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.     

A key functional role for the insulin-like growth factor 1 N-terminal pentapeptide.

In order to elucidate the role of the N-terminus of insulin-like growth factor 1 (IGF-1) with respect to its biological properties, we chemically synthesized analogues of IGF-1 truncated by one to five amino acid residues from the N-terminus. In a bioassay that measured the stimulation of protein synthesis in rat L6 myoblasts, the concentrations required to produce a half-maximal response were: IGF-1, 13 ng/ml; des-(1)-IGF-1, 10 ng/ml; des-(1-2)-IGF-1, 13 ng/ml; des-(1-3)-IGF-1, 1.5 ng/ml; des-(1-4)-IGF-1, 5.1 ng/ml; des-(1-5)-IGF-1, 1200 ng/ml. When tested for their abilities to compete with 125I-IGF-1 binding to L6 myoblasts at 3 degrees C, the concentrations required for 50% competition were: IGF-1, des-(1)-IGF-1 and des-(1-2)-IGF-1, 20 ng/ml; des-(1-3)-IGF-1, 14 ng/ml; des-(1-4)-IGF-1, 40 ng/ml; des-(1-5)-IGF-1, greater than 1000 ng/ml. Receptor-binding experiments at 25 degrees C, however, gave results suggesting that the myoblasts were secreting a binding protein selective for the three longest peptides. This interpretation was confirmed by binding studies with medium conditioned by the L6 myoblasts as well as binding protein purified from MDBK-cell-conditioned medium. In both cases IGF-1, des-(1)-IGF-1 and des-(1-2)-IGF-1 competed for tracer IGF-1 binding at least 60-fold better than did the three shorter peptides. The results obtained account for the increased potency of des-(1-3)-IGF-1 and des-(1-4)-IGF-1, since their activities are not attenuated by the binding protein, and the relatively lower potency of des-(1-4)-IGF-1 is a consequence of this peptide binding less well to the L6-myoblast receptor.     

Mutual regulation between IGF-1R and IGFBP-3 in human corneal epithelial cells

The insulin-like growth factor type 1 receptor (IGF-1R) is part of the receptor tyrosine kinase superfamily. The activation of IGF-1R regulates several key signaling pathways responsible for maintaining cellular homeostasis, including survival, growth, and proliferation. In addition to mediating signal transduction at the plasma membrane, in serum-based models, IGF-1R undergoes SUMOylation by SUMO 1 and translocates to the nucleus in response to IGF-1. In corneal epithelial cells grown in serum-free culture, however, IGF-1R has been shown to accumulate in the nucleus independent of IGF-1. In this study, we report that the insulin-like growth factor binding protein-3 (IGFBP-3) mediates nuclear translocation of IGF-1R in response to growth factor withdrawal. This occurs via SUMOylation by SUMO 2/3. Further, IGF-1R and IGFBP-3 undergo reciprocal regulation independent of PI3k/Akt signaling. Thus, under healthy growth conditions, IGFBP-3 functions as a gatekeeper to arrest the cell cycle in G0/G1, but does not alter mitochondrial respiration in cultured cells. When stressed, IGFBP-3 functions as a caretaker to maintain levels of IGF-1R in the nucleus. These results demonstrate mutual regulation between IGF-1R and IGFBP-3 to maintain cell survival under stress. This is the first study to show a direct relationship between IGF-1R and IGFBP-3 in the maintenance of corneal epithelial homeostasis.     

Relationships between IGF-1 and IGFBP-1 and adiposity in obese African-American and Latino adolescents

The purpose of this study was to examine interrelationships between insulin-like growth factor 1 (IGF-1), IGF binding proteins (IGFBPs), and adiposity in 49 African-American and 77 Latino obese adolescents (15.3 ± 0.1 and 15.4 ± 0.2 years; BMI: 33.0 ± 0.7 and 35.0 ± 1.0 kg/m(2), respectively). Immunoradiometric assays were used to measure IGF-1, IGFBP-1, and IGFBP-3. Total fat and soft lean tissue were measured by dual-energy X-ray absorptiometry and visceral adipose tissue (VAT), subcutaneous abdominal adipose tissue (SAAT), and hepatic fat fraction (HFF) were measured by magnetic resonance imaging. IGF-1 levels were 23.1% higher and IGFBP-1 were 40.4% higher in African Americans compared to Latinos after adjustment for total lean and total fat mass. IGF-1 and IGFBP-1 were inversely correlated with BMI, total fat mass, VAT, and HFF (r = -0.20 to -0.33, P < 0.05) while IGFBP-1 was inversely correlated with SAAT (r = -0.22, P < 0.05). These relationships did not differ by ethnicity, however, the relationship between IGF-1 and SAAT, as well as IGFBP-1 and HFF, differed by ethnicity. Predicted mean IGF-1 levels were 30.7% higher for African Americans at the 75th compared to 25th percentile of SAAT and only 11.7% higher for Latinos. Predicted mean IGFBP-1 levels were 158% higher for African Americans at the 25th compared to the 75th percentile of HFF while IGFBP-1 levels were 1.7% higher for Latinos at the 75th compared to the 25th percentile. These results demonstrate that the relationship between IGF-1 and SAAT as well as IGFBP-1 and HFF are different in African-American and Latino adolescents and may contribute to the higher IGF-1 levels in African-Americans.     

IGFBP-3 Inhibits the Proliferation of Neural Progenitor Cells

Insulin like growth factor-1 (IGF-1) plays an important role in the proliferation and differentiation of neural progenitor cells. The effects of IGF-1 can be regulated by insulin like growth factor binding protein-3 (IGFBP-3) which can either inhibit or stimulate the proliferation of cells depending on the expression of proteases that can release IGF-1 from IGF1-IGFBP3 complex. Although IGF-1 is essential for the development of brain, both IGFBP-3 and IGF-1 are elevated in the brains of children younger than 6 months of age. Likewise, IGFBP-3 is also upregulated following cerebral ischemia and hypoxia. However, the role of IGFBP-3 in neurogenesis is not clear. Using an in vitro culture system of rat neural progenitor cells, we demonstrate that IGFBP-3 specifically regulates the IGF-1 mediated neural progenitor cell proliferation via down regulation of phopho-Akt, and cyclin D1. In addition, IGFBP-3 also decreased the content of nestin in the neural progenitor cells indicating its potential role in neurogenesis.     

Classification of the insulin-like growth factor binding proteins into three distinct categories according to their binding specificities

Competitive binding experiments with insulin-like growth factor (IGF)-1, IGF-2 and des-(1-3)-IGF-1 have confirmed the interpretation based on limited amino-terminal sequence analysis that at least three types of IGF binding protein occur. In addition to the acid stable subunit of the large serum binding protein which exhibits des-(1-3)-IGF-1 binding only slightly less than IGF-1, the small IGF binding proteins can be separated into two classes based on differences in des-(1-3)-IGF-1 and IGF-2 binding potencies.     

Duplication and diversification of the hypoxia-inducible IGFBP-1 gene in zebrafish

Background

Gene 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 Findings

We 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/Significance

These 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.     

Insulin-like growth factor-1 and its binding proteins, IGFBP-1 and IGFBP-3, in adolescents with type-1 diabetes mellitus and microalbuminuria

BACKGROUND/AIMS: Numerous clinical and experimental studies suggest that growth factors may contribute to the development of diabetic microvascular complications. The aim of the study was to test the hypothesis that in adolescents with type-1 diabetes mellitus and microalbuminuria (MA) there are specific disorders of serum insulin-like growth factor-1 (IGF-1) and concentrations of its binding proteins, IGFBP-1 and IGFBP-3, that could be of importance in the pathogenesis of microvascular diabetic complications. METHODS: 25 adolescents with MA, 24 adolescents with diabetes without complications, and 17 controls were examined. There were no differences with regard to age, puberty stage, HbA1c and body mass index between the groups examined. Two of the patients in the first group also had diabetic retinopathy. Serum fasting concentrations of IGF-1 and overnight urine albumin concentrations were measured by radioimmunoassay, IGFBP-1 and IGFBP-3 concentrations by immunoradiometric assay and HbA1c by high-performance liquid chromatography methods. Diabetic patients were examined by an experienced ophthalmologist and neurologist. The data were analyzed using Kruskal-Wallis ANOVA and multiple regression analysis. RESULTS: Significantly lower IGF-1 concentrations were found in adolescents with diabetes and MA compared to diabetic patients without complications and healthy contemporaries. IGFBP-1 concentrations were significantly higher and IGFBP-3 concentrations were statistically lower in diabetic patients with MA than in patients without complications. CONCLUSIONS: The IGF-IGFBP system is deranged in adolescents with type-1 diabetes mellitus and MA. Our results suggest the participation of circulating IGFBP-1 in the origin of diabetic complications. It could be also possible that IGFBP-3 takes part in the protection from them.     

缺氧复氧对滑膜细胞IGF-IGFBP-3 以及线粒体的影响

目的:探讨氧波动环境对原代成纤维样滑膜细胞(fibroblast-like synoviocyte, FLS)胰岛素样生长因子-1(insulin growth factor-1,IGF-1)、胰岛素样生长因子结合蛋白-3(insulin-like growth factor binding protein-3, IGFBP-3)及线粒体的影响。方法:分离并鉴定正常人滑膜细胞,再对滑膜细胞进行分组:对照组、缺氧/再充氧(hypoxia/reoxygenation,H/R)干预组。采用实时定量PCR检测滑膜细胞中IGF-1、IGFBP-3的m RNA水平;Western blot检测滑膜细胞中IGF-1、IGFBP-3的蛋白水平;流式细胞仪检测线粒体膜电位(Mitochondrial membrane potential, MMP)以及线粒体通透性转换孔(Mitochondrial Permeability Transition Pore, MPTP)的变化。结果:与对照组比较,H/R干预组的相对IGF-1和IGFBP-3的m RNA水平和蛋白表达水平显著升高(P0.05),膜电位水平降低(P0.05),线粒体通透性转换孔开放。结论:氧波动环境可促进IGF-1和IGFBP-3的表达及细胞线粒体损伤,其可能是骨关节炎(OA)发病的重要机制之一。     

Crystal structure of human insulin-like growth factor-1: detergent binding inhibits binding protein interactions

Despite efforts spanning considerably more than a decade, a high-resolution view of the family of proteins known as insulin-like growth factors (IGFs) has remained elusive. IGF-1 consists of three helical segments which are connected by a 12-residue linker known as the C-region. NMR studies of members of this family reveal a dynamic structure with a topology resembling insulin but little structural definition in the C-region. We have crystallized IGF-1 in the presence of the detergent deoxy big CHAPS, and determined its structure at 1.8 A resolution by multiwavelength anomalous diffraction, exploiting the anomalous scattering of a single bromide ion and six of the seven sulfur atoms of IGF-1. The structure reveals a well-defined conformation for much of the C-region, which extends away from the core of IGF-1 and has residues known to be involved in receptor binding prominently displayed in a type II beta-turn. In the crystal, these residues form a dimer interface, but analytical ultracentrifugation experiments demonstrate that at physiological concentrations IGF-1 is monomeric. A single detergent molecule contacts residues known to be important for IGF-1 binding protein (IGFBP) interactions. Biophysical and biochemical data show that the detergent binds to IGF-1 specifically and blocks binding of IGFBP-1 and IGFBP-3.     

Insulin-like growth factor binding protein-5 (IGFBP-5) induces premature cell death in the mammary glands of transgenic mice

We have previously demonstrated that IGFBP-5 production by mammary epithelial cells increases dramatically during involution of the mammary gland. To demonstrate a causal relationship between IGFBP-5 and cell death we created transgenic mice expressing IGFBP-5 in the mammary gland using a mammary-specific promoter, beta-lactoglobulin. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Histological analysis indicated reduced numbers of alveolar end buds, with decreased ductal branching. Transgenic dams produced IGFBP-5 in their milk at concentrations similar to those achieved at the end of normal lactation. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. BrdU labelling was decreased, whereas DNA ladders were increased in transgenic animals on day 1 of lactation. On day 2 postpartum, the epithelial invasion of the mammary fat pad was clearly impaired in transgenic animals. The concentrations of the pro-apoptotic molecule caspase-3 and of plasmin were both increased in transgenic animals whilst the concentrations of 2 prosurvival molecules Bcl-2 and Bcl-x(L)were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I we examined IGF receptor phosphorylation and Akt phosphorylation and showed that both were inhibited. We attempted to "rescue" the transgenic phenotype by using growth hormone to increase endogenous IGF-I concentrations or by implanting minipumps delivering an IGF-1 analogue, R(3)-IGF-1, which binds weakly to IGFBP-5. Growth hormone treatment failed to affect mammary development suggesting that increased concentrations of endogenous IGF-1 are insufficient to overcome the high concentrations of IGFBP-5 produced by these transgenic animals. In contrast mammary development (gland weight and DNA content) was normalised by R3-IGF-I although milk production was only partially restored. This is the first demonstration that over-expression of IGFBP-5 can lead to; impaired mammary development, increased expression of the pro-apoptotic molecule caspase-3, increased plasmin generation and decreased expression of pro-survival molecules of the Bcl-2 family. It clearly demonstrates that IGF-I is an important developmental/survival factor for the mammary gland and, furthermore, this cell death programme may be utilised in a wide variety of tissues.     

Relationships between serum IGF-1, IGFBP-2, interleukin-1beta and interleukin-6 in inflammatory bowel disease

AIMS: To study the relationships between serum IGF-1, IGFBP-3 and IGFBP-2 and interleukin (IL)-1beta and IL-6 in inflammatory bowel disease (IBD). METHODS: Thirty-seven patients (18 males, 19 females, aged 8.8-26.1 years) with IBD (Crohn's disease, CD, n = 17, and ulcerative colitis, UC, n = 20) were studied. Patients were in relapse or remission according to established criteria. Serum IGF-1, IGFBP-3, IGFBP-2, IL-1beta and IL-6 levels were determined in patients and 15 healthy controls (aged 8.2-19.0 years). RESULTS: IGF-1 levels were lower in patients with CD in relapse compared with controls (p < 0.05). IGFBP-2 levels were higher in CD in relapse compared with other groups (all p < 0.05). In CD and UC patients (n = 37), IGF-1 levels were inversely correlated with the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). IGFBP-2 levels correlated positively with ESR and IL-1beta. IL-6 levels correlated positively with ESR and CRP. IL-1beta levels were elevated in CD in relapse compared to controls (p < 0.05) and were higher in UC in relapse than in other groups (all p < 0.05). In combined CD/UC patients in relapse (n = 20), IL-1beta levels were higher (p < 0.05) in patients with recto-sigmoiditis (n = 5) than in other patients. CONCLUSIONS: IGF-1, IGFBP-2 levels were related to IL levels, disease activity and anatomical distribution, consistent with active inflammation modifying the IGF-IGFBP system, possibly relevant to disturbance of growth.     

Contrasting effects of IGF binding protein-3 expression in mammary tumor cells and the tumor microenvironment

IGFBP-3 has both stimulatory and inhibitory effects on cancer progression. The growth of EO771 mammary carcinoma cells as syngeneic tumors in C57BL/6 mice is reduced in Igfbp3-null (BP3KO) mice, suggesting that systemic IGFBP-3 enhances tumor progression. In this study we assessed the growth of EO771 cells expressing human IGFBP-3 in BP3KO mice. Cells expressing hIGFBP-3 showed decreased proliferation in vitro and increased levels of IGF-1 receptor (IGF1R) protein but not mRNA, consistent with sequestration of endogenous IGF by IGFBP-3. The growth rate of these cells was restored by exposure to IGF-1 or analogues with reduced affinity for IGFBP-3 (long Arg³-IGF-1) or IGF1R (Leu²⁴-IGF-1). In EO771 cells implanted orthotopically into mice, hIGFBP-3 expression by the cells inhibited tumor establishment in BP3KO but not wild-type mice. For tumors that successfully established, final weight was not affected significantly by hIGFBP-3 expression. However, final tumor weight was inversely related to intratumoral T cell counts, and sera from BP3KO mice with tumors showed low-titer immunoreactivity against IGFBP-3. The contrasting effects on tumor establishment and progression of IGFBP-3 expressed by mammary carcinoma cells, compared to systemic stromal and circulating IGFBP-3, highlights the complexity of growth regulation by IGFBP-3 in mammary tumors.     

IGFBP-3, a marker of cellular senescence, is overexpressed in human papillomavirus-immortalized cervical cells and enhances IGF-1-induced mitogenesis

Human ectocervical cells, following retroviral transduction with the human papillomavirus type 16 E6/E7 oncogenes, are altered in their array of transcribed cellular genes, including increased mRNA for the insulin-like growth factor binding protein 3 (IGFBP-3). IGFBP-3 expression is associated with cellular senescence, and its addition to many cell types inhibits growth or induces apoptosis. By immunoblotting and enzyme-linked immunosorbent assay methods, we demonstrate that late-passage, immortalized E6/E7-transduced cells secrete high levels of IGFBP-3 (25 ng/ml), which represent a 500-fold increase compared to levels in early-passage, nonimmortalized transduced cells (<0.05 ng/ml). Concomitantly, these late-passage cervical cells exhibit an increase in sensitivity to IGF-1, including enhanced phosphorylation of the IGF receptor (IGF-R) and insulin receptor substrate as well as increased DNA synthesis (5-fold) and cell proliferation (3.7-fold). However, there was no change in the level of IGF-R in these cells (surface or total), and the cells did not synthesize IGF-1, indicating that these arms of the IGF pathway were independently regulated and not responsible for the augmented signaling. Consistent with a causal relationship between IGFBP-3 expression and enhanced IGF-1 responses, we found that early-passage cells could be converted to the late-passage, IGF-1-responsive phenotype by preincubation with IGFBP-3. Thus, in contrast to findings with some cell types, IGFBP-3 expression in cervical cells is associated with augmented IGF-1 signaling and cell proliferation and correlates with the timing of cellular immortalization.     

Duplication of the IGFBP-2 Gene in Teleost Fish: Protein Structure and Functionality Conservation and Gene Expression Divergence

Background

Insulin-like growth factor binding protein-2 (IGFBP-2) is a secreted protein that binds and regulates IGF actions in controlling growth, development, reproduction, and aging. Elevated expression of IGFBP-2 is often associated with progression of many types of cancers.

Methodology/Principal Findings

We report the identification and characterization of two IGFBP-2 genes in zebrafish and four other teleost fish. Comparative genomics and structural analyses suggest that they are co-orthologs of the human IGFBP-2 gene. Biochemical assays show that both zebrafish igfbp-2a and -2b encode secreted proteins that bind IGFs. These two genes exhibit distinct spatiotemporal expression patterns. During embryogenesis, IGFBP-2a mRNA is initially detected in the lens, then in the brain boundary vasculature, and subsequently becomes highly expressed in the liver. In the adult stage, liver has the highest levels of IGFBP-2a mRNA, followed by the brain. Low levels of IGFBP-2a mRNA were detected in muscle and in the gonad in male adults only. IGFBP-2b mRNA is detected initially in all tissues at low levels, but later becomes abundant in the liver. In adult males, IGFBP-2b mRNA is only detected in the liver. In adult females, it is also found in the gut, kidney, ovary, and muscle. To gain insights into how the IGFBP-2 genes may have evolved through partitioning of ancestral functions, functional and mechanistic studies were carried out. Expression of zebrafish IGFBP-2a and -2b caused significant decreases in the growth and developmental rates and their effects are comparable to that of human IGFBP-2. IGFBP-2 mutants with altered IGF binding-, RGD-, and heparin-binding sites were generated and their actions examined. While mutating the RGD and heparin binding sites had little effect, altering the IGF binding site abolished its biological activity.

Conclusions/Significance

These results suggest that IGFBP-2 is a conserved regulatory protein and it inhibits growth and development primarily by binding to and inhibiting IGF actions in vivo. The duplicated IGFBP-2 genes may provide additional flexibility in the regulation of IGF activities.     

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

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

Expression of the IGFBP-2 gene in post-implantation rat embryos.

The insulin-like growth factors (IGFs) stimulate mitogenesis in a variety of cell types both in vitro and in vivo. These effects are mediated by both IGF receptors and a family of IGF binding proteins (IGFBPs), which are found complexed with the IGFs in serum and tissue fluids. Here we compare the sites of expression during early rat embryogenesis of the genes encoding the RGD-containing IGF binding protein IGFBP-2 and IGF-II. At all ages from early post-implantation through mid-gestation, the expression of IGFBP-2 was highly complementary to IGF-II. IGFBP-2 mRNA was detected throughout the epiblast of the egg cylinder as early as e7, when IGF-II expression was restricted to trophectoderm and other extraembryonic cells. As gastrulation proceeded, IGFBP-2 expression ceased as IGF-II expression began in the newly formed embryonic and extra-embryonic mesoderm, but was retained in other epiblast derivatives including the surface ectoderm and neuroectoderm, throughout its rostral-caudal extent. By e10-e11, IGFBP-2 expression in neuroectoderm was restricted to the rostral brain of the primary neural tube and was found in the new population of neuroepithelium formed in the tail bud during secondary neurulation. IGFBP-2 expression remained high in the ventricular layer of the rostral brain into mid-gestation ages but decreased or disappeared as cells entered the mantle layer and began to express the neurofilament-related gene alpha-internexin. IGFBP-2 mRNA was abundant in surface ectoderm, particularly that of the branchial arches, and all ectodermal placodes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Rapid apoptosis induction by IGFBP-3 involves an insulin-like growth factor-independent nucleomitochondrial translocation of RXRalpha/Nur77

Insulin-like growth factor-binding protein-3 (IGFBP-3) induces apoptosis by its ability to bind insulin-like growth factors (IGFs) as well as its IGF-independent effects involving binding to other molecules including the retinoid X receptor-alpha (RXRalpha). Here we describe that in response to IGFBP-3, the RXRalpha binding partner nuclear receptor Nur77 rapidly undergoes translocation from the nucleus to the mitochondria, initiating an apoptotic cascade resulting in caspase activation within 6 h. This translocation is a type 1 IGF receptor-signaling independent event as IGFBP-3 induces Nur77 translocation in R-cells. IGFBP-3 and Nur77 are additive in inducing apoptosis. GFP-Nur77 transfection into RXRalpha wild-type and knock-out mouse embryonic fibroblasts and subsequent treatment with IGFBP-3 show that RXRalpha is required for IGFBP-3-induced Nur77 translocation and apoptosis. Addition of IGFBP-3 to 22RV1 cell lysates enhanced the ability of GST-RXRalpha to "pull down" Nur77, and overexpression of IGFBP-3 enhanced the accumulation of mitochondrial RXRalpha. This unique nongenotropic nuclear pathway supports an emerging role for IGFBP-3 as a novel, multicompartmental signaling molecule involved in induction of apoptosis in malignant cells.