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K. Yang, G. L. Hammond and J. R. G. Challis

ABSTRACT

Fetal sheep tissues possess glucocorticoid receptors (GR), and these change in number during the last two-thirds of gestation. There is, however, no information about developmental changes in tissue GR mRNA levels which might account for alterations in fetal GR content. We have therefore cloned and sequenced a 942 bp GR cDNA from a sheep liver cDNA library, and used it to study the relative abundance of GR mRNA in fetal and neonatal sheep tissues. Analysis of the cDNA revealed a partial sequence of the ovine GR which displayed over 80% identity with residues 143–453 in human GR and 163–472 in rat GR. Furthermore, the first zinc finger motif in these receptors was perfectly conserved among species. The relative abundance of GR mRNA was studied in hypothalami, anterior pituitary glands and adrenals in fetuses at days 60–70, 100–110, 125–130 and at term (approximately 145 days), and in newborn lambs. Total RNA extracts (20 μg) were analysed by Northern blot analysis. A single 5.6kb transcript was detected in all three fetal tissues, and its relative abundance did not change significantly throughout gestation. However, in newborn lambs, levels of GR mRNA increased significantly in the hypothalamus and pituitary gland but decreased to undetectable levels in the adrenal. These tissue-specific changes in the relative abundance of GR mRNA did not correlate with alterations in GR content in fetal tissues, which suggests that the latter may reflect alterations in GR mRNA translation, subsequent modifications and/or GR turnover. In addition, the pattern of developmental changes in GR mRNA content of the adrenal differs from that of the hypothalamus and pituitary gland in neonatal lambs, and indicates that tissue-specific factors may influence GR gene expression in neonatal sheep.

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A Fleury, L Ducharme and JG LeHoux

In this study, we report the cDNA cloning of hamster adrenal steroidogenic acute regulatory (StAR) protein and the effect of adrenocorticotrophin (ACTH) on its expression in vivo. A hamster adrenal cDNA library was screened using an 852 bp fragment obtained by polymerase chain reaction; this fragment corresponds to the entire coding sequence (CDS) of the hamster adrenal StAR cDNA. Ten clones of different lengths were isolated and sequenced. The longest clone was 1564 bp and contained 34 bp in the 5'-untranslated region, 852 bp in the CDS, and 678 bp in the 3'-untranslated region (3'-UTR). Two polyadenylation signal sequences were found in the 3'-UTR. The CDS of the ten isolated clones was identical, but six of these lacked the last 132 nucleotides in the 3'-UTR, thus indicating that they had used the first polyadenylation signal. The hamster StAR protein contains 284 amino acid residues, and is 91.9% homologous to mouse, 90.5% to rat, 86.4% to human, 85% to porcine, and 82.5% to bovine StAR protein. Southern blot analysis indicated the presence of only one StAR gene in the hamster genome. Northern blotting analysis revealed the presence of the StAR mRNA in male and female steroidogenic tissues, namely adrenals and gonads, but not in the liver or in the kidneys of either sex. Three mRNA species of 1.7, 3.1 and 5.3 kb were found in whole hamster adrenals. Administration of ACTH to hamsters provoked increases (two- to threefold) in the adrenal content of the StAR mRNA within 1 h in vivo. Western blotting analysis on adrenal mitochondria showed that the level of StAR protein was also significantly elevated (1.5-fold) 1 h after ACTH treatment.

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Kristine M Wadosky, Jessica M Berthiaume, Wei Tang, Makhosi Zungu, Michael A Portman, A Martin Gerdes and Monte S Willis

Thyroid hormone (TH) is recognized for its role in cellular metabolism and growth and participates in homeostasis of the heart. T3 activates pro-survival pathways including Akt and mTOR. Treatment with T3 after myocardial infarction is cardioprotective and promotes elements of physiological hypertrophic response after cardiac injury. Although T3 is known to benefit the heart, very little about its regulation at the molecular level has been described to date. The ubiquitin proteasome system (UPS) regulates nuclear hormone receptors such as estrogen, progesterone, androgen, and glucocorticoid receptors by both degradatory and non-degradatory mechanisms. However, how the UPS regulates T3-mediated activity is not well understood. In this study, we aim to determine the role of the muscle-specific ubiquitin ligase muscle ring finger-1 (MuRF1) in regulating T3-induced cardiomyocyte growth. An increase in MuRF1 expression inhibits T3-induced physiological cardiac hypertrophy, whereas a decrease in MuRF1 expression enhances T3's activity both in vitro and in cardiomyocytes in vivo. MuRF1 interacts directly with TRα to inhibit its activity by posttranslational ubiquitination in a non-canonical manner. We then demonstrated that a nuclear localization apparatus that regulates/inhibits nuclear receptors by sequestering them within a subcompartment of the nucleus was necessary for MuRF1 to inhibit T3 activity. This work implicates a novel mechanism that enhances the beneficial T3 activity specifically within the heart, thereby offering a potential target to enhance cardiac T3 activity in an organ-specific manner.

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CJ Kenyon, M Panarelli, L Zagato, L Torielli, RP Heeley, CD Holloway, R Fraser, G Casari, RG Sutcliffe and G Bianchi

The Milan hypertensive strain of rat (MHS) displays abnormalities in both renal function and adrenocortical activity. While the pressor role of the former has been studied in detail, the role of the latter has not yet been clearly evaluated. In the present study, glucocorticoid receptor (GR) binding characteristics in liver cytosol from adult MHS and Milan normotensive controls (MNS) have been investigated. Dexamethasone, aldosterone and corticosterone were bound with lower affinity to cytosol of MHS rats compared with that of MNS rats. This pattern of binding could explain the raised plasma corticosterone concentrations and adrenocortical hypertrophy previously noted in MHS. The coding sequence of MHS and MNS GR genes have been determined. The MHS gene differed in four respects from that of MNS: three silent point mutations and a polymorphic microsatellite region in exon 2. The latter polymorphism has been used in cosegregation studies of F2 hybrids of MHS x MNS. The MHS GR genotype was associated with hypercalciuria and lower blood pressure in female rats and lower body weight in male rats. Although the effect on blood pressure is small, it is consistent with the affinity data. MHS GR genotype cosegregated with lower blood pressure in F2 rats and displayed a lower affinity in binding studies. In conclusion, GR polymorphism may be responsible for differences of adrenocortical function between MHS and MNS. This may lead to a reduction in the blood pressure difference between the two strains.

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Aurimas Vinckevicius and Debabrata Chakravarti

Recent decades have been filled with groundbreaking research in the field of endocrine hormone signaling. Pivotal events like the isolation and purification of the estrogen receptor, the cloning of glucocorticoid receptor cDNA, or dissemination of nuclear hormone receptor (NHR) DNA binding sequences are well recognized for their contributions. However, the novel genome-wide and gene-specific information obtained over the last decade describing NHR association with chromatin, cofactors, and epigenetic modifications, as well as their role in gene regulation, has been largely facilitated by the adaptation of the chromatin immunoprecipitation (ChIP) technique. Use of ChIP-based technologies has taken the field of hormone signaling from speculating about the transcription-enabling properties of acetylated chromatin and putative transcription (co-)factor genomic occupancy to demonstrating the detailed, stepwise mechanisms of factor binding and transcriptional initiation; from treating hormone-induced transcription as a steady-state event to understanding its dynamic and cyclic nature; from looking at the DNA sequences recognized by various DNA-binding domains in vitro to analyzing the cell-specific genome-wide pattern of nuclear receptor binding and interpreting its physiological implications. Not only have these events propelled hormone research, but, as some of the pioneering studies, have also contributed tremendously to the field of molecular endocrinology as a whole. In this review, we give a brief summary of some of the most important discoveries in hormone signaling using ChIP and other derivative techniques and speculate on what the future may hold.

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Chunyi Li, Yan Li, Yinghui Li, Hong Liu, Zhijun Sun, Jingyu Lu and Yanyan Zhao

With-no-lysine (K) kinase-4 (WNK4) is a serine/threonine kinase that plays an essential role in the regulation of fluid and electrolyte homeostasis. The effects of glucocorticoids, key physiological regulators, on the WNK4 gene expression are still unknown. Here, we used dexamethasone (Dex) to treat the human embryo kidney 293 (HEK293) cells and found a decrease of human WNK4 (hWNK4) mRNA level by northern blot and real-time quantitative PCR. After an hWNK4 transcriptional initiation site was located by 5′ rapid amplification of cDNA end assay, a series of 5′-deleted hWNK4 promoter–luciferase constructs were generated by PCR. Transfection of these constructs in COS-7 and HEK293 cells revealed that Dex inhibited the hWNK4 transcriptional activity in glucocorticoid receptor (GR)-dependent pattern. Two negative glucocorticoid response elements (nGREs) were identified at −285 and −337 of the hWNK4 gene promoter and the GR binding activity to them was increased by Dex as shown by electrophoretic mobility shift assay and chromatin immunoprecipitation. In summary, these data demonstrated that hWNK4 was a new glucocorticoid-regulated gene whose expression was inhibited through the interaction of GR with nGREs in the promoter region.

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Salman Azhar, Dachuan Dong, Wen-Jun Shen, Zhigang Hu and Fredric B Kraemer

miRNAs are endogenous noncoding single-stranded small RNAs of ~22 nucleotides in length that post-transcriptionally repress the expression of their various target genes. They contribute to the regulation of a variety of physiologic processes including embryonic development, differentiation and proliferation, apoptosis, metabolism, hemostasis and inflammation. In addition, aberrant miRNA expression is implicated in the pathogenesis of numerous diseases including cancer, hepatitis, cardiovascular diseases and metabolic diseases. Steroid hormones regulate virtually every aspect of metabolism, and acute and chronic steroid hormone biosynthesis is primarily regulated by tissue-specific trophic hormones involving transcriptional and translational events. In addition, it is becoming increasingly clear that steroidogenic pathways are also subject to post-transcriptional and post-translational regulations including processes such as phosphorylation/dephosphorylation, protein‒protein interactions and regulation by specific miRNAs, although the latter is in its infancy state. Here, we summarize the recent advances in miRNA-mediated regulation of steroidogenesis with emphasis on adrenal and gonadal steroidogenesis.

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A J Conley, W E Rainey and J I Mason

ABSTRACT

This study examined fetal steroidogenic enzyme expression and function during pregnancy in the pig. Northern and Western analyses were performed to detect the cytochrome P450 enzyme 17α-hydroxylase/17–20 lyase (P450c17) and that for cholesterol side-chain cleavage (P450scc), as well as 3β-hydroxysteroid dehydrogenase (3β-HSD) expression in several porcine fetal tissues. The data demonstrate higher steroidogenic enzyme expression in the fetal adrenal glands and testes than in the placenta at all stages of development examined. Although steroidogenic enzyme expression was maintained throughout gestation in both the fetal adrenals and the testes, adrenal P450c17 expression was higher in the early and late stages when compared with the intermediate stages of fetal development. The stimulation of fetal adrenal steroidogenic enzyme expression in the later stage fetuses was accompanied by increased expression of P450c17 in both the fetal testes and placenta. The expression of 3β-HSD by porcine fetal testes was low compared with that of the fetal adrenal gland at all stages of development. Adrenal explants and cultured cells secreted cortisol and androstenedione but much lower amounts of corticosterone, dehydroepiandrosterone and aldosterone. Secretion of cortisol and androstenedione by adrenal explants was maintained by ACTH for 5 days of culture but declined in controls. In cultured porcine fetal adrenal cells, ACTH and angiotensin II stimulated the secretion of multiple steroids. Porcine fetal testis explants and cultured cells secreted testosterone, dehydroepiandrosterone and androstenedione, but were only moderately responsive to trophic stimulation by LH. In general, the data suggest that the fetal adrenal glands and the fetal testes have the potential to contribute significantly to the production of steroids during pregnancy in pigs.

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W E Farrell, M F Stewart, A J L Clark, S R Crosby, J R E Davis and A White

ABSTRACT

In the normal pituitary, glucocorticoids are the principal negative regulator of the pro-opiomelanocortin (POMC) gene which gives rise to the biologically active peptides ACTH and β-endorphin. In Cushing's syndrome, ACTH-secreting pituitary tumours show a degree of glucocorticoid resistance, whilst ACTH-secreting extra-pituitary tumours have an even greater resistance to glucocorticoid excess. In an attempt to understand the mechanism of this phenomenon, we have compared the effects of glucocorticoids on POMC mRNA and peptide secretion in human and mouse corticotroph adenoma cells and in small cell lung carcinoma (SCLC) cells. ACTH precursor peptides were inhibited within 24 h by 25–50 nm hydrocortisone in primary cultures from a human corticotroph adenoma. In the mouse corticotroph adenoma cell line (AtT20), inhibition of both ACTH precursors and ACTH was not observed after 24 h but, by 10 days, glucocorticoids suppressed peptide levels with a concentration causing 50% inhibition of 50 nm hydrocortisone and maximal inhibition at 500 nm hydrocortisone. In marked contrast, there was no response to 500 nm hydrocortisone in the five SCLC cell lines (COR L103, COR L42, COR L24, COR L31, DMS 79) all of which secrete ACTH precursors. However, two of the five SCLC cell lines (COR L31 and DMS 79) were responsive to 1000 nm hydrocortisone. POMC mRNA, quantitated by slot-blot analysis, gave similar results for the five SCLC cell lines, implying that the abnormality may occur at the level of gene expression. When one of the three resistant cell lines (COR L103) was incubated with 2000 nm hydrocortisone or 2000 nm dexamethasone a clear suppression of precursor peptides and POMC mRNA was observed. This suggests that the resistance to glucocorticoid inhibition is relative rather than absolute, implying that the normal mechanism is functioning but impaired. Furthermore, there is at least a 20-fold difference in the responsiveness to glucocorticoid inhibition between pituitary and extra-pituitary tumour cells in vitro, which may signify a difference in the underlying mechanism in these two cell types.

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ZN Wang, M Bassett and WE Rainey

Liver receptor homologue-1 (LRH-1, designated NR5A2) is a mammalian homologue of Drosophila fushi tarazu factor (dFTZ-F1) and structurally belongs to the orphan nuclear receptor superfamily. LRH-1 can recognize the DNA sequence 5'-AAGGTCA-3', the canonical recognition motif for steroidogenic factor 1 (SF-1). Herein, we hypothesized that LRH-1 might play a role in the regulation of human adrenal expression of steroidogenic enzymes. To test this hypothesis, LRH-1 expression in human adult and fetal adrenal glands was examined by RT-PCR analysis. The fetal and adult adrenal glands, as well as liver and pancreas, were observed to express LRH-1 mRNA using RT-PCR. The ability of LRH-1 to enhance transcription of the gene encoding human 11 beta- hydroxylase (hCYP11B1) was then examined using the H295R adrenal cell line. LRH-1 co-transfection with hCYP11B1 luciferase promoter constructs caused a 25-fold induction of luciferase activity. Furthermore, co-transfection of a hCYP11B1 reporter construct containing a mutation in the SF-1 binding cis-element abolished the stimulatory effect of both SF-1 and LRH-1. Electrophoretic mobility shift assay (EMSA) demonstrated that LRH-1 could bind to the SF-1 response element. Taken together, our data suggested that LRH-1 is expressed in the adrenal, and can substitute for SF-1 to enhance transcription of genes encoding certain of the steroid-metabolizing enzymes. A role for LRH-1 in the regulation of adrenal or gonadal steroid hormone production should be further studied.