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Abstract
The effect of glucocorticoids on the expression of intestinal cholecystokinin (CCK) was investigated both in vivo and in cell culture systems. In vivo, 2-day administration of methylprednisolone to adult male rats induced a decrease in CCK-like immunoreactivity (CCK-LI) and CCK mRNA levels in mucosal extracts. In two CCK-producing cell lines, RIN 1056E and STC-1 of pancreatic and intestinal origin respectively, dexamethasone induced dose-dependent decreases in both CCK-LI and steady-state CCK mRNA levels. The decrease in CCK mRNA was totally prevented by incubation of cells with an excess of RU 38486, a competitive inhibitor for the binding of glucocorticoids to their receptor. Actinomycin D, used to prevent RNA synthesis, did not modify CCK mRNA stability in dexamethasone-pretreated cells as compared with cells not exposed to dexamethasone. When cells were first incubated with actinomycin D, subsequent addition of dexamethasone left the steady-state CCK mRNA levels unaltered in both cell lines. Nuclear run-on assays performed in RIN 1056E cells showed that glucocorticoids decreased the rate of transcription of the CCK gene. In addition, cycloheximide, used to prevent protein synthesis, abolished the inhibitory effects of dexamethasone on steady-state CCK mRNA levels. These results demonstrate that glucocorticoids down-regulate CCK gene expression in the rat intestinal mucosa and in two CCK-producing cell lines. The effect is blocked by a glucocorticoid receptor antagonist. Inhibition of CCK gene expression may result from a decrease in the transcription rate, and probably involves one or several steps that depend on protein synthesis.
Journal of Endocrinology (1996) 151, 137–145
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Abstract
The prolactin receptor (PRLR) is a member of the cytokine/prolactin/GH receptor family, and it is widely expressed in various mammalian tissues. Expression of the two different forms of PRLR, differing in the length of their cytoplasmic domains, was studied in rat gonads during fetal and postnatal development. The two forms of PRLR mRNA were analyzed by reverse transcription (RT)-PCR using primer pairs specific for the different forms. The specificity of the cDNA species generated by RT-PCR was verified by Southern hybridization using nested 32P-labeled oligonucleotides. The results indicated that both forms of PRLR mRNA are expressed in the rat testis and ovary, which is in agreement with previous reports. The onset of expression of the two PRLR forms occurs on day 14·5 of fetal life in rat testis. In the ovary, the long form of PRLR mRNA appears 1 day before the short form, i.e. these forms begin to be expressed on fetal days 14·5 and 15·5 respectively. In situ hybridization with antisense cRNA probes specific to each form of the PRLR mRNAs demonstrated specific hybridization of both forms, localized in Leydig cells from day 18·5 of fetal life and at the postnatal ages studied. Compared with our previous findings concerning the ontogeny of LH receptor gene expression, PRLR gene expression starts earlier in development and exhibits no sexual dimorphism. The presence of two forms of PRLR mRNA in the fetal gonads suggest that they might play differential roles in gonadal development and function.
Journal of Endocrinology (1995) 147, 497–505
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ABSTRACT
GH receptor (GHR) mRNA has been identified in peripheral (liver and muscle) and central (brain and hypothalamus) tissues of sex-linked dwarf (SLD) Leghorn chickens. Total RNA was extracted from the tissues of immature (1 week, 4 week), pubertal (16 week) and adult (> 24 weeks) SLD and K (the normally growing strain) Leghorn chickens. In both groups and all tissues, an mRNA moiety of 4·4 kb hybridized with cRNA probes derived from the rabbit hepatic GHR sequence. An additional low-abundance transcript of 2·8 kb was also identified in some tissues. An age-related increase in expression was observed in K and SLD hepatic GHR mRNA, suggesting normal regulation of SLD GHR gene transcription. Amplification of cDNA from K and SLD tissues in the presence of oligonucleotide primers coding for the intracellular or extracellular domains of the chicken GHR generated electrophoretically separable fragments of expected size. Restriction enzyme digestion of the products with EcoRI, BstNI, HaeIII, NcoI or BamHI produced smaller moieties of expected sizes in both strains. These results demonstrate that, in contrast to broiler SLDs, a GHR gene deletion is not responsible for the GHR dysfunction in Leghorn SLDs. Although the actual defect in GHR gene expression in SLD Leghorns remains to be identified, this study demonstrates that sex-linked dwarfism, like Laron dwarfism, is due to a heterogeneity of lesions.
Journal of Endocrinology (1993) 137, 91–98
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The aim of this study was to determine whether expression of prostanoid receptor genes varied with gestational age or labour in the decidua and chorion of baboons. Tissues were obtained at the time of Caesarean hysterectomy in 15 baboons, 10 prior to the onset of labour in the last third of pregnancy and 5 in spontaneous labour at term. Expression of prostanoid receptor genes was determined using Northern blot analysis and the level of expression was related to each of three housekeeping genes. Expression of the genes encoding the EP(1) and FP receptor in decidua and the EP(4) receptor in chorion was lower with advancing gestational age. Expression of the EP(2) receptor gene was lower in labour in decidua, whereas expression of the IP receptor gene was higher in labour in both decidua (twofold) and chorion (fourfold). It is concluded that there is a complex pattern of change in expression of prostanoid receptor genes in chorion and decidua with advancing gestational age and in association with labour. It seems likely that direct effects of prostaglandins on the choriodecidua may have an important role in parturition in the primate.
Department of Pediatrics, Department of Molecular and Cellular Biology, National Hormone Peptide Program, Baylor College of Medicine, USDA/ARS Children's Nutrition Research Center, Houston, Texas 77030, USA
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mammary expression of the SOCS, CIS, and TPH1 genes. Materials and Methods Experimental animals All animals were studied in accordance with procedures outlined in the NIH Guide to Care and Use of Experimental Animals. These experiments were approved by the
Department of Agricultural Sciences, Imperial College London, Wye Campus, Ashford, Kent TN25 5AH, UK
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Department of Agricultural Sciences, Imperial College London, Wye Campus, Ashford, Kent TN25 5AH, UK
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Department of Agricultural Sciences, Imperial College London, Wye Campus, Ashford, Kent TN25 5AH, UK
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Department of Agricultural Sciences, Imperial College London, Wye Campus, Ashford, Kent TN25 5AH, UK
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Department of Agricultural Sciences, Imperial College London, Wye Campus, Ashford, Kent TN25 5AH, UK
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Department of Agricultural Sciences, Imperial College London, Wye Campus, Ashford, Kent TN25 5AH, UK
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Department of Agricultural Sciences, Imperial College London, Wye Campus, Ashford, Kent TN25 5AH, UK
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gene. PCR primer sequences are shown in Table 1 . Gene expressions were measured by the 2 −ΔCT method ( Livak & Schmittgen 2001 ). Levels of gene expression were expressed as relative values using commercial pig values as a reference (1
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Abstract
Stage-specific expression of the FSH receptor (FSHR) gene in the rat seminiferous epithelium was studied. Using transillumination-assisted microdissection for sample preparation and Northern hybridization for analysis of total RNA, we first reassessed the stage specificity of the FSHR gene expression in the adult rat testis. Sixfold higher FSHR mRNA levels were found in stages XIII–I compared with stage VI of the seminiferous epithelial cycle, which had the lowest signal level (P<0·01). The other stages had intermediate signal levels. In situ hybridization showed distribution of grains which confirmed the data obtained by Northern analysis. Prepubertal stage-specific FSHR gene expression was studied using in situ hybridization. Stage specificity could first be demonstrated at the age of 16 days when the average grain counts in stages I–IV were threefold higher than in stages VI–VII (P<0·01). The present data are in agreement with earlier findings on stage-specific FSH binding and FSHR gene expression using both microdissected and stage-synchronized seminiferous tubules. The onset of stage-specific FSHR gene expression is concomitant with maturation of the Sertoli cell population and completion of the first generation of spermatocytes. This supports the hypothesis that spermatogonia and spermatocytes may be involved in the regulation of FSHR gene expression.
Journal of Endocrinology (1996) 151, 29–35
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The experiments described herein were conducted to examine whether or not steroid administration allows in vivo induction of foreign gene expression in the oviduct of laying hens. The chloramphenicol acetyltransferase reporter gene driven by several viral and cellular promoters with or without steroid response elements was transfected by in vivo electroporation. The results indicated that in vivo, as observed in vitro, steroid administration induced transcriptional activities of the promoters with steroid response elements but it did not do so without steroid response elements. Our data implicate, therefore, that in vivo induction of foreign gene expression is possible in the oviduct of laying hens, and that the present in vivo gene transfer approach would serve as a useful tool to elucidate the mechanism of tissue-specific and steroid-induced transcription of chicken egg white genes.
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Estrogen receptor (ER) alpha is expressed during osteoblast differentiation; however, both its functional role in bone metabolism and its involvement in osteoporotic pathogenesis caused by estrogen deficiency are not well understood. Loss of ER alpha gene expression could be one of the mechanisms leading to osteoporosis. Therefore, we investigated a possible modulation of ER alpha gene expression in a human osteoblastic cell line and in four primary osteoblast cultures by using a decoy strategy. Double stranded DNA molecules, mimicking a regulatory region of the ER alpha gene promoter (DNA-102) and acting as a 'silencer' in breast cancer cells, were introduced into osteoblasts as 'decoy' cis-elements to bind and functionally inactivate a putative negative transcription factor, and thus to induce ER alpha gene expression. We found that the DNA-102 molecule was able to specifically bind osteoblast nuclear proteins. Before decoy treatment, absence or variable low levels of ER alpha RNAs in the different cultures were detected. When the cells were transfected with the DNA-102 decoy, an increase in expression of ER alpha and osteoblastic markers, such as osteopontin, was observed, indicating a more differentiated osteoblastic phenotype both in the cell line and in primary cultures. These results showed that the DNA-102 sequence competes with endogenous specific negative transcription factors that may be critical for a decrease in or lack of ER alpha gene transcription. Therefore, osteoblastic transfection with the DNA-102 decoy molecule may be considered a tempting model in a putative therapeutic approach for those pathologies, such as osteoporosis, in which the decrease or loss of ER alpha expression plays a critical role in bone function.
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ABSTRACT
The regulation of thyroglobulin (Tg) and its specific mRNA by interluekin-1 (IL-1) in cultured human thyrocytes was investigated. Specific binding of 125I-labelled IL-1 on thyrocytes was confirmed by solid-phase binding assay. Thyrocytes dispersed from Graves' thyroid tissues were incubated with TSH with or without recombinant human IL-1. TSH stimulated Tg release from cultured human thyrocytes in a dose- and time-dependent manner. Both IL-1α and β inhibited TSH-induced Tg release at concentrations ranging from 0·01 to 10 U/ml. The suppressive activities of IL-1α and β were similar. They did not alter the basal level of Tg release. Unstimulated human thyrocytes did not contain any detectable Tg mRNA, but TSH-stimulated thyrocytes expressed a single species of Tg mRNA (8·5 kb). Both IL-lα and β inhibited TSH-induced Tg mRNA in a dose-responsive manner. IL-1 (10 U/ml) caused maximal suppression of TSH-induced Tg mRNA to nearly basal levels. In contrast, the γ-actin mRNA hybridization signal was not altered in control or treated cells. Furthermore, IL-1 stimulated [3H]thymidine uptake into thyrocyte DNA. These results demonstrate that IL-1 directly inhibits TSH-induced Tg gene expression and provide further support for a functional role of IL-1 as a local modulator of thyroid hormone synthesis.
Journal of Endocrinology (1989) 122, 177–183
