IGFBP-3 mRNA Research Articles

Grade-Specific Expression of Insulin-like Growth Factor–Binding Proteins-2, -3, and -5 in Astrocytomas: IGFBP-3 Emerges as a Strong Predictor of Survival in Patients with Newly Diagnosed Glioblastoma

Insulin-like growth factor (IGF)-binding protein (IGFBP) isoforms have been implicated in the pathogenesis of human neoplasms including glioma. In view of this, we evaluated the expression of IGFBP isoforms (IGFBP-2, -3, and -5) during malignant progression of astrocytoma and their prognostic significance in glioblastoma. The expression of IGFBP isoforms was analyzed in diffusely infiltrating astrocytomas by real-time quantitative PCR (n = 203) and immunohistochemistry (n = 256). Statistical methods were used to assess their grade-specific expression pattern and mRNA-protein intercorrelation. Survival analyses were done on a uniformly treated, prospective cohort of adult patients with newly diagnosed glioblastoma (n = 136) by using Cox regression models. The mean transcript levels of IGFBP-2 and -3 were significantly higher in glioblastomas (GBM) relative to anaplastic astrocytoma (AA), diffuse astrocytoma (DA), and controls whereas IGFBP-5 mRNA was higher in GBM relative to AA and controls (P < 0.05). By immunohistochemistry, the mean labeling index of all isoforms was significantly higher in GBM compared with AA, DA, and control (P < 0.05). A strong positive correlation was observed between their respective mRNA and protein expressions (P < 0.01). Multivariate analysis revealed IGFBP-3 expression (hazard ratio, 1.021; P = 0.030) and patient age (hazard ratio, 1.027; P = 0.007) to be associated with shorter survival in glioblastoma. This study shows the associations of IGFBP-2, -3, and -5 expression with increasing grades of malignancy in astrocytomas. IGFBP-3 is identified as a novel prognostic glioblastoma biomarker. The strong correlation between their mRNA and protein expression patterns suggests their role in the pathogenesis of these tumors. IGFBP isoforms have emerged as biomarkers with diagnostic and prognostic utility in astrocytomas.

Skeletal muscle catabolism in trinitrobenzene sulfonic acid–induced murine colitis

The present study determined whether the muscle atrophy produced by colitis is associated with altered rates of muscle protein synthesis or degradation, as well as the potential role of the local (eg, muscle) insulin-like growth factor (IGF) system and muscle-specific ubiquitin E3 ligases atrogin-1 and MuRF1 in mediating altered muscle protein balance. Colitis was induced in C57BL/6 mice by intrarectal administration of trinitrobenzene sulfonic acid (TNBS), and blood and tissues were collected on day 10. Mice with inflammatory bowel disease demonstrated reduced skeletal muscle mass and protein content, whereas colonic segment weight and gross damage score were both increased in mice with colitis, compared with time-matched control values. There was no change in muscle protein synthesis in mice with inflammatory bowel disease; but there was an increased protein breakdown (45%), proteasome activity (85%), and messenger RNA (mRNA) expression for atrogin-1 and MuRF1 (200%-300%) in muscle. These changes were associated with a reduction in liver (but not muscle) IGF-I mRNA as well as a reduction in both total and free IGF-I in the blood. Colitis decreased the hepatic content of IGF binding protein (IGFBP)–3 mRNA by 40% and increased IGFBP-1 mRNA by 100%. In contrast, colitis did alter IGFBP mRNAs in muscle. The tumor necrosis factor– α, interleukin-6, and nitric oxide synthase 2 mRNA content of both liver and skeletal muscle was increased in TNBS-treated mice; and plasma tumor necrosis factor– α and interleukin-6 concentrations were also elevated. These data suggest that TNBS-induced colitis is independent of a change in muscle protein synthesis but dependent on stimulation of protein degradation via increased expression of muscle-specific atrogenes, which may be mediated in part by the reduction in circulating concentration of IGF-I and the concomitant increase in inflammatory mediators observed in the blood and muscle per se.

MRNA expression pattern of insulin-like growth factor components of granulosa cells and cumulus cells in women with and without polycystic ovary syndrome according to oocyte maturity

The mRNA expression of insulin-like growth factor (IGF) components was investigated in granulosa cells (GCs) and cumulus cells (CCs) sampled from immature and mature follicles in women with and without polycystic ovary syndrome (PCOS). Gene expression for IGF-binding protein (IGFBP) 3 in GCs definitely differed between normal women and women with PCOS, whereas increase in IGF-I and IGF receptor (IGFR) 2 mRNA in mature-follicle GCs, increase in IGF-II, IGFR-1, and IGFBP-4 mRNA in immature-follicle GCs, increase in IGFR-2 and IGFBP-2 mRNA in mature-follicle CCs, and increase IGFBP-6 mRNA in immature-follicle CCs were observed in both normal women and women with PCOS.

Oral inoculation with Salmonella enterica serovar Typhimurium or Choleraesuis promotes divergent responses in the somatotropic growth axis of swine1

Enteric disease and immune challenge are processes that have detrimental effects on the growth performance of young swine. The current study tested the hypothesis that salmonella-induced enteric disease would perturb the endocrine growth axis in a serovar-dependent fashion. Specifically, we evaluated the effects of Salmonella enterica serovar Typhimurium (Typhimurium) and serovar Choleraesuis (Choleraesuis) on critical regulatory components of growth in young swine. Weaned pigs were housed 2 per pen with ad libitum access to feed and water in a 14-d experiment. Pigs were then repeatedly fed 10(8) cfu of either Choleraesuis or Typhimurium in dough balls, with control pigs receiving dough without bacteria. Bacteria were refed twice weekly. Rectal temperatures were monitored daily from d 0 to 7 and ADFI was measured through d 14. Pigs were weighed and samples of serum were obtained for circulating IGF-I on d 0, 7, and 14. At the conclusion of the study, samples of semitendinosus muscle and liver were obtained and subsequently assayed for IGF-I, IGFBP-3, and IGFBP-5 mRNA. Rectal temperatures were elevated in pigs given Choleraesuis from d 2 through 7 (P < 0.05) when compared with control pigs and pigs fed Typhimurium. Pigs receiving Choleraesuis had a substantially decreased feed intake on d 2, 3, 4, 7, 8, 9, and 10 (P < 0.01), with a trend for a reduction on d 5 (P = 0.08), and they experienced an approximately 25% reduction in BW compared with control pigs and pigs given Typhimurium by the conclusion of the study. Pigs given Choleraesuis also experienced marked reductions in circulating IGF-I on d 7 (P < 0.01 vs. control and Typhimurium), with reductions of lesser magnitude on d 14 (P = 0.07 vs. control and P < 0.05 vs. Typhimurium). Inoculation tended to affect liver IGFBP-3 mRNA (P = 0.08), for which expression tended to be elevated in pigs given Typhimurium and Choleraesuis. In contrast, IGFBP-3 mRNA relative abundance was increased (P < 0.03) in pigs given Typhimurium compared with control pigs. Muscle IGF-I mRNA was reduced in pigs given Choleraesuis compared with control pigs and pigs given Typhimurium (P < 0.05). Treatment tended to affect muscle IGFBP-3 mRNA (P = 0.10). Oral inoculation of growing pigs with Choleraesuis disrupted feed intake and BW gain, and this was accompanied by decreases in circulating IGF-I and reduced muscle expression of mRNA for IGF-I and IGFBP-3.

IL-6 deficiency in mice neither impairs induction of metabolic genes in the liver nor affects blood glucose levels during fasting and moderately intense exercise

Fasting and exercise are strong physiological stimuli for hepatic glucose production. IL-6 has been implicated in the regulation of gluconeogenic genes, but the results are contradictory and the relevance of IL-6 for fasting- and exercise-induced hepatic glucose production is not clear. Investigations were performed in rat hepatoma cells, and on C57Bl6 and Il6(-/-) mice under the following conditions: IL-6 stimulation/injection, non-exhaustive exercise (60 min run on a treadmill) and fasting for 16 h. Metabolite analysis, quantitative real-time PCR and immunoblotting were performed. IL-6 stimulation of rat hepatoma cells led to higher glucose production. Injection of IL-6 in mice slightly increased hepatic Pepck (also known as Pck1) expression. Fasting of Il6(-/-) mice for 16 h did not alter glucose production compared with wild-type mice, since plasma glucose concentrations were similar and upregulation of phosphoenolpyruvate carboxykinase (PEPCK) and Pgc-1alpha (also known as Ppargc1a) expression was comparable. In the non-fasting state, Il6(-/-) mice showed a mild metabolic alteration including higher plasma glucose and insulin levels, lower NEFA concentrations and slightly increased hepatic PEPCK content. Moderately intense exercise resulted in elevated IL-6 plasma levels in wild-type mice. Despite that, plasma glucose, insulin, NEFA levels and hepatic glycogen content were not different in Il6(-/-) mice immediately after running, while expression of hepatic G6pc, Pgc-1alpha, Irs2 and Igfbp1 mRNA was similarly increased. These data suggest that in mice IL-6 is not essential for physiologically increased glucose production during fasting or non-exhaustive exercise.

Abstract 5478: Melatonin, a novel Sirt1 inhibitor, imparts anti-proliferative effects against PCa in vitro in cell culture and in vivo in transgenic adenocarcinoma of mouse prostate (TRAMP) model

Abstract Prostate cancer (PCa) is a major age-related malignancy because an increase in age correlates with increased risk for developing this neoplasm. Recently, we have shown that Sirt1, a member of the NAD+ dependent histone deacetylase family of sirtuin proteins, was overexpressed in PCa cells and human PCa tissues compared to normal prostate cells and the adjacent normal prostate tissue, respectively. We also demonstrated that chemical inhibition or genetic knockdown of Sirt1 resulted in a significant inhibition in the growth and viability of human PCa cells while having no effect on normal human prostate epithelial cells (PrEC). Studies have suggested a link between the cellular metabolic function of Sirt1 and the circadian rhythm, and the disruption of circadian rhythm has been linked with an increased risk for some cancers. Interestingly, a decreased production of the pineal hormone melatonin, a known regulator of circadian rhythm, has been shown to cause deregulation in the circadian rhythm and an increase in susceptibility to cancer. Further, disruption in circadian rhythmicity has also been associated with aging. It has been reported that as an individual ages, the ability to produce melatonin at night is decreased and PCa patients have been reported to have decreased levels of melatonin. Here we tested the hypothesis that the circadian rhythm regulator melatonin will impart anti-proliferative response against PCa via inhibiting Sirt1. Our data demonstrated that melatonin treatments (high, low acute, low chronic doses) significantly inhibited Sirt1 protein and activity in vitro in multiple PCa cell lines (LNCaP, 22Rv1, DU145, and PC3). Melatonin-mediated Sirt1 inhibition was accompanied with a significant decrease in the proliferative capacity of PCa cells while having no effect on PrEC, as assessed by Trypan blue and anchorage-independent growth assays. Further, we found that overexpression of Sirt1 in PCa cells was able to partially rescue the cells from the anti-proliferative effects of melatonin, suggesting that Sirt1 is a direct target of the anti-proliferative effects of melatonin. Finally, employing male TRAMP mice, we found that oral administration of melatonin at human achievable doses significantly inhibited PCa growth. Melatonin treatment was found to result in a significant decrease in (i) prostate and genitourinary weight, (ii) serum IGF-1/IGFBP3 ratio, (iii) mRNA and protein levels of the proliferation markers (PCNA, Ki-67), and (iv) a down-regulation of Sirt1 in prostatic tissue. Our data identified melatonin as a novel inhibitor of Sirt1 and suggested that this pineal hormone can inhibit PCa growth via Sirt1 inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5478.

Impact of treadmill locomotor training on skeletal muscle IGF1 and myogenic regulatory factors in spinal cord injured rats.

The objective of this study was to determine the impact of treadmill locomotor training on the expression of insulin-like growth factor I (IGF1) and changes in myogenic regulatory factors (MRFs) in rat soleus muscle following spinal cord injury (SCI). Moderate, midthoracic (T(8)) contusion SCIs were produced using a NYU (New York University) impactor. Animals were randomly assigned to treadmill training or untrained groups. Rats in the training group were trained starting at 1 week after SCI, for either 3 bouts of 20 min over 1.5 days or 10 bouts over 5 days. Five days of treadmill training completely prevented the decrease in soleus fiber size resulting from SCI. In addition, treadmill training triggered increases in IGF1, MGF and IGFBP4 mRNA expression, and a concurrent reduction of IGFBP5 mRNA in skeletal muscle. Locomotor training also caused an increase in markers of muscle regeneration, including small muscle fibers expressing embryonic myosin and Pax7 positive nuclei and increased expression of the MRFs, myogenin and MyoD. We concluded that treadmill locomotor training ameliorated muscle atrophy in moderate contusion SCI rats. Training-induced muscle regeneration and fiber hypertrophy following SCI was associated with an increase in IGF1, an increase in Pax7 positive nuclei, and upregulation of MRFs.

Modulating peripheral gonadotrophin levels affects follicular expression of mRNAs encoding insulin-like growth factor binding proteins in sheep

Evidence suggests that the insulin-like growth factor binding proteins (IGFBPs) are involved in modulating the role that IGF-I and -II play in regulating follicular growth and development in sheep. However, little information exists as to the role that key peripheral factors play in regulating the expression of IGFBP components within the follicle. The present study investigated the regulatory effects of FSH and LH on gene expression for IGFBP-2 to -6 in ovine follicles, using bovine follicular fluid (bFF) and gonadotrophin-releasing hormone antagonist (GnRHa) model systems to perturb endogenous gonadotrophin secretion. Gene expression studies were carried out using in situ hybridisation with sheep-specific ribonucleotide probes. Treatment of ewes with bFF had few consistent effects on IGFBP-3 and -4 mRNA expression in follicles, whereas bFF treatment resulted in significant decreases in IGFBP-2 and IGFBP-6 mRNA expression in only medium follicles 60 h after onset of bFF, and in follicles 12 h after the onset of bFF, respectively. Treatment of ewes with GnRHa resulted in transient significantly increased IGFBP-2 mRNA in healthy follicles in comparison to early atretic or atretic follicles 12 h post-GnRHa ( P < 0.05), in IGFBP-3 mRNA in early atretic and atretic follicles 36 h post-GnRHa ( P < 0.001), and IGFBP-5 mRNA in follicles 12 h post-GnRHa ( P < 0.001). In contrast, GnRHa treatment resulted in significant decreases in IGFBP-4 ( P < 0.001) and IGFBP-6 ( P < 0.01) mRNA expression in large follicles 36 h post-GnRHa, and atretic follicles by 60 h post-GnRHa, respectively. These data highlight that FSH and LH are involved, at least in part, in mediating the proliferative and differentiative changes in intrafollicular IGFBP levels that are observed during follicular growth and atresia in the sheep.

Effect of Prostaglandin F2 Alpha on Local Luteotropic and Angiogenic Factors During Induced Functional Luteolysis in the Bovine Corpus Luteum1

The essential role of endometrial prostaglandin F2 alpha (PTGF) for induction of the corpus luteum (CL) regression is well documented in the cow. However, the acute effects of PTGF on known local luteotropic factors (oxytocin [OXT] and its receptor, insulin-like growth factor [IGF] 1, and progesterone and its receptor), the principal angiogenic factor vascular endothelial growth factor (VEGF) A and the capillary destabilization factor angiopoietin (ANGPT) 2 were not thoroughly studied in detail. The aim of this study was therefore to evaluate the tissue concentration of these factors during PTGF induced luteolysis. In addition the mRNA expression of progesterone receptor (PGR), OXT receptor (OXTR), IGF1, IGFBP1, ANGPT1, and ANGPT2 was determined at different times after PTGF treatment. Cows (n = 5 per group) in the mid-luteal phase (Days 8-12, control group) were injected with the PTGF analog (cloprostenol), and CL were collected by transvaginal ovariectomy at 0.5, 2, 4, 12, 24, 48, and 64 h after injection. The mRNA expression was analyzed by quantitative real-time PCR, and the protein concentration was evaluated by enzyme immunoassay or radioimmunoassay. Progesterone concentrations, as well as mRNA expression of PGR, in CL tissue were significantly down-regulated by 12 h after PTGF. Tissue OXT peptide and OXTR mRNA decreased significantly after 2 h, followed by a continuous decrease of OXT mRNA. IGF1 and VEGFA protein already decreased after 0.5 h. By contrast, the IGFBP1 mRNA was up-regulated significantly after 2 h to a high plateau. ANGPT2 protein and mRNA significantly increased during the first 2 h, followed by a steep decrease after 4 h. The acute decrease of local luteotropic activity and acute changes of ANGPT2 and VEGFA suggest that modulation of vascular stability may be a key component in the cascade of events leading to functional luteolysis.

Altered placental expression of PAPPA2 does not affect birth weight in mice

BackgroundPregnancy-associated plasma protein A2 (PAPPA2) is an insulin-like growth factor binding protein (IGFBP) protease expressed in the placenta and upregulated in pregnancies complicated by pre-eclampsia. The mechanism linking PAPPA2 expression and pre-eclampsia and the consequences of altered PAPPA2 expression remain unknown. We previously identified PAPPA2 as a candidate gene for a quantitative trait locus (QTL) affecting growth in mice and in the present study examined whether this QTL affects placental PAPPA2 expression and, in turn, placental or embryonic growth.MethodsUsing a line of mice that are genetically homogenous apart from a 1 megabase QTL region containing the PAPPA2 gene, we bred mice homozygous for alternate QTL genotypes and collected and weighed placentae and embryos at E12.5. We used quantitative RT-PCR to measure the mRNA levels of PAPPA2, as well as mRNA levels of IGFBP-5 (PAPPA2's substrate), and PAPPA (a closely related IGFBP protease) to examine potential feedback and compensation effects. Western blotting was used to quantify PAPPA2 protein. Birth weight was measured in pregnancies allowed to proceed to parturition.ResultsPAPPA2 mRNA and protein expression levels in the placenta differed by a factor of 2.5 between genotypes, but we did not find a significant difference between genotypes in embryonic PAPPA2 mRNA levels. Placental IGFBP-5 and PAPPA mRNA expression levels were not altered in response to PAPPA2 levels, and we could not detect IGFBP-5 protein in the placenta by Western blotting. The observed difference in placental PAPPA2 expression had no significant effect on placental or embryonic mass at mid-gestation, birth weight or litter size.ConclusionsDespite a significant difference between genotypes in placental PAPPA2 expression similar in magnitude to the difference between pre-eclamptic and normal placentae previously reported, we observed no difference in embryonic, placental or birth weight. Our results suggest that elevated PAPPA2 levels are a consequence, rather than a cause, of pregnancy complications.

OBSL1mutations in 3-M syndrome are associated with a modulation ofIGFBP2andIGFBP5expression levels

3-M syndrome is an autosomal recessive disorder characterized by severe pre- and postnatal growth retardation and minor skeletal changes. We have previously identified CUL7 as a disease-causing gene but we have also provided evidence of genetic heterogeneity in the 3-M syndrome. By homozygosity mapping in two inbred families, we found a second disease locus on chromosome 2q35-36.1 in a 5.2-Mb interval that encompasses 60 genes. To select candidate genes, we performed microarray analysis of cultured skin fibroblast RNA from one patient, looking for genes with altered expression; we found decreased expression of IGFBP2 and increased expression of IGFBP5. However, direct sequencing of these two genes failed to detect any anomaly. We then considered other candidate genes by their function/location and found nine distinct mutations in the OBSL1 gene in 13 families including eight nonsense and one missense mutations. To further understand the links between OBSL1, CUL7, and insulin-like growth factor binding proteins (IGFBPs), we performed real-time quantitative PCR (RT-PCR) analysis for OBSL1, CUL7, IGFBP2, and IGFBP5, using cultured fibroblast RNAs from two patients with distinct OBSL1 mutations (p.F697G; p.H814RfsX15). We found normal CUL7 mRNA levels but abnormal IGFBP2 and IGFBP5 mRNA levels in the two patients, suggesting that OBSL1 modulates the expression of IGFBP proteins.

Downregulation of insulin-like growth factor binding protein 3 and 5 in nitrofen-induced pulmonary hypoplasia

The high mortality in congenital diaphragmatic hernia (CDH) is mainly attributed to pulmonary hypoplasia. Recent studies suggest that retinoid signaling pathway (RSP) is inhibited in the nitrofen-induced hypoplastic lung. The insulin-like growth factor (IGF) system plays a crucial role in fetal lung development by interaction of IGFBP-3 and IGFBP-5 with RSP. We hypothesized that pulmonary IGFBP-3 and IGFBP-5 gene expression levels are downregulated in the nitrofen-induced pulmonary hypoplasia. Pregnant rats were exposed to either olive oil or 100 mg nitrofen on day 9.5 (D9.5) of gestation. Fetal lungs were harvested on D18 and D21 and divided into control and nitrofen groups. IGFBP-3 and IGFBP-5 pulmonary gene and protein expression were determined using real-time RT-PCR and immunohistochemistry. Relative levels of IGFBP-3 mRNA were significantly decreased in the nitrofen group (8.00 +/- 14.44) in D21 compared to controls (14.81 +/- 16.11; p < 0.05). Expression levels of IGFBP-5 mRNA were also significantly decreased in nitrofen group (10.66 +/- 4.83) on D18 compared to controls (17.92 +/- 4.77). Immunohistochemistry showed decreased IGFBP-3 expression on D21 and decreased IGFBP-5 immunoreactivity on D18 in hypoplastic lungs compared to controls. Downregulation of IGFBP-3 and IGFBP-5 gene expression may cause pulmonary hypoplasia in the nitrofen-induced CDH model by interfering with retinoid signaling pathway.

Defects in embryonic development of EGLN1/PHD2 knockdown transgenic mice are associated with induction of Igfbp in the placenta

The HIF (hypoxia inducible factor) hydroxylases EGNL1/PHD2 has been implicated in embryonic development. Here we knocked down EGNL1 in vivo by injecting one-cell murine zygotes with lentivirus-containing RNAi. Progeny with demonstrated EGLN1 inhibition had elevated EPO production and erythropoiesis in vivo. The partial inhibition of EGLN1 in utero is embryonic lethal in some, but not all mice on gestation day 14, and is associated with defects in placental and heart development, similar to those noted in the EGLN1 knockout mouse. Importantly, the in utero inhibition of EGNL1 varied greatly between the embryo proper and the placenta. Using this as a tool we show that the embryopathic effects are associated with knockdown of EGNL1 and the associated induction of Igfbp1 (insulin-like growth factor binding protein-1) mRNA in the placenta, but not the embryo.

Diet-induced obesity is associated with hyperleptinemia, hyperinsulinemia, hepatic steatosis, and glomerulopathy in C57Bl/6J mice

Obesity and obesity-related illnesses are global epidemics impacting the health of adults and children. The purpose of the present work is to evaluate a genetically intact obese mouse model that more accurately reflects the impact of aging on diet-induced obesity and type 2 diabetes in humans. Male C57Bl/6J mice consumed either a control diet or one in which 60% kcal were due to lard beginning at 5-6 weeks of age. Body weight and fat measurements were obtained and necropsy performed at 15, 20, 30, and 40 weeks of age. Serum chemistry, histopathology, gene expression of the liver, and renal and hepatic function were also evaluated. In concert with significant increases in percent body fat and weight, mice fed the high-fat versus control diet had significantly increased levels of serum cholesterol. At ages 20 and 30 weeks, serum glucose was significantly higher in obese versus controls, while serum insulin levels were >/=4-fold higher in obese mice at ages 30 and 40 weeks. The effect of age exacerbated the effects of consuming a high-fat diet. In addition to being hyperinsulinemic and leptin resistant, older obese mice exhibited elevated hepatic PAI-1 and downregulation of GLUT4, G6PC, IGFBP-1, and leptin receptor mRNA in the liver, steatosis with subsequent inflammation, glomerular mesangial proliferation, elevated serum ALT, AST, and BUN, and increased numbers of pancreatic islets.

Effects of Maternal Global Nutrient Restriction on Fetal Baboon Hepatic Insulin-Like Growth Factor System Genes and Gene Products

Knowledge of altered maternal nutrition effects on growth-regulating systems is critical to understanding normal and abnormal fetal development. There are many reports of hepatic fetal IGF system responses to maternal nutrient restriction (MNR) during pregnancy in rodents and sheep but none in nonhuman primates. We determined effects of MNR on the fetal baboon hepatic IGF system. Social groups of female baboons were fed ad libitum, controls, or 70% controls (MNR) from 0.16 to 0.5 gestation and fetuses delivered by cesarean section. Fetal liver tissue was analyzed for IGF-I, IGF-II, and IGF binding protein (IGFBP)-3 mRNA by in situ hybridization and quantitative RT-PCR and protein by immunohistochemistry (IHC); IGF-I receptor, IGF-II receptor by quantitative RT-PCR and IHC and IGFBP-1 by in situ hybridization and IHC. MNR did not alter fetal body or liver weight. Fetal hepatic glycogen staining increased with MNR. MNR reduced fetal hepatic IGF-I and IGF-II and increased IGFBP-1 mRNA and decreased IGF-I, IGF-II, IGF-I receptor, and IGF-II receptor protein and increased protein for IGFBP-1 and IGFBP-3. MNR increased caspase-3, indicating apoptosis and decreased Akt staining, indicating decreased nutrient sensing. In conclusion, whereas fetal body and liver weights did not change in response to moderate MNR during the first half of baboon pregnancy, the major indices of function of the hepatic IGF system measured were all reduced.

Insulin-Like Growth Factor Binding Protein-1 in the Ruminant Uterus: Potential Endometrial Marker and Regulator of Conceptus Elongation

Establishment of pregnancy in ruminants requires conceptus elongation and production of interferon-tau (IFNT), the pregnancy recognition signal that maintains ovarian progesterone (P4) production. These studies determined temporal and spatial alterations in IGF binding protein (IGFBP)-1 and IGFBP3 in the ovine and bovine uterus; effects of P4 and IFNT on their expression in the ovine uterus; and effects of IGFBP1 on ovine trophectoderm cell proliferation, migration, and attachment. IGFBP1 and IGFBP3 were studied because they are the only IGFBPs specifically expressed by the endometrial luminal epithelia in sheep. In sheep, IGFBP1 and IGFBP3 expression was coordinate with the period of conceptus elongation, whereas only IGFBP1 expression was coordinate with conceptus elongation in cattle. IGFBP1 mRNA in the ovine endometria was between 5- and 29-fold more abundant between d 12 and 16 of pregnancy compared with the estrous cycle and greater on d 16 of pregnancy than nonpregnancy in the bovine uterus. In sheep, P4 induced and IFNT stimulated expression of IGFBP1 but not IGFBP3; however, the effect of IFNT did not mimic the abundant increase observed in pregnant ewes. Therefore, IGFBP1 expression in the endometrium is regulated by another factor from the conceptus. IGFBP1 did not affect the proliferation of ovine trophectoderm cells in vitro but did stimulate their migration and mediate their attachment. These studies reveal that IGFBP1 is a common endometrial marker of conceptus elongation in sheep and cattle and most likely regulates conceptus elongation by stimulating migration and attachment of the trophectoderm.

Expressions of IGFBP-5, cFLIP in cervical intraepithelial neoplasia, cervical carcinoma and their clinical significances: a molecular pathology

BackgroundInsulin-like growth factor binding protein (IGFBPs) have been as potential tumor suppressors in the occurrence and development of tumors. Cellular Fas-associated death domain-like interleukin-1β-converting enzyme (FLICE)-like inhibitory protein (cFLIP) contains a death effect domain (DED), which blocks death receptor pathway and inhibits apoptosis.MethodsWe collected normal cervical tissues from 28 subjects, CIN samples from 37 patients, and cervical cancer tissues from 40 patients. In these samples, we then measured the expression levels of IGFBP-5 and cFLIP via RT-PCR and immunohistochemistry, and we detected the presence of high-risk HPV by Hybrid capture II assays in cervical secretions provided by the subjects.Resultssignificant differences in the expression of IGFBP-5 protein among the normal, CIN, and CC tissues (P < 0.05). The highest expression of IGFBP-5 protein was found in CIN stage II and III tissues, whereas the expression of IGFBP-5 in CC samples was decreased relative to controls. The expression level was affected by factors such as clinical stage, pathological differentiation, and lymph node metastasis. Relative to the controls, IGFBP-5 mRNA content was higher in the CC group and lower in the CIN group (P < 0.05). No expression of cFLIP protein or mRNA was detected in normal cervical tissues. However, the degree of pathological changes correlated with increasing expression of cFLIP protein and mRNA, and significant differences were therefore detected between groups (P < 0.05). The HPV infection rates in the CIN and CC groups were much higher than in the normal group (P < 0.05).ConclusionIGFBP-5 expression is up-regulated in response to progression of CIN and down-regulated in invasive cervical carcinoma. Detection of IGFBP-5 and cFLIP expression levels, may prove particularly useful for diagnosing and differentiating CIN and CC.

In vivo and in vitro effect of recombinant salmon growth hormone treatment on IGF-I and IGFBPs in yellowtail Seriola quinqueradiata

The role of growth hormone (GH) in regulating hepatic mRNA expression of insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) in yellowtail Seriola quinqueradiata was examined using in vivo and in vitro assays. Yellowtail hepatic IGF-I, IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-5 mRNAs were measured by real-time quantitative RT-PCR. Intraperitoneal injection of recombinant GH of chum salmon Oncorhynchus keta (rsGH) at a dose of 1 μg/g body weight resulted in a significant increases in hepatic IGF-I, IGFBP-3, and IGFBP-5 mRNA levels, whereas significant reductions in hepatic IGFBP-1 and IGFBP-2 mRNA levels were observed. For in vitro assays, liver slices were incubated with rsGH at different concentrations (doses: 0, 1, 10, 100, 500, and 1,000 ng/ml). Liver slices incubated with 100 ng/ml rsGH elicited a significant increase in IGF-I mRNA level. Similarly, a slight increase in IGFBP-3 and IGFBP-5 mRNA levels were also observed in liver explants incubated with rsGH. In contrast, a significant decline in IGFBP-1 mRNA levels was observed in liver slices incubated with 1,000 ng/ml rsGH. A slight decline in the level of IGFBP-2 mRNA was noted in liver explants with rsGH treatment. This study demonstrates the modulating effect of GH on the IGF system.

Effects of fasting on IGF-I, IGF-II, and IGF-binding protein mRNA concentrations in channel catfish ( Ictalurus punctatus)

The effects of fasting on insulin-like growth factor (IGF)-I, IGF-II, and IGF-binding protein (IGFBPs) mRNA in channel catfish were examined. Fed control fish (Fed) were compared to fish that had been fasted for 30 d followed by 15 d of additional feeding (Restricted). Sequence alignment and similarity to orthologous proteins in other vertebrates provided structural evidence that the 3 catfish sequences identified in the present research were IGFBP-1, -2, and -3. Prolonged fasting (30 d) reduced body weight approximately 60% ( P < 0.001) and decreased IGF-I mRNA in the liver and muscle ( P < 0.01). Fifteen days of re-feeding restored concentrations of hepatic and muscle IGF-I mRNA. Liver IGF-II mRNA was not affected by fasting but was increased 2.2-fold after 15 d of re-feeding ( P < 0.05). Abundance of muscle IGF-II mRNA was similar between the fed control group and the restricted group throughout the experimental period. Fasting also increased liver IGFBP-1 mRNA ( P < 0.05) and decreased IGFBP-3 mRNA ( P < 0.01), whereas abundance of IGFBP-2 mRNA was not significantly affected. Interestingly, re-feeding for 15 d did not restore concentrations of IGFBP-1 and IGFBP-3 mRNA relative to fed control concentrations. The IGF results suggest that IGF-I and IGF-II are differently regulated by nutritional status and probably have a differential effect in promoting muscle growth during recovery from fasting. Similar to mammals, IGFBP-1 mRNA in catfish is increased during catabolism, whereas IGFBP-3 mRNA is decreased during inhibited somatic growth. The IGFBP results provide additional evidence of the conserved nature of the IGF-IGFBP-growth axis in catfish.

NKX3.1 Activates Expression of Insulin-like Growth Factor Binding Protein-3 to Mediate Insulin-like Growth Factor-I Signaling and Cell Proliferation

NKX3.1 is a homeobox gene that codes for a haploinsufficient prostate cancer tumor suppressor. NKX3.1 protein levels are down-regulated in the majority of primary prostate cancer tissues. NKX3.1 expression in PC-3 cells increased insulin-like growth factor binding protein-3 (IGFBP-3) mRNA expression 10-fold as determined by expression microarray analysis. In both stably and transiently transfected PC-3 cells and in LNCaP cells, NKX3.1 expression increased IGFBP-3 mRNA and protein expression. In prostates of Nkx3.1 gene-targeted mice Igfbp-3 mRNA levels correlated with Nkx3.1 copy number. NKX3.1 expression in PC-3 cells attenuated the ability of insulin-like growth factor-I (IGF-I) to induce phosphorylation of type I IGF receptor (IGF-IR), insulin receptor substrate 1, phosphatidylinositol 3-kinase, and AKT. The effect of NKX3.1 on IGF-I signaling was not seen when cells were exposed to long-R3-IGF-I, an IGF-I variant peptide that does not bind to IGFBP-3. Additionally, small interfering RNA-induced knockdown of IGFBP-3 expression partially reversed the attenuation of IGF-IR signaling by NKX3.1 and abrogated NKX3.1 suppression of PC-3 cell proliferation. Thus, there is a close relationship in vitro and in vivo between NKX3.1 and IGFBP-3. The growth-suppressive effects of NKX3.1 in prostate cells are mediated, in part, by activation of IGFBP-3 expression.