DcCDPK8 involved in abiotic stress such as low temperature and signal transduction of hormones ABA and MeJA,but the transcriptional regulation is still unclear. In order to study the core promoter region of DcCDPK8 gene in Dendrobium catenatum and explore its transcriptional regulation mechanism,the DcCDPK8 gene promoter sequence was cloned by PCR from D. catenatum. Promoter sequence function was studied by fusion of 5 'terminal deletion and GUS gene. The results showed that the promoter sequence of DcCDPK8 gene has a low-temperature responsive element( LTR) between~(-1) 749 bp and-614 bp,two MeJA responsive elements between~(-1) 749 bp and-230 bp,and one ABA responsive elements between-614 bp and-230 bp. Three 5'-end different deletion fragments were constructed to fuse the eukaryotic expression vectors p BI121 with GUS,which were transformed into tobacco leaves. The GUS activity under cold stress treatment was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. GUS activity under exogenous ABA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3,and GUS activity under exogenous MeJA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. It is speculated that the ABA response element( ARE) in the promoter sequences of DcCDPK8 is positive regulatory role in response to exogenous ABA,the MeJA cis-acting element plays a negative role in response to exogenous MeJA.
Abscisic Acid ; Acetates ; Cloning, Molecular ; Cold Temperature ; Cyclopentanes ; Dendrobium ; genetics ; Gene Expression Regulation, Plant ; Oxylipins ; Plant Proteins ; genetics ; Plants, Genetically Modified ; Promoter Regions, Genetic ; Response Elements ; Stress, Physiological ; Tobacco

OBJECTIVETo investigate the protective effect of extracts from Cichorium endivia (CEE) in H2O2-induced HepG2 cell oxidative stress injury, and explore the antioxidant mechanism of CEE in HepG2 cells.

METHODThe viability of H2O2-induced HepG2 cells and the intracellular ROS level were measured by MTT assay and DCFH-DA fluorescence staining assay. The antioxidant-response element (ARE)-Luciferase activity was tested in HepG2 cells stably transected by ARE reporter gene. The fluorescence quantitative RT-PCR was adopted to determine the mRNA expressions of genes containing ARE sequence in HepG2 cells.

RESULTThe cell viability reduced, while the ROS level increased after HepG2 cells were treated by H2O2. Different concentrations of CEE could be added to significantly improve the above results. After HepG2 cells transected by ARE reporter gene were treated with different concentrations of CEE, the intracellular ARE activity could increase in a concentration-dependent manner. In addition, the mRNA expressions of regulatory genesGCLC, GCLM and HMOX-1 containing ARE sequence in HepG2 cells were up-regulated in a concentration-dependent manner by CEE.

CONCLUSIONCEE inhibited the H2O2-injured HepG2 cells by reducing the ROS level. CEE's antioxidant mechanism for HepG2 cells may be closely related to the antioxidant defense system associated with its effect of activating Nrf2-ARE pathway in HepG2 cells.

Antioxidants ; isolation & purification ; pharmacology ; Asteraceae ; chemistry ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Hep G2 Cells ; Humans ; Hydrogen Peroxide ; pharmacology ; Reactive Oxygen Species ; metabolism ; Response Elements ; genetics

OBJECTIVESTo identify a NFB responsive element within the dimethylarginine dimethylaminohydrolase 2 gene (DDAH) promoter and demonstrate its role in DDAH2 transactivation.

METHODSDDAH2 promoter was analyzed with software to identify potential binding sites of transcription factors. A series of truncated DDAH2 promoter luciferase reporter plasmids were constructed and transfected into human embryonic kidney derived HEK293 cells. Luciferase assays were carried out to analyze the activity of the promoter. Electrophoresis mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were used to identify the NFB responsive element in vitro and in vivo. DDAH2 promoter luciferase reporter plasmid with mutated NFB site was constructed and transfected into cells, and its activity was compared with that of the wild-type plasmid.

RESULTSPotential bindings sites of many transcription factors were found within the DDAH2 promoter. The transcription activity of the DDAH2 promoter was high, and -530 to -437 was a positive regulating region. -476 to -469 of the DDAH2 promoter was a NFB responsive element, to which NFB can specifically bind. Mutation of the NFB element could significantly decrease the DDAH2 promoter activity.

CONCLUSION-476 to -469 of the DDAH2 promoter was a NFB responsive element and is important for the transactivation of DDAH2.

Amidohydrolases ; genetics ; metabolism ; Base Sequence ; Gene Expression Regulation, Enzymologic ; Humans ; Molecular Sequence Data ; NF-kappa B ; metabolism ; Promoter Regions, Genetic ; Protein Binding ; Response Elements

PURPOSE: SNF2L belongs to Imitation Switch family and plays an essential role in neural tissues and gonads. In our previous studies, we have demonstrated that the basal transcription of human SNF2L gene is regulated by two cis-elements, cAMP response element (CRE)- and Sp1-binding sites. Recent studies suggested that cyclic adenosine monophosphate (cAMP) stimulation significantly up-regulated SNF2L expression in ovarian granulose cells. These data suggested that protein kinase-mediated signal pathways might also regulate SNF2L expression in neural cells. We therefore investigated the effects of agents that activate protein kinases A on SNF2L gene expression in neural cells. MATERIALS AND METHODS: To increase intracellular cAMP levels, all neural cells were treated with forskolin and dbcAMP, two cAMP response activators. We exmined the effects of cAMP on the promoter activity of human SNF2L gene by luciferase reporter gene assays, and further examined the effects of cAMP on endogenous SNF2L mRNA levels by qPCR. RESULTS: Transient expression of a luciferase fusion gene under the control of the SNF2L promoter was significantly increased by treatment of rat primary neurons with forskolin or dbcAMP, but not PC12, C6 and SH-SY5Y cells. Consistently, treatment with forskolin or dbcAMP could enhance endogenous SNF2L mRNA levels also only in rat primary neurons. CONCLUSION: These results suggest that the CRE consensus sequence in the SNF2L proximal promoter most likely confers constitutive activation and regulation by cAMP in neural cells.
Animals ; Bucladesine/pharmacology ; Cell Line ; Colforsin/pharmacology ; Cyclic AMP/*metabolism ; DNA-Binding Proteins/chemistry/*genetics/metabolism ; *Gene Expression Regulation ; Humans ; Luciferases/analysis ; Neurons/*metabolism ; PC12 Cells ; Promoter Regions, Genetic ; RNA, Messenger/metabolism ; Rats ; Rats, Wistar ; Recombinant Fusion Proteins/analysis ; *Response Elements ; Transcription Factors/chemistry/*genetics/metabolism

OBJECTIVETo construct a luciferase reporter vector containing the response element of transcription protein AP2α for screening the effect of bone morphogenetic proteins (BMPs) on the transcriptional activity of AP2α.

METHODSFour tandem-linked response elements of AP2α were cloned to the pBGLuc luciferase reporter gene plasmid, which was digested with Bam HI and Mlu I to construct pBGLuc-AP2α-RE vector. The recombinant adenovirus Ad-AP2α and its dominant negative mutant Ad-dnAP2α were used to infect mouse mesenchymal stem cells C3H10; the changes in cellular AP2α mRNA and protein expressions were detected by real-time PCR and Western blotting, and electrophoretic mobility shift assay (EMSA) was carried out to assess the DNA-binding ability of AP2α. C3H10 cells were transfected with pBGLuc-AP2α-RE vector, and AP2α transcriptional activity was measured using luciferase reporter gene assay. In pBGLuc-AP2α-RE vector-transfected C3H10 cells infected with Ad-BMPs, luciferase reporter gene assay was performed to screen the effect of BMPs on AP2α transcriptional activity.

RESULTSThe results of PCR, enzyme digestion and sequencing all confirmed correct cloning of AP2α-RE into pBGLuc-AP2α-RE luciferase reporter vector, and Ad-AP2α infection significantly increased AP2α expression and its DNA binding ability. The dominant negative mutants expressed the corresponding mutants, and EMSA results showed that Ad-dnAP2α-δbHLH significantly lowered while Ad-dnAP2α-δTAD enhanced the DNA-binding ability of AP2α. AP2α over-expression promoted AP2α transcriptional activity, which was suppressed by the two dominant negative mutants. AP2α transcriptional activity increased in the cells infected with the recombinant adenovirus BMPs, especially in cells with BMP9 infection.

CONCLUSIONSThe luciferase reporter vector containing the response element of AP2α we constructed allows detection of AP2α transcriptional activity. BMP9 can significantly enhance AP2α transcriptional activity.

Adenoviridae ; Animals ; Bone Morphogenetic Proteins ; genetics ; metabolism ; Genes, Reporter ; Genetic Vectors ; Growth Differentiation Factor 2 ; genetics ; metabolism ; Luciferases ; genetics ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Osteogenesis ; Protein Binding ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Response Elements ; Transcription Factor AP-2 ; genetics ; metabolism ; Transcriptional Activation ; Transfection

The aim of this study is to investigate the protection effect of tanshinone IIA (Tan) against triptolide (TP)-induced liver injury and the mechanisms involved. Acute liver injury was induced by intraperitoneal injection of TP (1 mg x kg(-1)) in mice. The activities of AST, ALT and LDH in serum and the levels of GSH, GST, GSH-PX, SOD, CAT and MDA in liver tissue were detected. The histopathological changes of liver tissues were observed after HE staining. Nrf2 translocation in liver tissue was detected by Western blotting, and real-time PCR was used to measure the expression levels of GCLC, NQO1 and HO-1 mRNA. The results showed that pretreatment with Tan significantly prevented the TP induced liver injury as indicated by reducing the activities of AST, ALT and LDH (P < 0.01). Tan pretreatment also prevented TP-induced oxidative stress in the mice liver by inhibiting MDA and restoring the levels of GSH, GST, SOD and CAT (P < 0.05). Parallel to these changes, pretreatment with Tan could attenuate histopathologic changes induced by TP. Furthermore, the results indicated that Tan pretreatment caused nuclear accumulation of Nrf2 as well as induction of mRNA expression of antioxidant response element (ARE)-driven genes such as GCLC, NQO1 and HO-1. These results indicated that Tan could protect against TP-induced acute liver injury via the activation of Nrf2/ARE pathway.
Animals ; Antioxidant Response Elements ; drug effects ; Chemical and Drug Induced Liver Injury ; metabolism ; pathology ; Diterpenes ; toxicity ; Diterpenes, Abietane ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Epoxy Compounds ; toxicity ; Glutamate-Cysteine Ligase ; genetics ; metabolism ; Heme Oxygenase-1 ; genetics ; metabolism ; Liver ; metabolism ; pathology ; Male ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred C57BL ; NAD(P)H Dehydrogenase (Quinone) ; genetics ; metabolism ; NF-E2-Related Factor 2 ; metabolism ; Phenanthrenes ; toxicity ; RNA, Messenger ; metabolism ; Signal Transduction ; drug effects

Relative deficiency in production of glycoprotein hormone erythropoietin (Epo) is a major cause of renal anemia. This study planned to investigate whether the hypoxia-regulated system of Epo expression, constructed by fusing Epo gene to the chimeric phosphoglycerate kinase (PGK) hypoxia response elements (HRE) in combination with cytomegalovirus immediate-early (CMV IE) basal gene promoter and delivered by plasmid intramuscular injection, might provide a long-term physiologically regulated Epo secretion expression to correct the anemia in adenine-induced uremic rats. Plasmid vectors (pHRE-Epo) were synthesized by fusing human Epo cDNA to the HRE/CMV promoter. Hypoxia-inducible activity of this promoter was evaluated first in vitro and then in vivo in healthy and uremic rats (n = 30 per group). The vectors (pCMV-Epo) in which Epo expression was directed by a constitutive CMV gene promoter served as control. ANOVA and Student's t-test were used to analyze between-group differences. A high-level expression of Epo was induced by hypoxia in vitro and in vivo. Though both pHRE-Epo and pCMV-Epo corrected anemia, the hematocrit of the pCMV-Epo-treated rats exceeded the normal (P < 0.05), but that of the pHRE-Epo-treated rats didn't. Hypoxia-regulated system of Epo gene expression constructed by fusing Epo to the HRE/CMV promoter and delivered by plasmid intramuscular injection may provide a long-term and stable Epo expression and secretion in vivo to correct the anemia in adenine-induced uremic rats.
Anemia/blood/*therapy ; Animals ; Base Sequence ; Blood Urea Nitrogen ; Cell Hypoxia ; Creatinine/blood ; Erythropoietin/biosynthesis/*genetics/secretion ; Gene Expression Regulation ; Genes, Reporter ; Genetic Therapy ; HeLa Cells ; Humans ; Injections, Intramuscular ; Kidney/pathology ; Luciferases, Firefly/biosynthesis/genetics ; Molecular Sequence Data ; Plasmids/*genetics ; Promoter Regions, Genetic ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins/biosynthesis/genetics/secretion ; Response Elements ; Transcriptional Activation ; Uremia/blood/*therapy

Gastrointestinal tract carcinoma is one of the leading causes of cancer-related death in China. Chemoprevention has been considered as a potential approach to control this type of disease. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that protects cells from oxidative/electrophilic stresses by activating the expression of a battery of cytoprotective genes through the antioxidant response element (ARE). Recently, Nrf2 has emerged as a novel target for chemoprevention. Several natural or synthetic chemicals, which activate Nrf2/ARE signaling pathway, have showed effect in animal models, and promises in many ongoing clinical trials. This review summarizes the recent findings on the regulation of Nrf2/ARE signaling pathway, and the developments in both preclinical and clinical studies.
Animals ; Anticarcinogenic Agents ; pharmacology ; Antioxidants ; metabolism ; Chemoprevention ; Digestive System Neoplasms ; genetics ; metabolism ; prevention & control ; Humans ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Oxidative Stress ; drug effects ; Response Elements ; genetics ; Signal Transduction ; drug effects

OBJECTIVETo explore the regulation mechanism of the reversal of breast cancer resistance protein-mediated multidrug resistance by toremifene.

METHODSTwo recombinant plasmids (pcDNA3-promoter-BCRP and pcDNA3-CMV-BCRP) were designed to express the wild-type full-length BCRP cDNA enforced driven by its endogenous promoter containing a functional ERE and a CMV promoter as control, respectively. Two recombinant plasmids were transfected into ERα-positive MCF-7 and ERα-negative MDA-MB-231 breast cancer cell lines. Four kinds of BCRP expressing cell lines of MCF-7/Promoter-BCRP, MCF-7/CMV-BCRP, MDA-MB-231/Promoter-BCRP and MDA-MB-231/CMV-BCRP were established in which BCRP was promoted by the BCRP promoter and a CMV promoter as control, respectively. The drug resistant cells were treated with toremifene. Then RT-PCR, Western blot, mitoxantrone efflux assays and cytotoxicity assay were performed to detect the reversal function of BCRP by toremifene on the drug resistance cell lines.

RESULTSToremifene significantly downregulated BCRP mRNA levels in a dose-dependent manner in ERα-positive MCF-7/Promoter-BCRP cells than that of untreated control cells. In MCF-7/Promoter-BCRP cells, toremifene at the dose of 0.1, 1 and 10 µmol/L decreased BCRP mRNA expression by 29.5% (P < 0.05), 68.1% (P < 0.01) and 97.4% (P < 0.01), respectively. After being treated with toremifene and 17β-estradiol, the BCRP mRNA level in MCF-7/Promoter-BCRP cells was 64.2% ± 1.3%, significantly higher than that of toremifene treatment control cells (3.8% ± 0.2%,P < 0.01). Furthermore, the effect of toremifene on BCRP protein is similar in BCRP mRNA. Toremifene obviously increased the mitoxantrone fluorescence intensity and decreased the efflux activity by 47.3% (P < 0.05) in MCF-7/promoter-BCRP cells when compared with the untreated control, whereas intracellular accumulation of mitoxantrone obviously decreased and the efflux activity increased by 61.5% were observed in combination with 17β-estradiol when compared with toremifene treatment alone. The results therefore suggested that toremifene reversed mitoxantrone resistance in MCF-7/Promoter-BCRP cells. However, in MCF-7/CMV-BCRP, MDA-MB-231/Promoter-BCRP and MDA-MB-231/CMV-BCRP cells, toremifene or in combination with 17β-estradiol did not affect intracellular mitoxantrone uptake.

CONCLUSIONTaken together, our findings indicate that expression of BCRP is downregulated by toremifene, via a novel transcriptional mechanism which might be involved in the ERE of BCRP promoter through ER-mediated to inactivate the transcription of BCRP gene.

ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; genetics ; metabolism ; Antineoplastic Agents ; pharmacology ; Antineoplastic Agents, Hormonal ; administration & dosage ; pharmacology ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cytomegalovirus ; genetics ; Dose-Response Relationship, Drug ; Down-Regulation ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Estradiol ; pharmacology ; Estrogen Receptor alpha ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Mitoxantrone ; pharmacology ; Neoplasm Proteins ; genetics ; metabolism ; Plasmids ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Response Elements ; genetics ; Toremifene ; administration & dosage ; pharmacology

To establish a transgenic cell model based on anti-oxidative response element (ARE) and green fluorescence protein(GFP) reporter gene, the TK minimal promoter was amplified by PCR and cloned into pEGFP-N1 for constructing reporter vector pTK-GFP/Neo. Four synthetic oligonucleotide ARE motifs were annealed and purified and then were inserted into pTK-GFP/Neo one by one to construct the eukaryotic reporter vector p4ARE-TK-GFP/Neo. Two reconstruct eukaryotic reporter vectors were transfected into HepG2 cells mediated by lipofectamine. The positive clones were obtained by the screen of G418. The cell model was tested with PDTC and tBHQ, well known inducers of phase II enzymes, by determining GFP activity. The results showed that the expression level of GFP was significantly increased by PDTC and tBHQ, and a transgenic cell model based on ARE was established successfully.
Antioxidants ; metabolism ; Base Sequence ; Enhancer Elements, Genetic ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Hep G2 Cells ; Humans ; Molecular Sequence Data ; Response Elements ; genetics ; Thymidine Kinase ; biosynthesis ; genetics ; Transfection ; Transgenes