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Free access

M Watanabe, ER Simpson, N Pathirage, S Nakajin and CD Clyne

A number of clinical studies have highlighted the importance of estrogen in bone growth and maintenance in men and postmenopausal women. In these instances, estrogen is synthesized locally within bone tissue by aromatase, encoded by the CYP19 gene. The mechanisms regulating aromatase expression in bone, however, are unclear. In this work we characterized the expression of aromatase activity and gene transcripts in the human fetal osteoblastic cell line, SV-HFO. Aromatase activity and gene transcript expression were stimulated by dexamethasone. Oncostatin M strongly stimulated aromatase expression in synergy with dexamethasone. These factors induced CYP19 transcripts that included the sequence of exon I.4 in their 5'UTR. Consistent with this, a reporter construct harboring the genomic sequence of the promoter region of exon I.4 (promoter I.4) was also activated by dexamethasone and oncostatin M. 5' deletion and mutation analysis revealed important roles for a glucocorticoid response element, an interferon gamma activating sequence and a putative binding site for Sp1. Transfection of exogenous glucocorticoid receptor, STAT3 or Sp1 increased promoter activity, indicating a potential role for these transcription factors in regulating aromatase expression in SV-HFO cells. These data suggest that the SV-HFO cell line is a valuable model with which to elucidate the mechanisms regulating local estrogen synthesis in osteoblasts.

Free access

Anne-Marie O’Carroll, Stephen J Lolait and Gillian M Howell

The genomic structure and transcriptional regulation of the rat apelin receptor (APJR) were analysed by rapid amplification of 5′ cDNA ends (5′-RACE), transient expression assays and DNA–protein interaction. Analysis of the 5′-flanking region of a rat genomic clone shows no TATA box, but a putative CAAT box and several putative binding sites for transcription factors are present. Two transcriptional start sites were identified by 5′-RACE, RNase protection and primer extension analyses. Promoter activity was exhibited in the APJR- expressing SH-SY5Y cell line as well as in COS-7 and Chinese hamster ovary (CHO-K1) cells. Consecutive 5′-deletion analysis revealed the highest promoter activity in a region between bp −966 and −165. DNaseI footprint analysis revealed seven protected regions and electrophoretic mobility shift, super-shift and competition assays identified individual DNA–protein complexes capable of binding Sp1, estrogen receptor (ER)α, glucocorticoid receptor and CCAAT enhancer binding protein (C/EBP)γ transcription factors. Site-directed mutagenesis identified an individual Sp1 motif that plays a major role in activation of the APJR promoter and also demonstrated constitutive transcriptional regulation of the promoter by estrogen and glucocorticoid receptors. Promoter regulation by the cAMP-dependent signal cascade was also shown.

Free access

Jacqueline Brodie and Iain J McEwan

The androgen receptor (AR) is a ligand-activated transcription factor that recognises and binds to specific DNA response elements upon activation by the steroids testosterone or dihydrotestosterone. In vitro, two types of response element have been characterised - non-selective elements that bind the androgen, glucocorticoid and progesterone receptors, and androgen receptor-selective sequences. In the present study, the allosteric effects of DNA binding on the receptor amino-terminal domain (NTD) were studied. Binding to both types of DNA response element resulted in changes in the intrinsic fluorescence emission spectrum for four tryptophan residues within the AR-NTD and resulted in a more protease-resistant conformation. In binding experiments, it was observed that the presence of the AR-NTD reduced the affinity of receptor polypeptides for binding to both selective and non-selective DNA elements derived from the probasin, PEM and prostatin C3 genes respectively, without significantly altering the protein–base pair contacts. Taken together, these results highlight the role of intra-domain communications between the AR-NTD and the DNA binding domain in receptor structure and function.

Free access

NR Bury, A Sturm, P Le Rouzic, C Lethimonier, B Ducouret, Y Guiguen, M Robinson-Rechavi, V Laudet, ME Rafestin-Oblin and P Prunet

Using RT-PCR with degenerated primers followed by screening of a rainbow trout (Oncorhynchus mykiss) intestinal cDNA library, we have isolated from the rainbow trout a new corticosteroid receptor which shows high sequence homology with other glucocorticoid receptors (GRs), but is clearly different from the previous trout GR (named rtGR1). Phylogenetic analysis of these two sequences and other GRs known in mammals, amphibians and fishes indicate that the GR duplication is probably common to most teleost fish. The open reading frame of this new trout GR (named rtGR2) encodes a protein of 669 amino acids and in vitro translation produces a protein of 80 kDa that appears clearly different from rtGR1 protein (88 kDa). Using rtGR2 cDNA as a probe, a 7.3 kb transcript was observed in various tIssues suggesting that this gene would lead to expression of a steroid receptor. In vitro studies were used to further characterize this new corticosteroid receptor. Binding studies with recombinant rtGR1 and rtGR2 proteins show that the two receptors have a similar affinity for dexamethasone (GR1 K(d)=5.05+/-0.45 nM; GR2 K(d)=3.04+/-0.79 nM). Co-transfection of an rtGR1 or rtGR2 expression vector into CHO-K1 or COS-7 cells, along with a reporter plasmid containing multiple consensus glucocorticoid response elements, shows that both clones are able to induce transcriptional activity in the presence of cortisol and dexamethasone. Moreover, at 10(-)(6 )M 11-deoxycortisol and corticosterone partially induced rtGR2 transactivation activity but were without effect on rtGR1. The other major teleost reproductive hormones, as well as a number of their precursors or breakdown products of these and corticosteroid hormones, were without major effects on either receptor. Interestingly, rtGR2 transactivational activity was induced at far lower concentrations of dexamethasone or cortisol (cortisol EC(50)=0.72+/-0.87 nM) compared with rtGR1 (cortisol EC(50)=46+/-12 nM). Similarly, even though RU486 inhibited transactivation activity in both rtGR1 and rtGR2, rtGR1 was more sensitive to this GR antagonist. Altogether, these results indicate that these two GR sequences encode for two functionally distinct GRs acting as ligand-inducible transcription factors in rainbow trout.

Restricted access

N. Hanley, B. C. Williams, M. Nicol, I. M. Bird and S. W. Walker

ABSTRACT

Using tritiated-thymidine incorporation as a measure of cell growth, interleukin-1β stimulated the growth of bovine zona fasciculata/reticularis adrenocortical cells after 72h in primary culture. Within the range of 10–1000pg/ml, interleukin-1β produced over 40% of angiotensin II-stimulated [3H]thymidine incorporation (P<0.005 compared with basal for 10pg/ml and 1000pg/ml; P<0.05 for 100pg/ml; two-tailed unpaired Student's t-test). Interleukin-1β did not directly stimulate cortisol secretion.

By stimulating adrenocortical growth, the increase in interleukin-1 during fever provides a potential mechanism for chronically raising glucocorticoid output. This study is the first demonstration of a long-term effect involving interleukin-1β on the adrenal cortex.

Free access

SM Huang, CJ Huang, WM Wang, JC Kang and WC Hsu

The p160 coactivators, steroid receptor coactivator 1, glucocorticoid receptor interacting protein 1 (GRIP1) and the activator of thyroid and retinoic acid receptor, have two activation domains, AD1 and AD2, which transmit the activation signal from the DNA-bound nuclear receptor to the chromatin and/or transcription machinery. In screening for mammalian proteins that bind the AD2 of GRIP1, we identified a mouse actin-binding protein, alpha actinin 2 (mACTN2). mACTN2 was expressed in the heart, skeletal muscle, lung, brain and testis, but there was no expression in the spleen, liver or kidney. Interestingly, the expression level of mACTN2 in the developing embryo depended on the embryonic stage. We further demonstrated that mACTN2 could enhance two transactivation activities of GRIP1, which in turn could enhance the homodimerization of mACTN2. Importantly, mACTN2 not only served as a primary coactivator for androgen receptor, estrogen receptor and thyroid receptor activities, but also acted synergistically with GRIP1 to enhance these nuclear receptor (NR) functions. However, the NR binding motif, LXXLL, conserved in mACTN2 and other actinin family proteins, might be a dispensable domain for its coactivator roles in NRs. These findings suggested that mACTN2 might play an important role in GRIP1-induced NR coactivator functions.

Free access

ChengCheng Lin, Bei Shao, YuLei Zhou, XiaoTing Niu and YuanShao Lin

Diet-induced epigenetic modifications in early life could contribute to later health problem. However, it remains to be established whether high-fat diet (HFD) consumption during pregnancy and the suckling period could predispose the offspring to stroke. The present study investigated the influence of maternal HFD on stroke outcome in adult offspring. Female Sprague–Dawley rats were fed a normal diet (5.3% fat) or a HFD (25.7% fat), just before pregnancy until the end of lactation. Male offspring were fed with the control diet or the HFD after weaning, to form four groups (control offspring fed with the control diet (C/C) or the HFD (C/HFD) and offspring of fat-fed dams fed with the control diet (HFD/C) or the HFD (HFD/HFD)). The offspring received middle cerebral artery occlusion on day 120 followed by behavioral tests (neurological deficit score, staircase-reaching test and beam-traversing test), and infarct volumes were also calculated. We found that the HFD/C rats displayed larger infarct volume and aggravated functional deficits (all P<0.05), compared with the C/C rats, indicating that maternal fat-rich diet renders the brain more susceptible to the consequences of ischemic injury. Moreover, maternal HFD offspring displayed elevated glucocorticoid concentrations following stroke, and increased glucocorticoid receptor expression. In addition, adrenalectomy reversed the effects of maternal HFD on stroke outcome when corticosterone was replaced at baseline, but not ischemic, concentrations. Furthermore, expression of brain-derived neurotrophic factor (BDNF) in the ipsilateral hippocampus was decreased in the HFD/C offspring (P<0.05), compared with the C/C offspring. Taken together, maternal diet can substantially influence adult cerebrovascular health, through the programming of central BDNF expression and the hypothalamic–pituitary–adrenal axis.

Free access

Jun Yang and Morag J Young

The mineralocorticoid receptor (MR) is a member of the nuclear receptor superfamily and is essential for controlling sodium transport in epithelial tissues such as the kidney and colon. Moreover, it is also present in other non-epithelial tissues and is capable of activation by both mineralocorticoids and glucocorticoids. A challenge in understanding transcriptional regulation by the MR and other nuclear receptors is to determine how tissue- and ligand-specificity is achieved. Over the past decade, it has become clear that a heterogeneous group of non-receptor proteins termed as coregulators are required to either enhance or repress nuclear receptor-mediated transactivation of target genes. A subset of these coregulators may be expected to confer specificity to MR-mediated responses by virtue of their variable tissue expression and selectivity for different ligands. Specific coregulator–MR interactions may be a suitable target in the rational design of tissue-specific MR modulators as has been described for other steroid receptors. However, the number of coregulators identified to date for the MR is very limited compared with other nuclear receptors. Understanding the full complement of MR coregulators is essential for unraveling the complexity of MR signaling pathways and will facilitate the development of selective MR modulators.

Free access

O Nakabayashi, H Kikuchi, T Kikuchi and S Mizuno

In birds, differentiation of embryonic gonads is not as strictly determined by the genetic sex as it is in mammals, and can be influenced by early manipulation with a sex steroid hormone. Thus administration of an aromatase inhibitor induces testis development in the genetic female, and administration of estrogen induces a left ovotestis in the genetic male embryo. Another feature of avian gonadogenesis is that only the left ovary develops in most species. Molecular mechanisms underlying these features at the level of gene expression have not been elucidated. In this paper, we present evidence that a gene for aromatase cytochrome P-450, an enzyme required for the last step in the synthesis of estradiol-17beta, is expressed in medullae of the left and right gonads of a female chicken embryo, but not in those of a male chicken embryo, and that an estrogen receptor gene is expressed only in epithelium (and cortex later, in the female) of the left, not the right, gonad of both sexes, but the expression in the male left gonad is temporary and restricted to an early stage of development. Differential expression of these two genes serves well to explain the above features of gonadal development in birds. Furthermore, in ovo administration of estradiol-17beta from the 5th to the 14th day of incubation does not cause expression of the estrogen receptor gene in the right gonad of chicken embryos of either sex, suggesting that the absence of expression of the estrogen receptor gene in the right gonad is not the result of down-regulation, but may be regarded as an important cause of the unilateral ovarian development.

Free access

Jyotsna B Pippal and Peter J Fuller

The signature action of aldosterone in the regulation of electrolyte and fluid balance is well established. However, the role of aldosterone as an important contributor to morbidity and mortality in heart failure has gained a heightened interest in recent years, but the mechanisms of this action are not well understood. Aldosterone is the principal physiological ligand for the mineralocorticoid receptor (MR), a ligand-activated transcription factor, that also binds to the physiological glucocorticoid, cortisol. Both classes of hormones bind with similar affinity to the MR, but the molecular basis of selective and tissue-specific effects of MR ligands is not yet fully documented. The structural and functional determinants of MR function are described and their significance is discussed.