Endogenous IGF-I regulates IGF binding protein production in human intestinal smooth muscle cells

Human intestinal smooth muscle in culture produces insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3, IGFBP-4, and IGFBP-5, which modulate the effects of IGF-I. This study examined the regulation of IGFBP production by endogenous IGF-I. R3-IGF-I, an agonist unaffected by IGFBPs, elicited concentration-dependent increase in growth, measured by [(3)H]thymidine incorporation, and production of IGFBP-3, IGFBP-4, and IGFBP-5, measured by Western blot. Antagonists of the IGF-I receptor, IGF-I Analog or monoclonal antibody 1H7, elicited concentration-dependent inhibition of growth and decrease in IGFBP-3, IGFBP-4, and IGFBP-5 production, implying that endogenous IGF-I stimulated growth and IGFBP production. R3-IGF-I-induced increase in IGFBP-3, IGFBP-4, and IGFBP-5 production was partially inhibited by a mitogen-activated protein (MAP) kinase or a phosphatidylinositol-3-kinase (PI 3-kinase) inhibitor and abolished by the combination. We conclude that endogenous IGF-I stimulates growth and IGFBP-3, IGFBP-4, and IGFBP-5 production in human intestinal smooth muscle cells. Regulation of IGFBP production by IGF-I is mediated by activation of distinct MAP kinase and PI 3-kinase pathways, the same pathways through which IGF-I stimulates growth.     

Organ-specific expression of IGF-I during early development of bony fish as revealed in the tilapia, Oreochromis niloticus, by in situ hybridization and immunohistochemistry: indication for the particular importance of local IGF-I

The cellular sites of insulin-like growth factor I (IGF-I) synthesis in the early developing tilapia (0-140 days post fertilization, DPF) were investigated. IGF-I mRNA and peptide appeared in liver as early as 4 DPF and in gastro-intestinal epithelial cells between 5-9 DPF. In exocrine pancreas, the expression of IGF-I started at 4 DPF and continued until 90 DPF. IGF-I production was detected in islets at 6 DPF in non-insulin cells and occurred throughout life. In renal tubules and ducts, IGF-I production started at 8 DPF. IGF-I production in chondrocytes had its onset at 4 DPF, was more pronounced in growing regions and was also found in adults. IGF-I mRNA and peptide appeared in the cytoplasm of skeletal muscle cells at 4 DPF. In gill chloride cells, IGF-I production started at 6 DPF. At 13 DPF, IGF-I was detected in cardiac myocytes. IGF-I-producing epidermal cells appeared at 5 DPF. In brain and ganglia, IGF-I was expressed in virtually all neurones from 6 to 29 DPF, their number decreasing with age. Neurosecretory IGF-I-immunoreactive axons were first seen in the neurohypophysis around 17 DPF. Endocrine cells of the adenohypophysis exhibited IGF-I mRNA at 28 DPF and IGF-I immunoreactivity at 40 DPF. Thus, IGF-I appeared early (4-5 DPF), first in liver, the main source of endocrine IGF-I, and then in organs involved in growth or metabolism. The expression of IGF-I was more pronounced during development than in juvenile and adult life. Local IGF-I therefore seems to have a high functional impact in early growth, metabolism and organogenesis.This study was supported by the SNF (NRP 50, project 4050-66580).     

Process development for production of recombinant human insulin-like growth factor-I in Escherichia coli

Fed-batch cultures were carried out to overproduce human insulin-like growth factor I (IGF-I) in Escherichia coli. The effects of carbon sources (glucose or glycerol) and induction time on cell growth and IGF-I production were investigated in more detail. Glycerol was a better carbon source than glucose for IGF-I production in fed-batch culture. Induction at the mid-exponential phase with glycerol as a carbon source in the pH-stat fed-batch culture was optimal for IGF-I production. Under this condition, 2.8 g L⁻¹ of fusion IGF-I was produced as inclusion bodies. We have also developed downstream processing for preparative scale purification of IGF-I from the fusion protein produced by the fed-batch culture using glycerol as a carbon source. After the fusion protein expressed was solubilized in 8 M urea and cleaved with hydroxylamine, the released IGF-I was purified by cation exchange chromatography, refolding and preparative scale reverse phase HPLC (rp-HPLC) to give recombinant IGF-I of >98% purity. The biological activities of the purified IGF-I were measured and found to be identical to those of commercial IGF-I. Journal of Industrial Microbiology & Biotechnology (2000) 24, 94–99. Received 13 January 1999/ Accepted in revised form 02 October 1999     

Estradiol-17beta and dihydrotestosterone differentially regulate vitellogenin and insulin-like growth factor-I production in primary hepatocytes of the tilapia Oreochromis mossambicus

Effects of estradiol-17beta (E2) and 5alpha-dihydrotestosterone (DHT) on the production of vitellogenin (Vg), insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs) were examined in vitro using primary hepatocyte culture of the tilapia. Estradiol produced a significant and concentration-related stimulation of Vg release and concomitant, concentration-related reduction in IGF-I mRNA expression in both male and female hepatocytes. In male hepatocytes, DHT significantly increased IGF-I expression, whereas DHT inhibited IGF-I expression and stimulated Vg release in female hepatocytes. Estradiol treatment significantly reduced the release of 25 kDa IGFBP, while stimulating the release of 30 kDa IGFBP from male hepatocytes. In female hepatocytes, E2 significantly increased both 25 and 30 kDa IGFBPs. In male hepatocytes, DHT significantly reduced 25 kDa IGFBP without affecting 30 kDa IGFBP. Conversely, DHT treatment of hepatocytes from female fish significantly increased both the 25 and 30 kDa IGFBPs. The different growth rates observed between male and female tilapia may be a result of gonadal steroid hormones eliciting direct and antagonistic effects on production of IGF-I (growth) and Vg (reproduction) in the liver. Indeed, the different growth patterns likely result from a difference in the sensitivity of male and female hepatocytes to gonadal steroid hormones. These results also indicate direct effects of gonadal steroid hormones on production of IGFBPs, which may play a role in regulating IGF-I mediated growth.     

Human progenitor cells from bone marrow or adipose tissue produce VEGF, HGF, and IGF-I in response to TNF by a p38 MAPK-dependent mechanism

Accumulating evidence suggests that progenitor cells may decrease destructive inflammation and reduce tissue loss by antiapoptotic mechanisms. However, they remain poorly characterized, and many questions remain regarding the mechanisms by which they may positively affect wound healing, tissue remodeling, or tissue regeneration. It has been speculated that various growth factors are responsible, but what components of the wound milieu stimulate progenitor cell production of growth factors and by what mechanisms? We hypothesized that tumor necrosis factor-alpha (TNF-alpha) stimulated progenitor cell secretion of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor I (IGF-I) by a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. Human mesenchymal stem cells (hMSCs) and human adipose progenitor cells (hAPCs) were divided into four groups: control, p38 MAPK inhibitor (p38MKI), TNF, and TNF + p38MKI. After 24 h of incubation, supernatants were harvested for ELISA of VEGF, HGF, and IGF-I. Cells were collected for Western blot analysis of p38 MAPK activation. Secretion of VEGF, HGF, and IGF-I in hMSCs and hAPCs was significantly increased by stimulation with TNF and was associated with increased activation of p38 MAPK. The p38 MAPK inhibitor decreased production of TNF-stimulated VEGF, HGF, and IGF-I in hMSCs and hAPCs. However, p38 MAPK inhibitor alone had no effect on production of growth factors. These data demonstrate that progenitor cells are potent sources of VEGF, HGF, and IGF-I. TNF, a prominent tissue cytokine, strongly stimulated production of growth factors by hMSCs and hAPCs via a p38 MAPK-dependent mechanism.     

Dietary isoflavone increases insulin-like growth factor-I production, thereby promoting hair growth in mice

Sensory neurons release calcitonin gene-related peptide (CGRP) upon activation. We previously demonstrated that CGRP increases insulin-like growth factor-I (IGF-I) production in various tissues of mice including the skin. We demonstrated that isoflavone increases the CGRP synthesis in the dorsal root ganglion (DRG) neurons in rats. Since IGF-I plays a critical role in hair growth, we hypothesized that isoflavones may promote hair growth by increasing the IGF-I production in hair follicles. We examined this hypothesis using wild-type (WT) and CGRP-knockout (CGRP^−/−) mice. Isoflavone significantly increased the CGRP mRNA levels in DRG neurons isolated from WT mice (P<.01). Administration of isoflavone for 3 weeks increased the dermal levels of CGRP, IGF-I and IGF-I mRNA in WT mice, but not in CGRP^−/− mice. Isoflavone administration increased the immunohistochemical expression of IGF-I in hair follicle dermal papilla cells in WT mice. Significant enhancements of hair follicle morphogenesis, hair regrowth, and hair pigmentation were also observed in WT mice administered isoflavone. However, none of these effects in WT mice were observed in CGRP^−/− mice.These observations strongly suggest that isoflavone might increase IGF-I production in the hair follicle dermal papilla cells in mice through increasing CGRP production in the sensory neurons, thereby promoting hair growth associated with melanogenesis in mice.     

Regulation of insulin-like growth factor I binding in the rat uterus by growth hormone and thyroxine.

The insulin-like growth factor-I (IGF-I)-binding sites in the rat uterus were characterized further and the effects of growth hormone and thyroxine examined. The 125I-labelled IGF-I binding sites on uterine membranes demonstrated relative binding affinity of less than 20% for IGF-II, less than 1% for insulin and no affinity for an unrelated peptide, epidermal growth factor, compared with 100% for IGF-I confirming the specificity of these binding sites. Scatchard analysis of the specific binding data revealed the presence of a single class of high-affinity binding sites (Kd = 2.50 +/- 0.68 nmol l-1, with a binding capacity of 1.02 +/- 0.13 pmol mg-1 membrane protein in the uterus of the pituitary-intact ovariectomized rat. After hypophysectomy, the uteri from these rats had significantly (P less than 0.05) increased IGF-I binding sites, without significant changes in their affinity. Administration of growth hormone with or without L-thyroxine reversed this increase in IGF-I binding. Injection of thyroxine alone to the hypophysectomized ovariectomized rats had no significant effects on the uterine IGF-I binding sites. These data show that growth hormone, but not thyroxine, can regulate IGF-I binding sites in the rat uterus, possibly through regulating IGF-I production.     

Serum insulin-like growth factor I levels in growth hormone-deficient adults: influence of sex steroids

Measurement of serum insulin-like growth factor I (IGF-I) concentrations remains the single most important tool in the evaluation of growth hormone (GH) replacement in GH-deficient adults, and the therapeutic goal is to maintain the level within the age-adjusted normal range. In healthy adults, IGF-I levels do not differ between males and females, whereas spontaneous GH secretion is approximately twofold higher in females. Untreated GH-deficient women exhibit lower IGF-I levels compared with men, and the increase in serum IGF-I during GH replacement is also significantly less. Put together, these data suggest resistance to GH in women, which in healthy individuals is compensated for by increased GH secretion. Administration of oral oestrogen in healthy post-menopausal women suppresses hepatic IGF-I production and increases pituitary GH release, and oral oestrogen replacement in women with GH deficiency lowers IGF-I concentrations and increases the amount of GH necessary to obtain IGF-I target levels during treatment. These data clearly suggest that hepatic suppression of IGF-I production by oestrogen subserves the gender difference in GH sensitivity, but it is also likely that sex steroids may interact with the GH/IGF axis at further levels. There is also circumstantial evidence to indicate that testosterone stimulates IGF-I production, and it is speculated that a certain threshold level of androgens is essential to ensure hepatic IGF-I production. Whether these data should translate into earlier discontinuation of oestrogen replacement therapy in adult women with hypopituitarism merits consideration.     

Sex steroids and the growth hormone/insulin-like growth factor-I axis in adults

In healthy adults insulin-like growth factor (IGF)-I levels do not differ between males and females, whereas spontaneous growth hormone (GH) secretion is approximately twofold higher in females. Untreated GH-deficient (GHD) women exhibit lower IGF-I levels compared with men and the increase in serum IGF-I during GH replacement is also significantly less. These data suggest a resistance to GH in women, which in healthy subjects is compensated for by increased GH secretion. Administration of oral oestrogen in healthy postmenopausal women suppresses hepatic IGF-I production and increases pituitary GH release, and oral oestrogen replacement in women with GHD lowers IGF-I concentrations and increases the amount of GH necessary to achieve IGF-I target levels during treatment. These data clearly suggest that hepatic suppression of IGF-I production by oestrogen subserves the gender difference in GH sensitivity, but it is also likely that sex steroids may interact with the GH/IGF axis at other levels. There is also circumstantial evidence to indicate that testosterone stimulates IGF-I production and it is speculated that a certain threshold level of androgens is essential to ensure hepatic IGF-I production. Whether these data should translate into earlier discontinuation of oestrogen replacement therapy in women with hypopituitarism merits consideration.     

Insulin-like growth factors in sheep thyroid cells: action, receptors and production

Sheep thyroid cells cultured in serum-free medium were used to study the biologic activity, binding, and production of the insulin-like growth factors (IGFs). IGF-I, IGF-II, and insulin stimulated thyroid cell division. Abundant, specific IGF receptors on sheep thyroid cell membranes were identified by binding displacement studies. Maximal specific binding of [125I]-labeled IGF-I and IGF-II to 25 micrograms of membrane protein averaged 21% and 27% respectively. The presence of type I and type II IGF receptors was confirmed by polyacrylamide gel electrophoresis of [125I]IGFs covalently cross-linked to cell membranes. Under reducing conditions, [125I]IGF-I bound to a moiety of approximate Mr = 135,000 and [125I]IGF-II to a moiety of approximate Mr = 260,000. Cross-linking of [125I]IGF-I to medium conditioned by thyroid cells indicated the presence of four IGF binding proteins with apparent Mr = 34,000, 26,000, 19,000 and 14,000. Thyroid cells also secreted IGF-I and II into the medium. IGF synthesis was enhanced consistently by recombinant growth hormone. These data indicate that sheep thyroid cells are a site for IGF action, binding, and production and provide further evidence that IGFs may modulate thyroid gland growth in an autocrine or paracrine manner.     

Stimulation of macrophage growth and multinucleated cell formation in rat bone marrow cultures by insulin-like growth factor I

In this study the effects of rhIGF-I on macrophage differentiation and growth have been studied using liquid suspension cultures of rat bone marrow cells. IGF-I stimulated macrophage growth in a dose-dependent manner, a maximum response was found at a concentration of 20 ng/ml. IGF-I effects could be ascribed to stimulation of both postmitotic and proliferating cells. A remarkable finding was that IGF-I induced formation of multinucleated cells (MNC). The MNC resembled macrophage-like cells (AcP, NSE positive). A monoclonal antibody to rhIGF-I significantly inhibited IGF-stimulated macrophage growth and MNC formation. A specific antibody to mouse CSF-1 reduced IGF-stimulated macrophage growth in mouse bone marrow cultures indicating that IGF-I effects could, at least in part, be ascribed to endogenous production of CSF-1. These findings indicate that IGF-I in concert with locally induced CSF-1 can influence the differentiation and growth of bone marrow-derived macrophages.     

Regulation of IGFBP-4 levels in human intestinal muscle by an IGF-I-activated, confluence-dependent protease

Human intestinal smooth muscle cells in culture produce insulin-like growth factor-I (IGF-I), IGF binding protein-3 (IGFBP-3), IGFBP-4, and IGFBP-5, which can modulate the effects of IGF-I on growth. This study examined the role of IGFBP-4 on IGF-I-induced growth and the mechanisms regulating IGFBP-4 levels. IGFBP-4 inhibited IGF-I-induced [(3)H]thymidine incorporation. IGFBP-4 mRNA levels were not altered by IGF-I. IGF-I caused a concentration-dependent activation of an endogenous IGFBP-4 protease, resulting in time-dependent degradation of intact IGFBP-4 into inactive fragments. Protease activity was measured in a cell-free assay using smooth muscle cell conditioned medium containing the IGFBP-4 protease. The protease was inhibited by EDTA and benzamidine. Protease activity was highest in proliferating cells and lowest in postconfluent cells. The role of endogenous IGF-I in regulating IGFBP-4 degradation was confirmed by the ability of an IGF-I antagonist to inhibit IGF-I-activated IGFBP-4 proteolysis in intact cells. We conclude that in human intestinal smooth muscle cells levels of secreted IGFBP-4 are determined by the confluence-dependent production of a cation-dependent serine protease that is activated by endogenous IGF-I.     

Differential effects of follicle-stimulating hormone, insulin, and insulin-like growth factor I on hexose uptake and lactate production by rat Sertoli cells

The stimulatory effects of follicle-stimulating hormone (FSH), insulin, and insulin-like growth factor I (IGF-I) on lactate production and hexose uptake by Sertoli cells from immature rats were studied. The time-courses and the maximal stimulatory effects of FSH, insulin, and IGF-I on lactate production were virtually identical. When Sertoli cells were incubated in the presence of FSH in combination with insulin or IGF-I (submaximal doses), additive but no pronounced synergistic effects were observed. The stimulatory effects of FSH and insulin were not dependent on the presence of extracellular calcium. 2-Deoxy-D-glucose (2-DOG), an analogue of D-glucose, was used to investigate the hexose transport system of Sertoli cells. Uptake of 2-DOG was linear in time and virtually all of the intracellular 2-DOG was phosphorylated up to 30 min of incubation; 2-DOG uptake was inhibited by cytochalasin B, but not by cytochalasin E. D-glucose, but not D-galactose, appeared to be an effective competitor of 2-DOG uptake. The Km of 2-DOG uptake was not influenced by FSH, insulin, and IGF-I. FSH had no effect on the Vmax of 2-DOG uptake, whereas insulin and IGF-I caused a 30% stimulation of the Vmax. It is concluded that FSH, insulin, and IGF-I stimulate lactate production by cultured Sertoli cells, but that only insulin and IGF-I stimulate hexose transport. The insulin-like effect of FSH on Sertoli cells may principally involve stimulation of glycolytic enzyme activities.     

Modulation of heart fibroblast migration and collagen gel contraction by IGF-I

Dynamic interactions between cells and the extracellular matrix are essential in the regulation of a number of cellular processes including migration, adhesion, proliferation and differentiation. A variety of factors have been identified which modulate these interactions including transforming growth factor-beta, platelet-derived growth factor and others. Insulin-like growth factors have been shown to regulate collagen production by heart fibroblasts; however, the effects of this growth factor on the interactions of heart fibroblasts with the extracellular matrix have not been examined. The present studies were carried out to determine the effects of IGF-I on the ability of fibroblasts to interact with the extracellular matrix and to begin to determine the mechanisms of this response. These experiments illustrate that IGF-I treatment results in increased migration, collagen reorganization and gel contraction by heart fibroblasts. IGF-I has been shown to activate both the mitogen-activated protein kinase and phophatidylinositol-3 kinase pathways in isolated cells. Experiments with pharmacological antagonists of these pathways indicate that the mitogen-activated protein kinase pathway is essential for IGF-I stimulated collagen gel contraction by fibroblasts. These studies illustrate that IGF-I modulates the ability of fibroblasts to interact with the collagen matrix and that activation of multiple signaling pathways by IGF-I may produce distinct downstream responses in these cells.     

Modulation of Heart Fibroblast Migration and Collagen Gel Contraction by IGF-I

Dynamic interactions between cells and the extracellular matrix are essential in the regulation of a number of cellular processes including migration, adhesion, proliferation and differentiation. A variety of factors have been identified which modulate these interactions including transforming growth factor+, platelet-derived growth factor and others. Insulin-like growth factors have been shown to regulate collagen production by heart fibroblasts; however, the effects of this growth factor on the interactions of heart fibroblasts with the extracellular matrix have not been examined. The present studies were carried out to determine the effects of IGF-I on the ability of fibroblasts to interact with the extracellular matrix and to begin to determine the mechanisms of this response. These experiments illustrate that IGF-I treatment results in increased migration, collagen reorganization and gel contraction by heart fibroblasts. IGF-I has been shown to activate both the mitogen-activated protein kinase and phophatidylinositol-3 kinase pathways in isolated cells. Experiments with pharmacological antagonists of these pathways indicate that the mitogen-activated protein kinase pathway is essential for IGF-I stimulated collagen gel contraction by fibroblasts. These studies illustrate that IGF-I modulates the ability of fibroblasts to interact with the collagen matrix and that activation of multiple signaling pathways by IGF-I may produce distinct downstream responses in these cells.     

The effects of insulin-like growth factor-I and insulin on placental lactogen production by human term placental explants

The effects of insulin-like growth factor (IGF)-I and insulin on placental lactogen production (hPL) by term human placental explants were studied. The hPL content in medium and explant decreased rapidly after first 24 hours of culture. The decrease thereafter was gradual and reached a plateau by day 4 of culture. The decrease of HPL content in placental culture has previously been suggested being due to the depletion of a rapidly secreting preformed pool of hPL. Addition of IGF-I (0.1-10 micrograms/ml) and insulin (1-20 micrograms/ml) stimulated the decreased level of hPL in tissue and medium after 24 hours in culture. IGF-I was 10 times more potent than insulin in stimulating hPL. These findings suggest that IGF-I and insulin effects the production of hPL by placenta. The lower potency of insulin may indicate that the effect of insulin on hPL production is via IGF-I receptor.     

Synergistic Action of Transforming Growth Factor-β and Insulin-like Growth Factor-I Induces Expression of Type II Collagen and Aggrecan Genes in Adult Human Articular Chondrocytes

Reexpression of aggrecan and type II collagen genes in dedifferentiated adult human articular chondrocytes (AHAC) in suspension culture varied widely depending on the specific lot of bovine serum used to supplement the culture medium. Some lots of serum provided strong induction of aggrecan and type II collagen expression by AHAC while others did not stimulate significant production of these hyaline cartilage extracellular matrix molecules even following several weeks in culture. Addition of 50 ng/ml insulin-like growth factor-I (IGF-I) to a deficient serum lot significantly enhanced its ability to induce aggrecan and type II collagen mRNA. Given this observation, IGF-I and other growth factors were tested in defined serum-free media for their effects on the expression of these genes. Neither IGF-I nor insulin nor transforming growth factor β (TGF-β) alone stimulated induction of aggrecan or type II collagen production by dedifferentiated AHAC. However, TGF-β1 or TGF-β2 combined with IGF-I or insulin provided a strong induction as demonstrated by RNase protection and immunohistochemical assays. Interestingly, type I collagen, previously shown to be downregulated in serum supplemented suspension cultures of articular chondrocytes, persisted for up to 12 weeks in AHAC cultured in defined medium supplemented with TGF-β and IGF-I.