Insulin-like growth factor (IGF) binding protein-3 in pregnancy serum binds native IGF-I but not iodo-IGF-I.

Although serum immunoreactive insulin-like growth factor binding protein-3 (IGFBP-3) increases during pregnancy, radioligand binding methods such as ligand blotting with iodinated IGFs fail to detect the protein in pregnancy serum. Since IGFBP-3 must bind IGF-I or IGF-II to form a complex with the acid-labile subunit (alpha-subunit), we have used ternary complex formation from [125I]alpha-subunit as a measure of IGF binding to serum IGFBP-3. High-pressure liquid chromatography fractions containing IGFBP-3 from pregnancy serum did not bind [125I]IGF-I, although the equivalent fractions from nonpregnancy serum showed dose-dependent binding. In contrast, IGFBP-3 fractions from nonpregnancy and pregnancy sera both bound [125I]alpha-subunit in the presence of either exogenous IGF-I or endogenous serum IGFs, implying that non-iodinated IGFs could bind to the IGFBP-3. Substitution of nonradioactive iodo-IGF-I for native IGF-I in the complex formation assay confirmed that the pregnancy-induced alteration in IGFBP-3, probably resulting from proteolysis, prevents it from binding iodo-IGF-I while having little effect on its binding of the native peptide. This provides an explanation for the failure to detect IGFBP-3 in pregnancy by radioligand binding methods, and raises the question of the significance of proteolysis of IGFBP-3.     

Insulin-like growth factor (IGF)-binding protein-related protein-1: an autocrine/paracrine factor that inhibits skeletal myoblast differentiation but permits proliferation in response to IGF

Skeletal myogenic cells respond to the insulin-like growth factors (IGF-I and IGF-II) by differentiating or proliferating, which are mutually exclusive pathways. What determines which of these responses to IGF skeletal myoblast undergo is unclear. IGF-binding protein-related protein 1 (IGFBP-rP1) is a secreted protein with close homology to the IGF-binding proteins (IGFBPs) in the N-terminal region. IGFBP-rP1, previously called mac25 and IGFBP-7, is highly expressed in C2 skeletal myoblasts during the proliferative phase, but is down-regulated during myoblast differentiation. To determine the role of IGFBP-rP1 in myogenesis, IGFBP-rP1 was overexpressed in C2 myoblasts using a retroviral vector. Western blots indicated that the resulting C2-rP1 myoblasts secreted approximately 27-fold higher levels of IGFBP-rP1 than control C2-LX myoblasts that were transduced with a control vector (LXSN). Compared with C2-LX myoblasts, the differentiation responses of C2-rP1 myoblasts to IGF-I, IGF-II, insulin, and des(1-3)IGF-I were significantly reduced (P < 0.05). However, proliferation responses of C2-rP1 and C2-LX myoblasts to these same factors were not significantly different. Exposure of control C2-LX myoblasts to factors secreted by C2-rP1 myoblasts using a transwell coculture system reduced C2-LX myoblast differentiation significantly (P < 0.05). Experiments with the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 suggested that IGFBP-rP1 inhibits a MAPK-dependent differentiation pathway. In confirmation of this idea, levels of phosphorylated extracellular signal-regulated kinase-2 (a MAPK) were reduced in C2-rP1 myoblasts compared with those in C2-LX myoblasts. These findings indicate that IGFBP-rP1 may function as an autocrine/paracrine factor that specifies the proliferative response to the IGFs in myogenesis.     

Monitoring serum insulin-like growth factor-I (IGF-I), IGF binding protein-3 (IGFBP-3), IGF-I/IGFBP-3 molar ratio and leptin during growth hormone treatment for disordered growth

OBJECTIVE: Serum IGF-I levels are monitored during GH replacement treatment in adults with GH deficiency (GHD) to guide GH dose adjustment and to minimize occurrence of GH-related side-effects. This is not routine practice in children treated with GH. The aim of this study was to evaluate changes in (1) serum IGF-I, IGFBP-3 and IGF-I/IGFBP-3 molar ratio, and (2) serum leptin, an indirect marker of GH response, during the first year of GH treatment in children with disordered growth. DESIGN: An observational prospective longitudinal study with serial measurements at five time points during the first year of GH treatment was carried out. Each patient served as his/her own control. PATIENTS: The study included 31 patients, grouped as (1) GHD (n = 20) and (2) non-GHD (Turner syndrome n = 7; Noonan syndrome n = 4), who had not previously received GH treatment. MEASUREMENTS: Serum IGF-I, IGFBP-3 and leptin levels were measured before treatment and after 6 weeks, 3 months, 6 months and 12 months of GH treatment, with a mean dose of 0.5 IU/kg/wk in GHD and 0.7 IU/kg/wk in non-GHD groups. IGF-I, IGFBP-3 and the calculated IGF-I/IGFBP-3 molar ratio were expressed as SD scores using reference values from the local population. RESULTS: In the GHD group, IGF-I SDS before treatment was lower compared with the non-GHD (-5.4+/-2.5 vs. -1.8+/-1.0; P<0.001). IGF-I (-1.8 SDS +/- 2.2) and IGFBP-3 (-1.1 SDS +/- 0.6) levels and their molar ratios were highest at 6 weeks and remained relatively constant thereafter. In the non-GHD group, IGF-I levels increased throughout the year and were maximum at 12 months (0.3 SDS +/- 1.4) while IGFBP-3 (1.1 SDS +/- 0.9) and IGF-I/IGFBP-3 molar ratio peaked at 6 months. In both groups, IGF-I SDS and IGF-I/IGFBP-3 during treatment correlated with the dose of GH expressed as IU/m2/week (r-values 0. 77 to 0.89; P = 0.005) but not as IU/kg/week. Serum leptin levels decreased significantly during GH treatment in the GHD (median before treatment 4.0 microg/l; median after 12 months treatment 2.4 microg/l; P = 0.02) but not the non-GHD (median before treatment 3.0 microg/l; median after 12 months treatment 2.6 microg/l). In the GHD group, serum leptin before treatment correlated with 12 month change in height SDS (r = 0.70, P = 0.02). CONCLUSIONS: The pattern of IGF-I, IGFBP-3 and their molar ratio during the first year of GH treatment differed between the GHD and non-GHD groups. Calculation of GH dose by surface area may be preferable to calculating by body weight. As a GH dose-dependent increase in serum IGF-I and IGF-I/IGFBP-3 may be associated with adverse effects, serum IGF-I and IGFBP-3 should be monitored routinely during long-term GH treatment. Serum leptin was the only variable that correlated with first year growth response in GHD.     

Insulin-like growth factor (IGF)-binding protein-3 blocks IGF-I-induced receptor down-regulation and cell desensitization in cultured bovine fibroblasts.

Insulin-like growth factor-I (IGF-I) initiates its diverse biological effects by binding to type I IGF receptors on cells. In addition, IGF-I associates with distinct proteins that can modulate its actions. One of these IGF-binding proteins, IGFBP-3, is the major circulating form in adults and is produced by many cells in culture. We investigated the effect of purified bovine IGFBP-3 on IGF-I binding and IGF-I stimulation of amino acid uptake and DNA synthesis in cultured bovine fibroblasts, a cell culture system highly suitable for these types of studies. Incubation of cells with IGF-I resulted in time- and dose-dependent decreases in [125I]IGF-I binding and IGF-I stimulated [3H]aminoisobutyric acid uptake and [3H]thymidine incorporation. Preincubation with 4 nM IGF-I resulted in a 50-60% decrease in IGF-I receptor binding, accompanied by marked decreases in IGF-I-stimulated [3H]aminoisobutyric acid uptake (50-60%) and [3H]thymidine incorporation (80-90%). Preincubation with the IGF-I analog [QAYL]IGF-I (4 nM) or with 100 nM insulin, growth factors that bind and activate type I IGF receptor signalling but have little or no affinity for IGFBP-3, had effects comparable to IGF-I, decreasing both IGF-I binding and action 50-95%. The addition of IGFBP-3 during the preincubation period with IGF-I blocked the decrease in receptor availability and prevented the cells from becoming desensitized. IGFBP-3 did not prevent the [QAYL]IGF-I- or insulin-induced receptor loss and cellular resistance to IGF-I. These data indicate that IGFBP-3 can prevent IGF-I-induced receptor down-regulation, a process that renders cells refractory to further stimulation by IGF-I. Thus, cell-derived IGFBP-3 may function in a buffering capacity to restrict IGF-I and target cell interaction, thereby modulating the biological response to changes in local IGF-I levels.     

Pregnancy-associated plasma protein-A accounts for the insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) proteolytic activity in human pregnancy serum and enhances the mitogenic activity of IGF by degrading IGFBP-4 in vitro

Pregnancy-associated plasma protein-A (PAPP-A) has been identified as the insulin-like growth factor (IGF)-dependent IGF-binding protein-4 (IGFBP-4) protease produced by human fibroblasts. Recently, we found that serum proteases induced during human pregnancy cleaved IGFBP-4 in both an IGF-II-dependent and an IGF-II-independent fashion. This study sought to determine whether PAPP-A is the predominant IGFBP-4 protease in human pregnancy serum (PS) and to assess the in vitro role of serum PAPP-A. Immunoprecipitation with PAPP-A antibody effectively depleted PAPP-A from the PS and completely abolished both IGF-II-dependent and IGF-II-independent IGFBP-4 proteolytic activity in PS. Direct addition of PAPP-A antibody to PS completely blocked IGFBP-4 proteolysis and partially blocked IGFBP-5 proteolysis, but had no effect on IGFBP-3 proteolysis. To evaluate the role of serum PAPP-A, we tested whether PAPP-A in PS modulated the inhibitory activity of IGFBP-4 on IGF-II-induced cell proliferation in human osteosarcoma MG63 cells. The wild-type IGFBP-4 (WTBP-4; 200 ng/mL) failed to inhibit proliferation of the cells treated with PS (0.1% or 0.3%) alone or in combination with IGF-II (40 ng/mL), whereas the inhibitory effect of WTBP-4 was observed in the cells treated with nonpregnancy serum alone or in combination with IGF-II (P < 0.05). In contrast to WTBP-4, a protease-resistant IGFBP-4 was able to inhibit proliferation of the cells treated with PS alone or in combination with IGF-II (P < 0.05). In the presence of PAPP-A neutralizing antibody, the inhibitory effect of WTBP-4 on proliferation of the cells treated with IGF-II and PS was restored. In summary, these data demonstrate 1) that PAPP-A represents the predominant IGFBP-4 protease in PS; 2) that PAPP-A may in part contribute to IGFBP-5, but not IGFBP-3, proteolytic activity in PS; and 3) that PAPP-A enhances the bioactivity of IGFs in vitro by degrading IGFBP-4.     

Insulin-like growth factor (IGF)-binding protein release by human fetal fibroblasts: dependency on cell density and IGF peptides

Insulin-like growth factors (IGFs) are bound to specific binding proteins in extracellular fluids in vivo and when released by cells in vitro. One class of binding protein (IGF-BP), a peptide of 26 kDa purified from amniotic fluid, has been shown to modulate IGF bioactivity on isolated human fibroblasts. We have determined the factors that control release of IGF-BP from monolayers of human fetal fibroblasts using a radioimmunoassay, and have compared this with the effects of these factors on the release of IGF-I and -II. Separation of cell-conditioned cultured medium on SDS-PAGE, and subsequent immunoblotting with antibody against IGF-BP showed that fibroblasts released a single species of immunoreactive protein of an estimated molecular weight of 30 kDa. This was not the predominant binding protein released by cells since major bands of approximately 42 kDa and 39 kDa were visualized following separation by SDS-PAGE and ligand blotting with 125I-labelled IGF-I. The 30 kDa IGF-BP was released in parallel with radioimmunoassayable IGF-I and -II over 48 h. However, a significant inverse correlation was found between the release of IGF-BP, IGF-I, IGF-II and cell density. The exposure of fibroblasts to 1.3 nmol/l or greater of IGF-I or -II caused a significant release of IGF-BP. Maximum release was seen in sparse cultures with little or no release from confluent cultures. IGF-I and -II were approximately equipotent with a fourfold increase in IGF-BP release at 19.7 nmol/l. Insulin caused a release of IGF-BP and IGF-I and -II from fibroblasts at supraphysiological concentrations (16.7 nmol/l) which again was maximal on sparse cell cultures. Increases in IGF-BP, IGF-I and -II release were also found in the presence of human placental lactogen (23.3 nmol/l), but human GH, epidermal growth factor, fibroblast growth factor and platelet-derived growth factor were without effect. The results show that human fetal fibroblasts released an IGF-BP immunologically similar to that seen in amniotic fluid together with IGF-I and -II, that IGF-BP release was enhanced by exogenous addition of IGF peptides, and that the release of all three peptides was a property of sparsely plated, rapidly growing cells. These findings strengthen the hypothesis that the cellular expression of IGF-binding proteins may represent an important level of control in IGF physiology.     

Estrogens exert route- and dose-dependent effects on insulin-like growth factor (IGF)-binding protein-3 and the acid-labile subunit of the IGF ternary complex

We have previously shown that exogenous estrogens exert route-dependent effects on serum GH and insulin-like growth factor I (IGF-I) levels. IGF-I circulates as a ternary complex with IGF-binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). It is not known whether IGFBP-3 and ALS in blood are regulated by estrogen and, if so, whether this is also route dependent. In the present study we investigate the effects on IGFBP-3 and ALS of oral and transdermal estrogens (study 1), of different oral estrogen formulations (ethinyl estradiol, conjugated estrogen, and estradiol valerate; study 2), of different estrogen dosages (study 3) in normal postmenopausal women, and of oral estrogen in hypogonadal GH-deficient women (study 4). Administration of oral, but not transdermal, estrogen in normal postmenopausal women significantly decreased serum levels of IGFBP-3 and ALS (P < or = 0.005). The suppressive effects were similar with different oral estrogen formulations, and the degree of suppression increased with estrogen dosage. In hypogonadal GH-deficient women, oral estrogen treatment also significantly reduced IGFBP-3 and ALS (P = 0.02). The changes in IGF-I in each of the four studies paralleled the changes in both IGFBP-3 and ALS. In conclusion, exogenous estrogens suppress serum IGFBP-3 and ALS in a route- and dose-dependent manner, which are in parallel with the effects on serum IGF-I. These actions of oral estrogen are independent of endogenous GH status.     

Regulation of insulin-like growth factor (IGF)-I action by matrix metalloproteinase-3 involves selective disruption of IGF-I/IGF-binding protein-3 complexes

IGF-I and IGF-II play important roles in growth and development via interactions with cell-surface receptors; however, in nature, IGFs are sequestered by at least six soluble, high-affinity IGF-binding proteins (IGFBPs), namely IGFBPs 1-6. Herein, we demonstrate that the stromal cell-derived extracellular matrix-degrading metalloproteinase stromelysin 1 (matrix metalloproteinase 3) disrupts IGF/IGFBP-3 complexes and liberates free, intact IGFs, leading to phosphorylation of cell surface type 1 IGF receptors and cellular proliferation. Tissue inhibitor of metalloproteinases (TIMP-1) or an antibody to the type 1 IGF receptor mitigates IGF-mediated cellular proliferation. Thus, these studies suggest that matrix metalloproteinases, beyond their effects on extracellular matrix turnover, regulate cellular proliferation by modulating the bioavailability of IGFs, an event critical for such diverse phenomena as embryo development, morphogenesis, angiogenesis, and tumorigenesis.     

Mechanisms of Sertoli cell insulin-like growth factor (IGF)-binding protein-3 regulation by IGF-I and adenosine 3',5'-monophosphate.

FSH, which stimulates cAMP in the Sertoli cell, markedly lowers the concentration of insulin-like growth factor-binding protein-3 (IGFBP-3) in Sertoli cell-conditioned medium; in contrast, insulin-like growth factor-I (IGF-I) increases BP-3 expression. In this study, the mechanisms controlling the contrasting effects of cAMP and IGF-I were investigated. The abundance of BP-3 mRNA was dramatically lowered by (Bu)2cAMP, but was unaffected by IGF-I. Analyzed by ligand blot of conditioned medium, coincubation of (Bu)2cAMP and IGF-I largely eliminated the increase observed with IGF-I alone. Based on the following evidence, the effect of IGF-I appeared to be solely related to the capacity of IGF-I to interact directly with BP-3. 1) Insulin at micromolar concentrations failed to increase BP-3 abundance despite documentation by affinity cross-linking that insulin displaced [125I]IGF-I from the IGF-I receptor. 2) A synthetic IGF-I analog, [Leu24,1-62]IGF-I, which has reduced binding affinity for rat IGF-I receptor but displays high affinity for rat Sertoli cell-conditioned medium BPs, increased BP-3 abundance. 3) A synthetic IGF-I analog, B-chain mutant, which has reduced affinity for rat Sertoli cell BPs but displays normal affinity for the rat IGF-I receptor, failed to increase BP-3 abundance. 4) Human recombinant glycosylated [125I]BP-3 when added to cultured Sertoli cells was preserved in the medium when IGF-I or analogs with BP-3 affinity were present. 5) IGF-I, in dose-responsive manner, both retarded the disappearance from the medium of exogenously added human recombinant nonglycosylated BP-3 and decreased the amount of membrane-associated BP-3. These results indicate that whereas cAMP lowers BP-3 abundance in medium, most likely by markedly decreasing synthesis, IGF-I increases BP-3 accumulation by retarding its clearance by the Sertoli cell.     

Insulin-like growth factor (IGF) binding protein-3 inhibits type 1 IGF receptor activation independently of its IGF binding affinity

Insulin-like growth factor binding proteins (IGFBPs) regulate the cellular actions of the IGFs owing to their strong affinities, which are equal to or stronger than the affinity of the type 1 IGF receptor (IGF-IR), the mediator of IGF signal transduction. We recently found that IGFBP-3 modulates IGF-I binding to its receptor via a different mechanism possibly involving conformational alteration of the receptor. We have now investigated the effects of IGFBP-3 on the initial steps in the IGF signaling pathway. MCF-7 breast carcinoma cells were preincubated with increasing concentrations of IGFBP-3 and then stimulated with IGF-I, des(1-3)IGF-I, or [Q(3)A(4)Y(15)L(16)]-IGF-I, the latter two being IGF-I analogs with intact affinity for the type 1 IGF receptor, but weak or virtually no affinity for IGFBPs. Stimulation of autophosphorylation of the receptor and its tyrosine kinase activity was dose-dependently depressed. At 2.5 nM, IGFBP-3 provoked more than 50% inhibition of the stimulation induced by 3 nM des(1-3)IGF-1 and, at 10 nM, more than 80% inhibition. Similar results were obtained with [Q(3)A(4)Y(15)L(16)]-IGF-I. Cross-linking experiments using iodinated or unlabeled IGFBP-3 and anti-IGF-IR antibodies indicated that the inhibitory effects do not involve direct interaction between IGFBP-3 and IGF-IR. The inhibition appeared to be specific to IGFBP-3, because IGFBP-1 and IGFBP-5 at 10 nM had no significant effect. Also, inhibition was restricted to the IGF receptor, because IGFBP-3 failed to inhibit the tyrosine kinase activity of the insulin receptor stimulated by physiological concentrations of insulin. Our results provide the first demonstration that IGFBP-3 can specifically modulate the IGF-I signaling pathway independently of its IGF-I-binding ability. They also reveal a regulatory mechanism specific to the type 1 IGF receptor, with no effect on insulin receptor activation.     

Systemic administration of insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) increases bone formation parameters in mice by increasing IGF bioavailability via an IGFBP-4 protease-dependent mechanism

Insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) is a potent inhibitor of IGF actions in vitro. However, we found that systemic administration of IGFBP-4 at pharmacological doses caused a significant increase in bone formation parameters in mice by a mechanism that may involve increased IGF bioavailability via proteolysis of IGFBP-4. To evaluate the hypothesis that proteolysis of IGFBP-4 is essential for the stimulatory effects of systemically administered IGFBP-4, we produced wild-type, protease-resistant, and IGFBP-4 proteolytic fragments and evaluated their effects using biochemical markers. Protease-resistant IGFBP-4 was more potent than wild-type IGFBP-4 in inhibiting IGF-I-induced mouse osteoblast cell proliferation in vitro and in inhibiting IGF-I-induced increase in alkaline phosphatase (ALP) activity in bone extract after local administration in vivo. Systemic administration of wild-type IGFBP-4, but not protease-resistant IGFBP-4, increased serum osteocalcin, serum ALP, and ALP in skeletal extracts in a dose-dependent manner, with a maximal effect of 40% (P < 0.05) at 1.25 nmol/mouse. Systemic administration of wild-type, but not protease-resistant, IGFBP-4 increased free IGF-I levels in serum in normal mice. IGF-I, but not wild-type IGFBP-4, increased bone formation parameters in IGF-I-deficient mice. This study demonstrates that systemic administration of IGFBP-4 increases bone formation parameters in mice by increasing IGF bioavailability in the circulation via an IGFBP-4 protease-dependent mechanism.     

Insulin-like growth factor I (IGF-I)-binding protein complex is a better mitogen than free IGF-I

The insulin-like growth factors (IGF)-I and -II are bound to specific carrier proteins in the circulation. For investigation of their physiological role, the acid-stable subunit of the major binding protein (SmBP) was isolated from human plasma Cohn fraction IV. Its effect on the mitogenic activity of IGF-I was studied with baby hamster kidney fibroblasts (BHK-21) and human skin fibroblasts. While free IGF-I had no effect on thymidine incorporation into DNA with BHK-21 cells and only a moderate effect with human fibroblasts under standard conditions, DNA synthesis was significantly enhanced with both cell lines if IGF-I was complexed with SmBP before the experiment. The enhancement was optimal at an approximately equimolar ratio of both peptides. In contrast to experiments in which large concentrations of IGF-I were added at the beginning, repeated addition of small quantities of free IGF-I at hourly intervals clearly stimulated DNA synthesis in BHK-21 cells. Binding studies with radiolabeled SmBP revealed no evidence for direct interaction with either cell line. It is concluded that SmBP acts as a reservoir, releasing continuously low amounts of IGF-I and thereby creating a steady state situation of receptor occupancy, which appears to be a better mitogenic stimulus than temporary large concentrations of IGF-I.     

Vitronectin binding to IGF binding protein-5 (IGFBP-5) alters IGFBP-5 modulation of IGF-I actions.

IGF binding protein-5 (IGFBP-5) and vitronectin are matricellular proteins that are produced by smooth muscle cells and modulate their responsiveness to IGF-I. These studies were conducted to determine if vitronectin bound IGFBP-5 with high affinity and if this altered the ability of either protein to modify cellular responsiveness to IGF-I. Solution binding assays were used to determine that vitronectin bound to IGFBP-5 with high affinity. This binding was inhibitable with glycosaminoglycans. Synthetic peptides that contained four distinct regions of the IGFBP-5 sequence were used in competitive binding assays to determine the regions of IGFBP-5 that were necessary for vitronectin binding. The regions that mediated the interaction were determined to be between amino acids 201 and 218 and between amino acids 131 and 141. Mutation of specific basic residues within these regions resulted in significant reduction in vitronectin binding and residues R134, R136, K138, K139, R201, and K202 were shown to be the most important. When the combination of IGFBP-5 and IGF-I was added to smooth muscle cells that had been plated on a vitronectin-enriched matrix, the smooth muscle cell DNA synthesis and migration responses to IGF-I plus vitronectin were enhanced. In contrast, if mutant forms of IGFBP-5 that did not bind to vitronectin were used, the responses were decreased. These effects appeared to be due to modulation of IGF-I action because the addition of a mutant form of IGFBP-5 that did not bind to IGF-I had no additional effect over and above that noted with vitronectin alone. These findings suggest that localization of IGFBP-5 within the extracellular matrix by vitronectin results in modification of cellular responsiveness to IGF-I and that vitronectin may be an important component of the extracellular matrix that modulates IGFBP-5 actions.     

Insulin-like growth factors (IGFs) reduce IGF-binding protein-4 (IGFBP-4) concentration and stimulate IGFBP-3 independently of IGF receptors in human fibroblasts and epidermal cells.

Recent studies have provided a consensus that insulin-like growth factor-I (IGF-I) stimulates IGF-binding protein-3 (IGFBP-3) in vivo and in vitro. While it also appears well established that IGFBP-1 is inversely related to insulin concentrations, evidence regarding regulation of other IGFBP is inconclusive. Using immunoprecipitation and Western ligand blot, we have characterized the IGFBPs released into conditioned medium (CM) by cells from the adult human fibroblast cell line N3652 and the human epidermal squamous cell carcinoma line SCL-1. N3652 cells expressed IGFBP-3, IGFBP-2, a 24-kilodalton (kDa) IGFBP presumed to be IGFBP-4, and IGFBPs at 30 and 28 kDa. SCL-1 expressed IGFBP-3 and a putative IGFBP-4, with intermediate bands at 34 and 30 kDa. As determined by ligand blot of CM from confluent cells 72 h after the addition of peptides to serum-free medium, IGF-I and IGF-II potently stimulated IGFBP-3 in both cell lines, but otherwise IGFBP regulation in the two cells diverged. In N3652 cells, IGFBP-3 concentrations in CM increased to 700% and 800% of basal levels in the presence of IGF-I and IGF-II (at 100 ng/ml; n = 5 experiments), respectively. IGFBP-3 was not affected by insulin up to 10 micrograms/ml. In contrast, IGFBP-4 levels were diminished 54% and 73% by 100 ng/ml IGF-I and IGF-II, respectively, with no response to insulin. In SCL-1 cells, IGF-I and IGF-II were virtually identical in stimulating a mean 200% increase in IGFBP-3 (n = 5 experiments). Insulin was less potent, but caused a significant stimulation of IGFBP-3 levels. IGF-I, IGF-II, and insulin all stimulated an approximately 50% increase in IGFBP-4 concentrations. To test the hypothesis that IGF-induced alterations in IGFBP-3 and IGFBP-4 concentrations were regulated via the type 1 IGF receptor, we attempted to block IGFBP changes with type 1 IGF receptor antibody alpha IR-3 and to induce IGFBP changes with an IGF-II analog, [Leu27]IGF-II, with little affinity for the type 1 receptor. alpha IR-3 failed to block either the IGF-induced rise in IGFBP-3 in each cell line or the decline in IGFBP-4 in N3652 CM. [Leu27]IGF-II was as potent as IGF-II or IGF-I in inducing changes in IGFBP-3 and IGFBP-4 concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin-like growth factor (IGF)-I, IGF binding protein-3, and breast cancer risk: eight years on

Insulin-like growth factor (IGF)-I, and its main binding protein, IGFBP-3, are multi-functional regulatory peptides of cell growth and survival, attributes important for tumourigenesis. Following seminal work published in 1998, it has been hypothesised that circulating concentrations of these growth factors may be associated with cancer risk. Systematic reviews have reported that high normal range circulating levels of total IGF-I predict for pre- but not post-menopausal breast cancer. By contrast, associations with circulating IGFBP-3 have been inconsistent. A cumulative meta-analysis demonstrates that earlier reported positive associations between IGFBP-3 and pre-menopausal breast cancer risk now seem less clear as large-size cohorts are published. The reasons are complex and include differences in study design, lack of standardisation between assays, and variations in IGFBP-3 proteolytic activity--these are discussed in this commentary.     

Insulin-like growth factor (IGF) binding protein-3 attenuates prostate tumor growth by IGF-dependent and IGF-independent mechanisms

IGF binding protein (IGFBP)-3 inhibits cell growth and promotes apoptosis by sequestering free IGFs. In addition IGFBP-3 has IGF-independent, proapoptotic, antiproliferative effects on prostate cancer cells in vitro. Expression of the large T-antigen (Tag) under the long probasin promoter (LPB) in LPB-Tag mice results in prostate tumorigenesis. To investigate the IGF-dependent and IGF-independent effects of IGFBP-3 on prostate tumor growth, we crossed LPB-Tag mice with cytomegalovirus (CMVBP-3) and phosphoglycerate kinase (PGKBP-3) mice that overexpress IGFBP-3 under the cytomegalovirus promoter and the phosphoglycerate kinase promoter, respectively, and also I56G/L80G/L81G-mutant IGFBP-3 (PGKmBP-3) mice that express I56G/L80G/L81G-IGFBP-3, a mutant, that does not bind IGF-I but retains IGF-independent proapoptotic effects in vitro. Prostate tumor size and the steady-state level of p53 were attenuated in LPB-Tag/CMVBP-3 and LPB-Tag/PGKBP-3 mice, compared with LPB-Tag/wild-type (Wt) mice. A more marked effect was observed in LPB-Tag/CMVBP-3, compared with LPB-Tag/PGKBP-3, reflecting increased levels of transgene expression in CMVBP-3 prostate tissue. No attenuation of tumor growth was observed in LPB-Tag/PGKmBP-3 mice during the early tumor development, indicating that the inhibitory effects of IGFBP-3 were most likely IGF dependent during the initiation of tumorigenesis. At 15 wk of age, epidermal growth factor receptor expression was increased in LPB-Tag/Wt and LPB-Tag/PGKmBP-3 tissue, compared with LPB-Tag/PGKBP-3. IGF receptor was increased in all transgenic mice, but pAkt expression, a marker of downstream IGF-I action, was increased only in LPB-Tag/Wt and LPB-Tag/PGKmBP-3. After 15 wk of age, a marked reduction in tumor growth was apparent in LPB-Tag/PGKmBP-3 mice, indicating that the IGF-independent effects of IGFBP-3 may be important in inhibiting tumor progression.     

Insulin-like growth factor (IGF) I, -II, and IGF binding protein-3 and risk of ischemic stroke

BACKGROUND: Low IGF-I levels may be associated with the development of stroke; however, prospective data appear to be unavailable. METHODS: This was a nested case-control study within a Danish follow-up study, including 57,053 men and women. Baseline data included circulating IGF-I, IGF-II, and IGF binding protein (IGFBP)-3 concentrations as well as lifestyle factors and medical history. We identified 254 cases with incident ischemic stroke and 254 gender- and age-matched controls. RESULTS: Participants in the bottom quartiles of IGF-I and IGFBP-3 levels (median concentrations, 72 and 2937 ng/ml, respectively) were at increased risk of ischemic stroke, e.g. adjusted odds ratios (ORs) of 2.06 [95% confidence interval (CI), 1.05-4.03] and 2.29 (95% CI, 1.17-4.49), respectively, when compared with participants in the top quartiles (median concentrations, 125 and 4835 ng/ml, respectively). A negative, although weaker, association was also found for IGF-II (adjusted OR 1.44, 95% CI 0.79-2.64) when comparing the bottom quartile with the top quartile. No substantial associations were seen for IGF-I and IGF-II when also adjusting for IGFBP-3; adjusting IGFBP-3 for IGF-I and -II had only a minor impact on the risk estimates. CONCLUSION: These findings give some support to the hypothesis that the IGF axis is involved in the pathogenesis of ischemic stroke.     

Impaired blockade of insulin-like growth factor I (IGF-I)-induced hypoglycemia by IGF binding protein-3 analog with reduced ternary complex-forming ability

The hypoglycemic potential of circulating IGFs is thought to be regulated through the formation of ternary complexes consisting of an IGF, either IGF binding protein-3 (IGFBP-3) or IGFBP-5, and the acid-labile subunit. These high molecular weight complexes are confined to the circulation and represent a reservoir of IGF with a prolonged half-life. In this study, we show that hypoglycemia, induced by a bolus injection of recombinant human IGF-I into rats, can be blocked by coadministering equimolar concentrations of either recombinant glycosylated IGFBP-3 or nonglycosylated IGFBP-3 (IGFBP-3NG). In contrast, an IGFBP-3 mutant with reduced acid- labile subunit affinity (IGFBP-3MUT) only partially blocked the IGF-I hypoglycemic effect. IGFBP-3 and IGFBP-3NG significantly enhanced IGF-I retention in the circulation, whereas IGFBP-3MUT had a smaller effect. IGFBP-3MUT clearance was more rapid than that of the other IGFBP-3 forms, and the retention of all IGFBP-3 forms was greatly enhanced by coadministration of IGF-I. Characterization of the molecular mass distribution of the IGFBP-3 analogs indicated that 60% of IGFBP-3 and IGFBP-3NG was initially found in ternary complexes compared with 30% of IGFBP-3MUT. These data confirm the hypothesis that regulation of IGF-I bioactivity in vivo by IGFBP-3 depends on its ability to form ternary complexes.     

Insulin-like growth factor binding protein-1 (IGFBP-1) and IGF-I during oral and transdermal estrogen replacement therapy: relation to lipoprotein(a) levels

Low levels of insulin-like growth factor binding protein-1 (IGFBP-1) have recently been associated with several risk factors for cardiovascular disease. The effects of estrogen replacement therapy (ERT) on plasma IGFBP-1 levels are, however, unclear. A double-blind, placebo-controlled study for 6 months was conducted in 73 hysterectomized postmenopausal women randomized into two groups: oral estradiol (E2) valerate, 2 mg/day (n = 35) and transdermal E2 gel, 1 mg/day (n=38). Plasma IGFBP-1, insulin-like growth factor-I (IGF-I) and lipoprotein(a) (Lp(a)) were determined at baseline, 3 and 6 months. The groups were similar for age and BMI. The baseline levels of estrone (E1), E2, IGFBP-1, IGF-I and Lp(a) did not differ between the groups. During treatment, serum estradiol concentrations increased in both groups. During oral ERT, IGFBP-1 levels increased by 104% (P<0.001), whereas IGF-I levels decreased by 13% (mean, P<0.05). IGF-I and IGFBP-1 levels remained unchanged in the transdermal group. Lp(a) levels decreased by 23% (median, P<0.001) in the oral group, but were unaffected by transdermal therapy. The change in IGFBP-1 concentrations during oral ERT showed an inverse correlation to that in Lp(a) (r = -0.40, P<0.05, Spearman correlation). In conclusion, oral ERT seems to enhance plasma levels of IGFBP-1, which may be one reason for the reduced Lp(a) levels.