Synthesis and characterization of insulin-like growth factor (IGF)-1 photoprobes selective for the IGF-binding proteins (IGFBPS). photoaffinity labeling of the IGF-binding domain on IGFBP-2

Elevated insulin-like growth factor (IGF)-1 levels are prognostic for the development of prostate and breast cancers and exacerbate the complications of diabetes. In each case, perturbation of the balance between IGF-1/2, the IGF-1 receptor, and the IGF-binding proteins (IGFBPs) leads to elevated IGF-1 sensitivity. Blockade of IGF action in these diseases would be clinically significant. Unfortunately, effective IGF antagonists are currently unavailable. The IGFBPs exhibit high affinity and specificity for the IGFs and serve as natural IGF antagonists, limiting their mitogenic/anti-apoptotic effects. As an initial step in designing IGFBP-based agents that antagonize IGF action, we have begun to analyze the structure of the IGF-binding site on IGFBP-2. To this end, two IGF-1 photoprobes, N(alphaGly1)-(4-azidobenzoyl)-IGF-1 (abG(1)IGF-1) and N(alphaGly1)-([2-6-(biotinamido)-2(p-azidobenzamido)hexanoamido]ethyl-1,3'-dithiopropionoyl)-IGF-1 (bedG(1)IGF-1), selective for the IGFBPs were synthesized by derivatization of the alpha-amino group of Gly(1), known to be part of the IGFBP-binding domain. Mass spectrometric analysis of the reduced, alkylated, and trypsin-digested abG(1)IGF-1.recombinant human IGFBP-2 (rhIGFBP-2) complex indicated photoincorporation near the carboxyl terminus of rhIGFBP-2, between residues 266 and 287. Mass spectrometric analysis of avidin-purified tryptic peptides of the bedG(1)IGF-1.rhIGFBP-2 complex revealed photoincorporation within residues 212-227. Taken together, these data indicate that the IGFBP-binding domain on IGF-1 contacts the distal third of IGFBP-2, providing evidence that the IGF-1-binding domain is located within the C terminus of IGFBP-2.     

Recombinant human [Cys281]insulin-like growth factor-binding protein 2 inhibits both basal and insulin-like growth factor I-stimulated proliferation and collagen synthesis in fetal rat calvariae.

It is recognized that insulin-like growth factors (IGFs) are bound to specific high-affinity insulin-like growth factor-binding proteins (IGFBPs). The role of IGFBPs in bone metabolism is not well established. The effect of recombinant human [Cys281]IGFBP-2 ([Cys281]rhIGFBP-2) on bone formation in 21-day-old fetal rat calvariae was investigated. [Cys281]rhIGFBP-2 was expressed in and purified from conditioned medium of a clonal Chinese hamster ovary cell line. IGF-I-stimulated cell proliferation was inhibited dose dependently by [Cys281]rhIGFBP-2, with half-maximal inhibition observed at 2 x 10(-8) M. Suppression of the IGF-I-stimulated DNA synthesis was observed at an apparent dose ratio of 1:10. [Cys281]rhIGFBP-2 (10(-6) M) also inhibited the basal incorporation of [3H]thymidine into DNA by up to 45%. Insulin-stimulated cell proliferation was not affected in the presence of the binding protein. In addition, [Cys281]rhIGFBP-2 inhibited bone collagen synthesis under basal and IGF-I-stimulated conditions. In contrast, [Cys281]rhIGFBP-2 did not alter the parathyroid hormone-stimulated bone cell proliferation rate. In conclusion, binding of hIGF-I to rhIGFBP-2 results in an inhibition of the actions of free IGF-I on bone cell replication and matrix synthesis. Parathyroid hormone-stimulated cell proliferation is not mediated by an increase in free IGFs.     

Mitogenic and antiapoptotic effects of insulin-like growth factor binding protein-6 in the human osteoblastic osteosarcoma cell line Saos-2/B-10.

Insulin-like growth factor (IGF) I is a potent mitogen for human osteosarcoma cells such as the Saos-2/B-10 cell line. IGF binding proteins (IGFBPs) prevent stimulation of DNA synthesis by IGFs. In contrast to recombinant human (rh) IGFBP-2, -3, -4, and -5, 10-100 nM rhIGFBP-6 stimulated [(3)H]thymidine incorporation into DNA and multiplication of Saos-2/B-10 cells. Upon withdrawal of serum, 30 nM IGFBP-6 also decreased apoptosis (within 4 h) and increased protein content and sodium-dependent phosphate uptake (within 24 h), but less potently than IGF I. (125)I-labeled rhIGFBP-6 did not bind to the cells, and cold IGFBP-6 did not affect (125)I-labeled IGF I binding. Production of IGF I, IGF II, and IGFBP-6 by the cells or significant degradation of rhIGFBP-6 could not be detected within 24 h of incubation. Thus, among the rhIGFBPs tested, rhIGFBP-6 is unique in stimulating osteosarcoma cell growth. Furthermore, it has an antiapoptotic effect.     

Role of insulin-like growth factors and their binding proteins in growth control and carcinogenesis

Interest in the role of the insulin-like growth factor (IGF) axis in growth control and carcinogenesis has recently been increased by the finding of elevated serum insulin-like growth factor I (IGF-I) levels in association with three of the most prevalent cancers in the United States: prostate cancer, colorectal cancer, and lung cancer. IGFs serve as endocrine, autocrine, and paracrine stimulators of mitogenesis, survival, and cellular transformation. These actions are mediated through the type 1 IGF-receptor (IGF-1R), a tyrosine kinase that resembles the insulin receptor. The availability of free IGF for interaction with the IGF-1R is modulated by the insulin-like growth factor-binding proteins (IGFBPs). IGFBPs, especially IGFBP-3, also have IGF-independent effects on cell growth. IGF-independent growth inhibition by IGFBP-3 is believed to occur through IGFBP-3-specific cell surface association proteins or receptors and involves nuclear translocation. IGFBP-3-mediated apoptosis is controlled by numerous cell cycle regulators in both normal and disease processes. IGFBP activity is also regulated by IGFBP proteases, which affect the relative affinities of IGFBPs, IGFs and IGF-1R. Perturbations in each level of the IGF axis have been implicated in cancer formation and progression in various cell types.     

Cloning and functional expression in E. coli of a polyphenol oxidase transcript from Coreopsis grandiflora involved in aurone formation

Polyphenol oxidases are involved in aurone biosynthesis but the gene responsible for 4-deoxyaurone formation in Asteraceae was so far unknown. Three novel full-length cDNA sequences were isolated from Coreopsis grandiflora with sizes of 1.80 kb (cgAUS1) and 1.85 kb (cgAUS2a, 2b), encoding for proteins of 68–69 kDa, respectively. cgAUS1 is preferably expressed in young petals indicating a specific role in pigment formation. The 58.9 kDa AUS1 holoproenzyme, was recombinantly expressed in E. coli and purified to homogeneity. The enzyme shows only diphenolase activity, catalyzing the conversion of chalcones to aurones and was characterized by SDS–PAGE and shot-gun type nanoUHPLC–ESI-MS/MS.     

Expression of insulin-like growth factor binding proteins in human breast cancer correlates with estrogen receptor status

The insulin-like growth factors (IGFs) have been implicated in the growth regulation of human breast cancer. Since the IGFs are associated with specific binding proteins (IGFBPs) which may modulate receptor/ligand interactions, production of IGFBPs by breast cancer cells could alter their IGF-dependent growth. This study examined the expression of IGFBPs 4, 5, and 6 in eight breast cancer cell lines (BCCLs) using ribonuclease (RNase) protection assays. IGFBP-4 mRNA was detected in all BCCLs studied. IGFBP-5 expression was higher in estrogen receptor (ER) positive cells, while IGFBP-6 mRNA was detected in only two ER negative BCCLs. We also found that E₂ treatment enhanced the expression of IGFBPs 2, 4, and 5 in T47-D cells. We next studied IGFBP mRNA expression in 40 primary breast tumors. All tumors expressed mRNA for IGFBPs 2–6 but none expressed IGFBP-1 message. IGFBP-3 expression was higher in ER negative tumors, while that of IGFBP-4 and -5 was higher in ER positive specimens. These differences were statistically significant (P < .05). Ligand blot analysis of tumor extracts confirmed the presence of IGFBPs in breast cancer tissues. Thus, differential IGFBP expression in ER positive and negative tumors suggests an important role for this protein in breast cancer biology.     

Development of an efficient E. coli expression and purification system for a catalytically active, human Cullin3-RINGBox1 protein complex and elucidation of its quaternary structure with Keap1

The Cullin3-based E3 ubiquitin ligase complex is thought to play an important role in the cellular response to oxidative stress and xenobiotic assault. While limited biochemical studies of the ligase’s role in these complex signaling pathways are beginning to emerge, structural studies are lagging far behind due to the inability to acquire sufficient quantities of full-length, highly pure and active Cullin3. Here we describe the design and construction of an optimized expression and purification system for the full-length, human Cullin3-RINGBox 1 (Rbx1) protein complex from Escherichia coli. The dual-expression system is comprised of codon-optimized Cullin3 and Rbx1 genes co-expressed from a single pET-Duet-1 plasmid. Rapid purification of the Cullin3-Rbx1 complex is achieved in two steps via an affinity column followed by size-exclusion chromatography. Approximately 15 mg of highly pure and active Cullin3-Rbx1 protein from 1 L of E. coli culture can be achieved. Analysis of the quaternary structure of the Cullin3-Rbx1 and Cullin3-Rbx1-Keap1 complexes by size-exclusion chromatography and analytical ultracentrifugation indicates a 1:1 stoichiometry for the Cullin3-Rbx1 complex (MW = 111 kDa), and a 1:1:2 stoichiometry for the Cullin3-Rbx1-Keap1 complex (MW = 280 kDa). This latter complex has a novel quaternary structural organization for cullin E3 ligases, and it is fully active based on an in vitro Cullin3-Rbx1-Keap1-Nrf2 ubiquitination activity assay that was developed and optimized in this study.     

Analysis of four circulating complexes of insulin-like growth factor binding proteins in human blood during aging

The primary role of insulin-like growth factor binding proteins (IGFBPs) is to regulate availability of IGFs for interacting with receptors, but IGFBPs perform IGF-independent actions as well. The availability and activity of IGFBPs in the circulation is influenced primarily by their concentration and structural modifications, but possibly also by interaction with major plasma proteins such as transferrin, alpha-2-macroglobulin (α2M), and fibrinogen. Four types of circulating IGFBP complexes were examined in this study by immuno- and ligand-binding assays in adults of different age. The amounts of IGFBP-3/transferrin and IGFBP-1/fibrinogen complexes were similar in middle- and old-aged persons, whereas the amounts of IGFBP-1 (or -2)/α2M monomer complexes were lower in the old-aged group and negatively correlated with total IGFBP-1 (or -2) amounts in blood. In contrast to IGFBP-1, IGFBP-2 was present in significantly greater quantities in complexes with α2M dimer than α2M monomer in older individuals. IGFBP complexes did not bind ¹²⁵I-labeled IGF-I in amounts detectable by ligand blotting. According to the results of this study, the quantities of IGFBP-1 and IGFBP-2, which interact with α2M, are age-dependent and, in the case of complexes with α2M monomer, they are negatively correlated with the total circulating levels of these two IGFBPs.     

Association between Circulating Levels of IGF-1 and IGFBP-3 and Lung Cancer Risk: A Meta-Analysis

Background

The insulin-like growth factor (IGF) system was documented to play a predominant role in neoplasia. As lung cancer is one of the most malignant cancers, we conducted a meta-analysis in order to investigate the strength of association between circulating IGF-1 and IGFBP-3 levels and lung cancer.

Methodology/Principal Findings

A systematic literature search was conducted to identify all prospective case-control studies and case-control studies on circulating IGFs and IGFBPs levels. Six nested case-control studies (1 043 case subjects and 11 472 control participants) and eight case-control studies (401 case subjects and 343 control participants) were included in this meta-analysis. Pooled measure was calculated as the inverse variance-weighted mean of the natural logarithm of multivariate adjusted OR with 95% CIs for highest vs. lowest levels to assess the association of circulating IGF-1 and IGFBP-3 concentrations and lung cancer. Standard mean difference (SMD) was also calculated to indicate the difference of the circulating IGF-1 and IGFBP-3 concentrations between the lung cancer case group and the control group. Of the nested case-control studies, ORs for the highest vs. lowest levels of IGF-1 and IGFBP-3 were 1.047 (95% CI: [0.802,1.367], P = 0.736) and 0.960 (95%CI: [0.591,1.559], P = 0.868) respectively; and SMDs were −0.079 (95%CI:[ −0.169, 0.011], P = 0.086) and −0.097 (95%CI:[ −0.264,0.071], P = 0.258) for IGF-1 and IGFBP-3 respectively. As to the case-control studies, SMDs were 0.568 (95%CI:[ −0.035, 1.171], P = 0.065) and −0.780 (95%CI:[ −1.358, −0.201], P = 0.008) for IGF-1 and IGFBP-3 respectively.

Conclusions/Significance

Inverse association was shown between IGFBP-3 and lung cancer in the case-control studies,and the circulating level of IGFBP-3 underwent a decline during tumorogenesis and development of lung cancer, which suggested IGFBP-3 a promising candidate for the biomarker of lung cancer.     

Zinc partitions insulin-like growth factors (IGFs) from soluble IGF binding protein (IGFBP)-5 to the cell surface receptors of BC3H-1 muscle cells

Zinc (Zn(2+)) is a multifunctional micronutrient. The list of functions for this micronutrient expanded with the recent discovery that Zn(2+) retains insulin-like growth factors binding proteins (IGFBPs) on the surface of cultured cells, lowers the affinity of cell-associated IGFBPs, and increases the affinity of the cell surface insulin-like growth factor (IGF)-type 1 receptor (IGF-1R). However, currently there is no information concerning the effect of Zn(2+) on soluble IGFBPs. In the current study, the soluble IGFBP-5 secreted by BC(3)H-1 cells is shown to bind approximately 50% more [(125)I]-IGF-II than [(125)I]-IGF-I at pH 7.4. Zn(2+) is shown to depress the binding of both IGF-I and IGF-II to soluble secreted IGFBP-5; [(125)I]-IGF-I binding is affected more so than [(125)I]-IGF-II binding. Zn(2+) acts by lowering the affinity (K(a)) of IGFBP-5 for the IGFs. Scatchard plots are non-linear indicating the presence of high and low affinity binding sites; Zn(2+) affects only binding to the high affinity site. In contrast, Zn(2+) increases the affinity by which either [(125)I]-IGF-I or [(125)I]-R(3)-IGF-I binds to the IGF-1R, but depresses [(125)I]-IGF-II binding to the IGF-type 2 receptor (IGF-2R) on BC(3)H-1 cells. By depressing the association of the IGFs with soluble IGFBPs, Zn(2+) is shown to repartition either [(125)I]-IGF-I or [(125)I]-IGF-II from soluble IGFBP-5 onto cell surface IGF receptors. Zn(2+) was active at physiological doses depressing IGF binding to IGFBP-5 and the IGF-2R at 15-20 microM. Hence, a novel mechanism is further characterized by which the trace micronutrient Zn(2+) could regulate IGF activity.     

Biochemical characterization and structure–function relationship of two plant NCS2 proteins,the nucleobase transporters NAT3 and NAT12 from Arabidopsis thaliana

Nucleobase ascorbate transporters (NATs), also known as Nucleobase:Cation-Symporter 2 (NCS2) proteins, belong to an evolutionary widespread family of transport proteins with members in nearly all domains of life. We present the biochemical characterization of two NAT proteins, NAT3 and NAT12 from Arabidopsis thaliana after their heterologous expression in Escherichia coli UraA knockout mutants. Both proteins were shown to transport adenine, guanine and uracil with high affinities. The apparent K_M values were determined with 10.12 μM, 4.85 μM and 19.95 μM, respectively for NAT3 and 1.74 μM, 2.44 μM and 29.83 μM, respectively for NAT12. Competition studies with the three substrates suggest hypoxanthine as a further substrate of both transporters. Furthermore, the transport of nucleobases was markedly inhibited by low concentrations of a proton uncoupler indicating that NAT3 and NAT12 act as proton–nucleobase symporters. Transient expression studies of NAT-GFP fusion constructs revealed a localization of both proteins in the plasma membrane. Based on the structural information of the uracil permease UraA from E. coli, a three-dimensional experimentally validated homology model of NAT12 was created. The NAT12 structural model is composed of 14 TM segments and divided into two inverted repeats of TM1–7 and TM8–14. Docking studies and mutational analyses identified residues involved in NAT12 nucleobase binding including Ser-247, Phe-248, Asp-461, Thr-507 and Thr-508. This is the first study to provide insight into the structure–function of plant NAT proteins, which reveals differences from the other members of the NCS2 protein family.     

Diversity of human insulin-like growth factor (IGF) binding protein-2 fragments in plasma: primary structure, IGF-binding properties, and disulfide bonding pattern

The insulin-like growth factor binding proteins (IGFBPs) play a major role in the regulation of the effects and the bioavailability of the insulin-like growth factors (IGFs). IGFs are released from IGFBP-IGF complexes by proteolysis of IGFBPs generating fragments with reduced ligand-binding properties. To identify naturally occurring fragments of IGFBP-2, a peptide library generated from human hemofiltrate was immunologically screened. Purification of immunoreactive IGFBP-2 fragments was performed by consecutive chromatographic steps. A total of 18 different IGFBP-2 fragments was isolated and characterized. The peptides exhibited different N-terminal amino acid residues that were located in the variable midregion of IGFBP-2. Four major cleavage sites were determined to be between Tyr103 and Gly104, Leu152 and Ala153, Arg156 and Glu157, and Gln165 and Met166. The resulting fragments were further processed by amino and/or carboxy peptidases and comprised 37-185 amino acid residues. Ligand blotting, solution binding assays, and BIAcore analyses revealed that all tested fragments retained low IGF-binding capacity. The most abundant fragment IGFBP-2 (167-279) showed 10% of IGF-II binding compared to recombinant human (rh)IGFBP-2. Furthermore, the disulfide bonding pattern of the C-terminal domain of rhIGFBP-2 was defined, indicating linkages between cysteine residues 191-225, 236-247, and 249-270. This study provides the most comprehensive molecular characterization of human IGFBP-2 fragments formed in vivo, exhibiting both residual IGF-binding capacities and the integrin-binding sequence.     

Insulin-like growth factor binding protein-2 as a regulator of IGF actions in CNS: implications in multiple sclerosis

Insulin-like growth factors (IGFs) are indispensable peptide hormones for proper development of the central nervous system (CNS). Because IGF-1 exhibits neuroprotective and myelinogenetic effects, it possesses therapeutic potential in treating neurodegenerative demyelinating diseases such as multiple sclerosis (MS). However, IGF actions are largely dependant on high-affinity regulatory IGF binding proteins (IGFBPs), which are likely to interfere with therapeutic attempts at elevating IGF-1 levels in the CNS. In particular, IGFBP-2 plays a dominant role in IGF regulation in the CNS and is upregulated in several pathological conditions, including MS. The question remains as to whether IGFBPs should be considered "interfering" components of IGF treatment strategies or might possibly be utilized to clinical advantage. This review discusses our current understanding of biological functions of IGFBP-2 in the CNS and its implications in the demyelinating disease MS.     

Differential effects of thermal injury on circulating insulin-like growth factor binding proteins in burn patients

The results of this report provide evidence that insulin-like growth factor-1 binding proteins (IGFBPs) in human sera are differentially regulated as a result of severe burn injury. Using the ligand binding technique,¹²⁵I-IGF-1 visualizes 5 different protein bands corresponding to those previously reported for IGFBP-1 to 4 with apparent sizes of 23–42 kd in serum samples prepared from severely burned patients and healthy individuals. The level of IGFBP-3 was significantly decreased within 3–5 days of injury and remained depressed for up to 20 days post injury. The average level of this binding protein reached its lowest value within 3–5 days of the injury (3.8±1.48% relative to day 0–1 value, n=4, p<0.001). Serum samples from 3 to 4 patients showed no recovery within 20 days post injury and the level of IGFBP-3 remained significantly depressed (p<0.01). In contrast, the levels of IGFBP-2 and IGFBP-4 increased 2 and 3 fold in the same serum samples within 3–5 days of the burn injury, respectively. This increase returns to normal (day 0–1 value) within 7–10 days for IGFBP-2, but the level of IGFBP-4 remained elevated 4 fold relative to the day 0–1 (p<0.01). However, the abundance of IGFBP-1 in these serum samples was not significantly altered by the burn injury. By controlling for protein loading, these apparent alterations of IGFBPs in the sera of burned patients were not due to hemodilution. Similarly, significant reductions in IGFBP-3 were not likely due to IGFBP-3 specific protease activity in the sera of burn patients since incubation of sera from burn patients and normal individuals at 37° C did not alter the pattern of IGFBPs in sera obtained from normal individuals. Of interest, the level of IGF-1 protein in these samples was also markedly reduced following severe burn injury similar to IGFBP-3. The results of this study suggest that a marked reduction of serum IGF-1 seen in burn patients is associated with a significant reduction of IGFBP-3, a major IGF-1 binding protein in human serum.     

Interaction of Insulin-Like Growth Factor-Binding Protein 2 with α2-Macroglobulin in the Circulation

Insulin-like growth factors (IGFs) play active role in mitogenic and metabolic processes. In the peripheral circulation, they are mostly bound to specific IGF-binding proteins (IGFBPs). Proteolysis of IGFBPs releases free, active IGFs. IGFBP-2 is the second most abundant of the six binding proteins and its concentration increases in catabolic states. The possible interaction between IGFBP-2 and other proteins in the circulation was investigated in this study. Our results showed that IGFBP-2 associates with α2-macroglobulin (α2M), a protease inhibitor. Formation of IGFBP-2/α2M complexes most likely contributes to the regulation of IGFBP-2 proteolysis and, thus, the activity of IGFs.     

Insulin-like growth factor-binding protein-1: an evolutionarily conserved fine tuner of insulin-like growth factor action under catabolic and stressful conditions

The insulin-like growth factor-binding proteins (IGFBPs) are evolutionarily conserved components of the insulin-like growth factor (IGF) system. The six forms of IGFBPs (IGFBP-1–6) bind the IGF ligands (IGF-1 and -2) with high affinity and regulate the IGFs available to their receptors, therefore providing additional flexibilities in regulating IGF signalling. IGFBP-1, the first identified member of the IGFBP family is highly inducible under a variety of catabolic conditions, such as food deprivation, malnutrition, stress, injury and hypoxia. Recent in vivo studies have indicated that the induced IGFBP-1 serves as a molecular switch by restricting IGF signalling and diverts the limited energy resources away from growth and development towards those metabolic processes essential for survival. This article reviews the recent understandings of the molecular basis of IGFBP-1 regulation and its biological functions, as revealed through research in mammalian and fish models.     

Synthesis and characterization of biotinylated forms of insulin-like growth factor-1: topographical evaluation of the IGF-1/IGFBP-2 AND IGFBP-3 interface

Activation of the insulin-like growth factor-1 (IGF)-1 receptor signaling pathways by IGF-1 and IGF-2 results in mitogenic and anabolic effects. The bioavailability of the IGFs is regulated by six soluble binding proteins, the insulin-like growth factor binding proteins (IGFBPs), which bind with approximately 0.1 nM affinity to the IGFs and often serve as endogenous antagonists of IGF action. To identify key domains of IGF-1 involved in the interaction with IGFBP-2 and IGFBP-3, we employed IGF-1 selectively biotinylated on residues Gly 1, Lys 27, Lys 65, and Lys 68. All monobiotinylated species of IGF-1 exhibited high affinity ( approximately 0.1-0.2 nM) for IGFBP-2 and IGFBP-3 in solid-phase-binding assays. However, different labeling intensities were observed in ligand blot analysis of IGFBP-2 and IGFBP-3. The N(epsilon)(Lys65/68)(biotin)-IGF-1 (N(epsilon)(Lys65/68b)-IGF-1) probe exhibited the highest signal intensity, while N(alpha)(Gly1b)-IGF-1 and N(epsilon)(Lys27b)-IGF-1 demonstrated significantly lower signals. When taken together, these results suggest that, once bound to IGFBP-2 or IGFBP-3, the biotin moieties of N(alpha)(Gly1b)-IGF-1 and N(epsilon)(Lys27b)-IGF-1 are inaccessible to NeutrAvidin-peroxidase, the secondary binding component. Ligand blots using IGF-1 derivatized with a long chain form of the N-hydroxysuccinimide biotin (NHS-biotin) to yield N(alpha)(Gly1)(LC-biotin)-IGF-1 and N(epsilon)(Lys27)(LC-biotin)-IGF-1 demonstrated increased signal intensity compared with their NHS-biotin counterparts. In BIAcore analysis, IGFBP-2 and IGFBP-3 bound only to the N(epsilon)(Lys65/68b)-IGF-1-coated flowcell of a biosensor chip, confirming the inaccessibility of Gly 1 and Lys 27 when IGF-1 is bound to IGFBP-2 and IGFBP-3. These data confirm the involvement of the IGFBP-binding domain on IGF-1 in binding to IGFBP-2 and IGFBP-3 and support involvement of the IGF-1R-binding domain in IGFBP binding.     

Pharmacological disruption of insulin-like growth factor 1 binding to IGF-binding proteins restores anabolic responses in human osteoarthritic chondrocytes

Insulin-like growth factor 1 (IGF-1) has poor anabolic efficacy in cartilage in osteoarthritis (OA), partly because of its sequestration by abnormally high levels of extracellular IGF-binding proteins (IGFBPs). We studied the effect of NBI-31772, a small molecule that inhibits the binding of IGF-1 to IGFBPs, on the restoration of proteoglycan synthesis by human OA chondrocytes. IGFBPs secreted by human OA cartilage or cultured chondrocytes were analyzed by western ligand blot. The ability of NBI-31772 to displace IGF-1 from IGFBPs was measured by radiobinding assay. Anabolic responses in primary cultured chondrocytes were assessed by measuring the synthesis of proteoglycans in cetylpyridinium-chloride-precipitable fractions of cell-associated and secreted ³⁵S-labeled macromolecules. The penetration of NBI-31772 into cartilage was measured by its ability to displace ¹²⁵I-labeled IGF-1 from cartilage IGFBPs. We found that IGFBP-3 was the major IGFBP secreted by OA cartilage explants and cultured chondrocytes. NBI-31772 inhibited the binding of ¹²⁵I-labeled IGF-1 to IGFBP-3 at nanomolar concentrations. It antagonized the inhibitory effect of IGFBP-3 on IGF-1-dependent proteoglycan synthesis by rabbit chondrocytes. The addition of NBI-31772 to human OA chondrocytes resulted in the restoration or potentiation of IGF-1-dependent proteoglycan synthesis, depending on the IGF-1 concentrations. However, NBI-31772 did not penetrate into cartilage explants. This study shows that a new pharmacological approach that uses a small molecule inhibiting IGF-1/IGFBP interaction could restore or potentiate proteoglycan synthesis in OA chondrocytes, thereby opening exciting possibilities for the treatment of OA and, potentially, of other joint-related diseases.