the medium was changed to SFM. Conditioned medium was collected after a 24-h incubation period.

Immunoblot
Conditioned media (50 µl) from cocultures of human fibroblasts and transfected OV177 cells were processed by SDS-PAGE under reducing conditions (100 mM dithiothreitol) using a 7.5–15% linear gradient, and then transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.). Membranes were blocked with 5% milk for 3 h, incubated overnight at 4 C with pro-MBP monoclonal Ab (21) or nonspecific IgG at a 1:1000 dilution, washed, and then incubated for 2 h at room temperature with horseradish peroxidase-conjugated secondary antibody (1:2000 dilution). Antigen-antibody reactions were visualized using an enhanced chemiluminescent detection system (Amersham Pharmacia Biotech, Arlington Heights, IL).

Statistical analysis
Statistical comparisons were performed using ANOVA, followed by multiple comparisons. Results are considered statistically significant at P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phorbol ester tumor promoter-induced inhibition of IGFBP-4 protease activity
Treatment of normal human fibroblasts with the phorbol ester tumor promoter ß-PDD resulted in a time-dependent inhibition of the IGF-dependent IGFBP-4 protease activity in cell-conditioned medium. The [¹²⁵I]IGFBP-4 protease assay performed with these conditioned medium samples is shown in Fig. 2. Conditioned medium from untreated cells exhibited no IGFBP-4-degrading activity in the absence of IGF-II, but addition of IGF-II in the cell-free assay resulted in loss of intact [¹²⁵I]IGFBP-4 at 32 kDa and the generation of radiolabeled fragments at 18 and 14 kDa, as described previously (8, 10, 14, 15, 16). This proteolytic activity was apparent in control SFM within 2 h of incubation. Treatment of cells with ß-PDD (100 nM) resulted in inhibition of IGF-dependent proteolysis, which was evident at 6 h and maximal by 24 h of treatment. Direct addition of ß-PDD to the cell-free assay had no inhibitory effect (17), and ß-PDD treatment was not associated with decreased cell number (control vs. ß-PDD, 33,120 ± 681 vs. 33,813 ± 570; n = 6). To determine whether this loss of proteolytic activity was due to a decrease in the expression of the protease, PAPP-A, we assessed PAPP-A mRNA levels by real-time RT-PCR and PAPP-A protein levels in the cell-conditioned medium by a specific and sensitive ELISA. PAPP-A mRNA was constitutively expressed in control cells with detection at C_T = 25.3 compared with C_T = 18.5 for 28S (1:10 diluted). ß-PDD treatment had little effect on levels of PAPP-A mRNA, with a significant decrease only at the 24 h point (Fig. 3A). PAPP-A protein accumulated over time in the control conditioned medium, and levels were not affected by ß-PDD treatment (Table 2). Thus, a simple decrease in PAPP-A expression could not account for the rapid decrease in protease activity seen with ß-PDD treatment within 6 h. This was consistent with our original postulate that phorbol ester tumor promoters induced human fibroblasts to express and secrete an inhibitor of the IGF-dependent IGFBP-4 protease (17).

View larger version (31K): [in this window] [in a new window]   Figure 2. Regulation of IGFBP-4 protease activity: time-dependent effect of phorbol ester tumor promoters. Human fibroblasts were treated with 0.2% ethanol vehicle (C, control) or 100 nM ß-PDD for the indicated times. Conditioned media were collected and incubated with [125I]IGFBP-4 and without (-) or with (+) 5 nM IGF-II in a cell-free assay as described in Materials and Methods. Proteins were resolved by SDS-PAGE, and the gels were dried and exposed to film. Arrows indicate intact IGFBP-4 and radiolabeled fragments at 18 and 14 kDa.

View larger version (20K): [in this window] [in a new window]   Figure 3. PAPP-A and pro-MBP mRNA expression: effect of phorbol ester tumor promoters. Human fibroblasts were treated with 0.2% ethanol vehicle () or 100 nM ß-PDD () for the indicated times. Human fibroblast RNA was reverse transcribed and PCR-amplified using PAPP-A, pro-MBP, and the 28S primers and probes listed in Table 1. Expression of PAPP-A (A) and pro-MBP (B) was normalized for 28S expression. Results (mean ± SEM of triplicate determinations) are expressed as abundance relative to the 2 h control. Two-hour control CT values for PAPP-A, pro-MBP, and 28S (1:10) were 25.3 ± 0.07, 33.3 ± 0.37, and 18.5 ± 0.64, respectively. *, P < 0.05 vs. control.

View larger version (12K): [in this window] [in a new window]   Figure 4. PAPP-A and pro-MBP mRNA expression: dose-dependent effect of phorbol ester tumor promoters. Human fibroblasts were treated with vehicle (control) or ß-PDD (0.1–100 nM) for 10 h. RNA was reverse transcribed and PCR-amplified using PAPP-A, pro-MBP, and the 28S primers and probes listed in Table 1. Expression of PAPP-A () and pro-MBP () was normalized for 28S expression. Results (mean ± SEM of triplicate determinations) are expressed as abundance relative to the control. Control CT values for PAPP-A, pro-MBP, and 28S (1:10) were 30 ± 0.03, 37.4 ± 0.45, and 18.8 ± 0.15, respectively.

View larger version (43K): [in this window] [in a new window]   Figure 5. Regulation of IGFBP-4 protease activity by phorbol esters and forskolin. Human fibroblasts were treated without (control) and with 0.2% ethanol vehicle (EtOH), ß-PDD (10 nM), -PDD (10 nM), PMA (10 nM), or forskolin (10 µM) for 24 h. The IGF-dependent [125I]IGFBP-4 protease assay was performed on the conditioned media as described in Materials and Methods and Fig. 2. Arrows indicate intact IGFBP-4 and radiolabeled fragments at 18 and 14 kDa.

View larger version (20K): [in this window] [in a new window]   Figure 6. PAPP-A and pro-MBP mRNA expression: specificity of response. Human fibroblast RNA from the experiment in Fig. 4 was reverse transcribed and PCR-amplified using the specific primers and probes listed in Table 1. Expression of pro-MBP (A) and PAPP-A (B) was normalized for 28S expression. Results (mean ± SEM of triplicate determinations) are expressed as abundance relative to control (C). Control CT values for pro-MBP, PAPP-A and 28S (1:10) were 34.4 ± 0.63, 25.3 ± 0.19, and 19.5 ± 0.19, respectively. *, P < 0.05 vs. control.

View larger version (17K): [in this window] [in a new window]   Figure 7. ß-PDD regulation of PAPP-A and pro-MBP mRNA expression: effect of actinomycin D. Human fibroblasts were treated without (C, control) or with ß-PDD (100 nM). Thirty minutes before stimulation, actinomycin D (ActD; 1 µg/ml) was added. After 10 h, RNA was isolated, reverse transcribed, and PCR-amplified using the specific primers and probes listed in Table 1. Expression of pro-MBP (A) and PAPP-A (B) was normalized for 28S expression. Results (mean ± SEM of triplicate determinations) are expressed as abundance relative to control. Control CT values for pro-MBP, PAPP-A, and 28S (1:10) were 30.4 ± 0.16, 25.2 ± 0.15, and 21.1 ± 0.36, respectively. *, P < 0.05 vs. control.

View larger version (59K): [in this window] [in a new window]   Figure 8. Regulation of IGFBP-4 protease activity: effect of SV40 transformation. Conditioned media from parental (C) or SV40-transformed human fibroblast cultures were assayed for [125I]IGFBP-4 protease activity without (-) or with (+) 5 nM IGF-II as described in Materials and Methods and Fig. 2. Arrows indicate intact IGFBP-4 and radiolabeled fragments at 18 and 14 kDa.

View larger version (36K): [in this window] [in a new window]   Figure 9. Transient transfection pro-MBP cDNA. A, Conditioned media from cocultures of human fibroblasts with OV177 cells transfected with empty vector or pro-MBP cDNA construct were separated by SDS-PAGE, transferred to membrane, and immunoblotted with pro-MBP-specific monoclonal antibody as described in Materials and Methods. Migration positions of molecular size markers (in kilodaltons) are indicated on the left. The arrow indicates approximately 33-kDa pro-MBP. B, IGF-dependent [125I]IGFBP-4 protease assay was performed in conditioned media from cocultured human fibroblasts and OV177 cells transfected with empty vector or pro-MBP cDNA construct as described in Materials and Methods and Fig. 2. Arrows indicate intact IGFBP-4 and radiolabeled fragments at 18 and 14 kDa.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Pro-MBP might seem an unlikely inhibitor for an IGFBP-4 protease previously known as a pregnancy-associated plasma protein (14, 27). MBP is a cytotoxic and proinflammatory protein released from activated eosinophils (25). The pro piece is the highly acidic portion that neutralizes the basicity and presumably the toxicity of MBP in the granule of the eosinophil before processing (28). However, during pregnancy, pro-MBP is synthesized in placental X cells and is released into the circulation intact, where it forms a disulfide-bound complex with PAPP-A (29). We have recently shown that in this complex, PAPP-A’s proteolytic activity is masked (24). In this study we provide evidence for pro-MBP as a direct inhibitor of PAPP-A’s IGFBP-4 protease activity in vitro and outside of pregnancy: first, through the close temporal relationship between the induction of pro-MBP expression and the inhibition of proteolysis in cultured human fibroblasts, and second, through recombinant expression of pro-MBP resulting in inhibition of proteolysis in PAPP-A-producing cells. Although these data do not rule out the possibility that pro-MBP’s inhibitory effect is indirect through changes in other inhibitors or proteases, the results in pregnancy serum make this unlikely. It remains to be determined whether covalent bonding between pro-MBP and PAPP-A is required for pro-MBP’s inhibitory effect in this system. In pregnancy serum, PAPP-A and pro-MBP exist in a covalent 2:2 complex (29). A disulfide bridge that connects the pro-MBP and PAPP-A subunits is located close to the Zn²⁺ binding helix of PAPP-A, and, thus, pro-MBP could shield the active site (30). A subpopulation of PAPP-A may exist in a 2:1 complex with pro-MBP in pregnancy serum (24). PAPP-A normally exists as a dimer in human fibroblast-conditioned medium (14). It is of note that equimolar pro-MBP was not required for effective inhibition of PAPP-A’s proteolytic activity in this system. Indeed, taking into account the approximately 10-fold difference in molecular size of the two proteins (i.e. PAPP-A 200 kDa and pro-MBP 30 kDa), the molar ratio PAPP-A/pro-MBP was approximately 2:1 in 24-h conditioned medium from ß-PDD-treated fibroblasts. The exact biochemical nature of the interaction among the PAPP-A and pro-MBP molecules is currently under investigation.

The physiological relevance of increased pro-MBP expression and inhibition of IGFBP-4 proteolysis by early transformation effectors, i.e. phorbol ester tumor promoters and SV40 large T antigen, is unclear and needs further investigation. It is possible that constraints on IGFBP-4 proteolysis provide potential for preferential tumor growth. Indeed, transitory increases in the synthesis of tissue inhibitors of matrix metalloproteases have been shown to be characteristic of early stages of transformation (31).

Phorbol ester tumor promoters could also regulate pro-MBP expression through alterations in the PKC intracellular signaling pathway (32), which was not explored in this study. Along those lines, the results with forskolin were of interest. Forskolin increases intracellular cAMP, which activates the PKA pathway (33). The data suggest that signaling mediated by PKA could stimulate PAPP-A expression. This is the first demonstration of increased PAPP-A expression in any in vitro system and will need to be followed up with more extensive investigations.

These data represent the initial study of the regulation of the IGFBP-4 protease system at the molecular level. The results of this study in combination with those of previous studies indicate a complex regulation of gene expression for substrate (IGFBP-4), protease (PAPP-A), inhibitor (pro-MBP), and activator (IGF-II) as well as novel mechanisms of enzyme activation and inhibition. The PAPP-A/pro-MBP system is not pregnancy specific or limited to the circulation and has significant potential for local cell growth control.

	Footnotes

 
This work was supported in part by the Mayo Foundation (to C.A.C.), the Danish Medical Research Council (to M.T.O. and C.O.), and a fellowship grant from Diagnostics Systems Laboratories, Inc. (to B.-K.C.).

Abbreviations: C_T, Number of amplification cycles required by each gene to reach a fixed threshold of signal intensity; IGFBP-4, IGF-binding protein-4; PAPP-A, pregnancy-associated plasma protein A; ß-PDD, ß-phorbol 12,13-didecanoate; PMA, phorbol 12-myristate 13-acetate; pro-MBP, precursor form of major basic protein; SFM, serum-free medium; SV40, simian virus 40.

Received October 1, 2001.

Accepted for publication December 14, 2001.

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