Production Of IGF-binding Proteins Research Articles

Altered growth and insulin-like growth factor-binding protein-3 production in PC3 prostate carcinoma cells stably transfected with a constitutively active androgen receptor complementary deoxyribonucleic acid [published erratum appears in Endocrinology 1995 May;136(5):2319

Altered growth and insulin-like growth factor-binding protein-3 production in PC3 prostate carcinoma cells stably transfected with a constitutively active androgen receptor complementary deoxyribonucleic acid [published erratum appears in Endocrinology 1995 May;136(5):2319

Insulin-like growth factor-binding protein-3 production by MCF-7 breast cancer cells: stimulation by retinoic acid and cyclic adenosine monophosphate and differential effects of estradiol.

The insulin-like growth factors (IGFs) stimulate the proliferation of human breast cancer cells, including the estrogen-dependent cell line MCF-7. These cells secrete regulatory IGF-binding proteins (IGFBPs) which may enhance or attenuate IGF-stimulated cell proliferation. In this study, we have used RIA to quantify the production and regulation of IGFBP-3 and IGFBP-6 by MCF-7 cells in vitro. Under basal (serum- and phenol red-free) conditions, IGFBP-3 and IGFBP-6 accumulated in 72 h-conditioned MCF-7 medium to concentrations of approximately 0.18 nM and 0.02 nM, respectively. Treatment with retinoic acid (RA, 100 nM) increased medium concentrations of IGFBP-3 to 175 +/- 8% (mean +/- SE, n = 4), and IGFBP-6 to 217 +/- 20% of control values. Forskolin (0.5 microM) or dibutyryl cAMP (db-cAMP, 1 mM) increased both proteins 2- to 3-fold. In the presence of 100 nM RA, the stimulation elicited by these agents was enhanced, with IGFBP-3 levels increasing to 6-fold above that seen with RA alone. IGFBP-6 increased 12-fold with RA + forskolin and 20-fold with RA + dbcAMP. Estrogen (10 nM estradiol) reduced basal IGFBP-3 levels by 25% but increased IGFBP-6 1.5- to 2-fold. The stimulatory effect of RA + forskolin on IGFBP-3 was partially reversed by estrogen, whereas RA + forskolin-stimulated IGFBP-6 levels were further increased by estrogen. Increased IGFBP-3 and -6 production in response to RA + forskolin was accompanied by a decrease in IGF-stimulated thymidine incorporation into DNA; by contrast, the bioactivity of an IGF analog that does not bind with IGFBPs, [Gln3, Ala4, Tyr15, Leu16]IGF-I, was unchanged under these conditions. These data demonstrate that modulating the production of IGFBPs can lead to changes in the sensitivity of breast cancer cells to IGFs, and as a result change the cell proliferative effects of these growth factors. Further, IGFBP-3 and IGFBP-6 are differentially regulated by estrogen. Dissecting the roles of the individual IGFBPs is essential to understanding how such differential regulation will ultimately affect IGF-stimulated cell proliferation in breast cancer.

Insulin-like growth factor-binding protein-3 production by MCF-7 breast cancer cells: stimulation by retinoic acid and cyclic adenosine monophosphate and differential effects of estradiol

Insulin-like growth factor-binding protein-3 production by MCF-7 breast cancer cells: stimulation by retinoic acid and cyclic adenosine monophosphate and differential effects of estradiol

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.

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.

Does an IGF-binding protein (IGFBP) present in involuting rat mammary gland regulate apoptosis?

We examined the effects of GH and prolactin deficiency upon milk production, apoptosis and IGFBP production by the mammary gland. GH deficiency produced a 15% reduction in milk yield, prolactin a 50% reduction and combined prolactin- and GH-deficiency an 85% reduction in milk production. Litter removal led to complete inhibition of milk synthesis within 24 h owing in large part to milk accumulation. GH- and prolactin-deficiency also led to significant loss of mammary cells within 48 h and this was owing at least in part to apoptosis as judged by the appearance of characteristic DNA ladders. Prolactin replacement therapy could prevent all of these changes whilst GH was partially effective. The effects of GH are believed to be mediated via IGF-I, however, we were unable to mimic the effects of GH with IGF-I, IGF-II or a combination of IGF-I, IGF-II and IGFBP-3. We hypothesized that the cell-survival effects of exogenous IGFs might be blocked by an inhibitory IGFBP produced by the gland. Indeed the involuting mammary gland produces large concentrations of an IGFBP which Northern blotting identified as IGFBP-5. There was also a small increase in IGFBP-4 mRNA expression. Both appear to be produced by the secretory epithelial cells as judged by in situ hybridization. Preliminary studies using mouse “mammosphere” cultures suggest that they will be useful for investigating a potential causal relationship between IGFBP-5 synthesis and apoptosis.

Regulation of Insulin-Like Growth Factor Binding Protein-I During the 24-Hour Metabolic Clock and in Response to Hypoinsulinemia Induced by Fasting and Sandostatin in Normal Women

To establish the relation of insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (IGFBP-1) with 24-hour metabolic excursions in normal healthy women and in response to acute interruption of metabolic homeostasis by hypoinsulinemia. Hourly blood samples during the 24-hour metabolic clock were obtained from seven normally cycling women. Uniform dietary composition (50% carbohydrate, 35% fat, and 15% protein) and timing of meals (8 AM, 12 PM, and 6 PM) were prescribed. Daytime hypoinsulinemia was induced by omitting meals and by Sandostatin (100 micrograms) administration. Changes in serum levels of glucose, insulin, cortisol, IGF-I, and IGFBP-1 were measured. The diurnal pattern of serum IGFBP-1 levels during the 24-hour metabolic clock was characterized by a rapid fall during the feeding phase of the day and a progressive 3.5-fold rise during nocturnal fasting; IGF-I levels were unchanged. Changes in IGFBP-1 levels were in parallel to those of cortisol and were inversely related to increases in glucose (80%) and insulin (tenfold) levels after each meal and to their decline during nocturnal fasting. Daytime fasting and administration of Sandostatin were accompanied by rapid and sustained increases in IGFBP-1 when insulin levels declined to 54 +/- 20 pmol/L. With constant levels of IGF-I, the diurnal rhythm of IGFBP-1 may subserve a physiologic function by coordinating insulin and IGF-I action with substrate availability. Fluctuations of insulin levels during the 24-hour metabolic clock in normal women appear to serve as a signal, with an inhibitory effect on IGFBP-1 production when levels are above 70 pmol/L and a stimulatory effect at levels below 70 pmol/L. These findings provide a basis for future investigations in women with nutritionally related reproductive disorders.

Production of insulin-like growth factors and their binding proteins in primary cultures of rat liver parenchymal and nonparenchymal cells.

Regulation of the production of insulin-like growth factor (IGF)-I, IGF-II, IGF binding proteins (IGFBPs), and their related proteins by various hormones was investigated in primary cultures of rat liver parenchymal and nonparenchymal cells. Freshly isolated parenchymal cells contained mRNAs of IGF-I, IGF-II, IGFBP-1, IGFBP-4, growth hormone (GH) receptor, and the acid-labile subunit (ALS), which forms a ternary complex with IGF-I and IGFBP-3; however, parenchymal cells did not express the IGFBP-3 gene. In contrast, nonparenchymal cells contained IGFBP-3 mRNA exclusively, as we reported previously [Takenaka et al. Agric. Biol. Chem., 55, 1191-1193 (1991)]. Cultured rat parenchymal cells produced IGF-I, IGFBP-1, and IGFBP-4 prominently. In these cells, secretion of IGF-I and the content of IGF-I mRNA was greatly increased in the presence of GH in the medium. Insulin also increased the production of IGF-I. Secretion of IGFBP-1 into the medium was enhanced by treatment with glucagon, dibutyrylcyclic AMP (Bu2cAMP), and dexamethasone (Dex) and these enhancements with glucagon and Dex reflected the increase in its mRNA content. Insulin depressed the secretion of IGFBP-1. The content of IGFBP-4 in the parenchymal cells was increased by insulin, Bu2cAMP, and triiodothyronine (T3), thereby enhancing the production of IGFBP-4 and secretion into the medium. Cultured liver nonparenchymal cells of rats produced IGFBP-1, IGFBP-3, and IGFBP-4. Secretion of IGFBP-1 was increased by Bu2cAMP in the medium, that of IGFBP-3 by IGF-I, and that of IGFBP-4 by both IGF-I and Bu2cAMP. Regulation of the production of IGFBP-3 by IGF-I was demonstrated in these investigations. These results suggest that GH increases production of IGF-I in the parenchymal cells and this IGF-I, in turn, increases the production of IGFBP-3 in nonparenchymal cells. As we found GH also increases ALS production in parenchymal cells, by these mechanisms, GH increases the formation of the ternary complex of IGF-I, IGFBP-3, and ALS. This study clearly demonstrates the interrelationship between parenchymal and nonparenchymal cells in the production of IGF-I and IGFBPs in the liver.

A novel DNA/protein complex interacts with the insulin-like growth factor binding protein-1 (IGFBP-1) insulin response sequence and is required for maximal effects of insulin and glucocorticoids on promoter function

Glucocorticoids stimulate and insulin inhibits hepatic production of IGFBP-1 at the level of gene transcription. We previously identified contiguous insulin and glucocorticoid response sequences in the proximal rat IGFBP-1 promoter. This insulin response sequence (IRS) is palindromic ( CAAAACAAA TTATTTTG) and each half resembles an IRS in the phosphoenolpyruvate carboxykinase (PEPCK) gene. We have reported that both the IGFBP-1 and PEPCK IRSs bind hepatocyte nuclear factor-3 (HNF-3) proteins [1]. We now report that IRSs from the IGFBP-1 and PEPCK, as well as an IRS which also binds HNF-3 in the rat tyrosine aminotransferase (TAT) gene, also interact with another DNA/protein complex in gel shift studies. Further, methylation interferences studies, gel shift and transient transfection studies with site-specific mutations identified a single base in the first half of the IRS that is critical both for interactions with proteins in this complex, and for maximal effects of insulin and glucocorticoids, on promoter function. Of note, a 250-fold excess of an oligo containing a C/EBP binding site (but not other AT-rich sequences) inhibits the formation of this complex in gel shift assays. Nevertheless, interactions with this C/EBP site are negligible at lower titers (≤ 100-fold excess), and antibodies against known C/EBP proteins do not react with this complex. Similarly, preincubation with CHOP, a truncated member of the C/EBP family which contains a β-leucine zipper domain, does not prevent or alter the mobility of this novel DNA/protein complex, indicating that components of this complex do not form heterodimers with β-ZIP proteins. We conclude that HNF-3 proteins and this novel C/EBP-related DNA/protein complex may play an important role in mediating interactions between glucocorticoids and insulin in the regulation of IGFBP-1 and perhaps multiple hepatic genes.

Interactions between insulin-like growth factor-I (IGF-I) and the system of plasminogen activators and their inhibitors in the control of IGF-binding protein-3 production and proteolysis in human osteosarcoma cells

Limited proteolysis in vivo of insulin-like growth factor-binding protein-3 (IGFBP-3) by as yet unidentified serine proteases plays a key role in controlling the bioavailability of IGFBP-3-associated insulin-like growth factors (IGFs). Both the IGF system and the system of plasminogen activators (PAs) and their inhibitors (PAIs) are involved in bone remodeling, and plasmin has been shown to provoke dissociation of IGFBP-IGF complexes in cultured MG-63 human osteoblasts. The aim of this work was to investigate interactions between IGF-I and the PA/PAI system and their influence on IGFBP-3 production and proteolysis in this cell model. At confluency, MG-63 cells maintained for 3 days in serum-free medium constitutively secreted IGFBP-2 and small amounts of IGFBP-3 and IGFBP-4. As shown by Western ligand and immunoblot analyses of the culture medium and Northern blot analysis of IGFBP-3 messenger RNA, production of these IGFBPs, and of IGFBP-3 in particular, was dose dependently stimulated by the addition of 12.5-100 ng/ml recombinant human (rh) IGF-I. Increasing concentrations of plasminogen (0.05-3.5 micrograms/ml) added during the final 12 h of culture reduced the amounts of IGFBP detectable by Western ligand blotting, especially IGFBP-3. This reduction reflected proteolysis, as shown by immunoblotting, which revealed 30-, 20-, and 16-kilodalton fragments of IGFBP-3. In the presence of 25 ng/ml IGF-I, which stimulated IGFBP-3 production, proteolysis was reduced by approximately half. Incubation of glycosylated [125I]rh-IGFBP-3 as substrate in cell-free conditioned medium gave the same results. Addition of 50 ng/ml rhIGF-I to conditioned medium (to promote IGFBP-3-rhIGF-I complex formation) failed to diminish plasmin-induced proteolysis of IGFBP-3. Urokinase PA activity in the conditioned medium decreased significantly when the cells were cultured with rhIGF-I, indicating a direct action of IGF-I on urokinase PA and/or PAI production. Our results support the notion of a regulation loop whereby IGF-I controls its own bioavailability via its action on both IGFBP-3 production and the PA/PAI system, which regulates IGFBP-3 proteolysis. The proteolytic cleavages of IGFBP-3 caused by plasmin were the same as those caused in vivo by serine protease acting on this IGFBP.

Profile of IGF-binding proteins secreted by intestinal epithelial cells changes with differentiation.

Previous reports have shown that gastrointestinal epithelial cells produce insulin-like growth factor-binding proteins (IGF-BP), which modulate the actions of IGF. This study aims to examine the relationship between differentiation and IGF-BP secretion by human intestinal epithelial cells and the effect of growth factors on their production. Caco-2 cells were cultured in serum-free media. IGF-BP secretion into the incubation media was analyzed by Western ligand blotting and immunoblotting. Caco-2 cells produced IGF-BP-2, IGF-BP-3, and IGF-BP-4. Secretion of IGF-BP-2 and IGF-BP-3 increased with differentiation, but IGF-BP-4 secretion diminished. The effect of exogenous growth factors on IGF-BP secretion was maximal at earlier stages of differentiation. IGF-I stimulated mainly IGF-BP-3 production, but epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) stimulated predominantly IGF-BP-4 secretion. Adding an anti-EGF receptor antibody to block autocrine TGF-alpha activity inhibited IGF-BP-4 production but stimulated IGF-BP-2 and IGF-BP-3. In conclusion, the profile of IGF-BP secretion changes with differentiation. IGF-I and EGF (or TGF-alpha) stimulate different types of IGF-BP, with autocrine TGF-alpha activity being a factor affecting IGF-BP production during differentiation.

Localization of messenger ribonucleic acid for insulin-like growth factor-binding proteins in human skin by in situ hybridization.

The role of the insulin-like growth factors (IGFs) in human skin physiology has been increasingly recognized, although relatively little is known about the cell types involved or the cellular mechanisms that mediate these responses. Epidermal keratinocytes and dermal fibroblasts both possess IGF-I receptors and are responsive to IGF-I. IGF-binding proteins (IGFBPs), known modulators of IGF action, may also be responsible for targeting IGF-I to its receptors and are produced by both cultured keratinocytes and fibroblasts. To demonstrate sites of production of IGFBPs in human skin, we have used in situ hybridization to localize messenger ribonucleic acid (mRNA) for the six IGFBPs. Antisense and sense RNA probes for the IGFBPs (IGFBP-1 to -6) were produced, and 5-microns sections of normal adult human male chest skin were probed. The control probe used was keratin-5, which is known to hybridize to the basal keratinocytes of the epidermis. mRNAs for human IGFBP-2, -3, -4, and -5 were identified, with mRNAs for IGFBP-2 and IGFBP-4 localized in sebaceous glands and eccrine sweat glands (epidermal origin), IGFBP-3 mRNA in the basal layer of the epidermis and mRNAs for IGFBP-4, and IGFBP-5 found throughout the dermis. mRNAs for IGFBP-1 and -6 were not identified in human skin. These studies demonstrate specific localization of IGFBP mRNAs in adult human skin, suggesting that each IGFBP may play a specific role in targeting IGF-I to its receptor on responsive cells and, ultimately, in modulation of IGF-I action in skin.

Effect of insulin on the hepatic production of insulin-like growth factor-binding protein-1 (IGFBP-1), IGFBP-3, and IGF-I in insulin-dependent diabetes.

Insulin-like growth factors (IGFs) circulate attached to binding proteins (IGFBPs). Only the unbound form of IGF is suggested to be biological active. The main source of circulating IGF-I and IGFBP-1 is considered to be the liver, but that of circulating IGFBP-3 is not known. IGF-I and IGFBP-3 are GH dependent, whereas IGFBP-1 is insulin regulated. The aim of the present study was to examine the effect of insulin on the hepatic secretion of IGFBP-1, IGFBP-3, and IGF-I. Seven insulin-dependent diabetic patients in whom insulin was withheld for 12 h were studied in the overnight fasted state. Blood was sampled simultaneously from the hepatic vein, a peripheral vein, and an artery before and during insulin infusion for 3 h. The basal IGFBP-1 levels in the peripheral vein were several-fold elevated (249 +/- 44 micrograms/L) compared to those in healthy subjects (37 +/- 2 micrograms/L). Fasting IGFBP-1 concentrations were inversely correlated to the insulin levels (r = -0.918; P < 0.001). The mean IGF-I concentration (175 +/- 17 micrograms/L; -1.62 +/- 0.38 SD score) was decreased compared with that in age-matched healthy subjects. The basal IGFBP-3 levels in the peripheral vein (4.50 +/- 0.33 mg/L) were within the normal range. There was a significant correlation in the hepatic vein between fasting IGF-I and IGFBP-3 levels (r = 0.928; P < 0.001). Basal splanchnic IGFBP-1 production was 18 +/- 7 micrograms/min, whereas no basal net exchanges of IGF-I or IGFBP-3 were observed across the splanchnic area. Insulin inhibited splanchnic IGFBP-1 production within 120 min and glucose output within 20 min. Serum IGF-I, but not IGFBP-3, concentrations increased significantly during the insulin infusion. In summary, this study demonstrates the existence of considerable IGFBP-1 production from the liver during insulinopenia and the complete blocking of splanchnic IGFBP-1 production and increases in serum levels of IGF-I by insulin despite no effect on IGFBP-3 levels. Thus, insulin may play a role in determining the bioavailability of IGF-I.

Pancreatic hormones differentially regulate insulin-like growth factor (IGF)-I and IGF-binding protein production by primary rat hepatocytes.

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.

Porcine ovarian granulosa cells secrete insulin-like growth factor-binding proteins-4 and -5 and express their messenger ribonucleic acids: regulation by follicle-stimulating hormone and insulin-like growth factor-1.

Using ligand blotting, Western immunoblotting, and Northern analysis, we have characterized the insulin-like growth factor (IGF)-binding proteins (IGFBPs) produced by cultures of porcine granulosa cells. Ligand blot analysis of conditioned medium from untreated cultures of moderately differentiated granulosa cells (MDGCs; from 4-6-mm follicles) revealed mainly IGF-binding activity associated with IGFBP-2 (34 kDa) and IGFBP-3 (40/44-kDa doublet), which have previously been identified and characterized. In addition, these cultures secreted 30- and 22-kDa forms under some circumstances. The identification and regulation of these IGFBPs of lower molecular mass were the focus of the current studies. Treatment of these MDGCs with IGF-I dramatically stimulated the production (to a detectable level) of a 30-kDa IGFBP that was identified by immunoblotting with antiserum to IGFBP-5 but not antisera to IGFBP-1, -2, -3, -4, or -6. Production of IGFBP-5 was attenuated by concurrent treatment with FSH. IGFBP-5 mRNA in these cultures was correspondingly stimulated by IGF-I but unaffected by FSH. FSH increased the level of a minor 22-kDa IGFBP. Messenger RNAs for IGFBP-1, -4, and -6 were also examined but only IGFBP-4 mRNA was detectable, suggesting that the 22-kDa band was IGFBP-4. These results were compared to those in cultures of immature granulosa cells from 1-3-mm follicles, in which 22- and 30-kDa IGFBPs were readily detectable. An antiserum to IGFBP-4 precipitated the 22- and 30-kDa bands whereas deglycosylation shifted the 30-kDa IGFBP to 22 kDa, suggesting that both these bands represent glycosylation variants of IGFBP-4.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibroblast Growth Factor Regulates Insulin-like Growth Factor-Binding Protein Production by Vascular Smooth Muscle Cells

Insulin-like growth factor I is an important mitogen for vascular smooth muscle cells, and its effects are regulated by several binding proteins. Western ligand blotting of conditioned medium from rat aortic smooth muscle cells detected a 24 kDa binding protein and a 28 kDa glycosylated variant of this protein, consistent with insulin-like growth factor binding protein-4 by size. Low amounts of a glycosylated 38 to 42 kDa doublet (consistent with binding protein-3) and a 31 kDa non-glycosylated protein also were present. Basic fibroblast growth factor markedly increased secretion of the 24 kDa binding protein and its 28 kDa glycosylated variant. This effect was dose- and time-dependent and was inhibited by co-incubation with cycloheximide. Crosslinking of [125I]-insulin-like growth factor I to cell monolayers revealed no surface-associated binding proteins, either basally or after agonist treatment. Induction of binding protein production by fibroblast growth factor at sites of vascular injury may be important in vascular proliferative responses in vivo.

Effect of growth hormone on steroidogenesis, insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 production and DNA synthesis in cultured human luteinized granulosa cells.

Numerous clinical and experimental observations have suggested that GH is important in ovarian function. We have investigated the effect of GH alone and GH in combination with FSH on the secretion of oestradiol, progesterone, insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (IGFBP-1) and on [3H]thymidine incorporation in cultured human luteinized granulosa cells. Granulosa cells from patients undergoing treatment for in vitro fertilization were isolated and cultured for 2 days in culture medium with 10% serum. After this preincubation, the medium was removed and the cells were incubated with GH (1, 10 and 100 micrograms/l) with or without FSH in serum-free medium and in the presence of [3H]methylthymidine (2 microCi/ml). GH alone resulted in a significant dose-dependent increase of oestradiol (P < 0.05) and in IGFBP-1 (P < 0.002) in the medium. The release of IGF-I was undetectable and there was no increase in [3H]thymidine incorporation with GH alone. Neither GH nor FSH alone stimulated granulosa cell proliferation or progesterone release, while the combination induced increases (P < 0.001) in both. The stimulatory effect of GH on steroidogenesis, IGFBP-1 production and granulosa cell proliferation supports a putative role for GH in the regulation of ovarian function.

Regulation of insulin-like growth factor binding protein synthesis and secretion in human retinal pigment epithelial cells.

Cultured human retinal pigment epithelial cells (RPE) secrete insulin-like growth factor binding proteins (IGFBPs), a family of polypeptides which modulate the actions of the insulin-like growth factors. RPE cells secrete two IGFBPs with Mr estimates of 34,000 and 46,000, respectively. Treatment of RPE cells with IGF-I markedly stimulated the secretion of the 46,000 Mr form. This stimulation occurred via an IGF-I receptor independent mechanism because both [QAYL]IGF-I (an IGF-I analogue with decreased affinity for the IGFBPs but normal affinity for the IGF-I receptor) and alpha-IR3 (a blocking monoclonal antibody against the IGF-I receptor) had no effect on IGF-I stimulated increases in IGFBPs. Additionally, [QAYL]IGF-I enhanced RPE cell proliferation to the same magnitude as IGF-I. Treatment with IGF-I, [QAYL]IGF-I, or alpha-IR3 had no effect on steady-state levels of the 2.5 kb IGFBP-3 or the 1.3 kb IGFBP-6 mRNA transcripts as measured by Northern blotting and quantitative autoradiography. Forskolin and a group of candidate growth factors, including platelet-derived growth factor, epidermal growth factor, and acidic and basic fibroblast growth factor, modestly increased IGFBP secretion when compared to untreated cells, but these effects were small when compared to IGF-I treatment. Fetal calf serum enhanced the presence of the 2.5 kb IGFBP-3 mRNA transcript in a dose-dependent fashion but had no effect on the 1.3 kb IGFBP-6 mRNA transcript. IGF-I, forskolin, and the candidate growth factors had no effect on either IGFBP-3 or IGFBP-6 mRNA. These data suggest that the production of IGFBPs in human RPE cells is regulated by distinct mechanisms which include (1) an IGF-I receptor independent interaction of IGF-I with secreted IGFBPs and (2) de novo synthesis of IGFBPs by serum-containing factors.

IGF-II in the pathogenesis of rhabdomyosarcoma: a prototype of IGFs involvement in human tumorigenesis.

In recent years there has been a growing interest in the role of peptide growth factors in the regulation of the biologic behavior of several human malignancies. Among those peptides that might have importance in neoplastic pathogenesis are the insulin-like growth factors I and II (IGF-I and IGF-II). These proteins have been shown to stimulate myoblast proliferation and differentiation (1), to promote nutrient uptake and to inhibit proteolysis (2, 3). IGF-II mRNA is highly expressed in human skeletal fetal muscle tissue, but it is not detectable in normal adult skeletal muscle by standard Northern analysis and in situ hybridization (4). In mouse myoblasts, IGF-I and IGF-II mRNA levels increase transiently (IGF-II > > IGF-I) within 48-72 hours of the beginning of the myogenic differentiation process. The expression of mRNA is accompanied by secretion of the peptides in the culture medium that peaks at hour 92. IGF-I receptors increase transiently, doubling by 48 hours after the onset of differentiation, while IGF-II receptors increase and remain at a higher number throughout the differentiated state (5, 6). The production of IGF-binding proteins of Mr 29,000 to 32,000 is also induced throughout differentiation (7).

Concentrations of insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3), IGF, and IGFBP-3 protease activity in cerebrospinal fluid of children with leukemia, central nervous system tumor, or meningitis.

Insulin-like growth factor-II (IGF-II) is the major IGF in human cerebrospinal fluid (CSF), whereas IGF-I is only detectable in trace amounts. The major IGFBPs in CSF are IGFBP-2 and IGFBP-4. Normally, IGFBP-3 is a minor component in CSF of healthy subjects, but may be increased in pathological states. We investigated IGF-I, IGF-II, and IGFBP-3 levels by specific RIAs in CSF from patients with central nervous system (CNS) tumor or leukemia and compared them with values in patients with meningitis. Further, as proteolysis of IGFBP-3 is part of the modulation of IGF activity, IGFBP-3 fragmentation was quantified by densitometric analysis of [125I]IGFBP-3 protease assays. We examined CSFs of 23 children with malignant CNS tumors, 18 children with leukemia, and 13 children with meningitis. The CSF from 38 children who received lumbar punctures to exclude meningitis was used to define the normal range for IGF-I, IGF-II, IGFBP-3, and IGFBP-3 protease activity in CSF. CNS tumor and leukemia patients had normal levels of IGF-I and IGF-II in CSF, whereas the IGF-II concentration in CSF of meningitis patients was elevated (P < 0.0001). Only 2 of 13 (15%) meningitis patients had elevation of CSF IGFBP-3 concentrations, despite high numbers of inflammatory cells. By comparison, elevated IGFBP-3 concentrations were found in the CSF of 16 of 23 (70%) CNS tumor patients and 6 of 7 (86%) CNS tumor patients with microscopically detectable malignant cells in CSF. Twelve of 13 (92%) patients with medulloblastoma or ependymoma and all 7 medulloblastoma/ependymoma patients with malignant cells in CSF had elevated IGFBP-3 concentrations. The IGFBP-3 protease activity in CSF was elevated in 15 of 16 (94%) patients with CNS tumors of high grade histological malignancy. Five of 6 patients (83%) with acute leukemia and microscopically detectable malignant cells in CSF at the time of diagnosis showed elevated IGFBP-3 concentrations, with normalization after chemotherapy. Leukemia patients without malignant cells in CSF had normal IGFBP-3 concentrations. We conclude that in CSF of children with highly malignant CNS tumor or CNS leukemia, IGFBP-3 is elevated. This phenomenon could be caused by disruption of the blood-CSF barrier and entry of IGFBP-3 from serum, although this appears unlikely, especially for CNS leukemia. More likely possibilities are 1) local production of IGFBP-3 by CNS tumor tissue and secretion into the CSF, or 2) local production of IGFBP-3 by malignant cells within the CSF.

Insulin-like growth factor (IGF) suppression of IGFBP-1 production: evidence for mediation by the type I IGF receptor.

The regulation of insulin-like growth factor binding protein-1 (IGFBP-1) by its ligands, IGF-I and IGF-II, was studied in continuous cultures of HepG2 human hepatoma cells. Both IGF-I and IGF-II in concentrations as low as 1-10 nmol/l caused significant suppression of IGFBP-I protein levels. This suppression was accompanied by decreased IGFBP-1 mRNA levels occurring within 2-4 h of exposure to IGF-I or IGF-II, and by a significant decrease in IGFBP-1 promoter activity. IGF-I and IGF-II were equipotent in suppressing basal levels of IGFBP-1 protein, mRNA and promoter activity. IGF-I, IGF-II, and IGF-analogs with low IGFBP-1 affinity, (des 1-3)IGF-I and long R3IGF-I, all potently suppressed the previously characterized increase in IGFBP-1 protein levels and promoter activity induced by cAMP and theophylline. In contrast, [Leu-27]IGF-II, which interacts with the type II but not type I IGF receptor, had no effect on IGFBP-1 protein levels or promoter activity. Our data indicate that IGFBP-1 production is inhibited by its ligands, IGF-I and IGF-II, and that this effect is probably mediated at the transcriptional level. The effects of IGF-I and IGF-II apparently occur as a result of binding to the type I IGF receptor, and are similar to the previously characterized suppressive effects of insulin on IGFBP-1 transcription mediated through the insulin receptor. When considered with previous data regarding expression of IGFBP-1 and the type I IGF receptor, our results suggest that IGF regulation of IGFBP-1 may play an as yet undefined role in fetal development and postnatal hepatic regeneration.