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Originally published In Press as doi:10.1074/jbc.M112082200 on March 28, 2002

J. Biol. Chem., Vol. 277, Issue 24, 21285-21290, June 14, 2002
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Targeted Expression of a Protease-resistant IGFBP-4 Mutant in Smooth Muscle of Transgenic Mice Results in IGFBP-4 Stabilization and Smooth Muscle Hypotrophy*

Mingyu ZhangDagger , Eric P. SmithDagger , Hiroaki KurodaDagger , Walter Banach§, Steven D. Chernausek§, and James A. FaginDagger

From the Dagger  Divisions of Endocrinology and Metabolism, University of Cincinnati College of Medicine and the § Children's Hospital Medical Center, Cincinnati, Ohio 45267-0547

Received for publication, December 18, 2001, and in revised form, March 27, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The insulin-like growth factor-binding protein 4 (IGFBP-4), the most abundant IGF-binding protein produced by rodent smooth muscle cells (SMC), is degraded by specific protease(s) potentially releasing IGF-I for local bioactivity. IGFBP-4 protease(s) recognizes basic residues within the midregion of the molecule. We constructed a mutant IGFBP-4 with the cleavage domain substitution 119-KHMAKVRDRSKMK-133 to 119-AAMAAVADASAMA-133. Myc-tagged native and IGFBP-4.7A retained equivalent IGF-I binding affinity. Whereas native IGFBP-4 was cleaved by SMC-conditioned medium, IGFBP-4.7A was completely resistant to proteolysis. To explore the function of the protease-resistant IGFBP-4 in vivo, expression of the mutant and native proteins was targeted to SMC of transgenic mice by means of a smooth muscle alpha -actin promoter. Transgene expression was confined to SMC-rich tissues in all lines. Bladder and aortic immunoreactive IGFBP-4/transgene mRNA ratios in SMP8-BP4.7A mice were increased by 2- to 4-fold relative to SMP8-BP4 mice, indicating that the IGFBP-4.7A protein was stabilized in vivo. SMP8-BP4.7A mice had lower aortic, bladder, and stomach weight and intestinal length relative to SMP8-BP4 counterparts matched for protein expression by Western blotting. Thus, IGFBP-4.7A results in greater growth inhibition than equivalent levels of native IGFBP-4 in vivo, demonstrating a role for IGFBP-4 proteolysis in the regulation of IGF-I action.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin-like growth factors (IGFs)1 I and II regulate cellular proliferation, survival, and differentiation. These actions are carried out by locally produced IGFs, which are supplemented by IGFs derived from the circulation. Partition of IGFs between the vascular and cellular interstitial compartments is determined in part by members of a family of high affinity IGF-binding proteins (1, 2). IGFBPs modulate IGF-I and IGF-II bioavailability in part by competitive binding and sequestration and prevention of appropriate interaction with cell membrane receptors. In addition, some IGFBPs bind to components of the extracellular matrix (IGFBP-2, -3, and -5) or the cell membrane (IGFBP-1, -2, -3, and -5), thus providing a mechanism to concentrate IGFs within discrete tissue compartments (2). Free IGFs are released by proteolytic cleavage of the respective IGFBP through the action of specific proteases (3-6). Proteases that cleave IGFBP-2 (7), IGFBP-3 (8, 9), IGFBP-4 (4-6), and IGFBP-5 (10-12) have been reported.

IGFBP-4, which exists in biological fluids as a 28-kDa glycosylated and/or a 24-kDa non-glycosylated form (13, 14) is abundant in serum and expressed in many different tissues (2, 15). IGFBP-4 does not bind to the cell membrane and is found associated with connective tissue, although the precise nature of this interaction is not known. It consistently inhibits IGF-mediated cell proliferation of all cell types tested in vitro (13-16). Regulated proteolysis of IGFBP-4 results in fragments with reduced affinity for IGFs and therefore releases the growth factor for interaction with cell membranes (13-15, 17). IGFBP-4 is also the most abundant IGFBP expressed in the rat artery wall in vivo and is the dominant IGFBP secreted by rat adult vascular smooth muscle cells in vitro (18). After arterial injury, IGFBP-4 mRNA abundance increases, as does the activity of the IGFBP-4 protease, resulting in stable steady-state levels of the IGFBP-4 protein (18). Though increased expression of IGFBP-4 may serve to inhibit IGF-I, if this is coupled with increased proteolytic cleavage then this may ultimately serve to release increased amounts of active IGF-I. The protease(s) that cleave IGFBP-4 are found ubiquitously, including the conditioned medium of cultured fibroblasts, decidual cells, osteoblasts, and SMC and in biological fluids such as pregnancy serum and CSF (14, 19-21). Recently, pregnancy-associated plasma protein-A (PAPP-A) has been identified as a protease for IGFBP-4 (22), and this protein is expressed in smooth muscle cells (3, 23).

The biochemistry of IGFBP-4 cleavage has been examined through analyses of the fragments produced following exposure to IGFBP-4 protease from various sources and from the effects of limited mutations on protease susceptibility (6, 24-26). These experiments clearly identify a 13-amino acid domain within the middle of the molecule as the principal site of cleavage. This region is largely conserved among mammalian species of this binding protein but is unique to IGFBP-4 among all IGFBP family members, offering a biochemical explanation for the apparent specificity of the IGFBP-4 proteases. This region is rich in basic amino acid residues that could be subject to tryptic-like cleavage. Attempts to further define which bonds within this domain are initially cleaved have produced somewhat discordant results. Some reports indicate residues 120/121 are most critical while others describe the 130/132 basic amino acids as the primary cleavage site (6, 24, 25, 27-29). These differences could be caused by secondary site use by the protease, intraspecies differences, or more than one IGFBP-4 protease that attacks this highly charged domain.

Our objective was to examine the role of IGFBP-4 proteolysis in vivo, using a mutant IGFBP-4 that would be largely insensitive to degradation by physiological IGFBP-4 proteases. We show that substitution of the seven basic residues in the midregion of IGFBP-4 by alanine renders the protein resistant to proteolysis in vitro and in vivo, without affecting its IGF binding affinity. When expressed at equivalent levels in smooth muscle cells of transgenic mice, protease-resistant IGFBP-4 results in greater growth inhibition than native IGFBP-4, supporting a role for IGFBP-4 proteolysis in the regulation of IGF-1 action.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Production of IGFBP-4 Mutant cDNA (IGFBP-4.7A)-- A cDNA construct encoding a mutant IGFBP-4 in which all seven basic residues between Lys-120 and Lys-132 were exchanged for Ala residues was produced by stepwise mutagenesis of a cDNA construct originally provided by S. Shimasaki et al. (30) (Fig. 1). The previously created K120A IGFBP-4 (6) was mutated by PCR mutagenesis to K120A/H121A using the oligonucleotide 5'-ACTTTGGCCATAGCCGCCTGCAGGC-3' and transferred to pcDNA 3.0 (Invitrogen, Carlsbad, CA) using previously described methodology (6). Next the codon for residue Lys-124 was changed to Ala using the Chameleon double-stranded mutagenesis kit (Stratagene, Inc.), employing the mutagenesis primer 5'-TATGGCCGCGGTGAGAGATGCGAGCAAGATGA-3' that introduced a SacII restriction site. The final four residues were altered by generating a mutant PCR fragment that was cloned into the SacII and XbaI restriction sites of the aforementioned construct. The mutagenic oligonucleotide for this step was 5'-GGCTATGGCCGCGGTGGCAGATGCGAGCGCGATGGCGGTTGTGGGGACAC-3'. Fidelity of IGFBP-4.7A construct was verified by automated sequencing of the entire cDNA. This cDNA construct, IGFBP-4.7A, was ultimately expressed in vitro and in vivo for the experiments described.


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  		Fig. 1.   Development of protease-resistant IGFBP-4.7A. The midregion of rat IGFBP-4, including the sites cleaved by IGFBP-4 protease(s), is depicted. The expanded rectangular block displays amino acids 120-132. All basic residues within this domain (Lys-120, His-121, Lys-124, Arg-126, Arg-128, Lys-130, and Lys-132) were replaced with alanine by site-directed mutagenesis to generate IGFBP-4.7A.

Determination of Sensitivity of SMP8-IGFBP-4.7A to the SMC IGFBP-4 Protease-- To analyze the properties of the mutant IGFBP-4.7A protein, we used B104ns cells, a subclone of the B104 rat neuroblastoma cell line with low expression levels of endogenous native IGFBP-4 as host for transfection experiments (14). IGFBP-4 proteins that contained C-terminal Myc-His tags were produced by stably transfecting B104ns cells with pcDNA 3.1 plasmids (Invitrogen) into which native and IGFBP-4.7A cDNAs had been cloned. Plasmid-bearing cells were grown to confluency in medium containing 800 ng/ml G418, and conditioned medium was collected, heated to inactivate endogenous proteases, and concentrated by Centricon-10 filters (Millipore, Bedford, MA). An aliquot of the conditioned medium was exposed to adult SMC medium for 24 and 40 h in buffer (50 mM Tris, 2 mM CaCl2, 50 µM ZnSO4, 0.002% bovine serum albumin, pH 7.5) containing 100 ng/ml IGF-1. Susceptibility to proteolysis was assessed following SDS-PAGE of the reaction mixture by immunoblot using an anti-Myc-specific antibody.

Determination of Binding Affinity for IGF-I of Native IGFBP-4 and IGFBP-4.7A-- The IGFBP-4.7A cDNA was cloned into the pcDNA3.1 expression plasmid, which was subsequently stably transfected into B104ns cells. The expressed protein had a 9-amino acid C-terminal extension in addition to the 7 mutated residues. Cells were maintained in G418 and expressed IGFBP-4.7A was purified using affinity chromatography. This preparation was compared with native IGFBP-4 isolated from B104ns medium as previously described (13). 10 ng of the purified binding protein were then affinity cross-linked to 125I-IGF-I using the bifunctional cross-linking agent disuccinimidyl suberate in the presence of graded amounts of unlabeled IGF-I, as described (13). The products of the competitive inhibition experiment were analyzed by SDS-PAGE, and the signal corresponding to IGFBP-4 was quantified by phosphorimaging.

Generation of Transgenic Mice-- Mice with targeted overexpression of native IGFBP-4 in smooth muscle have been previously described (31). We used an identical approach to target expression of IGFBP-4 7A selectively to smooth muscle cells. Briefly, the cDNA for the IGFBP-4 protease-resistant mutant was cloned downstream of the SMP8 fragment of the mouse SM alpha -actin gene promoter, as previously reported (31). The SMP8-IGFBP-4.7A construct was released from Puc18 by digestion with KpnI and NotI and then gel-purified (31). Male pronuclei of fertilized eggs from FVB-N mouse strains were microinjected with 2 pl of DNA at the transgenic mouse facility of the University of Cincinnati. Microinjected eggs were implanted into the oviduct of pseudopregnant female mice and carried to term. Positive founders were identified by Southern blotting and bred to wild-type FVB-N mice for propagation of the line. Mouse tail tips were cut at 3 weeks of age, and the transgene identified by Southern blotting of EcoR1-digested DNA hybridized with a rat IGFBP-4 cDNA probe labeled by random priming (Prime-It, Stratagene). The transgene was identified by the presence of a unique ~3.0-kb band. Routine screening of established SMP8-BP-4 or SMP8-IGFBP-4.7A lines was performed by PCR of tail DNA using the following oligonucleotide primers: forward, 5'-CCTGTGACACTCCCGCTCTTTG-3'; reverse, 5'-AGGGCAGTGGATGGCTTCGTC-3'; endogenous c-Fos gene primers: forward, 5'-GGCTGGCCCTGTATTCCTGAT-3'; reverse, 5'-TCTTCTGACCCTTCCCTACTGAGC-3'. Amplification was performed under standard conditions using Redtaq polymerase (Sigma) with the following cycle parameters: initial hot-start denaturation, 95 °C, 5 min followed by 40 cycles of 94 °C, 1 min; 64 °C, 1 min; and 72 °C, 1 min.

Five separate SMP8-IGFBP-4.7A lines were propagated and three of them selected for more extensive analysis. Expression levels and phenotypic changes in SMP8-IGFBP-4.7A mice were compared with those in the previously reported SMP8-IGFBP-4 lines 23942, 23928, 23926, and 23945 (31).

Determination of Tissue Content of IGFBPs-- Transgene mRNA levels were determined by Northern blotting. Quantification was performed by phosphorimaging and adjusted to that of an appropriate internal standard. The same lines were then used for analysis of IGFBP-4 protein content by Western immunoblotting (31, 32). Briefly, tissues were homogenized in PBS with 12 mM EDTA and 1 mM phenylmethylsulfonyl fluoride in a Polytron (model PT3000) at full speed on ice. Extracts were centrifuged at 100,000 × g for 1 h at 4 °C, and the supernatant was further concentrated in a Centricon-3 device (Amicon, Inc., Beverly, MA). Aliquots of the concentrated extracts were taken for protein assay. The extracts were run on 10% Laemmli gels under reducing conditions. The blots were blocked with a Tris-buffered saline solution containing 0.05% Tween 20 and nonfat dry milk and incubated with either 1:5000 rabbit anti-rat IGFBP4 antibody (516.F) generated against residues 81-100 of the rodent sequence (6, 30) in the same blocking solution plus 0.01% thimerosal at room temperature for 16 h. IGFBP-4 was visualized by standard ECL techniques as described (33) and quantified by phosphorimager.

Phenotypic Analysis-- SMP8-IGFBP-4.7A mice from line 39208 had equivalent levels of IGFBP-4 in smooth muscle rich tissues to age and sex-matched SMP8-IGFBP-4 mice from line 23942, and these were selected for analysis of organ weights. To compare the two transgenic lines, animals were sacrificed at 10 weeks of age as previously described (31). Briefly, transgenic mice and their nontransgenic littermates were killed by CO2 asphyxiation. After obtaining body weight, blood was collected by cardiac puncture, and the serum was stored at -80 °C until use. Organs of interest were dissected, rinsed in ice-cold PBS, tissue-blotted, weighed, and immediately frozen in liquid nitrogen. Contents of stomach and small intestine were flushed out with PBS before weighing. A section of the arterial vessel from the aortic arch to the level of femoral bifurcation was excised and placed in PBS. Adhering fat and connective tissue from the adventitia were scraped off under surgical microscope, and the vessel was cleansed with PBS to remove any residual blood. Tissues were weighed after blotting away excess fluid.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Resistance of IGFBP-4.7A to Proteolytic Degradation-- We first explored the relative cleavage resistance of IGFBP-4 mutants with discrete substitutions across the putative protease recognition domain (amino acids 120-132). Purified alanine-substituted mutants IGFBP-4.K120A/H121A or IGFBP-4.K120A/H121A/K124A were only partially resistant to cleavage by protease activity from either osteoblast or smooth muscle cell-conditioned medium (not shown). We therefore developed an IGFBP-4 mutant in which all seven basic residues between Lys-120 and Lys-132 were substituted. As shown in Fig. 2, concentrated aliquots of smooth muscle cell-conditioned medium efficiently cleaved purified Myc-tagged native rat IGFBP-4 but failed to degrade IGBP-4.7A in the presence of IGF-1.


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  		Fig. 2.   Resistance of IGFBP-4.7A to cleavage by smooth muscle cell-conditioned medium. Concentrated aliquots of conditioned medium (0.5-ml equivalents) of primary cultures of rat adult arterial smooth muscle cells (SMC+) were incubated with either Myc-His-tagged IGFBP-4 (IGFBP-4MH) or IGFBP-4.7A for 24 and 40 h in the presence of IGF-1. The samples were size-separated by SDS-PAGE, and the resultant bands were identified by Western blotting with anti-Myc antibody. The conditioned medium had no proteolytic effect on IGFBP-4.7A but efficiently cleaved native IGFBP-4. Control samples were incubated in buffer alone. Lanes with media are slightly distorted due to high protein content of concentrated media. The calculated mass of Myc-His-IGFBP-4 fusion protein is indicated.

Binding of IGF-I by IGFBP-4.7A-- The goal of this study was to examine the contribution of IGFBP-4 degradation to IGF action in vivo. It was therefore important to demonstrate that the mutations introduced in the midregion of the molecule had not impaired IGF binding affinity. For this purpose, we purified IGFBP-4.7A and native IGFBP-4 from the conditioned medium of B104ns cells stably transfected with expression vectors for the respective binding protein. Equal amounts of native and mutant IGFBP-4 were then chemically cross-linked to 125I-IGF-1 in the presence of graded concentrations of unlabeled IGF-1. The samples were subjected to SDS-PAGE and quantified by phosphorimager. Linear regression analysis showed comparable displacement of tracer by unlabeled ligand for both the wild-type and mutant forms of IGFBP-4, indicating that IGFBP-4.7A bound IGF normally (Fig. 3).


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  		Fig. 3.   IGF-I affinity of IGFBP-4.7A relative to native IGFBP-4. 10 ng of IGFBP-4 and IGFBP-4.7A were chemically cross-linked to 125I-IGF-1 in the presence of graded concentrations of unlabeled IGF-1. Following SDS-PAGE, residual radioactivity bound to IGFBP-4 was determined by quantitative phosphorimaging. Linear regression analyses showed no significant difference between binding affinity for IGF-I of IGFBP-4 (open circles) and IGFBP-4.7A (closed circles).

Development of SMP8-IGFBP-4.7A Mice and Characterization of the Tissue Distribution of Expression-- To explore the function of the protease-resistant IGFBP-4 in vivo, we targeted its expression to smooth muscle cells of transgenic mice. We previously reported that targeted overexpression of native IGFBP-4 under the control of the SM alpha -actin gene promoter was associated with smooth muscle hypoplasia (31). The same approach was used to target expression of IGFBP-4.7A to smooth muscle cells. As shown in Fig. 4, tissue distribution of the IGFBP-4.7A mRNA transgene was restricted to smooth muscle cell-rich tissues such as the aorta, bladder, stomach, intestine, and spleen. This recapitulates the pattern of expression previously reported for the SMP8-IGFBP-4 mice (31) and that of smooth muscle alpha -actin (31,32). Endogenous IGFBP-4 mRNA was maximal in the liver, spleen, and kidney. Expression of the IGFBP-4 transgenes (identified through their faster migration) did not affect endogenous IGFBP-4 mRNA abundance. Serum IGFBP-4 levels were not increased in either SMP8-IGFBP-4 or SMP8-IGFBP-4.7A mice as determined by Western ligand blotting, confirming that the products of the respective transgenes are largely confined to tissues of origin (not shown).


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  		Fig. 4.   Tissue distribution of transgene expression in SMP8-IGFBP-4.7A mice. 5 µg of total RNA from the indicated tissues of representative wild-type FVB-N non-transgenic and SMP8-IGFBP-4.7A mice were gel-separated and hybridized to a rat IGFBP-4 cDNA probe. Endogenous IGFBP-4 mRNA (eIGFBP-4) is ubiquitous with maximal expression levels in the liver, aorta, bladder, spleen, and kidney. The IGFBP-4.7A transgene, identified because of its faster migration, is restricted to smooth muscle cell-rich tissues: i.e. aorta, bladder, stomach, intestine, and spleen. The blot was stripped and rehybridized with an 18 S ribosomal probe confirming similar RNA loading in each lane.

Increased Stability of the IGFBP-4.7A Protein in Vivo-- If IGFBP-4.7A were resistant to endogenous proteases in vivo, then the tissues in which it is expressed should show greater accumulation of the protein relative to the abundance of the respective mRNA. To test this premise, we first quantified IGFBP-4 mRNA abundance in bladders from representative age- and sex-matched mice from four native IGFBP-4 and three IGFBP-4.7A transgenic lines, respectively. As shown in Fig. 5, transgenic IGFBP-4 mRNA was highly expressed in bladder of all lines tested and far exceeded abundance of the endogenous transcript. Notably, bladders from mice derived from the three lines expressing the protease-resistant mutant contained markedly higher levels of immunoreactive IGFBP-4 than the lines overexpressing the native form of the protein (Fig. 6). The IGFBP-4.7A immunoreactive band migrated significantly faster than native IGFBP-4, which was likely due to different interaction with the gel matrix. Altered electrophoretic mobility was not due to differences in glycosylation of the mutant protein as it persisted following digestion with N-glycanase (not shown).


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  		Fig. 5.   Northern blot analysis of transgene expression in bladder of transgenic mice. Expression of transgene mRNA was compared in urinary bladders from representative mice of three SMP8-IGFBP-4.7A and four SMP8-IGFBP-4 lines. 5 µg of total RNA were gel-separated and hybridized to rat IGFBP-4 cDNA probe. Expression of transgenic IGFBP-4 mRNA was abundant in all lines tested. The blot was stripped and rehybridized with a human glyceraldehyde-3-phosphate dehydrogenase probe.


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  		Fig. 6.   Western blot analysis of tissue extracts from bladders of IGFBP-4.7A and IGFBP-4 transgenic animals. Top panel, Western blots of bladder tissue extracts from the indicated transgenic lines incubated with a specific IGFBP-4 antiserum. NT, bladder extract from non-transgenic littermate; far left lane, 6 ng of recombinant hIGFBP-4. The same blot stripped and reprobed with extracellular-regulated kinase (Erk) antisera revealed equivalent loading of samples.

Fig. 7 depicts the relative IGFBP-4 protein and IGFBP-4 mRNA levels in quantitative terms. Bladder immunoreactive IGFBP-4/transgene mRNA ratios in all three SMP8-IGFBP-4.7A lines were significantly higher than in SMP8-IGFBP-4 mice, indicating that the IGFBP-4.7A protein was stabilized in vivo. Stabilization of IGFBP-4.7A was also apparent in aortic tissue of mice from these lines (not shown).


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  		Fig. 7.   Relative levels of immunoreactive IGFBP-4 and IGFBP-4 mRNA in the bladder of SMP8-IGFBP-4.7A and SMP8-IGFBP-4 transgenic mice. Abundance of IGFBP-4 mRNA and protein were calculated by image analysis of Northern and Western blots of tissues from the indicated cell lines, after normalization to appropriate internal standards. Expression levels from at least three age- and sex-matched animals were examined for each line. Values were expressed relative to expression levels in the SMP8-IGFBP-4 line 23926. The IGFBP-4.7A overexpressing mice showed significantly higher immunoreactive IGFBP-4 levels relative to mRNA abundance, compared with mice overexpressing the wild-type form of the binding protein. *, p < 0.05.

Phenotype of the Native versus Mutant IGFBP-4 Transgenic Lines Selected for Similar Protein Expression-- We next explored whether IGFBP-4.7A exerted greater growth inhibitory effects than native IGFBP-4 when expressed at equivalent levels in smooth muscle cell-rich tissues. For this purpose, we studied SMP8-IGFBP-4 line 23942 and SMP8-IGBBP-4.7A line 39208, because they had comparable immunoreactive IGFBP-4 levels (Figs. 6 and 7). As previously reported, overexpression of native IGFBP-4 was associated with decreased wet weight of aorta, bladder, and stomach. Expression of comparable levels of the IGFBP-4 7A resulted in an even greater decrease of wet weight of the indicated tissues (Fig. 8A). There was no significant difference in total weight of the animals or weight of any other organ (data not shown). Overexpression of IGFBP-4.7A was also associated with a significant shortening of the small intestine, whereas native IGFBP-4 was without effect (Fig. 8B).


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  		Fig. 8.   Phenotypic comparison of SMC-rich tissues from SMP8-IGFBP-4.7A versus SMP8-IGFBP-4 transgenic mice. Transgenic mice overexpressing equivalent levels of either IGFBP-4 (line 23942) or IGFBP-4.7A (line 39208) were sacrificed at ~10 weeks of age. Tissues were collected and weighed as described under "Experimental Procedures." A, aorta (n = 12), bladder (n = 11), and stomach (n = 11) of SMP8-IGFBP-4 mice were lighter than those of non-transgenic controls. Aorta, bladder, and stomach from mice overexpressing the protease-resistant IGFBP-4.7A weighed less than those of mice expressing comparable levels of the wild-type form of the protein. **, p < values for SMP8-IGFBP-4 versus NT (aorta, 0.025; bladder, 0.000046; stomach, 0.010). *, p < values for SMP8-IGFBP-4.7A versus SMP8-IGFBP-4 (aorta, 0.005; bladder, 0.016; stomach, 0.013). B, length of the small intestines from the SMP8-IGFBP-4.7A mice were significantly shorter than those of SMP8-IGFBP-4 mice (*, p < 0.049) and non-transgenic controls (**, p < 0.00096).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

These studies demonstrate that basic residues between amino acids 120 and 135 are critical for IGFBP-4 proteolysis and can be replaced by alanine without significantly affecting IGF-I binding affinity. Preservation of IGF-I binding would be anticipated because the primary binding domain for IGF-I in IGFBP-4 was demonstrated to reside within a hydrophobic motif in the distal part of the conserved N terminus of the protein (29, 35), in a similar region required for IGFBP-5 binding to IGF-I (36). The decision to develop an IGFBP-4 mutant with multiple substitutions within the cleavage domain was based on evidence that mutants with more discrete replacements at the precise putative cleavage site(s) could still be degraded by IGFBP-4 protease(s) in the conditioned media of several cell types. Conover et al. (37) reported that the IGFBP-4 protease(s) secreted by human fibroblasts and osteoblasts cleaved hIGFBP-4 between Met-135 and Lys-136 (equivalent to Met-131/Lys-132 of rIGFBP-4) (37). Discrete substitutions at these sites resulted in an IGFBP-4 mutant with normal binding affinity to IGFs, and a relative, but not absolute, resistance to proteolysis. By contrast, in a previous report one of us found the primary cleavage domain of IGFBP-4 to reside between Lys-120/His-121. However, alanine substitution of these two residues alone resulted in only partial resistance to proteolysis (6) when B104 conditioned medium was the source of proteolytic activity.

The extended domain involved in proteolytic degradation raises questions about the nature of the IGFBP-4 proteases produced by smooth muscle. PAPP-A has recently been found to cleave IGFBP-4 in an IGF-dependent manner (22, 38). Although this zinc-binding metalloproteinase also cleaves IGFBP-5 (39), it is thought to represent the dominant IGFBP-4 protease in most systems examined so far (17, 22, 27, 40). PAPP-A is expressed in porcine vascular smooth muscle cells in culture, and immunoreactive PAPP-A increases in the medium following arterial injury in pigs (23). Furthermore, in a recent human study, PAPP-A protein abundance is increased in unstable coronary plaques and is detected by immunoassay at higher concentrations in patients with either unstable angina or acute myocardial infarction (41). PAPP-A mRNA is also expressed in rodent vascular smooth muscle cells and in aortic tissue, and at least some of the IGFBP-4 protease activity detected in rat smooth muscle cell cultures is likely to be accounted for by PAPP-A (18). However, porcine smooth muscle IGFBP-4 protease activity appears to cleave IGFBP-4 at a distinct site from PAPP-A and has a different inhibitor profile (24). Because of this discrepancy, and the presence of an extended cleavage domain in our studies, it is possible that there is more than one protease acting either individually or sequentially on IGFBP-4 to achieve complete degradation of the protein.

In accord with our previous report in which native IGFBP-4 was overexpressed under the control of the same SM alpha -actin promoter, the IGBP-4.7A mutant was expressed uniquely in smooth muscle-rich tissues (31). All IGFBP-4.7A-expressing lines had a higher ratio of immunoreactive IGFBP-4 to mRNA than their IGFBP-4 wild type-overexpressing counterparts. This points to a key post-translational step in the regulation of IGFBP-4 abundance in vivo, almost certainly through regulated proteolysis. This binding protein is also subject to significant pretranslational regulation in smooth muscle cells. Thus IGFBP-4 mRNA levels are regulated by PDGF (42), thrombin (43), and angiotensin (43) in smooth muscle cells in vitro, and following in vivo perturbations such as bladder outlet obstruction (44), arterial injury (18), and aortic coarctation (45). The evidence presented here indicates that IGFBP-4.7A results in greater growth inhibition than equivalent levels of wild type IGFBP-4 in vivo. These results support the concept that proteolysis of IGFBP-4 may be required for release of free IGF-I in smooth muscle and that the growth factor may be sequestered if the binding protein cannot be readily degraded.

Despite the fact that IGFBP-4 consistently inhibits IGF action in many cell types in vitro, preliminary data on IGFBP-4-null mice indicate that, contrary to expectation, these animals have lower weight at birth (10-15%) (46). One potential explanation is that the absence of IGFBP-4 diminishes tissue IGF storage capacity. This would predict that physiological levels of IGFBP-4 are required for normal growth, and that IGFs would be released through the action of PAPP-A and perhaps other IGFBP-4 proteases. When IGFBP-4 is selectively overexpressed in vivo tissue hypoplasia ensues due to ligand sequestration, because the degradation rate is exceeded by higher local production (31). Our present results further support this concept, because expression of a protease-resistant mutant-binding protein resulted in even greater growth suppression in the targeted tissue environments. Recent studies examining the effects of local infusions of IGFBP-4 in bone also support this notion. Injection of a protease-resistant IGFBP-4, in which the sequence His-121 to Pro-141 was deleted, directly into the parietal bone was more potent than wild-type IGFBP-4 in inhibiting the anabolic actions of IGF-I (26). IGFBP-4 also appears to play a role in transport of IGF-I across the endothelial barrier, an effect that may require proteolysis of the binding protein (26, 47, 48). As IGFBP-4 proteolysis is also a step amenable to regulation (18, 23); coordinated IGFBP-4 biosynthesis and degradation may be required to allow appropriate delivery of free IGF-I to target tissues.

    FOOTNOTES

* This work was supported by Public Health Service Grant DK54216.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, Vontz Center for Molecular Studies, 3125 Eden Ave., Cincinnati, OH 45267-0547. E-mail: James.Fagin@uc.edu.

Published, JBC Papers in Press, March 28, 2002, DOI 10.1074/jbc.M112082200

    ABBREVIATIONS

The abbreviations used are: IGF, insulin-like growth factor; SMC, smooth muscle cell; PAPP-A, pregnancy-associated plasma protein-A; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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