Molecular cloning of equine 17β-hydroxysteroid dehydrogenase type 1 and its downregulation during follicular luteinization in vivo

The type 1 form of 17β-hydroxysteroid dehydrogenase (17βHSD1) was the first isoform to be identified and is capable of converting estrone to 17β-estradiol. This study was aimed at characterizing the molecular structure of the equine 17βHSD1 gene and cDNA, as well as its molecular regulation during human chorionic gonadotropin (hCG)-induced follicular luteinization/ovulation in vivo. The equine 17βHSD1 gene was cloned from an equine genomic library and shown to have a conserved genomic structure composed of six exons. Its cDNA sequence was also identified and coded for a 308 amino acid protein, 72.1–74.5% homologous to other mammalian orthologs. RT-PCR/Southern blot analyses were performed to study the regulation of the 17βHSD1 transcript in equine preovulatory follicles isolated between 0 and 39 h after hCG treatment. Results demonstrated the presence of high 17βHSD1 mRNA expression prior to hCG treatment with a marked decrease observed 12 h after hCG (P < 0.05). Analyses on isolated preparations of granulosa and theca interna cells identified the granulosa cell layer as the site of 17βHSD1 transcript expression and downregulation (P < 0.05). A 1412 bp fragment of the equine 17βHSD1 proximal promoter was sequenced and shown to contain many putative transcription factor binding sites. Electromobility shift assays (EMSA) using a fragment of the proximal promoter (−230/−30) and nuclear extracts prepared from granulosa cells isolated prior to hCG (0 h post-hCG) revealed the presence of a major complex, and results from competition assays suggest that steroidogenic factor-1 (SF-1), NFκB, GATA, and Sp1 cis-elements are involved. Supershift assays using an antibody against the p65 subunit of NFκB led to the displacement of the binding nuclear proteins to the DNA probe, whereas the use of an anti-equine SF-1 antibody demonstrated the clear formation of a DNA–protein–antibody complex, confirming the potential role of these transcription factors in EMSA results. Interestingly, a notable decrease in DNA binding was observed when granulosa cell nuclear extracts isolated 30 h post-hCG were used, which paralleled the decrease in 17βHSD1 transcript after hCG treatment. Thus, this study is the first to report the gonadotropin-dependent downregulation of 17βHSD1 transcript expression in a monoovulatory species, the presence and regulation of protein/DNA interactions in the proximal region of the 17βHSD1 promoter during gonadotropin treatment, and the characterization of the primary structure of equine 17βHSD1 cDNA and gene.

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11β-Hydroxysteroid dehydrogenase type 1 inhibition in idiopathic intracranial hypertension: a double-blind randomized controlled trial

Brain Communications ◽

10.1093/braincomms/fcz050 ◽

2020 ◽

Vol 2 (1) ◽

Cited By ~ 7

Author(s):

Keira Markey ◽

James Mitchell ◽

Hannah Botfield ◽

Ryan S Ottridge ◽

Tim Matthews ◽

...

Keyword(s):

Intracranial Hypertension ◽

Lumbar Puncture ◽

Idiopathic Intracranial Hypertension ◽

Controlled Trial ◽

Hydroxysteroid Dehydrogenase ◽

Opening Pressure ◽

Double Blind ◽

Randomized Controlled

Abstract Treatment options for idiopathic intracranial hypertension are limited. The enzyme 11β-hydroxysteroid dehydrogenase type 1 has been implicated in regulating cerebrospinal fluid secretion, and its activity is associated with alterations in intracranial pressure in idiopathic intracranial hypertension. We assessed therapeutic efficacy, safety and tolerability and investigated indicators of in vivo efficacy of the 11β-hydroxysteroid dehydrogenase type 1 inhibitor AZD4017 compared with placebo in idiopathic intracranial hypertension. A multicenter, UK, 16-week phase II randomized, double-blind, placebo-controlled trial of 12-week treatment with AZD4017 or placebo was conducted. Women aged 18–55 years with active idiopathic intracranial hypertension (>25 cmH2O lumbar puncture opening pressure and active papilledema) were included. Participants received 400 mg of oral AZD4017 twice daily compared with matching placebo over 12 weeks. The outcome measures were initial efficacy, safety and tolerability. The primary clinical outcome was lumbar puncture opening pressure at 12 weeks analysed by intention-to-treat. Secondary clinical outcomes were symptoms, visual function, papilledema, headache and anthropometric measures. In vivo efficacy was evaluated in the central nervous system and systemically. A total of 31 subjects [mean age 31.2 (SD = 6.9) years and body mass index 39.2 (SD = 12.6) kg/m2] were randomized to AZD4017 (n = 17) or placebo (n = 14). At 12 weeks, lumbar puncture pressure was lower in the AZD4017 group (29.7 cmH2O) compared with placebo (31.3 cmH2O), but the difference between groups was not statistically significant (mean difference: −2.8, 95% confidence interval: −7.1 to 1.5; P = 0.2). An exploratory analysis assessing mean change in lumbar puncture pressure within each group found a significant decrease in the AZD4017 group [mean change: −4.3 cmH2O (SD = 5.7); P = 0.009] but not in the placebo group [mean change: −0.3 cmH2O (SD = 5.9); P = 0.8]. AZD4017 was safe, with no withdrawals related to adverse effects. Nine transient drug-related adverse events were reported. One serious adverse event occurred in the placebo group (deterioration requiring shunt surgery). In vivo biomarkers of 11β-hydroxysteroid dehydrogenase type 1 activity (urinary glucocorticoid metabolites, hepatic prednisolone generation, serum and cerebrospinal fluid cortisol:cortisone ratios) demonstrated significant enzyme inhibition with the reduction in serum cortisol:cortisone ratio correlating significantly with reduction in lumbar puncture pressure (P = 0.005, R = 0.70). This is the first phase II randomized controlled trial in idiopathic intracranial hypertension evaluating a novel therapeutic target. AZD4017 was safe and well tolerated and inhibited 11β-hydroxysteroid dehydrogenase type 1 activity in vivo. Reduction in serum cortisol:cortisone correlated with decreased intracranial pressure. Possible clinical benefits were noted in this small cohort. A longer, larger study would now be of interest.

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SAT0039 Endogenous Glucocorticoid Production by The Enzyme 11beta-Hydroxysteroid Dehydrogenase Type 1 Is Increased with Inflammation In Muscle, Where It Suppresses Inflammatory Cytokine Output and Protects against Muscle Wasting In Vivo

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2016-eular.4228 ◽

2016 ◽

Vol 75 (Suppl 2) ◽

pp. 677.3-678

Author(s):

R.S. Hardy ◽

Z. Hussain ◽

M. Pearson ◽

A. Filer ◽

C. Buckley ◽

...

Keyword(s):

Inflammatory Cytokine ◽

Muscle Wasting ◽

Hydroxysteroid Dehydrogenase ◽

Glucocorticoid Production

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Tat-SF1 Protein Associates with RAP30 and Human SPT5 Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.19.9.5960 ◽

1999 ◽

Vol 19 (9) ◽

pp. 5960-5968 ◽

Cited By ~ 36

Author(s):

Jae B. Kim ◽

Yuki Yamaguchi ◽

Tadashi Wada ◽

Hiroshi Handa ◽

Phillip A. Sharp

Keyword(s):

Recombinant Proteins ◽

Transcriptional Activity ◽

Transcription Activation ◽

Cellular Proteins ◽

Pol Ii ◽

Nuclear Extracts ◽

Cellular Factors ◽

Immunodeficiency Virus

ABSTRACT The potent transactivator Tat recognizes the transactivation response RNA element (TAR) of human immunodeficiency virus type 1 and stimulates the processivity of elongation of RNA polymerase (Pol) II complexes. The cellular proteins Tat-SF1 and human SPT5 (hSPT5) are required for Tat activation as shown by immunodepletion with specific sera and complementation with recombinant proteins. In nuclear extracts, small fractions of both hSPT5 and Pol II are associated with Tat-SF1 protein. Surprisingly, the RAP30 protein of the heterodimeric transcription TFIIF factor is associated with Tat-SF1, while the RAP74 subunit of TFIIF is not coimmunoprecipitated with Tat-SF1. Overexpression of Tat-SF1 and hSPT5 specifically stimulates the transcriptional activity of Tat in vivo. These results suggest that Tat-SF1 and hSPT5 are indispensable cellular factors supporting Tat-specific transcription activation and that they may interact with RAP30 in controlling elongation.

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siRNA-based breast cancer therapy by suppressing 17β-hydroxysteroid dehydrogenase type 1 in an optimized xenograft cell and molecular biology model in vivo

Drug Design Development and Therapy ◽

10.2147/dddt.s180836 ◽

2019 ◽

Vol Volume 13 ◽

pp. 757-766

Author(s):

Fang Li ◽

ZhiHan Zhu ◽

Man Xue ◽

WanHong He ◽

Ting Zhang ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Therapy ◽

Molecular Biology ◽

Hydroxysteroid Dehydrogenase ◽

Breast Cancer Therapy

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11β-Hydroxysteroid dehydrogenase type 1 contributes to the balance between 7-keto- and 7-hydroxy-oxysterols in vivo

Biochemical Pharmacology ◽

10.1016/j.bcp.2013.02.002 ◽

2013 ◽

Vol 86 (1) ◽

pp. 146-153 ◽

Cited By ~ 17

Author(s):

Tijana Mitić ◽

Steven Shave ◽

Nina Semjonous ◽

Iain McNae ◽

Diego F. Cobice ◽

...

Keyword(s):

Hydroxysteroid Dehydrogenase

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

Biochemical Journal ◽

10.1042/bj3170621 ◽

1996 ◽

Vol 317 (2) ◽

pp. 621-625 ◽

Cited By ~ 59

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.

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Elf-1 Contributes to the Function of the Complex Interleukin (IL)-2–responsive Enhancer in the Mouse IL-2 Receptor α Gene

Journal of Experimental Medicine ◽

10.1084/jem.185.7.1211 ◽

1997 ◽

Vol 185 (7) ◽

pp. 1211-1222 ◽

Cited By ~ 30

Author(s):

Irina Serdobova ◽

Maria Pla ◽

Patrick Reichenbach ◽

Peter Sperisen ◽

Jacques Ghysdael ◽

...

Keyword(s):

Transcriptional Control ◽

Proximal Region ◽

Binding Motif ◽

Nuclear Extracts ◽

Vitro Binding ◽

Ets Family ◽

Cd8 Cell ◽

Promoter Proximal Region

Lymphocytes regulate their responsiveness to IL-2 through the transcriptional control of the IL-2Rα gene, which encodes a component of the high affinity IL-2 receptor. In the mouse IL-2Rα gene this control is exerted via two regulatable elements, a promoter proximal region, and an IL-2–responsive enhancer (IL-2rE) 1.3 kb upstream. In vitro and in vivo functional analysis of the IL-2rE in the rodent thymic lymphoma-derived, CD4−CD8− cell line PC60 demonstrated that three separate elements, sites I, II, and III, were necessary for IL-2 responsiveness; these three sites demonstrate functional cooperation. Site III contains a consensus binding motif for members of the Ets family of transcription factors. Here we demonstrate that Elf-1, an Ets-like protein, binds to site III and participates in IL-2 responsiveness. In vitro site III forms a complex with a protein constitutively present in nuclear extracts from PC60 cells as well as from normal CD4−CD8− thymocytes. We have identified this molecule as Elf-1 according to a number of criteria. The complex possesses an identical electrophoretic mobility to that formed by recombinant Elf-1 protein and is super-shifted by anti–Elf-1 antibodies. Biotinylated IL-2rE probes precipitate Elf-1 from PC60 extracts provided site III is intact and both recombinant and PC60-derived proteins bind with the same relative affinities to different mutants of site III. In addition, by introducing mutations into the core of the site III Ets-like motif and comparing the corresponding effects on the in vitro binding of Elf-1 and the in vivo IL-2rE activity, we provide strong evidence that Elf-1 is directly involved in IL-2 responsiveness. The nature of the functional cooperativity observed between Elf-1 and the factors binding sites I and II remains unresolved; experiments presented here however suggest that this effect may not require direct interactions between the proteins binding these three elements.

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Tumor necrosis factor-α upregulates 11β-hydroxysteroid dehydrogenase type 1 expression by CCAAT/enhancer binding protein-β in HepG2 cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.90531.2008 ◽

2009 ◽

Vol 296 (2) ◽

pp. E367-E377 ◽

Cited By ~ 34

Author(s):

Irena D. Ignatova ◽

Radina M. Kostadinova ◽

Christopher E. Goldring ◽

Andrea R. Nawrocki ◽

Felix J. Frey ◽

...

Keyword(s):

P38 Mapk ◽

Hepg2 Cells ◽

Binding Protein ◽

Hydroxysteroid Dehydrogenase ◽

Expression Vectors ◽

Enhancer Binding Protein ◽

Tnf Α ◽

Ccaat Enhancer Binding Protein

The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes the conversion of inactive to active glucocorticoids. 11β-HSD1 plays a crucial role in the pathogenesis of obesity and controls glucocorticoid actions in inflammation. Several studies have demonstrated that TNF-α increases 11β-HSD1 mRNA and activity in various cell models. Here, we demonstrate that mRNA and activity of 11β-HSD1 is increased in liver tissue from transgenic mice overexpressing TNF-α, indicating that this effect also occurs in vivo. To dissect the molecular mechanism of this increase, we investigated basal and TNF-α-induced transcription of the 11β-HSD1 gene ( HSD11B1) in HepG2 cells. We found that TNF-α acts via p38 MAPK pathway. Transient transfections with variable lengths of human HSD11B1 promoter revealed highest activity with or without TNF-α in the proximal promoter region (−180 to +74). Cotransfection with human CCAAT/enhancer binding protein-α (C/EBPα) and C/EBPβ-LAP expression vectors activated the HSD11B1 promoter with the strongest effect within the same region. Gel shift and RNA interference assays revealed the involvement of mainly C/EBPα, but also C/EBPβ, in basal and only of C/EBPβ in the TNF-α-induced HSD11B1 expression. Chromatin immunoprecipitation assay confirmed in vivo the increased abundance of C/EBPβ on the proximal HSD11B1 promoter upon TNF-α treatment. In conclusion, C/EBPα and C/EBPβ control basal transcription, and TNF-α upregulates 11β-HSD1, most likely by p38 MAPK-mediated increased binding of C/EBPβ to the human HSD11B1 promoter. To our knowledge, this is the first study showing involvement of p38 MAPK in the TNF-α-mediated 11β-HSD1 regulation, and that TNF-α stimulates enzyme activity in vivo.

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