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Endocrinology Vol. 142, No. 10 4420-4427
Copyright © 2001 by The Endocrine Society

ARTICLES

IGF-Binding Protein-4 Expression and IGF-Binding Protein-4 Protease Activity Are Regulated Coordinately in Smooth Muscle During Postnatal Development and After Vascular Injury

E. P. Smith, A. Kamyar, W. Niu, J. Wang, B. Cercek, S. D. Chernausek and J. A. Fagin

Divisions of Endocrinology and Metabolism, University of Cincinnati College of Medicine (E.P.S., A.K., W.N., J.W., J.A.F.); and Division of Cardiology, Cedars-Sinai Medical Center (B.C.), Los Angeles, California 90048; and Children’s Hospital Medical Center (S.D.C.), Cincinnati, Ohio 45267

Address all correspondence and requests for reprints to: James A. Fagin, M.D., Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, Vontz Center for Molecular Studies, 3125 Eden Avenue, Cincinnati, Ohio 45267-0547. E-mail: james.fagin{at}uc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent studies support a critical role for the paracrine IGF/IGF-binding protein system in the regulation of vascular smooth muscle cell growth. In this study we have explored the hypothesis that the abundance of individual IGF-binding proteins in smooth muscle is subject to regulation during postnatal life and in response to injury. IGF-binding protein-2 was the predominant binding protein secreted by neonatal rat vascular smooth muscle cells, whereas IGF-binding protein-4 was most prevalent in adult vascular smooth muscle cells coincident with increased IGF-binding protein-4 protease activity. After arterial injury, IGF-binding protein-4 mRNA increased, associated with greater IGF-binding protein-4 proteolytic activity, resulting in stable steady state levels of the IGF-binding protein-4 protein. Expression of pregnancy-associated plasma protein A mRNA, recently identified as an IGF-binding protein-4 protease, was expressed at higher levels in adult than neonatal vascular smooth muscle cell lines, but did not change significantly after arterial injury. The peak of immunoreactive pregnancy-associated plasma protein A from hydrophobic interaction chromatography fractions of smooth muscle cell-conditioned medium coincided, but did not fully overlap, with the fractions containing maximal IGF-binding protein-4 protease activity. In conclusion, our data point to a developmental switch from IGF-binding protein-2 to IGF-binding protein-4 in vascular smooth muscle cells postnatally. Moreover, IGF-binding protein-4 expression is coregulated with IGF-binding protein-4 protease activity, suggesting that biosynthesis and degradation of this binding protein are coordinated events important for regulating biological activity of IGF-I.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ARTERIAL SMOOTH MUSCLE cells (SMC) are normally in a near-quiescent state within the media, with a rate of replication of about 0.02% (1). In their basal state they contract in response to vasoconstrictors and are relatively unresponsive to growth factors. However, SMC of the vessel wall will proliferate and remodel the extracellular matrix when subjected to a variety of pathophysiological challenges (2). For example, after vascular injury, arterial SMCs migrate from the tunica media into the intimal layer of the vessel wall, where they proliferate and synthesize extracellular matrix proteins (3). This neointimal proliferative response has been implicated in the pathogenesis of atherosclerosis and of restenosis after percutaneous balloon angioplasty (2, 4, 5).

The signals controlling neointimal proliferation are complex and involve in part the production of growth factors that act locally to orchestrate the response (1, 5). There is evidence that the IGF family of growth factors is prominently involved (6). IGF-I induces SMC differentiation (7) and migration (8) while inhibiting apoptosis (9). Furthermore, the growth of aortic SMC is inhibited by antisense IGF-I mRNA (10). We and others have shown that IGF-I gene expression is induced in the arterial wall after balloon injury (11) and that the type I IGF-I receptor is reciprocally down-regulated (12) coincident with the peak time of vascular SMC DNA synthesis (13). When this localized expression of IGF-I is inhibited, such as after hypophysectomy, in animals treated with the somatostatin analog octreotide or with administration of an IGF antagonist, neointimal formation is markedly decreased (14, 15). Perhaps the most direct evidence is the finding that transgenic animals engineered to overexpress IGF-I in vascular smooth muscle develop SMC hyperplasia (16).

Besides the abundance of IGF-I or its receptor, IGF action is determined by a family of structurally related secreted proteins (IGFBPs) that specifically bind IGFs and modulate IGF bioactivity in different tissue (17). Tissue IGFBP levels are, in turn, subject to regulation by processes that include altered synthesis and degradation (18, 19, 20). Previous studies of vascular SMC have demonstrated that adult SMC variably produce IGFBP-2, -3, -4, -5, and -6 depending on the species and origin of the cells (17). In rodent SMC, IGFBP-2 and IGFBP-4 predominate (20, 21, 22). There is no clear understanding of the individual functions that each of the IGFBPs may serve in vivo, particularly as most cells, SMC being typical, secrete several IGFBP that share at least some functional properties. IGFBP proteolysis may, in turn, contribute to IGF-I biological activity by releasing free IGFs to interact with membrane receptors, and there is evidence that IGFBP proteolysis itself may be subject to hormonal regulation (23, 24). In vascular smooth muscle, we and others have described an IGF-I-dependent cation-dependent IGFBP-4 protease activity (18, 20, 25). Recently, pregnancy-associated plasma protein A (PAPP-A) has been documented to be a specific IGF-I-dependent protease for IGFBP-4 (26, 27).

Preliminary data arising from IGFBP knockout mice have been interpreted as evidence that there may be considerable redundancy in the function of the IGFBPs (28). Alternatively, if each of the IGFBPs has unique functions, one would expect them to be differentially regulated in particular pathophysiological situations. Here, we determined whether expression of IGFBP-2 and IGFBP-4 and the activity of IGFBP-4 protease show a pattern of regulation that would be consistent with a distinct role for each of these binding proteins in the biology of vascular smooth muscle. To this end, we examined IGFBP expression and degradation in cultured SMCs derived from neonatal and adult rats (20) and in rat femoral arteries at various times after balloon artery injury (15).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and culture conditions
Pup, Pup I, and Pup II cultures are clonal cell isolates originally obtained from arteries of 9-d-old rats (29). Primary cultures of adult SMCs, C45, C49, and C53, were isolated from 4-month-old rats (20, 21). All cell lines were provided by Dr. Behrooz Sharifi (Cedars-Sinai Medical Center, Los Angeles, CA). Subcultures were grown in DMEM supplemented with 10% FCS, 200 mM L-glutamine, and antibiotics, with medium changes three times weekly. After being rinsed twice, cells were incubated with serum-free DMEM supplemented with L-glutamine, 5 µg/ml transferrin, 0.2 mM ascorbate, and antibiotics for 3 d. After this period of serum deprivation, the medium was replaced with or without addition of the indicated concentrations of IGF-I dissolved in a vehicle of 10 mM acetic acid and 0.2% BSA. For collection of conditioned medium to assay for protease activity, cells were grown to confluence, washed twice with PBS, and incubated for 48–72 h with DMEM with Lglutamine, 5 µg/ml transferrin, antibiotics, and 1 µg/ml insulin without BSA. The medium was stored at -20 C. Rodent B104 neuroblastoma cells were grown as previously described (30, 31).

Western ligand blots
For the studies measuring IGFBP by Western ligand blotting, cells were plated at 2 x 104 cells/cm2 onto six-well clusters and grown in DMEM with 10% FCS. After 3 d in serum-free medium, cells were incubated with the indicated reagents. At the appropriate times, medium was collected, immediately frozen, and stored until assayed. Two milliliters of conditioned media were concentrated 10x by ultrafiltration through a Centricon-10 microconcentrator (Millipore Corp., Bedford, MA), processed as previously described (20), and an aliquot was electrophoresed through an 11% discontinuous SDS-polyacrylamide gel under nonreducing conditions according to the method of Hossenlopp et al. (32). After transfer, the membrane was incubated with [125I]IGF-I as previously described (20), and the densities of the bands were determined by scanning densitometry.

Immunoprecipitation/Western ligand blotting
To confirm the identity of the IGFBP, 300 µl of an 8x concentrate of SMC-conditioned medium were incubated for 16 h at 4 C with 20 µl of one of the following polyclonal antibodies: anti-IGFBP-4 (Upstate Biotechnology, Inc., Lake Placid, NY), anti-IGFBP-2 (Upstate Biotechnology, Inc.), or nonimmune rabbit serum in the presence of 30 µl protein A (Repligen, Cambridge, MA). The anti-IGFBP-4 antiserum displays 0.1–1% cross-reactivity with IGFBP-1, -3, and -5 and 50% cross-reactivity with IGFBP-2. The anti-IGFBP-2 exhibits 0.1–0.5% cross-reactivity with IGFBP-1, -2, -3, and -5. After centrifugation, the pellets were washed, dried, resuspended, and Western ligand blotted as described above. Recombinant IGFBP-2 and IGFBP-4 were obtained from Austral Biologicals (San Ramon, CA).

Protease assays
In vitro. For comparison of protease activity from different smooth muscle cell lines, SMC-conditioned medium was collected as described above and stored at -20 C until assayed. To assess proteolytic activity, 200 µl medium were concentrated by centrifugation using a Centricon 10 concentrator, followed by reconstitution to a final volume of 100 µl in an incubation mixture containing 100 ng recombinant IGFBP-4, 100 ng IGF-I, 0.33 M Tris base (pH 7.5), 13.3 mM CaCl2, 0.67 mM ZnSO4, and 0.13% BSA. The reaction was incubated overnight, subjected to SDS-PAGE (12% resolving and 5% stacking), and Western blotted using rabbit polyclonal antisera specific for IGFBP-4 (33).

In vivo. Femoral arteries were snap-frozen in liquid N2, pulverized with mortar and pestle, and extracted on ice in a glass homogenizer in PBS with 0.1% Nonidet P-40 in the absence of protease inhibitors. Thirty micrograms of protein were then incubated with 80,000 cpm purified human [125I]IGFBP-4 for 12 h, and products were fractionated by PAGE, followed by autoradiography. The extent of degradation was quantified by analysis of remaining substrate.

Fractionation of adult SMC medium for IGFBP-4 protease and PAPP-A immunoblotting
Five hundred milliliters of conditioned medium from the C53 adult SMC line were made to 1 M ammonium sulfate, pH 7.5, and applied to a hydrophobic interaction chromatography column (Butyl Sepharose 4 Fast Flow, Pharmacia Biotech, Peapack, NJ). Five-milliliter fractions were eluted with a linear gradient from 1.0 M ammonium sulfate, 20 mM Tris to 20 mM Tris, pH 7.5, and 50-µl aliquots were assayed for protease activity by a charcoal assay as previously described (34). Briefly, individual HIC fractions from the SMC-conditioned medium were incubated overnight in standard protease assay buffer as described above with the addition of 100 ng rIGFBP-4 and 20,000 cpm [125I]IGF-I tracer. The mixture was then exposed to activated charcoal to remove free labeled IGF-I and was separated by centrifugation, and the supernatant-counted bound counts were inversely proportional to IGFBP-4 protease activity. The presence of PAPP-A protein in each fraction was assessed by Western blotting. Two hundred microliters of sample were subjected to 6% SDS-PAGE, transferred to nitrocellulose filter, and incubated with mouse anti-PAPP-A monoclonal antibody, Clone 6 (Research Diagnostics, Inc., Flanders, NJ), at a 1 µg/ml dilution according to standard procedures.

Northern blotting and semiquantitative RT-PCR
RNA was extracted from SMCs in culture by the method of Chirgwin et al. (35). Femoral artery RNA was extracted by the guanidine-LiCl method (16). For Northern blot analysis, gel electrophoresis of RNA was performed on 1% agarose gels containing 2.2 M formaldehyde (31). Probes were labeled with [{alpha}-32P]deoxy-CTP using the random primer technique following the manufacturer’s protocol (Stratagene, La Jolla, CA). The following rat probes were used: IGFBP-2, pRBP-2-501, EcoRI-HindIII fragment; IGFBP-4, pRBP-4-501, Smal-HindIII fragment; and cyclophylin, pCD15.8.1., BamHI fragment. A murine probe for PAPP-A mRNA was generated from mouse placenta cDNA, amplified with primers complementary to the PAPP-A cDNA mouse sequence (36); these were as follows: forward, 5'-ggacaaggaagccctaatg-3' (nucleotides 4813–4832); and reverse, 5'-cacggaagatgtgatagagg-3' (nucleotides 4921–4941). The PCR product was subcloned into Bluescript SK- and sequenced to confirm its identity. The PAPP-A insert derived from the plasmid was subsequently used for 32P labeling. The mouse cDNA was found to be greater than 98% homologous with the rat PAPPA based on sequencing of the appropriate fragment of the rat cDNA with the indicated primers.

Semiquantitative RT-PCR of PAPP-A mRNA was performed using the murine primers. ß-Actin mRNA was coamplified as an internal control using the following primers: forward, 5'-gagaccttcaacaccccagcc-3'; and reverse, 5'-ggccatctcttgctcgaagtc-3' (37). As the intron/exon boundaries for the mouse PAPP-A gene are not known, the specific PAPP-A primer pair was originally selected from a region flanking the putative zinc-binding domain, which is likely to encompass an intron/exon boundary based on the gene organization of other members of this gene family (38). These primers amplified a larger fragment from mouse genomic DNA.

The RT reaction was performed using Superscript TM II reverse transcriptase according to the manufacturer’s instructions with 1 µg total RNA. The PCR reaction was performed on 1 µl RT reaction product with 0.5 µM primer, 0.2 mM deoxy-NTP mix, 1 x reaction buffer, and 0.04 U/µl REDTaq polymerase (Sigma, St. Louis, MO) in a final volume of 50 µl. Initial screening was performed for 40 cycles as follows: hot start for 5 min at 95 C, denaturation at 94 C for 1 min, annealing at 55 C for 1 min, and extension at 72 C for 1 min. Subsequently, multiplex PCR was performed with the addition of ß-actin primers at a final concentration of 0.25 µM. Based on analysis of reaction products at 5-cycle increments to determine the linear phase of amplification, a total of 25 cycles was chosen for quantitation of ß-actin and 40 cycles for PAPP-A. The ethidium bromide-stained gel image was captured on a Kodak imager (Rochester, NY), and the relative PAPP-A DNA band intensity was determined and expressed as a ratio after normalization to ß-actin.

Injury of rat femoral artery
Adult male Sprague Dawley rats were anesthetized with pentobarbital, and a 2F balloon catheter was introduced from the left carotid artery into the ileofemoral artery. Injury was performed by pulling the catheter with an inflated balloon from the femoral artery to the aorta three times, as previously described (15). All animal studies were performed with the approval from the respective institutional animal care and use committee of Cedar Sinai Medical Center and University of Cincinnati.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Coordinate expression of IGFBP-4 and IGFBP-4 protease in SMC during postnatal development
Conditioned medium from neonatal (pup) rat aortic SMC is rich in IGFBP-2, but has no detectable IGFBP-4, whereas conditioned medium from adult rat SMC contains almost exclusively IGFBP-4 (Fig. 1Go). IGFBP-2 abundance in pup cells was not affected by IGF-I and was lower in medium from confluent compared with sparsely plated cells. The identity of the respective IGFBPs was further established by immunoprecipitation with antibodies to the respective binding proteins (Fig. 2Go). Northern analysis of RNA from confluent or sparsely grown neonatal and adult SMCs confirmed that neonatal SMC express only IGFBP-2 mRNA, and that its abundance is markedly decreased by cell confluence (Fig. 3Go). By contrast and as previously reported (20), adult rat SMC express primarily IGFBP-4 mRNA (Fig. 3Go).



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  		Figure 1. Western ligand blot of conditioned medium of two neonatal smooth muscle cell lines (Pup I and Pup II) and one adult isolate. Sparse and confluent cells were treated either without (C) or with 50 ng/ml IGF-I for 24 h. Lane 1, Recombinant hIGFBP-2; lane 2 (marked BP-4), recombinant hIGFBP-4.

 


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  		Figure 2. Immunological confirmation of the identity of the pup and adult SMC IGFBP. Conditioned medium was immunoprecipitated with antibodies against IGFBP-2 or IGFBP-4, and the products were analyzed by Western ligand blotting. Lane 1, Positive controls containing recombinant IGFBP-2 (31 kDa) and IGFBP-4 (24 kDa). Immunoprecipitation of recombinant IGFBPs with antibodies to IGFBP-2 or IGFBP-4 is shown in lanes 2 and 3.

 


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  		Figure 3. Northern blots of confluent or sparsely grown pup and adult SMCs treated with or without 50 ng/ml IGF-I. The representative filter was sequentially hybridized with IGFBP-2 (top) and IGFBP-4 cDNA. These data were confirmed in a second set of independent experiments.

 
The absence of detectable IGFBP-4 in medium conditioned by neonatal SMC cannot be attributed to proteolysis, as the activity of the IGFBP-4 protease appears to be coordinately regulated with the abundance of its substrate. Although there is detectable IGFBP-4 protease activity in conditioned medium from neonatal SMC, adult SMC-conditioned medium (Fig. 4Go) demonstrates consistently greater activity.



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  		Figure 4. Western blot analysis of IGFBP-4 protease activity in SMC-conditioned medium. Concentrated conditioned media from three pup cell lines (passage 7) and three adult cell lines (passages 2, 2, and 10, respectively) were incubated at 37 C overnight with 100 ng rat IGFBP-4 in the presence of IGF-I (100 ng/µl) as described in Materials and Methods. The products were resolved by 12% SDS-PAGE, followed by Western blot analysis using specific IGFBP-4 antisera. The conditioned medium of adult cells demonstrated greater IGFBP-4 proteolytic activity. This was verified in three separate independent experiments.

 
To address the possibility that the difference in protease activity in conditioned medium from neonatal vs. adult SMC could be accounted for by inhibitory effects of the greater abundance of IGFBP-2 in the neonatal cell-conditioned medium, the effect of addition of IGFBP-2 to adult SMC cell- and B104 cell-conditioned media on IGFBP-4 protease activity was assessed. Aliquots of C53-conditioned medium were concentrated and subjected to standard protease assay as described in Materials and Methods with and without added 1 µg IGFBP-2 (Fig. 5Go). The C53 medium was compared with conditioned medium from dexamethasone-treated B104 cells, a potent source of IGFBP-4 protease activity. There was no evidence that IGFBP-2 inhibited IGFBP-4 protease activity.



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  		Figure 5. Western blot analysis of the effect of added IGFBP-2 on IGFBP-4 protease activity from vascular SMC- and B104 neuroblastoma cell-conditioned media. IGFBP-4 (100 ng) was added to samples of SMC (C53)- and B104-conditioned media, followed by incubation overnight at 37 C in the presence or absence of 1 µg IGFBP-2. The samples were subjected to 12% SDS-PAGE, followed by Western blot analysis using IGFBP-4 antisera as described in Materials and Methods. The addition of IGBP-2 did not appreciably affect the abundance of intact IGFBP-4.

 
IGFBP-4 expression and proteolysis after balloon arterial injury
To determine whether the expression of IGFBP-4 and the activity of its protease are also regulated coordinately in vivo, we studied a model of femoral artery injury in the rat. We have previously established that IGF-I gene expression is markedly increased from 1–7 d after injury of rat aorta or femoral arteries (11, 14, 15). Balloon injury of rat femoral arteries was associated with a biphasic response: IGFBP-4 mRNA levels peaked between 6 and 24 h, followed by a sustained 2-fold increase through 14 d (Fig. 6Go). In contrast to the time-dependent fluctuations in IGFBP mRNA abundance, intact IGFBP-4 protein levels were unchanged over the same time course (Fig. 7Go). To determine whether this could be accounted for by concomitant changes in IGFBP-4 protease activity, femoral artery extracts were prepared and assayed for protease activity (Fig. 8Go). A significant increase in protease activity was observed within 12–24 h of injury, persisting for days, followed by a return to baseline by approximately 6 d. The sizes of the proteolytic fragments was comparable to those of fragments generated by the IGFBP-4 protease in conditioned medium from aortic SMC (data not shown).



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  		Figure 6. Regulation of arterial IGFBP-4 mRNA after balloon injury of the femoral artery. Injured vessels (four pooled per time point) were harvested at the indicated times after the procedure. Blots were hybridized with rat IGFBP-4 cDNA (top) and with an 18S RNA probe of low specific activity (bottom). A second experiment with a more limited time course (d 0, 7, and 14) yielded comparable results.

 


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  		Figure 7. Western ligand blot of tissue extracts from rat femoral arteries removed before or at the indicated times after balloon injury. Each lane represents a sample from an individual blood vessel. The only detectable band comigrates with authentic IGFBP-4 (BP-4). NRS, Normal rat serum. Arrows indicate size markers (40, 30, and 25 kDa).

 


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  		Figure 8. Determination of IGFBP-4 protease activity in femoral artery extracts derived from vessels removed at the indicated time points after arterial injury. Extracts were incubated with [125I]IGFBP-4 for 12 h, and the extent of degradation was quantified by analysis of the cleavage products. Each point represents the mean ± SD of one to three individual tissue samples.

 
Characterization of PAPP-A mRNA expression
Lawrence et al. (26) reported that the IGFBP-4 protease secreted by human fibroblasts is PAPP-A, based on its purification from conditioned medium, immunoneutralization of the protease with antibodies directed against PAPP-A, and evidence that purified PAPP-A cleaves IGFBP-4 to fragments of the appropriate predicted size in vitro. Subsequently, human recombinant PAPP-A was demonstrated to exhibit potent IGFBP-4 proteolytic activity (39). To determine whether PAPP-A may also account for the IGFBP-4 protease activity secreted by vascular SMC, we examined these cells for the expression of PAPP-A mRNA and the secretion of IGFBP-4 protease. Figure 9Go shows a Northern blot of RNA derived from rodent vascular SMC and B104 cells using a murine PAPP-A probe generated by primers flanking the 5'-region of the putative zinc binding domain. The levels of expression were considerably higher in B104 compared with SMC. A detectable transcript could be appreciated in SMC after longer exposure (data not shown). Because of the low levels of PAPP-A mRNA in SMC, its regulation was further examined by semiquantitative RT-PCR. PAPPA expression was increased in the adult relative to the pup SMCs (Fig. 10Go). Despite evidence that IGFBP-4 protease activity is regulated after arterial injury, PAPP-A mRNA levels in femoral arteries 2 d after injury were unchanged (data not shown).



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  		Figure 9. Northern analysis of PAPP-A expression. Total RNA (20 µg) from cultured SMC (neonatal cell, PUPI; adult cell, C53) and B104 cells were blotted and hybridized with a 32P-labeled mouse PAPP-A cDNA probe. Ethidium bromide staining of nylon membrane after transfer is depicted in the lower panel.

 


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  		Figure 10. Semiquantitative RT-PCR of PAPPA mRNA from cultured vascular SMCs. Cells from either neonatal or young adult animals (see Fig. 4Go) were grown to confluence and exposed to serum-free defined medium for 48 h. RT-PCR was performed on 1 µg total RNA under multiplex conditions using rat ß-actin and mouse homologous PAPP-A primers. The ethidium bromide-stained image of the PCR products was captured under UV illumination, and relative band intensity was determined. A, Ethidium bromide-stained image of amplified PCR products from neonatal (P, PI, and PII) and adult (C45, C49, and C53) cultured vascular SMC. B, Ratio of the relative PCR product band intensity of PAPP-A to ß-actin. Each bar represents the mean ± SD (n = 3 cell lines). *, P < 0.004.

 
To determine whether IGFBP-4 protease activity could be at least in part accounted for by PAPP-A, medium from the adult SMC line with greatest protease activity was fractionated through an HIC column. Fractions were subjected to both PAPP-A Western blot analysis and determination of IGFBP-4 protease activity by charcoal assay. Figure 11Go shows a discrete peak of immunoreactive PAPP-A migrating at approximately 400 kDa. Although the highest peak of IGFBP-4 protease activity coincided with the fractions most enriched for PAPP-A, there was a second significant peak that did not. Immunoneutralization experiments were inconclusive because the monoclonal antibody used was not able to neutralize IGFBP-4 protease activity from human or rodent sources (data not shown).



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  		Figure 11. Western blot analysis of PAPP-A and IGFBP-4 protease activity in HIC fractions of SMC-conditioned medium. C53 cellconditioned medium was applied to an HIC column and eluted. Individual fractions were assayed either for IGFBP-4 protease activity by a standard charcoal assay and activity was expressed as 1 - (bound - nonspecific bound/free - nonspecific bound) or for PAPP-A protein by Western blot analysis using a mouse monoclonal antibody to human PAPP-A. Top panel, The PAPP-A immunoreactive band associated with each fraction. Fractions with protease activity are indicated by the bars.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we show that IGFBP-2 is the dominant binding protein secreted by vascular SMC from neonatal rats, whereas in adult animals IGFBP-4 predominates. Moreover, the activity of the IGFBP-4 protease is higher in conditioned medium from adult animals. Similarly, IGFBP-4 expression and IGFBP-4 protease activity are induced coordinately in vivo after vessel wall injury. Although the precise nature of the protease activity(s) is not clear, these observations support the idea that there is coordinate activation of both biosynthesis and the necessary components to degrade IGFBP-4 at some stage in postnatal life and after perturbations such as vascular injury. These changes are likely to exert significant effects on IGF-I biological activity.

The predominant expression of IGFBP-2 in SMC from neonatal rats suggests that this protein may play a role in the development or growth of the vasculature and is consistent with the observation that IGFBP-2 is a major binding protein in fetal serum and fetal brain (40). IGFBP-2, unlike IGFBP-4, enhances IGF actions under some conditions (17) and can interact with the cell surface through Arg-Gly-Asp (RGD) sequences. Generalized overexpression of IGFBP-2 in transgenic mice results in a 10–13% reduction of total body weight in adult animals, with the principal organs affected being pancreas, spleen, and liver (41). By contrast, targeted disruption of the IGFBP-2 gene in mice has a minimal phenotype (42), manifesting as a reduction in weight of the spleen in adult males. Therefore, although IGFBP-2 deletion appears innocuous, perhaps due to IGFBP redundancy, when animals are exposed to supraphysiological levels of this binding protein, it appears to function as an antagonist of IGF-I action. The implications for the vasculature of predominant expression of IGFBP-2 in early postnatal life are not clear given the current inconclusive mouse transgenic studies.

In contrast to IGFBP-2, in vitro studies with IGFBP-4 have consistently demonstrated potent inhibition of IGF-I action (17, 23, 31). Coincubation of IGFBP-4 with IGF-I results in inhibition of IGF-I action on porcine SMC (43). One hypothesis is that the predominant expression of IGFBP-4 by mature SMC may contribute to the relatively quiescent state of the vasculature in the adult animal (1, 44). Yet, preliminary data on IGFBP-4 null mice indicate that, contrary to expectation, homozygous disruption of IGFBP-4 leads to lower postnatal weight (10–15%), an effect that becomes apparent by postnatal d 7 (42). A potential explanation is that in IGFBP-4-null mice, the IGF storage capacity is diminished, resulting in decreased local abundance of the ligand. However, overexpression of IGFBP-4 in SMC of transgenic mice is associated with medial arterial wall hypoplasia (45).

A potential explanation for these seemingly contradictory findings is that IGFBP-4 might serve as a tissue reservoir for IGF-I, which can only be released upon activation of an IGFBP-4 protease (17). In vascular SMCs IGFBP-4 is subject to proteolysis by a cation-dependent serine protease that cleaves it only in the presence of IGF-I (18, 20, 21, 31, 46, 47, 48, 49). If this were the case, one might expect that activation of expression of IGFBP-4 and its specific protease might be needed to increase the delivery of IGF-I to a specific tissue compartment, and that these two properties (i.e. IGFBP-4 synthesis and degradation) would be regulated coordinately. It is possible that when tissue IGFBP-4 expression levels exceed the ability of IGFBP-4 protease to release IGF-I, the binding protein functions primarily as an IGF antagonist. Regulated proteolysis of IGFBP-4 results in fragments that are either incapable of binding IGF or do so with very low affinity (46) and therefore release the growth factor for interaction with cell membranes. Our finding that IGFBP-4 proteins levels are stable after injury despite increases in IGFBP-4 mRNA and increased proteolysis supports a model in which the binding protein serves as a rapidly renewable reservoir to allow local accumulation of a releasable pool of IGF-I under the regulated activity of the IGFBP-4 protease.

The precise nature of the proteolytic activity observed in the SMC-conditioned medium and within the aortic extracts is uncertain. Despite the recent identification of PAPP-A as a distinct IGFBP-4 protease and the subsequent availability of mouse PAPP-A cDNA probes (27, 39, 49), we were unable to demonstrate definitively that the activity detected in our system can be exclusively accounted for by PAPP-A. PAPP-A mRNA levels correlated with IGFBP-4 protease activity in neonatal and adult arterial SMC, but not in injured vs. uninjured femoral artery extracts. On the other hand, recent data demonstrating higher PAPP-A immunohistochemical levels in injured porcine carotid arteries support the idea that this protein may account for the increased IGFBP-4 protease activity (50). Interpretation of the relationship between protease activity and PAPP-A mRNA levels is complicated by the low levels of expression of PAPP-A mRNA in most tissues (51) and the possibility that it may be regulated posttranslationally. A plausible candidate modulator of PAPP-A activity is eosinophil major basic protein (52). Pro-major basic protein binds in a 2:2 complex with PAPP-A and appears to function as a potent IGFBP-4 proteinase inhibitor (39). Like PAPP-A, pro-major basic protein is expressed ubiquitously, suggesting a mechanism for local regulation of PAPP-A.

Rodent SMC IGFBP-4 proteolytic activity has been demonstrated to be an IGF-I-enhanced, cation-dependent serine protease (20), whereas PAPP-A appears to have properties distinct from those of the serine class of proteases, i.e. zinc dependence with an inhibitor profile characteristic of a metalloproteinase inhibitor (23, 39, 48). Furthermore, Clemmons and colleagues have shown that the porcine SMC IGFBP-4 protease is a serine class protease of approximately 48 kDa that differs in size from the 200-kDa PAPPA (53). In addition, the cleavage site for the IGFBP-4 protease from porcine SMC is in the nonconserved middle domain of the IGFBP-4 protein at Lys120-His121 (53) rather than Met135-Lys146, the cleavage site for PAPP-A (49, 54, 55, 56). Moreover, the Lys120-His121 cleavage site appears to be the predominant target for the protease activity derived from B104 cell-conditioned medium (33). We were able to identify one commercially available monoclonal antibody raised against human PAPP-A that cross-reacted well with rodent PAPP-A in Western blot analysis but was not effective for immunoneutralization. Utilization of this antibody for characterization of HIC fractions revealed a significant, but not complete, overlap with the peak of IGFBP-4 protease activity. Thus, although PAPP-A mRNA and protein are present in rodent vascular smooth muscle, more studies are needed to clarify whether the predominant vascular SMC IGFBP-4 protease activity is indeed PAPPA.

In conclusion, our studies point to a postnatal switch from IGFBP-2 to IGFBP-4 in arterial smooth muscle. This argues, albeit indirectly, for an individual role for each of the binding proteins in the biology of smooth muscle. This is further supported by the observation that IGFBP-4 protease activity is coregulated with the expression of its substrate, a phenomenon also observed after arterial injury in vivo. We propose that this may serve as a regulated pathway to allow local concentrations of binding protein to release IGF-I within discrete tissue compartments of the vessel wall. This would predict that lack of IGFBP-4, by preventing local accumulation of IGFs, would cause growth retardation, which is supported by unpublished information on the phenotype of the IGFBP-4-null mice (42). When IGFBP-4 abundance exceeds the capacity of the IGFBP-4 protease system, IGF will remain in a bound state, and tissue hypoplasia ensues (45). Our data are not conclusive regarding the possible identity of the IGFBP-4 protease.


   Acknowledgments
 
The authors gratefully acknowledge the technical assistance of Water Banach, Children’s Hospital Medical Center (Cincinnati, OH), and the provision of the mouse PAPP-A partial cDNA sequence before its publication by Eli Y. Adashi, Department of Obstetrics and Gynecology, University of Utah Health Science Center (Salt Lake City, UT).


   Footnotes
 
This work was supported in part by USPHS Grant DK-54216.

Abbreviations: HIC, Hydrophobic interaction chromatography; IGFBP, IGF-binding protein; PAPP-A, pregnancy-associated plasma protein A; SMC, smooth muscle cell.

Received December 29, 2000.

Accepted for publication June 27, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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