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

DNA hypermethylation profiles associated with glioma subtypes and EZH2 and IGFBP2 mRNA expression

We explored the associations of aberrant DNA methylation patterns in 12 candidate genes with adult glioma subtype, patient survival, and gene expression of enhancer of zeste human homolog 2 (EZH2) and insulin-like growth factor-binding protein 2 (IGFBP2). We analyzed 154 primary glioma tumors (37 astrocytoma II and III, 52 primary glioblastoma multiforme (GBM), 11 secondary GBM, 54 oligodendroglioma/oligoastrocytoma II and III) and 13 nonmalignant brain tissues for aberrant methylation with quantitative methylation-specific PCR (qMS-PCR) and for EZH2 and IGFBP2 expression with quantitative reverse transcription PCR (qRT-PCR). Global methylation was assessed by measuring long interspersed nuclear element-1 (LINE1) methylation. Unsupervised clustering analyses yielded 3 methylation patterns (classes). Class 1 (MGMT, PTEN, RASSF1A, TMS1, ZNF342, EMP3, SOCS1, RFX1) was highly methylated in 82% (75/91) of lower-grade astrocytic and oligodendroglial tumors, 73% (8/11) of secondary GBMs, and 12% (6/52) of primary GBMs. The primary GBMs in this class were early onset (median age 37 years). Class 2 (HOXA9 and SLIT2) was highly methylated in 37% (19/52) of primary GBMs. None of the 10 genes for class 3 that were differentially methylated in classes 1 and 2 were hypermethylated in 92% (12/13) of nonmalignant brain tissues and 52% (27/52) of primary GBMs. Class 1 tumors had elevated EZH2 expression but not elevated IGFBP2; class 2 tumors had both high IGFBP2 and high EZH2 expressions. The gene-specific hypermethylation class correlated with higher levels of global LINE1 methylation and longer patient survival times. These findings indicate a generalized hypermethylation phenotype in glioma linked to improved survival and low IGFBP2. DNA methylation markers are useful in characterizing distinct glioma subtypes and may hold promise for clinical applications.

Dexamethasone stimulates the expression of leptin and 11β-HSD2 in primary human placental trophoblastic cells

Objectives Fetal glucocorticoid excess is thought to play an important role in early-life programming, promoting growth restriction and contributing to adult metabolic, cardiovascular and neuroendocrine disease. We hypothesized that dexamethasone incubation of primary trophoblastic cells from human healthy placentas at term might induce altered gene and protein expression of several endocrine placental regulators. Study design Primary villous trophoblastic cells were incubated with 10 μM dexamethasone for 6, 12, 24, 48 and 72 h. Non-incubated trophoblastic cells served as vehicle control. Gene expression of leptin, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) and insulin-like growth factor-binding protein-1 (IGFBP-1) was measured. Moreover, leptin, β-human chorionic gonadotropine (β-hCG) and lactate dehydrogenase (LDH) release into the culture medium was determined. Results Leptin gene expression was significantly increased in dexamethasone-incubated trophoblastic cells after 24, 48 and 72 h. There was a significant increase in leptin concentration in the medium of the cell culture after 48 h. Gene expression of 11β-HSD2 was significantly higher in dexamethasone-stimulated trophoblastic cells compared to vehicle controls after 72 h. The expression rate of IGFBP-1 mRNA was basal throughout the incubation period. The concentration of β-HCG in the supernatant increased significantly after 72 h of dexamethasone incubation, while LDH concentrations remained stable. Conclusion Our findings suggest that dexamethasone incubation stimulates leptin and 11β-HSD2 gene expression in primary villous trophoblastic cells of healthy human placentas, while enhancing cytotrophoblast differentiation.

Loss of the pregnancy-induced rise in cortisol concentrations in the ewe impairs the fetal insulin-like growth factor axis

Maternal cortisol levels increase during pregnancy. Although this change is important for optimal fetal growth, the mechanisms of the changes in growth remain unclear. The hypothesis examined was that alterations in maternal plasma cortisol concentrations are associated with changes in the fetal insulin-like growth factor (IGF) axis. Pregnant ewes in late gestation (115 ± 0.4 days) were studied: six control animals, five ewes given 1 mg kg(-1) day(-1) cortisol (high cortisol) and five adrenalectomised ewes given 0.5-0.6 mg kg(-1) day(-1) cortisol (low cortisol). Blood samples were taken throughout the experiment and at necropsy (130 ± 0.2 days) and fetal liver was frozen for mRNA analysis. Fetal IGF-I and insulin plasma concentrations were lower and insulin-like growth factor-binding protein-1 (IGFBP-1) concentrations were higher in the low cortisol group compared with those in the control group (P < 0.05). Fetal liver IGF-II and IGFBP-3 mRNA were decreased in low cortisol animals compared with controls (P < 0.05). There were no significant changes in these parameters in the high cortisol group, and there were no changes in fetal liver IGF-I, growth hormone receptor, IGF-I receptor, IGF-II receptor, IGFBP-1 or IGFBP-2 mRNA levels between the groups. These data suggest that reduced fetal IGF availability contributes to reduced fetal growth when maternal cortisol secretion is impaired, but not during exposure to moderate increases in cortisol.

IGFBP3 and BAG1 enhance radiation-induced apoptosis in squamous esophageal cancer cells

Identification of reliable markers of radiosensitivity and the key molecules that enhance the susceptibility of esophageal cancer cells to anticancer treatments would be highly desirable. To identify molecules that confer radiosensitivity to esophageal squamous carcinoma cells, we assessed the radiosensitivities of the TE-5, TE-9 and TE-12 cloneA1 cell lines. TE-12 cloneA1 cells showed significantly greater susceptibility to radiotherapy at 5 and 10 Gy than either TE-5 or TE-9 cells. Consistent with that finding, 24 h after irradiation (5 Gy), TE-12 cloneA1 cells showed higher levels of caspase 3/7 activity than TE-5 or TE-9 cells. When we used DNA microarrays to compare the gene expression profiles of TE-5 and TE-12 cloneA1 cells, we found that the mRNA and protein expression of insulin-like growth factor binding protein 3 (IGFBP3) and Bcl-2-associated athanogene 1 (BAG1) was five or more times higher in TE-12 cloneA1 cells than TE-5 cells. Conversely, knocking down expression of IGFBP3 and BAG1 mRNA in TE-12 cloneA1 cells using small interfering RNA (siRNA) significantly reduced radiosensitivity. These data suggest that IGFBP3 and BAG1 may be key markers of radiosensitivity that enhance the susceptibility of squamous cell esophageal cancer to radiotherapy. IGFBP3 and BAG1 may thus be useful targets for improved and more individualized treatments for patients with esophageal squamous cell carcinoma.

Effect of bisphenol A on human endometrial stromal fibroblasts in vitro

This study evaluated the effects of bisphenol A (BPA) on human endometrial stromal fibroblast (ESF) differentiation and expression of genes involved in oestrogen metabolism. Human ESF from eight hysterectomy specimens were cultured and treated with 5–100 μmol/l of BPA ± oestradiol or 8-br-cAMP for 48 h. mRNA expression was analysed by real-time reverse-transcription PCR. 8-br-cAMP-induced human ESF decidualization was confirmed by expression of insulin-like growth factor binding protein-1 (IGFBP1) and prolactin secretion. Short-term exposure (48 h) decreased human ESF proliferation ( P < 0.04) not due to apoptosis. High doses of BPA significantly induced IGFBP1 mRNA and protein, decreased P450scc mRNA, reversed the 8-br-cAMP-induced increase in HSD17B2 (oestradiol to oestrone conversion) in a dose-dependent manner and down-regulated HSD17B1 expression (oestrone to oestradiol conversion; P ⩽ 0.03). 8-br-cAMP significantly potentiated this effect ( P = 0.028). BPA had no significant effect on aromatase and PPAR γ expression. The oestrogen-receptor antagonist ICI had no effect on gene expression in BPA-treated cells, and oestrogen receptor α, but not oestrogen receptor β, was significantly down-regulated by high doses of BPA ( P = 0.028). BPA has an endocrine-disrupting effect on human ESF function and gene expression but the underlying mechanisms appear not to involve oestrogen-mediated pathways. Studies of the effects of bisphenol A (BPA), an endocrine disruptor, on the endometrium, a steroid hormone-sensitive tissue in which an embryo implants to establish pregnancy, are limited. Herein, we evaluated the effects of BPA on human endometrial stromal fibroblast (ESF) differentiation and expression of several genes involved in oestrogen metabolism. Human ESF were isolated from eight hysterectomy specimens, free from endometriosis or adenomyosis. They were cultured and treated with 5–100 μmol/l of BPA with or without oestradiol or 8-br-cAMP (a decidualization/differentiation stimulus) for 48 h. mRNA expression was analysed by real-time reverse-transcription PCR. 8-br-cAMP-induced human ESF decidualization (started 48 h prior to BPA) was confirmed by insulin-like growth factor binding protein 1 and prolactin secretion. Short-term exposure (48 h) of endometrial stromal cells to BPA significantly decreased human ESF proliferation, which was not due to increased apoptosis. High doses of BPA significantly induced differentiation in a dose-dependent manner to the same extent as 8-br-cAMP and significantly potentiated its effect. BPA had a significant effect on the expression of the enzyme that makes oestrogen, although the oestrogen-receptor antagonist had no effect on gene expression in BPA-treated cells. Interestingly, the main receptor that mediates oestrogen signalling, oestrogen receptor α, was significantly down-regulated by high doses of BPA. BPA thus has an endocrine-disrupting effect on human ESF function and gene expression. The effects on subsequent endometrial function remain to be determined.

Adipocytes IGFBP‐2 Expression in Prepubertal Obese Children

Aims of the study were to measure insulin-like growth factor-binding protein-2 (IGFBP-2) expression by abdominal subcutaneous adipocytes and to assess the relationship between IGFBP-2 expression, circulating IGFBP-2, obesity, and insulin sensitivity in obese children. Thirty-eight obese children were recruited. Insulin sensitivity was assessed by intravenous glucose tolerance test and body composition by total-body dual-energy X-ray absorptiometry. Serum free and total IGF-I, IGFBP-2, adiponectin, and leptin were measured. Relative quantification of IGFBP-2 mRNA by subcutaneous adipose tissue biopsies was obtained using real-time PCR. Circulating IGFBP-2 was positively associated with insulin sensitivity, in agreement with previous studies. IGFBP-2 expression was associated with fat mass percentage (r = 0.656; P < 0.02), insulin sensitivity (r = -0.604; P < 0.05), free IGF-I (r = 0.646; P < 0.05), and leptin (r = 0.603; P < 0.05), but not with circulating IGFBP-2 (r = 0.003, P = ns). The association between IGFBP-2 expression and adiposity (r = 0.648; P < 0.05) was independent of insulin sensitivity (covariate). In conclusion, circulating IGFBP-2 was positively associated with insulin sensitivity. IGFBP-2 was expressed by subcutaneous abdominal adipocytes of obese children and increased with adiposity, independently from the level of insulin sensitivity. IGFBP-2 expression may potentially be one of the local mechanisms used by adipocytes to limit further fat gain.

TGFβ1 attenuates expression of prolactin and IGFBP-1 in decidualized endometrial stromal cells by both SMAD-dependent and SMAD-independent pathways.

BackgroundDecidualization (differentiation) of the endometrial stromal cells during the secretory phase of the menstrual cycle is essential for successful implantation. Transforming Growth Factor β1 (TGFβ1) canonically propagates its actions via SMAD signalling. A role for TGFβ1 in decidualization remains to be established and published data concerning effects of TGFβ1 on markers of endometrial decidualization are inconsistent.Methodology/Principal FindingsNon-pregnant endometrial stromal cells (ESC) and first trimester decidual stromal cells (DSC) were cultured in the presence or absence of a decidualizing stimulus. Incubation of ESCs with TGFβ1 (10 ng/ml) down-regulated the expression of transcripts encoding the decidual marker proteins prolactin (PRL), insulin-like growth factor binding protein-1 (IGFBP-1) and tissue factor (TF). TGFβ1 also inhibited secretion of PRL and IGFBP-1 proteins by ESCs and surprisingly this response preceded down-regulation of their mRNAs. In contrast, DSCs were more refractory to the actions of TGFβ1, characterized by blunted and delayed down-regulation of PRL, IGFBP-1, and TF transcripts, which was not associated with a significant reduction in secretion of PRL or IGFBP-1 proteins. Addition of an antibody directed against TGFβ1 increased expression of IGFBP-1 mRNA in decidualised cells. Knockdown of SMAD 4 using siRNAs abrogated the effect of TGFβ1 on expression of PRL in ESCs but did not fully restore expression of IGFBP-1 mRNA and protein.Conclusions/SignificanceTGFβ1 inhibits the expression and secretion of decidual marker proteins. The impact of TGFβ1 on PRL is SMAD-dependent but the impact on IGFBP1 is via an alternative mechanism. In early pregnancy, resistance of DSC to the impact of TGFβ1 may be important to ensure tissue homeostasis.

Cyclical regulation of the insulin-like growth factor binding protein 3 gene in response to 1alpha,25-dihydroxyvitamin D3.

The nuclear receptor vitamin D receptor (VDR) is known to associate with two vitamin D response element (VDRE) containing chromatin regions of the insulin-like growth factor binding protein 3 (IGFBP3) gene. In non-malignant MCF-10A human mammary cells, we show that the natural VDR ligand 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) causes cyclical IGFBP3 mRNA accumulation with a periodicity of 60 min, while in the presence of the potent VDR agonist Gemini the mRNA is continuously accumulated. Accordingly, VDR also showed cyclical ligand-dependent association with the chromatin regions of both VDREs. Histone deacetylases (HDACs) play an important role both in VDR signalling and in transcriptional cycling. From the 11 HDAC gene family members, only HDAC4 and HDAC6 are up-regulated in a cyclical fashion in response to 1α,25(OH)2D3, while even these two genes do not respond to Gemini. Interestingly, HDAC4 and HDAC6 proteins show cyclical VDR ligand-induced association with both VDRE regions of the IGFBP3 gene, which coincides with histone H4 deacetylation on these regions. Moreover, combined silencing of HDAC4 and HDAC6 abolishes the cycling of the IGFBP3 gene. We assume that due to more efficient VDR interaction, Gemini induces longer lasting chromatin activation and therefore no transcriptional cycling but monotonically increasing IGFBP3 mRNA. In conclusion, 1α,25(OH)2D3 regulates IGFBP3 transcription through short-term cyclical association of VDR, HDAC4 and HDAC6 to both VDRE-containing chromatin regions.

Modulation of IGFBP2 mRNA Expression in White Adipose Tissue upon Aging and Obesity

The insulin/IGF-1 signaling pathway is a determinant of aging and age-related diseases. IGF-binding protein 2 (IGFBP2) is secreted by white adipocytes and contributes to the prevention of diet-induced obesity and age-related insulin resistance in mice. However, the expression levels of IGFBP2 in insulin resistance disorders have not been evaluated. The present study was aimed at determining IGFBP2 mRNA levels in adipose tissue in conditions of insulin resistance such as aging and obesity. In visceral white adipose tissue (WAT), but not in subcutaneous WAT, IGFBP2 mRNA levels were significantly lower in obese OB/OB, DB/DB and high fat-fed mice compared with those of their respective lean and chow-fed littermates. IGFBP2 mRNA levels were also decreased in visceral WAT of 12 and 24 months old mice compared with those of their 4 months old counterparts. Visceral WAT IGFBP2 expression was significantly associated with IGFBP2 circulating levels in mice, suggesting an important contribution from this tissue. The negative effect of aging on IGFBP2 mRNA levels in visceral WAT was confirmed in obese men. These findings demonstrate that the transcription of the IGFBP2 gene is modulated in a depot-specific fashion in obesity and aging in mice and men. Because IGFBP2 is an adipokine, an altered production from visceral WAT depots could impact on IGF-1 signaling and its downstream targets. This supports the need for further molecular and clinical studies to determine the factors regulating IGFBP2 expression and its relevance to metabolic diseases.

Insulin-like growth factor binding protein-2 (IGFBP-2) in orange-spotted grouper, Epinephelus coioides: Molecular characterization, expression profiles and regulation by 17β-estradiol in ovary

In the present study, a full-length cDNA encoding the insulin-like growth factor binding protein-2 (IGFBP-2) was cloned from the liver of orange-spotted grouper (Epinephelus coioides) by rapid amplification of cDNA ends technique. The IGFBP-2 cDNA sequence was 1413 bp long and had an open reading frame (ORF) of 813 bp encoding a predicted polypeptide of 270 amino acid residues. The deduced amino acid sequence contained a putative signal peptide of 22 amino acid residues resulting in a mature protein of 248 amino acids. Using semi-quantitative RT-PCR, tissue distribution pattern showed that IGFBP-2 mRNA was observed in all regions of brain with high levels, except lower levels in cerebellum and hypothalamus. In peripheral tissues, IGFBP-2 mRNA was most abundant in liver, although relatively high levels were observed in intestine, ovary and pituitary. Low or no IGFBP-2 mRNA expression was observed in other examined tissues. From the multicellular stage to the hatching stage, IGFBP-2 mRNA was expressed consecutively. Furthermore, 1 nM 17β-estradiol could inhibit the ovarian IGFBP-2 mRNA expression significantly in vitro. The mRNA expression and regulation profiles suggest that the IGFBP-2 may play important physiological roles in fish growth and reproduction, as well as cell growth and organ differentiation during the embryonic developmental stages.

Transcriptional down-regulation of IGFBP-3 in human hepatocellular carcinoma cells is mediated by the binding of TIA-1 to its AT-rich element in the 3′-untranslated region

Insulin-like growth factor binding protein-3 (IGFBP-3) plays key roles in regulating cell growth, differentiation, and apoptosis in a variety of cellular systems. We have observed significant down-regulation of IGFBP-3 expression in primary human hepatocellular carcinoma (HCC) tissues when compared to adjacent histologically normal tissues. In this study, we functionally mapped the entire 3′-UTR of the IGFBP-3 mRNA, spanning 1471nt and identified a 210bp fragment consisting of AT-rich elements at the distal downstream region preceding the consensus pre-mRNA polyadenylation signal that provide high affinity binding for TIA-1 to mediate the specific suppression of IGFBP-3 expression in human HCC cells.

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.

Effect of feeding level on serum IGF1 response to GH injection

This study was conducted to further understand the mechanism by which nutrition modulates GH-induced changes in serum IGF1 concentration in cattle. Cows were fed hay only or corn-based concentrates in addition to hay for 8 weeks. At week 8, serum concentrations of IGF1, IGF-binding protein 3 (IGFBP3), and acid-labile subunit (ALS) as well as their mRNA levels in liver were determined immediately before and 7 days after an injection of GH formulated for sustained release. GH injection caused greater increases in both serum IGF1 concentration and liver IGF1 mRNA expression in the cows fed concentrates than in those fed hay. In the cows fed concentrates, the magnitude of the GH-induced increase in serum IGF1 concentration was, however, much greater than that in liver IGF1 mRNA expression. This difference was not due to GH-induced IGF1 mRNA being translated more efficiently. GH injection also induced greater increases in serum IGFBP3 and ALS concentrations in the cows fed concentrates than fed hay. The injection, however, did not induce a greater increase in IGFBP3 or ALS mRNA in the cows fed concentrates than fed hay. These data and the fact that the ternary complex of IGF1/IGFBP3/ALS stabilizes each of the components in the blood suggest that in cows, increased nutrition enhances serum IGF1 response to GH not only by increasing IGF1 mRNA response to GH in the liver, but also by elevating IGFBP3 and ALS concentrations in the blood.

IGFBP-2 expression in MCF-7 cells is regulated by the PI3K/AKT/mTOR pathway through Sp1-induced increase in transcription

Insulin-like growth factor binding protein 2 (IGFBP-2) has been implicated in the pathophysiology of neoplasia. The PI3K/AKT/mTOR pathway has recently been shown to be a predominant regulator of IGFBP-2 at the protein level in MCF-7 breast cancer cells. However, there are gaps in knowledge with respect to the molecular mechanisms that underlie this regulation. Here, we show that the PI3K/AKT/mTOR pathway regulates IGFBP-2 protein levels by modulating IGFBP-2 mRNA abundance in MCF-7 cells. This change is achieved by regulating transcription through a critical region present in the first 200 bp upstream of the transcription initiation site where Sp1 transcription factor binds and drives transcription. IGF-1 treatment leads to increased nuclear abundance of Sp1 and increased IGFBP-2 mRNA and protein levels. Rapamycin and LY294002 induce a decline in Sp1 nuclear abundance and IGFBP-2 mRNA and protein levels. This work provides a mechanistic explanation for the observed effects of the PI3K/AKT/mTOR pathway on IGFBP-2 levels in MCF-7 cells.

IGFBP‐2 Expression in MCF‐7 Cells is Regulated by the PI3K/AKT/mTOR Pathway through Sp1 Induced Increase in Transcription

Insulin‐like growth factor binding protein 2 (IGFBP‐2) has been implicated in the pathophysiology of neoplasia. The PI3K/AKT/mTOR pathway has recently been implicated as a predominant regulator of IGFBP‐2 at the protein level in MCF‐7 breast cancer cells.ObjectiveTo elucidate the specific molecular mechanisms that underlie this regulation.ResultsWe show that the PI3K/AKT/mTOR pathway regulates IGFBP‐2 protein levels by modulating IGFBP‐2 mRNA abundance in MCF‐7 cells. This change is achieved by regulating transcription through a critical region present in the first 200 bp upstream of the transcription initiation site where Sp1 transcription factor binds and drives transcription. Manipulations of the PI3K/AKT/mTOR pathway lead to changes in posttranslational modifications and nuclear abundance of Sp1 transcription factor.Methods usedQRT‐PCR, ELISA, Western Blot, Luciferase reporter assays, Immunofluorescence, Chromatin immunoprecipitation.ConclusionThis work provides a mechanistic explanation for the observed effects of the PI3K/AKT/mTOR pathway on IGFBP‐2 levels in MCF‐7 cells.Matei Mireuta was supported by the graduate studentship from the Weekend to End Breast Cancer of the Jewish General Hospital Foundation.

Hepatic gene expression in multiparous Holstein cows treated with bovine somatotropin and fed n-3 fatty acids in early lactation

Multiparous cows were fed supplemental dietary fat and treated with bST to assess effects of n-3 fatty acid supply, bovine somatotropin (bST), and stage of lactation on hepatic gene expression. Cows were blocked by expected calving date and previous milk yield and assigned randomly to treatment. Supplemental dietary fat was provided from calving as either whole high-oil sunflower seeds (SS; 10% of dietary dry matter; n-6/n-3 ratio of 4.6) as a source of linoleic acid or a mixture of Alifet-High Energy and Alifet-Repro (AF; 3.5 and 1.5% of dietary dry matter, respectively; n-6/n-3 ratio of 2.6) as a source of protected n-3 fatty acids. Cows were treated with 0 (SSN, AFN) or 500 (SSY, AFY) mg of bST every 10 d from 12 to 70 d in milk (DIM) and at 14-d intervals thereafter. Liver biopsies were collected on −12, 10, 24, and 136 DIM for gene expression analysis. Growth hormone receptor (GHR), insulin-like growth factor-I (IGF-I), IGF-binding protein-3 (IGFBP3), hepatic nuclear factor 4α (HNF4α), fibroblast growth factor-21 (FGF-21), and peroxisome proliferator-activated receptor α (PPARα) were the target genes and hypoxanthine phosphoribosyltransferase (HPRT) was used as an endogenous control gene. Expression was measured by quantitative real-time reverse transcription-PCR analyses of 4 samples from each of 32 cows (8 complete blocks). Amounts of hepatic HPRT mRNA were not affected by bST or diet but were increased by approximately 3.8% in early lactation (3.42, 3.52, 3.54, and 3.41×104 message copies for −12, 10, 24, and 136 DIM, respectively). This small change had little detectable impact on the ability of HPRT to serve as an internal control gene. Amounts of hepatic GHR, IGF-I, and IGFBP3 mRNA were reduced by 1.5 to 2-fold after calving. Expression of GHR and IGF-I increased and IGFBP3 tended to increase within 12 d (by 24 DIM) of bST administration. These effects of bST persisted through 136 DIM. Hepatic HNF4α mRNA was not altered by DIM or any of the treatments. Abundance of PPARα mRNA was unchanged through 24 DIM but increased by 136 DIM. There was a trend for an interaction of bST, diet, and DIM on PPARα mRNA abundance from 24 to 136 DIM because the amount of PPARα mRNA increased in SSN, SSY, and AFN cows but was not altered in AFY cows. The amount of FGF-21 mRNA increased markedly in early lactation but, like HNF4α mRNA, was not affected by bST, diet, or their interactions. These results indicate 1) that bST induced increases in hepatic expression of GHR, IGF-I, and IGFBP3 when cows were in negative energy balance in early lactation, 2) there was no effect of reduced dietary n-6/n-3 content on hepatic gene expression, and 3) there was support for a potential homeorhetic role of hepatic FGF-21 via uncoupling the somatotropin-IGF-axis in early lactation.

Transforming growth factor-β1 modulates insulin-like growth factor binding protein-4 expression and proteolysis in cultured periosteal explants

Objective Periosteum is involved in bone growth and fracture healing and has been used as a cell source and tissue graft for tissue engineering and orthopedic reconstruction including joint resurfacing. Periosteum can be induced by transforming growth factor beta (TGF-β) or insulin-like growth factor-I (IGF-I) alone or in combination to form cartilage. However, little is known about the interaction between IGF and TGF-β signaling during periosteal chondrogenesis. The purpose of this study was to determine the effect of TGF-β1 on IGF binding protein-4 (IGFBP-4) and the IGFBP-4 protease pregnancy-associated plasma protein-A (PAPP-A) expression in cultured periosteal explants. Design Periosteal explants from rabbits were cultured with or without TGF-β1. IGFBP-4 and PAPP-A mRNA levels were determined by real-time quantitative PCR. Conditioned medium was analyzed for IGFBP-4 and PAPP-A protein levels and IGFBP-4 protease activity. Results TGF-β1-treated explants contained lower IGFBP-4 mRNA levels throughout the culture period with a maximum reduction of 70% on day 5 of culture. Lower levels of IGFBP-4 protein were also detected in the conditioned medium from TGF-β1-treated explants. PAPP-A mRNA levels were increased 1.6-fold, PAPP-A protein levels were increased threefold, and IGFBP-4 protease activity was increased 8.5-fold between 7 and 10 days of culture (the onset of cartilage formation in this model) in conditioned medium from TGF-β1-treated explants. Conclusions This study demonstrates that TGF-β1 modulates the expression of IGFBP-4 and PAPP-A in cultured periosteal explants.

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.

Common carp ( Cyprinus carpio) insulin-like growth factor binding protein-2 (IGFBP-2): Molecular cloning, expression profiles, and hormonal regulation in hepatocytes

In the present study, we cloned IGFBP-2 cDNA from common carp ( Cyprinus carpio) liver. The 1879 bp full-length cDNA encodes 274 amino acid residues containing a putative signal peptide of 22 residues. Two IGFBP-2 transcripts with estimated sizes of 2.2 and 1.5 kb have been detected with Northern blot analysis in liver. Relatively high levels of IGFBP-2 mRNA were observed in all regions of brain, liver, pituitary, ovary and testis. Intermediate levels were observed in white muscle, thymus gland and head kidney, while in retina, heart and other tissues IGFBP-2 mRNA levels were very low. A significant level of IGFBP-2 mRNA was firstly detected at lens formation stage, and it continued to increase to the highest level at blood cycling stage, and fell to a relatively high level until hatching. The expression pattern of IGFBP-2 mRNA was similar during different stages of testis and ovary. At recrudescing stage the expression level was extremely low, but it sharply increased to a high level at matured stage, and finally brought back to the very low level at regressed stage. Hepatocytes IGFBP-2 mRNA was greatly reduced by growth hormone but increased by insulin. PD-98059 and LY-294002, the specific inhibitor of MEK and PI3K, increased IGFBP-2 mRNA expression level and completely blocked the inhibitory effect of GH. It is suggested that the MAPK and PI3 kinase-signaling pathways were involved in the decrease of IGFBP-2 mRNA expression induced by GH in primary cultured hepatocytes.

Rexinoid-induced Expression of IGFBP-6 Requires RARβ-dependent Permissive Cooperation of Retinoid Receptors and AP-1

The synthetic rexinoid bexarotene (Targretin, LGD1069) inhibits the formation of both estrogen receptor-negative and estrogen receptor-positive breast cancer in preclinical models and controls the expression of growth-regulatory biomarkers, such as IGFBP-6 (insulin-like growth factor-binding protein 6), RARbeta, or cyclin D1. In this study, we identified a classical retinoic acid-responsive element in the first intron in the IGFBP-6 gene adjacent to a consensus AP-1 binding site, both elements essential for rexinoid-induced expression of IGFBP-6. In chromatin binding experiments, bexarotene increased the occupancy of the identified enhancer element by RXRalpha, RARbeta, cJun, cFos, and p300. In normal mammary epithelial cells and T47D breast cancer cells, small interfering RNA-mediated knockdown of all RXR isoforms or RARbeta, but not RARalpha or RARgamma alone, blocked the induction of IGFBP-6 by bexarotene. Simultaneous knockdown of RARalpha and RARgamma abrogated both the induction of RARbeta and the up-regulation and secretion of IGFBP-6. The suppression of either RARbeta or cJun by small interfering RNA blocked the recruitment of RXRalpha and cJun to the enhancer. These results demonstrate a novel cooperative interaction between retinoid receptors and AP-1 orchestrated by RARbeta and highlight a novel mechanism by which RARbeta can mediate the cancer-preventive effects of rexinoids.