Insulin and insulin-like growth factors (IGFs) stimulate production of IGF-binding proteins by ovarian granulosa cells.

Ligand blot analysis of granulosa cell (GC)-conditioned culture medium revealed several easily measurable insulin-like growth factor (IGF)-binding proteins (IGFBPs), including IGFBP-3 [40-44 kilodaltons (kDa)] and IGFBP-2 (34 kDa). In the present study, IGF-I, in a dose-dependent manner, significantly stimulated the production of these IGFBPs. Insulin, but not IGF-II, mimicked IGF-I's action on IGFBP-3 and -2 production, but was less potent. The synthetic IGF, long R3-IGF-I, which has very low affinity for IGFBPs and only slightly reduced affinity for the IGF-I (type I) receptor, had significantly greater potency in stimulating IGFBP-3 and -2 production compared to IGF-I. Des-(1-3)-IGF-I had similar effects. IGF-I, IGF-II, and the IGF-I analogs, but not insulin, also induced production of an unidentified 30-kDa IGFBP not normally detectable in these cultures. However, in the presence of epidermal growth factor (which was without independent effect on the 30-kDa IGFBP), insulin also induced this 30-kDa IGFBP. By Northern analysis the expression of IGFBP-3 mRNA was found to be significantly stimulated by IGF-I. In summary, insulin stimulated IGFBP-3 and -2 production in a manner that mimics that of IGF-I and the more potent long R3-IGF-I. However, its low potency suggested that IGFBP production is regulated via the IGF-I (type I) receptor. The much higher potency of long R3-IGF-I compared to that of IGF-I suggests that the IGFBPs themselves modulate the action of IGFs by sequestering exogenous IGFs. Thus, one cellular response to IGF stimulation is the production of IGFBPs, which, in turn, reduce or negate the biological activity of the IGFs. The effects of insulin-like peptides are exerted at least in part by increasing levels of mRNA for specific BPs.     

Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta 1 release IGF-binding protein-3 from human fibroblasts by different mechanisms.

Human neonatal fibroblasts in monolayer culture secrete insulin-like growth factor-binding proteins (IGFBPs), which may modulate IGF action. To examine whether an increase in extracellular concentrations of IGFBPs in response to IGF-I is due to the release of cell-associated IGFBPs, we measured secreted and cell-associated IGFBP-3 immunologically in fibroblast monolayers treated with IGF-I and IGF analogs with altered affinities for the IGF receptors and IGFBPs. IGFBP-3 in medium conditioned by fibroblasts treated with IGF-I was significantly increased (P < 0.05) compared with that in medium from untreated cultures; concomitantly, cell-associated IGFBP-3 was significantly decreased (P < 0.05). [Ser24]IGF-I (reduced affinity for IGF receptors) also increased secreted IGFBP-3 and decreased cell-associated IGFBP-3. In contrast, IGFBP-3 concentrations in medium conditioned by fibroblasts treated with B-chain IGF-I (reduced affinity for IGFBPs) were not significantly increased, and cell-associated IGFBP-3 was unchanged. Heparin, which releases proteins attached to cell surface proteoglycans, increased medium concentrations of IGFBP-3 and decreased IGFBP-3 binding to fibroblasts. An IGFBP of 29-31 kilodaltons (kDa) showed a pattern of regulation similar to that of IGFBP-3, while a third IGFBP, of 24 kDa, was decreased in IGF-I- and [Ser24]IGF-I-conditioned medium and unchanged by B-chain IGF-I and heparin. Preincubation with transforming growth factor-beta 1 (TGF beta 1), which stimulates fibroblast IGFBP-3 production, or human serum-derived IGFBP-3 did not increase cell-associated IGFBP-3. Analysis of total RNA isolated from fibroblasts revealed that IGFBP-3 mRNA was increased by TGF beta 1, but not by IGF-I. These data suggest that IGFs and TGF beta 1 release fibroblast IGFBPs by distinct mechanisms: IGFs by binding and subsequent release of cell-associated IGFBP-3 and 29- to 31-kDa IGFBP, and TGF beta 1 by increased de novo synthesis of IGFBP-3.     

Hormonal regulation of insulin-like growth factor (IGF)-binding proteins secreted by isolated sheep thyroid epithelial cells: relationship with iodine organification.

Isolated sheep thyroid follicles release specific insulin-like growth factor-binding proteins (IGFBPs). Since IGFBPs can modulate IGF bioactivity, at least in vitro, their presence in thyroid tissue may influence synergistic interactions between TSH and endogenous IGF-I or -II which are known to control both thyroid growth and function. We have examined the hormonal control of IGFBP release in relation to iodine organification. Sheep thyroid follicles were isolated by incubation with collagenase and differential centrifugation, grown in Coon's modified Ham's F12M medium with the addition of transferrin, glycylhistidyl-lysine, somatostatin (3H), TSH, cortisol and insulin (6H), and maintained in OH (hormone-free) or 3H medium with or without further supplements for 48 h. Conditioned culture medium was separated by 8% sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, transferred to nitrocellulose and incubated with 125I-labelled IGF-II followed by autoradiography (ligand blot). Additionally, the radioactive bands were cut from the filters and quantified by gamma-spectrometry. Iodine organification was assessed by incubation of follicles with 10(6) c.p.m. Na125I for 3 h before washing, solubilization in 0.1 mol NaOH/l and the precipitation of organified radioisotope with 10% (v/v) trichloroacetic acid. Cells conditioned in OH or 3H medium released specific IGFBPs of 46, 34, 28 and 19 kDa on ligand blot analysis. The proteins of 34 and 19 kDa were immunopositive on Western blot analysis using anti-bovine IGFBP-2 antiserum. The 46-kDa IGFBP was retained by Concanavalin A-Sepharose chromatography and demonstrated to be glycoprotein. This is probably ovine IGFBP-3. The addition of TSH, or TSH plus cortisol to OH or 3H medium significantly decreased the 125I-labelled IGF-II associated with the 34- and 28-kDa IGFBP species. All IGFBP species were substantially reduced in 6H medium, which was predominantly due to the effects of TSH and cortisol. When total 125I-labelled IGF-II associated with IGFBPs was considered, a significant (P less than 0.01) inverse correlation existed between IGFBP activity and iodine organification in the same cultures; the latter being greatest in OH or 3H medium supplemented with TSH and cortisol. None of these hormone additions altered the endogenous release of IGF-II by the cells. These results suggest that endogenous IGFs, under hormonal control, may modulate the action of endogenous IGF in the regulation of thyroid function.     

Steroid and peptide regulation of insulin-like growth factor-binding proteins secreted by human endometrial stromal cells is dependent on stromal differentiation.

Endometrial stromal cells undergo decidual transformation, in response to epidermal growth factor (EGF) and progesterone. Since insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) are believed to be involved in endometrial differentiation, and insulin regulates IGFBP production in a variety of cells, we have investigated the modulatory roles of EGF, progesterone, and insulin on IGFBP secretion by long term cultures of human endometrial stromal cells. Without insulin, the principal IGFBP secreted into conditioned medium, detected by Western ligand blotting, was a 28-kilodalton (kDa) IGFBP, identified by immunoprecipitation as IGFBP-1. This was observed only when the stromal cells were decidualized. With increasing insulin, IGFBP-1 decreased to undetectable levels. Concomitantly, IGFBP-2 increased, as did a 24-kDa IGFBP (believed to be IGFBP-4) and a 28-kDa IGFBP, shown to be a glycoprotein by endoglycosidase sensitivity (and believed to be glycosylated IGFBP-4). In the nondecidualized state, insulin increased the secretion of IGFBP-3, IGFBP-2, and the 24-kDa IGFBP, which were slightly inhibited by EGF and relatively unaffected by progesterone alone. In the absence of insulin, progesterone weakly stimulated IGFBP-1 secretion, which increased markedly when the cells were decidualized by combined treatment with EGF and progesterone. These data show that IGFBP-3, IGFBP-2, IGFBP-1, and presumably IGFBP-4 and its glycosylated form are differentially regulated by peptide and steroid hormones in endometrial stromal cells and that their regulation is a function of stromal differentiation.     

Insulin-like growth factor binding protein-3 and-5 are regulated by transforming growth factor-β and retinoic acid in the human prostate adenocarcinoma cell line PC-3

The family of insulin-like growth factor binding proteins (IGFBPs) can affect cell proliferation by modulating the availability and bioactivity of insulin-like growth factors (IGFs), or by mechanisms independent of IGFs. To understand better the role(s) of IGFBPs in prostate growth and malignancy, we examined the regulation of IGFBPs in PC-3 cells, a human prostatic adenocarcinoma epithelial cell line that is androgeninsensitive. Both transforming growth factor-β (TGF-β) and retinoic acid (RA), known inhibitors of cellular proliferation, significantly changed the IGFBP profile in PC-3 cells. In cells that were treated with transforming growth factor β-2 (TGF-β2) (0.5–10 ng/mL), IGFBP-3, and IGFBP-5 protein and mRNA increased in a time-and dose-dependent manner. At 10 ng/mL TGF-β, IGFBP-3, and IGFBP-5 protein concentrations were 14- and 9-fold, respectively, over that of controls. Cells treated with Ra (0–1 μM) also showed a time- and dose-dependent increase in IGFBP-3 protein and mRNA levels. However, in contrast to TGF-β2, high concentrations of RA (1 μM) negatively regulated IGFBP-5 expression, with IGFBP-5 mRNA levels downregulated to 20% of that of the control, and protein levels were decreased by 50%. Since both TGF-β and RA increased IGFBP-3 expression and both are known to inhibit prostate cell growth, we speculate that the inhibition of growth is mediated, at least in part, by IGFBP-3.     

Production of insulin-like growth factor binding proteins by human ovarian carcinoma cells

Cells of the human ovarian carcinoma lines EFO-21, EFO-27, MFO-35 and MFO-36 secrete binding proteins for insulin-like growth factors (IGFBPs) into their culture media. By sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and ligand blotting, seven groups of IGFBPs with molecular masses of 25, 30 (doublet), 34, 37, 40, 45, and 50 kDa were observed, depending on the cell line under investigation. By Northern blot analyses using cDNAs or oligonucleotides specific for the six types of IGFBP (IGFBP-1 to IGFBP-6), mRNA for all IGFBPs tested except for IGFBP-1 could be detected in the ovarian carcinoma cell extracts. In detail, analysis of EFO-21 protein products by SDS-PAGE yielded IGFBPs of 25, 34, and 50 kDa; extracts of EFO-21 cells contained mRNAs for IGFBP-2, -3, -4, and -6. EFO-27 cells produced IGFBPs of 40 kDa and 45 kDa as determined by SDS-PAGE, and mRNAs for IGFBP-3, -4, and -6 were detected. In the conditioned medium of MFO-35 cells, IGFBPs of 25, 30 (doublet), 34, 37, 40, and 45 kDa were observed by SDS-PAGE, while mRNAs for the five proteins IGFBP-2 to IGFBP-6 were found. MFO-36 cells produced IGFBPs of 34 kDa and 50 kDa as determined by SDS-PAGE, and the cells expressed mRNAs for IGFBP-2, -3, -4, and -6. In relation to published molecular mass data of the known IGFBPs, the size of the secreted proteins could be correlated to the mRNA patterns expressed by the ovarian carcinoma cells. It is concluded that ovarian carcinoma cells frequently express IGFBP-3, -4, and -6 and, to a lesser extent, IGFBP-2; the expression of IGFBP-5 appears as a rather rare event, while IGFBP-1 was not found to be expressed in ovarian carcinoma cells.Abbreviations BSA bovine serum albumin - FCS fetal bovine serum - PBS phosphate-buffered saline - SCC citrate-buffered saline - SFM serum-free medium - TRIS/NaCl TRIS-buffered saline Supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany (SFB 215, project Al), and by the Bundesministerium für Forschung und Technologie, Bonn, Germany (BMFT grant 01GA8715/9)     

Molecular characterization, tissue distribution patterns and nutritional regulation of IGFBP-1, -2, -3 and -5 in yellowtail, Seriola quinqueradiata

Insulin-like growth factor-binding proteins (IGFBPs) play a vital role in regulating the biological activities of IGFs. In this study, we cloned and determined full-length cDNA sequences of yellowtail IGFBP-1, -2, -3 and -5. Their tissue distribution was determined by real-time quantitative RT-PCR, which revealed that IGFBP-1, -2, -3 and -5 are widely distributed in yellowtail tissues. In yellowtail, both IGFBP-1 and –2 are highly expressed in the liver, IGFBP-3 is predominantly expressed in the heart and skin, with the lowest expression in the liver, and IGFBP-5 is highly expressed in the liver and kidneys. The widespread tissue expression of the yellowtail IGFBPs suggests that they may act in an autocrine and/or paracrine manner in the regulation of IGF activity. The effects of nutritional deprivation on yellowtail IGFBPs and IGF-I were also examined. During a 15-day starvation period, significant elevation was observed in hepatic yellowtail IGFBP-1. Refeeding restored its level to that of the control. No significant change was observed in the hepatic yellowtail IGFBP-2 mRNA levels in starved fish compared with control fish during the starvation period. Interestingly, during the early period of food deprivation, a significant increase was observed in hepatic yellowtail IGFBP-3 and -5 mRNA levels, concomitant to the significant elevation in hepatic IGF-I mRNA from day 3 to day 9. The unexpected increase in growth stimulatory IGFBPs and IGF-I during nutritional deprivation may represent a species-specific response to changes in nutritional condition.     

Production of insulin-like growth factor binding proteins (IGFBPs) by porcine granulosa cells: identification of IGFBP-2 and -3 and regulation by hormones and growth factors.

Using ligand blotting and immunoprecipitation we have characterized the insulin like growth factor binding proteins (IGFBPs) produced by cultured porcine granulosa cells. Ligand blot analysis of granulosa cell conditioned medium revealed 5 bands of IGF binding activity with apparent molecular sizes of 44, 40, 34, 29, and 22 kilodaltons (kDa). The 40-44 kDa bands of granulosa- conditioned medium were identified by immunoprecipitation with an antibody to porcine IGFBP-3, the acid-stable subunit of the 150 kDa GH-dependent serum IGFBP complex. The 34 kDa band was immunoprecipitated by an antibody to the rat IGFBP-2, the major IGFBP found in fetal rat serum and in BRL-3A cell cultures. To date we have been unable to immunoprecipitate the 29 and 22 kDa bands with any of the antibodies tested including a panel of monoclonal antibodies to human IGFBP-1, the amniotic fluid IGFBP. The pattern of secretion varied with size of the follicles from which granulosa cells were obtained and the culture conditions. With cells from small (2-4 mm) follicles, short term cultures secreted mainly IGFBP-3 and IGFBP-2, while IGFBP-2 and the 29 and 22 kDa bands were pronounced in longer term cultures. In short term culture, granulosa cells from medium sized (4-6 mm) porcine follicles produced IGFBPs in substantially greater amounts than did those from small (1-3 mm) follicles, but exhibited comparable band patterns. The production of IGFBPs was inhibited by cycloheximide. IGFBP production by granulosa cells in culture was regulated by hormones and growth factors. The most striking effects were the inhibition of IGFBP-3 secretion by transforming growth factor beta and FSH. In contrast, IGFBP-3 levels were enhanced by epidermal growth factor. The IGFBPs produced by cultured porcine granulosa cells are identical in size and immunoreactivity to those previously found in porcine follicular fluid. Thus, follicular cells may be the source of follicular fluid IGFBPs. The IGFBPs may be important modulators of the IGF autocrine/paracrine system in the ovary.     

Identification and characterization of insulin-like growth factor (IGF)-binding protein expression and secretion by adult human adrenocortical cells: differential regulation by IGFs and adrenocorticotropin

In previous studies we have shown that IGF-II stimulates basal as well as ACTH-induced cortisol secretion from adult human adrenocortical cells more potently than IGF-I, and that both IGFs predominantly stimulate androgen biosynthesis. The steroidogenic effect of IGF-I and IGF-II is mediated through interaction with the IGF-I receptor, and modified by locally produced IGF-binding proteins (IGFBPs). In the present study, we identified and characterized IGFBP synthesis in normal adult human adrenocortical cells in primary culture, and investigated the effect of ACTH and recombinant human IGF-I and -II on the regulation of IGFBP expression and secretion. Using RT-PCR, we identified the mRNA of all six high-affinity IGFBPs, in both adrenocortical tissue and monolayer cell cultures of adrenocortical cells. Using Western ligand and immunoblotting and two-dimensional Western ligand blotting we confirmed the secretion of IGFBP-1, -2, -3, -4 and -5 by adrenocortical cells in primary culture. The quantification of IGFBPs indicated that IGFBP-3 accounts for almost half the binding activity in conditioned medium of unstimulated cells (47%), followed by IGFBP-4 (20%), IGFBP-5 (15%), IGFBP-2 (12%) and IGFBP-1 (6%). After treatment with ACTH, the abundance of IGFBP-1 was upregulated significantly 2.6-fold, while IGFBP-3 was induced only slightly (1.3-fold). IGFBP-2, -4 and -5 remained unchanged. In contrast, IGF-I and -II (6.5 nM) predominantly induced the abundance of IGFBP-5 (2- and 1.6-fold respectively) and IGFBP-3 (2- and 1.7-fold respectively), while IGFBP-1, -2 and -4 were unaltered. The induction of IGFBP-1 and -5 by ACTH and IGFs, respectively, was paralleled by an increase in the amount of IGFBP-1 and -5 mRNA in these cells. In conclusion, all six high-affinity IGFBPs are expressed in the adult human adrenal gland, and the presence of at least five high-affinity IGFBPs has been demonstrated in conditioned medium of adult human adrenocortical cells. Furthermore, the expression and secretion of IGFBP-1 is upregulated by ACTH, whereas IGFBP-5 is induced by IGF-I and -II. Together with earlier findings, these results suggest that IGFBPs play an important modulatory role in the regulation of the differentiated adrenocortical function.     

Differential regulation of IGF-binding proteins in rabbit costal chondrocytes by IGF-I and dexamethasone

Cartilage is a primary target tissue for the IGFs. The mitogenic activity of these peptides is regulated by a family of high-affinity IGF-binding proteins (IGFBP-1 to -6). We characterized the IGFBPs produced by cultured chondrocytes derived from rib cartilage of prepubertal rabbits. Culture medium, which had been conditioned by these cells for 48 h showed bands of 22 kDa, 24 kDa and a 31/32 kDa doublet by Western ligand blotting with [(125)I]IGF-II. When the cells were grown in the presence of increasing amounts of IGF-I or IGF-II, the 31/32 kDa doublet increased in intensity (reaching a plateau of about 11-fold stimulation between 2 and 10 nM IGF-I). The 22 kDa and 24 kDa bands increased only slightly while a 26 kDa band became faintly visible. By Western immunoblotting the 31/32 kDa doublet was identified as IGFBP-5. An IGF-I analog with reduced affinity for IGFBPs, Long-R3 IGF-I, also induced IGFBP-5, while insulin was less effective (2.2-fold stimulation at 10 nM). IGF-I protected IGFBP-5 against proteolytic degradation by conditioned medium. IGF-I also enhanced the level of IGFBP-5 mRNA. LY294002, a specific inhibitor of the intracellular signaling molecule phosphatidylinositol 3-kinase, inhibited stimulation of IGFBP-5 by IGF-I. Dexamethasone suppressed IGFBP-5 (by 70% at 20 nM) but, at the same time, a 39/41 kDa doublet (presumably IGFBP-3) was induced. IGFBP-5 has been shown in several cell types to enhance the mitogenic activity of IGF-I. IGFBP-3 generally acts as a growth inhibitor. Therefore, the differential effects of dexamethasone on these regulatory proteins could account, at least in part, for the growth-arresting effect of this glucocorticoid.     

The role of the insulin-like growth factors and their binding proteins in glucose homeostasis

The insulin like growth factors (IGF-I and -II) are structurally and functionally related to insulin. While insulin is a key regulator of glucose homeostasis over the short term, emerging evidence suggests that the IGFs are involved in the longer term glucose homeostasis, possibly by modulating insulin sensitivity. Unlike insulin, the IGFs are present in most biological fluids as complexes with high affinity binding proteins, the insulin-like growth factor binding proteins (IGFBPs). The IGFBPs regulate the bioavailability of the IGFs. Of the six IGFBPs identified there is evidence from studies in transgenic mice that both IGFBP-1 and IGFBP-3 may have a role in glucose regulation.     

Basal and regulated secretion of insulin-like growth factor binding proteins in osteoblast-like cells is cell line specific.

Local secretion of insulin-like growth factor binding proteins (IGFBPs) may modulate the effects of IGF-I and IGF-II on bone cell metabolism and proliferation. Several osteoblast-derived cell lines are currently used as interchangeable models to study IGFBP production, although it is unknown whether findings in one cell line can be extrapolated to another cell line or to normal human osteoblasts. In this study, we examined by Western ligand blotting both basal and regulated secretion of IGFBPs in vitro in 1) normal human osteoblast-like (hOB) cells cultured from explants of human trabecular bone; 2) an SV40-transformed hOB (HOBIT) cell line; and 3) several human (U-2, MG-63, TE-85) and rat (ROS 17/2.8 and UMR-106-01) osteosarcoma cell lines. Constitutively, hOB and HOBIT cells produced a similar pattern of IGFBPs, while all other cell lines produced their own unique pattern of IGFBPs. The two rat cell lines differed from the human cell lines as well as from each other. The response to hormonal stimulation also varied among the cell lines. Treatment of hOB and HOBIT cells with IGF-I resulted in a 2-fold increase in medium levels of IGFBP-3; IGF-I decreased levels of 24-kilodalton (kDa) IGFBP in hOB cell-conditioned medium. In addition, IGF-I markedly increased levels of the 29/32/34 kDa IGFBP triplet in U-2 cells, but had little or no effect in the other human and rat osteosarcoma cell lines. PTH increased a 29-kDa IGFBP apparent only in UMR 106-01 cell-conditioned medium, whereas GH had no direct effect on IGFBP secretion in any of the osteoblast-like cells tested. We conclude that basal and regulated secretion of IGFBPs from osteoblast-like cells is cell-line specific. Spontaneously transformed human or rat osteoblast-like cells provide unique model systems to study features of distinct IGFBPs and their regulation; however, hOB cells and their derivatives may be more appropriate models for understanding the regulation of IGFs in human bone.     

Characterization of insulin-like growth factor-binding proteins from sheep thyroid cells

The insulin-like growth factors (IGFs) are bound by specific, high affinity binding proteins. Distinct classes of IGF-binding proteins have been described in human serum, amniotic fluid, cerebrospinal fluid, and conditioned medium from cultured cells. Sheep thyroid cells produce IGF-binding proteins under hormonal regulation. Cells grown without or with standard medium supplements (transferrin, glycyl-histidyl-lysine, hydrocortisone, somatostatin, insulin, and TSH) released binding proteins with apparent mol wt of 23, 29, and 32 kDa on Western ligand blot (nonreduced). Binding proteins from these cells appeared as 21, 26, 34, 36, and 41 kDa bands when cross-linked to [125I]IGF-I under reducing conditions. The addition of epidermal growth factor (EGF) or phorbol esters, thyroid cell mitogens stimulated the production of larger binding proteins with mol wt of 40-44 and 48-52 by ligand blot and cross-linking methods, respectively. Deglycosylation of conditioned medium cross-linked to [125I]IGF-I with endoglycosidase-F did not alter the size of the smaller binding proteins, but reduced EGF-stimulated binding proteins to 36-40 kDa. Similarly, tunicamycin treatment, which inhibits glycosylation, reduced only the size of this larger binding protein species. Polyclonal antisera directed against the human amniotic fluid binding protein (BP-28) immunoprecipitated the 32 kDa sheep thyroid binding protein seen on ligand blot and the cross-linked binding protein at 36-38 kDa. Antibody against the major human serum binding protein (BP-53) recognized only the larger EGF-stimulated binding proteins. In contrast to sheep thyroid cells, rat FRTL5 thyroid cells produced no detectable IGF-binding proteins. We conclude that the predominant binding proteins produced by sheep thyroid cells under standard culture conditions are non-glycosylated and immunoreact with antiserum directed against BP-28. EGF and phorbol esters stimulate production of larger glycosylated binding proteins antigenically related to BP-53.     

Metabolic hormones regulate insulin-like growth factor binding protein-1 mRNA levels in primary cultured salmon hepatocytes; lack of inhibition by insulin

IGF-binding proteins (IGFBPs) modulate the effects of the IGFs, major stimulators of vertebrate growth and development. In mammals, IGFBP-1 inhibits the actions of IGF-I. Rapid increases in circulating IGFBP-1 occur during catabolic states. Insulin and glucocorticoids are the primary regulators of circulating IGFBP-1 in mammals. Insulin inhibits and glucocorticoids stimulate hepatocyte IGFBP-1 gene expression and production. A 22 kDa IGFBP in salmon blood also increases during catabolic states and has recently been identified as an IGFBP-1 homolog. We examined the hormonal regulation of salmon IGFBP-1 mRNA levels and protein secretion in primary cultured salmon hepatocytes. The glucocorticoid agonist dexamethasone progressively increased hepatocyte IGFBP-1 mRNA levels (eightfold) and medium IGFBP-1 immunoreactivity over concentrations comparable with stressed circulating cortisol levels (10(-9) -10(-6) M). GH progressively reduced IGFBP-1 mRNA levels (0.3-fold) and medium IGFBP-1 immunoreactivity over physiological concentrations (5 x 10(-11)-5 x 10(-9) M). Unexpectedly, insulin slightly increased hepatocyte IGFBP-1 mRNA (1.4-fold) and did not change medium IGFBP-1 immunoreactivity over physiological concentrations and above (10(-9) -10(-6) M). Triiodothyronine had no effect on hepatocyte IGFBP-1 mRNA, whereas glucagon increased IGFBP-1 mRNA (2.2-fold) at supraphysiological concentrations (10(-6) M). This study suggests that the major inhibitory role of insulin in the regulation of liver IGFBP-1 production in mammals is not found in salmon. However, regulation of salmon liver IGFBP-1 production by other metabolic hormones is similar to what is found in mammals.     

Triiodothyronine (T3) stimulates insulin-like growth factor (IGF)-1 and IGF binding protein (IGFBP)-2 production by rat osteoblasts in vitro.

Osteoblast-like cells prepared from neonatal rat calvariae and grown under serum-free conditions produce IGF-1 and IGFBPs. In contrast to growth hormone, T3 and PTH increased both IGF-1 mRNA expression and net IGF-1 release in calvaria cells. In addition, they stimulated net production of IGFBP-3 and of an IGFBP with an apparent molecular weight of 32 kDa which was recognized by an antiserum against rat IGFBP-2. Bone cells expressed remarkably high levels of mRNA for IGFBP-2, the predominant IGFBP in serum of newborn rats. T3 at low physiological concentrations but not growth hormone stimulated IGFBP-2 mRNA expression and IGFBP-2 production in bone cells in vitro. Thus, IGFBPs are differentially regulated by these hormones and may play an autocrine/paracrine regulatory role in bone.     

1,25-Dihydroxyvitamin D3 stimulates the production of insulin-like growth factor-binding proteins-2, -3 and -4 in human bone marrow stromal cells

BACKGROUND: 1,25-Dihydroxyvitamin D3 (calcitriol) inhibits proliferation and stimulates differentiation of multiple cell types, including osteoblasts. Human (h) bone marrow stromal cells (MSCs) are a homogenous non-hematopoietic population of cells present in the bone marrow and exhibit a less differentiated osteoblastic phenotype. The IGF system, including IGFs-I, and -II and IGF binding proteins (IGFBPs), plays an important role in osteoblast cell proliferation and differentiation. OBJECTIVE: To examine the pattern of expression of the IGF system in hMSCss and its regulation by calcitriol. METHODS AND RESULTS: hMSCs express mRNA of both IGFs-I, and -II and IGFBPs-1 to -6 as shown by RT-PCR and northern blot analysis. As assessed by western ligand blotting (WLB) and western immmunoblot analysis, hMSCs secrete 38-42 kDa IGFBP-3, 24-28 kDa IGFBP-4 and a 33 kDa IGFBP-2. Calcitriol (dose range 10-10 mol/l) exerted no consistent dose-dependent effects on either IGF-I or IGF-II mRNA levels. In contrast, calcitriol treatment increased steady-state mRNA levels of IGFBPs-2, -3 and -4, but had no effect on IGFBP-5 or -6. Similarly, calcitriol increased the secretion of IGFBPs-2, -3 and -4 as determined by WLB. We found no detectable basal IGFBP-3 or IGFBP-4 protease activities in the absence or presence of calcitriol treatment. CONCLUSIONS: Our results demonstrate that hMSCs expressed a distinct pattern of IGFs and IGFBPs that may be related to their stage of differentiation. The observed increase in production of IGFBPs-2, -3 and -4 by hMSCs upon treatment with calcitriol may be an important mechanism mediating the effects of calcitriol on MSC proliferation and differentiation.     

Basic fibroblast growth factor induces proteolysis of secreted and cell membrane-associated insulin-like growth factor binding protein-2 in human neuroblastoma cells.

Insulin-like growth factor (IGF) action in the brain is modulated by IGF-binding proteins (IGFBPs) whose abundance can be altered by other locally expressed growth factors. However, the mechanisms involved are unclear. We here employed the neuroblastoma cell line SK-N-MC as a model to define the mechanisms involved in modulation of IGFBPs in neuronal cells. Western ligand blotting analysis and immunoprecipitation of conditioned media (CM) from SK-N-MC cells showed that in these cells, as in the brain, the most abundantly expressed IGFBP was IGFBP-2. However, IGFBP-2 was barely detectable in CM from cells treated with basic fibroblast growth factor (bFGF) without a change in IGFBP-2 messenger RNA (mRNA) abundance. These CM contained specific IGFBP-2 proteolytic activity, resulting in two IGFBP-2 fragments of 14 and 22 kDa. The activity was inhibited by EDTA/phenylmethylsulfonyl fluoride or aprotinin. Competitive binding studies indicated that IGFBP-2 fragments had reduced binding affinity for IGF-I. bFGF induced IGFBP-3 mRNA and protein. Affinity cross-linking of [125I]IGF-I to neuroblastoma cell membranes followed by immunoprecipitation revealed a approximately 38 kDa [125I]IGF-I/IGFBP-2 complex. Cell surface-associated IGFBP-2 was also susceptible to bFGF-induced proteolysis, with the appearance of a single cross-linked 21-kDa complex with low affinity for IGF-I. These findings indicate that intact IGFBP-2 and the 14-kDa, but not the 22-kDa fragment, bind to the cell surface. Our data suggest that induction of IGFBP-2 proteolysis on neuronal cell surface is a novel mechanism whereby IGF availability is modulated by the local growth factor bFGF.