Walker H. Busby

Cloning, characterization, and expression of a human insulin-like growth factor binding protein

Insulin-like growth factors (IGFs) bind to specific proteins present in extracellular fluids. One of these binding proteins (IGF-BP) was purified from human amniotic fluid and was shown to potentiate the effects of IGF-I in vitro (10). In these studies, a polyclonal antibody to this protein was used to isolate a cDNA clone from a human decidua library. This clone encodes a polypeptide of 25,832 daltons that includes the sequences of 9 tryptic peptides that had been prepared from the purified IGF-BP. The protein has 15 cysteines that are clustered at the amino and carboxy ends of the molecule. The protein has an RGD sequence near its C-terminus, which may account for its ability to attach to cells and to potentiate the biological actions of IGF-I.

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.

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.

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.

Differential regulation of insulin-like growth factor binding protein secretion from human decidual cells by IGF-I, insulin, and relaxin.

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.

Regulation of Vascular Smooth Muscle Cell Responses to Insulin- like Growth Factor (IGF)-I by Local IGF-binding Proteins*

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.

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 Gln66-Gln67 had been altered to Ala66-Ala67 (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.

A Monoclonal Antibody Against αVβ3 Integrin Inhibits Development of Atherosclerotic Lesions in Diabetic Pigs

An antibody directed at the C terminus of an integrin subunit can prevent the formation of atherosclerotic lesions in diabetic pigs. Lessons from the Pig: Diabetes-Induced Atherosclerosis When the hormonal controls on blood glucose go awry, diabetes develops. An unfortunate long-term consequence of this disease is atherosclerosis, which kills the majority of patients with type 2 diabetes. A result of the chronic hyperglycemia of diabetes, this type of atherosclerosis has no treatment beyond those for standard vascular disease. Maile et al. now use Yorkshire pigs with diabetes to show that very selective interference with insulin-like growth factor action is a good line of attack against this deadly complication of diabetes. During atherosclerosis plaque formation, IGF-1 is a driving force, causing smooth muscle cell division. Although IGF itself is not a good therapeutic target because it acts in many tissues, the αvβ3 integrin receptor pathway is also required for IGF-driven cell proliferation during plaque development. Maile and colleagues have therefore aimed their sights at αvβ3 integrin, choosing a region termed the C-loop as the target for a monoclonal antibody. This part of the protein is selectively activated in diabetes, so targeting this region avoids side effects from inhibiting other parts of the protein involved in other functions. When infused into diabetic pigs eating a high-fat diet for 3.5 months, this carefully designed monoclonal antibody inhibited the development of atherosclerotic lesions and reduced the number of dividing cells in the vascular wall. Phosphorylation markers of IGF-1 signaling confirmed that the antibody was inhibiting that pathway, as intended. The authors conclude that the C-loop of αvβ3 integrin is a critical region and possible therapeutic target for control of the atherosclerosis of diabetes. Atherosclerosis in pigs is much like the disease in humans. Unlike rodents, pigs exhibit diet-driven plaques that build up in vessels similar to those affected in humans, they develop spontaneous atherosclerosis with increased age, and their lipoprotein profiles and metabolism are similar to those of humans. In pigs made diabetic with streptozotocin (a toxin that kills β cells used in this study), a high-fat diet induces high triglycerides and calcified plaque development, as it does in humans. Thus, the identification of the αvβ3 integrin C-loop as a promising drug target for the atherosclerosis of diabetes in the diabetic pig bodes well for its application to humans. Atherosclerotic lesions develop and progress more rapidly in diabetic patients than in nondiabetic individuals. This may be caused by accelerated lesion formation in the high-glucose environment of diabetes. Smooth muscle cells (SMCs) cultured in high glucose are more responsive to growth factors such as insulin-like growth factor–1 (IGF-1). This enhanced response to IGF-1 is due in part to increased activation of the αVβ3 integrin. We tested whether αVβ3 integrin activation was increased in diabetic animals and whether an antibody to β3 would inhibit IGF-1 action and development of atherosclerosis. Eight male pigs were made diabetic with streptozotocin and fed a high-fat diet. A F(ab)2 antibody fragment directed at β3 was infused into one femoral artery, whereas the other artery received control F(ab)2 for 3.5 months. There was a 65 ± 8% reduction in atherosclerotic lesion area in the arteries treated with F(ab)2 antibody to β3. Phosphorylation of β3 was reduced by 75 ± 18% in vessels treated with the antibody. Shc and mitogen-activated protein kinase phosphorylation, which are required for IGF-1–stimulated SMC proliferation, were also significantly reduced. We conclude that activation of IGF-1 receptor and αVβ3-linked signaling pathways accelerates atherosclerosis in diabetes and that administration of an antibody to β3 to diabetic pigs inhibits αVβ3 activation, IGF-1–stimulated signaling, and atherosclerotic lesion development. This approach offers a potential therapeutic approach to the treatment of this disorder.

Aldosterone enhances IGF-I-mediated signaling and biological function in vascular smooth muscle cells.

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.

Insulin-like Growth Factor-I-stimulated Insulin Receptor Substrate-1 Negatively Regulates Src Homology 2 Domain-containing Protein-tyrosine Phosphatase Substrate-1 Function in Vascular Smooth Muscle Cells

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.