scholarly journals 11β-hydroxysteroid dehydrogenase type 1 expression in 2S FAZA hepatoma cells is hormonally regulated: a model system for the study of hepatic glucocorticoid metabolism

1996 ◽  
Vol 317 (2) ◽  
pp. 621-625 ◽  
Author(s):  
Michael W. VOICE ◽  
Jonathan R. SECKL ◽  
Christopher R. W. EDWARDS ◽  
Karen E. CHAPMAN
Keyword(s):  
Mrna Expression ◽  
Hydroxysteroid Dehydrogenase ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Transcription Start ◽  
Key Enzyme ◽  
Glucocorticoid Metabolism ◽  
Hepatic Cell Lines

11β-Hydroxysteroid dehydrogenase (11β-HSD) is a key enzyme in glucocorticoid metabolism, catalysing the conversion of active glucocorticoids into their inactive 11-keto metabolites, thus regulating glucocorticoid access to intracellular receptors. The type 1 isoform (11β-HSD 1) (EC 1.1.1.146) is widely distributed, with particularly high levels in liver, where accumulating evidence suggests that it acts as an 11β-reductase, regenerating active glucocorticoids. Investigation of the function and regulation of 11β-HSD 1 in liver has been hampered by the lack of hepatic cell lines which express 11β-HSD 1. Here, we describe 11β-HSD 1 mRNA expression and activity in 2S FAZA cells, a continuously cultured rat liver cell line. In intact 2S FAZA cells 11β-HSD 1 acts predominantly as a reductase, with very low dehydrogenase activity. In 2S FAZA cells 11β-HSD 1 activity and mRNA expression are regulated by hormones, with dexamethasone increasing activity and insulin, forskolin and insulin-like growth factor 1 decreasing it. Transfection of 2S FAZA cells with a luciferase reporter gene driven by the proximal promoter of the rat 11β-HSD 1 gene demonstrates that sequences which can mediate the responses to insulin, dexamethasone and forskolin all lie within 1800 bp of the transcription start site.

scholarly journals Role of glucocorticoid receptor and CCAAT/enhancer-binding protein α in the feed-forward induction of 11β-hydroxysteroid dehydrogenase type 1 expression by cortisol in human amnion fibroblasts

2007 ◽  
Vol 195 (2) ◽  
pp. 241-253 ◽  
Author(s):  
Zhen Yang ◽  
Chunming Guo ◽  
Ping Zhu ◽  
Wenjiao Li ◽  
Leslie Myatt ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Mrna Expression ◽  
Promoter Region ◽  
Molecular Mechanisms ◽  
Hydroxysteroid Dehydrogenase ◽  
Dominant Negative ◽  
Forward Induction ◽  
Human Amnion ◽  
Feed Forward

The amount of cortisol available to its receptors is increased by the pre-receptor enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which converts cortisone to cortisol. We examined the molecular mechanisms of the feedback effect of cortisol on 11β-HSD1 mRNA expression in human amnion fibroblasts. Our data showed that cortisol-induced 11β-HSD1 mRNA expression dose dependently in amnion fibroblasts, which could be completely blocked both by the mRNA transcription inhibitor 5,6-dichlorobenzimidazole riboside and by the glucocorticoid receptor (GR) antagonist RU486, and partially blocked by global inhibition of CCAAT/enhancer-binding proteins (C/EBPs) with transfection of C/EBP-specific dominant-negative expression CMV500 plasmid (AC/EBP) into the cells. Likewise, the induction of the promoter activity by cortisol could also be completely blocked by RU486 and partially by AC/EBP transfection. Progressive 5′ deletion of the 11β-HSD1promoter located the region responsible for cortisol’s induction within −204 bp upstream to the transcription start site. Specific nucleotide mutations of the putative glucocorticoid responsive element or CCAAT in this promoter region attenuated the induction by cortisol. Moreover, chromatin immunoprecipitation assay and electrophoretic mobility shift assay showed that GR and C/EBPα but not C/EBPβ could bind this promoter region upon cortisol stimulation of amnion fibroblasts. In conclusion, we demonstrated that GR and C/EBPα were involved in cortisol-induced 11β-HSD1 mRNA expression via binding to 11β-HSD1 promoter in amnion fibroblasts, which may cast a feed-forward production of cortisol in the fetal membranes at the end of gestation.

scholarly journals Anti-TAR Polyamide Nucleotide Analog Conjugated with a Membrane-Permeating Peptide Inhibits Human Immunodeficiency Virus Type 1 Production

2002 ◽  
Vol 76 (8) ◽  
pp. 3881-3891 ◽  
Author(s):  
Neerja Kaushik ◽  
Amartya Basu ◽  
Paul Palumbo ◽  
Rene L. Myers ◽  
Virendra N. Pandey
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Treatment ◽  
Significant Inhibition ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Nucleotide Analog ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The emergence of drug-resistant variants has posed a significant setback against effective antiviral treatment for human immunodeficiency virus (HIV) infections. The choice of a nonmutable region of the viral genome such as the conserved transactivation response element (TAR element) in the 5′ long terminal repeat (LTR) may potentially be an effective target for drug development. We have earlier demonstrated that a polyamide nucleotide analog (PNA) targeted to the TAR hairpin element, when transfected into cells, can effectively inhibit Tat-mediated transactivation of HIV type 1 (HIV-1) LTR (T. Mayhood et al., Biochemistry 39:11532-11539, 2000). Here we show that this anti-TAR PNA (PNATAR), upon conjugation with a membrane-permeating peptide vector (transportan) retained its affinity for TAR in vitro similar to the unconjugated analog. The conjugate was efficiently internalized into the cells when added to the culture medium. Examination of the functional efficacy of the PNATAR-transportan conjugate in cell culture using luciferase reporter gene constructs resulted in a significant inhibition of Tat-mediated transactivation of HIV-1 LTR. Furthermore, PNATAR-transportan conjugate substantially inhibited HIV-1 production in chronically HIV-1-infected H9 cells. The mechanism of this inhibition appeared to be regulated at the level of transcription. These results demonstrate the efficacy of PNATAR-transportan as a potential anti-HIV agent.

scholarly journals Enhancement of Cortisol-Induced 11β-Hydroxysteroid dehydrogenase Type 1 Expression by Interleukin 1β in Cultured Human Chorionic Trophoblast Cells

Endocrinology ◽  
2006 ◽  
Vol 147 (5) ◽  
pp. 2490-2495 ◽  
Author(s):  
Wenjiao Li ◽  
Lu Gao ◽  
Yan Wang ◽  
Tao Duan ◽  
Leslie Myatt ◽  
...  
Keyword(s):  
Gene Expression ◽  
Phospholipase A2 ◽  
Mrna Expression ◽  
Reporter Gene ◽  
Reductase Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Cytosolic Phospholipase A2 ◽  
Trophoblast Cells ◽  
Cytosolic Phospholipase

Chorion is the most abundant site of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) expression within intrauterine tissues. It is important to study the regulation of 11β-HSD1 expression in the chorion in terms of local cortisol production during pregnancy. Using real-time PCR and enzyme activity assay, we found that cortisol (1 μm) and IL-1β (10 ng/ml) for 24 h significantly increased 11β-HSD1 mRNA expression and reductase activity in cultured human chorionic trophoblasts. A further significant increase of 11β-HSD1 mRNA expression and reductase activity was observed with cotreatment of cortisol and IL-1β. To explore the mechanism of induction, 11β-HSD1 promoter was cloned into pGL3 plasmid expressing a luciferase reporter gene. By transfecting the constructed vector into WISH cells, an amnion-derived cell line, we found that cortisol (1 μm) or IL-1β (10 ng/ml) significantly increased reporter gene expression. Likewise, an additional increase in reporter gene expression was observed with cotreatment of cortisol and IL-β. To explore the physiological significance of 11β-HSD1 induction in the chorion, we studied the effect of cortisol on cytosolic phospholipase A2 and cyclooxygenase 2 expression. We found that treatment of chorionic trophoblast cells with cortisol (1 μm) induced both cytosolic phospholipase A2 and cyclooxygenase 2 mRNA expression. We conclude that cortisol up-regulates 11β-HSD1 expression through induction of promoter activity, and the effect was enhanced by IL-1β, suggesting that more biologically active glucocorticoids could be generated in the fetal membranes in the presence of infection, which may consequently feed forward in up-regulation of prostaglandin synthesis.

scholarly journals Getting to the heart of intracellular glucocorticoid regeneration: 11β-HSD1 in the myocardium

2017 ◽  
Vol 58 (1) ◽  
pp. R1-R13 ◽  
Author(s):  
Gillian A Gray ◽  
Christopher I White ◽  
Raphael F P Castellan ◽  
Sara J McSweeney ◽  
Karen E Chapman
Keyword(s):  
Myocardial Infarction ◽  
Heart Failure ◽  
Systemic Corticosteroid ◽  
Physiological Function ◽  
Pressure Overload ◽  
Hydroxysteroid Dehydrogenase ◽  
And Function ◽  
Glucocorticoid Metabolism

Corticosteroids influence the development and function of the heart and its response to injury and pressure overload via actions on glucocorticoid (GR) and mineralocorticoid (MR) receptors. Systemic corticosteroid concentration depends largely on the activity of the hypothalamic–pituitary–adrenal (HPA) axis, but glucocorticoid can also be regenerated from intrinsically inert metabolites by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), selectively increasing glucocorticoid levels within cells and tissues. Extensive studies have revealed the roles for glucocorticoid regeneration by 11β-HSD1 in liver, adipose, brain and other tissues, but until recently, there has been little focus on the heart. This article reviews the evidence for glucocorticoid metabolism by 11β-HSD1 in the heart and for a role of 11β-HSD1 activity in determining the myocardial growth and physiological function. We also consider the potential of 11β-HSD1 as a therapeutic target to enhance repair after myocardial infarction and to prevent the development of cardiac remodelling and heart failure.

Sp1-like activity mediates angiotensin-II-induced plasminogen-activator inhibitor type-1 (PAI-1) gene expression in mesangial cells

2000 ◽  
Vol 349 (2) ◽  
pp. 435-441 ◽  
Author(s):  
Masaru MOTOJIMA ◽  
Takao Ando ◽  
Toshimasa YOSHIOKA
Keyword(s):  
Transcription Factor ◽  
Mrna Expression ◽  
Mesangial Cells ◽  
Plasminogen Activator Inhibitor ◽  
Proximal Promoter ◽  
Ang Ii ◽  
Inhibitor Type ◽  
Activator Inhibitor Type ◽  
Pai 1

Angiotensin II (Ang II) up-regulates plasminogen-activator inhibitor type-1 (PAI-1) expression in mesangial cells to enhance extracellular matrix formation. The proximal promoter region (bp -87 to -45) of the human PAI-1 gene contains several potent binding sites for transcription factors [two phorbol-ester-response-element (TRE)-like sequences; D-box (-82 to -76) and P-box (-61 to 54), and one Sp1 binding site-like sequence, Sp1-box 1 (-72 to -67)]. We studied this region to determine the transcription factor(s) that mediates Ang-II-induced transcriptional activation of the PAI-1 gene. Various double-stranded decoy oligodeoxynucleotides (ODNs) corresponding to various sequences in the proximal promoter region were transfected to mesangial cells to examine the effects on Ang-II-induced PAI-1 mRNA expression. Transfection with the full-length decoy (bp -87 to -45, D-P-ODN) markedly attenuated Ang-II-induced PAI-1 mRNA expression by up to 70%. Transfection with D-ODN (-87 to -71) and P-ODN (-66 to -45), which correspond to each of the two TRE-like sequences, did not attenuate the expression. Gel-shift assays using nuclear extracts prepared from Ang-II-treated mesangial cells and D-P-ODN showed three specific complexes. The major complex was supershifted by anti-Sp1 antibody. The methylation-interference experiment demonstrated that human recombinant Sp1 bound to the so-called GT box (TGGGTGGGGCT, -78 to -69), which contains the Sp1-box 1. The complex that migrated with anti-Sp1 antibody was enhanced in the cells treated with Ang II. Further, D-Sp1-ODN (-85 to -63) containing the GT box attenuated up-regulation of PAI-1 mRNA expression induced by Ang II to a level (68±9% inhibition) comparable to D-P-ODN, whereas ODN with four mutations in the GT box had no effect. Our findings suggest that binding of Sp1 or an Sp1-like transcription factor to the GT box in the PAI-1 promoter up-regulates PAI-1 gene transcription in mesangial cells stimulated with Ang II. This transcription-factor binding site may be targeted to control Ang-II-dependent extracellular matrix formation by mesangial cells.

scholarly journals Functional analysis of the human annexin I and VI gene promoters

1998 ◽  
Vol 332 (3) ◽  
pp. 681-687 ◽  
Author(s):  
Shaun R. DONNELLY ◽  
Stephen E. MOSS
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Luciferase Reporter ◽  
Gene Promoters ◽  
Start Site ◽  
Luciferase Reporter Gene ◽  
Transcription Start ◽  
A431 Cells ◽  
Annexin I ◽  
Annexin Vi

To gain insight into the molecular basis of annexin gene expression we have analysed the annexin I and VI gene promoters. A previously described 881 bp sequence immediately upstream of the annexin I transcription start site and a similar size fragment proximal to the annexin VI transcription start site both drove expression of the luciferase reporter gene in fibroblasts and epithelial cells. Neither promoter displayed any sensitivity to dexamethasone, suggesting that the putative glucocorticoid response element in the annexin I promoter is non-functional. Consistent with this, endogenous annexin I gene expression was unaffected by dexamethasone at the mRNA and protein levels in A431 cells. A series of 5´ deletions of the two promoters were examined to define the minimal active sequences. For annexin I this corresponded to a sequence approx. 150 bp upstream of the transcription start site that included CAAT and TATA boxes. Unexpectedly, the annexin VI promoter, which also contains CAAT and TATA boxes, was fully active in the absence of these elements, a 53 bp sequence between these boxes and the transcription start site having maximal activity. Electrophoretic mobility-shift assays with nuclear extracts from A431 and HeLa cells with probes corresponding to this region revealed an SP1-binding site. These results show that the annexin I and VI genes have individual modes of transcriptional regulation and that if either annexin I or annexin VI has an anti-inflammatory role, then this is in the absence of steroid-induced gene expression.

scholarly journals A novel selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor prevents human adipogenesis

2008 ◽  
Vol 197 (2) ◽  
pp. 297-307 ◽  
Author(s):  
I J Bujalska ◽  
L L Gathercole ◽  
J W Tomlinson ◽  
C Darimont ◽  
J Ermolieff ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Mrna Expression ◽  
Adipocyte Differentiation ◽  
Reductase Activity ◽  
Primary Cultures ◽  
Hydroxysteroid Dehydrogenase ◽  
Novel Therapy ◽  
Fatty Acid Binding ◽  
Glucose Output

Glucocorticoid excess increases fat mass, preferentially within omental depots; yet circulating cortisol concentrations are normal in most patients with metabolic syndrome (MS). At a pre-receptor level, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates cortisol from cortisone locally within adipose tissue, and inhibition of 11β-HSD1 in liver and adipose tissue has been proposed as a novel therapy to treat MS by reducing hepatic glucose output and adiposity. Using a transformed human subcutaneous preadipocyte cell line (Chub-S7) and human primary preadipocytes, we have defined the role of glucocorticoids and 11β-HSD1 in regulating adipose tissue differentiation. Human cells were differentiated with 1.0 μM cortisol (F), or cortisone (E) with or without 100 nM of a highly selective 11β-HSD1 inhibitor PF-877423. 11β-HSD1 mRNA expression increased across adipocyte differentiation (P<0.001, n=4), which was paralleled by an increase in 11β-HSD1 oxo-reductase activity (from nil on day 0 to 5.9±1.9 pmol/mg per h on day 16, P<0.01, n=7). Cortisone enhanced adipocyte differentiation; fatty acid-binding protein 4 expression increased 312-fold (P<0.001) and glycerol-3-phosphate dehydrogenase 47-fold (P<0.001) versus controls. This was abolished by co-incubation with PF-877423. In addition, cellular lipid content decreased significantly. These findings were confirmed in the primary cultures of human subcutaneous preadipocytes. The increase in 11β-HSD1 mRNA expression and activity is essential for the induction of human adipogenesis. Blocking adipogenesis with a novel and specific 11β-HSD1 inhibitor may represent a novel approach to treat obesity in patients with MS.

scholarly journals Regulation of the Rat Follicle-Stimulating Hormone β-Subunit Promoter by Activin

2003 ◽  
Vol 17 (3) ◽  
pp. 318-332 ◽  
Author(s):  
Magdalena I. Suszko ◽  
Denise J. Lo ◽  
Hoonkyo Suh ◽  
Sally A. Camper ◽  
Teresa K. Woodruff
Keyword(s):  
Promoter Activity ◽  
Hormone Secretion ◽  
Signal Transduction Pathway ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Luciferase Reporter Gene ◽  
Additional Increase ◽  
Smad Binding Element ◽  
Stimulation Of

Abstract FSH is controlled by a variety of positive and negative stimuli, and the unique FSHβ-subunit is a major target for this regulation. Activin is a key modulator of FSHβ transcription and hormone secretion. The signal transduction pathway leading to FSH expression was previously unknown. Here, we show that the transcription factors Smad3 and Smad4 mediate activin-stimulated activity of the rat FSHβ promoter in a pituitary-derived cell line, LβT2. Cells were transiently transfected with the rat FSHβ promoter fused to a luciferase reporter gene (−338rFSHβ-Luc), and a minimal activin-responsive region was identified. Transfection of Smad3, but not the highly related Smad2, led to a ligand-independent stimulation of the FSHβ promoter activity. As expected, activin caused an additional increase of luciferase expression, which was blocked by cotreatment with follistatin. Although Smad4 alone had no effect on FSHβ transcription, it significantly augmented Smad3 and activin-mediated stimulation of the promoter. A palindromic consensus Smad-binding element in the proximal promoter was found to bind Smad4, and elimination of the region resulted in a loss of activin-mediated FSHβ transcription. The activin signaling pathway is conserved in a number of cells, but FSHβ expression is restricted to gonadotropes. A pituitary-specific transcription factor necessary for activin-dependent induction of the FSHβ promoter has been identified that permits FSHβ expression in nongonadotrope cells. Pitx2 is a member of Pitx subfamily of bicoid-related homeodomain factors that is required for pituitary development and is present in the adult pituitary. This factor was transfected into LβT2 cells, where it caused up-regulation of basal and activin-mediated FSHβ promoter activity. Furthermore, cotransfection of Pitx2c with Smad3 in kidney-derived TSA cells resulted in activin-regulated FSHβ response, suggesting its important role in tissue-restricted regulation of FSHβ by activin. A Pitx2c binding site was identified within the proximal promoter, and elimination of this region also resulted in a loss of activin-regulated FSHβ promoter activity. Taken together, these studies suggest that the regulation of FSHβ is dependent on activin-mediated signaling factors in concert with pituitary-derived nuclear regulatory proteins.

scholarly journals Ovarian modulators of 11beta-hydroxysteroid dehydrogenase (11betaHSD) activity in follicular fluid from gonadotrophin-stimulated assisted conception cycles

Reproduction ◽  
2002 ◽  
pp. 801-812 ◽  
Author(s):  
LM Thurston ◽  
DP Norgate ◽  
KC Jonas ◽  
C Chandras ◽  
HJ Kloosterboer ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Rat Kidney ◽  
Hydroxysteroid Dehydrogenase ◽  
Controlled Ovarian Hyperstimulation ◽  
Ovarian Hyperstimulation ◽  
Assisted Conception ◽  
Human Follicular Fluid ◽  
Glucocorticoid Metabolism ◽  
Stimulation Of

In the ovary, cortisol-cortisone interconversion is catalysed by isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD). The objective of this study was to establish whether human follicular fluid (hFF), obtained after controlled ovarian hyperstimulation, contains paracrine modulators of 11betaHSD activity. Of 274 hFF samples tested for effects in rat kidney homogenates, 206 hFF samples significantly inhibited NADP(+)-dependent oxidation of cortisol within 1 h (by 11-67% of control 11betaHSD activity), whereas 42 hFF samples significantly stimulated 11betaHSD activity (16-210% increase relative to control). Although charcoal-stripping of hFF prevented the inhibition and potentiated the stimulation of NADP(+)-dependent cortisol oxidation in a renal homogenate, effects of individual hFF samples on NADP(+)-dependent cortisol oxidation were independent of intrafollicular progesterone concentrations. Hydrophilic fractions of hFF samples, isolated by C18 column chromatography, stimulated both the NADP(+)-dependent oxidation of cortisol (by 55+/-5%, n=98) and the NADPH-dependent reduction of cortisone (by 86+/-22%, n= 5). In contrast, the hydrophobic fractions of hFF (eluted at 65-85% methanol) inhibited both NADP(+)-dependent 11beta-dehydrogenase and NADPH-dependent 11-ketosteroid reductase activities (by 63+/-2% and 74+/-4%, respectively). None of the C18 column fractions of 50 hFF samples had any significant effect on NAD(+)-dependent 11beta-dehydrogenase activities. The hydrophobic inhibitors of NADP(H)-dependent cortisol-cortisone metabolism did not co-elute with several candidate compounds (prostaglandins E(2) and F(2alpha), cortisol, cortisone, oestradiol, testosterone, progesterone, pregnenolone or cholesterol). Hence, hFF aspirated from women undergoing controlled ovarian hyperstimulation for assisted conception contains both hydrophilic stimuli and hydrophobic inhibitors of glucocorticoid metabolism which appear to be selective for the NADP(H)-dependent, type 1 isoform of 11betaHSD.

Sign in / Sign up

Export Citation Format

Share Document