scholarly journals Hormonal regulation of the human sterol 27-hydroxylase gene CYP27A1

2003 ◽  
Vol 372 (2) ◽  
pp. 529-534 ◽  
Author(s):  
Zufan ARAYA ◽  
Wanjin TANG ◽  
Kjell WIKVALL
Keyword(s):  
Reporter Gene ◽  
Hormonal Regulation ◽  
Promoter Activity ◽  
Luciferase Activity ◽  
Luciferase Reporter ◽  
Signal Transduction Pathways ◽  
Cytochrome P450 Enzyme ◽  
Luciferase Reporter Gene ◽  
Response Elements ◽  
P450 Enzyme

The mitochondrial sterol 27-hydroxylase (CYP27A1) is a multifunctional cytochrome P450 enzyme that catalyses important hydroxylations in the biosynthesis of bile acids and bioactivation of vitamin D3. Previous results [Babiker, Andersson, Lund, Xiu, Deeb, Reshef, Leitersdorf, Diczfalusy and Björkhem (1997) J. Biol. Chem. 272, 26253–26261] suggest that CYP27A1 plays an important role in cholesterol homoeostasis and affects atherogenesis. In the present study, the regulation of the human CYP27A1 gene by growth hormone (GH), insulin-like growth factor-1 (IGF-1), dexamethasone, thyroid hormones and PMA was studied. HepG2 cells were transfected transiently with luciferase reporter gene constructs containing DNA fragments flanking the 5′-region of the human CYP27A1 gene. GH, IGF-1 and dexamethasone increased the promoter activity by 2–3-fold, whereas thyroxine (T4) and PMA repressed the activity significantly when measured with luciferase activity expressed in the cells. The endogenous CYP27A1 enzyme activity in the cells was stimulated by GH, IGF-1 and dexamethasone, whereas T4 and PMA inhibited the activity. Experiments with progressive deletion/luciferase reporter gene constructs indicated that the response elements for GH may be localized in a region upstream to position −1094 bp. The putative response elements for dexamethasone were mapped to positions between −792 and −1095 bp. The −451 bp fragment of the human CYP27A1 gene was found to confer the activation by IGF-1, and the inhibition by T4 and PMA. Results of the present study suggest that CYP27A1 is regulated in human cells by hormones and signal-transduction pathways.

Phospholipase A 2 Metabolites Mediate Endothelin Stimulation of the Human Brain Natriuretic Peptide Promoter

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 721-721
Author(s):  
Quan He
Keyword(s):  
Gene Expression ◽  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Promoter Activity ◽  
Luciferase Activity ◽  
Phospholipase A ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
P450 Monooxygenase ◽  
Stimulation Of

P155 Brain natriuretic peptide (BNP) gene expression accompanies cardiac hypertrophy and heart failure. The vasoconstrictor endothelin-1 (ET)may be involved in the development of these diseases. ET has also been shown to activate phospholipase A 2 (PLA 2 ). Thus we studied whether ET and PLA 2 metabolites regulate BNP gene expression. The hBNP promoter (-1818 to + 100) coupled to a luciferase reporter gene was transferred into neonatal ventricular myocytes (NVM),and luciferase activity was measured as an index of promoter activity. ET (10 -7 M)induced BNP mRNA in NVM as assessed by Northern blot. It also stimulated the hBNP promoter 4-fold vs control, an effect completely inhibited by actinomycin D. To test the involvement of different PLA 2 isoforms, transfected cells were treated with the Ca ++ -independent PLA 2 (iPLA 2 )inhibitor bromoenol lactone (BEL), the cytosolic PLA 2 inhibitor methyl arachidonyl fluorophosphonate, or the secretory PLA 2 inhibitor ONO-RS-082 prior to stimulation with ET. Only the iPLA 2 inhibitor BEL prevented ET-stimulated hBNP promoter activity. The PLA 2 metabolite lysophosphatidic acid (LPA) also activated the hBNP promoter (2.2-fold; n = 3), but lysophosphatidylcholine did not. To test whether arachidonic acid metabolites are involved in ET’s effect, cells were pretreated with either a lipoxygenase (LO), cyclooxygenase, or p450 monooxygenase inhibitor. Only the LO inhibitor baicalein prevented ET stimulation of the hBNP promoter. Finally, we studied the involvement of cis elements in ET-stimulated hBNP promoter activity. Deletion of BNP promoter sequences from -1818 to -408 and from -408 to -40 reduced ET’s effect by 54% and 78%, respectively. Moreover, ET-stimulated luciferase activity was reduced by 53% when the GATA element (at position -85 relative to the start site of transcription) was mutated. These data suggest that: 1) ET activates the hBNP promoter through a transcriptional mechanism; 2) LPA, perhaps generated by a BEL-sensitive iPLA 2 , is involved in ET’s effect; 3) a LO pathway may also mediate ET signaling; and 4) ET regulation of the hBNP promoter targets both distal and proximal cis elements, including GATA.

scholarly journals Use of Leishmania donovani Field Isolates Expressing the Luciferase Reporter Gene in In Vitro Drug Screening

2005 ◽  
Vol 49 (9) ◽  
pp. 3776-3783 ◽  
Author(s):  
Ashutosh ◽  
Suman Gupta ◽  
Ramesh ◽  
Shyam Sundar ◽  
Neena Goyal
Keyword(s):  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Leishmania Donovani ◽  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Field Isolates

ABSTRACT Currently available primary screens for the selection of candidate antileishmanial compounds are not ideal. These techniques are time-consuming, laborious, and difficult to scale and require macrophages, which limit their use for high-throughput screening. We have developed Leishmania donovani field isolates that constitutively express the firefly luciferase reporter gene (luc) as a part of an episomal vector. An excellent correlation between parasite number and luciferase activity was observed. luc expression was stable, even in the absence of drug selection, for 4 weeks. The transfectants were infective to macrophages, and intracellular amastigotes exhibited luciferase activity. The suitability of these recombinant field isolates for in vitro screening of antileishmanial drugs was established. The luciferase-expressing sodium stibogluconate-resistant cell lines offer a model for the screening of compounds for resistance. The system is in routine use at the Central Drug Research Institute, Lucknow, India, for high-throughput screening of newly synthesized compounds.

scholarly journals Tissue- and time-dependent estrogen receptor activation in estrogen reporter mice

2004 ◽  
Vol 32 (3) ◽  
pp. 689-701 ◽  
Author(s):  
JG Lemmen ◽  
RJ Arends ◽  
AL van Boxtel ◽  
PT van der Saag ◽  
B van der Burg
Keyword(s):  
Reporter Gene ◽  
Imaging System ◽  
Luciferase Activity ◽  
Estrogenic Activity ◽  
Receptor Activation ◽  
Time Dependent ◽  
Luciferase Reporter ◽  
In Vivo Model ◽  
Luciferase Reporter Gene

With the aim of developing an in vivo model that directly detects activation of estrogen receptors (ERs), transgenic mice carrying a luciferase reporter gene were generated. The luciferase reporter gene was under the control of three consensus estrogen-responsive elements (EREs) coupled to a minimal TATA-box, with or without flanking chick beta-globin insulators. By using this model in combination with the IVIS imaging system, in vivo ER activation was measured. Dose- and time-dependent luciferase activity was induced in various organs of adult transgenic male mice exposed to diethylstilbestrol (DES) (10-1000 micro g/kg) and 17beta-estradiol dipropionate (EP) (10-1000 micro g/kg), when luciferase activity was measured ex vivo. The highest (>10 000-fold) induction of luciferase was measured in bone and kidney 24 h after exposure to 1000 micro g/kg EP. Other highly responsive organs include liver, testis, pituitary, brain, prostate and colon, which show different activity profiles. This in vivo model for detecting estrogenic activity can be used to assess tissue-specific action of ER agonists and antagonists. These could include selective ER modulators and environmental estrogens. In combination with the IVIS imaging system, this in vivo model is a powerful tool for assessing the kinetics of gene activation by estrogenic compounds.

scholarly journals Endothelial-specific gene expression directed by the tie gene promoter in vivo

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1828-1835 ◽  
Author(s):  
J Korhonen ◽  
I Lahtinen ◽  
M Halmekyto ◽  
L Alhonen ◽  
J Janne ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Promoter Activity ◽  
Cultured Cells ◽  
Luciferase Reporter ◽  
Specific Gene ◽  
Luciferase Reporter Gene ◽  
Human And Mouse

The tie gene encodes a receptor tyrosine kinase that is expressed in the endothelium of blood vessels, particularly during embryonic development and angiogenesis in adults. We have cloned and characterized the mouse tie gene and isolated the human and mouse tie promoters. The promoter activities of human and mouse tie were analyzed using luciferase reporter gene constructs in transfected cell lines and beta-galactosidase constructs in transgenic mice. In transfection assays of cultured cells, both human and mouse promoter DNA fragments showed activity that was not restricted to endothelial cells. In contrast, in transgenic mice both promoters directed expression of the reporter gene to endothelial cells undergoing vasculogenesis and angiogenesis. In adult mice, tie promoter activity in lung and many vessels of the kidney was as high as in the vessels of the corresponding embryonic tissues, whereas in the heart, brain and liver, tie promoter activity was downregulated and restricted to coronaries, cusps, capillaries, and arteries. Our results show that the endothelial cell-type specificity of the tie promoter in vivo can be transferred to heterologous genes by using relatively short promoter fragments. The tie promoter, thus, has useful properties for potential gene therapy.

scholarly journals MiR-545-3p Upregulated in Osteoporosis and Regulates The Apoptosis of Osteoblasts by Targeting SIRT6

2020 ◽  
Author(s):  
Qi-Ming Ma ◽  
Xiao-Jing Li ◽  
Shao-Bao Pei ◽  
Bo-Wen Li ◽  
Guo-Song Han ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Reporter Gene ◽  
Luciferase Activity ◽  
Luciferase Reporter ◽  
Osteogenic Medium ◽  
Luciferase Reporter Gene ◽  
Transfected Cells ◽  
Gene Assay ◽  
Related Proteins

Abstract Background: The imbalance of proliferation and apoptosis plays an important role in the pathogenesis of osteoporosis. MicroRNAs play an important role in the apoptosis of osteoblasts cells. However, the role and potential mechanism of miR-545-3p in regulating osteoblast apoptosis remain unclear. The purpose of this study was to investigate the effect of miR-545-3p on osteoblast cells apoptosis and explore the mechanism of osteoporosis.Methods: Osteogenic medium was used to induce the differentiation of osteoblasts MC3T3-E1 to construct the osteoporosis model, and the expression of ALP, Runx2, OCN were detected by western blot; miR-545-3p mRNA was detected by RT-PCR. Transfected with miR-545-3p mimics into MC3T3-E1, then flow cytometry was used to detected the changes of apoptosis status. The expression of apoptosis related proteins Bcl-2 and Bax were detected by western blot. Bioinformatics was used to analyze the binding protein of miR-545-3p, and luciferase reporter gene experiment was used to verify whether miR-545-3p directly targets SIRT6; RT-qPCR and western blot were used to detect the expression level of SIRT6 after transfection of miR-545-3p mimics or miR-545-3p inhibitor. After co-transfection of miR-545-3p mimics and pcDNA3.1-SIRT6, the apoptosis status of osteoblasts was analyzed by flow cytometry, and the expression of apoptosis related proteins Bcl-2 and Bax were detected by western blot.Results: The expression of miR-545-3p in patients with osteoporosis was significantly higher than that of normal in GEO database (P<0.05). After osteoblasts were cultured in osteogenic medium, the expression of ALP, Runx2 and OCN was increased, and the expression of miR-545-3p was decreased. Flow cytometry analysis showed that overexpression of miR-545-3p promoted the apoptosis of osteoblasts. Western blot results showed that overexpression of miR-545-3p promoted the expression of Bax and decreased the expression of Bcl-2. Bioinformatics analysis showed that miR-545-3p could target SIRT6. The results of real-time PCR and western blot showed that SIRT6 expression was significantly inhibited by miR-545-3p mimics (P<0.05). Luciferase reporter gene assay showed that miR-545-3p significantly inhibited luciferase activity of wild-type SIRT6-3'UTR plasmid transfected cells (P<0.05), but had no effect on luciferase activity of mutant SIRT6-3'UTR plasmid transfected cells (P<0.05). However, co-transfection of miR-545-3p mimics and pcDNA3.1-SIRT6 could reduce the apoptosis, and western blot results showed that co-transfection promoted the expression of Bcl-2 and decreased the expression of Bax.Conclusions: miR-545-3p can promote the apoptosis of osteoblasts by inhibiting the expression of SIRT6, which provides a certain idea for the treatment of osteoporosis.

scholarly journals Evaluation of the use of the luciferase-reporter-gene system for gene-regulation studies involving cyclic AMP-elevating agents

1995 ◽  
Vol 309 (2) ◽  
pp. 385-387 ◽  
Author(s):  
O Benzakour ◽  
C Kanthou ◽  
U Dennehy ◽  
A al Haq ◽  
L P Berg ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Reporter Gene ◽  
Transcriptional Activation ◽  
Luciferase Activity ◽  
Primary Cultures ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Promoter Elements ◽  
Gene System ◽  
Reporter Gene System

The effects of cyclic AMP (cAMP)-elevating agents on the activity of cis-acting gene promoter sequences are frequently studied using the luciferase-reporter-gene system. The aim of the present study was to assess whether cAMP-elevating agents induce any changes in the level of luciferase activity independently of a transcriptional activation of promoter elements. Chloramphenicol acteyltransferase (CAT) and luciferase reporter genes under the control of the same promoter elements were transiently expressed in primary cultures of human vascular smooth-muscle cells. Transfected cells were treated with a cell-permeable and non-hydrolysable cAMP analogue, 2′-O-monobutyryl-8-bromo cAMP, or with the cAMP-elevating agents forskolin and prostaglandin E1 (PGE1). Forskolin and PGE1 induced a significant increase in the level of luciferase activity, but had no effect on CAT activity. Conclusions based solely on the use of the luciferase-reporter-gene system in studies involving promoter activation by cAMP-elevating agents could therefore be misleading.

scholarly journals Unusual 5'-regulatory structure and regulation of the murine Mlc1 gene: Lack of promoter-specific functional elements

2011 ◽  
Vol 2 (1) ◽  
pp. 11
Author(s):  
Darja Henseler ◽  
Jonathan D. Turner ◽  
Matthias Eckhardt ◽  
Maaike Van der Mark ◽  
Yanina Revsin ◽  
...  
Keyword(s):  
Reporter Gene ◽  
In Silico ◽  
Promoter Activity ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Regulatory Structure ◽  
Luciferase Reporter Gene ◽  
Promoter Regions ◽  
Potential Promoter

<!--[if gte mso 9]><xml> <w:WordDocument> <w:View>Normal</w:View> <w:Zoom>0</w:Zoom> <w:HyphenationZone>21</w:HyphenationZone> <w:PunctuationKerning /> <w:ValidateAgainstSchemas /> <w:SaveIfXMLInvalid>false</w:SaveIfXMLInvalid> <w:IgnoreMixedContent>false</w:IgnoreMixedContent> <w:AlwaysShowPlaceholderText>false</w:AlwaysShowPlaceholderText> <w:Compatibility> <w:BreakWrappedTables /> <w:SnapToGridInCell /> <w:WrapTextWithPunct /> <w:UseAsianBreakRules /> <w:DontGrowAutofit /> </w:Compatibility> <w:BrowserLevel>MicrosoftInternetExplorer4</w:BrowserLevel> </w:WordDocument> </xml><![endif]--><!--[if gte mso 9]><xml> <w:LatentStyles DefLockedState="false" LatentStyleCount="156"> </w:LatentStyles> </xml><![endif]--><!--[if gte mso 10]> <mce:style><! /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Normale Tabelle"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-parent:""; mso-padding-alt:0pt 5.4pt 0pt 5.4pt; mso-para-margin:0pt; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-ansi-language:#0400; mso-fareast-language:#0400; mso-bidi-language:#0400;} --> <!--[endif]--> <p class="MsoNormal" style="text-align: justify;"><span style="color: black;" lang="EN-GB">The <em>MLC1</em> gene is involved in an autosomal recessive neurological disorder, megalencephalic leucoencephalopathy with subcortical cysts (MLC), which is characterized by macrocephaly during the first year of life and swollen white matter (leucoencephaly). Variants of <em>MLC1</em> have also been associated with psychiatric disorders such as schizophrenia, major depression and bipolar disorder. Currently, little is known about the encoded protein (MLC1). Judging from its similarity to other known proteins, it may serve as a trans-membrane transporter. However, the function of the encoded protein and its gene regulation has not been investigated successfully so far. We investigated the 5’ region of the murine <em>Mlc1</em> with respect to regulatory elements for gene expression. A promoter search and an <em>in silico</em> analysis were conducted. Luciferase reporter gene constructs with potential promoter regions were created to study promoter activity <em>in vitro</em>. We found two alternative first exons for the murine <em>Mlc1</em> but were not able to detect any promoter activity for the investigated reporter gene constructs in different cell lines, thus pointing to the presence of essential <em>cis</em>-acting elements far outside of the region. <em>In silico </em>analysis indicated an uncommon promoter structure for <em>Mlc1</em>, with CCAAT-boxes representing the only noticeable elements. </span></p>

Influence of chlorophyll and tannins in plant extracts on cell-based luciferase reporter gene assays

Planta Medica ◽  
2009 ◽  
Vol 75 (09) ◽  
Author(s):  
S Vogl ◽  
P Picker ◽  
N Fakhrudin ◽  
A Atanasov ◽  
E Heiß ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Plant Extracts ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Reporter Gene Assays

scholarly journals In vivo analysis of the myosin heavy chain IIB promoter region

1998 ◽  
Vol 274 (3) ◽  
pp. C681-C687 ◽  
Author(s):  
Steven J. Swoap
Keyword(s):  
Reporter Gene ◽  
Luciferase Activity ◽  
Fiber Type ◽  
Firefly Luciferase ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Base Pairs ◽  
Specific Expression ◽  
Mhc Iib

The myosin heavy chain (MHC) IIB gene is preferentially expressed in fast-twitch muscles of the hindlimb, such as the tibialis anterior (TA). The molecular mechanism(s) for this preferential expression are unknown. The goals of the current study were 1) to determine whether the cloned region of the MHC IIB promoter contains the necessary cis-acting element(s) to drive fiber-type-specific expression of this gene in vivo, 2) to determine which region within the promoter is responsible for fiber-type-specific expression, and 3) to determine whether transcription off of the cloned region of the MHC IIB promoter accurately mimics endogenous gene expression in a muscle undergoing a fiber-type transition. To accomplish these goals, a 2.6-kilobase fragment of the promoter-enhancer region of the MHC IIB gene was cloned upstream of the firefly luciferase reporter gene and coinjected with pRL-cytomegalovirus (CMV) (CMV promoter driving the renilla luciferase reporter) into the TA and the slow soleus muscle. Firefly luciferase activity relative to renilla luciferase activity within the TA was 35-fold greater than within the soleus. Deletional analysis demonstrated that only the proximal 295 base pairs (pGL3IIB0.3) were required to maintain this muscle-fiber-type specificity. Reporter gene expression of pGL3IIB0.3 construct was significantly upregulated twofold in unweighted soleus muscles compared with normal soleus muscles. Thus the region within the proximal 295 base pairs of the MHC IIB gene contains at least one element that can drive fiber-type-specific expression of a reporter gene.

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