Platelet-derived growth factor negatively regulates the insulin-like growth factor signaling pathway through the coordinated action of phosphatidylinositol 3-kinase and protein kinase C beta I.

We recently described that epidermal and fibroblast growth factors (EGF and FGF) regulate the IGF-I signaling pathway at the level of IRS-1 through the cooperative action of two independent signaling pathways; one dependent on phosphatidylinositol 3-kinase (PI 3-kinase) and the other on protein kinase D1 (PKD1) (Karam et al. [22]). To determine whether this mechanism could be generalized to another tyrosine kinase receptor-dependent growth factor, the effect of platelet-derived growth factor (PDGF) on the IGF-I signaling pathway was studied.

Phosphatidylinositol 3-kinase and protein kinase D1 specifically cooperate to negatively regulate the insulin-like growth factor signaling pathway.

Insulin receptor substrate-1 (IRS-1) is a key protein in the insulin-like growth factor (IGF) signaling whose tyrosine phosphorylation by the type 1 IGF receptor is necessary for the recruitment and activation of the downstream effectors. Through the analysis of cross-talks occurring between different tyrosine kinase receptor-dependent signaling pathways, we investigated how two growth factors [epidermal growth factor (EGF) and fibroblast growth factor (FGF)] could modulate the IGF-I-induced IRS-1 tyrosine phosphorylation and its downstream signaling. EGF and FGF inhibited IGF-I-stimulated tyrosine phosphorylation of IRS-1 and the subsequent IGF-I-induced phosphatidylinositol 3-kinase (PI 3-kinase) activity.

Growth factor-specific regulation of insulin receptor substrate-1 expression in MCF-7 breast carcinoma cells: effects on the insulin-like growth factor signaling pathway.

IGFs are potent mitogens that play a crucial role in cell proliferation and/or differentiation and tumorigenesis. Insulin receptor substrate-1 (IRS-1) is a key protein in the IGF signaling pathway in the estrogen-dependent MCF-7 breast carcinoma cell line. In this study, three growth factors [fibroblast growth factor (FGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF)] were tested for their ability to modulate IRS-1 protein expression and the IGF-I signaling pathway.

Insulin-like growth factor binding protein-6 inhibits neuroblastoma cell proliferation and tumour development.

In neuroblastoma cells, survival and proliferation are dependent upon the insulin-like growth factor (IGF) system. IGFs actively participate in cell growth, whereas IGFBP-6, is associated with the arrest of growth. With a view to blocking IGF-II action, we produced recombinant human IGFBP-6 capable of binding IGFs with affinities between 1.

Insulin-like growth factor binding protein-3 increases intracellular calcium concentrations in MCF-7 breast carcinoma cells.

Insulin-like growth factor binding protein-3, IGFBP-3, specifically binds to IGFs with high affinity, but it is also capable of modulating the IGF-I signalling pathway or inducing apoptosis independently of its binding to IGFs. The molecular mechanisms underlying the action of IGFBP-3 have not been elucidated. In this study, we have demonstrated that binding of IGFBP-3 to a cell surface receptor in MCF-7 breast carcinoma cells induces a rapid and transient increase in intracellular free calcium.

Use of the ligand immunofunctional assay for human insulin-like growth factor ((IGF) binding protein-3 (IGFBP-3) to analyze IGFBP-3 proteolysis and igf-i bioavailability in healthy adults, GH-deficient and acromegalic patients, and diabetics.

The ligand immunofunctional assay for plasma insulin-like growth factor (IGF) binding protein (IGFBP)-3 developed in our laboratory provides for specific measurement of intact, as opposed to proteolyzed, IGFBP-3. IGFBP-bound IGFs are dissociated and separated by acid pH ultrafiltration; thereafter, intact and proteolyzed IGFBP-3 are captured by a monoclonal antibody in a solid-phase assay and incubated with (125)I-IGF-I, which detects the intact protein but not its proteolytic fragments. This assay was combined with assays for IGF-I (RIA of the ultrafiltrate) and total IGFBP-3 (immunoradiometric assay) to quantify the percentage of proteolyzed IGFBP-3 (percent proteolyzed IGFBP-3) and to calculate the IGF-I/intact IGFBP-3 ratio as an index of the fraction of exchangeable IGF-I bound to IGFBP-3.

Measurement of intact insulin-like growth factor-binding protein-3 in human plasma using a ligand immunofunctional assay.

Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is a fundamental mechanism in the regulation of IGF-I bioavailability in the bloodstream. Its measurement by Western immunoblotting provides only semiquantitative estimation. We have developed a ligand immunofunctional assay (LIFA) for quantifying human (h) intact IGFBP-3 in biological fluids.

Growth retardation in constitutionally short children is related both to low serum levels of insulin-like growth factor-I and to its reduced bioavailability.

Measurements of serum levels of insulin-like growth factor (IGF)-I, IGF-II and IGF binding protein (IGFBP)-1 have been carried out in conjunction with Western ligand blot analysis of serum IGFBPs in 39 constitutionally short children and adolescents and compared with those of 27 age-matched normal subjects (and also with 23 hypopituitary patients). Estimated amounts of the two forms of IGFBP-3 (42 and 39 kDa) and of IGFBP-2 (34 kDa) were obtained by laser densitometry scanning. Mean serum levels of IGF-I were decreased by 46% +/- 5% in short, compared to normal, prepubertal children (P < 0.

A proteolytic fragment of insulin-like growth factor (IGF) binding protein-3 that fails to bind IGFs inhibits the mitogenic effects of IGF-I and insulin.

Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is increasingly becoming recognized as an essential mechanism in the regulation of insulin-like growth factor (IGF) bioavailability, both in the bloodstream and at cellular level. Plasmin generated on contact with various cell types provokes proteolytic cleavages that are similar to those induced in vivo by (as yet unidentified) IGFBP-3 proteases. Experimental conditions were determined to achieve plasmin-induced limited proteolysis of recombinant human nonglycosylated IGFBP-3.

Isolation and characterization of proteolytic fragments of insulin-like growth factor-binding protein-3.

Limited proteolysis of insulin-like growth factor-binding protein-3 (IGFBP-3) is a normal process in the regulation of insulin-like growth factor (IGF) activity, which we have reproduced in vitro using plasmin and recombinant human non-glycosylated IGFBP-3 in order to isolate and characterize the fragments obtained. Two major fragments of 22-25 and 16 kD were purified by RP-HPLC. The 22- to 25-kD fragment had severely reduced affinity for IGF-I, compared with intact IGFBP-3.

A proteolytic fragment of insulin-like growth factor (IGF) binding protein-3 that fails to bind IGF is a cell growth inhibitor.

Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is now recognized as a normal process in the regulation of insulin-like growth factor (IGF) activity, its major effect being to increase IGF bioavialability. In order to characterize the proteolytic fragments of IGFBP-3, we reproduced this proteolysis in vitro using plasmin which provokes cleavages that are similar to those induced in vivo by (unidentified) specific IGFBP-3 proteases. Two major peaks were purified by RP-HPLC.

Protease-induced alteration of insulin-like growth factor binding protein-3 as detected by radioimmunoassay. Agreement with ligand blotting data.

Structural alteration of insulin-like growth factor binding protein-3 (IGFBP-3) resulting from limited proteolysis by one or more serine proteases in vivo was first described in the serum of pregnant women and in certain pathological conditions. Western immunoblotting has since been employed to detect the phenomenon in normal serum, using a polyclonal antibody raised against recombinant human IGFBP-3 and a highly sensitive technique of visualization by chemiluminescence. The major proteolytic fragment of 30 kDa, which fails to be detected in native serum by ligand blotting owing to its weak affinity for IGFs, has proved clearly visible in all serum samples tested, sometimes accompanied by smaller fragments of 20 and 16 kDa.

Insulin-like growth factor binding protein-3 is functionally altered in pregnancy plasma.

In a variety of physio-pathological conditions, but also in the normal state, calcium-dependent serine protease(s) partially proteolyze proportions of circulating insulin-like growth factor binding protein-3 (IGFBP-3). This occurs without disrupting the 150-kilodalton (kDa) complexes in which IGFBP-3 carries of most of the IGF-I and IGF-II in serum. In this work we show that the 150-kDa complex is functionally altered during pregnancy, which is when the largest proportion of IGFBP-3 is proteolyzed.

Two insulin-like growth factor (IGF)-binding proteins are responsible for the selective affinity for IGF-II of cerebrospinal fluid binding proteins.

We have studied the relationships between the structure and affinity of two insulin-like growth factor-binding proteins (IGFBPs) purified from human cerebrospinal fluid (CSF). Competitive binding studies were performed using preparations of human recombinant IGF (rhIGF-I, rhIGF-II, and their labeled homologs) and the truncated variant form of IGF-I, rh-Des-(1-3)-IGF-I. One of these BPs, which is the most consistently detected in CSF, corresponds to IGFBP-2.

Serum insulin-like growth factors and insulin-like growth factor binding proteins in the human fetus. Relationships with growth in normal subjects and in subjects with intrauterine growth retardation.

IGF-I, IGF-II, and their binding proteins (BP) were studied in sera obtained by direct puncture of umbilical cords in utero between 20 and 37 wk of gestation in 103 normal fetuses and in 16 fetuses with intrauterine growth retardation, as well as in the cord blood of 37 normal newborns of 38- to 42-wk pregnancies. In normal fetuses, IGF-I levels were approximately 50 ng/mL and IGF-II levels approximately 350 ng/mL up to the 33rd wk of pregnancy. Thereafter, both increased to reach values two to three times higher at term.

Evidence of enzymatic degradation of insulin-like growth factor-binding proteins in the 150K complex during pregnancy.

Western ligand blot analysis of the different molecular forms of insulin-like growth factor-binding protein IGF-BP) in serum and plasma samples from 89 pregnant women has revealed a marked decrease, after the second month of pregnancy, in the 41.5 and 38.5K species (which are the binding units of the 150K complex) as well as in the 24K form.

Heterogeneity of insulin-like growth factor binding proteins and relationships between structure and affinity. 1. Circulating forms in man.

Circulating insulin-like growth factors (IGFs) are bound to specific, high-affinity binding proteins (BPs), and form complexes with relative molecular masses of about 150,000 ('large' complex) and 40,000 ('small' complex). The large complex appears to be under growth-hormone control and its proportions vary with those of the IGFs. Molecular heterogeneity among the binding proteins was revealed by polyacrylamide gel electrophoresis (SDS-PAGE) of serum in which they were cross-linked to 125I-labelled IGF I or II.

Specific assay for insulin-like growth factor (IGF) II using the IGF binding proteins extracted from human cerebrospinal fluid.

A protein-binding assay for insulin-like growth factor II (IGF II) is described. The assay uses IGF binding proteins extracted from human cerebrospinal fluid which have selective affinity for IGF II. IGF I was 9 times less potent than IGF II in displacing [125I]IGF II, and when mixtures of the IGFs were assayed at IGF I/IGF II ratios of 2, 5, and 10, interference from IGF I in the assay was 0%, 5%, and 9%, respectively.

Somatomedin (insulin-like growth factors)-binding proteins. Molecular forms and regulation.

The insulin-like growth factor (IGF)-binding proteins present in the human serum and various biological media have been characterized by several methods: gel filtration, sucrose gradient sedimentation, polyacrylamide gel electrophoresis and chromatofocusing. Several forms have been identified with molecular weights of approximately 42,000, 39,000, 34,000, 30,000 and 24,000 daltons. Results of competitive binding studies suggest that the different forms of binding proteins have different affinities for IGF-I and IGF-II.

Triiodothyronine stimulates the production of insulin-like growth factor (IGF) by fetal hypothalamus cells cultured in serum-free medium.

Grown in serum-free medium, dissociated cells from fetal mouse hypothalami release insulin-like growth factors (IGFs) and their binding proteins (IGF BPs) into the culture medium. Addition of triiodothyronine (10-12-10-8 M), which enhances neuron maturation, resulted in a significant increase in IGF concentration. By contrast, there was no significant effect on IGF BP.

Preferential measurement of insulin-like growth factor (IGF) I-related peptides in serum with the aid of IGF-binding proteins (IGF BPs) produced by rat liver in culture. Estimation of serum IGF BP levels.

A protein-binding assay for insulin-like growth factor (IGF) was developed which preferentially measures IGF I-related peptides in serum. Binding proteins (BPs) extracted from the culture media of livers from approximately 4-week-old rats were used in the assay, with pure IGF I as tracer and a partially purified IGF preparation as standard. Serum samples were gel filtered in acetic acid to separate the IGFs from their BPs.

Evidence for production by the liver of two IGF binding proteins with similar molecular weights but different affinities for IGF I and IGF II. Their relations with serum and cerebrospinal fluid IGF binding proteins.

Studies of the competitive binding of IGF I and IGF II and 125I-labelled IGF I and IGF II to the specific binding proteins (BPs) produced by the liver in culture suggest that two BPs exist, one with a selective affinity for IGF I and the other with a selective affinity for IGF II. The ratio of the former BP to the latter appeared to be higher in the culture medium of young rat livers and of fetal human livers than that in the culture medium of adult human livers. The two BPs form complexes with their IGFs with the same apparent molecular weight of approximately 40K.