scholarly journals Aromatase activity induction in human adipose fibroblasts by retinoic acids via retinoic acid receptor α

2013 ◽  
Vol 51 (2) ◽  
pp. 247-260 ◽  
Author(s):  
Jan Wilde ◽  
Maria Erdmann ◽  
Michael Mertens ◽  
Gabriele Eiselt ◽  
Martin Schmidt
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Retinoic Acid ◽  
Reporter Gene ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Aromatase Activity ◽  
Aromatase Expression ◽  
Estrogen Synthesis ◽  
Activity Induction

Estrogen synthesis in adipose tissue is associated with the development of breast cancer. Tumors are preferentially found in breast quadrants with strongest expression of the cytochrome P450 aromatase (encoded by the geneCYP19A1). Several promoters regulated by various hormonal factors drive aromatase expression in human breast adipose fibroblasts (BAFs). As adipose tissue is a major source of retinoids, in this study, we investigated their role in the regulation of aromatase expression. The retinoids all-trans-retinoic acid (at-RA) and 9-cis-RA induce aromatase activity in human BAFs. In BAFs, at-RA induces aromatase gene expression via promoter I.4. In 3T3-L1 cells, both retinoids specifically drive luciferase reporter gene expression under the control of aromatase promoter I.4, whereas other promoters active in human adipose tissue are insensitive. Activation by retinoids depends on a 467 bp fragment (−256/+211) of promoter I.4 containing four putative retinoic acid response elements (RAREs). Site-directed mutagenesis revealed that only RARE2 (+91/+105) mediates the retinoid-dependent induction of reporter gene activity. In 3T3-L1 preadipocytes and human BAFs, RA receptor α (RARα (RARA)) expression is predominant, whereas RARβ (RARB) or RARγ (RARG) expression is low. Electrophoretic mobility shift assays with nuclear extracts obtained from human BAFs and 3T3-L1 cells identified a specific RARE2-binding complex. Retinoids enhanced complex formation, whereas pre-incubation with anti-RARα antibodies prohibited the binding of RARα to RARE2. Chromatin immunoprecipitation showed RA-dependent binding of RARα to the RARE2-containing promoter regionin vivo. Furthermore, we provide evidence that RARE2 is also necessary for the basal activation of promoter I.4 in these cells. Taken together, these findings indicate a novel retinoid-dependent mechanism of aromatase activity induction in adipose tissue.

Abstract 117: MicroRNA-138 regulates S100A1 in Endothelial Cells

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Anagha Sen ◽  
Shumei Ren ◽  
Jianxin Sun ◽  
Patrick Most ◽  
Karsten Peppel
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Reporter Gene ◽  
Recombinant Adenovirus ◽  
Limb Ischemia ◽  
Bioinformatic Analysis ◽  
Reporter Gene Expression ◽  
Luciferase Reporter ◽  
C2c12 Myotubes ◽  
Luciferase Reporter Construct

Rationale: The EF-hand Ca2+ sensor S100A1 is essential for proper endothelial nitric oxide (NO) synthase (eNOS) activation. S100A1 levels are greatly reduced in endothelial cells (ECs) subjected to hypoxia, rendering them dysfunctional. Objective: To determine if the 3’UTR mediates the rapid hypoxia-induced downregulation of S100A1 in ECs. Methods and Results: ECs transfected with a S100A1 - 3’ untranslated region (UTR) luciferase reporter construct displayed significantly reduced gene expression when subjected to gas or chemical hypoxia. Bioinformatic analysis suggested that microRNA -138 (miR-138) could target the 3’UTR of S100A1. Hypoxia greatly increased miR-138 levels in ECs, but not in skeletal muscle C2C12 myotubes. Consistent with this finding, patients with critical limb ischemia (CLI) or mice subjected to femoral artery resection (FAR) displayed increased miR-138 levels. Transfection of a miR-138 mimic into ECs reduced S100A1 - 3 ‘UTR reporter gene expression, while transfection of an anti miR-138 (antagomir) prevented the hypoxia-induced downregulation of the reporter gene. The increased levels of miR-138 are dependent on Hif1-α activation as treatment with siRNA against Hif1-α prevented S100A1 reporter gene downregulation after hypoxia. Conversely, specific activation of Hif1-α by a selective prolyl-hydroxylase inhibitor (IOX2) reduced reporter gene expression. Finally, ECs transfected with miR-138 mimic displayed reduced tube formation when plated onto Matrigel matrix and expressed less NO when stimulated with VEGF. These effects were reversed by gene transfer of S100A1 using recombinant adenovirus. Conclusions: Our study shows that miR-138 is an essential mediator of EC dysfunction via its ability to target the 3’UTR of S100A1 in a hypoxia-induced manner. MiR-138 might thus be an attractive target for the treatment of pathologies that are linked to endothelial dysfunction.

Potency of isothiocyanates to induce luciferase reporter gene expression via the electrophile-responsive element from murine glutathione S-transferase Ya

2009 ◽  
Vol 23 (4) ◽  
pp. 617-621 ◽  
Author(s):  
Martijn Vermeulen ◽  
Anne-Marie M.J.F. Boerboom ◽  
Barry M.G. Blankvoort ◽  
Jac M.M.J.G. Aarts ◽  
Ivonne M.C.M. Rietjens ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Reporter Gene Expression ◽  
Luciferase Reporter ◽  
Glutathione S Transferase ◽  
Luciferase Reporter Gene ◽  
Responsive Element

scholarly journals Non-invasive somatotransgenic bioimaging in living animals

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 1216 ◽  
Author(s):  
Juliette M. Delhove ◽  
Rajvinder Karda ◽  
Lorna M. FitzPatrick ◽  
Suzanne M.K. Buckley ◽  
Simon N. Waddington ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Viral Vectors ◽  
Es Cells ◽  
Embryonic Stem ◽  
Whole Body ◽  
Luciferase Reporter ◽  
Gene Promoters ◽  
Luciferase Reporter Gene ◽  
Endogenous Gene

Bioluminescence imaging enables noninvasive quantification of luciferase reporter gene expression in transgenic tissues of living rodents. Luciferase transgene expression can be regulated by endogenous gene promoters after targeted knock-in of the reporter gene, usually within the first intron of the gene. Even using CRISPR/Cas9 mediated genome editing this can be a time consuming and costly process. The generation of germline transgenic (GLT) rodents by targeted genomic integration of a gene expression cassette in embryonic stem (ES) cells is commonplace but results in the wastage of large numbers of animals during colony generation, back-crossing and maintenance. Using a synthetic/truncated promoter-driven luciferase gene to study promoter activity in a given tissue or organ of a GLT also often results in unwanted background luciferase activity during whole-body bioluminescent imaging as every cell contains the reporter. We have developed somatotransgenic bioimaging; a method to generate tissue-restricted transcription factor activated luciferase reporter (TFAR) cassettes in rodents that substantially reduces the number of animals required for experimentation. Bespoke designed TFARs are delivered to newborn pups using viral vectors targeted to specific organs by tissue-tropic pseudotypes. Retention and proliferation of TFARs is facilitated by stem/progenitor cell transduction and immune tolerance to luciferase due to the naïve neonatal immune system. We have successfully applied both lentiviral and adeno-associated virus (AAV) vectors in longitudinal rodent studies, targeting TFARs to the liver and brain during normal development and in well-established disease models. Development of somatotransgenic animals has broad applicability to non-invasively determine mechanistic insights into homeostatic and disease states and assess toxicology and efficacy testing. Somatotransgenic bioimaging technology is superior to current whole-body, light-emitting transgenic models as it reduces the numbers of animals used by generating only the required number of animals. It is also a refinement over current technologies given the ability to use conscious, unrestrained animals.

In vivo regulation of β-MHC gene in rodent heart: role of T3 and evidence for an upstream enhancer

1999 ◽  
Vol 276 (4) ◽  
pp. C883-C891 ◽  
Author(s):  
Carola E. Wright ◽  
F. Haddad ◽  
A. X. Qin ◽  
P. W. Bodell ◽  
K. M. Baldwin
Keyword(s):  
Gene Expression ◽  
Thyroid Hormone ◽  
Reporter Gene ◽  
Normal Control ◽  
Transcriptional Activity ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Promoter Construct ◽  
Hormone Responsiveness

Cardiac β-myosin heavy chain (β-MHC) gene expression is mainly regulated through transcriptional processes. Although these results are based primarily on in vitro cell culture models, relatively little information is available concerning the interaction of key regulatory factors thought to modulate MHC expression in the intact rodent heart. Using a direct gene transfer approach, we studied the in vivo transcriptional activity of different-length β-MHC promoter fragments in normal control and in altered thyroid states. The test β-MHC promoter was fused to a firefly luciferase reporter gene, whereas the control α-MHC promoter was fused to the Renilla luciferase reporter gene and was used to account for variations in transfection efficiency. Absolute reporter gene activities showed that β- and α-MHC genes were individually and reciprocally regulated by thyroid hormone. The β-to-α ratios of reporter gene expression demonstrated an almost threefold larger β-MHC gene expression in the longest than in the shorter promoter fragments in normal control animals, implying the existence of an upstream enhancer. A mutation in the putative thyroid response element of the −408-bp β-MHC promoter construct caused transcriptional activity to drop to null. When studied in the −3,500-bp β-MHC promoter, construct activity was reduced (∼100-fold) while thyroid hormone responsiveness was retained. These findings suggest that, even though the bulk of the thyroid hormone responsiveness of the gene is contained within the first 215 bp of the β-MHC promoter sequence, the exact mechanism of triiodothyronine (T3) action remains to be elucidated.

scholarly journals Aromatase expression in the human fetal osteoblastic cell line SV-HFO

2004 ◽  
Vol 32 (2) ◽  
pp. 533-545 ◽  
Author(s):  
M Watanabe ◽  
ER Simpson ◽  
N Pathirage ◽  
S Nakajin ◽  
CD Clyne
Keyword(s):  
Cell Line ◽  
Bone Growth ◽  
Genomic Sequence ◽  
Oncostatin M ◽  
Osteoblastic Cell ◽  
Aromatase Activity ◽  
Gene Transcript ◽  
Aromatase Expression ◽  
Osteoblastic Cell Line ◽  
Estrogen Synthesis

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.

scholarly journals Aromatase expression in Xenopus oocytes: a three cell-type model for the ovarian estradiol synthesis

2011 ◽  
Vol 47 (2) ◽  
pp. 241-250 ◽  
Author(s):  
M Gohin ◽  
P Bodinier ◽  
A Fostier ◽  
J Bobe ◽  
F Chesnel
Keyword(s):  
Classical Model ◽  
Biological Significance ◽  
Type Model ◽  
Aromatase Activity ◽  
Steroid Synthesis ◽  
Aromatase Expression ◽  
Androgen Synthesis ◽  
Estrogen Synthesis ◽  
Estradiol Synthesis

In contrast to the classical model describing the synthesis of androgens and estrogens as restricted to somatic cells, a previous study demonstrated that Xenopus laevis oocytes participate in androgen synthesis. The objective of our study was to determine whether Xenopus oocytes are also involved in estrogen synthesis. More precisely, we analyzed aromatase expression by in situ hybridization and RT-QPCR and measured aromatase activity. Aromatase, the enzyme responsible for estrogen synthesis, appears to be expressed and active not only in the follicular cells but also in the vitellogenic oocytes. During late oogenesis, aromatase oocyte expression and activity decreased concomitantly with the trend observed in surrounding follicular layers. In order to investigate the role of estradiol-17β (E2), we studied its effect on oocyte meiotic resumption. It appears that, as in Rana pipiens, E2 inhibited the follicle-enclosed maturation of Xenopus oocytes, likely through inhibition of LH-induced maturation-inducing steroid synthesis. In addition, E2 exerted a slight enhancing action on denuded oocyte maturation whose biological significance remains unclear. Together, our results demonstrate that Xenopus oocyte significantly participates in ovarian E2 synthesis and this may be a common feature of vitellogenic vertebrates.

scholarly journals Regulation of GLUT4 gene expression by SREBP-1c in adipocytes

2006 ◽  
Vol 399 (1) ◽  
pp. 131-139 ◽  
Author(s):  
Seung-Soon Im ◽  
Sool-Ki Kwon ◽  
Seung-Youn Kang ◽  
Tae-Hyun Kim ◽  
Ha-Il Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Glucose Transporter ◽  
Regulatory Element ◽  
Diabetic Rats ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Response Element ◽  
Glut4 Gene Expression

Expression of the GLUT4 (glucose transporter type 4 isoform) gene in adipocytes is subject to hormonal or metabolic control. In the present study, we have characterized an adipose tissue transcription factor that is influenced by fasting/refeeding regimens and insulin. Northern blotting showed that refeeding increased GLUT4 mRNA levels for 24 h in adipose tissue. Consistent with an increased GLUT4 gene expression, the mRNA levels of SREBP (sterol-regulatory-element-binding protein)-1c in adipose tissue were also increased by refeeding. In streptozotocin-induced diabetic rats, insulin treatment increased the mRNA levels of GLUT4 in adipose tissue. Serial deletion, luciferase reporter assays and electrophoretic mobility-shift assay studies indicated that the putative sterol response element is located in the region between bases −109 and −100 of the human GLUT4 promoter. Transduction of the SREBP-1c dominant negative form to differentiated 3T3-L1 adipocytes caused a reduction in the mRNA levels of GLUT4, suggesting that SREBP-1c mediates the transcription of GLUT4. In vivo chromatin immunoprecipitation revealed that refeeding increased the binding of SREBP-1 to the putative sterol-response element in the GLUT4. Furthermore, treating streptozotocin-induced diabetic rats with insulin restored SREBP-1 binding. In addition, we have identified an Sp1 binding site adjacent to the functional sterol-response element in the GLUT4 promoter. The Sp1 site appears to play an additive role in SREBP-1c mediated GLUT4 gene upregulation. These results suggest that upregulation of GLUT4 gene transcription might be directly mediated by SREBP-1c in adipose tissue.

scholarly journals Regulation of thrombospondin-1 expression through AU-rich elements in the 3′UTR of the mRNA

2011 ◽  
Vol 16 (1) ◽  
Author(s):  
Asa Mcgray ◽  
Timothy Gingerich ◽  
James Petrik ◽  
Jonathan Lamarre
Keyword(s):  
Gene Expression ◽  
Mrna Stability ◽  
Half Life ◽  
Rna Binding ◽  
Binding Protein ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Thrombospondin 1 ◽  
Mrna Half Life

AbstractThrombospondin-1 (TSP-1) is a matricellular protein that participates in numerous normal and pathological tissue processes and is rapidly modulated by different stimuli. The presence of 8 highly-conserved AU rich elements (AREs) within the 3′-untranslated region (3′UTR) of the TSP-1 mRNA suggests that post-transcriptional regulation is likely to represent one mechanism by which TSP-1 gene expression is regulated. We investigated the roles of these AREs, and proteins which bind to them, in the control of TSP-1 mRNA stability. The endogenous TSP-1 mRNA half-life is approximately 2.0 hours in HEK293 cells. Luciferase reporter mRNAs containing the TSP-1 3′UTR show a similar rate of decay, suggesting that the 3′UTR influences the decay rate. Site-directed mutagenesis of individual and adjacent AREs prolonged reporter mRNA halflife to between 2.2 and 4.4 hours. Mutation of all AREs increased mRNA half life to 8.8 hours, suggesting that all AREs have some effect, but that specific AREs may have key roles in stability regulation. A labeled RNA oligonucleotide derived from the most influential ARE was utilized to purify TSP-1 AREbinding proteins. The AU-binding protein AUF1 was shown to associate with this motif. These studies reveal that AREs in the 3′UTR control TSP-1 mRNA stability and that the RNA binding protein AUF1 participates in this control. These studies suggest that ARE-dependent control of TSP-1 mRNA stability may represent an important component in the control of TSP-1 gene expression.

scholarly journals Effect of β-agonist on the dexamethasone-induced expression of aromatase by the human monocyte cells

2017 ◽  
Vol 6 (2) ◽  
pp. 82-88 ◽  
Author(s):  
Masatada Watanabe ◽  
Shuji Ohno ◽  
Hiroshi Wachi
Keyword(s):  
Atopic Dermatitis ◽  
Human Skin ◽  
Human Monocyte ◽  
Skin Thickness ◽  
Aromatase Activity ◽  
Gene Transcript ◽  
Aromatase Expression ◽  
Estrogen Synthesis ◽  
Peripheral Monocyte

Emerging evidence suggests that sex steroids are important for human skin health. In particular, estrogen improves skin thickness, elasticity and moisture of older women. The major source of circulating estrogen is the ovary; however, local estrogen synthesis and secretion have important roles in, for example, bone metabolism and breast cancer development. We hypothesized that infiltrated peripheral monocytes are one of the sources of estrogen in skin tissues. We also hypothesized that, during atopic dermatitis under stress, a decline in the hypothalamus–pituitary–adrenal axis (HPA) and facilitation of the (hypothalamus)–sympathetic–adrenomedullary system (SAM) attenuates estrogen secretion from monocytes. Based on this hypothesis, we tested aromatase expression in the human peripheral monocyte-derived cell line THP-1 in response to the synthetic glucocorticoid dexamethasone (Dex), the synthetic β-agonist isoproterenol (Iso) and the β-antagonist propranolol (Pro). Dex mimics glucocorticoid secreted during excitation of the HPA, and Iso mimics catecholamine secreted during excitation of the SAM. We found that aromatase activity and the CYP19A1 gene transcript were both upregulated in THP-1 cells in the presence of Dex. Addition of Iso induced their downregulation and further addition of Pro rescued aromatase expression. These results may suggest that attenuation of estrogen secretion from peripheral monocytes could be a part of the pathology of stress-caused deterioration of atopic dermatitis. Further examination using an in vitro human skin model including THP-1 cells might be a valuable tool for investigating the therapeutic efficacy and mechanism of estrogen treatment for skin health.

Sign in / Sign up

Export Citation Format

Share Document