Insulin-like Growth Factor Binding Protein-3 mRNA Research Articles

Growth cartilage expression of growth hormone/insulin-like growth factor I axis in spontaneous and growth hormone induced catch-up growth

IntroductionCatch-up growth following the cessation of a growth inhibiting cause occurs in humans and animals. Although its underlying regulatory mechanisms are not well understood, current hypothesis confer an increasing importance to local factors intrinsic to the long bones' growth plate (GP). AimThe present study was designed to analyze the growth-hormone (GH)-insulin-like growth factor I (IGF-I) axis in the epiphyseal cartilage of young rats exhibiting catch-up growth as well as to evaluate the effect of GH treatment on this process. Material and methodsFemale Sprague–Dawley rats were randomly grouped: controls (group C), 50% diet restriction for 3days+refeeding (group CR); 50% diet restriction for 3days+refeeding & GH treatment (group CRGH). Analysis of GH receptor (GHR), IGF-I, IGF-I receptor (IGF-IR) and IGF binding protein 5 (IGFBP5) expressions by real-time PCR was performed in tibial growth plates extracted at the time of catch-up growth, identified by osseous front advance greater than that of C animals. ResultsIn the absence of GH treatment, catch-up growth was associated with increased IGF-I and IGFBP5 mRNA levels, without changes in GHR or IGF-IR. GH treatment maintained the overexpression of IGF-I mRNA and induced an important increase in IGF-IR expression. ConclusionsCatch-up growth that happens after diet restriction might be related with a dual stimulating local effect of IGF-I in growth plate resulting from overexpression and increased bioavailability of IGF-I. GH treatment further enhanced expression of IGF-IR which likely resulted in a potentiation of local IGF-I actions. These findings point out to an important role of growth cartilage GH/IGF-I axis regulation in a rat model of catch-up growth.

Insulin-like growth factor and fibroblast growth factor expression profiles in growth-restricted fetal sheep pancreas

Placental insufficiency results in intrauterine growth restriction (IUGR), impaired fetal insulin secretion and less fetal pancreatic β-cell mass, partly due to lower β-cell proliferation rates. Insulin-like growth factors (IGFs) and fibroblast growth factors (FGFs) regulate fetal β-cell proliferation and pancreas development, along with transcription factors, such as pancreatic and duodenal homeobox 1 (PDX-1). We determined expression levels for these growth factors, their receptors and IGF binding proteins in ovine fetal pancreas and isolated islets. In the IUGR pancreas, relative mRNA expression levels of IGF-I, PDX-1, FGF7 and FGFR2IIIb were 64% (P < 0.01), 76% (P < 0.05), 76% (P < 0.05) and 52% (P < 0.01) lower, respectively, compared with control fetuses. Conversely, insulin-like growth factor binding protein 2 (IGFBP-2) mRNA and protein concentrations were 2.25- and 1.2-fold greater (P < 0.05) in the IUGR pancreas compared with controls. In isolated islets from IUGR fetuses, IGF-II and IGFBP-2 mRNA concentrations were 1.5- and 3.7-fold greater (P < 0.05), and insulin mRNA was 56% less (P < 0.05) than control islets. The growth factor expression profiles for IGF and FGF signaling pathways indicate that declines in β-cell mass are due to decreased growth factor signals for both pancreatic progenitor epithelial cell and mature β-cell replication.

Associations Between Bovine IGFBP2 Polymorphisms with Fertility, Milk Production, and Metabolic Status in UK Dairy Cows

Insulin-like growth factor binding protein-2 (IGFBP2) is a key regulator of IGF activity that has been associated with insulin resistance and obesity. In cows, IGFBP2 mRNA expression is differentially regulated according to nutritional status in different tissues including the liver, reproductive tract, and mammary gland. This study investigated associations between single nucleotide polymorphisms (SNPs) in bovine IGFBP2 with fertility, milk production, and metabolic traits in Holstein-Friesian dairy cows. Fertility was assessed in heifers by measuring age at first service, age at first conception, and age at first calving. During the first and second lactation, the number of postpartum days for commencement of luteal activity (based on milk progesterone profiles), days to first service, days to conception, average milk production per day, 305-day milk yield, total milk yield, and total days in milk were recorded. Blood samples were taken at -1, +1, and +8 weeks relative to first and second calving for assessment of metabolic status (IGF1, insulin, beta-hydroxybutyrate, and glucose). Five novel SNPs were identified in IGFBP2, two of which had significant associations with fertility (age at conception in heifers and commencement of luteal activity) and 305-day milk yield in lactation 1. Trends of association were also observed with the peripartum metabolic status, in particular the glucose, insulin, and beta-hydroxybutyrate concentrations around second calving. These results indicate that IGFBP2 SNPs may influence tissue mobilization in dairy cows and may thus be of interest for marker assisted selection.

Insulin stimulates IGFBP-2 expression in 3T3-L1 adipocytes through the PI3K/mTOR pathway

Insulin-like growth factor binding protein 2 (IGFBP-2) has been implicated in the etiology of several diseases, including the metabolic syndrome. Although IGFBP-2 derives mostly from the liver, recent evidence in mice and humans indicate that aging and obesity are associated with altered IGFBP-2 levels in white adipocytes. The present study was aimed at determining the mechanisms that control IGFBP-2 expression in mature adipocytes. IGFBP-2 mRNA and protein expression in serum-deprived 3T3-L1 adipocytes were twofold increased by acute insulin treatment. Co-treatments with the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin or the mammalian target of rapamycin (mTOR) inhibitor rapamycin blunted the effects of insulin. Coherently, IGFBP-2 mRNA levels were robustly increased in adipocytes lacking either TSC2 or 4E-BP1. Insulin triggered the recruitment of CAAT/enhancer binding protein α (C/EBPα) to the IGFBP-2 proximal promoter. These findings suggest that insulin upregulates IGFBP-2 expression through a PI3K/mTOR/C/EBPα pathway in white adipocytes.

Identification and expression analysis of IGFBP-1 gene from Japanese flounder (Paralichthys olivaceus)

Insulin-like growth factor binding protein-1 (IGFBP-1) plays an important role in IGF regulating vertebrate growth and development. In this study, we cloned IGFBP-1 cDNA from Japanese flounder (Paralichthys olivaceus) liver. The full-length cDNA is 1070 bp, including a 5'-untranslated region (UTR) of 69 bp, a 3'-UTR of 272 bp, and an open reading frame (ORF) of 729 bp encoding a polypeptide of 242 amino acids. Real-time quantitative PCR revealed that IGFBP-1 mRNA is mainly expressed in the liver, and a small amount of mRNAs was also found in other adult tissues. There are maternal transcripts of IGFBP-1 gene, and relatively low mRNA levels were observed in different embryonic stages. A higher level of IGFBP-1 mRNA was detected at 3 days post hatching (dph), and it got to the highest level at 29 dph (metamorphic climax), and finally brought back to a lower level at the end of metamorphosis. The expression of IGFBP-1 mRNA was greatly up-regulated in thyroid hormone (TH)-treated larvae, and declined after thiourea (TU) treatment. These results provide basic information for further studies on the role of IGF system in the P. olivaceus development and metamorphosis.

Functional relationship between high mobility group A1 (HMGA1) protein and insulin-like growth factor-binding protein 3 (IGFBP-3) in human chondrocytes

IntroductionInsulin-like growth factor I (IGF-I) regulates articular cartilage homeostasis. During osteoarthritis (OA), the anabolic responses of chondrocytes to IGF-I are likely to be prevented by the enhanced production of IGF-binding proteins (IGFBPs), especially IGFBP-3. The aim of this study is to evaluate whether the architectural transcription factor high mobility group A1 (HMGA1) influences IGFBP-3 overexpression in vitro, in cultured chondrocytic cell lines, and ex vivo, in human osteoarthritic cartilage compared to healthy human cartilage controls.MethodsQuantitative real-time reverse transcription-PCR (qRT-PCR) was performed to assess the relative transcript levels of HMGA1 and IGFBP-3 in vitro, in the human chondrocytic cell lines T/C-28a4 and C-28/I2. An electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) and transient transfection assays were performed to investigate the HMGA1-IGFBP-3 gene interaction. Samples of articular cartilage were harvested from osteoarthritic patients and controls and analyzed by qRT-PCR for HMGA1 and IGFBP-3 mRNA levels.ResultsA parallelism between HMGA1 protein levels and IGFBP-3 gene expression has been observed in T/C-28a4 and C-28/I2 cells. The interaction of HMGA1 with the IGFBP-3 gene promoter has been demonstrated by EMSA and ChIP. In transient transfections, IGFBP-3 promoter activity increased in cells overexpressing HMGA1 and decreased in cells pretreated with siRNA detected against HMGA1. IGFBP-3 mRNA expression was higher in cartilage from patients with OA, where the increased expression of IGFBP-3 closely paralleled the increased expression of HMGA1 mRNA.ConclusionsOur observations indicate that increased HMGA1 expression in human chondrocytes is associated with increased expression of IGFBP-3. It is tempting to speculate that, through the regulation of IGFBP3 expression, HMGA1 may act as a pathogenetic factor for OA.

Abstract P253: Transcriptional Regulation of the IGFBP-1 Gene by RTEF-1 in Endothelial Cells

Background and Objective: Cardiovascular disease is the most important complication of type 2 diabetes, however, its underlying mechanism(s) are poorly understood. Our lab previously reported that RTEF-1 is a transcription factor that promotes angiogenesis via up-regulating HIF-1α and VEGF. In addition to these studies, we recently determined that RTEF-1 upregulates insulin-like growth factor binding protein-1 (IGFBP-1) expression in endothelial cells in vitro and in vivo. IGFBPs are key regulators of insulin-like growth factor (IGF) type 1 and 2 bioavailability at the cellular level. In addition, IGFBP-1 is implicated in cardiovascular health, in terms of blood pressure and glucose tolerance. Methods and Results: RTEF-1 upregulates IGFBP-1 expression in endothelial cells in vitro and in vivo through selectively binding and promoting transcription from the insulin response element (IRE) site on the IGFBP-1 promoter, demonstrated by ChIP assays. In addition to insulin inhibition of IGFBP-1 levels, insulin also dose-dependently inhibits RTEF-1 promoter activity (-2.4 ± 0.3-fold) and expression. In vivo data suggest that Tie2-Cre:RTEF-1 flox/flox transgenic mice, RTEF-1 EC-specific knockout mice, show a significant decrease decrease in IGFBP-1 mRNA levels compared with controls (0.21 ± 0.09 vs. 1.0, respectively), while VE-Cadherin/RTEF-1 overexpressing transgenic mice have increased IGFBP-1 mRNA levels (25.4 ± 0.02-fold). Increased IGFBP-1 levels relate physiologically in our RTEF-1 EC-specific knockout by showing decreased blood glucose tolerance as well as increased insulin resistance as calculated by HOMA-IR compared with littermate controls, (4.1 ± 0.5 vs. 1.1 ± 0.2, respectively). Furthermore, the RTEF-1 EC-specific knockout mice showed a significant increase in systolic blood pressure (10mmHg ± 6mmHg) compared with littermate controls. Conclusion: These results demonstrate that RTEF-1-stimulated IGFBP-1 expression may be central to the mechanism by which RTEF-1 attenuates blood glucose levels and potential cardiovascular function. These findings provide the basis for novel insights into transcriptional regulation of IGFBP-1 and contribute to understanding the role of vascular endothelial cells in metabolism.

Dietary Monounsaturated Fat in Early Life Regulates IGFBP2: Implications for Fat Mass Accretion and Insulin Sensitivity

The aim of this study was to investigate effects of dietary supplementation with fat or sugar on body composition (BC) and insulin sensitivity (IS) in maturing pigs. Fifty newborn pigs randomized to a control diet or 18% saturated fat (SF), 18% monounsaturated fat (MUF), 18% mixed fat (MF), or 50% sucrose (SUC), from 1 to 16 weeks of age. Outcomes included weight gain, BC (dual energy X-ray absorptiometry, DXA), IS (fasting insulin and hyperinsulinaemic-euglycaemic clamps), fasting Non-Esterified Fatty Acid (NEFA) concentrations, and mRNA expression of genes involved in lipogenesis and IS in skeletal muscle (SM), subcutaneous (SAT), and visceral adipose tissue (VAT). In vitro studies examined direct effects of fatty acids on insulin-like growth factor-binding protein 2 (IGFBP2) mRNA in C2C12 myotubes. While SUC-fed pigs gained most weight (due to larger quantities consumed; P < 0.01), those fed fat-enriched diets exhibited more weight gain per unit energy intake (P < 0.001). Total (P = 0.03) and visceral (P = 0.04) adiposity were greatest in MUF-fed pigs. Whole-body IS was decreased in those fed fat (P = 0.04), with fasting insulin increased in MUF-fed pigs (P = 0.03). SM IGFBP2 mRNA was increased in MUF-fed pigs (P = 0.009) and, in all animals, SM IGFBP2 mRNA correlated with total (P = 0.007) and visceral (P = 0.001) fat, fasting insulin (r = 0.321; P = 0.03) and change in NEFA concentrations (r = 0.285; P = 0.047). Furthermore, exposure of in vitro cultured myotubes to MUF, but not SF, reduced IGFBP2 mRNA suggesting a converse direct effect. In conclusion, diets high in fat, but not sugar, promote visceral adiposity and insulin resistance in maturing pigs, with evidence that fatty acids have direct and indirect effects on IGFBP2 mRNA expression in muscle.

Evaluation of human first trimester decidual and telomerase-transformed endometrial stromal cells as model systems of in vitro decidualization.

BackgroundDecidualization, the differentiation process of maternal uterine stromal cells into secretory decidual cells, is a prerequisite for successful implantation and progression of pregnancy. For in vitro differentiation mostly primary human endometrial stromal cells (HESC) isolated from uterine samples after hysterectomy for benign gynaecological diseases are utilised. However, a continuous supply of endometrial tissue is often lacking. Hence, we analysed whether cultivated human decidual stromal cells (HDSC) prepared from first trimester pregnancy terminations may represent an alternative model system for in vitro decidualization. Moreover, based on the expression of critical marker genes these cells were compared to a previously established endometrial stromal cell line during in vitro differentiation.MethodsHDSC isolated from decidual tissue attached to first trimester placentae, and telomerase-transformed human endometrial stromal cells (THESC) were characterised by immunofluorescence and differentiated in vitro using either cyclic adenosine monophosphate (cAMP) and/or estrogen (E2)/progesterone (P4). Proliferation was measured by analyzing cumulative cell numbers. Expression of mRNAs encoding progesterone receptor (PR), prolactin (PRL), insulin-like growth factor binding protein-1 (IGFBP1), and Dickkopf-1 (DKK1) was evaluated using quantitative PCR after 3, 6, 9 and 12 days of in vitro differentiation. PRL and IGFBP-1 protein expression was investigated by enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. Furthermore, forkhead box O1A (FOXO1A), a critical transcription factor in decidualization, was analysed by immunofluorescence and Western blotting at two different time points of differentiation.ResultsTreatment with cAMP provoked morphological changes and growth arrest of THESC and HDSC, the latter showing loss of cells after 6 days of treatment. E2P4 stimulation did neither affect cell morphology nor proliferation of THESC and HDSC. Upon cAMP stimulation PR mRNA was suppressed in HDSC but not in THESC, whereas E2P4 did not alter transcript levels in both cell types. Protein expression of PR-A and PR-B was detectable in HDSC and diminished under cAMP, whereas THESC failed to produce the nuclear receptors. Supplementation of cAMP induced mRNA and protein expression of PRL and IGFBP-1 in both cell types at day 3, 6, 9, and 12 of treatment. In HDSC stimulation with E2P4 increased PRL and IGFBP-1 mRNA and protein production, whereas hormone treatment did not induce the two factors in THESC. E2P4 increased DKK1 mRNA at all time points in HDSC and cAMP provoked induction at day 9 and 12 of differentiation. In contrast, cAMP suppressed DKK1 mRNA in THESC, whereas E2P4 was ineffective. In both cell types combined treatments with cAMP and E2P4 provoked higher expression levels of PRL and IGFBP1 mRNA and protein as compared to cAMP stimulation alone. FOXO1A protein and its nuclear abundance were increased by cAMP in both cell types. However, reduction of its nuclear localisation upon E2P4 treatment could only be observed in HDSC.ConclusionBoth HDSC and THESC may represent suitable model systems for cAMP-dependent in vitro decidualization. Since cAMP decreases cell viability of HDSC after 6 days of incubation, this substance should be preferentially used in short-term experiments. Progesterone treatment of THESC might not be applicable since these cells lack progesterone response and PR protein. In contrast, stimulation of PR-expressing HDSC with E2P4 or cAMP/E2P4 may represent an appropriate protocol for human in vitro decidualization inducing and maintaining expression of critical marker genes in a time-dependent manner.

Increased Activation of the PI3K/AKT Pathway Compromises Decidualization of Stromal Cells from Endometriosis

Endometriosis affects approximately 10% of women in the United States and causes pain and infertility. Decidualization of endometrial stromal cells from women with endometriosis is aberrant. The objective of this study was to investigate a potential mechanism for the inadequate decidual response in stromal cells from ovarian endometriomas. Stromal cells of the endometrium from women without endometriosis (HSC) or from ovarian endometriomas (OsisSC) were grown in culture and treated with 10 μm LY294002 or 250 nm MK2206, 100 nm medroxyprogesterone acetate (M), and 0.5 mm dibutyryl cAMP (A) or infection with 100 multiplicity of infection adenoviral constructs containing wild-type Forkhead box O1 or triple-mutant FOXO1. Real-time PCR was used to measure the expression of FOXO1, IGF binding protein-1 (IGFBP1), and prolactin (PRL) mRNA, and Western blot and immunohistochemical staining were used to detect the levels of progesterone receptor (PR), FOXO1, AKT, and p(Ser473)-AKT protein in vitro or in vivo. Expression of the decidua-specific genes, IGFBP1 and PRL, were significantly lower in OsisSC compared with normal HSC in response to M+A treatment. Basal expression levels of PRA, PRB, and FOXO1 proteins were dramatically lower in OsisSC. Overexpression of triple-mutant FOXO1 increased mRNA levels of IGFBP1 and PRL in OsisSC in the presence of M+A, whereas the overexpression of wild-type FOXO1 had no effect. AKT was highly phosphorylated in OsisSC compared with HSC and inhibition of phosphatidylinositol 3-kinase, with LY294002, increased levels of FOXO1 protein as well as IGFBP1 mRNA in the presence of M+A. Moreover, inhibition of AKT with MK2206, an allosteric AKT inhibitor, dramatically increased the accumulation of nuclear FOXO1 as well as expression of IGFBP1. Finally, immunohistochemical staining demonstrated higher p(Ser473)-AKT and lower FOXO1 levels in endometriosis tissues, compared with normal endometrial tissues. In endometriotic stromal cells, overactivation of the phosphatidylinositol 3-kinase/AKT signaling pathway contributes to the reduced expression of the decidua-specific gene, IGFBP1, potentially through reduced levels of nuclear FOXO1.

High IGFBP2 Expression Correlates with Tumor Severity in Pediatric Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common childhood sarcoma and is identified as either the embryonal or alveolar (ARMS) subtype. In approximately 75% of cases, ARMSs are characterized by specific chromosomal translocations that involve PAX and FKHR genes. ARMS gene expression signatures vary, depending on the presence or absence of the translocations. Insulin-like growth factor-binding protein 2 (IGFBP2) is strongly overexpressed in translocation-negative RMS. Because IGFBP2 is associated with tumorigenesis, we investigated its functional role in RMS. An analysis of IGFBP2 distribution in RMS cell lines revealed a strong accumulation in the Golgi complex, in which morphological characteristics appeared peculiarly modified. After silencing IGFBP2 expression, our microarray analysis revealed mostly cell cycle and actin cytoskeleton gene modulations. In parallel, IGFBP2-silenced cells showed reduced cell cycle and rates of invasion and decreased seeding in the lungs after tail vein injections in immunodeficient mice. An analysis of IGFBP2 mRNA and protein localization in human tumors showed abnormal protein accumulation in the Golgi complex, mostly in PAX/FKHR-negative RMS. Moreover, an analysis of patients with RMS revealed the presence of conspicuous circulating levels of IGFBP2 proteins in children with highly aggressive RMS tumors. Taken together, our data provide evidence that IGFBP2 contributes to tumor progression and that it could be used as a marker to better classify clinical and biological risks in RMS.

Increased expression of insulin-like growth factor-binding protein-3 is implicated in erectile dysfunction in two-kidney one-clip hypertensive rats after propranolol treatment

This study aimed to investigate the role of insulin-like growth factor-binding protein-3 (IGFBP-3) in erectile dysfunction (ED) in two-kidney one-clip (2K-1C) hypertensive rats treated with the β-blocking agent propranolol. Adult male Wistar rats were randomly divided into three groups: a normal control group, a hypertensive control group and a propranolol treatment group (n=9). After 4 weeks of propranolol treatment, intracavernous pressure (ICP) responses to electrical stimulation of the cavernous nerves were evaluated. The expression of IGFBP-3 and insulin-like growth factor-1 (IGF-1) mRNA and protein in the rat cavernous tissue were detected by quantitative real-time PCR and Western blot, respectively. The concentration of cyclic guanosine monophosphate (cGMP) in the cavernous tissue was determined by enzyme-linked immunosorbent assay (ELISA). Cavernosal pressure in response to cavernous nerve stimulation was decreased 4 weeks after propranolol treatment (P<0.01, compared to the hypertensive control group). IGFBP-3 mRNA and protein expression was increased in the propranolol treatment group compared to the hypertensive control group (P<0.01), whereas IGF-1 expression was decreased in the propranolol treatment group compared to the hypertensive control group (P<0.01). In addition, cavernous cGMP concentration was decreased in the propranolol treatment group compared to the hypertensive control group (P<0.01). Taken together, these results suggest that the upregulation of IGFBP-3 may play a role in the development of ED in hypertensive rats.

Expression of insulin-like growth factor binding protein-4 (IGFBP-4) in acute pancreatitis induced by l-arginine in mice

The mechanisms of injury and regeneration after acute pancreatitis are still incompletely understood. Insulin-like growth factor binding proteins (IGFBPs) have been reported to play roles in various pancreatic diseases, but the involvement of insulin-like growth factor binding protein-4 (IGFBP-4) in acute pancreatitis is unknown. The aim of the study was to examine the expression of IGFBP-4 in mice with acute pancreatitis induced by two doses of l-arginine. IGFBP-4 expression was assayed by microarray test, real-time RT-PCR, Western blotting, ELISA and by an immunohistochemical assay. Microarray test of pancreatic mRNA showed that IGFBP-4 mRNA increased significantly after l-arginine treatment and the increase was confirmed by real-time RT-PCR. Western blotting and ELISA assay showed similar patterns of increase of IGFBP-4 in pancreatic tissues and serum. In the control pancreas, IGFBP-4 was mainly immunolocalized in the pancreatic islets. In the pancreatic tissues of mice with pancreatitis induced by l-arginine, the immunolocalization of IGFBP-4 was detected in both acinar cells and pancreatic islets. In conclusion, our results suggest that IGFBP-4 may play a potential role in pancreatic injury and regeneration in a murine model of acute pancreatitis induced by l-arginine.

IGF binding protein‐6 expression in vascular endothelial cells is induced by hypoxia and plays a negative role in tumor angiogenesis

Hypoxia stimulates tumor angiogenesis by inducing the expression of angiogenic molecules. The negative regulators of this process, however, are not well understood. Here, we report that hypoxia induced the expression of insulin-like growth factor binding protein-6 (IGFBP-6), a tumor repressor, in human and rodent vascular endothelial cells (VECs) via a hypoxia-inducible factor (HIF)-mediated mechanism. Addition of human IGFBP-6 to cultured human VECs inhibited angiogenesis in vitro. An IGFBP-6 mutant with at least 10,000-fold lower binding affinity for IGFs was an equally potent inhibitor of angiogenesis, suggesting that this action of IGFBP-6 is IGF-independent. The functional relationship between IGFBP-6 and vascular endothelial growth factor (VEGF), a major hypoxia-inducible angiogenic molecule, was examined. While VEGF alone increased angiogenesis in vitro, co-incubation with IGFBP-6 abolished VEGF-stimulated angiogenesis. The in vivo role of IGFBP-6 in angiogenesis was tested in flk1:GFP zebrafish embryos, which exhibit green fluorescence protein in developing vascular endothelium, permitting visualization of developing blood vessels. Injection of human IGFBP-6 mRNA reduced the number of embryonic inter-segmental blood vessels by ∼40%. This anti-angiogenic activity is conserved in zebrafish because expression of zebrafish IGFBP-6b had similar effects. To determine the anti-angiogenic effect of IGFBP-6 in a tumor model, human Rh30 rhabdomyosarcoma cells stably transfected with IGFBP-6 were inoculated into athymic BALB/c nude mice. Vessel density was 52% lower in IGFBP-6-transfected xenografts than in vector control xenografts. These results suggest that the expression of IGFBP-6 in VECs is up-regulated by hypoxia and IGFBP-6 inhibits angiogenesis in vitro and in vivo.

Characterization of three IGFBP mRNAs in Atlantic croaker and their regulation during hypoxic stress: potential mechanisms of their upregulation by hypoxia

Insulin-like growth factor-binding proteins (IGFBPs) play important roles in downregulating IGF activity and growth and development in vertebrates under hypoxic stress. However, the mechanisms of hypoxia regulation of IGFBPs in teleost fishes are unknown. The involvement of reactive oxygen species (ROS) and hypoxia-inducible factors (HIFs) in hypoxia upregulation of IGFBPs in Atlantic croaker were investigated. Three croaker IGFBPs, IGFBP-1, IGFBP-2, and IGFBP-5, were cloned and characterized. Chronic hypoxia exposure [dissolved oxygen (DO): 1.7 mg/l for 2-4 wk] caused significant increases in hepatic and neural IGFBP-1 mRNA expression compared with tissue mRNA levels in fish held under normoxic conditions (6.5 mg DO/l). Moreover, longer-term chronic hypoxia exposure (2-2.7 mg DO/l for 15-20 wk) caused significant increases in mRNA levels of all three IGFBPs in both liver and brain tissues. Hypoxia exposure also markedly increased superoxide radical (O(2)(·-), an index of ROS) production and HIF-1α mRNA and HIF-2α protein expression in croaker livers. Pharmacological treatment with an antioxidant attenuated the hypoxia-induced increases in O(2)(·-) production and HIFα mRNA and protein expression as well as the elevation of IGFBP-1 mRNA levels. These results suggest that the upregulation of IGFBP expression under hypoxia stress is due, in part, to alterations in the antioxidant status, which may involve ROS and HIFs.

Oncogenic Fusion Protein EWS/FLI1 Down-regulates Gene Expression by Both Transcriptional and Posttranscriptional Mechanisms

Ewing family tumors are characterized by a translocation between the RNA binding protein EWS and one of five ETS transcription factors, most commonly FLI1. The fusion protein produced by the translocation has been thought to act as an aberrant transcription factor, leading to changes in gene expression and cellular transformation. In this study, we investigated the specific processes EWS/FLI1 utilizes to alter gene expression. Using both heterologous NIH 3T3 and human Ewing Family Tumor cell lines, we have demonstrated by quantitative pre-mRNA analysis that EWS/FLI1 repressed the expression of previously validated direct target genes at the level of transcript synthesis. ChIP experiments showed that EWS/FLI1 decreases the amount of Pol II at the promoter of down-regulated genes in both murine and human model systems. However, in down-regulated target genes, there was a significant disparity between the modulation of cognate mRNA and pre-mRNAs, suggesting that these genes could also be regulated at a posttranscriptional level. Confirming this, we found that EWS/FLI1 decreased the transcript half-life of insulin-like growth factor binding protein 3, a down-regulated direct target gene in human tumor-derived Ewing's sarcoma cell lines. Additionally, we have shown through reexpression experiments that full EWS/FLI1-mediated transcriptional repression requires intact EWS and ETS domains. Together these data demonstrate that EWS/FLI1 can dictate steady-state target gene expression by modulating both transcript synthesis and degradation.

Insulin-Like Growth Factor-Binding Protein-3 Mediates High Glucose-Induced Apoptosis by Increasing Oxidative Stress in Proximal Tubular Epithelial Cells

IGF-binding protein-3 (IGFBP-3) is the major circulating carrier protein for IGF, and also acts as a potent antiproliferative agent in various cell types. Recently, IGFBP-3 was reported to mediate high glucose-induced apoptosis in mesangial cells and podocytes. In this study, we investigated the role of IGFBP-3 in high glucose-induced apoptosis in proximal tubular epithelial cells (PTEC). Expression of IGFBP-3 protein and mRNA in a porcine PTEC line (LLC-PK1 cells) was measured after exposure to either standard (5.5 mM) or high-glucose (30 mM) medium. We quantified apoptosis after treatment with small interfering RNA against IGFBP-3 (siRNA:IGFBP-3) in high-glucose medium or in cells that overexpressed IGFBP-3. Oxidative stress was measured in high-glucose medium, in the presence of siRNA:IGFBP-3, or in IGFBP-3-overexpressing cells. IGFBP-3 protein and mRNA expression in LLC-PK1 cells was higher in high-glucose medium than in standard-glucose medium. Exposure to high-glucose medium increased apoptosis, and high-glucose-induced apoptosis was abolished by siRNA:IGFBP-3. IGFBP-3 overexpression induced apoptosis in LLC-PK1 cells. Both high-glucose medium and IGFBP-3 overexpression increased reactive oxygen species, and siRNA:IGFBP-3 reduced this increase. Antioxidant treatment decreased IGFBP-3 expression and apoptosis, whereas oxidative stress from hydrogen peroxide increased IGFBP-3 expression, suggesting that oxidative stress increases IGFBP-3 expression. Our results suggest that increased IGFBP-3 expression by high glucose mediates high-glucose-induced apoptosis in PTEC. Increased oxidative stress from high glucose enhances IGFBP-3 expression, inducing apoptosis. Increased expression of IGFBP-3 by high glucose induces additional oxidative stress, which may result in amplification of hyperglycemic damage.

Insulin-like growth factor-binding protein-2 promotes prostate cancer cell growth via IGF-dependent or -independent mechanisms and reduces the efficacy of docetaxel

Background:The development of androgen independence, chemo-, and radioresistance are critical markers of prostate cancer progression and the predominant reasons for its high mortality. Understanding the resistance to therapy could aid the development of more effective treatments.Aim:The aim of this study is to investigate the effects of insulin-like growth factor-binding protein-2 (IGFBP-2) on prostate cancer cell proliferation and its effects on the response to docetaxel.Methods:DU145 and PC3 cells were treated with IGFBP-2, insulin-like growth factor I (IGF-I) alone or in combination with blockade of the IGF-I receptor or integrin receptors. Cells were also treated with IGFBP-2 short interfering ribonucleic acid with or without a PTEN (phosphatase and tensin homologue deleted on chromosome 10) inhibitor or docetaxel. Tritiated thymidine incorporation was used to measure cell proliferation and Trypan blue cell counting for cell death. Levels of IGFBP-2 mRNA were measured using RT–PCR. Abundance and phosphorylation of proteins were assessed using western immunoblotting.Results:The IGFBP-2 promoted cell growth in both cell lines but with PC3 cells this was in an IGF-dependent manner, whereas with DU145 cells the effect was independent of IGF receptor activation. This IGF-independent effect of IGFBP-2 was mediated by interaction with β-1-containing integrins and a consequent increase in PTEN phosphorylation. We also determined that silencing IGFBP-2 in both cell lines increased the sensitivity of the cells to docetaxel.Conclusion:The IGFBP-2 has a key role in the growth of prostate cancer cells, and silencing IGFBP-2 expression reduced the resistance of these cells to docetaxel. Targeting IGFBP-2 may increase the efficacy of docetaxel.

IGF binding protein 1 is correlated with hypoxia-induced growth reduce and developmental defects in grass carp (Ctenopharyngodon idellus) embryos

The effects of hypoxia on embryonic development and the underlying cellular and molecular mechanisms are poorly understood in teleost fish, although the hypoxic effects on embryonic growth and development have been reported in the zebrafish model. Here, we found that hypoxia caused significant developmental delay and growth retardation during grass carp embryogenesis. Placing the embryos in hypoxic conditions at different developmental stages strongly induced the mRNA expression of insulin-like growth factor-binding protein 1 (IGFBP1), an inhibitory protein that binds to IGF and inhibits its subsequent actions in vivo. Further gain-of-function analysis results provided strong evidence to support the hypothesis that IGFBP1 plays an important role in mediating hypoxic-induced growth and developmental defects. Overexpression of IGFBP1 mRNA reduced the growth rate to a degree that was similar to hypoxia. Additionally, overexpression of IGFBP1 caused significant developmental defects in the formation of midline, somite and hindbrain structures during grass carp embryogenesis. Taken together, our studies suggest that IGFBP1 plays a key role in mediating these hypoxia-induced embryonic growth retardation and developmental delay in grass carp.

IGFBP-1 is expressed specifically in ovarian clear cell adenocarcinoma

To investigate the specific expression of insulin-like growth factor binding protein-1 (IGFBP-1) in ovarian clear cell adenocarcinoma (CCA). Immunohistochemistry and in situ hybridization for IGFBP-1 were performed in normal endometrium, placenta, and 100 surgically resected cases of ovarian cancer including 31 CCAs and 69 non-CCAs. Immunohistochemistry for hepatocyte nuclear factor-1 (HNF-1β) was also examined in all cases. Specific expression of IGFBP-1 was confirmed in secretory endometrium, decidua of placenta and Arias-Stella glands of miscarriage material. Among ovarian cancers, almost all cases of CCA showed expression of both IGFBP-1 protein and mRNA, but non-CCA hardly expressed IGFBP-1. There was a significant difference between CCA and non-CCA in the expression of IGFBP-1 protein and mRNA. No correlation was found between the rate of IGFBP-1 expression and pathological T and N factors of the tumour-node-metastasis (TNM) classification. All CCA cases except for one exhibited expression of HNF-1β protein, whereas only 15.9% of non-CCAs did so. The expression of IGFBP-1 in CCA is more specific than that of HNF-1β. IGFBP-1 shows expression by decidual endometrium and Arias-Stella glands, and CCA also exhibits characteristic expression. These results indicate that IGFBP-1 is a immunohistochemical marker for CCA.