scholarly journals Expression of taurine transporter is regulated through the TonE (tonicity-responsive element)/TonEBP (TonE-binding protein) pathway and contributes to cytoprotection in HepG2 cells

2004 ◽  
Vol 382 (1) ◽  
pp. 177-182 ◽  
Author(s):  
Takashi ITO ◽  
Yasushi FUJIO ◽  
Mayo HIRATA ◽  
Tomoka TAKATANI ◽  
Takahisa MATSUDA ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Hepg2 Cells ◽  
Promoter Activity ◽  
Binding Protein ◽  
Dominant Negative ◽  
Taurine Transporter ◽  
Hypertonic Medium ◽  
Hypertonic Stress ◽  
Gene Assay ◽  
Responsive Element

In hypertonic environment, taurine accumulates in cells via activation of TauT (taurine transporter) as an adaptive regulation. Recent studies revealed that TonE (tonicity-responsive element)/TonEBP (TonE-binding protein) pathway regulated the expression of various molecules which protect cells against hypertonic stress. In the present study, we investigated the osmoregulatory mechanisms of TauT expression. TauT was up-regulated at both functional and transcriptional levels in HepG2 under hypertonic condition. The TonE site was identified in the promoter region of TauT gene. Reporter gene assay revealed that promoter activity was increased under hypertonic conditions, whereas deletion or mutation of TonE sequence abolished the induction of the promoter activity in response to hypertonicity. By using the reporter gene plasmids containing a TonE site of TauT promoter (p2xTonE-Luc), it was demonstrated that a TonE site was sufficient for the hypertonicity-mediated activation of TauT promoter. Importantly, co-transfection of TauT promoter gene plasmid with wild-type TonEBP expression vector enhanced promoter activity under isotonic conditions, whereas dominant-negative TonEBP abrogated the TauT promoter activity induced by hypertonicity. Finally, treatment with taurine prevented HepG2 cells from cell death induced by hypertonic medium. These findings suggested that induction of TauT by hypertonicity is mediated by the activation of the TonE/TonEBP pathway and confers resistance to hypertonic stress.

scholarly journals Interaction of Adenosine 3′,5′-Cyclic Monophosphate and Tumor Necrosis Factor-α on Serum Amyloid A3 Expression in Mouse Granulosa Cells: Dependence on CCAAT-Enhancing Binding Protein-β Isoform

Endocrinology ◽  
2010 ◽  
Vol 151 (7) ◽  
pp. 3407-3419 ◽  
Author(s):  
Deok-Soo Son ◽  
Paul F. Terranova ◽  
Katherine F. Roby
Keyword(s):  
Granulosa Cells ◽  
Promoter Activity ◽  
Binding Protein ◽  
Camp Response Element Binding ◽  
Dominant Negative ◽  
Serum Amyloid ◽  
Element Binding Protein ◽  
Granulose Cells ◽  
Factor Α ◽  
Interfering Rna

TNFα is an inflammatory-related cytokine that has inhibitory effects on gonadotropin- and cAMP-stimulated steroidogenesis and folliculogenesis. Because ovulation is an inflammatory reaction and TNF specifically induces serum amyloid A3 (SAA3) in mouse granulosa cells, the effect of cAMP on TNF-induced SAA3 promoter activity, mRNA and protein was investigated. Granulosa cells from immature mice were cultured with TNF and/or cAMP. TNF increased SAA3 promoter activity, mRNA, and protein, which were further increased by cAMP. cAMP alone increased SAA3 promoter activity, but SAA3 mRNA and protein remained undetectable. Thus, there appeared to be different mechanisms by which TNF and cAMP regulated SAA3 expression. SAA3 promoters lacking a nuclear factor (NF)-κB-like site or containing its mutant were not responsive to TNF but were responsive to cAMP. Among four CCAAT-enhancing binding protein (C/EBP) sites in the SAA3 promoter, the C/EBP site nearest the NF-κB-like site was required for TNF-induced SAA3. The C/EBP site at −75/−67 was necessary for responsiveness to cAMP. Dominant-negative C/EBP and cAMP response element-binding protein or short interfering RNA of C/EBPβ blocked TNF- or cAMP-induced SAA3 promoter activity. The combination of TNF and cAMP increased C/EBPβ protein above that induced by TNF or cAMP alone. Thus, cAMP in combination with TNF specifically induced C/EBPβ protein, leading to enhanced SAA3 expression but requiring NF-κB in mouse granulose cells. In addition, like TNF, SAA inhibited cAMP-induced estradiol accumulation and CYP19 levels. These data indicate SAA may play a role in events occurring during the ovulation process.

scholarly journals Epidermal Growth Factor-Induced GnRH-II Synthesis Contributes to Ovarian Cancer Cell Invasion

2009 ◽  
Vol 94 (9) ◽  
pp. 3618-3618
Author(s):  
Song Ling Poon ◽  
Gareth T. Hammond ◽  
Peter C. K. Leung
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Promoter Activity ◽  
Binding Protein ◽  
Ovarian Cancer Cells ◽  
Mrna Levels ◽  
Upstream Regulator ◽  
Enhancer Binding Protein ◽  
Responsive Element ◽  
Interfering Rna

GnRH-II modulates ovarian cancer cells invasion and is expressed in normal ovary and ovarian epithelial cancer cells; however, the upstream regulator(s) of GnRH-II expression in these cells remains unclear. We now demonstrate that epidermal growth factor (EGF) increases GnRH-II mRNA levels in several human ovarian carcinoma cell lines and up-regulates GnRH-II promoter activity in OVCAR-3 cells in a dose-dependent manner, whereas an EGF receptor inhibitor (AG148) abolishes EGF-induced increases in GnRH-II promoter activity and GnRH-II mRNA levels. EGF increases the phosphorylation of cAMP-responsive element-binding protein (p-CREB) and its association with the coregulator, CCAAT/enhancer binding protein β, whereas blocking the EGF-induced ERK1/2 phosphorylation with MAPK inhibitors (PD98059/U0126) markedly reduced these effects. Moreover, depletion of CREB using small interfering RNA attenuated EGF-induced GnRH-II promoter activity. Chromatin immunoprecipitation assays demonstrated that EGF induces p-CREB binding to a cAMP responsive-element within the GnRH-II promoter, likely in association with CCAAT/enhancer binding protein β, and mutagenesis of this cAMP responsive-element prevented EGF-induced GnRH-II promoter activity in OVCAR-3 cells. Importantly, GnRH-II acts additively with EGF to promote invasion of OVCAR-3 and CaOV-3 cells, but not SKOV-3 cells that express low levels of GnRH receptor (GnRHR). Treatment with GnRHR small interfering RNA also partially inhibited the EGF-induced invasion of OVCAR-3 and CaOV-3 cells. Furthermore, EGF treatment transiently increases GnRHR levels in OVCAR-3 and CaOV-3, which likely accentuates the effects of increase GnRH-II production on cell invasion. These results provide evidence that EGF is an upstream regulator of the autocrine actions of GnRH-II on the invasive properties of ovarian cancer cells.

scholarly journals Peroxisome-Proliferator Receptor γ Represses Hepatic Sex Hormone-Binding Globulin Expression

Endocrinology ◽  
2009 ◽  
Vol 150 (5) ◽  
pp. 2183-2189 ◽  
Author(s):  
David M. Selva ◽  
Geoffrey L. Hammond
Keyword(s):  
Hepg2 Cells ◽  
Promoter Activity ◽  
Binding Activity ◽  
Sex Hormone Binding Globulin ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Luciferase Reporter Gene ◽  
Pparγ Agonist ◽  
Gene Assay

Plasma SHBG production by the liver is influenced by its metabolic state, and hepatocyte nuclear factor-4α regulates SHBG expression in response to changes in lipogenesis. Peroxisome-proliferator receptors (PPARs) also regulate glucose homeostasis and fatty acid metabolism. The human SHBG promoter contains a PPAR-response element (PPAR-RE), and plasma SHBG levels increase in polycystic ovarian syndrome patients treated with the PPARγ agonist, rosiglitazone. In addition, plasma SHBG levels are associated with a genetic polymorphism in the PPARγ-2 coding sequence that alters its transcriptional activity. Therefore, we set out to determine whether PPARγ influences hepatic production of SHBG by using human HepG2 hepatoblastoma cells as an in vitro model. Surprisingly, treatment of HepG2 cells with rosiglitazone reduced SHBG production and SHBG promoter activity (as assessed in a luciferase reporter gene assay) by 20–25%, whereas the PPARγ antagonist, GW9662, increased both by 2- to 3-fold. The effects of PPARγ agonists and antagonists on SHBG promoter activity were substantially diminished when the PPAR-RE in the SHBG promoter was mutated. A PPARγ small interfering RNA also increased SHBG production by HepG2 cells as well as SHBG promoter activity, and the latter was accentuated by cotreatment with GW9662. Importantly, overexpression of a PPARγ-2 Pro12 variant in HepG2 cells was more effective at reducing SHBG promoter activity, when compared with PPARγ-2 Ala12, consistent with its superior PPAR-RE binding activity. We conclude that PPARγ represses human SHBG expression in liver cells, and that differences in PPARγ levels and activity contribute directly to variations in plasma SHBG levels.

scholarly journals Regulation of the human involucrin gene promoter by co-activator proteins

2004 ◽  
Vol 381 (1) ◽  
pp. 267-273 ◽  
Author(s):  
Nhu Q. TRAN ◽  
David L. CROWE
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Promoter Activity ◽  
Binding Protein ◽  
Regulatory Region ◽  
Terminal Differentiation ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Activator Proteins ◽  
Involucrin Promoter

Stratified squamous epithelial cells undergo an orderly process of terminal differentiation that is characterized by specific molecular and morphological changes, including expression of the cornified envelope protein involucrin. Significant progress has been made in characterizing the upstream regulatory region of the involucrin gene. Binding sites for AP-1 (activator protein 1) and Sp1 transcription factors were shown to be important for involucrin promoter activity and tissue-specific expression. Defective terminal differentiation is often characterized by decreased or lack of involucrin expression. Recently, a dominant-negative construct of the transcriptional co-activator P/CAF [p300/CBP-associated factor, where CBP stands for CREB (cAMP-response-element-binding protein)-binding protein] was shown to inhibit involucrin expression in immortalized keratinocytes [Kawabata, Kawahara, Kanekura, Araya, Daitoku, Hata, Miura, Fukamizu, Kanzaki, Maruyama and Nakajima (2002) J. Biol. Chem. 277, 8099–8105]. Loss of expression or inactivation of other co-activators has also been demonstrated [Suganuma, Kawabata, Ohshima, and Ikeda (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 13073–13078]. In the present study, we re-expressed CBP and P/CAF in immortalized keratinocyte lines that had lost expression of these co-activator proteins. Re-expression of these proteins restored calcium- and RA (retinoic acid)-responsive involucrin expression in these cells. RA and calcium signalling induced exchange of CBP and P/CAF occupancy at the AP-1 sites of the involucrin promoter. CBP and P/CAF inductions of the involucrin expression were not dependent on MEK (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase), p38, protein kinase C or CaM kinase (calcium/calmodulin-dependent kinase) signalling. Kinase-induced changes in involucrin promoter activity directly resulted from changes in AP-1 protein expression. We concluded that CBP and P/CAF are important regulators of involucrin expression in stratified squamous epithelial cells.

scholarly journals Hepatic Expression of the SPOT 14 (S14) Paralog S14-Related (Mid1 Interacting Protein) Is Regulated by Dietary Carbohydrate

Endocrinology ◽  
2008 ◽  
Vol 149 (10) ◽  
pp. 5155-5161 ◽  
Author(s):  
Nikolas G. Tsatsos ◽  
Lance B. Augustin ◽  
Grant W. Anderson ◽  
Howard C. Towle ◽  
Cary N. Mariash
Keyword(s):  
Mammary Gland ◽  
Binding Protein ◽  
Dominant Negative ◽  
Null Mouse ◽  
Mrna Levels ◽  
Glucose Response ◽  
Hepatic Expression ◽  
Spot 14 ◽  
Element Binding Protein ◽  
Responsive Element

The Spot 14 (S14) gene is rapidly up-regulated by signals that induce lipogenesis such as enhanced glucose metabolism and thyroid hormone administration. Previous studies in S14 null mice show that S14 is required for normal lipogenesis in the lactating mammary gland, but not the liver. We speculated that the lack of a hepatic phenotype was due to the expression of a compensatory gene. We recently reported that this gene is likely an S14 paralog that we named S14-Related (S14-R). S14-R is expressed in the liver, but not in the mammary gland. If S14-R compensates for the absence of S14 in the liver, we hypothesized that, like S14, S14-R expression should be glucose responsive. Here, we report that hepatic S14-R mRNA levels increase with high-carbohydrate feeding in mice or within 2 h of treating cultured hepatocytes with elevated glucose. A potential carbohydrate response element (ChoRE) was identified at position −458 of the S14-R promoter. Deletion of or point mutations within the putative S14-R ChoRE led to 50–95% inhibition of the glucose response. Gel-shift analysis revealed that the glucose-activated transcription complex carbohydrate responsive element-binding protein/Max-like protein X (Mlx) binds to the S14-R ChoRE. Finally, S14-R glucose induction is completely blocked when a dominant-negative form of Mlx is overexpressed in primary hepatocytes. In conclusion, our results indicate that the S14-R gene is a glucose-responsive target of carbohydrate responsive element-binding protein/Mlx and suggest that the S14-R protein is a compensatory factor, at least partially responsible for the normal liver lipogenesis observed in the S14 null mouse.

scholarly journals Effects of 2-Methoxyestradiol, a Main Metabolite of Estradiol on Hepatic ABCA1 Expression in HepG2 Cells

Nutrients ◽  
2022 ◽  
Vol 14 (2) ◽  
pp. 288
Author(s):  
Tomohiro Ibata ◽  
Jingya Lyu ◽  
Hitomi Imachi ◽  
Kensaku Fukunaga ◽  
Seisuke Sato ◽  
...  
Keyword(s):  
Fatty Liver ◽  
Lipid Content ◽  
Hepg2 Cells ◽  
Promoter Region ◽  
Promoter Activity ◽  
Pi3k Pathway ◽  
Dominant Negative ◽  
Nuclear Accumulation ◽  
Hepatic Lipid ◽  
Abca1 Expression

ATP-binding cassette transporter A1 (ABCA1) is a key regulator of lipid efflux, and the absence of ABCA1 induces hepatic lipid accumulation, which is one of the major causes of fatty liver. 2-Methoxyestradiol (2-ME2) has been demonstrated to protect against fatty liver. In this study, we investigated the effects of 2-ME2 on the hepatic lipid content and ABCA1 expression. We found that 2-ME2 dose-dependently increased ABCA1 expression, and therefore, the lipid content was significantly decreased in HepG2 cells. 2-ME2 enhanced the ABCA1 promoter activity; however, this effect was reduced after the inhibition of the PI3K pathway. The overexpression of Akt or p110 induced ABCA1 promoter activity, while dominant-negative Akt diminished the ability of 2-ME2 on ABCA1 promoter activity. Further, 2-ME2 stimulated the rapid phosphorylation of Akt and FoxO1 and reduced the nuclear accumulation of FoxO1. Chromatin immunoprecipitation confirmed that FoxO1 bonded to the ABCA1 promoter region. The binding was reduced by 2-ME2, which facilitated ABCA1 gene transcription. Furthermore, mutating FoxO1-binding sites in the ABCA1 promoter region or treatment with FoxO1-specific siRNA disrupted the effect of 2-ME2 on ABCA1 expression. All of our results demonstrated that 2-ME2 might upregulate ABCA1 expression via the PI3K/Akt/FoxO1 pathway, which thus reduces the lipid content in hepatocytes.

Combining an in vitro reporter gene assay with metabolomics to identify tomato phytochemicals responsible for inducing electrophile-responsive element (EpRE)-mediated gene transcription

Metabolomics ◽  
2014 ◽  
Vol 11 (2) ◽  
pp. 302-311 ◽  
Author(s):  
Henriëtte D. L. M. van Eekelen ◽  
Linda Gijsbers ◽  
Chris A. Maliepaard ◽  
Robert A. M. Vreeburg ◽  
Richard Finkers ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Gene Transcription ◽  
Reporter Gene Assay ◽  
Gene Assay ◽  
Responsive Element

scholarly journals 12-O-tetradecanoylphorbol-13-acetate activation of the MDR1 promoter is mediated by EGR1.

1995 ◽  
Vol 15 (11) ◽  
pp. 6100-6108 ◽  
Author(s):  
C McCoy ◽  
D E Smith ◽  
M M Cornwell
Keyword(s):  
Promoter Activity ◽  
Time Course ◽  
Efflux Pump ◽  
Hematopoietic Cells ◽  
K562 Cells ◽  
Dominant Negative ◽  
Multidrug Resistance Gene ◽  
Promoter Construct ◽  
Responsive Element ◽  
Mdr1 Promoter

P-glycoprotein, the product of the MDR1 gene (multidrug resistance gene 1), is an energy-dependent efflux pump associated with treatment failure in some hematopoietic malignancies. Its expression is regulated during normal hematopoietic differentiation, although its function in normal hematopoietic cells is unknown. To identify cellular factors that regulate the expression of MDR1 in hematopoietic cells, we characterized the cis- and trans-acting factors mediating 12-O-tetradecanoylphorbol-13-acetate (TPA) activation of the MDR1 promoter in K562 cells. Transient-transfection assays demonstrated that an MDR1 promoter construct containing nucleotides -69 to +20 conferred a TPA response equal to that of a construct containing nucleotides -434 to +105. TPA induced EGR1 binding to the -69/+20 promoter sequences over a time course which correlated with increased MDR1 promoter activity and increased steady-state MDR1 RNA levels. The -69/+20 promoter region contains an overlapping SP1/EGR site. The TPA-responsive element was localized to the overlapping SP1/EGR site by using a synthetic reporter construct. A mutation in this site that inhibited EGR protein binding blocked the -69/+20 MDR1 promoter response to TPA. The expression of a dominant negative EGR protein also blocked the TPA response of the -69/+20 promoter construct. Finally, the expression of EGR1 was sufficient to activate a construct containing tandem MDR1 promoter SP1/EGR sites. These data suggest a role for EGR1 in modulating MDR1 promoter activity in hematopoietic cells.

Sign in / Sign up

Export Citation Format

Share Document