Regulation of constitutive gene expression through interactions of Sp1 protein with the nuclear aryl hydrocarbon receptor complex.

The region of residues -145 to -119 (CD/L) of the cathepsin D gene promoter contains a GC-rich motif that binds Sp1 protein and an adjacent pentanucleotide (CACGC) that corresponds to the core sequence of a dioxin responsive element (DRE) and binds the aryl hydrocarbon receptor (AhR)-AhR nuclear translocator (Arnt) complex. This Sp1(N)(4)DRE(core) motif has been identified in promoters of several genes in which Sp1 plays an important role in basal gene expression. In transient transfection assays with MCF-7 human breast cancer cells using wild-type pCD/L and constructs mutated in the core DRE (pCD/L(m1)) and Sp1 (pCD/L(m2)) sites, it was shown that both motifs were required for maximal basal activity. The requirements for AhR-Arnt interactions with Sp1 protein for maximal activity of pCD/L were confirmed in wild-type MCF-7 and Hepa 1c1c7 cells and Arnt-deficient Hepa 1c1c7 cells using antisense Arnt and Arnt expression plasmids. The functional interactions of Sp1 with AhR-Arnt were paralleled by physical interactions showing that AhR-Arnt and Sp1 proteins were co-immunoprecipitated and AhR-Arnt enhanced Sp1-[(32)P]CD/L binding in electrophoretic mobility shift assays. The physical and functional interactions of Sp1 with AhR-Arnt proteins bound to the Sp1(N)(4)DRE(core) motif were also dependent on the proximity of these sites, and both the activity and the extent of Sp1-DNA binding decreased as the number of intervening nucleotides increased from 4 to 20. These studies show that regulation of basal expression of some genes by Sp1 may also require interactions with AhR-Arnt.     

Effects of dexamethasone on the production of insulin-like growth factor-I and insulin-like growth factor binding proteins in primary cultures of rat hepatocytes.

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

Effect of celiac disease and gluten-free diet on growth hormone-binding protein, insulin-like growth factor-I, and insulin-like growth factor-binding proteins

Failure to thrive is common in children with celiac disease. As alterations in the growth hormone-insulin-like growth factor I (GH-IGF-I) growth axis have been reported in these patients, we studied the behavior of growth hormone-binding proteins (GH-BPs I and II), IGF-I and its binding proteins in 14 children with celiac disease, either before or after a 6-month gluten-free diet. GH-BP II levels were significantly lower in patients during the active phase of the disease than after the diet or in comparison with control subjects, appropriate for age and sex. There was no difference in the GH-BP-I levels of patients and controls, nor did they change after the diet. Blood levels of IGF-I and IGFBP-3 were reduced before the diet in all patients while ligand blotting showed that IGFBP-2 and 1 were increased. All of these parameters normalized after the gluten-free diet. IGFBP-4 was not greatly influenced by the disease. Furthermore, we found a significant, positive correlation between GH-BP II and IGF-I or IGFBP-3 levels. The height standard deviation scores and body mass indices of the patients improved significantly after the diet. The body mass index significantly and positively correlated with GH-BP II, IGF-I or IGFBP-3 levels. In conclusion, our data show that celiac children had multiple alterations in the growth axis during the active phase of the disease which disappeared during the gluten-free diet.     

Interleukin-1 inhibits the induction of insulin-like growth factor-I by growth hormone in CWSV-1 hepatocytes

Sepsis results in hepatic "growth hormone (GH) resistance" with reductions in plasma IGF-I despite a two- to fourfold increase in circulating GH. In this study, we examine the effects of IL-1 on GH receptor (GHR) expression, GH signaling (via the JAK/STAT and MAPK pathways), and the induction of gene expression [IGF-I mRNA and serine protease inhibitor (Spi) 2.1] by GH in CWSV-1 hepatocytes. Incubation of cells with IL-1beta (10 ng/ml, 24 h) had no effect on the relative abundance of GHR or signaling proteins JAK2, STAT5b, and ERK1/2 in cell lysates. Baseline phosphorylation of GHR, JAK2, STAT5b, and ERK1/2 was minimal. After GH stimulation, tyrosine phosphorylation of GHR, JAK2, STAT5b, and ERK1/2 increased 2- to 10-fold. However, neither the time course nor the magnitude of GHR, JAK2, and ERK1/2 phosphorylation by GH were significantly altered by IL-1. The GH-induced translocation of STAT5b to the nucleus was not prevented by IL-1. Although phosphorylated STAT5 in nuclear extracts from GH + IL-1 cells was decreased by 24% (vs. controls) 15 min after GH stimulation, this did not result in reduced STAT5-DNA binding activity. Pretreatment with IL-1 did not significantly decrease IGF-I mRNA stability. We conclude that IL-1 only minimally affects the time course of JAK2/STAT5 and MAPK signaling by GH. Therefore, an inhibitory effect of IL-1 on IGF-I and Spi 2.1 mRNA synthesis by GH represents the most likely mechanism for IL-1-mediated GH resistance.     

Hyperlipidemia is associated with altered levels of insulin-like growth factor-I

Previous studies revealed altered levels of the circulating insulin-like growth factor-I (IGF-I) and of its binding protein-3 (IGFBP-3) in subjects with coronary atherosclerosis, metabolic syndrome and premature atherosclerosis. Hyperlipidemia is a powerful risk factor of atherosclerosis. We expected IGF-I and IGFBP-3 alterations in subjects with moderate/severe hyperlipidemia but without any clinical manifestation of atherosclerosis. Total IGF-I and IGFBP-3 were assessed in 56 patients with mixed hyperlipidemia (MHL; cholesterol >6.0 mmol/l, triglycerides >2.0 mmol/l), in 33 patients with isolated hypercholesterolemia (IHC; cholesterol >6.0 mmol/l, triglycerides <2.0 mmol/l), and in 29 healthy controls (cholesterol<6.0 mmol/l, triglycerides<2.0 mmol/l). The molar ratio of IGF-I/IGFBP-3 was used as a measure of free IGF-I. IHC subjects differed from controls by lower total IGF-I (164+/-60 vs. 209+/-73 ng/ml, p=0.01) and IGF-I /IGFBP-3 ratio (0.14+/-0.05 vs. 0.17+/-0.04, p=0.04). Compared to controls, MHL subjects had lower total IGF-I (153+/-54 ng/ml, p=0.0002) and IGFBP-3 (2.8+/-0.6 mg/ml, p<0.0001), but higher IGF-I/IGFBP-3 ratio (0.25+/-0.06, p<0.0001). Differences remained significant after the adjustment for clinical and biochemical covariates, except for triglycerides. Patients with both IHC and MHL have lower total IGF-I compared to controls. The mechanism is presumably different in IHC and MHL. Because of prominent reduction of IGFBP-3 in patients with MHL, they have reduced total IGF-I despite the actual elevation IGF-I/IGFBP-3 ratio as a surrogate of free IGF-I.     

C-terminal Src kinase associates with ligand-stimulated insulin-like growth factor-I receptor

Increased expression of the insulin-like growth factor-I receptor (IGF-IR) protein-tyrosine kinase occurs in several kinds of cancer and induces neoplastic transformation in fibroblast cell lines. The transformed phenotype can be reversed by interfering with the function of the IGF-IR. The IGF-IR is required for transformation by a number of viral and cellular oncoproteins, including SV40 large T antigen, Ras, Raf, and Src. The IGF-IR is a substrate for Src in vitro and is phosphorylated in v-Src-transformed cells. We observed that the IGF-IR and IR associated with the C-terminal Src kinase (CSK) following ligand stimulation. We found that the SH2 domain of CSK binds to the tyrosine-phosphorylated form of IGF-IR and IR. We determined the tyrosine residues in the IGF-IR and in the IR responsible for this interaction. We also observed that fibroblasts stimulated with IGF-I or insulin showed a rapid and transient decrease in c-Src tyrosine kinase activity. The results suggest that c-Src and CSK are involved in IGF-IR and IR signaling and that the interaction of CSK with the IGF-IR may play a role in the decrease in c-Src activity following IGF-I stimulation.     

Pharmacodynamic considerations with recombinant human insulin-like growth factor-I in children

AIM: To report effects of weight-based recombinant human insulin-like growth factor-I (rhIGF-I) on IGF axis parameters in children with hyperinsulinism. METHODS: Open label trial with subcutaneous rhIGF-I (40 microg/kg/dose). Patients studied were children (1 month to 11 years) with diffuse hyperinsulinism (n = 7). Serial serum IGF and insulin-like growth factor binding protein (IGFBP) concentrations were measured by RIA and analyzed by linear Pearson regression. RESULTS: Following the initial rhIGF-I dose, total insulin-like growth factor-I (IGF-I) rose by 56% at 30 min (p < 0.01) and 85% at 120 min (p < 0.02). Serum IGF-II, IGFBP-2, and IGFBP-3 levels did not change. Peak serum IGF-I levels within 12 h of the initial rhIGF-I dose were 167-700 mg/ml. The variable peak IGF-I response is attributable in part to IGFBP-3 differences across this pediatric age range. Models of rhIGF-I dosing based upon body surface area (BSA) or initial IGFBP-3 resulted in predictable peak serum IGF-I levels (r = 0.78; p < 0.03). Recalculating rhIGF-I dosing based upon the BSA . IGFBP-3 product correlated closely with peak IGF-I level (r = 0.85; p < 0.007). CONCLUSIONS: Weight-based IGF-I dosing in this cohort resulted in variable IGF-I levels. Considering BSA and serum IGFBP-3 concentration in children is appropriate for subcutaneous IGF-I administration. A combination of these values may yield predictable individualization of rhIGF-I dosing.     

Stimulatory effects of insulin-like growth factor-I on growth plate chondrogenesis are mediated by nuclear factor-kappaB p65

Insulin-like growth factor-I (IGF-I) is an important regulator of endochondral ossification. However, little is known about the signaling pathways activated by IGF-I in growth plate chondrocytes. We have previously shown that NF-kappaB-p65 facilitates growth plate chondrogenesis. In this study, we first cultured rat metatarsal bones with IGF-I and/or pyrrolidine dithiocarbamate (PDTC), a known NF-kappaB inhibitor. The IGF-I-mediated stimulation of metatarsal growth and growth plate chondrogenesis was neutralized by PDTC. In rat growth plate chondrocytes, IGF-I induced NF-kappaB-p65 nuclear translocation. The inhibition of NF-kappaB-p65 expression and activity (by p65 short interfering RNA and PDTC, respectively) in chondrocytes reversed the IGF-I-mediated induction of cell proliferation and differentiation and the IGF-I-mediated prevention of cell apoptosis. Moreover, the inhibition of the phosphatidylinositol 3-kinase and Akt abolished the effects of IGF-I on NF-kappaB activation. In conclusion, our findings indicate that IGF-I stimulates growth plate chondrogenesis by activating NF-kappaB-p65 in chondrocytes.     

Genetic disorders in the growth hormone - insulin-like growth factor-I axis

In the last few years, our knowledge of genetically determined causes of short stature has greatly increased by reports of challenging patients, who offered the opportunity to study genes that play a role in growth. Since the first paper that showed the etiology of Laron syndrome [Godowski PJ, et al: Proc Natl Acad Sci USA 1989;86:8083-8087], many mutations in the growth hormone (GH) receptor have been identified. Recently, new mutations or deletions have been found in several components of the GH-insulin-like growth factor-I (IGF-I) axis: a homozygous mutation of the GH1 gene, resulting in a bio-inactive GH; mutations in the STAT5b gene, which plays a major role in the GH signal transduction; a homozygous missense mutation in the IGF-I gene; heterozygous mutations in the IGF-I receptor gene and a homozygous deletion of the acid-labile subunit gene. In this mini review, we describe the clinical and biochemical features of these genetic defects. Genetic analysis has become essential in the diagnostic workup of a patient with short stature. However, regarding the time consuming nature of molecular analysis, it is important to carefully select the patient for specific genetic evaluation. To help in this selection process, we developed flowcharts, based on the recently described patients, that can be used as guidelines in the diagnostic process of patients with severe short stature of unknown origin.     

Insulin-like growth factor-I, insulin-like growth factor-binding proteins, and gonadotropins in the hypothalamic-pituitary axis and serum of nutrient-restricted ewes.

Body condition scores (BCS) of ovariectomized estradiol-treated ewes were controlled to examine effects of suboptimum BCS on insulin-like growth factor (IGF)-I, IGF-binding proteins (IGFBPs), and LH in the anterior pituitary gland, hypophyseal stalk-median eminence (SME), and circulation. Serum LH increased in ewes with BCS (1 = emaciated, 9 = obese) > 3 (HIGH-BCS), but not in ewes with BCS 相似文献