Pregnant Women's perceptions of exposure to brominated flame retardants.

BACKGROUND: Recent media reports on human studies associating brominated flame retardants (BFRs) in household products in pregnancy with urogenital anomalies in boys and endocrine disruption in both sexes. We sought to explore the perceptions of pregnant women of brominated flame retardant (BFR) exposure, in light of recent media reports on the adverse health effects of BFR exposure prenatally. METHODS: Pregnant women were recruited for interviews through posters and pamphlets in prenatal clinics, prenatal fairs and community centres. Interviews were audiotaped and transcribed verbatim for Charmaz-based qualitative analysis supported by NVIVO 10™. RESULTS: Theoretical sufficiency was reached after analyzing the interviews of 23 pregnant women. Themes co-constructed were: I-Lack of Awareness of BFRs; II-Factors Influencing BFR Exposure; III-Responsibility; IV-Informed Choice. Almost all participants felt it was difficult to make informed choices to avoid BFRs, and wanted communication from clinicians and regulation from governments regarding decreasing BFR exposure. CONCLUSION: Pregnant women in Canada may be unaware of the potential risks of exposure to BFRs. Professional organizations and governments should further study risk associated with BFR exposure in pregnancy and provide educational materials for pregnant women and clinicians regarding BFR exposure.

Parental genomic imprinting of the human IGF2 gene.

The mouse igf2 gene, coding for the insulin-like growth factor II (IGF-II) is parentally imprinted, only the gene copy derived from the father is expressed. To know whether IGF2, the human homologue, is also imprinted, we used an ApaI polymorphism at the 3' untranslated region in order to distinguish between mRNA derived from each copy of the gene in placentae from heterozygote human fetuses, studied after careful removal of the decidua. Six term and two pre-term placentae of heterozygotes were studied, and in each case the cDNA contained only one of the two alleles present in the genomic DNA. In three cases the mother was homozygous for the non-expressed allele, allowing assignment of paternal origin to the transcribed gene copy. We conclude that, as in the mouse, human IGF2 is parentally imprinted.

Insulin expression in human thymus is modulated by INS VNTR alleles at the IDDM2 locus.

Type 1 diabetes or insulin-dependent diabetes mellitus (IDDM) is due to autoimmune destruction of pancreatic beta-cells. Genetic susceptibility to IDDM is encoded by several loci, one of which (IDDM2) maps to a variable number of tandem repeats (VNTR) minisatellite, upstream of the insulin gene (INS). The short class I VNTR alleles (26-63 repeats) predispose to IDDM, while class III alleles (140-210 repeats) have a dominant protective effect. We have reported that, in human adult and fetal pancreas in vivo, class III alleles are associated with marginally lower INS mRNA levels than class I, suggesting transcriptional effects of the VNTR. These may be related to type 1 diabetes pathogenesis, as insulin is the only known beta-cell specific IDDM autoantigen. In search of a more plausible mechanism for the dominant effect of class III alleles, we analysed expression of insulin in human fetal thymus, a critical site for tolerance induction to self proteins. Insulin was detected in all thymus tissues examined and class III VNTR alleles were associated with 2- to 3-fold higher INS mRNA levels than class I. We therefore propose higher levels of thymic INS expression, facilitating immune tolerance induction, as a mechanism for the dominant protective effect of class III alleles.

Human growth hormone receptor (GHR) expression in obesity: II. Regulation of the human GHR gene by obesity-related factors.

BACKGROUND AND METHODS: In our previous analyses, we found significantly lower levels of growth hormone receptor (GHR) mRNA in adipose tissues of obese than in those of lean individuals, suggesting that idiopathic obesity involves GH resistance due to decreased GHR availability. To understand the mechanism(s) behind this downregulation, we performed an in silico analysis of the three most relevant GHR gene promoters, which revealed putative response elements (REs) for a number of obesity adipose-associated factors, including tumor necrosis factor-alpha (TNFα), hypoxia-inducible factor-1-alpha (HIF-1α) and glucocorticoids. We then characterized the dose-dependent effects of these factors on GHR expression in HEK293 cells and in mature human SGBS (Simpson-Golabi-Behmel syndrome) adipocytes using quantitative reverse transcriptase-PCR and assessed the function of their putative REs by luciferase-reporter assays, site-directed mutagenesis and chromatin immunoprecipitation (ChIP) assays. RESULTS: TNFα treatments significantly reduced GHR mRNA levels and GHR promoter activities at doses ≥ 10 ng ml(-1) in both cell lines. Transient overexpression of HIF-1α or exposure to the hypoxia mimetic CoCl(2) significantly increased GHR mRNA levels and promoter activities. Dexamethasone had biphasic effects: there was a significant increase in GHR mRNA levels at 10(-10) M and in promoter activities at 10(-10) and 10(-8) M, whereas a significant decrease in both mRNA levels and promoter activities occurred at 10(-6) M. Site-directed mutagenesis of the putative nuclear factor-κB, HIF-1α and glucocorticoid REs resulted in the loss of these effects, whereas ChIP analysis confirmed specific transcription factor-promoter interactions. CONCLUSIONS: Our results suggest that the increased activity of TNFα, HIF-1α and glucocorticoids in obese adipose tissues could alter GHR gene transcription through specific REs and that TNFα may be involved in the development of GH resistance.

Human growth hormone receptor (GHR) expression in obesity: I. GHR mRNA expression in omental and subcutaneous adipose tissues of obese women.

OBJECTIVES: Growth hormone (GH)-deficient individuals display increased adiposity that can be effectively reduced by GH therapy because of GH's lipolytic effects. However, similar GH treatments of individuals with idiopathic obesity (not associated with an endocrinopathy/syndrome) have had little success. We hypothesized that this form of obesity may be associated with GH resistance at the level of the adipocyte because of reduced GH receptor (GHR) expression. SUBJECTS AND METHODS: We studied GHR expression in omental and subcutaneous fat tissues from a cohort of 55 women ranging from lean to obese by various adiposity parameters. mRNA levels of total GHR and the dominant-negative truncated GHR(1-279) (trGHR) form were assayed by quantitative reverse transcriptase-PCR. Associations between adiposity measures and GHR levels as well as trGHR/GHR ratios were analyzed. RESULTS: Total GHR mRNA expression was 2-3-fold lower in omental as well as subcutaneous adipose tissues of obese compared with lean women (P ≤ 0.05-0.001). Lean individuals expressed higher GHR mRNA levels in omental fat compared with subcutaneous (P ≤ 0.01); in obese women, this depot-specific difference was lost. Omental and subcutaneous adipose GHR mRNA levels displayed significant negative correlations with a spectrum of indicators of obesity while, in subcutaneous fat, there was a significantly higher trGHR/GHR ratio with increasing adiposity (P ≤ 0.05). CONCLUSION: These results support our hypothesis that, with obesity, there is lower GHR expression in the adipocyte, and suggest one possible explanation why GH supplementation is not an effective treatment for individuals with idiopathic obesity.

Effect of forkhead box O1 (FOXO1) on beta cell development in the human fetal pancreas.

AIMS/HYPOTHESIS: Recent studies have demonstrated that in adult murine beta cells the forkhead box O1 (FOXO1) transcription factor regulates proliferation and stress resistance. However, the role of FOXO1 during pancreatic development remains largely unknown. The present study aimed to characterise the expression of the FOXO1 transcription factor in the early to mid-gestation human fetal pancreas and to understand its role in islet cell development. METHODS: Human (8-21 week fetal age) pancreases were examined using immunohistological, quantitative RT-PCR and western blotting. Isolated human (18-21 week) fetal islet epithelial cell clusters were treated with insulin or glucose, or transfected with FOXO1 small interfering RNA (siRNA). RESULTS: Nuclear and cytoplasmic FOXO1 were widely produced during human fetal endocrine pancreatic development, co-localising in cells with the transcription factors pancreatic and duodenal homeobox 1 (PDX-1) and neurogenin 3 (NGN3) as well as cytokeratin 19 (CK19), insulin and glucagon. Treatment with exogenous insulin (50 nmol/l) induced the nuclear exclusion of FOXO1 in both cytokeratin 19 (CK19)(+) (p < 0.01) and insulin(+) cells (p < 0.05) in parallel with increased phospho-Akt (p < 0.05) production. siRNA knockdown of FOXO1 significantly increased the number of NGN3(+) (p < 0.01) and NK6 homeobox 1 (NKX6-1)(+) (p < 0.05) cells in parallel with increases in insulin gene expression (p < 0.03) and C-peptide(+) cells (p < 0.05) and reduced levels of hairy and enhancer of split 1 (HES1) (p < 0.01). CONCLUSIONS/INTERPRETATION: Our results indicate that FOXO1 may negatively regulate beta cell differentiation in the human fetal pancreas by controlling critical transcription factors, including NGN3 and NKX6-1. These data suggest that the manipulation of FOXO1 levels may be a useful tool for improving cell-based strategies for the treatment of diabetes.

SGNE1/7B2 is epigenetically altered and transcriptionally downregulated in human medulloblastomas.

In a genome-wide screen using differential methylation hybridization (DMH), we have identified a CpG island within the 5' region and untranslated first exon of the secretory granule neuroendocrine protein 1 gene (SGNE1/7B2) that showed hypermethylation in medulloblastomas compared to fetal cerebellum. Bisulfite sequencing and combined bisulfite restriction assay were performed to confirm the methylation status of this CpG island in primary medulloblastomas and medulloblastoma cell lines. Hypermethylation was detected in 16/23 (70%) biopsies and 7/8 (87%) medulloblastoma cell lines, but not in non-neoplastic fetal (n=8) cerebellum. Expression of SGNE1 was investigated by semi-quantitative competitive reverse transcription-polymerase chain reaction and found to be significantly downregulated or absent in all, but one primary medulloblastomas and all cell lines compared to fetal cerebellum. After treatment of medulloblastoma cell lines with 5-aza-2'-deoxycytidine, transcription of SGNE1 was restored. No mutation was found in the coding region of SGNE1 by single-strand conformation polymorphism analysis. Reintroduction of SGNE1 into the medulloblastoma cell line D283Med led to a significant growth suppression and reduced colony formation. In summary, we have identified SGNE1 as a novel epigenetically silenced gene in medulloblastomas. Its frequent inactivation, as well as its inhibitory effect on tumor cell proliferation and focus formation strongly argues for a significant role in medulloblastoma development.

Caspase-1 activation of caspase-6 in human apoptotic neurons.

Active caspase-6 (Csp-6) induces cell death in primary cultures of human neurons and is abundant in the neuropathological lesions of Alzheimer's disease. However, the mode of Csp-6 activation is not known. Here, we show that the Csp-1 inhibitor, Z-YVAD-fmk specifically prevents activation of Csp-6 and cell death in human neurons. A transient increase in Csp-1-like activity and an increase in the p23Csp-1 subunit occur early after serum deprivation. Recombinant active Csp-1 (R-Csp-1) cleaves recombinant and neuronal pro-Csp-6 in vitro resulting in Csp-6 activity. However, R-Csp-1 does not induce cell death when microinjected in human neurons despite the inhibition of serum-deprivation induced cell death with a Csp-1 dominant negative construct. These results show that Csp-1 is an upstream positive regulator of Csp-6-mediated cell death in primary human neurons. Furthermore, these results suggest that the activation of Csp-1 must be accompanied by an apoptotic insult to induce Csp-6-mediated cell death.

Pex/PEX tissue distribution and evidence for a deletion in the 3' region of the Pex gene in X-linked hypophosphatemic mice.

PEX, a phosphate-regulating gene with homology to endopeptidases on the X chromosome, was recently identified as the candidate gene for X-linked hypophosphatemia. In the present study, we cloned mouse and human Pex/PEX cDNAs encoding part of the 5' untranslated region, the protein coding region, and the entire 3' untranslated region, determined the tissue distribution of Pex/PEX mRNA, and characterized the Pex mutation in the murine Hyp homologue of the human disease. Using the reverse transcriptase/polymerase chain reaction (RT/PCR) and ribonuclease protection assays, we found that Pex/PEX mRNA is expressed predominantly in human fetal and adult mouse calvaria and long bone. With RNA from Hyp mouse bone, an RT/PCR product was generated with 5' but not 3' Pex primer pairs and a protected Pex mRNA fragment was detected with 5' but not 3' Pex riboprobes by ribonuclease protection assay. Analysis of the RT/PCR product derived from Hyp bone RNA revealed an aberrant Pex transcript with retention of intron sequence downstream from nucleotide 1302 of the Pex cDNA. Pex mRNA was not detected on Northern blots of poly (A)+ RNA from Hyp bone, while a low-abundance Pex transcript of approximately 7 kb was apparent in normal bone. Southern analysis of genomic DNA from Hyp mice revealed the absence of hybridizing bands with cDNA probes from the 3' region of the Pex cDNA. We conclude that Pex/PEX is a low-abundance transcript that is expressed predominantly in bone of mice and humans and that a large deletion in the 3' region of the Pex gene is present in the murine Hyp homologue of X-linked hypophosphatemia.

Developmental upregulation of human parathyroid hormone (PTH)/PTH-related peptide receptor gene expression from conserved and human-specific promoters.

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR) functions in skeletal development and mediates an array of other physiological responses modulated by PTH and PTHrP. PTHR gene transcription in mouse is controlled by two promoters: P1, which is highly and selectively active in kidney; and P2, which functions in a variety of tissues. P1 and P2 are conserved in human tissue; however, P1 activity in kidney is weak. We have now identified a third human promoter, P3, which is widely expressed and accounts for approximately 80% of renal PTHR transcripts in the adult. No P3 activity was detected in mouse kidney, indicating that renal PTHR gene expression is controlled by different signals in human and mouse. During development, only P2 is active at midgestation in many human tissues, including calvaria and long bone. This strongly suggests that factors regulating well conserved P2 control PTHR gene expression during skeletal development. Our results indicate that human PTHR gene transcription is upregulated late in development with the induction of both P1 and P3 promoter activities. In addition, P2-specific transcripts are differentially spliced in a number of human cell lines and adult tissues, but not in fetal tissues, giving rise to a shorter and less structured 5' UTR. Thus, our studies show that both human PTHR gene transcription and mRNA splicing are developmentally regulated. Moreover, our data indicate that renal and nonrenal PTHR gene expression are tightly coordinated in humans.

Cellular prion protein inhibits proapoptotic Bax conformational change in human neurons and in breast carcinoma MCF-7 cells.

Prion protein (PrP) prevents Bcl-2-associated protein X (Bax)-mediated cell death, but the step at which PrP inhibits is not known. We first show that PrP is very specific for Bax and cannot prevent Bak (Bcl-2 antagonist killer 1)-, tBid-, staurosporine- or thapsigargin-mediated cell death. As Bax activation involves Bax conformational change, mitochondrial translocation, cytochrome c release and caspase activation, we investigated which of these events was inhibited by PrP. PrP inhibits Bax conformational change, cytochrome c release and cell death in human primary neurons and MCF-7 cells. Serum deprivation-induced Bax conformational change is more rapid in PrP-null cells. PrP does not prevent active caspase-mediated cell death. PrP does not colocalize with Bax in normal or apoptotic primary neurons and cannot prevent Bax-mediated cytochrome c release in a mitochondrial cell-free system. We conclude that PrP protects against Bax-mediated cell death by preventing the Bax proapoptotic conformational change that occurs initially in Bax activation.

Fetal- and tumor-specific regulation of growth hormone receptor messenger RNA expression in human liver.

Eight different 5'-untranslated region variants of the human growth hormone receptor (hGHR) mRNA have been identified in adult liver (V1-V8). We have compared the expression of two of these variants (V1 and V3) in several human fetal and postnatal tissues (including liver) as well as in hepatoblastomas (HBs) and hepatocellular carcinomas (HCCs). Using reverse transcription-PCR assays, followed by Southern blotting to confirm the specificity of the amplified fragments, we found that V3 was expressed in all fetal and postnatal liver (n = 13 fetal and 5 postnatal), kidney (n = 4 fetal and 4 postnatal), lung (n = 4 fetal and 2 postnatal), intestine (n = 8 fetal and 4 postnatal), skeletal muscle (n = 1 fetal and 1 postnatal), and adrenal (n = 1 fetal and 1 postnatal) samples. In contrast, V1 was expressed only in postnatal liver. We then screened for V1 and V3 in HBs (n = 17, ages 6-36 months, including 5 with paired normal liver), and HCCs (n = 4, ages 50-75 years, with paired normal liver). V1 was undetectable in 15 of 17 HBs, including all HBs paired with (V1-expressing) normal liver; the absence of V1 did not correlate with patient age, sex, HB subtype, +/- chemotherapy, exon 3-retaining and -deficient hGHR mRNA isoform pattern, or loss of heterozygosity at 11p, 1p, and 1q. The four HCCs showed marked (>20-fold; n = 2) or complete (n = 2) suppression of V1 as compared to paired normal liver. V3 was expressed in all HBs, HCCs, and paired normal livers. Interestingly, V3, but not V1, was detected in two Wilms' tumor and paired normal kidney specimens. Our findings suggest that, in the human, there is tissue-, fetal- and tumor-specific regulation of V1 hGHR mRNA.

17-beta-estradiol induces an inhibitor of active caspases.

We have shown previously that caspase-6 activity is lethal to human neurons (LeBlanc et al., 1999; Zhang et al., 2000). Here we find that 17-beta-estradiol but not 17-alpha-estradiol prevents caspase-6-mediated neuronal cell death. 17-beta-estradiol-treated neuronal extracts directly inhibit recombinant active caspase-6, caspase-3, caspase-7, and caspase-8 in vitro. We conclude that 17-beta-estradiol induces a caspase inhibitory factor (CIF) that is preventing neuronal apoptosis. The induction of CIF occurs within 10 min of 17-beta-estradiol exposure to neurons, does not require de novo protein synthesis, and involves mitogen-activated protein kinase activation. The effect is antagonized by the estrogen receptor antagonist tamoxifen. In contrast, 17-beta-estradiol does not induce CIF or prevent caspase-mediated cell death in cultured astrocytes. CIF does not act through oxidation of the caspase active site. CIF activity copurifies with proteins of between 12 and 14 kDa in size. Our results indicate that 17-beta-estradiol induces an inhibitor of active caspases through a receptor-mediated nongenomic pathway and provide an additional mechanism for the neuroprotective action of 17-beta-estradiol that is likely highly relevant to the understanding of the role of estrogen against Alzheimer's disease.

A functional analysis of the role of IGF2 in IDDM2-encoded susceptibility to type 1 diabetes.

Genetic studies have identified a number of loci demonstrating linkage to type 1 diabetes. One of the largest single contributors to genetic susceptibility, after the major histocompatability complex, is the IDDM2 locus, which maps to a nontranscribed variable number of tandem repeats (VNTR) minisatellite upstream of the insulin (INS) and insulin-like growth factor 2 (IGF2) genes. In a progression from population to functional studies, recent reports have shown that VNTR susceptibility alleles (class I) have different transcriptional effects on INS than protective VNTR alleles (class III) in thymus and pancreas, two tissues important in the pathogenesis of the disease. Similar VNTR transcriptional effects on IGF2 have also been proposed as a mechanism by which the IDDM2 locus confers susceptibility in addition to, or instead of, effects on INS. We evaluated this hypothesis by comparing IGF2 expression levels from chromosomes with the protective class III alleles to those with class I alleles in tissues relevant to type 1 diabetes pathogenesis. In thymus, class III alleles were associated with an IGF2 mRNA level of 4.7 +/- 0.9 (mean +/- SE, arbitrary units, n = 12) compared with 4.7 +/- 1.3 for class I alleles (n = 17). The same absence of a significant difference was found in pancreas, where class III alleles were associated with a level of 28.4 +/- 4.2 (n = 7) and class I alleles with a level of 29.5 +/- 5.2 (n = 6). There was a significant correlation between fetal age and IGF2 in both tissues, but fetal ages were not different in the genotype groups compared. We therefore did not detect any significant difference in IGF2 mRNA levels associated with the protective class of VNTR alleles as compared with the predisposing class. This is evidence against the hypotheses that have suggested IGF2 is a mediator of IDDM2-encoded susceptibility and corroborates previous studies suggesting insulin is the gene involved.

Variable imprinting of H19 and IGF2 in fetal cerebellum and medulloblastoma.

Only the maternal or paternal allele of an imprinted gene is expressed in somatic cells. The gene for insulin-like growth factor II (IGF2) and the H19 gene (a putative tumor suppressor gene) are imprinted in humans with monoallelic paternal and maternal expression, respectively. Loss of imprinting (LOI) (i.e., biallelic expression) of IGF2 occurs in some tumors and may promote tumor growth. We examined imprinting of IGF2 and H19 in 6 fetal cerebella, 1 adult cerebellum, 15 medulloblastomas, and 7 medulloblastoma cell lines using polymerase chain reaction (PCR) and reverse transcription-PCR of exonic polymorphisms. Loss of imprinting of IGF2 occurred in 2 out of 3 informative fetal cerebella, 3 out of 7 informative medulloblastomas, and 1 out of 4 informative cell lines. Loss of imprinting of H19 occurred in 0 out of 4 informative fetal cerebella, 0 out of 1 informative adult cerebellum, 4 out of 8 informative medulloblastomas, and 1 out of 4 informative cell lines. The biallelic expression of H19 was only partial in two medulloblastomas, however, with one allele being significantly weaker than the other. Loss of imprinting of IGF2 occurs in medulloblastomas or medulloblastoma cell lines but can also occur in normal fetal cerebellum. Its occurrence in medulloblastomas may therefore reflect the tumors' embryonal nature rather than representing a primary pathogenetic mechanism. Our data also indicate that both genes can be imprinted and expressed independently of each another, both in normal cerebellum and medulloblastomas.

In vitro regulation of growth hormone (GH) release from ovine pituitary cells during fetal and neonatal development: effects of GH-releasing factor, somatostatin, and insulin-like growth factor I.

Using a monolayer approach, we have examined the acute (3 h) effects of GRF, somatostatin (SRIF), and insulin-like growth factor I (IGF-I) on GH release from pituitary cells of male and female 70-, 100-, and 130-day-old fetuses and newborn lambs and of prepubertal male lambs. GRF stimulated basal GH release in a dose-dependent (10(-12)-10(-8) M) manner at each stage in development. There was no linear relationship between maximal response and increasing age of the donor animals. The ED50 values for GRF were similar in all groups, except in the pituitaries from male and female 130-day-old fetuses, where the ED50 values were significantly higher. SRIF elicited a dose-related (10(-10)-10(-6) M) inhibition of basal GH secretion at each stage of fetal life and in the prepubertal period; although the response was lower in the youngest fetal pituitaries, there was no significant change in maximal response during the fetal or prepubertal period. No effect of SRIF on basal GH secretion was observed in newborn lambs. However, SRIF (10(-7) M) was able to block GRF (10(-8) M)-stimulated GH release in 100- and 130-day-old fetal and prepubertal as well as newborn lamb pituitary cells. Plasma IGF-I concentrations increased from 15.0 +/- 0.7 (mean +/- SE) and 13.8 +/- 0.9 ng/ml for male and female animals, respectively, at 70 days gestation to 55.8 +/- 3.2 and 51.8 +/- 11.1 ng/ml at the time of birth. The increase was much more pronounced in prepubertal lambs, especially in male animals, where IGF-I levels reached 300.8 +/- 37.7 ng/ml. IGF-I (100 ng/ml) had no effect on basal GH release in 70- and 100-day-old fetal, newborn, and prepubertal lamb pituitary cultures, but significantly inhibited basal GH secretion from 130-day-old fetal cells. This dose of IGF-I had no effect on GRF (10(-9) M)-stimulated GH release at 70 days gestation. It significantly inhibited this effect at 100 days and in prepubertal lamb cells. In 130-day-old fetal and newborn lamb pituitary cultures, IGF-I completely blocked the GH response to GRF.(ABSTRACT TRUNCATED AT 400 WORDS)

Organization and evolution of the human growth hormone receptor gene 5'-flanking region.

Previous studies have identified eight variant human GH receptor (hGHR) messenger RNA (mRNAs; V1-V8), that differ in their 5'-untranslated regions (5'UTRs) but splice into the same site just upstream of the translation start site in exon 2; thus, they encode the same protein. Here we report a novel variant, V9, and describe the mapping of all nine 5'UTR sequences within 40 kb upstream of exon 2. A cluster of three sequences, V2-V9-V3 (termed module A), lies furthest 5', and approximately 16 kb downstream is a second cluster of four exons, V7-V1-V4-V8 (module B). V6 is midway between modules A and B. Module B is about 18 kb upstream of V5, which lies adjacent to exon 2. hGHR expression is under developmental- and tissue-specific regulation, and expression of the variant mRNAs is related to their position within the 5'-flanking region; whereas module A (V2,V9,V3) and V5 variants are widely expressed, module B (V7,V1,V4,V8) and V6 variant mRNAs are detectable only in postnatal liver. Transcriptional start sites for V1 and V9 (representing the two different modules) were identified, showing that postnatal liver-specific expression of V1 is driven from two TATA boxes, whereas the ubiquitous V9 transcript has a single start site and a TATA-less promoter. V9 promoter activity was shown by in vivo and in vitro transfection assays, and an NF-Y binding site was demonstrated by electromobility shift assay. Thus, the regulatory regions of the hGHR gene are complex, and the clustering of seven 5'UTR exons within two modules with distinctly different mRNA expression patterns is the most striking feature.

Characterization of insulin-like growth factor receptors in rat anterior pituitary, hypothalamus, and brain.

Studies were undertaken to determine whether the insulin-like growth factors (IGF-I and -II), bind to specific membrane receptors in the pituitary and brain. Anterior pituitary glands, hypothalami, and brains (minus hypothalami) were obtained from adult male Sprague-Dawley rats (225-300 g) and 15,000 X g membranes prepared by differential centrifugation. Binding of 125I-IGF-I and 125I-IGF-II to all three membrane preparations was specific, time and temperature dependent, reversible, and increased in proportion to increasing concentrations of membrane protein or labeled ligand. Neither the pH of the assay buffer (6.5-8.5) nor the presence or absence of 1 mg/ml bacitracin had any significant effect on the levels of specific binding. In all three membrane preparations IGF-II specific binding was 3-5 times higher than that observed for IGF-I, and unlabeled IGF-II displaced either 125I-IGF-I or 125I-IGF-II better than comparable concentrations of IGF-I. All three membrane preparations showed similar low specific binding of 125I-insulin (1.3-2.2%) and negligible specific binding of 125I-rat GH (less than 0.5%). The presence of specific IGF and insulin receptors in rat anterior pituitary, hypothalamic, and brain tissue is additional evidence that IGFs and insulin are involved in modulating brain and pituitary function.

Effects of insulin-like growth factors on adult male rat pituitary function in tissue culture.

UNLABELLED: To determine the direct effects of insulin-like growth factors (IGFs) on pituitary secretion of GH, PRL, and ACTH, adult male rat pituitary explant cultures were tested with acute (3-4 h) or chronic (24 h) exposure to a semipurified preparation of IGF peptides, free of immunoreactive insulin, containing IGF-I and IGF-II in a ratio of approximately 1:4. To examine the effect of serum binding proteins on IGF bioactivity, certain experiments were run in parallel using culture medium supplemented with 10% fetal bovine serum or 1% purified BSA. To compare IGF effects with those of known regulators of pituitary function, cultures were also tested with SRIF, TRH, human pancreatic GH-releasing factor, insulin, and human GH (hGH). IGFs, at 10-100 ngeq/ml, were able to inhibit significantly both basal and (1 mM) theophylline-stimulated rat GH (rGH) and rat PRL (rPRL) release during acute (3-4 h) exposure. Only the higher concentration (100 ngeq/ml) was consistently effective in inhibiting rGH and rPRL output after 24 h in culture, due to gradual metabolism of IGF peptides by the cells. Parallel experiments carried out in medium containing 10% fetal bovine serum or 1% BSA gave similar results, demonstrating that IGF serum binding proteins did not interfere with IGF bioactivity in this test system. Chronic 5-day exposure to IGFs, at 100 ngeq/ml, resulted in a significant inhibition of rGH release for the entire 5-day period and rPRL release for the first 3 days. IGFs (10-100 ngeq/ml) had no acute or chronic effect on basal or theophylline-stimulated ACTH release. Purified IGF-I (50 ng/ml) and IGF-II (50 ng/ml) gave approximately equivalent effects on basal rGH and rPRL release during an acute (3 h) exposure suggesting that both IGFs can exert inhibitory influence on pituitary function. Ten thousand nanograms per ml insulin and 10(-9) M SRIF had acute inhibitory effects on rGH and rPRL release similar to what were observed for 100 ngeq/ml semipurified IGFs. hGH (200 and 1000 ng/ml) had no effect on rGH, rPRL, or ACTH release when administered either acutely (3-4 h) or chronically (24 h). CONCLUSIONS: These studies demonstrate that IGFs, administered acutely or chronically, directly inhibit basal as well as theophylline-stimulated rGH and rPRL output by the rat pituitary; ACTH release remains unaltered. Insulin, at high concentrations, can mimic these effects, whereas hGH has no effect either acutely or chronically.

Effects of growth hormone (GH)-releasing factor and somatostatin on GH secretion from early to midgestation human fetal pituitaries.

Using explant cultures of human anterior pituitary glands (9-19 weeks fetal age) and an acute (3-h) test protocol, we investigated fetal somatotrope responsiveness to human GH-releasing factor [hGRF-(1-44)] and somatostatin [SRIF-(1-14) and SRIF-(1-28)] as a function of age. Ontogenic data were analyzed using three age groups: 9-10, 12-13, and 15-19 weeks fetal age. Both daily (24-h) and acute test (3-h) basal GH secretion increases as a function of fetal age, with the greatest increase occurring after 12-13 weeks; however, the 3 h/24 h secretion ratio remains unchanged, at approximately 12%. GRF (0.1-10 nM) stimulates GH release in a dose-related fashion, regardless of fetal age; there is a significant increase in the response to both 1 nM (P < 0.05) and 10 nM (P < 0.01) GRF between 9-10 and 15-19 weeks fetal age and to 10 nM GRF (P < 0.05) between 12-13 and 15-19 weeks. Pretreatment of cultures (9-19 weeks) with 1 or 10 nM GRF for 24 h does not alter basal 3-h GH secretion, but significantly decreases subsequent responses to 1 or 10 nM GRF, respectively (P < 0.01). Pretreatment with 1 nM GRF does not alter a subsequent 3-h response to 10 nM GRF. SRIF-(1-14) (1-100 nM) causes a dose-related inhibition of basal GH secretion from as early as the ninth week of fetal life; there is a small age-related increase in the somatotrope response to 100 nM SRIF-(1-14) between 12-13 and 15-19 weeks fetal age (P < 0.05). In a group of 11- to 14-week-old fetal pituitaries, SRIF-(1-28) had a significantly (P < 0.05) greater inhibitory effect than SRIF-(1-14) at both 1 and 10 nM; the two peptides decreased basal GH secretion to a similar extent at 100 nM (52.8 +/- 4.0% of control value; P < 0.01). SRIF-(1-14) (10 and 100 nM) does not significantly alter 10 nM GRF-stimulated GH release from 9- to 10-week-old fetal pituitaries. However, by 12-13 weeks, 10 nM SRIF-(1-14) reduces GRF-stimulated GH secretion by 60% (P < 0.01), while 100 nM SRIF-(1-14) decreases it by 80% (P < 0.01); similar inhibitory effects are observed with 15- to 19-week-old fetal somatotropes.(ABSTRACT TRUNCATED AT 400 WORDS)