The alpha 2u-globulins are the major urinary proteins in adult male rats. They are encoded by a gene family, the expression of which is under multihormonal control in the liver. Glucocorticoids are positive regulatory hormones and we have analyzed the contribution of 5'-upstream sequences to the induction by dexamethasone of two cloned members of the family transfected into mouse L-cells. The results demonstrate that sequences from -762 bp to -226 bp of clone 91 are required for the 24-fold level of induction that was observed. Addition of 5.5 kb of upstream sequence beyond -762 bp did not alter the level of induction significantly, whereas deletion of the DNA between -762 bp and -226 bp reduced inducibility to about 4-fold. Sequencing of this region revealed that an element, 5'-AGAACAggtTTCAAA-3', similar to the 15 bp consensus glucocorticoid response element 5'-AGAACAnnnTGTACC-3', is situated 513 bp upstream of the transcription start site. We infer that this element or its left half site is necessary for the dexamethasone-induced expression of clone 91 from the observation that a second gene, clone 2, that contained a base substitution at position 5 in the left half site was not inducible. It now appears that at least three distinct cis-acting regulatory regions, all of which bind to the glucocorticoid receptor in vitro, may contribute to the full induction of clone 91 by dexamethasone. These are: the distal upstream region identified by this study, a proximal upstream region that binds not only the receptor but also alpha 2uNF1, a constitutively expressed nuclear protein required for induction and a region within the fourth intron that contains five tandem receptor binding sites.
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I J Bujalska, M Quinkler, J W Tomlinson, C T Montague, D M Smith and P M Stewart
Obesity is associated with increased morbidity and mortality from cardiovascular disease, diabetes and cancer. Although obesity is a multi-factorial heterogeneous condition, fat accumulation in visceral depots is most highly associated with these risks. Pathological glucocorticoid excess (i.e. in Cushing’s syndrome) is a recognised, reversible cause of visceral fat accumulation. The aim of this study was to identify depot-specific glucocorticoid-target genes in adipocyte precursor cells (preadipocytes) using Affymetrix microarray technique. Confluent preadipocytes from subcutaneous (SC) and omental (OM) adipose tissue collected from five female patients were treated for 24 h with 100 nM cortisol (F), RNA was pooled and hybridised to the Affymetrix U133 microarray set. We identified 72 upregulated and 30 downregulated genes by F in SC cells. In OM preadipocytes, 56 genes were increased and 19 were decreased. Among the most interesting were transcription factors, markers of adipocyte differentiation and glucose metabolism, cell adhesion and growth arrest protein factors involved in G-coupled and Wnt signalling. The Affymetrix data have been confirmed by quantitative real-time PCR for ten specific genes, including HSD11B1, GR, C/EBPα, C/EBPβ, IL-6, FABP4, APOD, IRS2, AGTR1 and GHR. One of the most upregulated genes in OM but not in SC cells was HSD11B1. The GR was similarly expressed and not regulated by glucocorticoids in SC and OM human preadipocytes. C/EBPα was expressed in SC preadipocytes and upregulated by F, but was below the detection level in OM cells. C/EBPβ was highly expressed both in SC and in OM preadipocytes, but was not regulated by F. Our results provide insight into the genes involved in the regulation of adipocyte differentiation by cortisol, highlighting the depot specifically in human adipose tissue.
A Cote-Vélez, L Pérez-Martínez, M Y Díaz-Gallardo, C Pérez-Monter, A Carreón-Rodríguez, J-L Charli and P Joseph-Bravo
Hypothalamic proTRH mRNA levels are rapidly increased (at 1 h) in vivo by cold exposure or suckling, and in vitro by 8Br-cAMP or glucocorticoids. The aim of this work was to study whether these effects occurred at the transcriptional level. Hypothalamic cells transfected with rat TRH promoter (− 776/+85) linked to the luciferase reporter showed increased transcription by protein kinase (PK) A and PKC activators, or by dexamethasone (dex), but co-incubation with dex and 8Br-cAMP decreased their stimulatory effect (as observed for proTRH mRNA levels). These effects were also observed in NIH-3T3-transfected cells supporting a characteristic of TRH promoter and not of hypothalamic cells. Transcriptional regulation by 8Br-cAMP was mimicked by noradrenaline which increased proTRH mRNA levels, but not in the presence of dex. PKA inhibition by H89 avoided 8Br-cAMP or noradrenaline stimulation. TRH promoter sequences, cAMP response element (CRE)-like (− 101/− 94 and − 59/− 52) and glucocorticoid response element (GRE) half-site (− 210/− 205), were analyzed by electrophoretic mobility shift assays with nuclear extracts from hypothalamic or neuroblastoma cultures. PKA stimulation increased binding to CRE (− 101/− 94) but not to CRE (− 59/− 52); dex or 12-O-tetradecanoylphorbol-13-acetate (TPA) increased binding to GRE, a composite site flanked by a perfect and an imperfect activator protein (AP-1) site in the complementary strand. Interference was observed in the binding of CRE or GRE with nuclear extracts from cells co-incubated for 3 h with 8Br-cAMP and dex; from cells incubated for 1 h, only the binding to GRE showed interference. Rapid cross-talk of glucocorticoids with PKA signaling pathways regulating TRH transcription constitutes another example of neuroendocrine integration.
Kelly L Short, A Daniel Bird, Bennet K L Seow, Judy Ng, Annie R A McDougall, Megan J Wallace, Stuart B Hooper and Timothy J Cole
Glucocorticoid (GC) signaling via the glucocorticoid receptor (GR) is essential for lung maturation in mammals. Previous studies using global or conditional mouse model knockouts of the GR gene have established that GR-mediated signaling in the interstitial mesenchyme of the fetal lung is critical for normal lung development. Screens for downstream GC-targets in conditional mesenchymal GR deficient mouse lung (GRmesKO) identified Versican (Vcan), an important extracellular matrix component and cell proliferation regulator, as a potential GR-regulated target. We show that, of the five major VCAN isoforms, the VCAN-V1 isoform containing the GAGβ domain is the predominant VCAN isoform in the fetal mouse lung distal mesenchyme at both E16.5 and E18.5, whereas the GAGα-specific VCAN-V2 isoform was only localized to the smooth muscle surrounding proximal airways. Both Vcan-V1 mRNA and protein levels were strongly overexpressed in the GRmesKO lung at E18.5. Finally, we investigated the GC regulation of the ECM protease ADAMTS 12 and showed that Adamts 12 mRNA levels were markedly reduced at E18.5 in GRmesKO fetal mouse lung and were strongly induced by both cortisol and betamethasone in cultures of primary rat fetal lung fibroblasts. ADAMTS12 protein immunoreactivity was also strongly increased in the distal lung at E18.5, after dexamethasone treatment in utero. In summary, glucocorticoid signaling via GR represses GAGβ domain-containing VCAN isoforms in distal lung mesenchyme in vivo by repressing Vcan gene expression and, in part, by inducing the ECM protease ADAMTS12, thereby contributing to the control of ECM remodelling and lung cell proliferation prior to birth.
The nuclear receptor family responds to a diverse group of ligands, including steroids, retinoids, thyroid hormone, prostaglandins and fatty acids. Previous sequence analyses of adrenal and sex steroid receptors indicate that they form a clade separate from other nuclear receptors. However, the relationships of adrenal and sex steroid receptors to each other and to their ancestors are not fully understood. We have used new information from androgen, estrogen, mineralocorticoid and progesterone receptors in fish to better resolve the phylogeny of adrenal and sex steroid receptors. Sequence divergence between fish and mammalian steroid receptors correlates with differences in steroid specificity, suggesting that phylogeny needs to be considered in evaluating the endocrine effects of xenobiotics. Among the vertebrate steroid receptors, the most ancient is the estrogen receptor. The phylogeny indicates that adrenal and sex steroid receptors arose in a jawless fish or a protochordate and that changes in the sequence of the hormone-binding domain have slowed considerably in land vertebrates. The retinoid X receptor clade is closest to the adrenal and sex steroid receptor clade. Retinoid X receptor is noteworthy for its ability to form dimers with other nuclear receptors, an important mechanism for regulating the action of retinoid X receptor and its dimerization partners. In contrast, the adrenal and sex steroid receptors bind to DNA as homodimers. Moreover, unliganded adrenal and sex steroid receptors form complexes with heat shock protein 90. Thus, the evolution of adrenal and sex steroid receptors involved changes in protein-protein interactions as well as ligand recognition.
Gábor Bánhegyi, Miklós Csala and Angelo Benedetti
Hexose-6-phosphate dehydrogenase (H6PD) got into the focus of interest due to its role in the prereceptorial activation of glucocorticoids, which has been implicated in the pathomechanism of metabolic syndrome. Genetic observations, results gained in H6PD knockout mice, and studies on differentiating adipocytes demonstrated the importance of the enzyme in metabolic regulation. A nutrient-sensing function can be postulated for the enzyme, which links metabolism to endocrinology in the endoplasmic reticulum. This review provides an overview of the recent developments concerning the enzyme and its impact on various branches of the intermediary metabolism, which make it an important subject for the research on obesity, diabetes, and metabolic syndrome.
Chung Thong Lim, Blerina Kola and Márta Korbonits
AMP-activated protein kinase (AMPK) is a key molecular player in energy homeostasis at both cellular and whole-body levels. AMPK has been shown to mediate the metabolic effects of hormones such as leptin, ghrelin, adiponectin, glucocorticoids and insulin as well as cannabinoids. Generally, activated AMPK stimulates catabolic pathways (glycolysis, fatty acid oxidation and mitochondrial biogenesis) and inhibits anabolic pathways (gluconeogenesis, glycogen, fatty acid and protein synthesis), and has a direct appetite-regulating effect in the hypothalamus. Drugs that activate AMPK, namely metformin and thiazolidinediones, are often used to treat metabolic disorders. Thus, AMPK is now recognised as a potential target for the treatment of obesity and associated co-morbidities.
Hyeyeon Ko, WooDong Park, Dae-Jung Kim, Makito Kobayashi and Young Chang Sohn
Gonadotropins (GTHs), FSH and LH, play central roles in vertebrate reproduction. Here, we report the production of biologically-active recombinant FSH (r-mtFSH) and LH (r-mtLH) of an endangered salmon species, Manchurian trout (Brachymystax lenok), by baculovirus in silkworm (Bombyx mori) larvae. The biological activities of the recombinant hormones were analyzed using COS-7 cell line transiently expressing either amago salmon FSH or LH receptor. The steroidogenic potency of the r-mtFSH and r-mtLH was examined by a culture system using rainbow trout follicles in vitro. In vivo, bioactivity was assessed by measuring ovarian weight, oocyte diameter, and plasma steroid hormone levels in female rainbow trout. Moreover, inducing potency of milt production were examined in vivo using goldfish. Our results demonstrated that the r-mtFSH and r-mtLH were successfully produced in the baculovirus-silkworm system and recognized by their cognate receptors specifically in vitro. The production of estradiol-17β (E2) and testosterone (T) was stimulated by the r-mtFSH and r-mtLH respectively, from the full-grown follicles of rainbow trout, whereas both E2 and T were increased by relatively higher doses of the recombinant hormones from the follicles of the maturing stage. In in vivo assay, injection of the r-mtFSH but not r-mtLH increased ovarian weight, oocyte diameter, and plasma E2 levels in immature rainbow trout. Injection of both r-mtFSH and r-mtLH induced milt production in male goldfish. In conclusion, the present study strongly suggests that the r-mtFSH and r-mtLH have distinct biological properties, such as a specific responsiveness for the cognate receptor, steroidogenic, and vitellogenic activities for ovarian follicles in salmonids. These recombinant FSH and LH may be applied for future studies on the gonadal development and maturation in fishes as well as the endangered salmon species.
Colin R Jefcoate and Jinwoo Lee
Cholesterol is an important regulator of cell signaling, both through direct impacts on cell membranes and through oxy-metabolites that activate specific receptors (steroids, hydroxy-cholesterols, bile acids). Cholesterol moves slowly through and between cell membranes with the assistance of specific binding proteins and transfer processes. The prototype cholesterol regulator is the Steroidogenesis Acute Regulatory (STAR), which moves cholesterol into mitochondria, where steroid synthesis is initiated by cytochrome P450 11A1 in multiple endocrine cell types. CYP27A1 generates hydroxyl cholesterol metabolites that activate LXR nuclear receptors to control cholesterol homeostatic and transport mechanisms. LXR regulation of cholesterol transport and storage as cholesterol ester droplets is shared by both steroid-producing cells and macrophage. This cholesterol signaling which is crucial to brain neuron regulation by astrocytes and microglial macrophage, is mediated by ApoE and is sensitive to disruption by β-amyloid plaques. sm-FISH delivers appreciable insights into signaling in single cells, by resolving single RNA molecules as mRNA and by quantifying pre-mRNA at gene loci. sm-FISH has been applied to problems in physiology, embryo development and cancer biology, where single cell features have critical impacts. sm-FISH identifies novel features of STAR transcription in adrenal and testis cells, including asymmetric expression at individual gene loci, delayed splicing and 1:1 association of mRNA with mitochondria. This may represent a functional unit for the translation-dependent cholesterol transfer directed by STAR, which integrates into mitochondrial fusion dynamics. Similar cholesterol dynamics repeat with different players in the cycling of cholesterol between astrocytes and neurons in the brain, which may be abnormal in neurodegenerative diseases.
T. Imai, H. Seo, Y. Murata, M. Ohno, Y. Satoh, H. Funahashi, H. Takagi and N. Matsui
The changes in steady-state levels of mRNA for cholesterol side-chain cleavage cytochrome P-450 (P-450scc) and steroid 21-hydroxylase cytochrome P-450 (P-450c21) caused by hypophysectomy and ACTH treatment were determined in rat adrenals. Hypophysectomy caused marked decreases in adrenal weight and total RNA per gland. Administration of ACTH resulted in increases in adrenal weight and total RNA. A significant correlation between the amount of RNA and adrenal weight was observed. Both P-450scc and P-450c21 mRNAs were decreased by hypophysectomy and increased by ACTH treatment. P-450scc mRNA decreased to 20% and P-450c21 mRNA to 76% of control values 1 day after hypophysectomy. ACTH caused a significant increase in P-450scc mRNA after 3 h. However, a significant increase in P-450c21 mRNA was observed 12 h after administration of ACTH. These results are concordant with previous studies in vitro utilizing cultured adrenocortical cells. Moreover, the induction of steady-state levels of P-450scc mRNA was faster than that observed by other investigators in studies in vitro. These results may indicate that integrity of the adrenal gland in vivo is important for the action of ACTH.