SMART MODE:

NORMAL GENOMIC

• Horney MJ, Evangelista CA, Rosenzweig SA
• 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.
• J Biol Chem. 2001; 276: 2880-9
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• 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.

• Zeslawski W et al.
• The interaction of insulin-like growth factor-I with the N-terminal domain of IGFBP-5.
• EMBO J. 2001; 20: 3638-44
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• Insulin-like growth factors (IGFs) are key regulators of cell proliferation, differentiation and transformation, and are thus pivotal in cancer, especially breast, prostate and colon neoplasms. They are also important in many neurological and bone disorders. Their potent mitogenic and anti-apoptotic actions depend primarily on their availability to bind to the cell surface IGF-I receptor. In circulation and interstitial fluids, IGFs are largely unavailable as they are tightly associated with IGF-binding proteins (IGFBPs) and are released after IGFBP proteolysis. Here we report the 2.1 A crystal structure of the complex of IGF-I bound to the N-terminal IGF-binding domain of IGFBP-5 (mini-IGFBP-5), a prototype interaction for all N-terminal domains of the IGFBP family. The principal interactions in the complex comprise interlaced hydrophobic side chains that protrude from both IGF-I and the IGFBP-5 fragment and a surrounding network of polar interactions. A solvent-exposed hydrophobic patch is located on the IGF-I pole opposite to the mini-IGFBP-5 binding region and marks the IGF-I receptor binding site.

• Matsumoto T, Tsurumoto T, Goldring MB, Shindo H
• Differential effects of IGF-binding proteins, IGFBP-3 and IGFBP-5, on IGF-I action and binding to cell membranes of immortalized human chondrocytes.
• J Endocrinol. 2000; 166: 29-37
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• Insulin-like growth factor-I (IGF-I) is an important anabolic factor for cartilage tissue and its action is, in part, regulated by IGF-binding proteins (IGFBPs). The object of this study was to investigate the effects of IGFBPs on IGF-I action and on binding of IGF-I to cells using a reproducible immortalized human chondrocyte culture model. Treatment of the C-28/I2 cells with IGF-I or des(1-3)IGF-I in serum-free medium stimulated cell proliferation in a dose-dependent manner. However, the effect of des(1-3)IGF-I was more potent, thereby suggesting that endogenously produced IGFBPs inhibited IGF action. The stimulatory effect of IGF-I was inhibited significantly by addition of IGFBP-3 but enhanced slightly by IGFBP-5. However, neither IGFBP-3 nor IGFBP-5 had an effect on basal cell growth. Binding of (125)I-labeled IGF-I to the cells was displaced by both IGFBP-3 and IGFBP-5, although higher concentrations of unlabeled IGFBP-5 were required to displace IGF-I to the same extent as IGFBP-3. Treatment of the cells with IGF-I increased the levels of IGFBP-5 protein measured by Western ligand blotting, and stimulated a corresponding increase in IGFBP-5 mRNA while increasing type II collagen mRNA. Our findings indicate that the balance between IGFBP-3 and IGFBP-5 influences IGF receptor binding and its action on chondrocyte proliferation, and may thereby modulate cartilage metabolism.

• Baxter RC
• Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities.
• Am J Physiol Endocrinol Metab. 2000; 278: 96776-96776
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• The insulin-like growth factor (IGF)-binding proteins (IGFBPs) are a family of six homologous proteins with high binding affinity for IGF-I and IGF-II. Information from NMR and mutagenesis studies is advancing knowledge of the key residues involved in these interactions. IGF binding may be modulated by IGFBP modifications, such as phosphorylation and proteolysis, and by cell or matrix association of the IGFBPs. All six IGFBPs have been shown to inhibit IGF action, but stimulatory effects have also been established for IGFBP-1, -3, and -5. These generally involve a decrease in IGFBP affinity and may require cell association of the IGFBP, but precise mechanisms are unknown. The same three IGFBPs have well established effects that are independent of type I IGF receptor signaling. IGFBP-1 exerts these effects by signaling through alpha(5)beta(1)-integrin, whereas IGFBP-3 and -5 may have specific cell-surface receptors with serine kinase activity. The regulation of cell sensitivity to inhibitory IGFBP signaling may play a role in the growth control of malignant cells.

• Bhakta NR, Garcia AM, Frank EH, Grodzinsky AJ, Morales TI
• The insulin-like growth factors (IGFs) I and II bind to articular cartilage via the IGF-binding proteins.
• J Biol Chem. 2000; 275: 5860-6
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• Bovine articular cartilage discs (3 mm diameter x 400 micrometer thick) were equilibrated in buffer containing (125)I-insulin-like growth factor (IGF)-I (4 degrees C) +/- unlabeled IGF-I or IGF-II. Competition for binding to cartilage discs by each unlabeled IGF was concentration-dependent, with ED(50) values for inhibition of (125)I-IGF-I binding of 11 and 10 nM for IGF-I and -II, respectively, and saturation by 50 nM. By contrast, an analog of IGF-I with very low affinity for the insulin-like growth factor-binding proteins (IGF-BPs), des-(1-3)-IGF-I, was not competitive with (125)I-IGF-I for cartilage binding even at 100-400 nM. Binding of the (125)I-labeled IGF-II isoform to cartilage was competed for by unlabeled IGF-I or -II, with ED(50)s of 160 and 8 nM, respectively. This probably reflected the differential affinities of the endogenous IGF-BPs (IGF-BP-6 and -2) for IGF-II/IGF-I. Transport of (125)I-IGF-I was also measured in an apparatus that allows diffusion only across the discs (400 micrometer), by addition to one side and continuous monitoring of efflux on the other side. The time lag for transport of (125)I-IGF was 266 min, an order of magnitude longer than the theoretical prediction for free diffusion in the matrix. (125)I-IGF-I transport then reached a steady state rate (% efflux of total added (125)I-IGF/unit time), which was subsequently accelerated approximately 2-fold by addition of an excess of unlabeled IGF-I. Taken together, these results indicate that IGF binding to cartilage, mostly through the IGF-BPs, regulates the transport of IGFs in articular cartilage, probably contributing to the control of their paracrine activities.

• Rosenfeld RG et al.
• The insulin-like growth factor binding protein superfamily: new perspectives.
• Pediatrics. 1999; 104: 1018-21
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• The insulin-like growth factor (IGF) binding proteins (IGFBPs) were initially identified as carrier proteins for IGF-I and IGF-II in a variety of biologic fluids. Their presumed function was to protect IGF peptides from degradation and clearance, increase the half-life of the IGFs, and deliver them to appropriate tissue receptors. The concept of IGFBPs as simple carrier proteins has been complicated, however, by a number of observations: 1) the six IGFBPs vary in their tissue expression and their regulation by other hormones and growth factors; 2) the IGFBPs are subjected to proteolytic degradation, thereby altering their affinities for the IGFs; 3) IGFBP-3 and IGFBP-5, in addition to binding IGFs, also can associate with an acid-labile subunit, thereby increasing further the half-life of the IGFs; 4) in addition to modifying the access of IGF peptides to IGF and insulin receptors, several of the IGFBPs may be capable of increasing IGF action; 5) some of the IGFBPs may be capable of IGF-independent regulation of cell growth; 6) some of the IGFBPs are associated with cell membranes or possibly with membrane receptors; and 7) some of the IGFBPs have nuclear recognition sites and may be found within the nucleus. Additionally, a number of cDNAs identified recently have been found to encode proteins that bind IGFs, but with substantially lower affinities than is the case with IGFBPs. The N-terminal regions of the predicted proteins are structurally homologous to the classic IGFBPs, with conservation of the cysteine-rich region. These observations suggest that these low-affinity binders are members of an IGFBP superfamily, capable of regulating cell growth by both IGF-dependent and IGF-independent mechanisms.insulin-like growth factor, insulin-like growth factor binding proteins.

• Lee DY
• Identification of insulin-like growth factor (IGF)-I and IGF-binding protein in chylous ascites.
• J Korean Med Sci. 1998; 13: 17-20
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• Insulin-like growth factors (IGFs) are bound by several IGF-binding proteins (IGFBPs) that appear to regulate IGF transportation, receptor binding and action. In adult human serum, most of IGFs are bound in a 150 kDa complex which could not cross the capillary wall. We measured IGF-I and IGFBPs in chyle by radioimmunoassay and western ligand blot. The concentration of IGF-I in chyle was only 15% of the corresponding serum level and most of IGF-I was found in 50 kDa complex. The IGFBPs profile in chyle, especially IGFBP-3, was different from that of serum. The concentration of IGFBP-3 in chyle was much less than in serum and the size of glycosylated IGFBP-3 was different from that of serum. However, the size and relative amount of IGFBP-1 and -2 in chyle were similar to serum. This finding indicates that IGF-I and IGFBPs in chyle to a large extent originate in the vascular system and only the 50 kDa complex can cross the capillary barrier.

• Lee DY, Park SK, Kim JS
• Insulin-like growth factor-I (IGF-I) and IGF-binding proteins in children with nephrotic syndrome.
• J Clin Endocrinol Metab. 1996; 81: 1856-60
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• Growth failure appears to be a major problem for nephrotic children who fail to respond to steroid therapy. Recently altered serum insulin-like growth factor (IGF) and IGF-binding protein (IGFBP) profiles are reported in renal failure and glomerulonephritis. In this study, the serum IGFBP profile was evaluated by Western ligand blot and RIA in 22 patients with the nephrotic syndrome. Serum IGFBP-3 was decreased, whereas IGFBP-2 was increased in most patients with the nephrotic syndrome. The mean serum IGFBP-3 level was 2123 +/- 531 ng/mL in active states and was increased to a normal level (3593 +/- 407 ng/mL) in remission states. We also measured serum IGF-I by RIA. The serum concentration of IGF-I (mean +/- SD) was 67.4 +/- 23.2 ng/mL in active states and was increased to 127.1 +/- 21.8 ng/mL in remission states, but was still lower than that in control subjects (180.4 +/- 15.8 ng/mL). IGF-I and IGFBP-3 levels were not correlated with primary renal diseases or the amount of proteinuria. For serum IGF-IGFBP complexes, 150-kDa complexes were significantly decreased in patients with the nephrotic syndrome compared with those in control subjects. In urine from nephrotic syndrome patients, 150- and 50-kDa complexes were found, whereas these complexes did not exist in the urine of control subjects. We speculate that low serum IGF-I and IGFBP-3 levels would be partially due to the increased urinary losses of serum IGF-IGFBP complexes, especially that of 150 kDa, and these changes may contribute to growth failure in persistent nephrotic syndrome.

• Grellier P, Feliers D, Yee D, Woodruff K, Abboud SL
• Interaction between insulin-like growth factor-I and insulin-like growth factor-binding proteins in TC-1 stromal cells.
• J Endocrinol. 1996; 149: 519-29
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• IGF-I and -II play an important role in regulating bone formation. Bone marrow stromal cells, particularly those with osteoblast-like features, may act in concert with osteoblasts to increase IGF-I and -II levels in the bone microenvironment. Local bioavailability of IGFs, however, is modulated by IGF binding proteins (IGFBPs). We have previously demonstrated that murine TC-1 stromal cells constitutively secrete IGF-I and IGFBPs. In the present study, we determined the phenotype of these cells and used them as a model to explore the effect of IGFBPs on IGF-I-induced mitogenesis. The effect of IGF-I on IGFBPs expressed by TC-1 was also determined. When grown under conditions that promote osteogenic differentiation, TC-1 cells showed high alkaline phosphatase activity and mRNA levels, weakly expressed osteocalcin mRNA, and formed mineralized bone-like nodules. TC-1 cells expressed IGF-I and IGF-II mRNAs, while other stromal phenotypes preferentially expressed IGF-I. IGF-I stimulated TC-1 DNA synthesis in a dose-dependent manner and this effect was inhibited by recombinant IGFBP-1 and -4. Since IGF-I may regulate IGFBP production, the effect of IGF-I on IGFBPs expressed by TC-1 cells was determined. IGF-I increased the abundance of IGFBP-3, -4 and -5 in TC-1 conditioned medium; this correlated with induction of IGFBP-3 mRNA, but not with that of IGFBP-4 or -5 mRNAs. The findings demonstrate that most stromal cells express IGF-I which may act in an autocrine and/or paracrine fashion. The local effects of IGF-I, however, may be blocked by IGFBP-1 or -4. IGF-I regulates the relative abundance of IGFBPs in stromal cells which, in turn, may influence IGF-I-mediated effects on bone remodeling.

• Bereket A et al.
• Effect of short-term fasting on free/dissociable insulin-like growth factor I concentrations in normal human serum.
• J Clin Endocrinol Metab. 1996; 81: 4379-84
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• A small portion of circulating insulin-like growth factor I (IGF-I) is detected in the free or readily dissociable state, which is thought to be the metabolically active form. The amount of free/dissociable IGF-I in serum is dependent on a complex interplay between the production rate and the concentrations of IGF-I and IGF-binding proteins (IGFBPs). IGF availability is also influenced by posttranslational changes in IGFBPs that affect the affinity of IGFBPs for IGF-I. In the present study, we examined whether a short term fast (approximately 12 h) alters the serum concentration of free/dissociable IGF-I, and whether these changes are associated with alterations in IGFBP-1 and the proteolysis status of IGFBP-3. Circulating free/dissociable IGF-I concentrations, as assessed by a two-site immunoradiometric assay, did not differ between fasting and 4 h after a morning meal (1.48 +/- 0.07 vs. 1.50 +/- 0.07 microgram/L, respectively). Likewise, free/dissociable IGF-I levels measured by RIA after separation by centrifugal ultrafiltration were not different between the two groups (1.43 +/- 0.14 vs. 1.38 +/- 0.18 microgram/L, respectively). IGF-I bioactivity, as measured by thymidine incorporation by fibroblasts, did not differ in fasting and 4-h postprandial sera. There was no difference in IGFBP-3 and total acid-ethanol-extractable IGF-I concentrations in serum from fasted and fed subjects. In contrast, the concentration of IGFBP-1 in the serum was increased approximately 5-fold in the fasted state compared to fed values. IGFBP-1 existed in a highly phosphorylated form under fasting conditions. There was no change in IGFBP-3 proteolysis assessed either in vivo or in vitro between the fasting and fed states. The results indicate that a physiologically relevant short term overnight fast does not alter the circulating levels of free/dissociable IGF-I despite a marked elevation in IGFBP-1.

• Arai T, Clarke J, Parker A, Busby W Jr, Nam T, Clemmons DR
• Substitution of specific amino acids in insulin-like growth factor (IGF) binding protein 5 alters heparin binding and its change in affinity for IGF-I response to heparin.
• J Biol Chem. 1996; 271: 6099-106
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• Heparin binding to insulin-like growth factor (IGF)-binding protein 5 (IGFBP-5) leads to a 17-fold decrease in its affinity for IGF-I, and a region that contains several basic amino acids (Arg201-Arg218) may be involved in this affinity shift. In the present study, mutagenesis was used to analyze the effect of substitutions for basic amino acids in the Arg201-Arg218 region of IGFBP-5 on heparin-binding and the heparin-induced affinity shift. Nine mutant forms were prepared. Their association constants (Ka) for IGF-I were similar to native IGFBP-5. When 10 microg/ml of heparin was added, the Ka of native IGFBP-5 decreased 17-fold, and the Ka of the K134A/R136A mutant decreased 16-fold. In contrast, substitutions for specific basic amino acids in the Arg2O1-Arg218 region decrease the affinity shift to 1.1-3.2-fold. Lys 211 was especially important. When a mutant containing that single substitution was tested, heparin caused only a 2.5-fold reduction in IGF-I affinity. Affinity cross-linking studies showed that heparin was equipotent in inhibiting the formation of 125I-IGF-I-K134A/Rl36A mutant complexes compared to native IGFBP-5. In contrast, heparin had minimal effects on the formation of complexes between 125I-IGF-I and the other mutants. The heparin-binding activity of each mutant was determined. Four mutants, R201A/K202N, K202A/K206A/R207A, R201A/K202N/K206N/K208N, and K211N/R214A/K217A/R218A, had reduced heparin binding compared to native IGFBP-5. The other five mutants, including the K21IN mutant, showed no change in heparin binding. The four mutants with reduced heparin binding could be dissociated from heparin-Sepharose with much lower NaCl concentrations, indicating that they had reduced affinity. These findings suggest that Arg201 Lys202, LysS206, and Arg214 are important for heparin binding. In contrast, LyS211 is not important for the binding of IGFBP-5 to heparin, but substitution for it reduced the heparin-induced affinity shift.

• Ewton DZ, Florini JR
• IGF binding proteins-4, -5 and -6 may play specialized roles during L6 myoblast proliferation and differentiation.
• J Endocrinol. 1995; 144: 539-53
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• It is well known that IGFs-I and -II stimulate both the proliferation and differentiation of myoblasts, but the role of the IGF binding proteins (IGFBPs) during these processes has not been established. In this study we show that IGF-I analogs with greatly reduced affinity for IGFBPs exhibited about a 10-fold increase in potency in stimulating proliferation (as in other cell types), but up to a 100-fold greater potency than native IGF-I in stimulating L6A1c differentiation. Analysis of conditioned media revealed that L6 cells secrete significant levels of IGFBPs that react with antisera to IGFBP-4, -5 and -6. Steady-state levels of IGFBP-4 mRNA were highest in proliferating myoblasts, while IGFBP-5 mRNA could not be detected in myoblasts although its levels were dramatically increased during IGF- or insulin-stimulated differentiation of myoblasts into myotubes. Elevated IGFBP-6 mRNA levels were found in quiescent cells in serum-free medium. IGF-I and IGF-II treatment elevated IGFBP-5 in conditioned media, but longR3IGF-I and insulin, which do not bind to IGFBPs, had smaller effects. This complex regulation of expression of different IGFBPs not only during different stages of muscle growth and differentiation, but also upon stimulation by IGFs or insulin, suggests that the IGFBPs play a specific and significant role in modulating the actions of the IGFs during myogenesis.

• Conover CA, Clarkson JT, Bale LK
• Effect of glucocorticoid on insulin-like growth factor (IGF) regulation of IGF-binding protein expression in fibroblasts.
• Endocrinology. 1995; 136: 1403-10
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• Insulin-like growth factor (IGF)-binding proteins (IGFBPs) can determine IGF biological competence at the cellular level. IGF-I itself has been shown to be an important peptide regulator of local IGFBP availability. Glucocorticoid also has major effects on IGFBP expression. In the present study, we assessed integrated IGF-I and glucocorticoid regulation of IGFBP messenger RNA (mRNA) and protein expression in two fibroblast model systems. In bovine fibroblasts, IGF-I treatment induced IGFBP-3 and IGFBP-5 mRNA and protein secretion, and had a moderate effect on IGFBP-4 expression. Dexamethasone had little effect on the IGF-induced increase in IGFBP-3, but completely blocked the increase in IGFBP-5 expression. Basal IGFBP-4 expression was inhibited by dexamethasone, and this effect was counteracted by IGF-I. IGFBP-2 expression did not vary with IGF-I or dexamethasone treatment in these cells; IGFBP-1 mRNA was not detectable, and IGFBP-6 mRNA was low and inconsistent. In human fibroblasts, IGF-I treatment increased levels of IGFBP-3 and decreased levels of IGFBP-4 without influencing mRNA expression. IGF-I also increased steady state levels of IGFBP-5 mRNA. Dexamethasone alone decreased IGFBP-3, IGFBP-4, and IGFBP-5 mRNA, but it had no significant effect on IGFBP-3, -4, or -5 expression in the presence of IGF-I. Human fibroblast IGFBP-6 expression was stable under the different culture conditions; IGFBP-1 and -2 mRNA were not detected. These data demonstrate that IGF peptide and glucocorticoid individually modulate IGFBP expression and indicate that glucocorticoid has distinct effects on IGF regulation of IGFBP depending upon the particular IGFBP and the underlying mechanism of IGF regulation. Bovine fibroblasts may provide a useful model system to probe the molecular mechanisms of IGFBP-3, -4, and -5 gene expression and regulation by IGF-I and glucocorticoid, whereas human fibroblasts may be suitable for studying posttranscriptional interactions.

• Valentinis B, Bhala A, DeAngelis T, Baserga R, Cohen P
• The human insulin-like growth factor (IGF) binding protein-3 inhibits the growth of fibroblasts with a targeted disruption of the IGF-I receptor gene.
• Mol Endocrinol. 1995; 9: 361-7
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• The insulin-like growth factors (IGFs) are important mitogens that exert their proliferative effects on cells via the type I IGF receptors (IGF-R). The IGFs also associate with IGF binding proteins (IGFBPs). IGF-inhibitory, IGF-stimulatory, and IGF-independent effects of IGFBPs on cell growth have been reported. We have asked whether the IGFBP-3 has an effect on cell growth, which is independent of its ability to bind IGF-I and thus inhibit the activation of the IGF-I receptor. For this purpose, we have used a fibroblast cell line (R- cells) derived from mouse embryos homozygous for a targeted disruption of the IGF-R gene. When compared with wild type cells (W), which bind IGF-I with high affinity and specificity, R- cells transfected with a mammalian expression vector containing the human (h) IG-FBP-3 cDNA were selected (R-/BP3) and found to express hIGFBP-3 mRNA (detected by Northern blots) and to secrete hIGFBP-3 protein [detected by Western ligand blotting (WLB), immunoblotting, and immunoprecipitation as well as immunofluorescence confocal microscopy]. Growth of five different R- cells, and 10-fold slower compared with W cells, grown under identical conditions. Confluent R- cells. These experiments show that the overexpression of IGFBP-3 has an inhibitory effect on cell growth which does not involve IGF binding to, or signal transduction via, the type I IGF-R. We conclude that the cellular production of IGFBPs serves as a negative regulator of cell proliferation which involves a cellular signaling pathway separate from the IGF-IGF-R system.

• Fawcett J, Rabkin R
• The processing of insulin-like growth factor-I (IGF-I) by a cultured kidney cell line is altered by IGF-binding protein-3.
• Endocrinology. 1995; 136: 1340-7
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• The proximal renal tubule is a common site of peptide hormone metabolism, including that of insulin-like growth factor-I (IGF-I). To further explore the renal uptake and processing of IGF-I, a study was carried out with the proximal-like cultured opossum kidney (OK) cell line. [125I]IGF-I associated with these cells in a specific manner. Association was competitively inhibited by IGF-I. Des(1-3)-IGF-I was equally effective, insulin had only a small effect, and the unrelated peptides, glucagon and GH, were without effect. Degradation was inhibited in similar manner. Comparisons of [125I]IGF-I with [125I]insulin revealed comparable cell association, but degradation of internalized IGF-I was several-fold slower. Furthermore, IGF-I degradation was less sensitive, by half, to the inhibitory effect of chloroquine. When OK cells were exposed to [125I]IGF-I in the presence of IGF-binding protein-3 (IGFBP-3) cell association (binding and internalization) was reduced significantly. Of note, total cell degradation was reduced (P < 0.01), but the IGF-I that was internalized was degraded more rapidly than in control cells. Gel filtration and reverse phase HPLC revealed that the products of IGF-I degradation included large IGF-I-size intermediates in addition to trichloroacetic acid-soluble material. This product profile was not altered by IGFBP-3. Thus, as previously described for insulin, cultured OK cells possess specific IGF-I receptors and degrade internalized IGF-I. However, IGF-I processing differs from that of insulin, in that degradation is slower and relatively insensitive to competition by insulin. This study also shows that IGFBP-3 inhibits the binding and uptake of [125I]IGF-I by these kidney cells. However, once IGF-I is internalized, IGFBP-3 enhances degradation. Although the mechanism of this paradoxical action requires further study, analysis of the products of degradation suggests that the same enzymes are involved in IGF-I degradation regardless of whether IGFBP-3 is present.

• Schmid C, Schlapfer I, Keller A, Waldvogel M, Froesch ER, Zapf J
• Effects of insulin-like growth factor (IGF) binding proteins (BPs) -3 and -6 on DNA synthesis of rat osteoblasts: further evidence for a role of auto-/paracrine IGF I but not IGF II in stimulating osteoblast growth.
• Biochem Biophys Res Commun. 1995; 212: 242-8
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• IGFBP-3 and IGFBP-6 were used to study whether both IGF I and IGF II play a role in auto-/paracrine stimulation of rat osteoblast growth. Both IGFBPs decreased basal DNA synthesis in neonatal rat calvaria cells but with different potencies. Consistent with their IGF binding affinities, IGFBP-3 blocked both IGF I- and IGF II-stimulated DNA synthesis, whereas IGFBP-6 preferentially blocked IGF II-stimulated DNA synthesis. These inhibitory effects of the two IGFBPs can be fully explained by the sequestration of IGFs. Because IGFBP-6 preferentially binds IGF II and is much less potent than IGFBP-3 in decreasing basal DNA synthesis in calvaria cells, IGF I but not IGF II appears to be an important auto-/paracrine stimulator of DNA synthesis.

• Coverley JA, Baxter RC
• Regulation of insulin-like growth factor (IGF) binding protein-3 phosphorylation by IGF-I.
• Endocrinology. 1995; 136: 5778-81
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• Insulin-like growth factor (IGF) action is modulated by six IGF-binding proteins (IGFBP-1 to -6). IGFBP-3 is the main IGFBP in serum and is produced by many cell types, which can modify it post-translationally to yield glycosylation, proteolysis and phosphorylation products. This study investigates the regulation of IGFBP-3 phosphorylation by IGF-I in human neonatal skin fibroblasts. Fibroblasts were incubated with IGF peptides and 32P-orthophosphate for 4 h, and phosphorylated IGFBP-3 (P-IGFBP-3) was immunoprecipitated from the medium and analysed by SDS-PAGE. Media collected from parallel experiments without radioactivity were assayed for immunoreactive IGFBP-3 (I-IGFBP-3). IGF-I (50 ng/ml) increased levels of P-IGFBP-3 and I-IGFBP-3 in conditioned medium to 205 +/- 9% and 198 +/- 10% of control, respectively (n = 5). Stimulation of I-IGFBP-3 was consistent with IGF-mediated release of cell-associated IGFBP-3, since treatment with an IGF-I analogue with reduced affinity for IGFBPs did not increase I-IGFBP-3 levels, whereas treatment with an analogue with reduced affinity for receptor but normal affinity for binding proteins did. In contrast, stimulation of P-IGFBP-3 occurred independently of IGF binding to IGFBP, instead requiring interaction of IGF-I with its receptor. While the functional significance of IGFBP-3 phosphorylation is unclear, we propose that it plays a regulatory role in IGFBP-3 action.

• Hirschberg R, Kaysen GA
• Insulin-like growth factor I and its binding proteins in the experimental nephrotic syndrome.
• Endocrinology. 1995; 136: 1565-71
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• Insulin-like growth factor I (IGF-I) is present in serum in association with specific IGF-binding proteins (IGFBPs) primarily in a large (approximately 150K) ternary or a smaller (approximately 50K) binary protein complex or in the free form (< or = 1%). We hypothesized that glomerular proteinuria results in urinary excretion of IGF-I/IGF-binding protein complexes and that the nephrotic syndrome induces abnormal serum distribution and liver synthesis of IGF-binding proteins. In nephrotic rats, serum IGF-I levels are reduced compared with pair-fed control animals. In nephrotic rat serum, binding to IGFBP-3 is reduced and Western immune analysis demonstrates an approximately 27K fragment that does not bind IGF-I, suggesting in vivo proteolysis of IGFBP-3. In contrast, binding and serum levels of IGFBP-2 are increased in nephrotic rats, which results from increased synthesis in the liver. In Nagase analbuminemic rats, the IGF-I levels and IGFBP-distribution in serum are normal suggesting that the reduced albumin levels in the nephrotic syndrome do not cause the increased liver synthesis and serum levels of IGFBP-2. Nephrotic rat urine contains IGFBP-3 and IGFBP-2 as well as strong activity of an IGFBP-3 protease. Because the 150K ternary complex in serum but not the smaller binding protein complex is restricted to the intravascular space, the shift of binding from IGFBP-3 (ternary complex) to IGFBP-2 (binary complex) in nephrotic rat serum may help to maintain tissue availability despite the reduction in serum IGF-I levels.

• Sunic D, Belford DA, McNeil JD, Wiebkin OW
• Insulin-like growth factor binding proteins (IGF-BPs) in bovine articular and ovine growth-plate chondrocyte cultures: their regulation by IGFs and modulation of proteoglycan synthesis.
• Biochim Biophys Acta. 1995; 1245: 43-8
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• Cultured chondrocytes respond to insulin-like growth factors (IGFs) by increasing the production of proteoglycans and insulin-like growth factor binding proteins (IGF-BPs). To investigate the biological effects of IGFs and IGF-BPs, isolated bovine articular and ovine growth-plate chondrocytes were cultured at high density in the presence of IGF-1, and its truncated form, des (1-3) IGF-I. Both growth factors stimulated the production of IGF-BPs in articular and growth-plate chondrocyte monolayers. Western ligand blots showed that bovine articular chondrocytes released two forms of IGF-BPs into conditioned medium with molecular weights of 29 and 31 kDa. Ovine growth-plate chondrocytes released four different forms of IGF-BPs of approx. 22, 24; 29-30 and 34 kDa. IGF-I and des (1-3) IGF-I stimulated total proteoglycan synthesis by articular chondrocytes up to 1.5-fold. The truncated analogue was more potent at lower concentrations, particularly in stimulating incorporation of newly synthesized proteoglycans into the cell-layer. The maximal stimulation of proteoglycan synthesis in ovine growth-plate chondrocyte culture was 3-fold with des (1-3) IGF-I, while IGF-I enhanced proteoglycan production by only 2-fold over the concentrations used. Our results suggest that endogenous IGF-BPs in chondrocyte cultures act as a part of a feed-back mechanism which diminishes the bioactivity of IGF-I.

• Mohan S et al.
• Studies on the mechanisms by which insulin-like growth factor (IGF) binding protein-4 (IGFBP-4) and IGFBP-5 modulate IGF actions in bone cells.
• J Biol Chem. 1995; 270: 20424-31
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• The growth potentiating effects of the insulin-like growth factor (IGF)-I and IGF-II are modulated by a family of six insulin-like growth factor binding proteins (IGFBPs). Despite the similarity in amino acid sequences of the IGFBPs, their effects on the growth of bone cells differ. Studies on the molecular mechanisms for IGFBP-4 actions revealed that coincubation of bone cells with IGFBP-4 and 125I-IGF-I or 125I-IGF-II decreased the binding of both of these ligands in a dose-dependent manner. In addition, IGFBP-4 decreased the binding of IGF-I tracer to purified type I IGF receptor. These data in conjunction with data showing that IGFBP-4 had no effect on cell proliferation induced by analogs of IGF-I or IGF-II, which exhibited > 100-fold reduced affinity for binding to IGFBP-4 suggest that IGFBP-4 may inhibit IGF action by preventing the binding of ligand to its membrane receptor. In contrast to IGFBP-4, IGFBP-5 treatment increased the binding of IGF tracer to bone cells but did not increase the binding of 125I-IGF-I to type I IGF receptor. Studies on the mechanism by which IGFBP-5 increased the binding of 125I-IGF tracer to bone cells suggest that IGFBP-5 could facilitate IGF binding by a mechanism in which IGFBP-5 has cell surface binding sites independent of IGF receptors. These data in conjunction with the findings that IGFBP-5 potentiated cell proliferation even in the presence of those same IGF analogs that exhibited > 200-fold reduced affinity for binding to IGFBP-5, suggest that IGFBP-5 may in part stimulate bone cell proliferation by an IGF-independent mechanism involving IGFBP-5-specific cell surface binding sites.

• Gockerman A, Prevette T, Jones JI, Clemmons DR
• Insulin-like growth factor (IGF)-binding proteins inhibit the smooth muscle cell migration responses to IGF-I and IGF-II.
• Endocrinology. 1995; 136: 4168-73
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• Smooth muscle cells have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the role of IGF-binding proteins (IGFBPs) in this process has not been determined. As IGFBPs are synthesized and secreted by smooth muscle cells and bind to IGFs with high affinity, this study was undertaken to determine the ability of IGFBP-1 and -2 to modulate cellular migration in response to IGF-I and -II. Confluent monolayers of porcine vascular smooth muscle cells were wounded with a razor blade. After wounding, IGF-I and -II induced increases of 159 +/- 49% (mean +/- SD) and 108 +/- 33%, respectively, above control values in the number of cells migrating over a fixed distance in 4 days. The addition of IGFBP-1 caused a 19-21% inhibition of IGF-I- or IGF-II-stimulated migration, whereas the addition of IGFBP-2 inhibited the effect of both by greater than 60%. The addition of IGFBP-2 alone had no effect, whereas IGFBP-1 addition was associated with a 42 +/- 12% increase. In contrast, [Trp221]IGFBP-1 in which the RGD sequence was changed to WGD, thus eliminating its capacity to bind to the alpha 5 beta 1 integrin, inhibited IGF-I-stimulated migration by 67 +/- 17%. An IGF analog that has a reduced affinity for IGFBP-2-stimulated migration equally well as IGF-I alone even in the presence of IGFBP-2. Likewise, the addition of insulin, which cannot bind to IGFBPs, at supraphysiological concentrations that are adequate to activate the IGF-I receptor resulted in a similar increase in migration. In summary, IGF-I and -II stimulate smooth muscle cell migration after wounding. This migratory response is modulated by IGFBPs. Both IGFBP-1 and IGFBP-2 appear to neutralize the effects of the IGFs by inhibiting their interaction with IGF receptors, but IGFBP-1 also has a direct stimulatory effect that requires an intact RGD integrin recognition sequence.

• Wilson KF et al.
• Long-term effects of insulin-like growth factor (IGF)-I treatment on serum IGFs and IGF binding proteins in adolescent patients with growth hormone receptor deficiency.
• Clin Endocrinol (Oxf). 1995; 42: 399-407
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• OBJECTIVE: The aim of this investigation was to study the effect of relatively high dose IGF-I therapy given for several months, on serum levels of IGF-I, IGF-II and IGFBP-3, and on IGF-I pharmacokinetics in patients with growth hormone insensitivity due to GH receptor dysfunction. DESIGN AND PATIENTS: Two adolescent subjects from Ecuador were treated with recombinant IGF-I at a dosage of 120 micrograms/kg s.c. twice daily, in combination with a GnRH analogue for 8 months. MEASUREMENTS: Serum was sampled at baseline and at 3-8 months, for determination of IGF-I, IGF-II and IGFBP-3 by radioimmunoassay, and for evaluation of IGFBPs and IGFBP-3 protease activity by Western ligand blot and protease assay, respectively. RESULTS: Peak serum IGF-I levels ranged from 272 to 492 micrograms/l. Mean serum IGF-II levels were decreased concurrently with the increase in IGF-I. Serum IGFBP-3 levels failed to rise with prolonged IGF-I treatment. There was no apparent change in the half-life of IGF-I during the treatment period. CONCLUSIONS: IGF-I administration does not increase serum levels of IGFBP-3 or significantly alter IGF-I pharmacokinetics.

• Ovesen P, Flyvbjerg A, Orskov H
• Insulin-like growth factor I (IGF-I) and IGF binding proteins in seminal plasma before and after vasectomy in normal men.
• Fertil Steril. 1995; 63: 913-8
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• OBJECTIVE: To examine the levels and origins of insulin-like growth factor I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) in the human male genital tract. DESIGN: Examining seminal plasma before and 3 months after vasectomy. SUBJECTS: Fifteen men who were candidates for vasectomy were included in the study. MAIN OUTCOME MEASURES: Seminal plasma and serum levels of IGF-I, IGFBP-1, and IGFBP-3 were determined by commercially available assays, furthermore, samples were subjected to Western ligand blotting. RESULTS: Seminal plasma concentrations of IGF-I were significantly lower after vasectomy: 18.0 +/- 2.4 micrograms/L (before) and 12.5 +/- 1.2 micrograms/L (after). When the total ejaculate content of IGF-I was calculated, the figures were reduced by 50% after vasectomy: 45.64 +/- 7.8 ng (before) and 23.45 +/- 3.8 ng (after). The patterns observed for seminal plasma IGFBP-3 concentrations were 844.9 +/- 59 micrograms/L (before) and 816.5 +/- 65 micrograms/L (after). When the total ejaculate IGFBP-3 content was calculated there was a 36% reduction after vasectomy: 2,300 +/- 251 ng (before) and 1,474 +/- 217 ng (after). CONCLUSIONS: A considerable amount of seminal plasma IGF-I and IGFBP-3 may be of testicular origin. Although the physiological significance of IGF-I and IGFBPs in the male reproductive system still remains uncertain, the demonstration of their presence in the testes add support to a functional role in the regulation of gonadal function.

• Silverman LA, Cheng ZQ, Hsiao D, Rosenthal SM
• Skeletal muscle cell-derived insulin-like growth factor (IGF) binding proteins inhibit IGF-I-induced myogenesis in rat L6E9 cells.
• Endocrinology. 1995; 136: 720-6
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• The insulin-like growth factors (IGFs) stimulate the differentiation of skeletal muscle cells. IGF binding proteins (IGFBPs), which are expressed by skeletal muscle cells, may enhance or inhibit IGF actions. To explore the role of skeletal muscle-derived IGFBPs in IGF-induced myogenesis, we compared the differentiation-inducing effects of IGF-I and des(1-3)IGF-I in rat L6E9 skeletal myoblasts. Des(1-3)IGF-I is a naturally occurring IGF-I analog with markedly reduced affinity for IGFBPs but with an affinity for the IGF-I receptor that is comparable to that for native IGF-I. We find that rat L6E9 cells produce principally IGFBP-4 and BP-6, with a minor component of IGFBP-5. Both IGFBP-4 and BP-6 accumulate during differentiation and increase further in response to IGF-I or des(1-3)IGF-I treatment. We find that an IGF-I analog with reduced affinity for IGFBPs is significantly more potent than native IGF-I in stimulating myogenesis (as assessed by myogenin messenger RNA abundance and muscle creatine kinase activity), indicating that IGFBPs expressed by skeletal muscle cells inhibit differentiation induced by IGF-I. In view of the relative abundance of IGFBP-4, its relatively high affinity for IGF-I and the low affinity of IGFBP-6 for IGF-I, it is likely that the inhibitory effect of rat skeletal muscle-derived IGFBPs on IGF-I-induced myogenesis is mediated principally by IGFBP-4.

• Duclos MJ, Chevalier B, Simon J
• Preferential binding of insulin-like growth factors to a binding protein rather than to receptors on chicken hepatoma cell (LMH) membranes.
• Growth Regul. 1994; 4: 155-63
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• [125I]IGF-I binding to chicken hepatoma cell (LMH) membranes was displaced by unlabelled IGF-I or IGF-II, but not by insulin. Cross-linking revealed specific binding sites of 128 and 28-31 kDa, which following solubilization could be separated by wheat germ agglutinin (WGA) chromatography. [125I]IGF-I binding to the WGA eluate (128 kDa) could be displaced by insulin although with a 30-fold lower potency than IGF-I. Binding to the WGA flow-through (28-31 kDa) was not inhibited by insulin. This suggested that IGF binding to LMH was due mainly to membrane bound IGFBP rather than to type 1 IGF receptors. A reverse proportion was observed in normal chicken liver. A predominant 28 kDa IGFBP was synthesized and secreted by LMH cells, together with an unusual 60 kDa IGF binding entity which only bound [125I]IGF-II (with weak affinity). This process was not affected by the presence or absence of glucose, dexamethasone, glucagon, insulin or IGF-I.

• Adashi EY, Resnick CE, Rosenfeld RG
• IGF-I stimulates granulosa cell-derived insulin-like growth factor binding protein-5: evidence for medication via type I IGF receptors.
• Mol Cell Endocrinol. 1994; 99: 279-84
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• Although the precise role of insulin-like growth factor binding proteins (IGFBPs) in ovarian physiology remains a matter of study, existing data suggest a possible antigonadotropic role in the context of follicular atresia. Given the above and the need for improved understanding of the regulation of ovarian IGFBPs, we have set out to explore the ability of IGF-I to modulate IGFBP levels in cultured rat granulosa cells. Specifically, granulosa cells (5 x 10(5) viable cells/dish) from immature (23-25 days old), estrogen-primed rats were cultured under serum-free conditions for 72 h in the absence or presence of IGF-I. At the conclusion of this incubation period, media samples were collected and subjected to Western ligand blotting. Treatment with IGF-I (100 ng/ml) resulted in a substantial (P < 0.05) increase in the accumulation of IGFBP-5, the major 28-29 kDa IGFBP species. Subsequent studies revealed this effect of IGF-I to be both dose- and time-dependent. A similar effect was noted for insulin at dose levels 1-10 micrograms/ml at which cross-reaction with the type I IGF receptor (but not with IGFBPs) has been amply documented. Des (1-3) IGF-I, a type I receptor-selective ligand with markedly reduced avidity for IGFBPs, proved substantially more potent (as a promoter of IGFBP-5 accumulation) than its native counterpart. In contrast, treatment with IGF-II or [Leu27]IGF-II, type II IGF receptor-selective ligands, yielded a more limited effect on IGFBP-5 accumulation in keeping with an overall rank order of potency of des (1-3) IGF-I > IGF-I > IGF-II > or = [Leu27]IGF-II.(ABSTRACT TRUNCATED AT 250 WORDS)

• Denver RJ, Nicoll CS
• Pancreatic hormones differentially regulate insulin-like growth factor (IGF)-I and IGF-binding protein production by primary rat hepatocytes.
• J Endocrinol. 1994; 142: 299-310
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• We investigated the influence of and interactions among pancreatic hormones on the secretion of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IG-FBPs) by treating primary hepatocytes from young male Long-Evans rats with insulin or glucagon in combination with rat GH (rGH). The concentration of IGF-I secreted into the medium was estimated by radioimmunoassay after formic acid-acetone cryoextraction, and secreted IGFBPs were analysed by Western ligand blot and immunoblot; accumulation of IGF-I mRNA was analysed by Northern blot. Both insulin (0.1-100 nmol/l) and rGH (0.5, 5 and 50 pmol/l) produced a dose-dependent stimulation of IGF-I secretion over a 24-h incubation period. In contrast, glucagon (0.1-100 nmol/l) inhibited IGF-I production in a dose-related manner. Glucagon (10 nmol/l) also inhibited IGF-I secretion stimulated by rGH (5 pmol/l) and insulin (10 nmol/l). Northern blot analysis of total RNA isolated from rat hepatocytes revealed that rGH (5 pmol/l) elevated IGF-I mRNA levels, glucagon (10 nmol/l) alone had no effect on this parameter, but glucagon significantly reduced IGF-I transcript accumulation in response to rGH. IGFBPs secreted by rat hepatocytes run in two molecular weight ranges on SDS-PAGE: approximately 25 kDa (IGFBP-4) and approximately 29-31 kDa (IGFBP-1 and -2); the predominant hormonally regulated IGFBP was identified as IGFBP-1. Insulin produced a dose-dependent inhibition of production of IGFBP-1, while glucagon was stimulatory; when given together at an equivalent concentration (1 nmol/l), the effects of insulin were dominant to glucagon on IGFBP-1. These observations provide support for significant opposite roles for the pancreatic hormones, insulin and glucagon, in the regulation of liver IGF-I and IGFBP-1 production. As the production of pancreatic hormones is influenced by nutritional status, these polypeptides may mediate the effects of changing nutritional state on the hormonal control of protein anabolism and glucose homeostasis by directly influencing the circulating level of liver-derived IGF-I and its binding proteins.

• Ramagnolo D, Akers RM, Byatt JC, Wong EA, Turner JD
• IGF-I-induced IGFBP-3 potentiates the mitogenic actions of IGF-I in mammary epithelial MD-IGF-I cells.
• Mol Cell Endocrinol. 1994; 102: 131-9
• Display abstract

• Limited information is available concerning the molecular and cellular mechanisms that regulate expression of insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs) in bovine mammary epithelial cells. Here, we report on the autocrine mechanisms of action of IGF-I and hormonal regulation of expression of IGFBPs in bovine mammary epithelial MD-IGF-I cells which express recombinant IGF-I under the control of the glucocorticoid-inducible mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Levels of IGFBP-3 mRNA and secretion of IGFBP-3 by MD-IGF-I cells were stimulated by IGF-I, insulin (INS), and IGF-I analogs but not prolactin (PRL). Conversely, parental MAC-T cells expressed little IGF-I and secreted primarily IGFBP-2 (29-32 kDa) in response to stimulation with INS, dexamethasone (DEX), or IGF-I analogs. Secretion of recombinant IGF-I caused a 26.5-fold increase in secretion of IGFBP-3, as measured by densitometric analysis of ligand blots, which was associated with a 1.7-fold increase in total DNA. Conditioned media (CM) from MD-IGF-I cells induced with DEX stimulated a 2.8-fold increase in [3H]thymidine incorporation into DNA of parental MAC-T cells, compared with uninduced cells. Moreover, inclusion of exogenous IGF-I with CM from MD-IGF-I cells triggered an additional 3.0-fold increase in label incorporation, but only a 1.6-fold increase in the presence of IGFBP-2-containing media conditions by MAC-T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

• Kratz G, Lake M, Gidlund M
• Insulin like growth factor-1 and -2 and their role in the re-epithelialisation of wounds; interactions with insulin like growth factor binding protein type 1.
• Scand J Plast Reconstr Surg Hand Surg. 1994; 28: 107-12
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• Insulin like growth factor (IGF) 1 and 2 which are present and actively synthesised in the wound fluid stimulate several cell types involved in the process of wound healing. To investigate the role of IGF-1 and 2 and in addition, the association between IGF and their carrier proteins, IGF binding proteins (IGFBP), we have used a newly established model for human wound healing in fresh biopsy material. Histological examination shows that IGF-1 stimulates efficient reepithelialisation of the wounds both alone and in the presence of recombinant IGFBP-1. In contrast, IGF-2 stimulates healing only when used in combination with IGFBP-1. These findings suggest that the two IGFs and their carrier proteins may function during different phases of wound healing and that both IGF-1 and 2 act as potent inducers of wound healing; this may have direct clinical implications.

• Lord AP, Martin AA, Ballard FJ, Read LC
• Transfer of insulin-like growth factor (IGF)-I from blood to intestine: comparison with IGFs that bind poorly to IGF-binding proteins.
• J Endocrinol. 1994; 141: 505-15
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• The net transfer of 125I-labelled insulin-like growth factor (IGF)-I from the blood to the distal small intestine was measured in anaesthetized lambs using a non-recirculating vascular-perfused intestine. To determine whether IGF-binding proteins (IGFBPs) reduce net IGF transfer, radiolabelled IGF-I was compared with two analogues, des(1-3)IGF-I and LR3IGF-I, which show reduced affinity for IGFBPs. Radiolabelled IGF-I, des(1-3)IGF-I or LR3IGF-I (1 ng/ml plasma) was infused for 45 min into the arterial supply of a 10 cm intestinal segment, either in the absence of added unlabelled peptide (high specific activity) or in the presence of a 100-fold excess of unlabelled homologous peptide (low specific activity) to achieve different proportions of free and complexed peptide. Very little degradation of radiolabelled peptides was detected in plasma, with 3-10% degradation in the intestinal tissue. Less than 5% of radiolabelled IGF-I remained as free peptide in the efferent venous plasma of the perfused segment at both specific activities. Bound radiolabelled IGF-I was found by size-exclusion chromatography mainly in the 30-50 kDa region, with a smaller proportion in the 150 kDa peak. The net intestinal transfer of IGF-I, calculated as the sum of the proportions of infused tracer recovered from intestinal tissue, luminal contents and lymph, was 3.46 +/- 0.22% (S.E.M.) and 3.49 +/- 0.93% when infused at high and low specific activities respectively. The analogues differed from IGF-I with up to ninefold higher concentrations of free radiolabelled peptide in venous plasma of the perfused intestinal segment, and corresponding decreases in binding to the 30-50 kDa binding proteins. Notwithstanding these marked differences in the plasma levels of free peptide, net intestinal transfer was very similar for the three peptides, as was the extent of degradation in the intestinal tissue. The lack of correlation between binding to 30-50 kDa binding proteins and net intestinal transfer suggests that association with 30-50 kDa plasma binding proteins is not a rate-limiting determinant of net IGF transfer to intestinal tissue.

• Bang P, Westgren M, Schwander J, Blum WF, Rosenfeld RG, Stangenberg M
• Ontogeny of insulin-like growth factor-binding protein-1, -2, and -3: quantitative measurements by radioimmunoassay in human fetal serum.
• Pediatr Res. 1994; 36: 528-36
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• There is evidence for a role for IGF-I in the endocrine control of human fetal growth despite the low serum IGF-I concentrations. The formation in serum of binary complexes between IGF-I or -II and either of six IGF binding proteins (IGFBP-1 to -6) and, in particular, of long-lived ternary complexes between IGF-I or -II, IGFBP-3, and acid-labile subunit is thought to regulate IGF-I bioavailability by increasing its serum half-life. The present study assesses the bioavailability of circulating IGF-I in 19- to 35-wk gestation human fetuses in utero 1) by quantitative RIA measurements of IGF and IGFBP in serum and 2) by examining whether serum proteolysis of IGFBP-3 may further increase IGF-I bioavailability. Fetal serum concentrations of IGFBP-3, IGF-I, and IGF-II were low with marked or only modest increases with gestational age (p < 0.001, p < 0.005, and p < 0.05, respectively). The mean molar ratio between IGF-I plus -II and IGFBP-3 demonstrated a molar excess of IGF (50%) similar to that in adolescents but in contrast to the 1:1 molar ratio in adults. The median IGFBP-2 concentration was 3-fold elevated to a molar concentration similar to that of IGFBP-3 (adult serum displays 10-fold higher IGFBP-3 concentrations). The median serum IGFBP-1 concentration was not elevated as previously reported in newborns. IGFBP-3 protease activity was not increased in fetal serum, in contrast to pregnancy serum and amniotic fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

• Lord AP, Bastian SE, Read LC, Walton PE, Ballard FJ
• Differences in the association of insulin-like growth factor-I (IGF-I) and IGF-I variants with rat, sheep, pig, human and chicken plasma-binding proteins.
• J Endocrinol. 1994; 140: 475-82
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• Associations between labelled insulin-like growth factors (IGFs) and IGF-binding proteins in plasma have been compared in the rat, sheep, human, pig and chicken. The IGFs tested were recombinant human IGF-I, the truncated variant, des(1-3)IGF-I, and LR3IGF-I, an extended form that had been engineered so as to minimize interactions with IGF-binding proteins. Marked species differences were demonstrated, notably that the IGF-I variants which exhibited extremely weak binding in rat plasma bound significantly in plasma from the other species. This result was shown both by size-exclusion chromatography of labelled IGFs added to plasma, in which the extent of variant IGF-I binding decreased in the order sheep > human > pig = chicken > rat, and by competition for labelled IGF-I binding in vitro, in which the order was pig = chicken > sheep > human > rat. Notwithstanding these differences, the two IGF-I variants showed only slight between-species binding differences when tested with purified rat, sheep and human IGF-binding protein-3. Ligand blotting experiments with plasma from the five species similarly showed a consistent pattern in that IGF-I binding was much greater than des(1-3)IGF-I binding, which in turn was greater than LR3IGF-I binding.(ABSTRACT TRUNCATED AT 250 WORDS)

• Grimes RW, Hammond JM
• Proteolytic degradation of insulin-like growth factor (IGF)-binding protein-3 by porcine ovarian granulosa cells in culture: regulation by IGF-I.
• Endocrinology. 1994; 134: 337-43
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• Porcine ovarian granulosa cells in culture secrete glycosylated insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3), which inhibits gonadotropin and IGF action in the ovary. Synthesis of IGFBP-3 is stimulated by IGF-I and attenuated by gonadotropin. The purpose of the present study was to determine whether IGFBP-3 levels were also regulated via proteolysis. Exogenously added nonglycosylated recombinant human IGFBP-3 (rhIGFBP-3) was significantly degraded over time by a soluble serine-specific protease, similar to plasmin, in control cultures and those treated with FSH, insulin, or several other classes of hormones. In contrast, degradation was greatly attenuated by the IGFs. Degraded rhIGFBP-3 exhibited much reduced affinity for [125I]IGF-II, suggesting that degradation could make available IGFs for cellular interaction. The mechanism of IGFBP-3 protease inhibition by IGFs is unclear. Mediation by IGF receptors is unlikely, as insulin at a dose that activated both insulin and type I IGF receptors did not alter intrinsic degradation of IGFBP-3 (as does IGF). Additionally, IGF-I attenuation of IGFBP-3 degradation was not inhibited by antagonism of receptor action with a tyrosine kinase inhibitor. Further, IGF-I inhibited degradation in cell-free conditioned medium. Direct stabilization of IGFBP-3 via binding of IGFs was suggested from these results. However, long R3 IGF-I attenuated IGFBP-3 degradation even though it has low affinity for IGFBPs. Inhibition of the protease by IGFs is also possible. We conclude that IGFs inhibit the degradation of exogenous nonglycosylated rhIGFBP-3. If active in vivo, this may serve to increase endogenous IGFBP-3 levels in follicular fluid.

• Leighton JK, Grimes RW, Canning SF, Hammond JM
• IGF-binding proteins are differentially regulated in an ovarian granulosa cell line.
• Mol Cell Endocrinol. 1994; 106: 75-80
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• We have recently established an immortalized granulosa cell line as a model system to investigate ovarian function, with particular emphasis on the insulin-like growth factor (IGF) regulatory system. Previous results have shown that these cells express mRNAs for IGF-binding proteins (IGFBPs)-2 to -5. These IGFBPs are also detected by ligand blots. The current work evaluated the regulation by the IGFs and cAMP on the IGFBPs and their mRNAs and compared the findings to that in primary culture. Our results indicate that levels of the IGFBPs are controlled, in part, by expression of the mRNAs. However, evidence for post-transcriptional regulation was also discovered. IGFBP-3 was stimulated by IGF-I, IGFBP-4 by forskolin, and IGFBP-5 by IGF-I. IGFBP-2, -3, and -4 are expressed under basal conditions whereas IGFBP-5 is only detectable after IGF-I induction. An alteration in the biphasic actions of cAMP in this cell line, as compared to primary culture, was evident.

• Hadsell DL, Gibson CA, Baumrucker CR
• Insulin-like growth factor (IGF) binding to a murine mammary epithelial cell line.
• J Cell Physiol. 1994; 161: 435-40
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• The goal of this study was to relate insulin-like growth factor (IGF) binding with IGF-stimulated growth in a murine mammary epithelial (COMMA-D/MME) cell line. Affinity crosslinking with [125I]IGF-I showed major bands at 224,000 and 148,000 Mr that were ablated by the inclusion of IGF-I or -II at 130 nM. Scatchard-transformed [125I]IGF-I binding data was best fit by a two-site model, with 24,000 sites possessing a Kd of 0.33 nM and 1,000 sites possessing a Kd of 8.09 nM per cell. Competition analysis showed ED50 values for IGF-I, -II, and insulin to be 0.90 +/- 0.03, 7.15 +/- 4.27, and 335 +/- 104 nM, respectively. Affinity crosslinking of [125I]IGF-II showed three major bands of 230,000, 148,000, and 61,000 to 58,000 Mr. Unlabeled IGF-II ablated all bands, while IGF-I and insulin ablated only the 148,000 Mr band. Competition analysis showed ED50 values for unlabeled IGF-I and -II to be 0.10 +/- 0.01 and 5.31 +/- 2.04 nM, respectively. In spite of the receptor affinity differences, no significant difference was noted in IGF-I and -II in capacity to stimulate cell growth. These data indicate that COMMA-D/MME cells express IGF receptors and that both IGFs are mitogenic.

• Baxter RC
• Insulin-like growth factor binding proteins in the human circulation: a review.
• Horm Res. 1994; 42: 140-4
• Display abstract

• The actions and bioavailability of the insulin-like growth factors (IGFs) are regulated by a family of six IGF binding proteins. IGFBP-3, the major circulating IGFBP, is unique in combining with a glycoprotein, the acid-labile subunit (ALS), to form a ternary complex with IGF-I or IGF-II. Each component of this trimer is growth hormone-dependent, and the hypoglycemic potential of circulating IGFs appears neutralized in this form. IGFs in the complex have a greatly extended circulating half-life, their stability being conferred by the presence of ALS, which is itself very stable in the circulation. IGFBP-1 does not appear to be a carrier of IGFs, but to act as an acute modulator of their metabolic activities. In this role it can be viewed as an insulin counter-regulator, blocking 'free' insulin-like activity during fasting or hypoglycemia. IGF-I administration causes complex changes in circulating IGFBPs, so that a detailed knowledge of their regulation is essential if the therapeutic potential of IGF-I is to be optimised.

• Underwood LE, Thissen JP, Lemozy S, Ketelslegers JM, Clemmons DR
• Hormonal and nutritional regulation of IGF-I and its binding proteins.
• Horm Res. 1994; 42: 145-51
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• The influence of nutrition on growth seems likely to be mediated in part through IGF-I. Restriction of dietary nutrients adversely effects IGF-I synthesis and action at multiple steps, including decreased GH receptors, postreceptor defects in GH action, decreased steady state levels of IGF-I mRNA, and attenuation of IGF-I action. In addition, undernutrition affects the IGF binding proteins in ways that vary from one tissue to another. Understanding the alterations in IGFBPs that are caused by nutritional insufficiency may provide insight into the actions of the IGFBPs.

• Gargosky SE et al.
• Effects of insulin-like growth factor I treatment on the molecular distribution of insulin-like growth factors among different binding proteins.
• Acta Paediatr Suppl. 1994; 399: 159-62
• Display abstract

• The molecular distribution of insulin-like growth factor I (IGF-I) and IGF-II among the IGF binding proteins (IGFBPs) was studied before and during IGF-I therapy in Ecuadorean adults with growth hormone receptor deficiency (GHRD). Of the total circulating IGF-I and IGF-II, 70% was carried by the 150 kDa complex in normal subjects, while in patients with GHRD, 50% of serum IGF-I, but only 30-35% of serum IGF-II, was measured within the 150 kDa IGFBP-3 region. Administration of IGF-I altered the concentration of IGF-I and IGF-II, although the percentage of total IGF measured within each IGFBP region was not affected, as the increase in IGF-I and the decrease in IGF-II were proportional. Similarly, serum concentrations of IGFBP-3 and the acid-labile subunit, measured by radioimmunoassay, were unaltered. Thus, administration of IGF-I to patients with GHRD was unable to correct the aberrant distribution of IGFs among the IGFBPs.

• Yee D, Jackson JG, Kozelsky TW, Figueroa JA
• Insulin-like growth factor binding protein 1 expression inhibits insulin-like growth factor I action in MCF-7 breast cancer cells.
• Cell Growth Differ. 1994; 5: 73-7
• Display abstract

• Interactions between insulin-like growth factor I (IGF-I) and the type of I IGF receptor may be affected by high-affinity extracellular binding proteins. To date, six distinct IGF binding proteins (IGFBPs) have been identified, but their physiological roles are not well understood. For example, depending on experimental conditions, IGFBP-1 has been shown to both enhance and inhibit IGF-I mediated mitogenesis. We have previously shown that excess exogenous IGFBP-1 inhibited IGF-I mediated growth of MCF-7 cells. In this study, we examined whether or not endogenously expressed IGFBP-1 could interfere with IGF-I mediated growth of MCF-7 cells. Cells were stably transfected with an IGFBP-1 expression vector. IGFBP-1 mRNA was produced by the cells, and protein was detected in the conditioned media by ligand blot and immunoblot. Type I IGF receptor could not be phosphorylated by IGF-I in cells expressing IGFBP-1; however, an IGF-I analogue (Arg-3-IGF-I), which cannot complex with IGFBPs, stimulated receptor phosphorylation. IGF-I did not stimulate cell growth in IGFBP-1 expressing cells. These results suggest that IGFBP-1 expression in MCF-7 breast cancer cells inhibits IGF-I induced growth by interrupting the interaction between IGF-I and its receptor.

• Fielder PJ et al.
• Serum profiles of insulin-like growth factors and their binding proteins in adults with growth hormone receptor deficiency treated with insulin-like growth factor I.
• Acta Paediatr Suppl. 1993; 388: 40-3
• Display abstract

• Six adult patients with growth hormone receptor deficiency (GHRD) (2 men, 4 women) with an identical defect in the growth hormone receptor (GHR) gene, were treated with recombinant human insulin-like growth factor I (IGF-I), 40 micrograms/kg s.c. twice daily, for 7 days. Serum concentrations of IGF peptide and IGF binding protein-3 (IGFBP-3) were measured by specific radioimmunoassays; serum IGFBPs were also measured by Western ligand blotting. The size distribution of both IGF-I and IGF-II was measured in serum following size-exclusion fast-performance liquid chromatography. IGF-I treatment resulted in a normalization of serum IGF-I levels on days 1-7 of treatment and a decrease in serum IGF-II levels. The fall in IGF-II levels and the simultaneous rise in IGF-I levels, however, resulted in an unchanged total serum IGF level. The low IGFBP-3 values did not significantly change during treatment, whereas there was a slight increase in IGFBP-2 levels. Preliminary analysis of size-fractionated sera suggested an increase in IGF-I levels in the 40 and 150 kDa regions at the expense of IGF-II levels. The results suggest that despite the failure of IGF-I treatment to increase IGFBPs significantly, serum IGFBP concentrations were sufficient to maintain normal levels of IGF-I.

• Tham A, Nordberg A, Grissom FE, Carlsson-Skwirut C, Viitanen M, Sara VR
• Insulin-like growth factors and insulin-like growth factor binding proteins in cerebrospinal fluid and serum of patients with dementia of the Alzheimer type.
• J Neural Transm Park Dis Dement Sect. 1993; 5: 165-76
• Display abstract

• After acid gel-chromatography cerebrospinal fluid and serum levels of immunoreactive insulin-like growth factor 1 and 2 (IGF-1 and IGF-2) were determined in patients with dementia of the Alzheimer type (AD) and in healthy subjects. The AD CSF levels of immunoreactive IGF-1 did not differ from the subjects but the levels of immunoreactive IGF-2 was significantly elevated in both serum and CSF in the AD patient group. Additionally immunoreactive IGF-1 in AD serum was found to be significantly elevated. To characterize the CSF IGF binding protein activity (IGFBP), ligand blotting was performed on whole CSF from AD patients and subjects. The results demonstrate two major forms of IGFBP in CSF with approximate molecular weights of 33 KDa and 30 KDa. The two IGFBP forms are suggested to represent IGFBP-2 and IGFBP-6. A highly significant increase in both the IGFBPs was observed in the CSF of the AD patients compared to the healthy subjects.

• Baxter RC, Hizuka N, Takano K, Holman SR, Asakawa K
• Responses of insulin-like growth factor binding protein-1 (IGFBP-1) and the IGFBP-3 complex to administration of insulin-like growth factor-I.
• Acta Endocrinol (Copenh). 1993; 128: 101-8
• Display abstract

• The importance of insulin-like growth factor binding proteins (IGFBPs) in modulating the bioactivity of administered IGFs is poorly understood. This study examines responses of IGFBP-1 and the IGFBP-3 complex to recombinant human IGF-I. Eight fasted subjects received a single dose of 0.1-0.125 mg/kg IGF-I sc. This caused a 10-fold rise in IGFBP-1 over 6 h, falling rapidly after food intake. Peak (6-h) IGFBP-1 values were highly correlated with peak post-prandial (8-h) glucose values (r = 0.941). IGFBP-3 showed little response, decreasing slightly over the 48-h period following IGF-I. Adaptive changes in IGFBPs were studied in fed adults injected daily for 7 days with IGF-I, 0.1 mg/kg sc. Following the first injection, IGFBP-1 had a markedly blunted response compared to that in fasted subjects. However, after the seventh IGF-I injection, a 3.5-fold greater IGFBP-1 response to the same IGF-I dose was seen. Concomitantly with the increased IGFBP-1 responsiveness, mean immunoreactive IGFBP-3 and acid-labile subunit levels decreased significantly (p < 0.005), whereas IGFBP-2 detected by immunoblotting increased. Thus IGF-I administration causes changes in IGFBPs which may be important in regulating IGF-I bioavailability.

• Oh Y, Muller HL, Lee DY, Fielder PJ, Rosenfeld RG
• Characterization of the affinities of insulin-like growth factor (IGF)-binding proteins 1-4 for IGF-I, IGF-II, IGF-I/insulin hybrid, and IGF-I analogs.
• Endocrinology. 1993; 132: 1337-44
• Display abstract

• Insulin-like growth factor (IGF)-binding proteins (BPs) bind IGF-I and IGF-II with high affinity and modify the activity of IGF peptides in a complex manner. We have characterized the affinities of IGFBP-1-4 for IGF-I and -II by employing 1) purified IGFBP preparations, 2) both [125I]IGF-I and [125I]IGF-II as radioligands, and 3) multiple IGF analogs designed to have altered affinities for IGFBPs. To this end, human (h) IGFBP-1, hIGFBP-2, and rat (r) IGFBP-4 have been purified to homogeneity from human amniotic fluid, human prostate epithelial cell culture, and B104 rat neuroblastoma cells; for human IGFBP-3, the glycosylated recombinant form (rec-hIGFBP-3), produced in Chinese hamster ovary cells, was employed. The IC50 values of IGF-I for hIGFBP-1, hIGFBP-2, rec-hIGFBP-3, rIGFBP-4, and human serum IGFBPs were 0.05 +/- 0.01, 5.0 +/- 0.01, 0.25 +/- 0.20, 0.6 +/- 0.4, and 0.1 +/- 0.01 ng/ml, respectively. While hIGFBP-1 and rIGFBP-4 had virtually equivalent affinities for IGF-I and IGF-II, hIGFBP-2 and rec-hIGFBP-3 demonstrated 2- to 5-fold higher affinities for IGF-II than for IGF-I. Studies with [Gln3,Ala4,Tyr15,Leu16]IGF-I and Des-(1-3)-IGF-I indicate that specific residues in the first 16 amino acids of the B domain of IGF-I appear to be critical for binding to all of the IGFBPs tested, but not to IGF receptors. However, severe modifications in the B domain disrupt binding affinity, not only for IGFBPs, but also for receptors (IGF-I/insulin hybrid and B-chain mutant). Interestingly, modifications in the A domain of IGF-I, which is believed to contain residues critical for binding to IGF-I and insulin receptors, show differential effects on binding affinity to BPs. [Thr49,Ser50,Ile51]IGF-I, which has normal affinity for the type I IGF receptor, shows at least a 500-fold decreased affinity for hIGFBP-1 and recombinant hIGFBP-3, in contrast to 50- to 100-fold reduced affinity for hIGFBP-2 and rIGFBP-4, and 5- to 10-fold reduced affinity for purified human serum IGFBP-3. More significantly, [1-27,Gly4,38-70]IGF-I shows a 30-fold decreased affinity for the type I IGF receptor and 10- and 2.5-fold reduced affinities for hIGFBP-1 and rec-hIGFBP-3, respectively, but no reduction in affinity for hIGFBP-2 or rIGFBP-4.(ABSTRACT TRUNCATED AT 400 WORDS)

• Myers SE, Cheung PT, Handwerger S, Chernausek SD
• Insulin-like growth factor-I (IGF-I) enhanced proteolysis of IGF-binding protein-4 in conditioned medium from primary cultures of human decidua: independence from IGF receptor binding.
• Endocrinology. 1993; 133: 1525-31
• Display abstract

• Previous studies demonstrated that human decidual cells release insulin-like growth factor-binding protein (IGFBP)-1, IGFBP-2, and a 24-kilodalton (kDa) IGFBP in culture. The accumulation of 24-kDa IGFBP, as assessed by ligand blot analysis, decreased when the cells were exposed to IGF-I, but the mechanism was not explored. In the present study, we observed that the IGF-I-mediated decrease in IGFBP-4 accumulation could be explained by increased IGFBP-4 proteolysis. Analysis by IGFBP-4 immunoblotting demonstrated a decline in 24-kDa IGFBP-4 accompanied by a marked increase in a 17- to 18.5-kDa IGFBP-4 fragment(s). In addition, when medium from IGF-I-treated cells was incubated with rat IGFBP-4, the decrease in IGFBP-4 was inhibited by chelators of divalent cations and inhibitors of serine proteases. IGF-I enhancement of IGFBP-4 proteolysis occurs independent of the type I IGF receptor. [Leu24,1-62]IGF-I, an analog with reduced receptor affinity, mimicked the effect of native IGF-I in cell culture. Additionally, alpha-IR3, a monoclonal antibody to the type I IGF receptor, did not block the effect of IGF-I. When IGF-I was incubated with medium from control cells, there was a marked decrease in 24-kDa IGFBP-4 levels and a concomitant increase in levels of a 17- to 18.5-kDa fragment(s), suggesting that IGFBP-4 complexed with IGF-I is more susceptible to proteolysis than IGFBP-4 alone. Together, these findings suggest a novel mechanism for regulation of IGF-I action in the decidua.

• Lee TC et al.
• Normal human mesothelial cells and mesothelioma cell lines express insulin-like growth factor I and associated molecules.
• Cancer Res. 1993; 53: 2858-64
• Display abstract

• Insulin-like growth factor (IGF) I has important growth regulatory functions in normal growth and development. IGF-I is also a mitogen for a number of cancer cell lines; however, its autocrine effect has not been well established. In this study, the expression of IGF-I, its receptor, and its major serum-binding protein were examined in 5 normal human mesothelial (NHM) cell samples and 11 pleural mesothelioma cell lines. All NHM cells and mesothelioma cell lines expressed IGF-I, IGF-binding protein 3 (IGFBP-3), and IGF-I receptor mRNA by either Northern blot or reverse transcription polymerase chain reaction analysis. IGF-I (0.136 +/- 0.024 ng/ml, mean +/- SEM) and IGFBP-3 (18.5 +/- 3.2 ng/ml) proteins were readily detected in the conditioned medium of mesothelioma cell lines but were not greater than corresponding measurements in that of NHM cells (IGF-I, 0.120 +/- 0.080 ng/ml; IGFBP-3, 15.9 +/- 1.3 ng/ml). Exogenous recombinant IGF-I stimulated cell proliferation of NHM cells, demonstrating the presence of a functional IGF-I receptor. Our results suggest that IGF-I may function as an autocrine growth stimulus in normal proliferating mesothelial cells, which may contribute to their malignant transformation.

• Gajdusek CM, Luo Z, Mayberg MR
• Sequestration and secretion of insulin-like growth factor-I by bovine aortic endothelial cells.
• J Cell Physiol. 1993; 154: 192-8
• Display abstract

• Endothelial cells elaborate growth promoting activities in culture medium that support limited smooth muscle cell and fibroblast growth in vitro in the absence of serum. We investigated whether insulin-like growth factor-I (IGF-I) was synthesized and secreted by bovine aortic endothelial cells in vitro. Subconfluent endothelial cell cultures in serum-free medium secreted severalfold higher IGF-I levels than confluent cultures by acid-sizing chromatography and IGF-I radioimmunoassay. The IGF-I secretory level was not sustained during a second serum-free incubation. In contrast, secretion of IGF binding proteins persisted and was maintained at constant levels throughout the same observation periods. Analysis of poly(A+)RNA by northern blots revealed hybridization of an IGF-I cDNA to a 7.5- to 7.0-kb transcript and superinduction of the 7.5-7.0-kb mRNA by the translational inhibitor, cyclohexamide. However, no endogenously labeled IGF-I was detected in conditioned media after incubation of cultures with [35S]cysteine or [3H]leucine. When cultures were incubated in the presence of serum supplemented with IGF-I, subconfluent cultures sequestered and released more IGF-I than confluent cultures. We concluded that the majority of IGF-I secreted in vitro was sequestered from serum.

• Masters BA, Raizada MK
• Insulin-like growth factor I receptors and IGF-I actions in neuronal cultures from the brain.
• Ann N Y Acad Sci. 1993; 692: 89-101
• Display abstract

• Neurons in primary culture have been used to study IGF-I receptors and IGF-I-induced cellular actions in the brain. Intact neurons in culture specifically bind [125I]IGF with high affinity. The potency for the competition of [125I]IGF-I binding was IGF-I > IGF-II > insulin. A curvilinear Scatchard plot represented high-affinity (0.15 nM) and low-affinity (3 nM) binding sites with a Bmax of 142 fmol and 618 fmol/mg protein, respectively. These binding sites are predominantly localized on neurites with relatively few sites on the cell soma. IGF-I induced synthesis of protein(s) in the M(r) range of 48,000-50,000 with pI values of 6.1-6.4. These observations show that IGF-I receptor mediates induction of specific proteins and suggest that these proteins may be involved in the neurotrophic activity of IGF-I in the brain.

• Bhaumick B
• Insulin-like growth factor (IGF) binding proteins and insulin-like growth factor secretion by cultured chondrocyte cells: identification, characterization and ontogeny during cell differentiation.
• Regul Pept. 1993; 48: 113-22
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• Insulin-like growth factor binding proteins (IGF BP) and insulin-like growth factors (IGF) secretion by differentiating chondrocytes, derived from mouse embryonic limb bud and responsive to both IGF-I and -II [23], was investigated. The Western ligand blot analysis of the conditioned medium (CM) from days 1, 3, 5 and 7 of culture revealed the secretion of IGF BP of approx. 35-40, 28-30 and 24-26 kDa. The 35-40 kDa protein which comigrated with the 40 kDa protein in CM of trophoblast cells identified as IGF BP-3. The 28-30 kDa protein was identified as IGF BP-2 by Western immunoblotting with alpha-IGF BP-2 antisera. The 24-26 kDa protein was consistent with the nonglycosylated form of IGF BP-4. Secretion of three IGF BPs were increased with the age of the culture. This suggested that the major IGF BP secreted by differentiating chondrocytes in culture are IGF BP-2, -3 and -4. All three of these IGF BPs were stimulated by both IGF-I and -II. IGF-I was approx. 2-fold more potent than IGF-II. The investigation of the localized production of IGF revealed that chondrocytes, similar to IGF BP, secreted IGF-II in differentiation dependent manner. No IGF-I secretion was identified. Examination of the secretion of solubilized IGF-II receptor by the chondrocytes, in contrast to trophoblasts, failed to reveal the presence of IGF-II receptor in the CM. This suggested that, unlike many other cells, including trophoblasts, chondrocytes do not secrete solubilized IGF-II receptor. In summary, the present results suggested an interactive autocrine/paracrine action of IGF BP and IGF-II in the chondrocytes, while the IGF-I action is predominantly endocrine.

• Irwin JC, de las Fuentes L, Dsupin BA, Giudice LC
• Insulin-like growth factor regulation of human endometrial stromal cell function: coordinate effects on insulin-like growth factor binding protein-1, cell proliferation and prolactin secretion.
• Regul Pept. 1993; 48: 165-77
• Display abstract

• The insulin-like growth factor (IGF) autocrine/paracrine system is believed to be involved in endometrial differentiation, but there is limited information on the specific cellular functions regulated by IGFs in uterine tissues and their regulation of IGF-binding proteins (IGFBPs). We have investigated the regulation by insulin, IGF-I, and IGF-II, of IGFBP secretion in human endometrial stromal cells decidualized in vitro, and examined the interrelationship between the induced changes in IGFBP levels and the biological responses of stromal cells to IGFs. IGFBPs in conditioned media were analyzed by Western ligand blotting, and IGFBP-1 was quantified by an immunoenzymometric assay (IEMA). In the absence of peptides, decidualized stromal cells secreted 25.5 +/- 3.2 micrograms/day per 10(6) cells of IGFBP-1. Insulin caused a dose-dependent reduction of IGFBP-1 secretion (half-maximal inhibition at < 1 ng/ml) to a maximum of 1% of control values. Northern analysis using a specific cDNA probe showed the expression in decidualized stromal cells of a single 1.5 kb transcript for IGFBP-1, which was absent in insulin-treated cells. The effects of IGF-I and IGF-II on IGFBP-1 secretion were biphasic, with initial stimulation (200-250%) that peaked at 1 and 10 ng/ml, respectively, followed by inhibition at higher concentrations (half maximal inhibition at 3 ng/ml and 30 ng/ml, respectively). The decrease in IGFBP-1 levels in decidualized stromal cultures was associated with the induction of mitogenesis by IGF-I and IGF-II, while IGF effects on prolactin secretion paralleled those of IGFBP-1 secretion, with stimulation (243-324%) in the low concentration range followed by inhibition at higher concentrations. These data indicate that endometrial stromal cell IGFBP-1 is regulated by insulin, at concentrations that are compatible with insulin acting via its own receptor, while the effects of IGF-I and IGF-II on IGFBP-1 secretion, are suggestive of their acting probably through the type I IGF receptor. The present study describes distinct effects of the IGFs on stromal cell IGFBPs, that correlate with changes in the proliferative and secretory responses of decidualized stromal cells to the IGFs. Our findings suggest that complex IGF-IGFBP interactions may participate in the regulation of endometrial cell function, and support a role for IGF-II in stromal cell mitogenesis during decidualization, and as a local regulator of decidual cell function during the late secretory phase and early pregnancy.

• Cascieri MA, Bayne ML
• Analysis of the interaction of IGF-I analogs with the IGF-I receptor and IGF binding proteins.
• Adv Exp Med Biol. 1993; 343: 33-40
• Display abstract

• Distinct domains of IGF-I are important for maintaining high affinity for the IGF-I receptor and for the various species of IGFBPs. The analogs that selectively bind the receptor have proven useful in determining the relative importance of IGFBPs in the regulation of the biological activity of IGF-I. Analogs with poor affinity for the receptor have also been useful in order to demonstrate that a given activity of IGF-I is mediated by the type 1 IGF receptor. These studies confirm that the role of these various proteins in IGF-I action is complex, and may be cell or tissue-type specific.

• Reeve JG, Morgan J, Schwander J, Bleehen NM
• Role for membrane and secreted insulin-like growth factor-binding protein-2 in the regulation of insulin-like growth factor action in lung tumors.
• Cancer Res. 1993; 53: 4680-5
• Display abstract

• The insulin-like growth factors (IGFs) have been implicated in the autocrine and/or paracrine growth of a number of tumor types, including lung tumors. Importantly, insulin-like growth factor-binding proteins (IGFBPs), which both enhance and inhibit the physiological and biological actions of the IGFs, have been shown to be secreted in vitro by a wide range of tumors. In particular, IGFBP-2 is frequently produced by human tumor cells, suggesting that this protein may be an important determinant of IGF action in tumors. In the present study, we investigated IGFBP-2 effects in lung tumor cells by examining the influence of IGFBP-2 on IGF-receptor interaction and the biological actions of IGF-I and IGF-II. Affinity cross-linking studies demonstrated expression of type-I and type-II IGF receptors on small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells and the presence of abundant membrane-associated IGFBP in SCLC cells but not in NSCLC cells. An antiserum specific for IGFBP-2 was used in immunoprecipitation and immunoblotting studies which demonstrated that the membrane-associated IGFBP identified by affinity cross-linking in SCLC cells is IGFBP-2. In NSCLC cells, both IGF-I and IGF-II bound predominantly to IGF-I receptors, whereas in SCLC cells binding was principally to surface-associated IGFBP-2. SCLC cells failed to respond to IGF-I and -II stimulation in a DNA synthesis assay. For NSCLC cells, IGF-II was a more potent stimulator of DNA synthesis than IGF-I. Soluble IGFBP-2 inhibited the binding of radiolabeled IGF-I and -II to both SCLC and NSCLC cells in a concentration-dependent manner and inhibited IGF-stimulated DNA synthesis in NSCLC cells. These observations indicate that both soluble and membrane-associated IGFBP-2 compete with IGF receptors for ligand binding and, thus, are likely to be important determinants of IGF responsiveness. The findings of the present study also indicate that the type-I receptor on NSCLC cells contains a high-affinity binding site for IGF-II which presumably mediates the biological effects of IGF-II in these cells, thereby implicating IGF-II in the autocrine/paracrine growth of NSCLC.

• Schuller AG, Lindenbergh-Kortleve DJ, de Boer WI, Zwarthoff EC, Drop SL
• Localization of the epitope of a monoclonal antibody against human insulin-like growth factor binding protein-1, functionally interfering with insulin-like growth factor binding.
• Growth Regul. 1993; 3: 32-4
• Display abstract

• In order to identify regions in insulin-like growth factor binding protein-1 involved in the binding of IGFs, we tested three monoclonal antibodies, designated MAb A, B, and C on their interference with IGF-binding. Monoclonal A interfered with the binding of IGF to IGFBP-1 as determined by immunoprecipitation whereas monoclonal B and C did not. Furthermore MAb A was found to abolish IGFBP-1 inhibition of IGF stimulation in an in vitro proliferation assay. The epitopes of all three monoclonal antibodies were found to be located within the C-terminal part of IGFBP-1. The regions surrounding residue 188-196 and 222-227 are especially important for antibody recognition. These results indicate that MAb A functionally interferes with the binding of IGF to IGFBP-1. Furthermore, we suggest that part of the epitope of MAb A is located at or sterically near the IGF binding domain of IGFBP-1.

• Jones JI, Busby WH Jr, Wright G, Clemmons DR
• Human IGFBP-1 is phosphorylated on 3 serine residues: effects of site-directed mutagenesis of the major phosphoserine.
• Growth Regul. 1993; 3: 37-40
• Display abstract

• Human IGFBP-1 is phosphorylated by cells in culture and is present in both phosphorylated and nonphosphorylated forms in human fetal serum and amniotic fluid. We have found immunoprecipitable [32P]IGFBP-1 in the conditioned media of both Chinese hamster ovary (CHO) cells (stabley transfected and secreting human IGFBP-1) and human hepatoma (HepG2) cells metabolically labelled with [32P]orthophosphate. Phosphoamino acid analysis of this [32P]IGFBP-1 demonstrates that only serine residues are phosphorylated. Four phosphorylated isoforms of IGFBP-1 can be separated from one nonphosphorylated form by nondenaturing gel electrophoresis. Since we have shown that the nonphosphorylated form of IGFBP-1 has a lower affinity for IGF-I compared to phosphorylated forms and a greater potentiating effect of IGF-I actions, we determined which serine residues in human IGFBP-1 are phosphorylated. After metabolically labelling IGFBP-1 with 32P, the purified phosphoprotein was digested first with trypsin and then with endoproteinase Glu-C. By radiosequencing the resulting 32P-labelled phosphopeptides, we found 3 serine residues to be phosphorylated. Approximately 70% of incorporated 32P was attributed to Ser101, while Ser169 accounted for approximately 25% and Ser119 for 5%. To investigate the physiologic importance of Ser101, this residue (and the nonphosphorylated Ser98) were changed to alanine by site directed mutagenesis of a human IGFBP-1 expression vector, followed by transfection into CHO cells. The [Ala98,101]IGFBP-1 purified from the conditioned media of these cells had the following characteristics: 1) when labelled with [32P]orthophosphate, it contained 63% less radioactivity than wild type IGFBP-1; 2) when analyzed by nondenaturing gel electrophoresis, it contained none of the most rapidly migrating and most rapidly migrating and most highly phosphorylated isoform, more of the nonphosphorylated isoform, and more of the most slowly migrating phosphorylated isoform; and 3) its affinity for IGF-I was reduced 2.5-fold and was midway between wild type IGFBP-1 from transfected CHO cells and dephosphorylated IGFBP-1. We conclude that Ser101 represents the major site of phosphorylation of IGFBP-1 and that while phosphorylation of Ser101 increases affinity of IGFBP-1 for IGF-I, phosphorylation of Ser169 and/or Ser119 also contributes to the high affinity of fully phosphorylated IGFBP-1.

• Shambaugh G 3rd, Glick R, Radosevich J, Unterman T
• Insulin-like growth factor-I and binding protein-1 can modulate fetal brain cell growth during maternal starvation.
• Ann N Y Acad Sci. 1993; 692: 270-2
• Display abstract

• 1. Stimulation of brain cell growth by fetal plasma is reduced when levels of IGF-I are low and IGFBP-1 are high. 2. Fetal brain cells respond to IGFs in cell culture, and effects of plasma are reduced by addition of IGFBP-1. 3. Increased levels of IGFBP-1 and reduced availability of IGF-I may contribute to reduced brain growth when maternal nutrition is impaired.

• Yateman ME, Claffey DC, Cwyfan Hughes SC, Frost VJ, Wass JA, Holly JM
• Cytokines modulate the sensitivity of human fibroblasts to stimulation with insulin-like growth factor-I (IGF-I) by altering endogenous IGF-binding protein production.
• J Endocrinol. 1993; 137: 151-9
• Display abstract

• Human dermal fibroblasts produce a number of insulin-like growth factor-binding proteins (IGFBPs) including the main circulating form, IGFBP-3. It has been suggested that the regulation of IGFBP secretion may play a major role in modulating insulin-like growth factor (IGF) bioactivity. We have quantified the effects of two cytokines, transforming growth factor-beta 1 (TGF-beta 1) and tumour necrosis factor-alpha (TNF-alpha) which have opposing actions on fibroblast IGFBP-3 production, and examined their subsequent role in IGF-I mitogenesis. TGF-beta 1 caused a dose-dependent increase in IGFBP-3 in serum-free fibroblast-conditioned media. TGF-beta 1 (1 microgram/l) resulted in immunoreactive IGFBP-3 levels reaching 286.5 +/- 22.4% of control after 20 h, the increase being confirmed by Western ligand blot. TNF-alpha caused a dose-dependent decrease in fibroblast IGFBP-3 secretion, 1 microgram TNF-alpha/l reducing IGFBP-3 levels to 32.1 +/- 11.% of control. This effect was not due to cytotoxicity and was not cell-density-dependent. Fibroblast proliferation was examined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric cytochemical bioassay. The addition of IGF-I resulted in dose-dependent growth stimulation after 48 h, the effective range being 20-100 micrograms/l. The IGF-I analogue Long-R3-IGF-I which has little affinity for the IGFBPs was approximately 20-fold more potent in this assay, and was unaffected by exogenous IGFBP-3.(ABSTRACT TRUNCATED AT 250 WORDS)

• Hizuka N et al.
• Effects of insulin-like growth factor I (IGF-I) administrations on serum IGF binding proteins (IGFBPs) in patients with growth hormone deficiency.
• Adv Exp Med Biol. 1993; 343: 301-9

• Bach LA, Hsieh S, Sakano K, Fujiwara H, Perdue JF, Rechler MM
• Towards identification of a binding site on insulin-like growth factor-II for IGF-binding proteins.
• Adv Exp Med Biol. 1993; 343: 55-61

• Shemer J et al.
• Regulation of insulin-like growth factor (IGF) binding protein-5 in the T47D human breast carcinoma cell line by IGF-I and retinoic acid.
• J Clin Endocrinol Metab. 1993; 77: 1246-50
• Display abstract

• The T47D human breast carcinoma cell line has been shown to synthesize insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs) and IGF-I receptors, and to exhibit a mitogenic response to exogenous IGF-I. We have used T47D cells to investigate the regulation of IGFBPs by IGF-I and retinoic acid (RA), agents that affect cell proliferation and have been shown to regulate IGFBP levels in other cell types. Exposure of T47D cells to IGF-I resulted in the appearance of IGFBP-2, -4, and -5 in conditioned medium but had no effect on the levels of IGFBPs in Triton X-100-extracted cells. This effect was most pronounced for IGFBP-5 and was also elicited by an IGF-I analog that retains affinity for IGFBPs but not by insulin or IGF analogs that have decreased affinity for IGFBPs. Additionally, this effect was not associated with a change in IGFBP-5 messenger RNA (mRNA) levels; however, the appearance of IGFBP-5 in the conditioned medium was inhibited by an anti-IGF-I receptor antibody (alpha IR-3). RA decreased IGFBP-5 mRNA levels and cell-associated IGFBP-5 in both the presence and absence of IGF-I and inhibited the IGF-I-stimulated secretion of IGFBP-5 into T47D cell conditioned medium. These results suggest that IGF-I increases IGFBP-5 levels in the T47D cell line both through direct interaction with IGFBP-5 as well as through a receptor-mediated process that does not require direct interaction with IGFBPs. The latter results are consistent with an effect of IGF-I on a factor that may modulate an IGFBP protease activity. The inhibitory effect of RA, on the other hand, appears to be due primarily to regulation of IGFBP-5 mRNA levels. Thus, IGFBP-5 accumulation appears to be positively regulated by IGF-I, potentially at the level of susceptibility to proteolysis, and negatively regulated at the level of gene expression by RA.

• Bang P, Degerblad M, Thoren M, Schwander J, Blum W, Hall K
• Insulin-like growth factor (IGF) I and II and IGF binding protein (IGFBP) 1, 2 and 3 in serum from patients with Cushing's syndrome.
• Acta Endocrinol (Copenh). 1993; 128: 397-404
• Display abstract

• In the present study of twenty-two patients with Cushing's syndrome, serum insulin-like growth factor (IGF)-I concentrations were normal to high with an increased mean IGF-I concentration, 40% above that of healthy subjects of the same age (p < 0.001). Serum IGF-II concentrations were normal. The morning serum IGF binding protein (IGFBP)-1 concentrations were within the range of healthy controls. IGFBP-1 was inversely correlated to the IGF-I concentration (p < 0.001) and to the 24 h urinary cortisol excretion (p < 0.005) with a combined R squared value of 0.58. In contrast to IGFBP-1, serum IGFBP-2 and IGFBP-3 concentrations were elevated by 1.89 +/- 1.78 SD and 0.92 +/- 0.78 SD (mean +/- 2 SD), respectively. Although increased, the serum IGFBP-2 concentration was inversely correlated to the IGF-I concentration (r = -0.67, p < 0.001). Immunoreactive IGFBP-3 was increased in proportion to IGF-I and the molar ratio [IGFBP-3]:[IGF-I] + [IGF-II] was close to unity (1.04 +/- 0.14), as that of healthy subjects. In serum from patients with Cushing's syndrome, with increased immunoreactive IGFBP-3, there was a corresponding increase in intact glycosylated 40-43 kDa IGFBP-3 as determined by Western ligand blotting. Neutral size chromatography of serum from patients with Cushing's syndrome showed that IGF-I and IGFBP-3 immunoreactivity were predominantly found at the elution volume of the ternary 150 kDa IGF-I/IGFBP-3/acid labile subunit complex and a similar pattern was displayed by normal serum.(ABSTRACT TRUNCATED AT 250 WORDS)

• Lindgren BF, Isaksson M, Stern I, Hall K
• Insulin-like growth factor binding protein-1 from Hep G2 cells is potently inhibited by the truncated IGF-I analogue des-(1-3) IGF-I.
• Acta Endocrinol (Copenh). 1993; 128: 81-7
• Display abstract

• Des-(1-3) insulin-like growth factor-I (IGF-I) is an IGF analogue lacking aminoacid 1 to 3 which displays reduced binding to insulin-like growth factor binding protein-1 (IGFBP-1). A greater inhibition of immunoreactive IGFBP-1 was obtained with des-(1-3) IGF-I (10 ng/ml) in Hep G2 medium when incubated in Eagle's Modified Essential Medium (EMEM) without phenolred compared to EMEM with phenolred; EMEM without phenolred was chosen for further experiments. Des-(1-3)IGF-I decreases dose dependently the concentration of IGFBP-1, with a maximal effect at 3-10 micrograms/l when incubated for 24 h; 10 micrograms/l of des-(1-3)IGF-I caused a small but significant inhibition of IGFBP-1 after 8 h incubation and this inhibition was 41% and 33% of controls after 14 and 19 h incubation. The relative potencies at 16 h of incubation of IGF-I and insulin in suppressing IGFBP-1 in comparison to des-(1-3)IGF-I were 0.41 (0.25-0.78) and 0.08 (0.01-0.26), respectively. A dose-dependent decrease of IGFBP-1 mRNA to 30% of control was observed after 4 h incubation with 0.1-10 micrograms/l des-(1-3)IGF-I. Changes of glucose concentration (0-20 mmol/l) in the medium did not affect the IGFBP-1 concentration in the medium. In summary: Des-(1-3)IGF-I was tenfold more potent than insulin, and threefold more potent than IGF-I in decreasing IGFBP-1 concentration in medium conditioned by Hep G2 cells.

• Pao CI et al.
• Regulation of insulin-like growth factor-I (IGF-I) and IGF-binding protein 1 gene transcription by hormones and provision of amino acids in rat hepatocytes.
• Mol Endocrinol. 1993; 7: 1561-8
• Display abstract

• Synthesis of insulin-like growth factor-I (IGF-I) and IGF binding protein-1 (IGFBP-1) is altered in diabetes and malnutrition, but underlying processes are poorly understood. To study molecular mechanisms, we examined regulation of IGF-I and IGFBP-1 gene transcription in primary cultures of rat hepatocytes. Transcription of the IGF-I and IGFBP-1 genes was measured as incorporation of [alpha-32P]UTP into preinitiated message in isolated nuclei. IGFBP-1 gene transcription was not sensitive to reduction in amino acid concentration from 5x to 0.5x rat arterial plasma levels. However, IGF-I gene transcription fell 60-70% in response to reduced provision of amino acids. Culture with 10(-9) M insulin lowered IGFBP-1 gene transcription 50% below control levels (10-11 M) but did not affect IGF-I gene transcription; 10(-6) M insulin raised IGF-I gene transcription 2-fold. After an acute reduction in insulin concentration, IGFBP-1 transcription began to rise within 30 min, but IGF-I gene transcription was unchanged over 120 min. Similarly, 3-6 h were required for stimulation of IGF-I gene transcription by insulin, but a 40% decrease in IGFBP-1 gene transcription could be detected within 15 min after adding 10(-6) M insulin, and suppression of IGFBP-1 transcription by insulin was unaffected by the presence of cycloheximide. Effects of insulin on IGFBP-1 gene transcription were not mimicked or antagonized by phorbol ester.(ABSTRACT TRUNCATED AT 250 WORDS)

• Rutishauser J, Schmid C, Hauri C, Froesch ER, Zapf J
• Growth hormone, but not insulin-like growth factor I, induces a serum protease activity for insulin-like growth factor binding protein-3 in hypophysectomized rats in vivo.
• FEBS Lett. 1993; 334: 23-6
• Display abstract

• Insulin-like growth factor binding proteins (IGFBPs) modulate IGF action. Proteolytic cleavage of IGFBPs yields lower molecular forms with reduced ability to bind IGFs, thereby increasing IGF bioavailability. In serum from normal adult rats, we found a proteolytic activity for IGFBP-3, presumably a cation-dependent serine protease. It is lacking in serum from hypophysectomized rats and restored by infusion of growth hormone (GH), but not IGF I. Thus, IGF I does not appear to mediate the GH effect on IGFBP-3 proteolysis. Rather, GH seems to modulate IGF action indirectly via alteration of IGFBP-3 structure.

• Laron Z
• Effect of insulin-like growth factor I on IGF binding proteins.
• J Pediatr. 1993; 123: 169-70

• Miura Y, Higashi Y, Kato H, Takahashi S, Noguchi T
• Effects of dexamethasone on the production of insulin-like growth factor-I and insulin-like growth factor binding proteins in primary cultures of rat hepatocytes.
• Biosci Biotechnol Biochem. 1992; 56: 1396-400
• Display abstract

• The effects of dexamethasone (Dex) on insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-1 production were investigated in primary cultures of rat hepatocytes. Dex enhanced the secretion of IGFBP-1 as measured by ligand blot analysis but did not show any prominent effect on immunoreactive IGF-I secretion. EC50 of Dex on IGFBP-1 secretion was calculated to be 3 x 10(-8) M. The content of IGFBP-1 mRNA in the cells increased greatly in the presence of Dex but the IGF-I mRNA content did not change significantly under the same conditions. Insulin showed the opposite effect of Dex by decreasing the production of IGFBP-1 and the cellular content of IGFBP-1 mRNA. This effect of insulin was observed also with Dex in the medium. These results show that the gene expression of IGF-I and IGFBP-1 is differently regulated by glucocorticoids and insulin in primary cultures of rat hepatocytes. The results most possibly explain the in vivo effects of glucocorticoids and insulin in regulation of IGF-I and IGFBP-1 production by liver.

• Dor J et al.
• Insulin-like growth factor-I and follicle-stimulating hormone suppress insulin-like growth factor binding protein-1 secretion by human granulosa-luteal cells.
• J Clin Endocrinol Metab. 1992; 75: 969-71
• Display abstract

• Local regulation of insulin-like growth factor binding protein-1 (IGFBP-1) production in the human ovarian follicle was investigated using cultured human granulosa-luteal cells. Both insulin-like growth factor-I (IGF-I) and follicle-stimulating hormone (FSH) exerted a dual effect on granulosa cells: while estradiol (E2) production was increased by both stimulants, the addition of either of the two hormones led to a reduction in IGFBP-1 secretion by more than 50%. Inhibition of IGFBP-1 production in response to IGF-I was dose-dependent,with the highest effect observed at 5 nM IGF-I. A significant correlation was found between the increase in E2 and inhibition of IGFBP-1 secretion in response to IGF-I. These observations may suggest a novel mechanism, at the follicular level, by which FSH and IGF-I amplify the IGF-I effect in the ovarian follicular cells.

• Smith EP, Cheung PT, Ferguson A, Chernausek SD
• Mechanisms of Sertoli cell insulin-like growth factor (IGF)-binding protein-3 regulation by IGF-I and adenosine 3',5'-monophosphate.
• Endocrinology. 1992; 131: 2733-41
• Display abstract

• 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.

• Adashi EY et al.
• Granulosa cell-derived insulin-like growth factor (IGF) binding proteins are inhibitory to IGF-I hormonal action. Evidence derived from the use of a truncated IGF-I analogue.
• J Clin Invest. 1992; 90: 1593-9
• Display abstract

• An increasing body of information now suggests that insulin-like growth factor (IGF) binding proteins (BPs) may serve as antigonadotropins at the level of the ovary. It is the objective of the present communication to evaluate the functional role of endogenous (granulosa cell-derived) IGFBPs by exploiting the unique properties of des(1-3)IGF-I, a naturally occurring IGF-I analogue characterized as a weak ligand of IGFBPs but not of type I IGF receptors. Given IGFBP-replete circumstances, des(1-3)IGF-I proved more potent (10-fold) than its intact counterpart in promoting the follicle stimulating hormone (FSH)-stimulated accumulation of progesterone by cultured rat granulosa cells. In contrast, des(1-3)IGF-I proved virtually equipotent to the unmodified principle under IGFBP-deplete circumstances. Taken together, these findings are in keeping with the notion and that the apparently enhanced potency of des(1-3)IGF-I (under IGFBP-replete conditions) is due to its diminished affinity for endogenously generated IGFBPs and that rat granulosa cell-derived IGFBPs are inhibitory to IGF (and thus inevitably to gonadotropin) hormonal action. Accordingly, the reported ability of gonadotropins to attenuate IGFBP release by granulosa cells may be designed to enhance the bioavailability of endogenously generated IGFs in the best interest of ovarian steroidogenesis.

• Young SC, Underwood LE, Celniker A, Clemmons DR
• Effects of recombinant insulin-like growth factor-I (IGF-I) and growth hormone on serum IGF-binding proteins in calorically restricted adults.
• J Clin Endocrinol Metab. 1992; 75: 603-8
• Display abstract

• To determine the effects of exogenous insulin-like growth factor-I (IGF-I) and GH on IGF-binding proteins (IGFBP)-1, -2, and -3, six healthy nonobese adult volunteers underwent two 2-week periods of diet restriction (20 Cal/kg.day), and during the last 6 days of the first period received either IGF-I (12 micrograms/kg.h by iv infusion over 16 h) or GH (0.05 mg/kg.day by sc injection). During the second 2-week study period, the alternate hormone was given. IGFBP-1 and -2 concentrations were determined by specific RIA, and changes in IGFBP-3 were assessed by ligand blotting. Free IGF-I concentrations were measured by size-exclusion high pressure liquid chromatography, followed by RIA. Diet restriction alone did not affect either IGFBP-1 or -2 significantly. IGF-I treatment increased IGFBP-1 from 78 +/- 46 ng/mL (mean pretreatment) to 137 +/- 64 ng/mL (P less than 0.001; mean for the last 4 days of IGF-I). IGF-I also caused an increase in IGFBP-2 from 315 +/- 136 to 675 +/- 304 ng/mL (P less than 0.001). GH injections caused a modest decline in IGFBP-1 concentrations but had no effect on IGFBP-2 concentrations. By ligand blotting, both IGF-I and GH caused a modest increase in IGFBP-3 band intensity. In three subjects diet restriction alone caused a small decrease in IGFBP-3 hand intensity, and this was reversed by hormone treatment. Free IGF-I concentrations in serum were increased from 1.6% to 4.4% of the total IGF-I during IGF-I infusions. GH injections caused a smaller increase in free IGF-I concentrations. The results show significant increases in IGFBP-1 and -2 during IGF-I infusion. The change in IGFBP-3, while significant, is quantitatively less than that in experimental animals that have been given IGF-I while undergoing dietary restriction. The net effect of the changes in these three forms of IGFBPs is not sufficient to maintain a normal IGF-I-binding capacity in serum, because free IGF-I levels were increased disproportionately during the IGF-I infusions. Because hypoglycemia was noted in these subjects despite insulin suppression, these alterations in IGFBPs might have changed the tissue bioavailability of IGF-I and facilitated its hypoglycemic effects.

• van Haren L, Flinterman JF, Orly J, Rommerts FF
• Luteinizing hormone induction of the cholesterol side-chain cleavage enzyme in cultured immature rat Leydig cells: no role of insulin-like growth factor-I?
• Mol Cell Endocrinol. 1992; 87: 57-67
• Display abstract

• Long-term inductive effects of luteinizing hormone (LH) on cholesterol side-chain cleavage (CSCC) enzyme activity were studied, using cultured Leydig cells isolated from 21-day-old rats. Particular reference was given to the role of insulin-like growth factor-I (IGF-I) as an autocrine or paracrine modulator or as an essential extracellular mediator of LH action. The CSCC enzyme activity was measured using an excess of 22(R)-hydroxycholesterol as substrate to saturate the enzyme, and inhibitors of pregnenolone metabolism to concentrate all the products of the enzyme reaction in pregnenolone. The rate of sterol conversion into pregnenolone (CSCC enzyme activity) reflected the amount of cytochrome P-450scc (P-450scc), as was shown by Western blotting. In cells cultured without LH, the CSCC enzyme activity decreased to 10% on day 7 of the culture period. In the presence of various doses of LH ranging from 0.01 to 100 ng/ml, the CSCC enzyme activity also diminished during the first 3 days of culture, but during the following days, the amount of CSCC enzyme was stimulated by LH. In contrast to the absence of any LH effect on the activity of the CSCC enzyme during the first days of the culture, the endogenous steroid production (no added 22(R)-hydroxycholesterol) could be stimulated at least 10-fold by high doses of LH. When LH (1 ng/ml) was added to cells which had been cultured for 7 days without hormones, CSCC enzyme activity was elevated 8-fold after 4 days of exposure of LH. These effects of LH could be mimicked by dbcAMP (0.5 mM). No evidence could be provided that IGF-I plays any role in the LH induction of the CSCC enzyme; neither the addition of exogenous IGF-I or analogs that do not bind to IGF-I binding proteins (IGFBPs) nor the inactivation of endogenous IGF-I action (through binding to IGFBP and antibodies to IGF-I or via masking of IGF-I receptor by antibodies) could influence the LH induced CSCC enzyme activity. The present data raise the question under which conditions IGF-I is capable of modulating Leydig cell steroidogenesis.

• Cortizo AM, Gagliardino JJ
• Protein glycation: its role in the changes induced by diabetes in the properties of the serum insulin-like growth factor-I binding proteins.
• J Endocrinol. 1991; 131: 33-8
• Display abstract

• The purpose of this work was to study the effect of diabetes on 125I-labelled insulin-like growth factor (IGF) binding to specific serum binding proteins (IGFBPs) and the possible role of protein glycation in such an effect. Accordingly, ligand blotting and fructosamine assays were performed in serum samples from diabetic and non-diabetic eSS rats as well as in samples of normal rat serum previously incubated with different concentrations of glucose. IGFBPs with molecular weights of 24, 30 and 40 kDa were identified in samples from diabetic and non-diabetic rats. 125I-Labelled IGF-I binding to each of these fractions increased significantly in the serum of diabetic rats. IGF-I binding to IGFBP-40 increased significantly as a function of the degree of glycation of serum proteins. Conversely, the increased binding of IGFBP-24 and IGFBP-30 was related only to the glucose concentration attained at 120 min during the oral glucose tolerance test. Glycation of proteins of normal serum and the binding of labelled IGF-I increased as a function of glucose concentration in the incubation media. In these in-vitro glycated normal sera, only the binding to IGFBP-40 increased significantly; this increase was closely related to the amount of protein glycation. No clear and reproducible changes occurred with the binding of 125I-labelled IGF-I to IGFBP-24 and IGFBP-30 fractions. These results confirm the increase in the binding capacity of IGFBPs reported in diabetic animals. They also show that the increase in IGF-I binding to each IGFBP fraction is regulated by a different mechanism; whereas protein glycation induces changes in IGFBP-40, this mechanism does not affect the binding properties of the other two IGFBPs. The increased binding of IGFBP might affect the availability of free IGF-I, and the consequent alterations in IGF-I-dependent metabolic processes could explain the role of this growth factor in the pathogenesis of chronic complications of diabetes.

• Tollefsen SE, Heath-Monnig E, Cascieri MA, Bayne ML, Daughaday WH
• Endogenous insulin-like growth factor (IGF) binding proteins cause IGF-1 resistance in cultured fibroblasts from a patient with short stature.
• J Clin Invest. 1991; 87: 1241-50
• Display abstract

• The ED50 of insulin-like growth factor (IGF)-I-stimulated alpha-aminoisobutyric acid (AIB) uptake (mean +/- SD) in cultured fibroblasts from a child with short stature that we have reported (1.40 +/- 0.24 nM), is significantly higher than the ED50 of IGF-I-stimulated AIB uptake in fibroblasts from 11 normal subjects (0.42 +/- 0.12 nM) and from 127 short children (0.35 +/- 0.11 nM). Similarly, the ED50 of IGF-I-stimulated thymidine incorporation in fibroblasts from this child is 2.8 times higher than that in fibroblasts from four normal subjects. To minimize potential modulation of IGF-I action by endogenous IGF binding proteins in these assays, fibroblast responsiveness to [Q3,A4,Y15,L16]IGF-I, an IGF-I variant that has a 600-fold reduced affinity for serum IGF binding proteins, has been examined. The biological activity of this variant is comparable in the patient's and normal fibroblasts, suggesting that the resistance to IGF-I action cannot be attributed to a defective IGF-I receptor. To investigate directly the possibility that IGF-I sensitivity in the patient's fibroblasts is reduced by endogenous IGF binding proteins (IGFBP), binding proteins that are secreted into AIB assay buffer during a 3-h collection and that are cell-associated at the end of the collection have been analyzed. Ligand blot analysis of conditioned AIB assay buffer demonstrates that fibroblasts from the patient secrete 1.3-2.2 times more of Mr 46,400/42,900, 32,000, and 26,800 binding proteins than normal fibroblasts. The major difference between fibroblasts from the patient and from normal subjects is a striking 10-fold increase in the amount of a cell surface Mr 32,000 binding protein in the patient's fibroblasts. The Mr 32,000 binding protein is similar in size to IGFB-1 and different from IGFBP-2 and IGFBP-3, but it does not cross-react with an antibody against IGFBP-1. We conclude that the resistance to IGF-I action in the patient's fibroblasts is caused by an abnormal production and/or cell association of IGF binding proteins.

• Campbell PG, Skaar TC, Vega JR, Baumrucker CR
• Secretion of insulin-like growth factor-I (IGF-I) and IGF-binding proteins from bovine mammary tissue in vitro.
• J Endocrinol. 1991; 128: 219-28
• Display abstract

• In vitro, insulin-like growth factor-I (IGF-I) promotes both growth and development of bovine mammary tissue. In vivo, the effects of IGF-I may encompass endocrine, paracrine or autocrine mediation. We addressed the possibility of paracrine/autocrine effects of IGF-I in the mammary gland by examining the in-vitro secretion of IGF-I and IGF-binding proteins (IGFBPs) from bovine mammary tissue. Bovine mammary explants from pregnant non-lactating and lactating non-pregnant animals were found to synthesize and secrete IGF-I and IGFBPs. Mammary acini cultures, representative of mammary secretory epithelia, secreted both IGF-I and IGFBP, but synthesized only IGFBP. Concentrations of IGF-I in conditioned media from explants were 1.54 and 0.72 fmol/micrograms DNA for pregnant and lactating animals respectively. Concentrations of IGFBPs in conditioned media from explants were similar for both physiological states at 2529 pmol 125I-labelled IGF-I bound/micrograms DNA. Ligand/Western blotting procedures identified four IGFBPs of 29, 33, 37 and 44 kDa for acini cultures and five IGFBPs of 28, 31, 36, 44 and 46 kDa for explant cultures. Similar affinities for IGF-I and IGF-II were shown by IGFBP, using 125I-labelled recombinant human IGF-I as the competing ligand (median effective dose (ED50) of 0.085 pmol). When 125I-labelled bovine IGF-II was used as the ligand, only bovine IGF-II (ED50 of 0.25 pmol) inhibited binding. The addition of prolactin, insulin and cortisol, with or without GH, did not affect secretion of either IGF-I or IGFBP. This report describes the ability of normal mammary tissue to synthesize and secrete IGF-I and IGFBPs.

• Cohick WS, Clemmons DR
• Regulation of insulin-like growth factor binding protein synthesis and secretion in a bovine epithelial cell line.
• Endocrinology. 1991; 129: 1347-54
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• The Madin-Darby bovine kidney cell line was used to examine regulation of insulin-like growth factor binding protein (IGFBP) synthesis by epithelial cells. Ligand and immunoblot analysis of conditioned media indicated that IGFBP-2 was the predominant IGFBP secreted by untreated cells. Treatment with forskolin decreased secretion of IGFBP-2 by 75 +/- 3% and induced the appearance of IGFBP-3 and 24,000 Mr IGFBP. Although insulin alone did not induce the appearance of either band, in the presence of forskolin it increased the IGFBP-3 and 24,000 Mr bands 4.2 +/- 1.1 and 7.3 +/- 0.9-fold, respectively, above the values for forskolin treatment alone. Exposure to forskolin resulted in a 3-fold decrease in the abundance of IGFBP-2 messenger RNA (mRNA), and a 30-fold increase in IGFBP-3 mRNA. An additional 2- to 3-fold increase in IGFBP-3 mRNA was observed when cells were treated with insulin plus forskolin. Treatment with insulin plus forskolin increased cell number 2-fold, compared to small increases (26%) observed with forskolin treatment alone. Since treatment with IGF-I or -II did not result in similar responses to those of insulin, IGF analogs with differing affinities for IGFBP and IGF type I receptor were tested. B-chain IGF-I (decreased affinity for IGFBP) increased cell number and enhanced forskolin's effects on IGFBP-3 secretion and mRNA abundance to the same extent as insulin, whereas [Leu24,1-62]IGF-I (decreased affinity for the type I IGF receptor) did not. Therefore, activation of the type I IGF receptor was required to elicit increases in cell number and IGFBP synthesis and secretion, and the actions of IGF-I and II were likely blocked by binding to the large amounts of IGFBP-2 that were secreted. These results are in direct contrast to studies with human fibroblasts in which IGF-I and [Leu24,1-62]IGF-I stimulate IGFBP-3 secretion, whereas B-chain IGF-I has only a minimal effect. The ability to differentially regulate secretion of different forms of IGFBPs by epithelial cells and the finding that regulation is distinct from that of fibroblasts may have important implications for understanding mechanisms by which IGFs and IGFBPs interact to regulate epithelial cell growth.

• Ceda GP et al.
• Differential effects of insulin-like growth factor (IGF)-I and IGF-II on the expression of IGF binding proteins (IGFBPs) in a rat neuroblastoma cell line: isolation and characterization of two forms of IGFBP-4.
• Endocrinology. 1991; 128: 2815-24
• Display abstract

• The isolation and hormonal regulation of two low molecular weight insulin-like growth factor binding proteins (IGFBPs) present in the conditioned medium (CM) of the rat neuroblastoma cell line B104 cells has been performed. IGFBPs were purified by ZnSO4 precipitation, insulin-like growth factor-I 1IGF-I) affinity chromatography, and reverse phase HPLC. Final isolation and N-terminal analysis was accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotting to polyvinylidene difluoride membranes, and sequencing of the bound proteins. Two IGFBPs, with apparent Mr of 28K and 24K were coisolated and sequenced. Both proteins had identical N-terminal sequences and appear to be two forms of IGFBP-4. Treatment of the IGFBPs with endoglycosidase-F caused a shift in the apparent Mr of the 28K IGFBP to 24K. However, there was no change in the apparent Mr of the 24K IGFBP. The data from this study suggest that the IGFBP-4 exists as both a glycosylated and nonglycosylated protein. Treatment of B104 cells with IGF-I increased the expression of both the 24K and 28K IGFBPs and also resulted in the appearance of IGFBP-3 and an unknown IGFBP at 29K. When added to subconfluent cells, IGF-I was also mitogenic in B104 cells. Similar to IGF-I, IGF-II treatment increased cell number and resulted in the appearance of IGFBP-3 and the 29K IGFBP. However, IGF-II treatment resulted in a significant decrease (approximately 50%) in the 24K IGFBP and also decreased the 28K IGFBP. This decrease in the expression of the 24K and 28K IGFBPs was dose-dependent and was blocked by addition of IGF-I to the cells. When an IGF-II receptor antibody was added to the cells it mimicked the effects of IGF-II on B104 cells, suggesting that the inhibitory effects of IGF-II are mediated through the type II IGF receptor. Although both IGF-I and IGF-II affected the amount of the 24K IGFBP in the CM, neither peptide affected the expression of the messenger RNA for the 24K IGFBP. In conclusion, we have isolated two IGFBPs from the CM of B104 cells. Both the 24K and 28K IGFBPs appear to be isoforms of the same protein, and sequence data suggest these proteins are two forms of IGFBP-4. IGF-I increases the expression of both of these IGFBPs, whereas IGF-II decreases their expression.(ABSTRACT TRUNCATED AT 400 WORDS)

• Conover CA
• Glycosylation of insulin-like growth factor binding protein-3 (IGFBP-3) is not required for potentiation of IGF-I action: evidence for processing of cell-bound IGFBP-3.
• Endocrinology. 1991; 129: 3259-68
• Display abstract

• Insulin-like growth factor binding protein-3 (IGFBP-3) is unique among the IGF binding proteins in its extensive glycosylation in the native state. To determine the functional significance of carbohydrate moieties on IGFBP-3, we examined the effects of nonglycosylated Escherichia coli-derived recombinant human IGFBP-3 (hIGFBP-3E. coli) and glycosylated Chinese hamster ovary cell-derived hIGFBP-3 (hIGFBP-3CHO) on IGF-I action in cultured bovine fibroblasts. Both hIGFBP-3 preparations bound IGF-I with high affinity and were approximately 5-fold more potent than unlabeled IGF-I in inhibiting [125I]IGF-I binding to bovine fibroblasts. Coincubation of IGF-I and hIGFBP-3E. coli or hIGFBP-3CHO produced a dose-dependent inhibition of IGF-I but not insulin-stimulated [3H]aminoisobutyric acid (AIB) uptake. In contrast, preincubation of bovine fibroblasts with hIGAFBP-3E. coli or hIGFBP-3CHO potentiated subsequent IGF-I-stimulated [3H]AIB uptake. When cells were preincubated with 50 nM hIGFBP-3E. coli for 24 h, [125I]IGF-I binding to bovine fibroblasts increased 2.4-fold, whereas responsiveness to IGF-I was increased only 25%. After a 72-h preincubation, IGF-I cell binding remained increased 2-fold with commensurate enhancement of IGF-I-stimulated [3H]AIB uptake. The increase in [125I]IGF-I binding to bovine fibroblast monolayers was primarily due to association of hIGFBP-3E. coli with the cell surface; there was no significant change in IGF-I receptor number or affinity under these conditions. Affinity cross-linking experiments indicated that intense binding of [125I]IGF-I to cell-associated 29,000 Mr hIGFBP-3E. coli seen after 24 h of incubation was reduced approximately 70% after 72 h, concomitant with the appearance of smaller bands indicating hIGFBP-3E. coli forms of 12,000-27,000 Mr. Cell-associated IGFBP-3E. coli (72 h preincubation conditions) had a 10-fold lower affinity for IGF-I compared to hIGFBP-3E. coli in solution and a 2-fold lower affinity compared to the IGF-I receptor. These data demonstrate that glycosylation is not obligatory for biologically functional IGFBP-3. Furthermore, they suggest that processing of cell-associated IGFBP-3 to forms with altered affinity for IGF-I peptide may underly the potentiating effect of IGFBP-3 on IGF-I action.

• McCusker RH, Busby WH, Dehoff MH, Camacho-Hubner C, Clemmons DR
• Insulin-like growth factor (IGF) binding to cell monolayers is directly modulated by the addition of IGF-binding proteins.
• Endocrinology. 1991; 129: 939-49
• Display abstract

• Insulin-like growth factor-I (IGF-I) binds to specific receptors and IGF-binding proteins (IGFBPs) that are present on cell surfaces. The analysis of [125I]IGF-I binding to human fibroblasts is complicated by IGFBPs on the cell surface and their release into the medium during the binding assay. This release alters the distribution of [125I]IGF-I between type I IGF receptors and both soluble as well as cell surface-associated IGFBPs. In the present study we have determined the effects of three different forms of IGFBPs on [125I]IGF-I binding to cell surface binding sites of human fetal fibroblasts (GM10 cells) and porcine smooth muscle cells. Human 29,000 mol wt (Mr; IGFBP-1), bovine 34,000 Mr (IGFBP-2), and bovine 46,000 Mr (IGFBP-3) forms of IGFBP were compared. Each of the three IGFBPs inhibited [125I]IGF-I binding to the cell surface of both cell types. This effect was due to increased binding of [125I]IGF-I by the IGFBPs in the assay buffer. At equimolar concentrations, IGFBP-3 was more effective than either IGFBP-1 or IGFBP-2 in blocking cell surface binding. The addition of increasing concentrations of unlabeled IGF-I in the presence of each IGFBP showed that either IGFBP-1 or IGFBP-3, but not IGFBP-2, resulted in a paradoxical increase in [125I]IGF-I binding to the cell surface. The paradoxical increase occurred in the presence of excess insulin, indicating that unsaturated type I IGF receptors are not required to demonstrate this phenomenon. In a physiological salt solution, the order of affinity of the IGFBPs for IGF-I was IGFBP-3 greater than IGFBP-1 greater than IGFBP-2. These differences in affinity appear to account for the differences in IGF-I competition for binding that are seen when each of the three proteins is added. Thus, IGFBPs have the potential to alter the partitioning of IGF-I between cell surface-associated IGFBPs, membrane receptors, and the IGFBPs in extracellular fluids. The various forms of IGFBP affect IGF cell surface binding differently, and therefore, each may have distinct effects on IGF target cell actions.

• Conover CA, Powell DR
• Insulin-like growth factor (IGF)-binding protein-3 blocks IGF-I-induced receptor down-regulation and cell desensitization in cultured bovine fibroblasts.
• Endocrinology. 1991; 129: 710-6
• Display abstract

• 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.

• Gourmelen M, Perin L, Binoux M
• Effects of exogenous insulin-like growth factor I on insulin-like growth factor binding proteins in a case of growth hormone insensitivity (Laron-type).
• Acta Paediatr Scand Suppl. 1991; 377: 115-7
• Display abstract

• The electrophoretic profiles of serum insulin-like growth factor binding proteins (IGFBPs) from an 18-year-old patient with growth hormone (GH) insensitivity were analysed by Western ligand blotting before and after administration of recombinant human insulin-like growth factor I (IGF-I). Under basal conditions, the profile was the same as that observed in all cases of GH deficiency or insensitivity, that is, low IGFBP-3 (seen as two bands of 41.5 and 38.5 kDa) and enhanced IGFBP-2 and, to a lesser extent, IGFBP-1. Subcutaneous injection of IGF-I, 40 micrograms/kg, provoked an increase in serum IGF-I levels to close to the lower limits of the normal range and a small increase in IGFBP-3, suggesting that IGF-I has a direct effect on the synthesis of this GH/IGF-I-dependent IGFBP. Moderate increases were also observed in IGFBP-2 and IGFBP-4. Repeated doses of IGF-I over 7 days had no further effect on these changes, which persisted for a few days after the last injection.

• Conover CA
• Regulation of insulin-like growth factor (IGF)-binding protein synthesis by insulin and IGF-I in cultured bovine fibroblasts.
• Endocrinology. 1990; 126: 3139-45
• Display abstract

• Specific insulin-like growth factor-binding proteins (IGFBPs) are synthesized and secreted by bovine fibroblasts in vitro. By Western ligand blotting, three molecular forms of IGFBP were identified in conditioned medium from control cultures with mol wt (Mr) of 34,000, 28,000, and 24,000. Concentrations of these three IGFBP forms increased with time in serum-free conditioned medium without benefit of hormonal supplementation. Insulin and IGF-I were potent stimuli for IGFBP production by bovine fibroblasts, whereas bovine GH, epidermal growth factor, or steroid treatment had little or no effect. Insulin and IGF-I enhanced the production of 24,000, 28,000, and 34,000 Mr IGFBPs in a dose-dependent fashion. Moreover, addition of low nanomolar concentrations of insulin or IGF-I to bovine fibroblast cultures specifically induced the secretion of a 42,000/38,000 Mr species of IGFBP, which corresponded in size to the IGF-binding subunit of the principal 150,000 Mr IGFBP complex in serum. After stimulation with insulin or IGF-I, bovine fibroblasts (3 X 10(5) cells) secreted approximately 30 ng/24 h 42,000/38,000 Mr IGFBP. Subunits of 42,000/38,000 Mr in bovine fibroblast-conditioned medium did not form macromolecular complexes in either the absence or presence of bovine GH.

• Gargosky SE, Walton PE, Owens PC, Wallace JC, Ballard FJ
• Insulin-like growth factor-I (IGF-I) and IGF-binding proteins both decline in the rat during late pregnancy.
• J Endocrinol. 1990; 127: 383-90
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• Insulin-like growth factor-I (IGF-I), IGF-II and IGF-binding proteins (IGFBP) were examined in rat serum during pregnancy and lactation. IGF-I concentrations determined after acid column chromatography of serum were low during the last third of pregnancy. IGF-II was undetectable in pregnant and non-pregnant rats. IGF-binding protein (IGFBP) concentrations, measured as high molecular mass activity in the IGF-I RIA and the IGF-II RRA of acid column fractions, paralleled the changes observed with IGF-I. Western ligand blot analysis of serum from non-pregnant rats revealed a 40-50 kDa IGFBP aligning with IGFBP-3, a smaller 28-30 kDa doublet and 24 kDa IGFBP. Serum from rats in late pregnancy lacked IGFBP-3, whereas the smaller IGFBP persisted during late pregnancy. IGFBP-3 reappeared in postpartum animals. The fall in serum IGF-I is consistent with a maternal catabolic state during late pregnancy which may maximize substrate availability for the developing fetus.

• Thraikill KM, Clemmons DR, Busby WH Jr, Handwerger S
• Differential regulation of insulin-like growth factor binding protein secretion from human decidual cells by IGF-I, insulin, and relaxin.
• J Clin Invest. 1990; 86: 878-83
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• Several growth hormone-independent 25-31,000 kD insulin-like growth factor binding proteins (IGF-BPs) have been identified in plasma, extravascular fluids, and various cell-conditioned media. Cultured human decidual cells release three IGF-BPs with 24,000, 30,000, and 34,000 Mr. Using ligand blot analysis and an RIA for the 30,000-Mr form (IGF-BP-1), we examined the effects of IGF-I (10-1,000 ng/ml), insulin (10-10,000 ng/ml), and relaxin (10-250 ng/ml) on decidual cell IGF-BP release after 120 h of hormone exposure. IGF-I inhibited release of both IGF-BP-1 and the 24,000 Mr form. Inhibition of IGF-BP-1 release was noted after 48 h of treatment and was progressive throughout the subsequent 120 h. Insulin stimulated a fourfold increase in release of the 24,000-Mr protein while inhibiting IGF-BP-1 release comparable to IGF-I, alpha-IR3, a monoclonal antibody to the IGF-I receptor, blocked approximately 33% of the IGF-I response but had no effect on insulin-mediated IGF-BP-1 inhibition. Relaxin stimulated a 2.4-fold increase in release of the 24,000-Mr form and a 16-fold increase in the 30,000-Mr protein after 120 h. Stimulation of the 30,000-Mr protein was inhibited by the addition of cycloheximide (50 micrograms/ml). Both IGF-I and insulin also blocked the relaxin-mediated increase in IGF-BP-1. These studies suggest that three structurally related proteins differentially regulate IGF-BP secretion possibly via activation of distinct receptor subtypes.

• Ahmed SR, Manni A, Gray G, Hammond JM
• Characterization and hormonal regulation of radioimmunoassayable IGF-I (insulin-like growth factor I) like activity and IGF-binding proteins secreted by human breast cancer cells.
• Anticancer Res. 1990; 10: 1217-23
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• Insulin-like growth factor-I (IGF-I) is considered an important local mitogenic growth factor involved in autocrine/paracrine regulation of human breast cancer cell proliferation. We have characterized the IGF-I-like activity and studied its hormonal regulation by estradiol in the MCF-7 human breast cancer cell line. We found that the radioimmunoassayable IGF-I-like activity measured in conditioned medium (CM) is predominantly due to the presence of IGF-binding proteins (IGFBP). Acid chromatography demonstrated that most of the IGF-I-like activity eluted in the high molecular weight fractions and less than 10% co-eluted with authentic IGF-I (mol wt 7500). Binding protein activity measured by a 125I-IGF-I-ligand binding IGFBP-assay was present in these same high molecular weight fractions. SDS-polyacrylamide gel electrophoresis and 125I-IGF-I-ligand blot analysis of the CM showed the presence of two species of binding proteins of 29 kDa and 41 kDa molecular weight which demonstrated specific 125I-IGF-I binding activity. Estradiol did not stimulate IGFBP activity as assessed by the IGFBP-assay and as indirectly reflected by the IGF-I-like activity in the high molecular weight fractions. We conclude that the IGF-I-like activity in CM from human breast cancer cell cultures is predominantly due to the presence of IGFBP. Binding proteins of apparent molecular weight 29 kDa and 41 kDa are present in CM from MCF-7 cells. Assessment of their hormonal regulation showed that estradiol did not stimulate IGFBP. However, this needs to be assessed more stringently using better quantitative estimations for BP. The IGF-binding proteins may have an important role in the regulation of tumor cell growth by influencing the local concentrations and receptor mediated actions of IGF-I.

• Gopinath R, Walton PE, Etherton TD
• An acid-stable insulin-like growth factor (IGF)-binding protein from pig serum inhibits binding of IGF-I and IGF-II to vascular endothelial cells.
• J Endocrinol. 1989; 120: 231-6
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• The effects of a porcine insulin-like growth factor (IGF)-binding protein on binding of IGF-I and IGF-II to porcine aortic endothelial cells (PAEC) were determined. Binding of 125I-labelled IGF-I and -II to IGF receptors was inhibited by IGF-binding protein. IGF-binding protein inhibited binding of IGF-I and -II in a dose-dependent manner with half-maximal inhibition occurring at 5.43 and 108 micrograms/l respectively. A 125I-labelled IGF-I--IGF-binding protein complex, formed by incubating 125I-labelled IGF-I with IGF-binding protein overnight at 4 degrees C, did not effectively bind to endothelial IGF receptors. Addition of IGF-binding protein to PAEC previously incubated with IGF-I caused a marked dissociation of bound IGF-I (47% dissociation within 12h). These results indicate that the acid-stable IGF-binding protein which appears to be a part of the 150 kDa GH-dependent binding protein, blocks binding of IGF-I and -II by the IGF receptors and appears to exhibit a higher affinity for IGF-I than the endothelial type-I IGF receptor. The ramifications of this latter point with respect to transfer of circulating IGFs (bound to their IGF-binding proteins) across the vascular endothelium are not clear.

• Lee YL, Hintz RL, James PM, Lee PD, Shively JE, Powell DR
• Insulin-like growth factor (IGF) binding protein complementary deoxyribonucleic acid from human HEP G2 hepatoma cells: predicted protein sequence suggests an IGF binding domain different from those of the IGF-I and IGF-II receptors.
• Mol Endocrinol. 1988; 2: 404-11
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• The primary structure of an insulin-like growth factor (IGF) binding protein produced by human HEP G2 hepatoma cells has been deduced from the cDNA sequence. The 234 amino acid protein has a predicted molecular mass of 25,274 and contains a single, distinctive cysteine-rich region. The N-terminal sequence of this protein is quite similar to the limited sequence data available for a rat IGF binding protein produced by BRL-3A cells and suggests a common ancestral origin. In contrast, the HEP G2 IGF binding protein sequence bears no similarity to the N-terminal 15 amino acids of a 53 kilodalton binding protein purified from human plasma. Comparison of full-length protein sequences for the IGF-I and IGF-II receptors with that of the HEP G2 IGF binding protein also fails to demonstrate any significant similarities among these three proteins, and suggests that each contains a unique binding domain for the IGF peptides.

• Ritvos O et al.
• Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells.
• Endocrinology. 1988; 122: 2150-7
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• The placenta expresses genes for insulin-like growth factors (IGFs) and possesses IGF-receptors, suggesting that placental growth is regulated by IGFs in an autocrine manner. We have previously shown that human decidua, but not placenta, synthesizes and secretes a 34 K IGF-binding protein (34 K IGF-BP) called placental protein 12. We now used human choriocarcinoma JEG-3 cell monolayer cultures and recombinant (Thr59)IGF-I as a model to study whether the decidual 34 K IGF-BP is able to modulate the receptor binding and biological activity of IGFs in trophoblasts. JEG-3 cells, which possess type I IGF receptors, were unable to produce IGF-BPs. Purified 34 K IGF-BP specifically bound [125I]iodo-(Thr59)IGF-I. Multiplication-stimulating activity had 2.5% the potency of (Thr59)IGF-I, and insulin had no effect on the binding of [125I] iodo-(Thr59)IGF-I. 34 K IGF-BP inhibited the binding of [125I] iodo-(Thr59)IGF-I to JEG-3 monolayers in a concentration-dependent manner by forming with the tracer a soluble complex that could not bind to the cell surface as demonstrated by competitive binding and cross-linking experiments. After incubating the cell monolayers with [125I]iodo-(Thr59)IGF-I in the presence of purified binding protein, followed by cross-linking, no affinity labeled bands were seen on autoradiography. In contrast, an intensely labeled band at 40 K was detected when the incubation medium was analyzed, suggesting that (Thr59)IGF-I and 34 K IGF-BP formed a complex in a 1:1 molar ratio. Also, 34 K IGF-BP inhibited both basal and IGF-I-stimulated uptake of alpha-[3H]aminoisobutyric acid in JEG-3 cells. RNA analysis revealed that IGF-II is expressed in JEG-3 cells. We conclude that decidual 34 K IGF-BP inhibits the cellular binding and biological action of IGFs in JEG-3 cells. Our data show that JEG-3 cells represent a cell type that can produce IGF, but not IGF-BPs. These cells may thus provide a useful model system for a better understanding of autocrine growth regulation mediated by the IGFs.

• Bar RS et al.
• Production of IGF-binding proteins by vascular endothelial cells.
• Biochem Biophys Res Commun. 1987; 148: 734-9
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• Conditioned serum-free media from cultured human, bovine and rodent endothelial cells contained binding proteins with high affinity for the insulin-like growth factors (IGFs). After partial purifications on heparin or Multiplication Stimulating Activity (MSA)-affinity columns, 2 species of binding protein were identified, a major protein having Mr approximately 35,000 and a minor 22-28,000 protein. The binding proteins had greater affinity for IGF-I than IGF-II with no affinity for insulin or proinsulin. Substantial amounts of the binding proteins remained cell-associated, loosely bound to the outer cell surface of the endothelial cell. Binding protein(s) from human endothelial cells cross-reacted with antibodies to the 53,000 dalton acid-stable human serum binding protein. Production of endothelial binding proteins was not stimulated by growth hormone or insulin. We conclude that endothelial cells in culture produce large quantities of specific IGF binding proteins. Such binding proteins should be relevant in understanding the complex metabolism and function of the IGFs in the intact host.

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