'Liver-type' 11β-hydroxysteroid dehydrogenase cDNA encodes reductase but not dehydrogenase activity in intact mammalian COS-7 cells

1994 ◽  
Vol 13 (2) ◽  
pp. 167-174 ◽  
Author(s):  
S C Low ◽  
K E Chapman ◽  
C R W Edwards ◽  
J R Seckl
Keyword(s):  
Dehydrogenase Activity ◽  
Rat Liver ◽  
Mammalian Cells ◽  
Reductase Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Intact Cells ◽  
Mmtv Ltr

ABSTRACT 11β-Hydroxysteroid dehydrogenase (11β-HSD) catalyses the metabolism of corticosterone to inert 11-dehydrocorticosterone, thus preventing glucocorticoid access to otherwise non-selective renal mineralocorticoid receptors (MRs), producing aldosterone selectivity in vivo. At least two isoforms of 11β-HSD exist. One isoform (11β-HSD1) has been purified from rat liver and an encoding cDNA cloned from a rat liver library. Transfection of rat 11β-HSD1 cDNA into amphibian cells with a mineralocorticoid phenotype encodes 11 β-reductase activity (activation of inert 11-dehydrocorticosterone) suggesting that 11β-HSD1 does not have the necessary properties to protect renal MRs from exposure to glucocorticoids. This function is likely to reside in a second 11β-HSD isoform. 11β-HSD1 is co-localized with glucocorticoid receptors (GRs) and may modulate glucocorticoid access to this receptor type. To examine the predominant direction of 11β-HSD1 activity in intact mammalian cells, and the possible role of 11β-HSD in regulating glucocorticoid access to GRs, we transfected rat 11β-HSD1 cDNA into a mammalian kidney-derived cell system (COS-7) which has little endogenous 11β-HSD activity or mRNA expression. Homogenates of COS-7 cells transfected with increasing amounts of 11β-HSD cDNA exhibited a dose-related increase in 11 β-dehydrogenase activity. In contrast, intact cells did not convert corticosterone to 11-dehydrocorticosterone over 24 h, but showed a clear dose-related 11β-reductase activity, apparent within 4 h of addition of 11-dehydrocorticosterone to the medium. To demonstrate that this reflected a change in functional intracellular glucocorticoids, COS-7 cells were co-transfected with an expression vector encoding GR and a glucocorticoid-inducible MMTV-LTR luciferase reporter construct, with or without 11β-HSD. Corticosterone induced MMTV-LTR luciferase expression in the presence or absence of 11β-HSD. 11-Dehydrocorticosterone was without activity in the absence of 11β-HSD, but induced MMTV-LTR luciferase activity in the presence of 11β-HSD. These results indicate that rat 11β-HSD1 can behave exclusively as a reductase in intact mammalian cells. Thus in some tissues in vivo, 11β-HSD1 may regulate ligand access to GRs by reactivating inert glucocorticoids.

Intracellular regulation of 17β-hydroxysteroid dehydrogenase type 2 catalytic activity in A431 cells

1997 ◽  
Vol 153 (3) ◽  
pp. 453-464 ◽  
Author(s):  
C H Blomquist ◽  
B S Leung ◽  
C Beaudoin ◽  
D Poirier ◽  
Y Tremblay
Keyword(s):  
Reductase Activity ◽  
Maximum Velocity ◽  
Hydroxysteroid Dehydrogenase ◽  
A431 Cells ◽  
Intact Cells ◽  
Cell Monolayers ◽  
Intracellular Regulation

Abstract There is growing evidence that various isoforms of 17β-hydroxysteroid dehydrogenase (17-HSD) are regulated at the level of catalysis in intact cells. A number of investigators have proposed that the NAD(P)/NAD(P)H ratio may control the direction of reaction. In a previous study, we obtained evidence that A431 cells, derived from an epidermoid carcinoma of the vulva, are enriched in 17-HSD type 2, a membrane-bound isoform reactive with C18 and C19 17β-hydroxysteroids and 17-ketosteroids. The present investigation was undertaken to confirm the presence of 17-HSD type 2 in A431 cells and to assess intracellular regulation of 17-HSD at the level of catalysis by comparing the activity of homogenates and microsomes with that of cell monolayers. Northern blot analysis confirmed the presence of 17-HSD type 2 mRNA. Exposure of cells to epidermal growth factor resulted in an increase in type 2 mRNA and, for microsomes, increases in maximum velocity (Vmax) with no change in Michaelis constant (Km) for testosterone and androstenedione, resulting in equivalent increases in the Vmax/Km ratio consistent with the presence of a single enzyme. Initial velocity data and inhibition patterns were consistent with a highly ordered reaction sequence in vitro in which testosterone and androstenedione bind only to either an enzyme–NAD or an enzyme–NADH complex respectively. Microsomal dehydrogenase activity with testosterone was 2- to 3-fold higher than reductase activity with androstenedione. In contrast, although cell monolayers rapidly converted testosterone to androstenedione, reductase activity with androstenedione or dehydroepiandrosterone (DHEA) was barely detectable. Lactate but not glucose, pyruvate or isocitrate stimulated the conversion of androstenedione to testosterone by monolayers, suggesting that cytoplasmic NADH may be the cofactor for 17-HSD type 2 reductase activity with androstenedione. However, exposure to lactate did not result in a significant change in the NAD/NADH ratio of cell monolayers. It appears that within A431 cells 17-HSD type 2 is regulated at the level of catalysis to function almost exclusively as a dehydrogenase. These findings give further support to the concept that 17-HSD type 2 functions in vivo principally as a dehydrogenase and that its role as a reductase in testosterone formation by either the Δ4 or Δ5 pathway is limited. Journal of Endocrinology (1997) 153, 453–464

scholarly journals Hexose-6-phosphate dehydrogenase confers oxo-reductase activity upon 11β-hydroxysteroid dehydrogenase type 1

2005 ◽  
Vol 34 (3) ◽  
pp. 675-684 ◽  
Author(s):  
Iwona J Bujalska ◽  
Nicole Draper ◽  
Zoi Michailidou ◽  
Jeremy W Tomlinson ◽  
Perrin C White ◽  
...  
Keyword(s):  
Dehydrogenase Activity ◽  
Reductase Activity ◽  
Chinese Hamster ◽  
Hydroxysteroid Dehydrogenase ◽  
Mrna Levels ◽  
Gene Encoding ◽  
Intact Cells ◽  
Hek 293 ◽  
Treatment Of Obesity ◽  
Novel Target

Two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD) interconvert active cortisol and inactive cortisone. 11β-HSD2 (renal) acts only as a dehydrogenase, converting cortisol to cortisone. 11β-HSD1 (liver) is a bi-directional enzyme in cell homogenates, whereas in intact cells it typically displays oxo-reductase activity, generating cortisol from cortisone. We recently established that cortisone reductase deficiency is a digenic disease requiring mutations in both the gene encoding 11β-HSD1 and in the gene for a novel enzyme located within the lumen of the endoplasmic reticulum (ER), hexose-6-phosphate dehydrogenase (H6PDH). This latter enzyme generates NADPH, the co-factor required for oxo-reductase activity. Therefore, we hypothesized that H6PDH expression may be an important determinant of 11β-HSD1 oxo-reductase activity. Transient transfection of chinese hamster ovary (CHO) cells with 11β-HSD1 resulted in the appearance of both oxo-reductase and dehydrogenase activities in intact cells. Co-transfection of 11β-HSD1 with H6PDH increased oxo-reductase activity whilst virtually eliminating dehydrogenase activity. In contrast, H6PDH had no effect on reaction direction of 11β-HSD2, nor did the cytosolic enzyme, glucose-6-phosphate dehydrogenase (G6PD) affect 11β-HSD1 oxo-reductase activity. Conversely in HEK 293 cells stably transfected with 11β-HSD1 cDNA, transfection of an H6PDH siRNA reduced 11β-HSD1 oxo-reductase activity whilst simultaneously increasing 11β-HSD1 dehydrogenase activity. In human omental preadipocytes obtained from 15 females of variable body mass index (BMI), H6PDH mRNA levels positively correlated with 11β-HSD1 oxo-reductase activity, independent of 11β-HSD1 mRNA levels. H6PDH expression increased 5.3-fold across adipocyte differentiation (P<0.05) and was associated with a switch from 11β-HSD1 dehydrogenase to oxo-reductase activity. In conclusion, H6PDH is a crucial determinant of 11β-HSD1 oxo-reductase activity in intact cells. Through its interaction with 11β-HSD1, H6PDH may represent a novel target in the pathogenesis and treatment of obesity.

scholarly journals 11 beta-hydroxysteroid dehydrogenase type 1 is a predominant 11 beta-reductase in the intact perfused rat liver

2000 ◽  
Vol 165 (3) ◽  
pp. 685-692 ◽  
Author(s):  
PM Jamieson ◽  
BR Walker ◽  
KE Chapman ◽  
R Andrew ◽  
S Rossiter ◽  
...  
Keyword(s):  
Rat Liver ◽  
Reductase Activity ◽  
Chronic Administration ◽  
Selective Inhibition ◽  
Hydroxysteroid Dehydrogenase ◽  
Rat Plasma ◽  
Male Rat ◽  
Perfused Rat Liver

11 beta-Hydroxysteroid dehydrogenase type 1 (11 beta-HSD-1), a regulator of intrahepatocellular glucocorticoid activity, is bidirectional in homogenates but catalyses 11 beta-reduction (regenerating glucocorticoid) in intact primary hepatocytes in culture. To examine this discrepancy at the whole-organ level, we examined 11 beta-HSD-1 activity in the intact bivascularly perfused rat liver. On a single pass through male rat liver, 44+/-5% of 11-dehydrocorticosterone (11-DHC) recovered was 11 beta-reduced to corticosterone, whereas 10+/-1% of corticosterone was 11 beta-dehydrogenated to 11-DHC. 11 beta-Reduction was less in female liver (21+/-2%, P<0.01) and was significantly greater with perfusion of all substrate via the portal vein (50+/-3%) than via the hepatic artery (30+/-2%, P<0.05). 11 beta-Reductase activity was not saturated by 11-DHC (10(-)(9)-10(-)(6) M). Perfusion with carbenoxolone (CBX, 10(-)(6)-10(-)(3 )M) did not alter 11 beta-reduction of 11-DHC. In contrast, pretreatment with CBX in vivo (10 mg/day) for 7 days inhibited 11 beta-reductase (19+/-4% conversion, P<0.01). Concentrations of 11-DHC in male rat plasma were 44+/-6 nM. Thus 11 beta-HSD-1 is predominantly an 11 beta-reductase in the intact rat liver and is only inhibited by chronic administration of CBX. The substantial concentrations of plasma 11-DHC as substrate suggest that 11 beta-HSD-1 activity and its potential selective inhibition could modify glucocorticoid action in vivo.

scholarly journals Minireview: Hexose-6-Phosphate Dehydrogenase and Redox Control of 11β-Hydroxysteroid Dehydrogenase Type 1 Activity

Endocrinology ◽  
2005 ◽  
Vol 146 (6) ◽  
pp. 2539-2543 ◽  
Author(s):  
Kylie N. Hewitt ◽  
Elizabeth A. Walker ◽  
Paul M. Stewart
Keyword(s):  
Reductase Activity ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Hydroxysteroid Dehydrogenase ◽  
Pentose Phosphate ◽  
Intact Cells ◽  
The Metabolic Syndrome ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
Glucose 6 Phosphate Dehydrogenase

Abstract Hexose-6-phosphate dehydrogenase (H6PDH) is a microsomal enzyme that is able to catalyze the first two reactions of an endoluminal pentose phosphate pathway, thereby generating reduced nicotinamide adenine dinucleotide phosphate (NADPH) within the endoplasmic reticulum. It is distinct from the cytosolic enzyme, glucose-6-phosphate dehydrogenase (G6PDH), using a separate pool of NAD(P)+ and capable of oxidizing several phosphorylated hexoses. It has been proposed to be a NADPH regenerating system for steroid hormone and drug metabolism, specifically in determining the set point of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activity, the enzyme responsible for the activation and inactivation of glucocorticoids. 11β-HSD1 is a bidirectional enzyme, but in intact cells displays predominately oxo-reductase activity, a reaction requiring NADPH and leading to activation of glucocorticoids. However, in cellular homogenates or in purified preparations, 11β-HSD1 is exclusively a dehydrogenase. Because H6PDH and 11β-HSD1 are coexpressed in the inner microsomal compartment of cells, we hypothesized that H6PDH may provide 11β-HSD1 with NADPH, thus promoting oxo-reductase activity in vivo. Recently, several studies have confirmed this functional cooperation, indicating the importance of intracellular redox mechanisms for the prereceptor control of glucocorticoid availability. With the increased interest in 11β-HSD1 oxo-reductase activity in the pathogenesis and treatment of several human diseases including insulin resistance and the metabolic syndrome, H6PDH represents an additional novel candidate for intervention.

Evidence that mammalian ribonucleotide reductase is a nuclear membrane associated glycoprotein

1990 ◽  
Vol 68 (5) ◽  
pp. 880-888 ◽  
Author(s):  
Marianna Sikorska ◽  
Linda M. Brewer ◽  
Tony Youdale ◽  
Robert Richards ◽  
James F. Whitfield ◽  
...  
Keyword(s):  
Rat Liver ◽  
Ribonucleotide Reductase ◽  
Nuclear Membrane ◽  
Mammalian Cells ◽  
Reductase Activity ◽  
Cell Types ◽  
Liver Cells ◽  
Affinity Column ◽  
Rat Liver Cells

Epitope-specific antibodies to the M1 and M2 subunits of mammalian ribonucleotide reductase were prepared using peptides predicted to have a high antigenic index. Western blotting demonstrated that the anti-M1 antibody was specific for the 89-kilodalton M1 subunit (and its degradation fragments) and the anti-M2 antibody specifically recognized the 45-kilodalton M2 subunit. Both antibodies inhibited the CDP-reductase activity of the holoenzyme. Using these antibodies, both the M1 and M2 subunits were shown to be localized in the cytoplasm and in the nuclear regions of a number of cell types, including B77 avian sarcoma virus transformed NRK cells, T51B rat liver cells, 5123tc hepatoma cells, and rat liver cells in vivo. In addition, the M1 subunit was found to be localized as a halo around isolated rat liver nuclei. Biochemical analysis of the cytoplasmic fraction of liver cells and a Triton X-100 wash of nuclei from these cells confirmed the location of the enzyme activity in these cellular compartments. The M1 subunit appears to be glycosylated, as indicated by its retention on a Affi-Gel – concanavalin A affinity column. Therefore, in mammalian cells ribonucleotide reductase appears to be not only in the cytoplasm, but is also associated with the nuclear membrane or nuclear lamina. The activity of the enzyme in the membrane fraction changes dynamically during the cell cycle.Key words: replication, DNA, synthesis, glycosylation, liver.

scholarly journals Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells

1991 ◽  
Vol 276 (3) ◽  
pp. 637-641 ◽  
Author(s):  
F F Craig ◽  
A C Simmonds ◽  
D Watmore ◽  
F McCapra ◽  
M R H White
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Luciferase Expression ◽  
Intact Cells ◽  
Cos Cells ◽  
Escherichia Coli Cells ◽  
Luminescent Signal ◽  
The Difference ◽  
Kinetics Of

Five esters of luciferin were synthesized and compared with native luciferin as substrates for firefly luciferase expressed in live intact mammalian cells. The esters themselves were not substrates for purified luciferase, but four were substrates for a purified esterase and all appeared to be hydrolysed to luciferin within mammalian cells. At a substrate concentration of 0.01 mM, the peak luminescence from the cos cells expressing luciferase was up to 6-fold greater with the esters than with unmodified luciferin. At 0.1 mM, the difference between luciferin and the esters was decreased. The kinetics of the luminescent signal with the different luciferin esters varied significantly, indicating possible differences in the rates of uptake, breakdown and enzyme inhibition. The esters did not support luminescence from Escherichia coli cells expressing firefly luciferase, suggesting a lack of appropriate esterase activity in this particular strain. The esters could be useful for the assay of luciferase expression in intact mammalian cells when luciferin levels are limiting, for example in tissues, and in plants. Alternative luciferin derivatives may allow further improvements in sensitivity.

In vivo evidence that human adrenal glands possess 11β-hydroxysteroid dehydrogenase activity

Life Sciences ◽  
1999 ◽  
Vol 65 (26) ◽  
pp. 2823-2827 ◽  
Author(s):  
Cinzia Tortorella ◽  
Francesco Aragona ◽  
Gastone G. Nussdorfer
Keyword(s):  
Dehydrogenase Activity ◽  
Adrenal Glands ◽  
Hydroxysteroid Dehydrogenase

scholarly journals In vivo non-invasive monitoring of dystrophin correction in a new Duchenne muscular dystrophy reporter mouse

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Leonela Amoasii ◽  
Hui Li ◽  
Yu Zhang ◽  
Yi-Li Min ◽  
Efrain Sanchez-Ortiz ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Genetic Disorder ◽  
Dystrophin Gene ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Gene Correction ◽  
Reading Frame ◽  
Non Invasive

Abstract Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by mutations in the dystrophin gene. To enable the non-invasive analysis of DMD gene correction strategies in vivo, we introduced a luciferase reporter in-frame with the C-terminus of the dystrophin gene in mice. Expression of this reporter mimics endogenous dystrophin expression and DMD mutations that disrupt the dystrophin open reading frame extinguish luciferase expression. We evaluated the correction of the dystrophin reading frame coupled to luciferase in mice lacking exon 50, a common mutational hotspot, after delivery of CRISPR/Cas9 gene editing machinery with adeno-associated virus. Bioluminescence monitoring revealed efficient and rapid restoration of dystrophin protein expression in affected skeletal muscles and the heart. Our results provide a sensitive non-invasive means of monitoring dystrophin correction in mouse models of DMD and offer a platform for testing different strategies for amelioration of DMD pathogenesis.

scholarly journals MicroRNA-98 reduces nerve growth factor expression in nicotine-induced airway remodeling

2020 ◽  
Vol 295 (52) ◽  
pp. 18051-18064
Author(s):  
Cherry Wongtrakool ◽  
Junsuk Ko ◽  
Andrew J. Jang ◽  
Kora Grooms ◽  
Sarah Chang ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Airway Remodeling ◽  
Luciferase Reporter ◽  
Mouse Lung ◽  
Luciferase Expression ◽  
Nerve Growth ◽  
Pparγ Agonist

Evolving evidence suggests that nicotine may contribute to impaired asthma control by stimulating expression of nerve growth factor (NGF), a neurotrophin associated with airway remodeling and airway hyperresponsiveness. We explored the hypothesis that nicotine increases NGF by reducing lung fibroblast (LF) microRNA-98 (miR-98) and PPARγ levels, thus promoting airway remodeling. Levels of NGF, miR-98, PPARγ, fibronectin 1 (FN1), endothelin-1 (EDN1, herein referred to as ET-1), and collagen (COL1A1 and COL3A1) were measured in human LFs isolated from smoking donors, in mouse primary LFs exposed to nicotine (50 μg/ml), and in whole lung homogenates from mice chronically exposed to nicotine (100 μg/ml) in the drinking water. In selected studies, these pathways were manipulated in LFs with miR-98 inhibitor (anti-miR-98), miR-98 overexpression (miR-98 mimic), or the PPARγ agonist rosiglitazone. Compared with unexposed controls, nicotine increased NGF, FN1, ET-1, COL1A1, and COL3A1 expression in human and mouse LFs and mouse lung homogenates. In contrast, nicotine reduced miR-98 levels in LFs in vitro and in lung homogenates in vivo. Treatment with anti-miR-98 alone was sufficient to recapitulate increases in NGF, FN1, and ET-1, whereas treatment with a miR-98 mimic significantly suppressed luciferase expression in cells transfected with a luciferase reporter linked to the putative seed sequence in the NGF 3′UTR and also abrogated nicotine-induced increases in NGF, FN1, and ET-1 in LFs. Similarly, rosiglitazone increased miR-98 and reversed nicotine-induced increases in NGF, FN1, and ET-1. Taken together, these findings demonstrate that nicotine-induced increases in NGF and other markers of airway remodeling are negatively regulated by miR-98.

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