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Background: Insulin-resistant subjects develop more severe and diffuse coronary artery atherosclerosis than insulin-sensitive controls but the mechanisms that mediate this atherosclerosis phenotype are unknown., Research Objective: To determine the metabolic parameters that associate with the severity of coronary atherosclerosis in insulin resistant pigs fed a high fat/high NaCl diet., Key Methods: The primary endpoint was severity of coronary atherosclerosis in adult pigs (Sus scrofa, n = 37) fed a high fat diet that also contained high NaCl (56% above recommended levels) for 1 year., Principal Findings: Twenty pigs developed severe and diffuse distal coronary artery atherosclerosis (i.e., severe = intimal area as a percent medial area > 200% in at least 2 coronary artery cross sections and diffuse distal = intimal area as a percent medial area ≥ 150% over 3 sections separated by 2 cm in the distal half of the coronary artery). The other 17 pigs had substantially less coronary artery atherosclerosis. All 37 pigs had blood pressure in a range that would be considered hypertensive in humans and developed elevations in total and LDL and HDL cholesterol, weight gain, increased backfat, and increased insulin resistance (Bergman Si) without overt diabetes. Insulin resistance was not associated with atherosclerosis severity. Five additional pigs fed regular pig chow also developed increased insulin resistance but essentially no change in the other variables and little to no detectible coronary atherosclerosis. Most importantly, the 20 high fat/high NaCl diet-fed pigs with severe and diffuse distal coronary artery atherosclerosis had substantially greater increases (p< 0.05) in oxidized LDL (oxLDL) and fructosamine consistent with increased protein glycation., Conclusion: In pigs fed a high fat/high NaCl diet, glycated proteins are induced in the absence of overt diabetes and this degree of increase is associated with the development of severe and diffuse distal coronary artery atherosclerosis.

The IGF-I pathway and renin-angiotensin-aldosterone axis are both involved in the pathogenesis of hypertension and atherosclerosis, but no information is available about IGF-I and aldosterone interaction or their potential synergistic effects in vascular smooth muscle cells (VSMCs). The aims of this study were to investigate whether aldosterone influences IGF-I signaling and to determine the mechanism(s) by which aldosterone affects IGF-I function. Aldosterone resulted in significant increases in the Akt (1.87 ± 0.24, P < 0.001), MAPK (1.78 ± 0.13, P < 0.001), p70S6kinase (1.92 ± 0.15, P < 0.001), IGF-I receptor (1.69 ± 0.05, P < 0.01), and insulin receptor substrate-1 (1.7 ± 0.04, P < 0.01) (fold increase, mean ± SEM, n = 3) phosphorylation responses to IGF-I compared with IGF-I treatment alone. There were also significant increases in VSMC proliferation, migration, and protein synthesis (1.63 ± 0.03-, 1.56 ± 0.08-, and 1.51 ± 0.04-fold increases compared with IGF-I alone, respectively, n = 3, P < 0.001). Aldosterone induced osteopontin (OPN) mRNA expression and activation of αVβ3-integrin as well as an increase in the synthesis of IGF-I receptor. The enhancing effects of aldosterone were inhibited by eplerenone (10 μmol/liter), actinomycin-D (20 nmol/liter), and an anti-αVβ3-integrin antibody that blocks OPN binding. The antioxidant N-acetylcysteine (2 mmol/liter) completely inhibited the ability of aldosterone to induce any of these changes. In conclusion, our results show that aldosterone enhances IGF-I signaling and biological actions in VSMCs through induction of OPN followed by its subsequent activation of the αVβ3-integrin and by increasing IGF-I receptor. These changes are mediated in part through increased oxidative stress. The findings suggest a new mechanism by which aldosterone could accelerate the development of atherosclerosis.

Vascular smooth muscle cells maintained in normal (5.6 mm) glucose respond to insulin-like growth factor-I (IGF-I) with increased protein synthesis but do not proliferate. In contrast, hyperglycemia alters responsiveness to IGF-I, resulting in increased SHPS-1 phosphorylation and assembly of a signaling complex that enhances MAPK and phosphatidylinositol 3-kinase pathways. Hyperglycemia also reduces the basal IRS-1 concentration and IGF-I-stimulated IRS-1-linked signaling. To determine if failure to down-regulate IRS-1 alters vascular smooth muscle cell (VSMC) responses to IGF-I, we overexpressed IRS-1 in VSMCs maintained in high glucose. These cultures showed reduced SHPS-1 phosphorylation, transfer of SHP-2 to SHPS-1, and impaired Shc and MAPK phosphorylation and cell proliferation in response to IGF-I. In vitro studies demonstrated that SHPS-1 was a substrate for type I IGF receptor (IGF-IR) and that IRS-1 competitively inhibited SHPS-1 phosphorylation. Exposure of VSMC cultures to a peptide that inhibited IRS-1/IGF-IR interaction showed that IRS-1 binding to IGF-IR impairs SHPS-1 phosphorylation in vivo. IRS-1 also sequestered SHP-2. Expression of an IRS-1 mutant (Y1179F/Y1229F) reduced IRS-1/SHP-2 association, and exposure of cells expressing the mutant to the inhibitory peptide enhanced SHPS-1 phosphorylation and SHP-2 transfer. This result was confirmed by expressing an IRS-1 mutant that had both impaired binding to IGF-IR and to SHP-2 IGF-I increased SHPS-1 phosphorylation, SHP-2 association with SHPS-1, Shc MAPK phosphorylation, and proliferation in cells expressing the mutant. We conclude that IRS-1 is an important factor for maintaining VSMCs in the non-proliferative state and that its down-regulation is a component of the VSMC response to hyperglycemic stress that results in an enhanced response to IGF-I.

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.

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.

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.

Insulin-like growth factor binding protein-5 (IGFBP-5) and thrombospondin-1 (TS-1) are both present in extracellular matrix (ECM). Both proteins have been shown to bind to one another with high affinity. The purpose of these studies was to determine how the interaction between IGFBP-5 and TS-1 modulates IGF-I actions in porcine aortic smooth muscle cells (pSMC) in culture. The addition of increasing concentrations of TS-1 to pSMC cultures enhanced the protein synthesis and cell migration responses to IGF-I; whereas the addition of IGFBP-5 alone resulted in minimal changes. In contrast, the addition of IGFBP-5 to cultures that were also exposed to IGF-I and TS-1 resulted in inhibition of protein synthesis. When the cell migration response was assessed, the response to IGF-I plus TS-1 was also significantly inhibited by the addition of IGFBP-5, whereas 1.0 microg/ml of IGFBP-5 alone had no effect on the response to IGF-I. To determine the molecular mechanism by which this inhibition occurred, a mutant form of IGFBP-5 that does not bind to IGF-I was tested. This mutant was equipotent compared to native IGFBP-5 in its ability to inhibit both protein synthesis and cell migration responses to IGF-I plus TS-1 thus excluding the possibility that IGFBP-5 was inhibiting the response to TS-1 and IGF-I by inhibiting IGF-I binding to the IGF-I receptor. To determine if an interaction between TS-1 and IGFBP-5 was the primary determinant of the inhibitory effect of IGFBP-5, an IGFBP-5 mutant that bound poorly to TS-1 was utilized. The addition of 1.0 microg/ml of this mutant did not inhibit the protein synthesis or cell migration responses to IGF-I plus TS-1. To determine the mechanism by which IGFBP-5 binding to TS-1 inhibited cellular responses to TS-1 plus IGF-I, TS-1 binding to integrin associated protein (IAP) was assessed. The addition of IGFBP-5 (1.0 microg/ml) inhibited TS-1-IAP association. In contrast, a mutant form of IGFBP-5 that bound poorly to TS-1 had a minimal effect on TS-1 binding to IAP. Further analysis showed that IGFBP-5 addition altered the ability of TS-1 to modulate the SHPS-1/IAP interaction. When the IGFBP-5 mutant that did not bind to IGF-I was incubated with TS-1 and IGF-I, it inhibited the capacity of TS-1 to enhance the IGF-I receptor phosphorylation and MAP kinase activation in response to IGF-I. In contrast, the IGFBP-5 mutant that did not bind to TS-1 had no effect on IGF-I stimulated IGF-I receptor phosphorylation or MAP kinase activation. These results indicate that IGFBP-5 inhibits the binding of TS-1 to IAP, and this results in an alteration of the ability of TS-1 to modulate the disruption of the IAP/SHPS-1 interaction which leads to attenuation of the ability of TS-1 to enhance cellular responsiveness to IGF-I., (Copyright 2005 Wiley-Liss, Inc.)

Insulin-like growth factor (IGF)-I is a pleiotropic hormone that regulates vascular smooth muscle cell (VSMC) migration, proliferation, apoptosis, and differentiation. These actions are mediated by the IGF-I receptor. How activation of the same receptor by the same ligand leads to these diverse cellular responses is not well understood. Here we describe a novel mechanism specifying VSMC responses to IGF-I stimulation, distinctive for the pivotal roles of local IGF-binding proteins (IGFBPs). The role of local IGFBPs was indicated by comparing the activities of IGF-I and des-1-3-IGF-I, an IGF-I analog with reduced binding affinity to IGFBPs. Compared with IGF-I, des-1-3-IGF-I was more potent in stimulating DNA synthesis but much less potent in inducing directed migration of VSMCs. When the effects of individual IGFBPs were tested, IGFBP-2 and IGFBP-4 were found to inhibit IGF-I-stimulated DNA synthesis and migration. IGFBP-5 had an inhibitory effect on IGF-I-stimulated DNA synthesis, but it strongly potentiated IGF-I-induced VSMC migration. By using a non-IGF-binding IGFBP-5 mutant and an IGF-I-neutralizing antibody, it was demonstrated that IGFBP-5 also stimulates VSMC migration in an IGF-independent manner. This effect of IGFBP-5 was inhibited by soluble heparin and by treating cells with heparinase. Mutation of the heparin-binding motif of IGFBP-5 reduced its migration promoting activity. These findings suggest that local IGFBPs are important determinants of cellular responses to IGF-I stimulation, and a key player in this paradigm is IGFBP-5. IGFBP-5 not only modulates IGF-I actions, but it also stimulates cell migration by interacting with cell-surface heparan sulfate proteoglycans.

IGF binding protein-5 (IGFBP-5) is an important trophic factor for controlling the actions of IGF-I in human dermal fibroblasts and porcine aortic smooth muscle cells. When IGFBP-5 is associated with extracellular matrix, it acts to enhance the cell growth response to IGF-I. The amount of IGFBP-5 within the extracellular matrix is related in part to the amount that is present in conditioned medium, which is related to its rate of synthesis and degradation. A serine protease that degrades IGFBP-5 is present in the conditioned medium of both of these cell types. Because the IGFBP-5 protease activity that is secreted by fibroblasts has been shown to be due to the complement components C1r and C1s, these studies were undertaken to determine whether smooth muscle cells also secreted these proteases and to identify some of the factors that regulate their secretion by both cell types. Both smooth muscle cells and human fibroblasts were shown to release C1r and C1s into conditioned medium. Both C1r and C1s were detected as activated forms, as determined by SDS-PAGE using reducing conditions. The addition of increasing concentrations of either IL-1beta or TNFalpha resulted in increased synthesis of C1s by fibroblasts and smooth muscle cells, and they each increased C1r release. TNFalpha (50 ng/ml) and IL-1beta (20 ng/ml) resulted in maximum stimulation of release of both proteases. In contrast dexamethasone (10(-7) M) had no effect on C1s release and stimulated C1r release only by smooth muscle cells. To determine the physiological significance of this increase in C1r and C1s, the amount of IGFBP-5 protease activity that was present in conditioned medium was determined before and after exposure to TNFalpha, IL-beta, and dexamethasone. All three compounds resulted in an increase in the amount of IGFBP-5 proteolytic activity. Dexamethasone inhibited the release of C(1) inhibitor from fibroblasts, and this contributed to the net increase in proteolytic activity. TNFalpha inhibited the smooth muscle cell DNA synthesis response to IGF-I, but the effect of IGF-I was partially restored by the addition of C1 inhibitor. In conclusion, both C1r and C1s are released by cultured fibroblasts, and the release of each into fibroblast or porcine smooth muscle cells medium is stimulated by TNFalpha and IL-1beta. This increase results in a net increase in IGFBP-5 proteolysis, which has the potential to modify IGF-I and IGFBP-5 actions.

Objective: The complement component C1s is present in dog joint fluid in an activated state. Since C1s degrades insulin-like growth factor binding protein 5 (IGFBP-5), we undertook to determine whether inhibiting C1s in joint fluid would result in an increase in the amount of intact IGFBP-5 and IGF-1 in cartilage and joint fluid, and whether C1s inhibition would be associated with a reduction in cartilage destruction during the development of osteoarthritis (OA)., Methods: Twenty-two dogs were randomized to 3 treatment groups. All dogs underwent anterior cruciate ligament transection and were exercised. Dogs received 1 of 3 treatments: buffer alone (controls; n = 6); PB-145, a peptide derived from the sequence of antithrombin III (n = 9); and pentosan polysulfate (PPS; n = 7). PB-145 or saline was injected into the joint space 3 times per week for 3 weeks. PPS was injected intramuscularly weekly for 3 weeks., Results: Joint histology showed preservation of chondrocytes and a smooth joint surface in the animals treated with PB-145 and PPS. Mankin scoring showed statistically significant reductions in joint destruction with PB-145 and PPS treatments (P < 0.01) compared with buffer control. Mean active collagenase concentrations were decreased by these two treatments. Immunoblotting of joint fluid showed that both treatments increased concentrations of intact IGFBP-5. Direct analysis of IGFBP-3 and IGFBP-5 protease activity showed that IGFBP-5 was degraded more rapidly and that PB-145 and PPS inhibited the degradation of both proteins. Total IGF-1 concentrations in joint fluid were increased 5.6-5.8-fold by these two treatments. Analysis showed that C1s was being activated in joint fluid and that its activation was inhibited by the addition of PB-145 or PPS., Conclusion: The findings suggest that direct inhibition of the serine protease C1s results in increased concentrations of intact IGFBP-5 and that proteolysis of IGFBP-3 is also inhibited, probably by the inhibition of some other protease. This increase in concentrations of intact IGFBP-3 and IGFBP-5 leads to an increase in IGF-1 which is associated with an improvement in joint architecture during the development of OA.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Insulin-like growth factor (IGF)-binding protein 1 (IGFBP-1) contains an Arg-Gly-Asp (RGD) integrin recognition sequence. In vitro mutagenesis was used to alter this RGD sequence to Trp-Gly-Asp (WGD). Migration of Chinese hamster ovary (CHO) cells expressing the wild-type protein was more than 3-fold greater in 48 hr compared with cells expressing the WGD mutant form of IGFBP-1. Similarly, wild-type IGFBP-1 added to the media of control CHO cells stimulated migration 2-fold compared with the WGD protein. A synthetic RGD-containing peptide, when added to the medium with wild-type IGFBP-1, blocked the effect of IGFBP-1 on cell migration. The addition of IGF-I to the culture medium had no effect on the migration of cells expressing IGFBP-1 or vector alone. Affinity chromatography of 125I-labeled CHO cell membrane proteins, using IGFBP-1 coupled to agarose, identified the alpha 5 beta 1 integrin (fibronectin receptor) as the only cell surface molecule capable of binding IGFBP-1 in an RGD-dependent manner. Furthermore, wild-type IGFBP-1, but not the WGD mutant form, could be coprecipitated from CHO cells with an antibody directed against the alpha 5 integrin subunit. These studies demonstrate that IGFBP-1 stimulates CHO cell migration and binds to the alpha 5 beta 1 integrin receptor, both by an RGD-dependent mechanism. The effect of IGFBP-1 on migration is independent of IGF-I and is probably mediated through the alpha 5 beta 1 integrin.

Insulin-like growth factor-binding proteins (IGFBPs) are a family of secreted proteins that bind insulin-like growth factors I and II (IGFs I and II) and are capable of modulating IGF actions on target cells. We have shown previously that C2 myoblasts secrete a single approximately 29-kDa IGFBP during their terminal differentiation (Tollefsen, S. E., Lajara, R., McCusker, R. H., Clemmons, D. R., and Rotwein, P. (1989) J. Biol. Chem. 264, 13810-13817). In this study, we have purified the protein from C2 cell-conditioned media by conventional and IGF-affinity chromatography, cloned its cDNA by PCR-based and traditional library screening, and identified it as mouse IGFBP-5. The resultant 5561 nucleotide cDNA encodes a 252-amino acid mature protein (predicted M(r) approximately 28,400) that is 97% identical to rat and human IGFBP-5. In differentiating C2 myoblasts and in F3 azamyoblasts the > 6-kilobase IGFBP-5 mRNA accumulates concomitantly with induction of myogen mRNA, an early marker of muscle differentiation. Ligand blot analysis shows that IGFBP-5 protein is secreted within 12 h of the onset of differentiation in these cells and that it is the only IGFBP produced in several fusing skeletal muscle cell lines. In vivo, IGFBP-5 transcripts are expressed in a variety of mouse tissues including striated muscle, but, unlike other IGFBPs, it is barely detectable in liver. IGFBP-5 is more conserved than other IGFBPs in mammals; its conserved structure and sequence also extends to non-mammalian vertebrates. Hybridization of a mouse BP5 coding region probe to RNA from several chicken and Xenopus tissues demonstrated similarly sized transcripts in these species. A partial Xenopus cDNA is identical in 38/45 deduced amino acids to the mammalian proteins. Identification of an IGF-binding protein that is produced during myoblast differentiation provides a model system in which to study the potential modulatory role of IGFBPs in development.

Insulin-like growth factor binding proteins (IGFBPs) have been shown to serve as carrier proteins for the insulin-like growth factors (IGFs) and to modulate their biologic effects. Since extracellular matrix (ECM) has been shown to be a reservoir for IGF-I and IGF-II, we examined the ECM of cultured human fetal fibroblasts and found that IGFBP-5 was incorporated intact into ECM, while mostly inert proteolytic fragments were found in the medium. In contrast, two other forms of IGFBP that are secreted by these cells were either present in ECM in minimal amounts (IGFBP-3) or not detected (IGFBP-4). Likewise, when purified IGFBPs were incubated with ECM, IGFBP-5 bound preferentially. IGFBP-5 was found to bind to types III and IV collagen, laminin, and fibronectin. Increasing salt concentrations inhibited the binding of IGFBP-5 to ECM and accelerated the release of IGFBP-5 from ECM, suggesting an ionic basis for this interaction. ECM-associated IGFBP-5 had a sevenfold decrease in affinity for IGF-I compared to IGFBP-5 in solution. Furthermore, when IGFBP-5 was present in cell culture substrata, it potentiated the growth stimulatory effects of IGF-I on fibroblasts. When IGFBP-5 was present only in the medium, it was degraded to a 22-kD fragment and had no effect on IGF-I-stimulated growth. We conclude that IGFBP-5 is present in fibroblast ECM, where it is protected from degradation and can potentiate the biologic actions of IGF-I. These findings provide a molecular explanation for the association of the IGF's with the extracellular matrix, and suggest that the binding of the IGF's to matrix, via IGFBP-5, may be important in mediating the cellular growth response to these growth factors.

Human IGFBP-1 is phosphorylated by cells in culture and is present in both phosphorylated and nonphosphorylated forms in human fetal serum and amniotic fluid. We have found immunoprecipitable [32P]IGFBP-1 in the conditioned media of both Chinese hamster ovary (CHO) cells (stabley transfected and secreting human IGFBP-1) and human hepatoma (HepG2) cells metabolically labelled with [32P]orthophosphate. Phosphoamino acid analysis of this [32P]IGFBP-1 demonstrates that only serine residues are phosphorylated. Four phosphorylated isoforms of IGFBP-1 can be separated from one nonphosphorylated form by nondenaturing gel electrophoresis. Since we have shown that the nonphosphorylated form of IGFBP-1 has a lower affinity for IGF-I compared to phosphorylated forms and a greater potentiating effect of IGF-I actions, we determined which serine residues in human IGFBP-1 are phosphorylated. After metabolically labelling IGFBP-1 with 32P, the purified phosphoprotein was digested first with trypsin and then with endoproteinase Glu-C. By radiosequencing the resulting 32P-labelled phosphopeptides, we found 3 serine residues to be phosphorylated. Approximately 70% of incorporated 32P was attributed to Ser101, while Ser169 accounted for approximately 25% and Ser119 for 5%. To investigate the physiologic importance of Ser101, this residue (and the nonphosphorylated Ser98) were changed to alanine by site directed mutagenesis of a human IGFBP-1 expression vector, followed by transfection into CHO cells. The [Ala98,101]IGFBP-1 purified from the conditioned media of these cells had the following characteristics: 1) when labelled with [32P]orthophosphate, it contained 63% less radioactivity than wild type IGFBP-1; 2) when analyzed by nondenaturing gel electrophoresis, it contained none of the most rapidly migrating and most rapidly migrating and most highly phosphorylated isoform, more of the nonphosphorylated isoform, and more of the most slowly migrating phosphorylated isoform; and 3) its affinity for IGF-I was reduced 2.5-fold and was midway between wild type IGFBP-1 from transfected CHO cells and dephosphorylated IGFBP-1. We conclude that Ser101 represents the major site of phosphorylation of IGFBP-1 and that while phosphorylation of Ser101 increases affinity of IGFBP-1 for IGF-I, phosphorylation of Ser169 and/or Ser119 also contributes to the high affinity of fully phosphorylated IGFBP-1.

Serine phosphorylation of insulin-like growth factor binding protein-1 (IGFBP-1) has been shown to alter its affinity for the insulin-like growth factors (IGF-I and IGF-II) and to modify its capacity to modulate cellular responses to the IGFs. Because of this, we determined the sites of serine phosphorylation. Purification of 32P-labeled IGFBP-1 was followed by digestion with trypsin and endoproteinase Glu-C and radiosequencing of labeled peptides. Three serines were found to be phosphorylated, with Ser101, Ser119, and Ser169 containing 70%, 5%, and 25% of the incorporated 32P, respectively. A mutated IGFBP-1, substituting alanine for serine at positions 98 and 101, was expressed in CHO cells. On nondenaturing gels, the wild type protein migrated as five isoforms (one non-phosphorylated and four phosphorylated). However, in the mutated protein, the most rapidly migrating band (a phosphorylated form) was not present. The cells containing the mutated cDNA incorporated 60% less 32P into immunoprecipitable IGFBP-1. The mutated protein had a 3-fold reduction in affinity for IGF-I compared to the wild type protein. We conclude that Ser101 represents the major site of phosphorylation containing 63% of the total 32P incorporated and that phosphorylation of Ser101 is important for maintenance of high affinity binding for this growth factor.

Human fibroblasts secrete insulin-like growth factor-binding proteins (IGFBPs) that can modify insulin-like growth factor (IGF) I action. We have determined the molecular identities of three forms of IGFBPs that are secreted by human fibroblasts in vitro. Ligand blot analysis of fibroblast conditioned media revealed that the M(r) 43,000 and 39,000 forms were the most abundant, but that M(r) 31,000 and 24,000 forms were also present. An antiserum that was specific for IGFBP-5 reacted with the M(r) 31,000 form, and an IGFBP-4-specific antiserum recognized only the M(r) 24,000 form. The M(r) 39,000 and 43,000 forms were detected by IGFBP-3 antiserum. Further proof that fibroblasts synthesized these forms of IGFBPs was obtained by Northern blotting. A cDNA probe for IGFBP-3 hybridized with a 2.4-kilobase (kb) transcript, whereas a cDNA probe for IGFBP-5 recognized a single 6.0-kb transcript, and an IGFBP-4 cDNA probe recognized 2.2- and 2.0-kb transcripts. IGF-I and -II caused a minimal (less than 43%) increase in IGFBP-5 mRNA abundance and had no effect on IGFBP-4 mRNA abundance. IGF-I and -II (100 ng/ml) stimulated 6-8-fold increases in IGFBP-5 levels, whereas IGFBP-4 was inhibited. Insulin failed to elicit any change in IGFBP-5, suggesting that binding of the IGFs to IGFBPs was required to detect the increase. Immunoblotting for IGFBP-5 revealed an M(r) 23,000 (non-IGF-I-binding) fragment. To determine if the IGFs were influencing proteolytic degradation of IGFBP-5, pure IGFBP-5 was added to fibroblast cultures and incubated for 4 h at 37 degrees C. The amount of fragment formation was attenuated by the presence of IGF-I and -II, but not insulin, suggesting that this is a mechanism by which the IGFs act to modulate IGFBP-5 concentration. In contrast to the IGFs, forskolin, which increased IGFBP-4 and -5 mRNA abundance and secretion, had no effect on fragment formation. The results show that human fibroblasts synthesize and secrete IGFBP-3, -4, and -5 and that changes in intracellular cAMP regulate synthesis, whereas the IGFs regulate IGFBP-4 and -5 levels by post-transcriptional mechanisms.

Insulin-like growth factor binding proteins (IGFBPs) are secreted by several cell types and can modify IGF actions. Mandin-Darby Bovine Kidney (MDBK) cells have been shown to secrete a 34,000 Da form of IGF binding protein whose N-terminal sequence is similar to a form of IGFBP purified from rat BRL-3A cells that has recently been named IGFBP-2. These studies report the complete amino acid sequence of bovine IGFBP-2 and compare its functional properties with human IGFBP-1. The protein is 81% identical to rat IGFBP-2. When compared with both rat IGFBP-2 and human IGFBP-1, the positions of all 18 cysteine residues are conserved. Similarly an RGD sequence is present near the carboxyl terminus in both proteins. IGFBP-2 has a higher affinity for IGF-II than for IGF-I and its affinity for both forms of IGF is greater than for human IGFBP-1. Like IGFBP-1 the protein can enhance the DNA synthesis response of porcine aortic smooth muscle cells to IGF-I; however, IGFBP-2 was much less potent. The maximum potentiation of the IGF-mediated mitogenic response that could be achieved was approximately 42% that of IGFBP-1. This potentiation is dependent upon a factor contained in platelet poor plasma and if this factor is omitted from the incubation medium, IGFBP-2 inhibits DNA synthesis. The purification of IGFBP-2 will allow more detailed comparisons to be made between it and other forms of IGFBPs in physiologic test systems.

Insulin-like growth factor binding protein-2 (IGFBP-2) is one of a family of IGFBPs that are present in extracellular fluids, and binds both IGF-II and IGF-I with high affinity. These studies were conducted to determine the nutritional and hormonal variables that regulate plasma IGFBP-2 concentrations in humans. The mean plasma IGFBP-2 concentration for 38 normal adult subjects was 150 +/- 61 micrograms/L and was 4.7-fold greater than their mean fasting IGFBP-1 value. Mean IGFBP-2 values in cord sera of 26 normal term infants was 3.8-fold greater than the normal adult mean value. Likewise, the mean value for 44 hypopituitary adults was increased 2-fold compared to normal. There was no suppression of IGFBP-2 values in acromegaly. Normal adult subjects showed minimal fluctuations (less than 2-fold changes) in plasma IGFBP-2 concentrations during a 48-h sampling period. These changes were significantly less than the changes that occurred in plasma IGFBP-1 during the same interval. Plasma IGFBP-2 did not change significantly post prandially or after a glucose infusion. Extreme insulin deficiency, after 9 days of fasting, was associated with a 1.7-fold increase in plasma IGFBP-2. Administration of GH, which is known to cause a major decrease in plasma IGFBP-1 and in IGFBP-2 in hypophysectomized animals, did not result in a change in calorically restricted normal adult subjects, suggesting that a normal caloric intake is required for GH to suppress IGFBP-2. In summary, these results show that plasma IGFBP-2 is regulated differently than IGFBP-1. Acute stimulation of insulin secretion does not suppress IGFBP-2, and there is much less daily fluctuation compared to IGFBP-1. These findings suggest that plasma IGFBP-2 levels are more stable than IGFBP-1, and therefore IGFBP-2 may serve as a larger reservior that is available for IGF transport.

Several growth hormone-independent 25-31,000 kD insulin-like growth factor binding proteins (IGF-BPs) have been identified in plasma, extravascular fluids, and various cell-conditioned media. Cultured human decidual cells release three IGF-BPs with 24,000, 30,000, and 34,000 Mr. Using ligand blot analysis and an RIA for the 30,000-Mr form (IGF-BP-1), we examined the effects of IGF-I (10-1,000 ng/ml), insulin (10-10,000 ng/ml), and relaxin (10-250 ng/ml) on decidual cell IGF-BP release after 120 h of hormone exposure. IGF-I inhibited release of both IGF-BP-1 and the 24,000 Mr form. Inhibition of IGF-BP-1 release was noted after 48 h of treatment and was progressive throughout the subsequent 120 h. Insulin stimulated a fourfold increase in release of the 24,000-Mr protein while inhibiting IGF-BP-1 release comparable to IGF-I, alpha-IR3, a monoclonal antibody to the IGF-I receptor, blocked approximately 33% of the IGF-I response but had no effect on insulin-mediated IGF-BP-1 inhibition. Relaxin stimulated a 2.4-fold increase in release of the 24,000-Mr form and a 16-fold increase in the 30,000-Mr protein after 120 h. Stimulation of the 30,000-Mr protein was inhibited by the addition of cycloheximide (50 micrograms/ml). Both IGF-I and insulin also blocked the relaxin-mediated increase in IGF-BP-1. These studies suggest that three structurally related proteins differentially regulate IGF-BP secretion possibly via activation of distinct receptor subtypes.

Recent studies have shown that human decidual explants synthesize a 25,272 dalton form of insulin-like growth factor binding protein (IGFBP-1) that is identical to the most abundant form of IGFBP detected in human amniotic fluid. To determine whether decidual cells also secrete other structurally distinct forms of IGFBP, conditioned medium obtained from human decidual cells was analyzed by ligand blotting and immunoblotting using antisera that were specific for IGFBP-1 or 2. IGFBP-2 is a form of binding protein that is structurally distinct from IGFBP-1. Ligand blotting analysis (which detects all forms of IGFBPs) showed that 3 forms of IGFBP with Mr estimates of 34,000, 30,000 and 24,000 were present in basal, unstimulated, conditioned media. Immunoblotting showed that the 30,000 Mr form reacted with an antibody that is specific for IGFBP-1, whereas the 34,000 Mr form reacted with an antibody that is specific for IGFBP-2. The 24,000 Mr band did not react with antisera to either IGFBP-1 or 2. Basal IGFBP-1 release from human decidual cultures, as measured by RIA, showed a significant decrease during the 5 day period from 34 +/- 1.6 on day 1 to 12 +/- 1.9 ng/ml on day 5. Exposure of the cells to (Bu)2cAMP (1.0 mM) for 5 days had no significant effect on IGFBP-1 release during the first day of exposure, but caused a progressive stimulation of release during the subsequent 4 days. By day 5, exposure to (Bu)2cAMP induced a 5- to 6-fold increase in IGFBP-1 release that was completely inhibited by exposure to cycloheximide. Exposure of the cells to cholera toxin (0.1 micrograms/ml) for 4 days caused a 3.7-fold increase in IGFBP-1 release. The stimulation of IGFBP-1 release by (Bu)2cAMP was completely inhibited by simultaneous exposure to insulin (100 ng/ml) and/or IGF-I (100 ng/ml) (day 5 control = 35 ng/ml, (Bu)2cAMP alone = 248 ng/ml, insulin + (Bu)2cAMP = 36 ng/ml, or IGF-I + (Bu)2cAMP = 10.1 ng/ml). Hydrocortisone and the phorbol ester (phorbol myristate acetate) caused no significant changes in basal IGFBP-1 release but prevented the decrease in the basal rate of release that occurred between day 1 and 5. Ligand blotting studies showed that the 24,000 Mr form of binding protein was also stimulated by (Bu)2cAMP (3.3-fold increase); however, cholera toxin and phorbol myristate acetate were without effect. In contrast, the release of the 34,000 Mr form was unaffected by exposure to any of these agents, suggesting that regulation of this form of IGFBP is distinct from the other 2 forms.(ABSTRACT TRUNCATED AT 400 WORDS)

A reliable technique for purification of crude tissue extracts and analysis for TRH by HPLC, TLC, radioimmunoassay and bioassay is presented. We conclude that authentic TRH is present throughout the mammalian brain and accounts for much of the TRH-like immunoreactivity in several peripheral organs, but that it is not present in urine, placenta, or pineal, and its concentration in blood is some 250-fold less than a value obtained from a direct radioimmunoassay of a sample of blood.

The insulin-like growth factors IGF-I and IGF-II circulate in blood bound to carrier proteins. The higher molecular mass IGF-binding protein complex (150 kDa) is composed of subunits, and one subunit that forms this complex is growth hormone dependent. In addition, many cell types and tissues secrete another form of IGF binding protein that is not growth hormone dependent. Both forms of the IGF binding protein are believed to inactivate the IGFs and to function as delivery systems to tissues. This conclusion was based on studies that determined the effects of impure preparations of these binding proteins or that examined the effect of these proteins only on the insulin-like actions of the IGFs. We report here that a pure preparation of the extracellular form of the IGF binding protein (purified from human amniotic fluid) markedly potentiated replication of several cell types in response to human IGF-I. Secondary cultures of human, mouse, and chicken embryo fibroblasts as well as porcine aortic smooth muscle cells showed marked enhancement of their DNA synthesis response (2.8- to 4.4-fold increases) to IGF-I in the presence of this protein. These responses were synergistic since the sum of the responses to either IGF-I or to the binding protein alone was between 8 and 17% of the increase obtained in cultures exposed to both peptides. The binding protein not only potentiated the DNA synthesis response but also enhanced the increase in cell number in response to IGF-I. This stimulation is specific for growth factors that bind to the binding protein since incubation with insulin, which binds to the type I IGF receptor but not to the binding protein, did not result in potentiation of this response. We conclude that a form of IGF binding protein that is present in extracellular fluids and is secreted by many types of cells can markedly potentiate the cellular response to IGF-I.

Current assays for glycine-directed, peptide-amidating enzymes have several shortcomings. In this report, we describe a rapid, sensitive microassay for amidating activity which overcomes these disadvantages. Tissue homogenates are incubated in the presence of D-Tyr-Val-Gly-OH and the product, D-Tyr-Val-NH2, is measured by radioimmunoassay using an antiserum with an affinity for the product, D-Tyr-Val-NH2 (Kaff 2 X 10(8) L/M), 4 orders of magnitude higher than for the substrate, D-Tyr-Val-Gly-OH (Kaff 4 X 10(4) L/M), and 3 orders of magnitude higher than for the deamidated product D-Tyr-Val-OH (Kaff 8 X 10(5) L/M). Addition of N-ethylmaleimide (0.5 mM) to the enzyme incubates prevents the degradation of D-Tyr-Val-NH2 and permits the measurement of enzymatic activity in crude homogenates. This assay is sensitive enough to permit the measurement of amidating activity in crude rat brain homogenates containing as little as 4-8 micrograms protein.

1. Artiodactyla haptoglobins (Hps), goat, sheep and cattle (family Bovidae), and pig (family Suidae) were structurally characterized. 2. The polymeric Hp systems of goat, sheep and cattle were similar to the polymeric human Hp system, while the monomeric system of pig was more comparable to the monomeric human form. 3. All members of the Artiodactyla (family Bovidae) examined exhibited a large polypeptide subunit, comparable to that of the beta subunit of human Hp. 4. In addition, a small subunit, similar in molecular weight to the human alpha 2 subunit, was demonstrated. Pig Hp was shown to have two subunits, one slightly larger than the human beta subunit and the other intermediate in size to the human alpha 1 and alpha 2 subunits. 5. Immunoelectrophoretic and immunodiffusion studies indicated complete cross reactivity among the polymeric Artiodactyla Hps. 6. The polymeric Hps do not, however, cross react with the monomeric pig Hp.

The enzymes catalyzing the breakdown of TRH are soluble and dependent upon active sulfhydryl groups. One of these enzymes is a pyroglutamyl aminopeptidase (EC 3.4.11.8) (apparent molecular weight 60,000 daltons) with a specificity restricted to pyroglutamyl peptide bonds. The second enzyme is a prolyl endopeptidase (apparent molecular weight 70,000 daltons) with a specificity restricted to proline peptide bonds wherein the proline is preceded by an alpha adjacent amino acid possessing a blocked N-terminus. Substrate requirements for this latter enzyme are identical to those reported previously for the TRH deamidase of rat brain, the prolyl endopeptidase of rabbit brain and the post proline cleaving enzyme of rat brain. We conclude that this enzymatic activity variously described as TRH deamidase, post proline cleaving enzyme, and prolyl endopeptidase is that of a single enzyme best denoted as a prolyl endopeptidase (EC 3.4.22.16, proposed). The pyroglutamyl aminopeptidase has a Km for TRH of 45 micro M as compared to a Km for TRH of 2400 micro M for the prolyl endopeptidase. Total enzymatic activity of the prolyl endopeptidase, however, is approximately 3500 nmol/h/rat brain with the total enzymatic activity of the pyroglutamyl aminopeptidase being approximately 600 nmol/h/rat brain. The 5- to 6-fold higher affinity of the pyroglutamyl aminopeptidase for TRH suggests that of these two catabolic pathways, removal of the N-terminal pyroglutamyl moiety is likely to be the more important pathway for the initial catabolism of TRH in rat brain. Analysis of substrate specificity permitted the synthesis of several effective competitive inhibitors of the two enzymes. Of these, the most effective inhibitors of the pyroglutamyl aminopeptidase were p-glu-NH2 (Ki 185 micro M) and p-glu-his-OCH3 (Ki 16.5 micro M). The most effective inhibitors of the prolyl endopeptidase were Ac-gly-pro-NH2 (Ki 1143 micro M) and Z-gly-pro-NH2 (Ki 442 micro M).

Human amniotic fluid has been shown to contain a protein that binds insulin-like growth factor I and II (IGF-I and IGF-II). Partially purified preparations of this protein have been reported to inhibit the biologic actions of the IGFs. In these studies our laboratory has used a modified purification procedure to obtain a homogeneous preparation of this protein as determined by polyacrylamide gel electrophoresis and amino acid sequence analysis. During purification the ion exchange chromatography step resulted in two peaks of material with IGF binding activity termed peaks B and C. Each peak was purified separately to homogeneity. Both peaks were estimated to be 31,000 daltons by polyacrylamide gel electrophoresis and their amino acid compositions were nearly identical. Amino acid sequence analysis showed that both peaks had identical N-terminal sequences through the first 28 residues. Neither protein had detectable carbohydrate side chains and each had a similar affinity for radiolabeled IGF-I (1.7-2.2 x 10(10) liters/mol). In contrast, these two forms had marked differences in bioactivity. Concentrations of peak C material between 2 and 20 ng/ml inhibited IGF-I stimulation of [3H]thymidine incorporation into smooth muscle cell DNA. In contrast, when peak B (100 ng/ml) was incubated with IGF-I there was a 4.4-fold enhancement of stimulation of DNA synthesis. Additionally, pure peak B was shown to adhere to cell surfaces, whereas peak C was not adherent. The non-adherent peak C inhibited IGF-I binding to its receptor and to adherent peak B. We conclude that human amniotic fluid contains two forms of IGF binding protein that have very similar physiochemical characteristics but markedly different biologic actions. Since both have similar if not identical amino acid compositions, N-terminal sequences, and do not contain carbohydrate, we conclude that they differ in some other as yet undefined post-translational modification.

An abiotic system is described which chemically catalyzes the formation of less than Glu-His-Pro-NH2 (thyrotropin-releasing hormone) from less than Glu-His-Pro-amino acid in the presence of copper, ascorbate, and molecular oxygen. Evidence is presented to support the participation of hydroxyl and carbon radicals as reaction intermediates in the production of a peptide amide and an aldehyde or ketone. The characteristics of this model system closely mimic the characteristics of enzymatic peptide amidation, and an oxidative, free-radical mechanism for enzymatic peptide amidation is proposed as an alternative to the mechanism for enzymatic amidation offered by Bradbury et al. (Bradbury, A. F., Finnie, M. D. A., and Smyth, D. G. (1982) Nature 298, 686-688).

The mechanism for the post-translational conversion of glutamine to pyroglutamic acid on the N terminus of newly synthesized peptides and proteins is unknown. An assay is reported that permits measurement of the rate of conversion of Gln-His-Pro-NH2 to pyroGlu-His-Pro-NH2 (TRH). Using this assay, we demonstrate that the spontaneous cyclization of the N-terminal glutamine of this peptide occurs only slowly under physiological conditions. Furthermore, we describe the presence in rat brain, porcine pituitary, and human B lymphocytes of an enzyme(s) which converts Gln-His-Pro-NH2 into pyroGlu-His-Pro-NH2. The enzyme(s) appears to be a glycoprotein, is maximally active at neutral pH, has a Mr of 55,000, and contains catalytically significant sulfhydryl groups. The product of the enzymatic reaction was confirmed by high resolution fast atom bombardment-mass spectrometry. In preliminary studies, we find that over 90% of the enzyme in bovine adrenal medulla is contained in the soluble chromaffin vesicle fraction. These findings indicate that in vivo the post-translational conversion of a glutaminyl-peptide into a pyroglutamyl-peptide is neither spontaneous nor abiotic as has been previously proposed.

To study the possibility of glycine-directed amidation in rat brain, we synthesized the substrate p-Glu-His-Pro-Gly-OH. Adult and neonatal rat brain and adult rat pituitary were sonicated, frozen and thawed, and fractionated by gel permeation chromatography, and fractions from each tissue were assayed for enzymatic activity capable of converting this model substrate into thyrotropin-releasing hormone. We report the presence in rat brain and rat pituitary of two enzymes catalyzing conversion of p-Glu-His-Pro-Gly-OH into thyrotropin-releasing hormone. Based on the differing chemical and physical properties of these two enzymes and their differing affinities for a number of p-Glu-His-Pro-aa-OH analogs (in which aa = glycine, beta-alanine, gamma-butyric acid, and delta-aminovaleric acid), we conclude that there are two distinct enzymatic processes for the terminal amidation of peptides in brain and that COOH-terminal extensions other than glycine are capable of directing COOH-terminal amidation.

A peptide alpha-amidating enzyme was purified to apparent homogeneity from porcine pituitary. This enzyme is a glycoprotein with a mol wt of 64,000, a metal prosthetic group, and a dependence upon ascorbate and molecular oxygen. The purified enzyme has a strong preference for peptides ending in glycine. It also catalyzes the oxidation of valylglycine bonds more rapidly than prolylglycine bonds, and demonstrates a primary isotope effect greater than 5 when the alpha-hydrogens of glycine are replaced by deuterium. Kinetic analysis is consistent with a ping-pong or double displacement catalytic mechanism in which both the peptide substrate and ascorbate are competitive inhibitors with respect to each other. With respect to its kinetic properties, catalytic mechanism, and cofactor requirements, the purified amidating enzyme is very similar to dopamine beta-hydroxylase, a finding which supports the previous suggestion that the peptide alpha-amidating enzyme be classified as a peptidyl glycine monooxygenase.