An anti-αVß3 antibody inhibits coronary artery atherosclerosis in diabetic pigs.

BACKGROUND AND AIMS: Diabetes is a major risk factor for the development of atherosclerosis. Hyperglycemia stimulates vascular smooth muscle cells (VSMC) to secrete ligands that bind to the αVß3 integrin, a receptor that regulates VSMC proliferation and migration. This study determined whether an antibody that had previously been shown to block αVß3 activation and to inhibit VSMC proliferation and migration in vitro, inhibited the development of atherosclerosis in diabetic pigs. METHODS: Twenty diabetic pigs were maintained on a high fat diet for 22 weeks. Ten received injections of anti-ß3 F(ab)2 and ten received control F(ab)2 for 18 weeks. RESULTS: The active antibody group showed reduction of atherosclerosis of 91 ± 9% in the left main, 71 ± 11%, in left anterior descending, 80 ± 10.2% in circumflex, and 76 ± 25% in right coronary artery, (p < 0.01 compared to lesions areas from corresponding control treated arteries). There were significant reductions in both cell number and extracellular matrix. Histologic analysis showed neointimal hyperplasia with macrophage infiltration, calcifications and cholesterol clefts. Antibody treatment significantly reduced number of macrophages contained within lesions, suggesting that this change contributed to the decrease in lesion cellularity. Analysis of the biochemical changes within the femoral arteries that received the active antibody showed a 46 ± 12% (p < 0.05) reduction in the tyrosine phosphorylation of the ß3 subunit of αVß3 and a 40 ± 14% (p < 0.05) reduction in MAP kinase activation. CONCLUSIONS: Blocking ligand binding to the αVß3 integrin inhibits its activation and attenuates increased VSMC proliferation that is induced by chronic hyperglycemia. These changes result in significant decreases in atherosclerotic lesion size in the coronary arteries. The results suggest that this approach may have efficacy in treating the proliferative phase of atherosclerosis in patients with diabetes.

Complement 1s is the serine protease that cleaves IGFBP-5 in human osteoarthritic joint fluid.

UNLABELLED: Insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) are trophic factors for cartilage and have been shown to be chondroprotective in animal models of osteoarthritis (OA). IGFBP-5 is degraded in joint fluid and inhibition of IGFBP-5 degradation has been shown to enhance the trophic effects of IGF-I. OBJECTIVE: To determine the identity of IGFBP-5 protease activity in human OA joint fluid. METHOD: OA joint fluid was purified and the purified material was analyzed by IGFBP-5 zymography. RESULTS: Both crude joint fluid and purified material contained a single band of proteolytic activity that cleaved IGFBP-5. Immunoblotting of joint fluid for complement 1s (C1s) showed a band that had the same Mr estimate, e.g., 88 kDa. In gel tryptic digestion and subsequent peptide analysis by LC-MS/MS showed that the band contained human C1s. A panel of protease inhibitors was tested for their ability to inhibit IGFBP-5 cleavage by the purified protease. Three serine protease inhibitors, FUT175 and CP-143217 and CB-349547 had IC50's between 1 and 6 microM. Two other serine protease inhibitors had intermediate activity (e.g., IC50's 20-40 microM) and MMP inhibitors had no detectible activity at concentrations up to 300 microM. CONCLUSION: Human OA fluid contains a serine protease that cleaves IGFBP-5. Zymography, immunoblotting and LC-MS/MS analysis indicate that C1s is the protease that accounts for this activity.

Protease-resistant insulin-like growth factor (IGF)-binding protein-4 inhibits IGF-I actions and neointimal expansion in a porcine model of neointimal hyperplasia.

IGF-I has been shown to play a role in the progression of atherosclerosis in experimental animal models. IGF-binding protein-4 (IGFBP-4) binds to IGF-I and prevents its association with receptors. Overexpression of a protease-resistant form of IGFBP-4 has been shown to inhibit the ability of IGF-I to stimulate normal smooth muscle cell growth in mice. Based on these observations, we prepared a protease-resistant form of IGFBP-4 and infused it into hypercholesterolemic pigs. Infusion of the protease-resistant mutant inhibited lesion development by 53.3 +/- 6.1% (n = 6; P < 0.01). Control vessels that received an equimolar concentration of IGF-I and the protease-resistant IGFBP-4 showed no reduction in lesion size compared with control lesions that were infused with vehicle. Infusion of a nonmutated form of IGFBP-4 did not significantly inhibit lesion development. Proliferating cell nuclear antigen analysis showed that the mutant IGFBP-4 appeared to inhibit cell proliferation. The area occupied by extracellular matrix was also reduced proportionally compared with total lesion area. Immunoblotting revealed that the mutant IGFBP-4 remained intact, whereas the wild-type IGFBP-4 that was infused was proteolytically cleaved. Further analysis of the lesions revealed that a marker protein, IGFBP-5, whose synthesis is stimulated by IGF-I, was decreased in the lesions that received the protease-resistant, IGFBP-4 mutant, whereas there was no change in lesions that received wild-type IGFBP-4 or the mutant protein plus IGF-I. These findings clearly illustrate that infusion of protease-resistant IGFBP-4 into the perilesion environment results in inhibition of cell proliferation and attenuation of the development of neointima. The findings support the hypothesis that inhibiting IGFBP-4 proteolysis in the lesion microenvironment could be an effective means for regulating neointimal expansion.

Recombinant, nonglycosylated human insulin-like growth factor-binding protein-3 (IGFBP-3) is degraded preferentially after administration to type II diabetics, resulting in increased endogenous glycosylated IGFBP-3.

CONTEXT: Administration of IGF-binding protein-3 (IGFBP-3) with IGF-I stabilizes IGF-I concentrations and prolongs its half-life. One determinant of IGFBP-3 stability is proteolysis. Normal subjects have minimal IGFBP-3 protease activity; however, with pregnancy, acute catabolic illness, or diabetes, IGFBP-3 protease activity is increased. OBJECTIVE: This study was conducted to determine the degree of proteolysis that occurs in glycosylated, endogenous serum IGFBP-3 and nonglycosylated IGFBP-3 after administration of an IGF-I/IGFBP-3 combination to patients with diabetes. DESIGN: Thirty-two patients received either 1 (n = 8) or 2 (n = 24) mg/kg x d IGF-I/IGFBP-3 by bolus s.c. injection (n = 16) or continuous s.c. infusion (n = 16). RESULTS: When nonglycosylated IGFBP-3 was given, the abundance of both glycosylated and nonglycosylated forms of IGFBP-3 in serum was increased. Incubation of nonglycosylated IGFBP-3 with diabetic serum in vitro resulted in more rapid degradation compared with glycosylated IGFBP-3. When the serum obtained from subjects who had received nonglycosylated IGFBP-3 was analyzed, significant differences in the stability of glycosylated and nonglycosylated IGFBP-3 were present. The addition of increasing concentrations of nonglycosylated IGFBP-3 to diabetic serum resulted in a dose-dependent increase in the abundance of endogenous, glycosylated IGFBP-3. Administration of IGF-I and nonglycosylated IGFBP-3 for 2 wk to 32 subjects increased glycosylated IGF-I/IGFBP-3 by 20-40%. The increases were the greatest in the groups that received IGFBP-3 by infusion (e.g. 31% and 40%). CONCLUSIONS: After administration to diabetics, nonglycosylated IGFBP-3 is degraded more rapidly than glycosylated IGFBP-3. By acting as a preferential substrate for the IGFBP-3 protease, nonglycosylated IGFBP-3 protects endogenous, glycosylated IGFBP-3 from degradation, allowing total IGFBP-3 concentrations to increase.

Biochemical and functional analysis of a conserved IGF-binding protein isolated from rainbow trout (Oncorhynchus mykiss) hepatoma cells.

Rainbow trout (Oncorhynchus mykiss) serum contains several IGF-binding proteins (IGFBPs) that specifically bind to IGFs. The structures of these fish IGFBPs have not been determined and their physiological functions are poorly defined. In this study, we identified a 30 kDa IGFBP present in rainbow trout serum and secreted by cultured trout hepatoma cells. This IGFBP binds to IGFs but not to insulin. This IGFBP was purified to homogeneity using a three-step procedure involving Phenyl-Sepharose chromatography, IGF-I affinity chromatography and reverse-phase HPLC. Affinity cross-linking studies indicated that this IGFBP binds to IGF-I with a higher affinity than to IGF-II. N-terminal sequence analysis of the trout IGFBP suggests that it shares high sequence identity with that of human IGFBP-1 in the N-terminal region. When added to cultured fish and human cells, the trout IGFBP inhibited IGF-I-stimulated DNA synthesis and cell proliferation in a concentration-dependent manner. The inhibitory effect of the fish IGFBP was comparable to those of human IGFBP-1 and -4. These results indicate that the IGFBP molecule is structurally and functionally conserved in evolutionarily ancient vertebrate species such as bony fish.

Tissue transglutaminase facilitates the polymerization of insulin-like growth factor-binding protein-1 (IGFBP-1) and leads to loss of IGFBP-1's ability to inhibit insulin-like growth factor-I-stimulated protein synthesis.

Insulin-like growth factor-binding protein-1 (IGFBP-1) binds to insulin-like growth factors (IGFs) and has been shown to inhibit or stimulate cellular responses to IGF-I in vitro. This capacity of IGFBP-1 to inhibit or stimulate IGF-I actions correlates with its ability to form stable high molecular weight multimers. Since the ability of some proteins to polymerize is dependent upon transglutamination, we determined if tissue transglutaminase could catalyze this reaction and the effect of polymerization of IGFBP-1 upon IGF-I action. Following incubation with pure tissue transglutaminase (Tg), IGFBP-1 formed covalently linked multimers that were stable during SDS-polyacrylamide gel electrophoresis using reducing conditions. Dephosphorylated IGFBP-1 polymerized more rapidly and to a greater extent compared with native (phosphorylated) IGFBP-1. Exposure to IGF-I stimulated transglutamination of IGFBP-1 in vitro. An IGFBP-1 mutant in which Gln(66)-Gln(67) had been altered to Ala(66)-Ala(67) (Q66A/Q67A) was relatively resistant to polymerization by Tg compared with native IGFBP-1. Tg localized in fibroblast membranes was also shown to catalyze the formation of native IGFBP-1 multimers, however, Q66A/Q67A IGFBP-1 failed to polymerize. Although the mutant IGFBP-1 potently inhibited IGF-I stimulated protein synthesis in pSMC cultures, the same concentration of native IGFBP-1 had no inhibitory effect. The addition of higher concentrations of native IGFBP-1 did inhibit the protein synthesis response, and this degree of inhibition correlated with the amount of monomeric IGFBP-1 that was present. In conclusion, IGFBP-1 is a substrate for tissue transglutaminase and Tg leads to the formation of high molecular weight covalently linked multimers. Polymerization is an important post-translational modification of IGFBP-1 that regulates cellular responses to IGF-I.

The complement component C1s is the protease that accounts for cleavage of insulin-like growth factor-binding protein-5 in fibroblast medium.

Cultured fibroblasts secrete an 88-kDa serine protease that cleaves insulin-like growth factor binding protein-5 (IGFBP-5). Because IGFBP-5 has been shown to regulate IGF-I actions, understanding the chemical identity and regulation of this protease is important for understanding how IGF-I stimulates anabolic functions. The protease was purified from human fibroblast-conditioned medium by hydrophobic interaction, lectin affinity, and heparin Sepharose affinity chromatography followed by SDS-polyacrylamide gel electrophoresis. An 88-kDa band was excised and digested with lysyl-endopeptidase. Sequencing of the high pressure liquid chromatography-purified peptides yielded the complement components C1r and C1s. To confirm that C1r/C1s accounted for the proteolytic activity in the medium, immunoaffinity chromatography was performed. Most of the protease activity adhered to the column, and the eluant was fully active in cleaving IGFBP-5. SDS-polyacrylamide gel electrophoresis with silver staining showed two bands, and IGFBP-5 zymography showed a single 88-kDa band. Amino acid sequencing confirmed that the 88-kDa band contained only C1r and C1s. C1r in the fibroblast medium underwent autoactivation, and the activated form cleaved C1s. C1s purified from the conditioned medium cleaved C(4), a naturally occurring substrate. The purified protease cleaved IGFBP-5 but had no activity against IGFBP-1 through -4. C1 inhibitor, a protein known to inhibit activated C1s, was shown to inhibit the cleavage of IGFBP-5 by the protease in the conditioned medium. In summary, human fibroblasts secrete C1r and C1s that actively cleave IGFBP-5. The findings define a mechanism for cleaving IGFBP-5 in the culture medium, thus allowing release of IGF-I to cell surface receptors.

Substitutions for hydrophobic amino acids in the N-terminal domains of IGFBP-3 and -5 markedly reduce IGF-I binding and alter their biologic actions.

Insulin-like growth factor-binding protein-3 and -5 (IGFBP-3 and -5) have been shown to bind insulin-like growth factor-I and -II (IGF-I and -II) with high affinity. Previous studies have proposed that the N-terminal region of IGFBP-5 contains a hydrophobic patch between residues 49 and 74 that is required for high affinity binding. These studies were undertaken to determine if mutagenesis of several of these residues resulted in a reduction of the affinity of IGFBP-3 and -5 for IGF-I. Substitutions for residues 68, 69, 70, 73, and 74 in IGFBP-5 (changing one charged residue, Lys(68), to a neutral one and the four hydrophobic residues to nonhydrophobic residues) resulted in an approximately 1000-fold reduction in the affinity of IGFBP-5 for IGF-I. Substitutions for homologous residues in IGFBP-3 also resulted in a >1000-fold reduction in affinity. The physiologic consequence of this reduction was that IGFBP-3 and -5 became very weak inhibitors of IGF-I-stimulated cell migration and DNA synthesis. Likewise, the ability of IGFBP-5 to inhibit IGF-I-stimulated receptor phosphorylation was attenuated. These changes did not appear to be because of alterations in protein folding induced by mutagenesis, because the IGFBP-5 mutant was fully susceptible to proteolytic cleavage by a specific IGFBP-5 protease. In summary, residues 68, 69, 70, 73, and 74 in IGFBP-5 appear to be critical for high affinity binding to IGF-I. Homologous residues in IGFBP-3 are also required, suggesting that they form a similar binding pocket and that for both proteins these residues form an important component of the core binding site. The availability of these mutants will make it possible to determine if there are direct, non-IGF-I-dependent effects of IGFBP-3 and -5 on cellular physiologic processes in cell types that secrete IGF-I.

Thrombospondin and osteopontin bind to insulin-like growth factor (IGF)-binding protein-5 leading to an alteration in IGF-I-stimulated cell growth.

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) has been shown to bind to extracellular matrix (ECM) with relatively high affinity, but the ECM components that mediate this interaction have not been identified. These studies show that radiolabeled IGFBP-5 specifically coprecipitates with two ECM proteins, thrombospondin-1 (TSP-1) and osteopontin (OPN). As TSP-1 binds avidly to heparin, as does IGFBP-5, the effect of glycosaminoglycans on the TSP-1/IGFBP-5 interaction was analyzed. Heparan and dermatan sulfate inhibited binding, whereas heparin increased binding. Chondroitin sulfate A and B had no effect. In contrast, both heparin and heparan sulfate significantly inhibited the OPN-IGFBP-5 interaction and chondroitin sulfate A, B, and C had no effect. To determine the region of IGFBP-5 that was involved in each interaction, synthetic peptides that spanned several regions of IGFBP-5 were tested for their capacity to competitively inhibit coprecipitation. A peptide that contained the amino acids between positions 201 and 218 resulted in 76% and 86% inhibition of binding to TSP-1 and OPN, respectively. Three other synthetic peptides that spanned regions ofIGFBP-5 with several charged residues had no effect. IGFBP-5 mutants that contained substitutions for basic residues in the 201-218 region were tested for their ability to bind to TSP-1 or OPN. A mutant with substitutions for amino acids at positions R201 and K202 and a mutant with substitutions for K211, R214, K217, and R218 had the greatest reduction in binding to TSP-1. Mutants containing substitutions for R214 alone and the combined K217A, R218A mutant had the greatest reductions in OPN binding. When the smooth muscle cell growth response to these components was assessed, IGF-I plus IGFBP-5 or the combination of TSP-1 or OPN with IGF-I potentiated the IGF-I effect. The addition of IGFBP-5 to these combinations resulted in further significant growth stimulation. Both OPN and TSP-1 specifically bind to IGFBP-5 with high affinity. These interactions may be important for concentrating intact IGFBP-5 in extracellular matrix and for modulating the cooperative interaction between the IGF-I receptor and integrin receptor signaling pathways.

A protease-resistant form of insulin-like growth factor (IGF) binding protein 4 inhibits IGF-1 actions.

Smooth muscle cells (SMC) secrete a serine protease that cleaves insulin-like growth factor (IGF) binding protein (IGFBP)-4 into fragments that have low affinity for IGF-1. When IGFBP-4 is added to monolayer cultures of cell types that do not secrete this protease, IGF-1 stimulation of DNA synthesis is significantly inhibited. In contrast, if cell types that secrete this protease are used, IGFBP-4 is a much less potent inhibitor. These studies were conducted to determine whether proteolysis of IGFBP-4 accounted for its reduced capacity to inhibit IGF-1-stimulated DNA synthesis. The cleavage site in IGFBP-4 that the SMC protease uses was determined to be lysine120, histidine121. A protease-resistant mutant form of IGFBP-4 was prepared, expressed, purified, and tested for biologic activity using porcine SMC cultures. Addition of the protease-resistant mutant resulted in inhibition of DNA and cell migration responses to IGF-1. The inhibition was concentration dependent and was maximal when 500 ng/ml (20 nM) of the mutant was added with 20 ng/ml (2.8 nM) of IGF-1. When the mutant was added in the absence of IGF-1, it had no activity. The results show that cleavage of IGFBP-4 at lysine120, histidine121 results in inactivation of the ability of IGFBP-4 to bind to IGF-1. Creation of a mutant form of IGFBP-4 that was not cleaved by the protease resulted in inhibition of IGF-1-stimulated actions. The results suggest that IGFBP-4 can act as a potent inhibitor of the anabolic effects of IGF-1 and that the variables that regulate protease activity may indirectly regulate IGF-1 actions.

Binding of insulin-like growth factor (IGF)-binding protein-5 to smooth-muscle cell extracellular matrix is a major determinant of the cellular response to IGF-I.

Insulin-like growth factor-binding protein-5 (IGFBP-5) has been shown to bind to fibroblast extracellular matrix (ECM). Extracellular matrix binding of IGFBP-5 leads to a decrease in its affinity for insulin-like growth factor-I (IGF-I), which allows IGF-I to better equilibrate with IGF receptors. When the amount of IGFBP-5 that is bound to ECM is increased by exogenous addition, IGF-I's effect on fibroblast growth is enhanced. In this study we identified the specific basic residues in IGFBP-5 that mediate its binding to porcine smooth-muscle cell (pSMC) ECM. An IGFBP-5 mutant containing alterations of basic residues at positions 211, 214, 217, and 218 had the greatest reduction in ECM binding, although three other mutants, R214A, R207A/K211N, and K202A/R206N/R207A, also had major decreases. In contrast, three other mutants, R201A/K202N/R206N/R208A, and K217N/R218A and K211N, had only minimal reductions in ECM binding. This suggested that residues R207 and R214 were the most important for binding, whereas alterations in K211 and R218, which align near them, had minimal effects. To determine the effect of a reduction in ECM binding on the cellular replication response to IGF-I, pSMCs were transfected with the mutant cDNAs that encoded the forms of IGFBPs with the greatest changes in ECM binding. The ECM content of IGFBP-5 from cultures expressing the K211N, R214A, R217A/R218A, and K202A/R206N/R207A mutants was reduced by 79.6 and 71.7%, respectively, compared with cells expressing the wild-type protein. In contrast, abundance of the R201A/K202N/R206N/R208A mutant was reduced by only 14%. Cells expressing the two mutants with reduced ECM binding had decreased DNA synthesis responses to IGF-I, but the cells expressing the R201A/K202N/R206N/R208A mutant responded well to IGF-I. The findings suggest that specific basic amino acids at positions 207 and 214 mediate the binding of IGFBP-5 to pSMC/ECM. Smooth-muscle cells that constitutively express the mutants that bind weakly to ECM are less responsive to IGF-I, suggesting that ECM binding of IGFBP-5 is an important variable that determines cellular responsiveness.

Insulin-like growth factor-binding protein-5 is cleaved by physiological concentrations of thrombin.

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) is cleaved by a serine protease that is secreted by fibroblasts and porcine smooth muscle cells (pSMC) in culture. To investigate whether other serine proteases could cleave this substrate at physiologically relevant concentrations, we determined the proteolytic effects of thrombin on IGFBP-5. Human alpha-thrombin (0.0008 NIH U/ml) cleaved IGFBP-5 into 24-, 23-, and 20-kDa non-IGF-I-binding fragments. Cleavage occurred at a physiologically relevant thrombin concentration. The effect was specific for IGFBP-5, as other forms of IGFBPs, e.g. IGFBP-1, IGFBP-2, and IGFBP-4 were not cleaved by thrombin. Although IGFBP-3 was cleaved by thrombin, this effect required a 50-fold greater thrombin concentration. [35S]Methionine labeling followed by immunoprecipitation confirmed that IGFBP-5 that was constitutively synthesized by pSMC cultures was also degraded by thrombin into 24-, 23-, and 20-kDa fragments. The binding of IGF-I to IGFBP-5 partially inhibited IGFBP-5 degradation by thrombin, and an IGF analog that does not bind to IGFBP-5 had no effect. Thrombin did not account for the serine protease activity that had been shown previously to be present in pSMC-conditioned medium. This was proven by showing that 1) no immunoreactive thrombin could be detected in the pSMC-conditioned medium; 2) the IGFBP-5 fragments that were generated by thrombin showed three cleavage sites (Arg192-Ala193, Arg156-Ile157, and Lys120-His121), whereas the serine protease in conditioned medium cleaves IGFBP-5 at a different site; and 3) hirudin had no effect on IGFBP-5 cleavage by the protease in pSMC medium; however, it inhibited IGFBP-5 degradation by thrombin. To determine the physiological significance of IGFBP-5 cleavage, the effect of an IGFBP-5 mutant that is resistant to cleavage by the pSMC protease and has been shown to inhibit IGF-I actions in pSMC was determined. This mutant inhibited IGF-I-stimulated DNA synthesis, but if thrombin was added simultaneously, IGF-I was fully active. In summary, physiological concentrations of thrombin degrade IGFBP-5. Degradation can be blocked by hirudin and is partially inhibited by IGF-I binding. Generation of active thrombin in vessel walls may be a physiologically relevant mechanism for controlling IGF-I bioactivity.

Protease-resistant form of insulin-like growth factor-binding protein 5 is an inhibitor of insulin-like growth factor-I actions on porcine smooth muscle cells in culture.

IGFs are pleiotrophic mitogens for porcine smooth muscle cells (pSMC) in culture. The effects of IGFs on cells are modulated by various insulin-like growth factor-binding proteins (IGFBP). IGFBP-5 is synthesized by pSMC and binds to the extracellular matrix. However, IGFBP-5 is also secreted into conditioned medium of cultured cells and is cleaved into fragments by a concomitantly produced protease. These fragments have reduced affinity for the IGFs and cleavage makes it difficult to assess the role of intact IGFBP-5. To study the consequence of accumulation of intact IGFBP-5 in medium, we determined the cleavage site in IGFBP-5 and prepared a protease resistant mutant. Amino acid sequencing of purified IGFBP-5 fragments suggested Arg138-Arg139 as the primary cleavage site. Arg138-Arg139-->Asn138-Asn139 mutations were introduced to create protease-resistant IGFBP-5, which has the same affinity for IGF-I as the native protein. This mutant IGFBP-5 remained intact even after 24 h of incubation and it inhibited several IGF-I actions when added to pSMC culture medium. The mutant IGFBP-5 (500 ng/ml) decreased IGF-I stimulated cellular DNA synthesis by 84%, protein synthesis by 77%, and it inhibited IGF-I stimulated migration of pSMC by 77%. It also inhibited IGF-I stimulation of IRS-1 phosphorylation. In contrast, the same amount of native IGFBP-5 did not inhibit IGF-I actions. The significance of inhibitory effects of the protease resistant IGFBP-5 was further demonstrated in pSMC transfected with mutant or native IGFBP-5 cDNAs. The mutant IGFBP-5 accumulated in culture medium of transfected cells, while native IGFBP-5 was degraded into fragments, PSMC overexpressing the mutant IGFBP-5 also responded poorly to IGF-I compared with mock transfected cells. IGF-I (5 ng/ml) increased [35S]methionine incorporation into control cells by 36% above the basal level, but it did not significantly change (4%) in pSMC cultures that were producing the mutant IGFBP-5. In conclusion, the accumulation of protease-resistant IGFBP-5 in the medium was inhibitory to IGF-I actions on pSMC. This suggests that proteolysis can prevent IGFBP-5 from acting as an inhibitor of IGF-I-stimulated effects and that it serves as an important mechanism for regulating cellular responsiveness to IGF-I.

Characterization and determination of the relative abundance of two types of insulin-like growth factor binding protein-5 proteases that are secreted by human fibroblasts.

We previously reported that human fibroblasts secrete a protease into their conditioned medium that cleaves insulin-like growth factor binding protein-5 (IGFBP-5) into non-IGF-I binding fragments. Because the protease activity in the fibroblast medium has characteristics of both serine and metalloproteases, the activity was purified and analyzed to determine whether it retained serine or metalloprotease properties. The protease was purified by heparin Sepharose affinity chromatography followed by alpha1 antichymotrypsin affinity or gelatin agarose chromatography. The heparin Sepharose purified material degraded IGFBP-5 into 22-, 17-, and 16-kDa fragments. Amino acid sequencing showed that the 22-kDa fragment contained the amino-terminus of the protein. The protease activity in the fibroblast conditioned medium that was purified by heparin Sepharose was inhibited by both serine and metalloprotease inhibitors. To attempt to separate these activities, the heparin Sepharose purified activity was further purified by gelatin agarose chromatography. The IGFBP-5 protease activity that did not bind to gelatin agarose was inhibited by serine protease inhibitors, such as 3,4 dichloroisocoumain (3,4 DCI), whereas tissue inhibitor metalloprotease-1 (TIMP-1) had minimal activity. When this same pool of protease activity that had been eluted from heparin Sepharose was applied to an alpha1 antichymotrypsin peptide affinity column, the protease activity that bound to the column was inhibited by 3,4 dichloroisocoumain, but was not inhibited by TIMP-1. In contrast, the activity that did not adhere to this column was inhibited by TIMP-1. IGFBP-5 zymography showed that the Mr estimate of the protease that was inhibited by serine protease inhibitors was 92 kDa, whereas gelatin zymography showed that the metalloproteases had Mr estimates of 72, 69, and 55 kDa. When the protease activity in the crude conditioned medium was analyzed by zymography, almost all of the detectable protease had an Mr estimate of 92 kDa, suggesting that the metalloproteases that were detected in the partially purified fractions were inactive in the medium. In summary, fibroblasts secrete a 92-kDa protease that cleaves IGFBP-5 into 22-, 17-, and 16-kDa fragments. The protease inhibitor specificity results, chromatographic characteristics, and zymographic analyses suggest that this is a serine protease. Although metalloproteases are secreted by these cells, the 92-kDa serine protease is the predominate form of activity in the conditioned medium that cleaves IGFBP-5.

Identification of the extracellular matrix binding sites for insulin-like growth factor-binding protein 5.

Fibroblast extracellular matrix (ECM) contains two forms of insulin-like growth factor-binding proteins (IGFBPs), IGFBP-3 and IGFBP-5. These studies were undertaken to identify the regions within IGFBP-5 that mediate its binding to fibroblast ECM. Synthetic peptides were prepared that were homologous with two regions of basic amino acids within IGFBP-5 (Arg201-Arg218 and Ala131-Thr141). Increasing concentrations of both peptides competed with IGFBP-5 for binding to ECM but the Arg201-Arg218 peptide was more potent. Mutagenesis was used to define the effect of substituting for these basic residues on ECM binding. Substitution for two peptide B residues K134A and R136A reduced binding by 40%. Substitution of a single basic residue within the peptide A region (K211N) reduced binding to ECM by 49%. Substitution for K211N, K134A, and R136A reduced binding by 52%. More extensive substitutions in the peptide A region, e.g. K211N,R214A,K217A,R218N, resulted in a greater (e.g. 88%) decrease. The positional location of basic residues appeared to be more important than the total number of substitutions since the mutant K202N,K206A,R207A had a 79% reduction in ECM binding. Two basic regions of IGFBP-5 contribute to its binding to ECM, but the region containing amino acids 201-218 has a greater contribution. ECM binding is mediated by charged residues and acts to stabilize IGFBP-5 by protecting it from proteolysis.

Properties of an insulin-like growth factor-binding protein-4 protease that is secreted by smooth muscle cells.

Smooth muscle cells (SMC) secrete insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) and an IGFBP-4 protease. The purpose of this study was to determine the characteristics of this IGFBP-4 protease and to compare its inhibitor profile to those of IGFBP-5 and IGFBP-2 proteases, which are also present in SMC-conditioned medium. Cultured SMC were exposed to serum-free medium for periods of 24-72 h, and the amount of proteolytic activity in the conditioned medium was assessed by its capacity to degrade pure IGFBP-4. Minimal activity (e.g. < 20% of IGFBP-4 degraded in 24 h at 37 C) was present in conditioned medium unless IGF-I or IGF-II was added. This resulted in more than 60% of the intact IGFBP-4 being degraded in 14 h. The activity was a calcium-dependent serine protease and was inhibited by EDTA or 3,4-dicloroisocoumarin. Calcium, but not zinc, could restore proteolytic activity. Heparin alone inhibited IGFBP-4 proteolysis by more than 60%. When heparin cofactor-II and antithrombin-III (AT-III) were added alone, they each had an effect. The combination of heparin plus AT-III was no more active than heparin alone, but the combination of heparin cofactor-II and heparin resulted in near complete inhibition. Peptides that contained the active sites of AT-III or alpha 1-antichymotrypsin were potent inhibitors of the IGFBP-4 protease. The medium also contained proteolytic activities for IGFBP-2 and IGFBP-5. Comparison of the inhibitor profiles for the IGFBP-4 and IGFBP-5 proteolytic activities revealed major differences, but the IGFBP-2 proteolytic activity was very similar to that of the IGFBP-4 protease. IGFBP-4 zymography showed a band with a molecular mass estimate of 48 kilodaltons. In contrast, when IGFBP-2 was used as the substrate, a single band at 36 kilodaltons was visualized. These data taken together with the protease inhibitor results suggest that the IGFBP-2, IGFBP-4, and IGFBP-5 proteases are members of a similar family of calcium-dependent serine proteases, but they are distinct proteases. As IGFBP-4 is a potent inhibitor of IGF action, and the activity of this protease is regulated by IGF exposure, the protease represents a novel system for regulating the actions of IGF-I in this cell type.

Proteolytic cleavage of insulin-like growth factor binding protein 4 (IGFBP-4). Localization of cleavage site to non-homologous region of native IGFBP-4.

Insulin-like growth factor binding protein 4 (IGFBP-4) is a 24-kDa protein that binds insulin-like growth factor 1 (IGF-1) and IGF-2 with high affinity and inhibits IGF action in vitro. We recently described a protease produced by the B104 neuronal cell line that cleaves IGFBP-4, yielding an approximate 16-kDa immunoreactive protein that binds IGFs with reduced affinity. We analyzed fragments produced by exposing pure IGFBP-4 to the protease to determine potential cleavage sites. Electrospray mass spectrometry and amino acid sequencing indicated the 16-kDa fragment spanned the NH2 terminus of native IGFBP-4 through Lys-120. There was evidence for an additional proteolytic fragment beginning at amino acid 132 and continuing to the COOH terminus. Proteolysis could be blocked by a synthetic peptide that spanned amino acids 117-126 but not by peptides that contained flanking sequences 111-120 or 125-135. Mutagenesis was used to alter the basic residue at position 120. The expressed mutant IGFBP-4 (K120A) was relatively resistant to cleavage, strongly suggesting that residues 120-121 represent the cleavage site. This region of IGFBP-4 is not homologous with other IGFBPs, explaining the apparent specificity of the protease for IGFBP-4. The 16-kDa IGFBP-4 fragment no longer inhibited IGF-1-stimulated thymidine uptake in vitro, suggesting that proteolytic processing of IGFBP-4 may have important functional consequences in vivo.

Role of insulin-like growth factor binding proteins in the control of IGF actions.

The insulin-like growth factor binding proteins have been shown to modify IGF actions. IGFBP-5 binds to extracellular matrix (ECM) and its ability to potentiate IGF activity is dependent upon the amount that is ECM associated. To determine the specific regions of IGFBP-5 that are required for ECM association, site directed mutagenesis has been used to prepare several forms of IGFBP-5. Mutants that have had the amino acids between positions 201 and 218 altered have been useful. Mutation of the lysine 211 resulted in no change in the affinity of IGFBP-5 for ECM or heparin Sepharose; however, it resulted in a major reduction in affinity for IGF-I following heparin binding. Other mutations which disrupted heparin binding also resulted in loss of this affinity shift. Most distruptive were mutations of amino acids 211, 214, 217 and 218 and 202, 206 and 207. Mutation of residues 201 plus 202 had some effect, but substitution for 207, 211, 217 and 218 had no effect. When binding to intact ECM was analyzed, similar results were obtained. This suggests that amino acids 202, 206 and 214 are definitely involved in heparin and ECM binding. When binding to proteoglycans such as tenascin and heparin sulfate proteoglycan was analyzed, similar results were obtained. IGFBP-5 also binds to other proteins in ECM, including type IV collagen and plasminogen activator inhibitor-I. Specific antisera for plasminogen activator inhibitor-1 can coprecipitate IGFBP-5. IGFBPs are degraded by specific proteases. Three proteases that degrade IGFBP-2, -4 and -5 have been characterized. They are serine proteases that cleave these proteins at basic residues. Although several well characterized serine proteases cleave IGFBP-4 or -5, the proteases in cell conditioned media appear to be distinct.

Glycosaminoglycans inhibit degradation of insulin-like growth factor-binding protein-5.

Human dermal fibroblasts secrete insulin-like growth factor-binding protein-3 (IGFBP-3), -4, and -5. Fibroblast-conditioned medium contains minimal intact IGFBP-5, and this form of IGFBP is predominantely a 23-kilodalton fragment, suggesting that the IGFBP-5 fragment is derived from intact IGFBP-5 by proteolysis. In this study we investigated the effects of glycosaminoglycans on IGFBP-5 degradation in fibroblast-conditioned medium. The addition of heparin, heparan sulfate, and dermatan sulfate (100 micrograms/ml) to the medium of fibroblast monolayer cultures inhibited IGFBP-5 degradation, as determined by the conversion of intact IGFBP-5 to a 23-kilodalton fragment. In contrast, hyaluronic acid, keratan sulfate, and chondroitin sulfate-A and -C had no effect. Heparin and heparan sulfate inhibited IGFBP-5 degradation at concentrations of 1 or 2.5 micrograms/ml, but 100 micrograms/ml dermatan sulfate were required. Heparin was also inhibitory in vitro, that is when conditioned medium and heparin were incubated without cells. Experiments with modified forms of heparin showed that O-sulfate groups in the 2 or 3 carbon position were required for heparin to be inhibitory. Completely desulfated heparin had no activity, and N-resulfation of desulfated heparin had only a minimal effect. Dextran sulfate, pentosan polysulfate, and fucoidan, which are composed of different saccharide units but contain O-sulfate groups in the 2 or 3 carbon positions, also inhibited IGFBP-5 degradation. These results demonstrate that heparin-like molecules are important regulators of IGFBP-5 degradation. O-Sulfation of the 2 or 3 position of the saccharide ring is required for inhibitory activity. As glycosaminoglycan side-chains are present in proteoglycans that are present in extracellular matrix and on cell surfaces, these side-chains represent a potential mechanism for regulating IGFBP-5 proteolysis in vivo.

Human fibroblasts secrete a serine protease that cleaves insulin-like growth factor-binding protein-5.

We have previously reported the presence of proteolytic activity in conditioned medium from human fibroblast cultures that cleaves insulin-like growth factor-binding protein-5 (IGFBP-5) into non-IGF-I-binding fragments. Coincubation of IGF-I or IGF-II and IGFBP-5 with fibroblast cultures decreased proteolysis. The protease was purified by heparin-Sepharose affinity chromatography. The purified protease cleaved IGFBP-5 into 22-, 20-, and 17-kilodalton non-IGF-I-binding fragments. Protease inhibitor profiles obtained using partially purified enzyme showed that it was a calcium-dependent serine protease. After chelation with EDTA, the activity could only be partially restored with zinc, indicating that it was probably not a metalloprotease. The protease was specific for IGFBP-5 and did not cleave pure IGFBP-1, -2, -3, or -4. IGF-I and IGF-II caused minimal inhibition of proteolysis in vitro. This suggests that the IGF-I-induced increase in IGFBP-5 in fibroblast medium is only partially due to direct protease inhibition. Heparin, antithrombin-III (AT-III), and heparin cofactor-II had inhibitory activity, and heparin potentiated the activity of AT-III. Synthetic peptides, that contained the active sites of AT-III and alpha 1-antichymotrypsin, were also inhibitory. Peptides containing sequences found in two basic regions of IGFBP-5 were tested, and one had inhibitory activity. In summary, fibroblasts secrete a serine protease that cleaves IGFBP-5 and is specific for this form of IGFBP. The protease has properties that are similar to kallikreins, a family of serine proteases that is known to cleave epidermal and nerve growth factor-binding proteins.