Reporter Gene Activity In Cells Research Articles

Induction of cyclooxygenase-2 by ginsenoside Rd via activation of CCAAT-enhancer binding proteins and cyclic AMP response binding protein

Panax ginseng is a widely used herbal medicine in East Asia and is reported to have a variety of pharmacological effects against cardiovascular diseases and cancers. Here we show a unique effect of ginsenoside Rd (Rd) on cyclooxygenase-2 (COX-2) expression in RAW264.7 macrophages. Rd (100 μg/ml), but not other ginsenosides induced COX-2 and increased prostaglandin E 2 production. Gel shift and Western blot analyses using nuclear fractions revealed that Rd increased both the DNA binding of and the nuclear levels of CCAAT/enhancer binding protein (C/EBP)α/β and cyclic AMP response element binding protein (CREB), but not of p65, in RAW264.7 cells. Moreover, Rd increased the luciferase reporter gene activity in cells transfected with a 574-bp mouse COX-2 promoter construct. Site-specific mutation analyses confirmed that Rd-mediated transcriptional activation of COX-2 gene was regulated by C/EBP and CREB. These results provide evidence that Rd activated C/EBP and CREB, and that the activation of C/EBP and CREB appears to be essential for induction of COX-2 in RAW264.7 cells.

Activation of cAMP-Dependent Signaling Pathway Induces Mouse Organic Anion Transporting Polypeptide 2 Expression

Rodent Oatp2 is a hepatic uptake transporter for such compounds as cardiac glycosides. In the present study, we found that fasting resulted in a 2-fold induction of Oatp2 expression in liver of mice. Because the cAMP-protein kinase A (PKA) signaling pathway is activated during fasting, the role of this pathway in Oatp2 induction during fasting was examined. In Hepa-1c1c7 cells, adenylyl cyclase activator forskolin as well as two cellular membrane-permeable cAMP analogs, dibutyryl cAMP and 8-bromo-cAMP, induced Oatp2 mRNA expression in a time- and dose-dependent manner. These three chemicals induced reporter gene activity in cells transfected with a luciferase reporter gene construct containing a 7.6-kilobase (kb) 5'-flanking region of mouse Oatp2. Transient transfection of cells with 5'-deletion constructs derived from the 7.6-kb Oatp2 promoter reporter gene construct, as well as 7.6-kb constructs in which a consensus cAMP response element (CRE) half-site CGTCA (-1808/-1804 bp) was mutated or deleted, confirms that this CRE site was required for the induction of luciferase activity by forskolin. Luciferase activity driven by the Oatp2 promoter containing this CRE site was induced in cells cotransfected with a plasmid encoding the protein kinase A catalytic subunit. Cotransfection of cells with a plasmid encoding the dominant-negative CRE binding protein (CREB) completely abolished the inducibility of the reporter gene activity by forskolin. In conclusion, induction of Oatp2 expression in liver of fasted mice may be caused by activation of the cAMP-dependent signaling pathway, with the CRE site (-1808/-1804) and CREB being the cis- and trans-acting factors mediating the induction, respectively.

Molecular Mechanism of Inhibitory Aryl Hydrocarbon Receptor—Estrogen Receptor/Sp1 Cross Talk in Breast Cancer Cells

The trifunctional carbamoylphosphate synthetase/aspartate transcarbamyltransferase/dihydroorotase (CAD) gene is hormone responsive in MCF-7 and ZR-75 breast cancer cells, and this response is inhibited by the aryl hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Estrogen-dependent induction of CAD mRNA and reporter gene activity in cells transfected with constructs (pCAD) containing hormone-responsive GC-rich CAD promoter inserts involves estrogen receptor alpha (ERalpha)/Sp1 interactions with these proximal GC-rich motifs. TCDD also inhibits hormone-induced transactivation in MCF-7 and ZR-75 cells transfected with pCAD constructs. The mechanism of inhibitory AhR-ERalpha/Sp1 cross talk was further investigated by chromatin immunoprecipitation (ChIP), and the results show that ERalpha/Sp1 and the AhR are constitutively bound to the CAD gene promoter and only minor changes are observed after treatment with 17beta-estradiol, TCDD, or their combination. However, examination of interactions of these transcription factors by fluorescence resonance energy transfer shows that E2 enhances ERalpha-Sp1 interactions, whereas cotreatment with TCDD significantly decreases interaction of these proteins. These results suggest that inhibitory AhR-ERalpha/Sp1 cross talk is due, in part, to enhanced association of AhR and ERalpha (also determined by fluorescence resonance energy transfer), which coordinately dissociates ER and Sp1 and decreases ERalpha/Sp1-mediated transactivation, whereas remaining associated with the CAD promoter. This represents a novel interaction between two ligand activated receptors where one receptor inhibits activation of the second receptor.

Bifunctional effect of resveratrol on the expression of ErbB2 in human breast cancer cell

This study evaluated the effect of resveratrol on the expression of ErbB2 in a human breast cancer cell line, MCF-7. Low concentrations of resveratrol (1–10μM) reduced the basal expression level of ErbB2 in MCF-7 cells cultured in an estrogen-free medium. When cells were cultured in a medium containing estrogen, resveratrol increased the ErbB2 protein levels in a dose-dependent manner. Resveratrol increased the luciferase reporter gene activity in cells transfected with the −756bp flanking region of the human erbB2 gene. Resveratrol increased the nuclear levels of AP-2α and AP-2γ, and the induction of the luciferase reporter gene by resveratrol was inhibited by a mutation of two AP-2 binding sites in the promoter region of the human erbB2 gene. Blocking the ERK, p38 kinase or PI3-kinase activity had no effect on the resveratrol-inducible transactivation of the erbB2 gene and the ErbB2 expression level.

Egr-1 is activated by 17beta-estradiol in MCF-7 cells by mitogen-activated protein kinase-dependent phosphorylation of ELK-1.

Early growth response-1 (Egr-1) is an immediate-early gene induced by E2 in the rodent uterus and breast cancer cells. E2 induces Egr-1 mRNA and protein levels in MCF-7 human breast cancer cells and reporter gene activity in cells transfected with pEgr-1A, a construct containing the -600 to +12 region of the Egr-1 promoter linked to the firefly luciferase gene. Deletion analysis of the Egr-1 promoter identified a minimal E2-responsive region of the promoter that contained serum response element (SRE)3 (-376 to -350) which bound Elk-1 and serum response factor (SRF) in gel mobility shift assays. Hormone-responsiveness of Egr-1 in MCF-7 cells was specifically inhibited by PD98059, a mitogen-activated protein kinase kinase inhibitor, but not by LY294002, an inhibitor of phosphatidylinositol-3-kinase (PI3-K). These results contrasted with hormone-dependent activation of the SRE in the c-fos promoter, which was inhibited by both PD98059 and LY294002. Differences in activation of the SREs in Egr-1 and c-fos were related to promoter sequence, which defines the affinities of Elk-1 and SRF to their respective binding sites. Thus, Egr-1, like c-fos, is activated through non-genomic (extranuclear) pathways of estrogen action in breast cancer cells.

Activation of CCAAT/enhancer-binding protein beta by 2'-amino-3'-methoxyflavone (PD98059) leads to the induction of glutathione S-transferase A2.

The induction of glutathione S-transferases by flavonoids is associated with cancer chemopreventive effects. We reported that 2'-amino-3'-methoxyflavone (PD98059), an MKK1 inhibitor, induces glutathione S-transferase A2 (rGSTA2). This report comparatively examines the role of CCAAT/enhancer-binding protein (C/EBP) and Nrf2 in the induction of rGSTA2 by PD98059. We first assessed whether the MKK1/ERK1/2 pathway regulated rGSTA2 induction. Northern and western blot analyses showed that PD98059 at the concentrations effective for the inhibition of MKK1 increased the rGSTA2 protein and mRNA levels in H4IIE cells. PD98059 also induced rGSTA2 in cells stably transfected with dominant-negative mutant of MKK1(-), which provided evidence that the inhibition of MKK1/ERK1/2 by PD98059 was not responsible for rGSTA2 induction. Gel shift assay and immunoblot analysis of subcellular fractions revealed that PD98059 caused nuclear translocation of C/EBP beta and increased C/EBP DNA binding, which was super-shifted with anti-C/EBP beta antibody. Nrf2 was not activated by PD98059. PD98059 increased the luciferase reporter gene activity in cells transfected with the C/EBP-containing -1.65 kb flanking region of the rGSTA2 gene. Deletion of the C/EBP-binding site or over-expression of dominant-negative mutant of C/EBP abolished the reporter gene activity. Flavone, a backbone structure of PD98059, also induced nuclear translocation of C/EBP beta and C/EBP-mediated rGSTA2 gene induction. Inhibition of phosphatidylinositol 3-kinase abolished C/EBP beta-mediated rGSTA2 induction by PD98059. These results provide evidence that PD98059 and flavone induce nuclear translocation of C/EBP beta and activate the C/EBP-binding site in the rGSTA2 gene, which constitutes the distinct pathway for the enzyme induction irrespective of the inhibition of MKK1/ERK activity.

Increased Protein Stability as a Mechanism That Enhances Nrf2-mediated Transcriptional Activation of the Antioxidant Response Element: DEGRADATION OF Nrf2 BY THE 26 S PROTEASOME

Nrf2 (NF-E2-related factor 2) is a central transcription factor involved in the transcriptional activation of many genes encoding phase II drug-metabolizing enzymes via the antioxidant response element. Nrf2 has previously been found to undergo nuclear translocation by a phosphorylation-dependent mechanism mediated by protein kinase C in HepG2 cells treated with tert-butylhydroquinone, beta-naphthoflavone, or 12-O-tetradecanoylphorbol-13-acetate. In the present report, we have found that the levels of Nrf2 were increased in cells treated with tert-butylhydroquinone or beta-naphthoflavone by a post-transcriptional mechanism. Treatment of HepG2 cells with cycloheximide resulted in the loss of Nrf2 within 30 min. By contrast, treatment with the proteasome inhibitors (lactacystin or MG-132) caused an accumulation of Nrf2 as well as an induction of reporter gene activity in cells transfected with the GSTA2 antioxidant response element-chloramphenicol acetyl transferase construct. Similarly, the protein phosphatase inhibitor okadaic acid also caused an accumulation of Nrf2, whereas the reverse effects were observed with PD 98059 and U 0126, two compounds that block the activation of the MAPK/ERK signaling cascade. These data suggest that Nrf2 is degraded by the ubiquitin-dependent pathway and that phosphorylation of Nrf2 leads to an increase in its stability and subsequent transactivation activity.

Identification of a cis-element regulating transcriptional activity in response to fluid shear stress in bovine aortic endothelial cells.

Fluid shear stress exerts numerous effects on gene expression in endothelial cells. To investigate the regulatory mechanisms involved, we designed oligonucleotides composed of a 20-bp core containing the classical shear stress response element (SSRE+) or of a 20-bp core, in which base pairs flanking the SSRE were mutated (SSRE-). Hexamers of the oligonucleotides were cloned in front of reporter genes, transfected in bovine aortic endothelial cells (BAECs), and subjected to either a continuous low shear stress (3 dynes cm(-2)) or to a stepwise increase in shear stress from 3 dynes cm(-2) to 12 dynes cm(-2) (16/4 h). Shear stress increased reporter gene activity in cells transfected with pSSRE-, but not with pSSRE+. Cyclic strain (6%, 1 Hz, 4 h) did not significantly affect reporter gene activity. In gel retardation assays, more proteins bound to SSRE- than to SSRE+ in response to high shear stress. In competition experiments, a cAMP response element-binding (CREB) protein-specific oligonucleotide suppressed protein binding to the SSRE-; however, an antibody directed against CREB itself did not affect protein binding to the SSRE+. Our results indicate that the sequence ACC(G)/(T)AGACCAG represents a novel SSRE and that a protein that binds a CREB-specific oligonucleotide is part of the complex implicated in response to shear stress.

Involvement of a post-transcriptional mechanism in the inhibition of CYP1A1 expression by resveratrol in breast cancer cells

Resveratrol decreases basal and induced CYP1A1 mRNA/protein levels in both in vitro and in vivo models, and some studies suggest that resveratrol acts as an aryl hydrocarbon receptor (AhR) antagonist. Treatment of T47D or MCF-7 cells with 10 μM resveratrol inhibited induction of CYP1A1 mRNA and CYP1A1-dependent activity after treatment with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), as previously reported. In contrast, resveratrol did not inhibit TCDD-induced reporter gene activity in cells transfected with an Ah-responsive construct containing a human CYP1A1 gene promoter insert, whereas 3′-methoxy-4′-nitroflavone, a “pure” AhR antagonist, inhibited this response. Resveratrol induced transformation of the rat cytosolic AhR and, after treatment of T47D and MCF-7 cells with resveratrol, a transformed nuclear AhR complex was observed. In contrast to 3′-methoxy-4′-nitroflavone, resveratrol did not block TCDD-induced AhR transformation in vitro or nuclear uptake of the AhR complex in breast cancer cells. Thus, the action of resveratrol on the AhR was consistent with that of an AhR agonist; however, resveratrol did not exhibit functional AhR agonist or antagonist activities in breast cancer cells. Actinomycin D chase experiments in T47D cells showed that resveratrol and dehydroepiandrosterone both increased the rate of CYP1A1 mRNA degradation, whereas resveratrol did not affect CYP1A1-dependent activity in cells pretreated with TCDD for 18 hr. These data suggest that resveratrol inhibits CYP1A1 via an AhR-independent post-transcriptional pathway.

3',4'-dimethoxyflavone as an aryl hydrocarbon receptor antagonist in human breast cancer cells.

Treatment of MCF-7 and T47D human breast cancer cells with 3', 4'-dimethoxyflavone (3',4'-DMF) alone did not induce CYP1A1-dependent ethoxyresorufin O:-deethylase (EROD) activity or reporter gene activity in cells transfected with an aryl hydrocarbon (Ah)-responsive construct (pRNH11c). In contrast, 1 nM 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) induced up to a 50- to 80-fold increase in EROD and reporter gene activity in MCF-7 and T47D cells. In cells cotreated with 1 nM TCDD plus 0.1-10 microM 3',4'-DMF, there was a concentration-dependent decrease in the TCDD-induced responses, with 100% inhibition observed at the 10 microM concentration. Gel mobility shift assays using rat liver cytosol and breast cancer cell nuclear extracts showed that 3',4'-DMF alone did not transform the AhR to its nuclear binding form, but inhibited TCDD-induced AhR transformation in rat liver cytosol and blocked TCDD-induced formation of the nuclear AhR complex in MCF-7 and T47D cells. TCDD also inhibited estrogen-induced transactivation in MCF-7 cells, and this response was also blocked by 3',4'-DMF, confirming the AhR antagonist activity of this compound in breast cancer cells.

Transcriptional activation of cathepsin D gene expression by growth factors.

Insulin-like growth factor-I (IGF-I), transforming growth factor alpha (TGFalpha) and epidermal growth factor (EGF) induced cathepsin D gene expression and reporter gene activity in MCF-7 human breast cancer cells transiently transfected with a construct (pCD1) containing a -2576 to -124 cathepsin D gene promoter insert. In contrast, IGF-I, but not TGFalpha or EGF, induced reporter gene activity in cells cotransfected with wild-type estrogen receptor (ER) expression plasmid and a construct (pCD2) containing estrogen-responsive downstream elements from -208 to -101. Promoter deletion and mutational analysis experiments identified four GC-rich sites and an imperfect palindromic estrogen responsive element required for IGF-I activation of the ER (ligand-independent). Subsequent studies with the mitogen-activated protein kinase (MAPK) inhibitor, PD98059, and a serine(118(-ER mutant confirmed the role of the MAPK pathway for IGF-I activation of the ER in MCF-7 cells. Thus, growth factor activation of ER can mediate transactivation vs ER/Sp1 binding to GC-rich sites and represents a novel pathway for ligand-independent ER action. The divergent pathways for IGF-I and TGFalpha/EGF activation of the ER observed in MCF-7 cells contrast with previous data indicating that pathways for growth factor activation of the ER are dependent on the gene and/or gene promoter and on cell context.

Nonisotopic Assays for Reporter Gene Activity

This unit describes two nonisotopic systems for reporter gene activity in cells transfected with the firefly luciferase.

Estrogen and aryl hydrocarbon responsiveness of ECC-1 endometrial cancer cells

ECC-1 endometrial cancer cells express estrogen receptor α (ER α), and 17β-estradiol (E2) induces cell proliferation, cathepsin D mRNA levels, and reporter gene activity in cells transiently transfected with constructs derived from the human cathepsin D and creatine kinase B (pCD and pCKB, respectively) gene promoters. The comparative antiestrogenic activity of aryl hydrocarbon receptor (AhR) agonists and ER α antagonists were also determined in these endometrial cancer cells. A functional AhR was expressed in ECC-1 cells and AhR agonists including 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) inhibited E2-induced cell proliferation and transactivation. This was comparable to inhibitory AhR-ER crosstalk in breast cancer cell lines. The pure ER antagonist ICI 182,780 also exhibited antiestrogenic activity in ECC-1 cells; however, the results obtained for 4′-hydroxytamoxifen were response-specific. 4′-Hydroxytamoxifen alone did not induce ECC-1 cell proliferation but completely inhibited E2-induced cell proliferation. 4′-Hydroxytamoxifen primarily exhibited ER antagonist activities in transactivation assays and this contrasted to the predominant ER agonist responses observed in other endometrial cancer cell lines. The unique cellular context of ECC-1 cells was confirmed using pCKB and constructs expressing wild-type ER or ER variants expressing activation function 1 (AF1) or AF2 (ER-AF1 and ER-AF2, respectively). 4′-Hydroxytamoxifen did not induce reporter gene activity in cells cotransfected with pCKB and ER-AF1 or ER-AF2; however, in cotreatment studies (4′-hydroxytamoxifen plus E2), 4′-hydroxytamoxifen inhibited E2-induced transcriptional activation by ER-AF1 or ER-AF2. Thus, the primarily antiestrogenic activity observed for 4′-hydroxytamoxifen in ECC-1 cells may be related to the inability to activate gene expression through AF1-dependent pathways.

Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of diindolylmethane.

Phytochemicals such as indole-3-carbinol (I3C) and sulforaphane are components of cruciferous vegetables which exhibit antitumorigenic activity associated with altered carcinogen metabolism and detoxification. Diindolylmethane (DIM) is a major acid-catalyzed metabolite of I3C formed in the gut that binds to the aryl hydrocarbon receptor (AhR) and treatment of MCF-7 human breast cancer cells with 10-50 microM DIM resulted in rapid formation of the nuclear AhR complex and induction of CYP1A1 gene expression was observed at concentrations >50 microM. Previous studies have demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a high affinity AhR ligand, inhibits 17beta-estradiol (E2)-induced responses in MCF-7 cells and growth of E2-dependent 7,12-dimethylbenzanthracene (DMBA)-induced mammary tumors in female Sprague-Dawley rats. Results of this study show that like TCDD, DIM inhibits E2-induced proliferation of MCF-7 cells, reporter gene activity in cells transiently transfected with an E2-responsive plasmid (containing a frog vitellogenin A2 gene promoter insert) and down-regulates the nuclear estrogen receptor. Moreover, DIM (5 mg/kg every other day) also inhibits DMBA-induced mammary tumor growth in Sprague-Dawley rats and this was not accompanied by induction of hepatic CYP1A1-dependent activity. Thus, DIM represents a new class of relatively non-toxic AhR-based antiestrogens that inhibit E2-dependent tumor growth in rodents and current studies are focused on development of analogs for clinical treatment of breast cancer.

Estrogen-induced c-fos protooncogene expression in MCF-7 human breast cancer cells: role of estrogen receptor Sp1 complex formation.

17Beta-estradiol (E2) induces c-fos protooncogene expression in MCF-7 human breast cancer cells, and previous studies in HeLa cells identified an imperfect palindromic estrogen-responsive element (-1212 to -1200) that was required for trans-activation. In contrast, the estrogen-responsive element was not required for E2 responsiveness in MCF-7 cells, and using a series of constructs containing wild-type (pF1) and mutant 5'-flanking sequences (-1220 to -1155) from the c-fos protooncogene promoter in transient transfection assays, it was shown that a GC-rich motif (5'-GGGGCGTGG) containing an imperfect Sp1-binding site was required for hormone-induced activity. This sequence also bound Sp1 protein in gel mobility shift assays, and coincubation with the estrogen receptor (ER) enhanced Sp1-DNA binding. E2 and 4'-hydroxytamoxifen, but not ICI 164,384, induced reporter gene activity in cells transiently transfected with pF1. E2 induced reporter gene activity in MDA-MB-231 breast cancer cells transiently cotransfected with pF1 and wild-type ER or variant ER in which the DNA-binding domain was deleted (HE11); plasmids expressing N-terminal or C-terminal domains of the ER containing activator function-1 or -2, respectively, were inactive in these assays. In contrast, only wild-type ER mediated 4'-hydroxytamoxifen-induced activity. Induction of c-fos protooncogene expression by E2 in MCF-7 cells is dependent on the formation of a transcriptionally active ER/Sp1 complex that binds to a GC-rich enhancer element.

Estrogen- and antiestrogen-responsiveness of HEC1A endometrial adenocarcinoma cells in culture

HEC1A endometrial cancer cells express the wild-type form of the estrogen receptor (ER) and 17 β-estradiol (E2) induces proliferation of these cells. In contrast, tamoxifen only causes a minimal increase (<20%) in cell proliferation. In HEC1A cells transiently transfected with the C3-Luc plasmid derived from the complement C3 gene, both E2 and tamoxifen exhibited ER agonist activity and tamoxifen was also a partial antagonist for this response. The relative ER agonist/antagonist activities of E2, tamoxifen and ICI 182,780 were also investigated in HEC1A1 cells transiently transfected with two E2-responsive plasmids, pCATHD-CAT and pCKB-CAT which contain 5′-promoter inserts from the cathepsin D and creatine kinase B genes, respectively. The results showed that E2 and tamoxifen induced reporter gene activity in cells transiently transfected with both constructs. ICI 182,780 exhibited partial ER agonist activity only in cells transiently transfected with pCKB-CAT and antagonized E2-induced reporter gene activity using both the CKB- and CATHD-derived constructs. These results demonstrate that HEC1A endometrial cancer cells are E2-responsive and represent a useful cell culture model for understanding hormone/antihormone-induced endometrial cell responses.

Nonisotopic Assays for Reporter Gene Activity

This unit describes two nonisotopic systems for reporter gene activity in cells transfected with the firefly luciferase expression plasmid or the beta-galactosidase expression plasmid. Both of these chemiluminescent assays have the advantages of high sensitivity and broad linear range. In the chemiluminescent detection procedures given in this unit, both luciferase activity and beta-galactosidase activity can be measured with either a luminometer or a scintillation counter.

An inducible nuclear factor binds to a growth hormone-regulated gene.

Transcription of the serine protease inhibitor (Spi) 2.1 gene, a member of the serine protease inhibitor family, is induced by growth hormone (GH) in rat liver. To further study the mechanism involved in this process, we have isolated and characterized the Spi 2.1 gene from a rat genomic library. Examination of the 5'-flanking region of the Spi 2.1 gene from normal animals revealed the presence of a DNase I hypersensitive site within 500 base pairs of the transcriptional initiation site, which was not detectable in hypophysectomized animals. Portions of the 5'-flanking region of the Spi 2.1 gene were fused to a heterologous promoter and reporter gene and introduced into primary rat hepatocytes by lipofection. Spi 2.1 sequences from -275 to -54 gave a 2-3-fold induction of reporter gene activity in cells grown in the presence of GH, similar to the level of induction of the endogenous Spi 2.1 mRNA in isolated hepatocytes. Further definition of the essential sequences revealed that a segment from -147 to -102 could confer GH responsiveness when linked in tandem copies in front of a heterologous promoter. Using the gel shift assay, a nuclear factor(s) from normal rat liver was identified which could interact with this minimal response fragment. The importance of this activity to GH regulation was suggested by the fact that it was absent in hypophysectomized animals but reappeared by 1 h after treatment of such animals with GH. The appearance of this activity was not blocked by pretreatment of animals with an inhibitor of protein synthesis, suggesting a preexisting factor is modified by GH to yield an activity which interacts with the Spi 2.1 gene.