Affinity For IGF-I Research Articles

Targeting Insulin Receptor in Breast Cancer Using Small Engineered Protein Scaffolds.

Insulin receptor (InsR) and the type I insulin-like growth factor (IGF1R) are homologous receptors necessary for signal transduction by their cognate ligands insulin, IGF-I and IGF-II. IGF1R mAbs, intended to inhibit malignant phenotypic signaling, failed to show benefit in patients with endocrine-resistant tumors in phase III clinical trials. Our previous work showed that in tamoxifen-resistant cells, IGF1R expression was lacking, but InsR inhibition effectively blocked growth. In endocrine-sensitive breast cancer cells, insulin was not growth stimulatory, likely due to the presence of hybrid InsR/IGF1R, which has high affinity for IGF-I, but not insulin. Combination inhibition of InsR and IGF1R showed complete suppression of the system in endocrine-sensitive breast cancer cells. To develop InsR-binding agents, we employed a small protein scaffold, T7 phage gene 2 protein (Gp2) with the long-term goal of creating effective InsR inhibitors and diagnostics. Using yeast display and directed evolution, we identified three Gp2 variants (Gp2 #1, #5, and #10) with low nanomolar affinity and specific binding to cell surface InsR. These Gp2 variants inhibited insulin-mediated monolayer proliferation in both endocrine-sensitive and resistant breast cancer, but did not downregulate InsR expression. Gp2 #5 and Gp2 #10 disrupted InsR function by inhibiting ligand-induced receptor activation. In contrast, Gp2 #1 did not block InsR phosphorylation. Notably, Gp2 #1 binding was enhanced by pretreatment of cells with insulin, suggesting a unique receptor-ligand-binding mode. These Gp2 variants are the first nonimmunoglobulin protein scaffolds to target insulin receptor and present compelling opportunity for modulation of InsR signaling. Mol Cancer Ther; 16(7); 1324-34. ©2017 AACR.

The role and regulation of IGFBP-1 phosphorylation in fetal growth restriction.

Fetal growth restriction (FGR) increases the risk of perinatal complications and predisposes the infantto developing metabolic, cardiovascular, and neurological diseases in childhood and adulthood. The pathophysiology underlying FGR remains poorly understood and there is no specific treatment available. Biomarkers for early detection are also lacking. The insulin-like growth factor (IGF) system is an important regulator of fetal growth. IGF-I is the primary regulator of fetal growth, and fetal circulating levels of IGF-I are decreased in FGR. IGF-I activity is influenced by a family of IGF binding proteins (IGFBPs), which bind to IGF-I and decrease its bioavailability. During fetal development the predominant IGF-I binding protein in fetal circulation is IGFBP-1, which is primarily secreted by the fetal liver. IGFBP-1 binds IGF-I and thereby inhibits its bioactivity. Fetal circulating levels of IGF-I are decreased and concentrations of IGFBP-1 are increased in FGR. Phosphorylation of human IGFBP-1 at specific sites markedly increases its binding affinity for IGF-I, further limiting IGF-I bioactivity. Recent experimental evidence suggests that IGFBP-1 phosphorylation is markedly increased in the circulation of FGR fetuses suggesting an important role of IGFBP-1 phosphorylation in the regulation of fetal growth. Understanding of the significance of site-specific IGFBP-1 phosphorylation and how it is regulated to contribute to fetal growth will be an important step in designing strategies for preventing, managing, and/or treating FGR. Furthermore, IGFBP-1 hyperphosphorylation at unique sites may serve as a valuable biomarker for FGR.

Abstract LB-238: Antitumor activity of mini-IGFBP-3, a protease-resistant derivative of human IGF binding protein-3

Abstract Insulin-like growth factors (IGF) are polypeptide hormones with potent anabolic and mitogenic properties that regulate cell growth and differentiation. Dysregulation of the IGF axis has been well documented in the development and progression of multiple types of cancer. Consequently, targeting the IGF axis has become an area of intense preclinical and clinical research through the development of 3 major classes of therapeutic compounds: antibodies that target IGF-IR, dual IGF-IR and IR tyrosine kinase inhibitors (TKI), and IGF ligands (IGF-I and IGF-II) neutralizing antibodies. Due to their high affinities for IGF-I and IGF-II, six IGFBPs regulate IGF bioavailability and IGFBP-3 is the main carrier of circulating IGFs in postnatal serum. In order to efficiently neutralize the excess of free IGF ligand maintaining tumor progression, we have produced, using the baculovirus insect cell system, a protease-resistant mutein of human IGFBP-3 with conserved affinities towards both IGF-I and IGF-II that was fused to the human IgG1 Fc fragment. In vitro, the resulting immunoadhesin (mIGFBP-3-Fc), evaluated in both regular and 3D culture, was found to block proliferation, arrest cell cycle and induce apoptosis in various types of cancer cell lines (Colon Adenocarcinoma, Ewing's Sarcoma, Adrenocortical Carcinoma and Multiple Myeloma (MM). mIGFBP-3-Fc antitumor activity was evaluated in vivo using RPMI-8226 luciferase-expressing MM cells xenografts. When MM cells were grafted subcutaneouly, daily mIGFBP-3-Fc injections drastically reduced tumor growth and induced tumor cell apoptosis. Similarly, after iv tumor cell injection, bone marrow localization of MM cells was substantially reduced in the mIGFBP-3-Fc-treated group. Furthermore, when used in association with the proteasome inhibitor Bortezomib, mIGFBP-3-Fc was shown to enhance the antitumor effect of Bortezomib used as single agent. Altogether, these data support the concept that mIGFBP-3-Fc could improve MM treatment either as a single agent or in association with proteasome inhibitors. Citation Format: François Godeau, Patricia Leneuve, Laetitia Da Costa, Yves Le Bouc, Jack-Michel Renoir. Antitumor activity of mini-IGFBP-3, a protease-resistant derivative of human IGF binding protein-3. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-238. doi:10.1158/1538-7445.AM2014-LB-238

Ternary complex formation and IGFBP-3 proteolytic activity during childhood: age-dependent changes.

IGF-I is mainly sequestered in a 150-kDa ternary complex with IGF binding protein (IGFBP)-3 and the acid-labile subunit. Data on complex formation and factors influencing formation have not been established. Dissociation of IGF-I from the ternary complex is in part regulated by proteolysis of IGFBP-3, which reduces its affinity for IGF-I. Short small for gestational age (SGA) children have lower IGF-I and IGFBP-3 levels compared with healthy peers. The objective of the study was to determine complex formation in healthy normal-statured children and assess variables influencing complex formation. Second, we determined complex formation in short SGA children. Complex formation was assessed using (125)I-hIGF-I column chromatography in 70 controls (40 boys), median age 10.6 years, and 40 short SGA children (25 boys), median age 8.6 years. IGFBP-3 was determined by Western immunoblotting. (125)I-hIGF-I complex formation showed an age-specific pattern in healthy controls. Variables positively influencing ternary complex formation were higher serum IGF-I levels compared with IGFBP-3 levels (P < .001) and lower serum IGF-II (P < .001) and IGFBP-1 levels (P < .001). In addition, a higher presence of proteolyzed IGFBP-3 negatively influenced 150-kDa complex formation (P = .006). At a young age, healthy children showed considerable IGFBP-3 proteolytic activity, which declined with aging (P < .001). IGFBP-3 proteolytic activity was negatively correlated with IGF-I levels (P < .001). Compared with healthy controls, short SGA children showed reduced IGF-I levels (-1.3 vs 0.1 SD score) and increased proteolyzed IGFBP-3 (35.1% vs 12.2%). Age-specific normative values for (125)I-hIGF-I 150-kDa ternary complex formation are presented. A decrease in IGF-I and an increase in IGF-II, IGFBP-1, and IGFBP-3 proteolytic activity associate with reduced (125)I-hIGF-I ternary complex formation. Our results suggest that in conditions in which IGF-I levels are low, such as young age and in short SGA children, IGFBP-3 proteolytic activity is increased to ensure IGF-I bioavailability.

Exogenous administration of protease-resistant, non-matrix-binding IGFBP-2 inhibits tumour growth in a murine model of breast cancer.

Background:Insulin-like growth factors (IGF-I and IGF-II) signal via the type 1 IGF receptor (IGF-1R) and IGF-II also activates the insulin receptor isoform A (IR-A). Signalling via both receptors promotes tumour growth, survival and metastasis. In some instances IGF-II action via the IR-A also promotes resistance to anti-IGF-1R inhibitors. This study assessed the efficacy of two novel modified IGF-binding protein-2 (IGFBP-2) proteins that were designed to sequester both IGFs. The two modified IGFBP-2 proteins were either protease resistant alone or also lacked the ability to bind extracellular matrix (ECM).Methods:The modified IGFBP-2 proteins were tested in vitro for their abilities to inhibit cancer cell proliferation and in vivo to inhibit MCF-7 breast tumour xenograft growth.Results:Both mutants retained low nanomolar affinity for IGF-I and IGF-II (0.8–2.1-fold lower than IGFBP-2) and inhibited cancer cell proliferation in vitro. However, the combined protease resistant, non-matrix-binding mutant was more effective in inhibiting MCF-7 tumour xenograft growth and led to inhibition of angiogenesis.Conclusions:By removing protease cleavage and matrix-binding sites, modified IGFBP-2 was effective in inhibiting tumour growth and reducing tumour angiogenesis.

Placental alkaline phosphatase de-phosphorylates insulin-like growth factor (IGF)-binding protein-1

BackgroundInsulin-like growth factors (IGF) regulate fetal growth through their effects on placenta. Their actions are influenced by IGF binding protein-1. Phosphorylated IGFBP-1 (pIGFBP-1) has high affinity for IGF-I and usually inhibits IGF-I activity but during pregnancy, it is de-phosphorylated to generate lower affinity isoforms and consequently, increased IGF bioavailability. Here we investigate the role of placenta in this process. ResultsOur data show that term human placental explants, but not their conditioned medium, can de-phosphorylate IGFBP-1 through the action of placental alkaline phosphatase (PLAP). DiscussionPLAP-mediated de-phosphorylation of IGFBP-1 may provide a mechanism for controlling IGF-I bioavailability and action at the maternal/fetal interface.

Phosphorylation of IGFBP-1 at Discrete Sites Elicits Variable Effects on IGF-I Receptor Autophosphorylation

We previously demonstrated that hypoxia and leucine deprivation cause hyperphosphorylation of IGF-binding protein-1 (IGFBP-1) at discrete sites that markedly enhanced IGF-I affinity and inhibited IGF-I-stimulated cell growth. In this study we investigated the functional role of these phosphorylation sites using mutagenesis. We created three IGFBP-1 mutants in which individual serine (S119/S169/S98) residues were substituted with alanine and S101A was recreated for comparison. The wild-type (WT) and mutant IGFBP-1 were expressed in Chinese hamster ovary cells and IGFBP-1 in cell media was isolated using isoelectric-focusing-free-flow electrophoresis. BIACore analysis indicated that the changes in IGF-I affinity for S98A and S169A were moderate, whereas S119A greatly reduced the affinity of IGFBP-1 for IGF-I (100-fold, P < .0001). Similar results were obtained with S101A. The IGF-I affinity changes of the mutants were reflected in their ability to inhibit IGF-I-induced receptor autophosphorylation. Employing receptor-stimulation assay using IGF-IR-overexpressing P6 cells, we found that WT-IGFBP-1 inhibited IGF-IRβ autophosphorylation (~2-fold, P < .001), possibly attributable to sequestration of IGF-I. Relative to WT, S98A and S169A mutants did not inhibit receptor autophosphorylation. S119A, on the other hand, greatly stimulated the receptor (2.3-fold, P < .05). The data with S101A matched S119A. In summary, we show that phosphorylation at S98 and S169 resulted in milder changes in IGF-I action; nonetheless most dramatic inhibitory effects on the biological activity of IGF-I were due to IGFBP-1 phosphorylation at S119. Our results provide novel demonstration that IGFBP-1 phosphorylation at S119 can enhance affinity for IGF-I possibly through stabilization of the IGF-IGFBP-1 complex. These data also propose that the synergistic interaction of distinct phosphorylation sites may be important in eliciting more pronounced effects on IGF-I affinity that needs further investigation.

Sugar and Bone: A Not-So Sweet Story

I predict that most readers of the article by Moyer-Mileur and colleagues in this month’s Journal of Bone and Mineral Research will be challenged not just by the results of their study, but also by the long term implications of their findings. In this relatively small, but well done CRU (Clinical Research Unit) investigation, several indices of the growth hormone- IGF-I axis in adolescent girls with at least 5 years of Type I diabetes mellitus (TIDM), were compared to appropriately age and tanner staged- matched controls (1). These parameters were then regressed against measures of skeletal size and content using areal and volumetric bone mass measurements. On first inspection, the results are not surprising, and are consistent with earlier studies from this group and others; i.e. adolescent girls with long standing TIDM have lower serum IGF-I, higher IGFBP-1, and greater growth hormone concentrations than their age-matched controls. Moreover, those girls with poor metabolic control had the lowest circulating IGF-I and highest IGFBP-1 concentrations. In respect to skeletal structure, increased urinary magnesium in the diabetic girls predicted a smaller, lighter skeleton, and overall disease duration accounted for the greatest variability in bone mineral content and cross sectional area. Higher blood glucoses and greater urinary magnesium were also associated with measures of increased bone resorption. Low serum IGF-I was a major predictor of tibial bone strength, as determined by pQCT.

Hypoxia and Leucine Deprivation Induce Human Insulin-Like Growth Factor Binding Protein-1 Hyperphosphorylation and Increase Its Biological Activity

Fetal growth restriction is often caused by uteroplacental insufficiency that leads to fetal hypoxia and nutrient deprivation. Elevated IGF binding protein (IGFBP)-1 expression associated with fetal growth restriction has been documented. In this study we tested the hypothesis that hypoxia and nutrient deprivation induce IGFBP-1 phosphorylation and increase its biological potency in inhibiting IGF actions. HepG2 cells were subjected to hypoxia and leucine deprivation to mimic the deprivation of metabolic substrates. The total IGFBP-1 levels measured by ELISA were approximately 2- to 2.5-fold higher in hypoxia and leucine deprivation-treated cells compared with the controls. Two-dimensional immunoblotting showed that whereas the nonphosphorylated isoform is the predominant IGFBP-1 in the controls, the highly phosphorylated isoforms were dominant in hypoxia and leucine deprivation-treated cells. Liquid chromatography-tandem mass spectrometry analysis revealed four serine phosphorylation sites: three known sites (pSer 101, pSer 119, and pSer 169); and a novel site (pSer 98). Liquid chromatography-mass spectrometry was used to estimate the changes of phosphorylation upon treatment. Biacore analysis indicated that the highly phosphorylated IGFBP-1 isoforms found in hypoxia and leucine deprivation-treated cells had greater affinity for IGF-I [dissociation constant 5.83E (times 10 to the power)--0 m and 6.40E-09 m] relative to the IGFBP-1 from the controls (dissociation constant approximately 1.54E-07 m). Furthermore, the highly phosphorylated IGFBP-1 had a stronger effect in inhibiting IGF-I-stimulated cell proliferation. These findings suggest that IGFBP-1 phosphorylation may be a novel mechanism of fetal adaptive response to hypoxia and nutrient restriction.

Duplication and Diversification of the Hypoxia-Inducible IGFBP-1 Gene in Zebrafish

BackgroundGene duplication is the primary force of new gene evolution. Deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its amenability to genetic and experimental manipulation and because it possess a large number of duplicated genes.Methodology/Principal FindingsWe report the identification and characterization of two hypoxia-inducible genes in zebrafish that are co-ortholgs of human IGF binding protein-1 (IGFBP-1). IGFBP-1 is a secreted protein that binds to IGF and modulates IGF actions in somatic growth, development, and aging. Like their human and mouse counterparts, in adult zebrafish igfbp-1a and igfbp-1b are exclusively expressed in the liver. During embryogenesis, the two genes are expressed in overlapping spatial domains but with distinct temporal patterns. While zebrafish IGFBP-1a mRNA was easily detected throughout embryogenesis, IGFBP-1b mRNA was detectable only in advanced stages. Hypoxia induces igfbp-1a expression in early embryogenesis, but induces the igfbp-1b expression later in embryogenesis. Both IGFBP-1a and -b are capable of IGF binding, but IGFBP-1b has much lower affinities for IGF-I and -II because of greater dissociation rates. Overexpression of IGFBP-1a and -1b in zebrafish embryos caused significant decreases in growth and developmental rates. When tested in cultured zebrafish embryonic cells, IGFBP-1a and -1b both inhibited IGF-1-induced cell proliferation but the activity of IGFBP-1b was significantly weaker.Conclusions/SignificanceThese results indicate subfunction partitioning of the duplicated IGFBP-1 genes at the levels of gene expression, physiological regulation, protein structure, and biological actions. The duplicated IGFBP-1 may provide additional flexibility in fine-tuning IGF signaling activities under hypoxia and other catabolic conditions.

Inhibition of the Proteolytic Activity of Pregnancy-associated Plasma Protein-A by Targeting Substrate Exosite Binding

The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves both insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4) and -5 at a single site in their central domain causing the release of bioactive IGF. Inhibition of IGF signaling is relevant in human disease, and several drugs in development target the IGF receptor. However, inhibition of PAPP-A activity may be a valuable alternative. We have generated monoclonal phage-derived single chain fragment variable (scFv) antibodies which selectively inhibit the cleavage of IGFBP-4 by PAPP-A, relevant under conditions where cleavage of IGFBP-4 represents the final step in the delivery of IGF to the IGF receptor. None of the antibodies inhibited the homologous proteinase PAPP-A2, which allowed mapping of antibody binding by means of chimeras between PAPP-A and PAPP-A2 to the C-terminal Lin12-Notch repeat module, separated from the proteolytic domain by almost 1000 amino acids. Hence, the antibodies define a substrate binding exosite that can be targeted for the selective inhibition of PAPP-A proteolytic activity against IGFBP-4. In addition, we show that the Lin12-Notch repeat module reversibly binds a calcium ion and that bound calcium is required for antibody binding, providing a strategy for the further development of selective inhibitory compounds. To our knowledge these data represent the first example of differential inhibition of cleavage of natural proteinase substrates by exosite targeting. Generally, exosite inhibitors are less likely to affect the activity of related proteolytic enzymes with similar active site environments. In the case of PAPP-A, selective inhibition of IGFBP-4 cleavage by interference with exosite binding is a further advantage, as the activity against other known or unknown PAPP-A substrates, whose cleavage may not depend on binding to the same exosite, is not targeted.

An N-terminal fragment of insulin-like growth factor binding protein-3 (IGFBP-3) induces apoptosis in human prostate cancer cells in an IGF-independent manner

ObjectiveIGF-binding protein-3 (IGFBP-3) can induce apoptosis in human prostate cancer cells by direct, IGF-independent mechanisms that are poorly understood. IGFBP-3 undergoes limited proteolysis by plasmin and other proteases to generate small N-terminal fragments (e.g., amino acids 1–97) that have lost their affinity for IGF-I and IGF-II yet still can inhibit mitogenesis. The present study examines whether the N-terminal 1–97-IGFBP-3 fragment can induce apoptosis in human prostate cancer cells in an IGF-independent manner. DesignN-terminal 1–97-IGFBP-3 with or without a signal prepeptide was fused to yellow fluorescent protein (YFP) and expressed in PC-3 human prostate cancer cells. In some cases, the N-terminal IGF-binding site was mutated. Subcellular localization was determined by confocal microscopy. Loss of cell viability was determined by Annexin V-APC staining in the presence and absence of a general caspase inhibitor, z-VAD-fmk. ResultsAll of the fusion proteins, including those synthesized with a signal peptide, were predominantly intracellular, suggesting that they had been internalized following secretion. YFP-1–97-IGFBP-3 is present at comparable concentrations in the nucleus and cytoplasm, indicating that it does not contain a nuclear localization signal. Cells transfected with YFP-1–97-IGFBP-3 lost viability. Cell death was blocked by incubation with a caspase inhibitor suggesting that it resulted from apoptosis. Similar results were obtained with YFP-1–97-IGFBP-3 mutants that do not bind IGFs. ConclusionsThe N-terminal 1–97-IGFBP-3 fragment induces apoptosis in human prostate cancer cells in an IGF-independent manner. Generation of the fragment might contribute to the proapoptotic activity of IGFBP-3 in vivo.

The in Vivo Phosphorylation and Glycosylation of Human Insulin-like Growth Factor-binding Protein-5

Mass spectrometry is often used to determine post-translational modifications by analysis of tryptic digests of proteins. Here we demonstrate that the analysis of tryptic peptides together with analysis of the full-length protein provided optimal characterization of insulin-like growth factor-binding protein-5 (IGFBP-5) phosphorylation and glycosylation. IGFBP-5 binds insulin-like growth factors with high affinity and has important roles in cell survival, differentiation, and apoptosis. Until now, the primary structure of IGFBP-5 has been incompletely defined. We analyzed human IGFBP-5 from T47D cells by mass spectrometry to determine all of the in vivo post-translational modifications. In full-length IGFBP-5, 31% of the protein was unmodified, 37% was monophosphorylated, and 4% was diphosphorylated with no other modification. The remaining 27% was glycosylated, more than half of which was also monophosphorylated. The major phosphorylation site was Ser(96) in the central domain, and a minor phosphorylation site was Ser(248) near the C terminus. Neither site was phosphorylated in vitro by casein kinase 2, ruling it out as the in vivo kinase. An in vivo phosphorylation site was also found in IGFBP-2 at an analogous position, Ser(106). IGFBP-5 was heterogeneously O-glycosylated mainly by sialylated core 1 type glycans. The most abundant structure contained N-acetylhexosamine, hexose, and two N-acetylneuraminic acid carbohydrates. A small amount of sialylated core 2 type glycan was also present. Phosphorylation and O-glycosylation both affected IGFBP-5 binding to heparin but not insulin-like growth factor binding or ternary complex formation with the acid-labile subunit. The results reveal the first description of the in vivo phosphorylation of IGFBP-5 and its glycan composition.

Promotion of Cancer Cell Migration: AN INSULIN-LIKE GROWTH FACTOR (IGF)-INDEPENDENT ACTION OF IGF-BINDING PROTEIN-6

A family of six high affinity IGF-binding proteins (IGFBPs 1-6) plays an important role in modulating IGF activities. Recent studies suggest that some IGFBPs may have IGF-independent effects, including induction of apoptosis and modulation of cell migration. However, very little is known about possible IGF-independent actions of IGFBP-6. We have generated a non-IGF-binding IGFBP-6 mutant by substituting Ala for four amino acid residues (Pro(93)/Leu(94)/Leu(97)/Leu(98)) in its N-domain IGF-binding site. A >10,000-fold loss of binding affinity for IGF-I and IGF-II was observed using charcoal solution binding assay, BIAcore biosensor, and ligand blotting. Wild-type and mutant IGFBP-6, as well as IGF-II, induced cell migration in RD rhabdomyosarcoma and LIM 1215 colon cancer cells. Cell migration was mediated by the C-domain of IGFBP-6. Transient p38 phosphorylation was observed in RD cells after treatment with IGFBP-6, whereas no change was seen in phospho-ERK1/2 levels. Phospho-JNK was not detected. IGFBP-6-induced cell migration was inhibited by SB203580, an inhibitor of p38 MAPK, and PD98059, an inhibitor of ERK1/2 MAPK activation. In contrast, SP600125, a JNK MAPK inhibitor, had no effect on migration. Knockdown of p38 MAPK using short interfering RNA blocked IGFBP-6-induced migration of RD cells. These results indicate that p38 MAPK is involved in IGFBP-6-induced IGF-independent RD cell migration.

A 30-kDa fragment of insulin-like growth factor (IGF) binding protein-3 in human pregnancy serum with strongly reduced IGF-I binding.

Proteolytic cleavage of insulin-like growth factor (IGF) binding protein (IGFBP)-3 during pregnancy is likely to have both IGF-dependent and -independent effects on maternal, placental and fetal growth and metabolism. A 30-kDa proteolytic IGFBP-3 fragment was isolated from third trimester pregnancy human serum and identified by N- and C-terminal amino acid sequence analysis and mass spectrometry to correspond to residues 1-212 of the parent protein. This fragment is the dominating IGFBP-3 immunoreactive species in pregnancy serum. The 30-kDa fragment was also detected in serum of non-pregnant women where it coexists with intact IGFBP-3. Using biosensor technology, (1-212)IGFBP-3 was found to have 11-fold lower affinity for IGF-I compared to intact IGFBP-3, while a 4-fold decrease in affinity was found for IGF-II. Tests with des(1-3)IGF-I suggest fast binding of IGF-I to the N-terminal region of IGFBP-3 and similar affinity to a slow binding site in the C-terminal region.

Insulin-like Growth Factor-binding Protein-5 Activates Plasminogen by Interaction with Tissue Plasminogen Activator, Independently of Its Ability to Bind to Plasminogen Activator Inhibitor-1, Insulin-like Growth Factor-I, or Heparin

Transgenic mice expressing IGFBP-5 in the mammary gland exhibit increased cell death and plasmin generation. Because IGFBP-5 has been reported to bind to plasminogen activator inhibitor-1 (PAI-1), we determined the effects of this interaction in HC11 cells. PAI-1 prevented plasmin generation from plasminogen and inhibited cleavage of focal adhesions, expression of caspase 3, and cell death. IGFBP-5 could in turn prevent the effects of PAI-1. IGFBP-5 mutants with reduced affinity for IGF-I (N-term) or deficient in heparin binding (HEP- and C-term E and F) were also effective. This was surprising because IGFBP-5 reportedly interacts with PAI-1 via its heparin-binding domain. Biosensor analysis confirmed that, although wild-type IGFBP-5 and N-term both bound to PAI-1, the C-term E had greatly decreased interaction with PAI-1. This suggests that IGFBP-5 does not antagonize the actions of PAI-1 by a direct molecular interaction. In a cell-free system, using tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) to activate plasminogen, PAI-1 inhibited plasmin generation induced by both activators, whereas IGFBP-5 prevented the effects of PAI-1 on tPA but not uPA. Furthermore, we noted that IGFBP-5 activated plasminogen to a greater extent than could be explained solely by inhibition of PAI-1, suggesting that IGFBP-5 could directly activate tPA. Indeed, IGFBP-5 and the C-term E and F were all able to enhance the activity of tPA but not uPA. These data demonstrate that IGFBP-5 can enhance the activity of tPA and that this can result in cell death induced by cleavage of focal adhesions. Thus IGFBP-5 can induce cell death by both sequestering IGF-I and enhancing plasmin generation.

Serine Phosphorylated IGF Binding Protein-1 (IGFBP-1) Accumulates in the Serum Following Burn Injury

Burn injury is associated with early apoptotic death of T cells and Insulin-like growth factor-1 (IGF-I) is able to protect T cells from apoptosis. Association of IGF-I with its IGFBP (Binding Protein)-1 limits its bioavailability and serine phosphorylation of IGFBP-1 makes this worse because of increased affinity for IGF-I. The level of phosphorylated IGFBP-1 has been shown to increase in pediatric burn patients. Thus we hypothesize that a longitudinal study of burn patients will demonstrate 1) increased IGFBP-1 levels 2) increased IGFBP-1 phosphorylation 3) and decreased IGF-I levels over time. We obtained peripheral blood from burn patients within the first 24 hours after injury and daily thereafter. We used ELISA to measure the serum levels of IGF-I and IGFBP-1 and compared data using Student's t-test. In addition, we incubated samples containing equivalent amounts of IGFBP-1 with or without 50U calf intestinal phosphatase (CIP) and performed Western blots for IGFBP-1. There...

Cumulative Mutagenesis of the Basic Residues in the 201–218 Region of Insulin-Like Growth Factor (IGF)-Binding Protein-5 Results in Progressive Loss of Both IGF-I Binding and Inhibition of IGF-I Biological Action

We have reported previously that mutation of two conserved nonbasic amino acids (G203 and Q209) within the highly basic 201-218 region in the C-terminal domain of IGF-binding protein-5 (IGFBP-5) decreases binding to IGFs. This study reveals that cumulative mutagenesis of the 10 basic residues in this region, to create the C-Term series of mutants, ultimately results in a 15-fold decrease in the affinity for IGF-I and a major loss in heparin binding. We examined the ability of mutants to inhibit IGF-mediated survival of MCF-7 cells and were able to demonstrate that this depended not only upon the affinity for IGF-I, but also the kinetics of this interaction, because IGFBP-5 mutants with similar affinity constants (K(D)) values, but with different association (Ka) and dissociation (Kd) rate values, had markedly different inhibitory properties. In contrast, the affinity for IGF-I provided no predictive value in terms of the ability of these mutants to enhance IGF action when bound to the substratum. Instead, these C-Term mutants appeared to enhance the actions of IGF-I by a combination of increased dissociation of IGF-IGFBP complexes from the substratum, together with dissociation of IGF-I from IGFBP-5 bound to the substratum. These effects of the IGFBPs were dependent upon binding to IGF-I, because a non-IGF binding mutant (N-Term) was unable to inhibit or enhance the actions of IGF-I. These results emphasize the importance of the kinetics of association/dissociation in determining the enhancing or inhibiting effects of IGFBP-5 and demonstrate the ability to generate an IGFBP-5 mutant with exclusively IGF-enhancing activity.

Essential roles of IGFBP-3 and IGFBP-rP1 in breast cancer

Insulin and insulin-like growth factors (IGFs) have critical functions in growth regulatory signalling pathways. They are part of a tightly controlled network of ligands, receptors, binding proteins and their proteases. However, the system becomes uncontrolled in neoplasia. The insulin-like growth factor binding protein 3 (IGFBP-3) and the insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) have unique properties among the sixteen known members of the IGFBP superfamily. IGFBP-3 has very high affinity for IGFs ( k d ∼ 10 −10 M), it transports > 75% of serum IGF-I and -II, whereas it’s affinity for insulin is very low. On the other hand, IGFBP-rP1 binds insulin with very high affinity (500-fold higher compared to other IGFBPs), but has low affinity for IGF-I and -II proteins ( k d = 3 × 10 −8 M). In this review, we have examined the roles of IGFBP-3 and IGFBP-rP1 in breast cancer, and discuss the potential impact of these two proteins in mammary carcinoma risk assessment and the development of treatments for breast cancer.

Effect of maternal diabetes on phosphorylation of insulin‐like growth factor binding protein‐1 in cord serum

The insulin-like growth factor (IGF) system is considered important in the regulation of fetal growth. Binding of IGFs to specific binding proteins (IGFBPs) modifies their actions. In fetal blood, IGFBP-1 is the primary IGF binding protein whose phosphorylation generates proteins with different affinities for IGF-I. We studied cord serum IGFBP-1 phosphoisoform profiles in normal pregnancies and in diabetic pregnancies, which are frequently complicated by macrosomia. Cord serum IGFBP-1 phosphoisoform concentrations were measured at birth by two immunoenzymometric assays in 67 pregnancies complicated by Type 1 diabetes, in 28 pregnancies complicated by insulin-treated gestational diabetes, and in 62 normal pregnancies. Cord serum highly phosphorylated IGFBP-1 (hpIGFBP-1) concentrations were lower in pregnancies complicated by Type 1 diabetes (204 +/- 36 microg/l, P = 0.032) and in pregnancies complicated by gestational diabetes (170 +/- 28 microg/l, P = 0.031) than in controls (316 +/- 34 microg/l). Cord serum lesser phosphorylated IGFBP-1 (lpIGFBP-1) concentrations were similar in diabetic and normal pregnancies (P = 0.692 between groups by analysis of variance). Relative birth weight correlated negatively with cord serum hpIGFBP-1 and lpIGFBP-1 in diabetic pregnancies, and with cord serum lpIGFBP-1 in normal pregnancies. Maternal diabetes is associated with suppressed hpIGFBP-1 but unaltered lpIGFBP-1 concentrations in cord serum, suggesting that IGFBP-1 phosphoisoforms are differentially regulated in the fetus. Because hpIGFBP-1 has a higher affinity for IGF-I than does lpIGFBP-1, diabetes-related changes in fetal IGFBP-1 phosphorylation may increase IGF-I bioavailability and, consequently, stimulate fetal growth. This may partly explain the increased occurrence of macrosomia in diabetic pregnancies.