Dose-dependent Increase In Luciferase Activity Research Articles

Development and Evaluation of Olive Flounder cyp1a1-Luciferase Assay for Effective Detection of CYP1A-Inducing Contaminants in Coastal Sediments.

Flounders have been widely used as indicator species for monitoring the benthic environment of marine coastal regions owing to their habitat and feeding preferences in or on sandy sediments. Here, a single-step, sensitive, specific, and simple luciferase assay was developed, using the olive flounder cyp1a1 gene, for effective detection of CYP1A-inducing contaminants in coastal sediments. The developed cyp1a1-luciferase assay was highly sensitive to the widely used CYP1A inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo[a]pyrene (B[a]P), and 3,3',4,4',5-pentachlorobiphenyl (PCB 126). In the case of TCDD, significant dose-dependent increases in luciferase activity (0.3-300 ng/L) were detected. The assay was more sensitive to PCB 126 than to B[a]P. The assay also involved the highly sensitive expression of luciferase to extracted mixtures of PCBs and polycyclic aromatic hydrocarbons (PAHs) collected from coastal sediments. PCBs were more capable of cyp1a1 induction in the assay system at small doses than PAHs in environmental samples. Using the cyp1a1-luciferase assay along with water or sediment chemistry will certainly aid in diagnosing CYP1A-inducing contaminants in coastal environments.

SAT-419 Mechanisms Underlying Glucocorticoid-Induced Suppression of Pulsatile LH: Insights from Immortalized Kisspeptin Cells

Stress inhibits fertility at least in part by inhibiting gonadotropin secretion. Part of the stress response is an increase in circulating glucocorticoid concentrations, specifically corticosterone in mice. We have demonstrated that continuously elevated corticosterone concentrations can inhibit ovarian cyclicity and suppress luteinizing hormone (LH) pulse frequency in female mice (ICN 2018, 310). Since the gonadotropin releasing hormone (GnRH) and LH pulse generator is likely formed by the kisspeptin neuron population in the arcuate nucleus of the hypothalamus, this suppression by corticosterone may be mediated by kisspeptin cells. Our immunohistochemical data indicating that arcuate kisspeptin neurons contain glucocorticoid receptor (GR) in mice (ICN 2018, 312), provides evidence in support of this hypothesis. To investigate direct actions of glucocorticoids in kisspeptin neurons, we utilized an immortalized mouse arcuate kisspeptin neuron cell line (KTaR cells). RT-PCR data indicated the presence of mRNA for GR in KTaR cells. KTaR cells were subsequently transfected with a mouse mamillary tumor virus (MMTV)-luciferase plasmid, which contains a positive glucocorticoid response element, and treated with varying concentrations of a synthetic glucocorticoid, dexamethasone. The MMTV promoter exhibited a dose-dependent increase in luciferase activity, with doses in the μM to nM range producing a significant increase in promoter activity compared to vehicle treated samples (n = 3 independent experiments). Together, these data indicate that functional GR is present in KTaR cells and that this cell type could be valuable in understanding glucocorticoid effects on LH pulse dynamics. Arcuate kisspeptin cells in mice (and other mammals) also contain neurokinin B and dynorphin, and are termed KNDy cells. The KNDy hypothesis for GnRH/LH pulse generation posits that each pulse of GnRH is caused by a pulse of kisspeptin secretion and that each pulse of kisspeptin is turned on (stimulated) by neurokinin B (NKB; encoded by Tac2) and terminated (inhibited) by dynorphin (encoded by pDyn). To determine if these genes are regulated by glucocorticoids, we treated KTaR cells with 1μM dexamethasone for 24 hrs and quantified transcript abundance with qPCR. Dexamethasone treatment did not alter mRNA abundance of Kiss1 or pDyn themselves or the receptors for NKB or dynorphin (Tacr1, Tacr2, Tacr3, and Oprk1). However, dexamethasone treatment caused a significant decrease in Tac2 mRNA abundance compared to vehicle treatment (51.7 ± 10.0% reduced). These experiments demonstrate that KTaR cells have functional GR and that dexamethasone treatment suppresses NKB transcript abundance. These data raise the possibility that the inhibitory actions of glucocorticoids on LH pulse frequency that we recently demonstrated in mice are mediated by an inhibition of NKB, a factor implicated in GnRH pulse onset.

Cytotoxicity and genotoxicity of nanosilver in stable GADD45α promoter‐driven luciferase reporter HepG2 and A549 cells

The intense commercial application of silver nanoparticles (AgNPs) has been raising concerns about their potential adverse health effects to human. This study aimed to explore the potency of AgNPs to induce GADD45α gene, an important stress sensor, and its relationships with the cytotoxicity and genotoxicity elicited by AgNPs. Two established HepG2 and A549 cell lines containing the GADD45α promoter-driven luciferase reporter were treated with increasing concentrations of AgNPs for 48 hours. After the treatment, transcriptional activation of GADD45α indicated by luciferase activity, cell viability, cell cycle arrest, and levels of genotoxicity were determined. The uptake and intracellular localization of AgNPs, cellular Ag doses as well as Ag+ release were also detected. AgNPs could activate GADD45α gene at the transcriptional level as demonstrated by the dose-dependent increases in luciferase activity in both the reporter cells. The relative luciferase activity was greater than 12× the control level in HepG2-luciferase cells at the highest concentration tested where the cell viability decreased to 17.0% of the control. These results was generally in accordance with the positive responses in cytotoxicity, cell cycle arrest of Sub G1 and G2/M phase, Olive tail moment, micronuclei frequency, and the cellular Ag content. The cytotoxicity and genotoxicity of AgNPs seems to occur mainly via particles uptake and the subsequent liberation of ions inside the cells. And furthermore, the GADD45α promoter-driven luciferase reporter cells, especially the HepG2-luciferase cells, could provide a new and valuable tool for predicting nanomaterials genotoxicity in humans.

Extracellular ATP activates NFAT-dependent gene expression in neuronal PC12 cells via P2X receptors

BackgroundTreatment of neuronal PC12 cells with ATP induces depolarisation and increases intracellular calcium levels via purinergic receptors. In many cell types, sustained elevation of intracellular calcium levels cause changes in gene expression via activation of the transcription factor NFAT (nuclear factor of activated T cells). We have therefore characterised the signalling pathway by which ATP regulates NFAT-dependent gene expression in PC12 cells.ResultsThe activation of NFAT transcriptional activity by extracellular ATP was characterised with the help of reporter gene assays. Treatment of PC12 cells with ATP elicited a dose-dependent increase in luciferase activity (EC50 = 78 μM). UTP, 4-benzoylbenzoyl ATP and α,β-methylene ATP did not mimic the effect of ATP, which was abolished by treatment with the P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS). This pharmacological characterisation provides evidence for a critical role of ionotropic P2X receptors. Blockade of L-type voltage-dependent calcium channels by nifedipine reduced the response of NFAT to ATP, indicating that a depolarisation-mediated calcium influx was required for maximal NFAT activation. Inhibition of store-operated calcium entry by the pyrazole derivative BTP2 also diminished ATP-dependent NFAT activation. Furthermore, ATP-induced NFAT activation was associated with the activation of the mitogen-activated protein kinases ERK1/2. Finally, treatment with ATP increased the levels of the NFAT target transcripts, RCAN1-4 (regulator of calcineurin) and BDNF (brain derived neurotrophic factor).ConclusionThe present data show that ATP induces NFAT-dependent changes in gene expression in PC12 cells by acting on P2X receptors. Maximal NFAT activation depends on both depolarisation-induced calcium influx and store-operated calcium entry and requires the activity of the protein phosphatase calcineurin and the mitogen-activated protein kinase cascade.

Transcriptional Regulation of γ-Globin Gene Expression by KLF4

AbstractAbstract 645Kruppel-like factors (KLFs) are a family of Cys2His2 zinc-finger DNA binding proteins that regulate gene expression through CACCC/GC/GT box binding in various gene promoters. The CACCC element is also critical for developmental regulation of the human γ-globin and β-globin genes; therefore studies to identify transcription factors that bind the CACCC element to alter gene expression are desirable. By microarray-based gene profiling, we identified two Kruppel-like factors, KLF4 and KLF12 whose expression levels decreased simultaneously with γ-globin silencing during in vitro erythroid maturation. Subsequent reverse transcription quantitative PCR (RT-qPCR) analysis confirmed KLF4 and KLF12 mRNA levels decreased 56-fold and 16-fold respectively in erythroid progenitors from day 7 to day 28 with over 90% γ-globin gene silencing. The effects of known fetal hemoglobin inducers hemin (50μM) and sodium butyrate (2mM) on KLF factor expression was tested in K562 cells. Hemin and sodium butyrate increased KLF4 3-fold (p<0.05) and 13-fold (p<0.01) respectively while KLF12 was only induced by butyrate. Likewise, hemin treatment of KU812 leukemia cells, which actively express γ-globin and β-globin, produced a 7-fold increase in KLF4 (p<0.05) while KLF12 levels were not changed suggesting KLF4 may be directly involved in γ-globin gene regulation. To characterize its role further siRNA-mediated loss of function studies were performed in K562 cells. A 60% knockdown of KLF4 expression produced 40% attenuation of γ-globin transcription (p<0.05). To confirm this effect, rescue experiments were performed as follows: K562 cells were treated with 100nM siKLF4 alone or in combination with the pMT3-KLF4 expression vector (10 and 20μg) for 48 hrs. The 40% knockdown of γ-globin expression produced by siKLF4 was rescued to baseline levels after enforced pMT3-KLF4 expression (p<0.05). To establish whether KLF4 directly stimulates γ-globin promoter activity, we performed co-transfection of pMT3-KLF4 and the Gγ-promoter (-1500 to +36) cloned into the pGL4.17 Luc2/neo vector; a dose-dependent increase in luciferase activity (2- to 5-fold; p<0.001) was observed. Furthermore, enforced expression of pMT3-KLF4 augmented endogenous γ-globin expression 2-fold (p<0.01). Collectively, these studies suggest that KLF4 acts as a trans-activator of γ-globin gene transcription. To address the physiological relevance of these findings, studies were extended to human primary erythroid cells grown in a two-phase liquid culture system. At day 11 when γ-globin gene expression was maximal, siKLF4 treatment produced a 60% decrease in γ/β-globin mRNA levels (p<0.001). By contrast, enforced pMT3-KLF4 expression enhanced γ/β-globin 1.5-fold at day 11 and day 28 (after γ-globin silencing); HbF levels were induced 1.5-fold (p<0.05) which was demonstrated by enzyme-linked immunosorbent assay. To gain insights into the molecular mechanism of KLF4-mediated γ-globin regulation, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) were completed. Since CREB binding protein (CBP) is known to function as a co-activator for KLF1, 4 and 13, we also tested its role in γ-globin gene regulation. EMSA performed with K562 nuclear extract and a [γ-32P] labeled γ-CACC probe (-155 to -132 relative to the γ-globin cap site) produced three DNA-protein complexes; the addition of KLF4 or CBP antibody resulted in a marked decrease in intensity of all complexes suggesting these factors bind the γ-CACC element. ChIP assay demonstrated 10-fold and 20-fold chromatin enrichment with KLF4 and CBP antibody respectively (p<0.001) confirming in vivo binding at the γ-CACC region. Lastly, co-immunoprecipitation established protein-protein interaction between KLF4 and CBP in K562 cells. Future studies will investigate the role of CBP in KLF4-mediated γ-globin regulation which will provide molecular targets for fetal hemoglobin induction and treatment of sickle cell anemia and β-thalassemia. Disclosures:No relevant conflicts of interest to declare.

Glucocorticoid Responsiveness of the Porcine GnRH Receptor (GnRHR) Gene Is Conferred by an Element(s) Located Between -290/-270 bp of Proximal Promoter.

The binding of GnRH to its receptor results in the synthesis and secretion of the gonadotropins, as well as stimulation of the gene encoding its own receptor. Thus, the interaction between GnRH and GnRHR represents a central point for regulation of reproductive function. Glucocorticoids can alter reproduction by reducing GnRH responsiveness of gonadotropes within the anterior pituitary gland, potentially via transcriptional regulation of the GnRHR gene. In addition, transcription of the murine GnRHR gene is stimulated by glucocorticoids. To determine the effect of glucocorticoids on porcine GnRHR gene expression, we isolated 5118 bp of 5' flanking sequence for the porcine GnRHR gene and produced reporter constructs containing the GnRHR promoter fused to the cDNA encoding luciferase (-5118LUC). The gonadotrope-derived αT3-1 cell line was transiently transfected with -5118LUC for 12 h and treated with increasing concentrations of the glucocorticoid agonist, dexamethasone (1, 10, 100 and 1,000 nM) for an additional 12 h prior to harvest. A dose-dependent increase in luciferase activity was observed with maximal induction noted at 100 nM dexamethasone (2-fold over vehicle; P < 0.05). The dexamethasone induction of the -5118 promoter was blocked by the glucocorticoid antagonist, mifepristone (100 pM). To determine the location of the glucocorticoid response element(s) within the GnRHR promoter, we performed transient transfection assays with luciferase reporter constructs containing progressively less 5' flanking region for the porcine GnRHR gene. Dexamethasone-stimulated luciferase activity was maintained following reduction of the full length GnRHR promoter to 323 bp upstream of the translational start site. However, further deletion to 274 bp of proximal promoter eliminated glucocorticoid responsiveness, suggesting the presence of a glucocorticoid response element(s) within this region. Electrophoretic mobility shift assays using 32P-labeled oligomers spanning this region revealed increased binding of nuclear extracts from αT3-1 cells treated with 100 nM dexamethasone compared to vehicle treated cells to the -290/-270 bp oligonucleotide. Sequence analysis of this region revealed putative binding sites for PR, ER, GR, COUP-TF and GATA, as well as RXR α, β, and γ. Inhibition of p38 MAPK and ERK 1/2 pathways significantly decreased dexamethasone-induced promoter activity, indicating the involvement of p38 MAPK and ERK 1/2 pathways for glucocorticoid responsiveness of the promoter. Further, our Western blot data revealed that the JNK pathway is also involved in dexamethasone-stimulated promoter activity. In summary, glucocorticoid responsiveness of the porcine GnRHR gene is conferred by an element(s) located between 270 and 290 bp of proximal promoter that is activated by p38 MAPK, ERK 1/2, and JNK pathways. (platform)

Development of a cell‐based assay for screening of phosphodiesterase 10A (PDE10A) inhibitors using a stable recombinant HEK‐293 cell line expressing high levels of PDE10A

The cDNA encoding PDE10A (phosphodiesterase 10A) was cloned and a stable recombinant HEK-293 (human embryonic kidney-293) cell line expressing high levels of PDE10A was generated. Transient transfection of pCRE-Luc plasmid, harbouring the luciferase reporter gene under the control of CRE (cAMP-response element)-binding sequence, into the stable recombinant cell line, followed by treatment with PDE10 inhibitor, resulted in a dose-dependent increase in luciferase activity. This method provides a simple and sensitive cell-based assay for screening of PDE10 inhibitors for development of novel therapeutics for the treatment of neurological disorders.

Cloning, stable expression of human phosphodiesterase 7A and development of an assay for screening of PDE7 selective inhibitors

Phosphodiesterases (PDEs) constitute a superfamily of enzymes that plays an important role in signal transduction by catalysing the hydrolysis of cAMP and cGMP. cDNA encoding PDE7A1 subtype was cloned and a stable recombinant HEK 293 cell line expressing high levels of PDE7A1 was generated. Transient transfection of pCRE-Luc plasmid, harboring luciferase reporter gene into the stable recombinant cell line and subsequent treatment with PDE7 inhibitor, resulted in a dose-dependent increase in luciferase activity. This method provides a simple and sensitive cell-based assay for screening of PDE7 selective inhibitors for the treatment of T cell mediated diseases.

Modulation of the expression of the π class of glutathione S-transferase by Andrographis paniculata extracts and andrographolide

Andrographis paniculata (Ap) is a commonly used herb for traditional medicine in many Southeast Asian countries. In the present study, we investigated the effect of Ap on the expression of the pi class of glutathione S-transferase (GSTP) in rat primary hepatocytes. Hepatocytes were treated with 25 or 50 μg/mL of ethanol or ethyl acetate extracts of Ap (ApEE or ApEAE) or 10 or 20 μM andrographolide, which is the major active diterpene lactone of Ap, for 48 h. ApEE, ApEAE, and andrographolide dose-dependently induced GSTP protein and mRNA expression. In a GST activity assay, GST activity was significantly higher in cells treated with the maximum concentrations of ApEE, ApEAE, and andrographolide than in control cells ( P < 0.05). The pTA-2713 luciferase reporter construct containing rat GSTP enhancer 1 (GPE1) was transiently transfected into Clone 9 liver cells. Cells treated with ApEE, ApEAE, and andrographolide showed a dose-dependent increase in luciferase activity. GPE1 deletion abolished the induction efficiency of Ap. Also, the induction of GSTP expression by Ap was inhibited by wortmannin, which is an inhibitor of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. These results indicate that ApEE, ApEAE, and andrographolide induce GSTP expression. This induction is likely related to the PI3K/Akt pathway, and GPE1, an enhancer element in GSTP promoter, is essential for the induction.

Dexamethasone-Mediated Up-Regulation of HumanCYP2A6Involves the Glucocorticoid Receptor and Increased Binding of Hepatic Nuclear Factor 4α to the Proximal Promoter

Human cytochrome P450 2A6 (CYP2A6) metabolizes various clinically relevant compounds, including nicotine- and tobacco-specific procarcinogens; however, transcriptional regulation of this gene is poorly understood. We investigated the role of the glucocorticoid receptor (GR) in transcriptional regulation of CYP2A6. Dexamethasone (DEX) increased CYP2A6 mRNA and protein levels in human hepatocytes in primary culture. This effect was attenuated by the GR receptor antagonist mifepristone (RU486; 17beta-hydroxy-11beta-[4-dimethylamino phenyl]-17alpha-[1-propynyl]estra-4,9-dien-3-one), suggesting that induction of CYP2A6 by DEX was mediated by the GR. In gene reporter assays, DEX caused dose-dependent increases in luciferase activity that was also prevented by RU486 and progressive truncations of the CYP2A6 promoter delineated DEX-responsiveness to a -95 to +12 region containing an hepatic nuclear factor 4 (HNF4) alpha response element (HNF4-RE). Mutation of the HNF4-RE abrogated HNF4alpha- and DEX-mediated transactivation of CYP2A6. In addition, overexpression of HNF4alpha increased CYP2A6 transcriptional activity by 3-fold. DEX increased HNF4alpha mRNA levels by 4-fold; however, the amount of HNF4alpha nuclear protein was unaltered. Electrophoretic mobility shift, chromatin immunoprecipitation (ChIP), and streptavidin DNA binding assays revealed that DEX increased binding of HNF4alpha to the HNF4-RE and that an interaction of GR and HNF4alpha occurred at this site. Moreover, ChIP assays indicated that histone H4 acetylation of the CYP2A6 proximal promoter chromatin was increased by DEX that may allow for increased binding of HNF4alpha to the HNF4-RE in human hepatocytes. These findings indicate that increased expression of CYP2A6 by DEX is mediated by the GR via a nonconventional transcriptional mechanism involving interaction of HNF4alpha with an HNF4-RE rather than a glucocorticoid response element.

Loss of Huntingtin Function Complemented by Small Molecules Acting as Repressor Element 1/Neuron Restrictive Silencer Element Silencer Modulators

Increased levels of the repressor element 1/neuron restrictive silencer element (RE1/NRSE) silencing activity promoter, and a consequent reduction in the transcription of many RE1/NRSE-bearing neuronal genes, including brain-derived neurotrophic factor (BDNF), have been demonstrated in Huntington disease (HD) and represent one possible effector of its selective neuronal vulnerability. Restoring the expression levels of neuronal genes in diseased neurons therefore seems to be an attractive therapeutic approach. To this end, we have developed a cell-based reporter assay for monitoring RE1/NRSE silencing activity and validated it by genetically inactivating the RE1/NRSE or pharmacologically stimulating global transcription. In a pilot compound screen, we identified three closely related structural analogues that up-regulate reporter expression at low nanomolar concentrations, and follow-up studies have shown that they efficaciously increase endogenous BDNF levels in HD cells. Moreover, one of the compounds increases the viability of HD cells. Our findings suggest a new avenue for the development of drugs for HD and other neurodegenerative disorders based on the pharmacological up-regulation of the production of the neuronal survival factor BDNF and of other RE1/NRSE-regulated neuronal genes.

Short Communication: A Milk Trait-Associated Polymorphism in the Bovine Growth Hormone Receptor Gene Does Not Affect Receptor Signaling

Growth hormone (GH), also known as somatotropin, stimulates milk production in cows. At the tissue level, the action of GH is mediated by the GH receptor (GHR) and the receptor-activated intracellular signaling pathway involving Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5). A T/A nucleotide variation in exon 8 of the bovine GHR gene, resulting in a phenylalanine to tyrosine change in the transmembrane domain of the GHR protein, has been reported to be associated with a major effect on milk yield in cows. The objective of this study was to determine whether the 2 versions of GHR differ in mediating GH-induced STAT5 activation of gene expression. We created cDNA expression plasmids for the 2 versions of GHR and cotransfected each of them with a STAT5 expression plasmid and a luciferase reporter gene construct containing STAT5 binding sites into 2 different cell lines. Treatment of the transfected cells with various concentrations of GH triggered a dose-dependent increase in luciferase activity. However, the GH-induced luciferase activity was not different between the 2 GHR expression plasmids, indicating that the 2 GHR forms did not differ in mediating GH-induced STAT5 activation of gene expression. Thus, if the T/A polymorphism in exon 8 of the GHR gene has a causative effect on milk production, this effect is unlikely to be mediated by the JAK2-STAT5 pathway, the currently known major signaling pathway from the growth hormone receptor.

Primary mouse hepatocytes for systems biology approaches: a standardized in vitro system for modelling of signal transduction pathways

Complex cellular networks regulate regeneration, detoxification and differentiation of hepatocytes. By combining experimental data with mathematical modelling, systems biology holds great promises to elucidate the key regulatory mechanisms involved and predict targets for efficient intervention. For the generation of high-quality quantitative data suitable for mathematical modelling a standardised in vitro system is essential. Therefore the authors developed standard operating procedures for the preparation and cultivation of primary mouse hepatocytes. To reliably monitor the dynamic induction of signalling pathways, the authors established starvation conditions and evaluated the extent of starvation-associated stress by quantifying several metabolic functions of cultured primary hepatocytes, namely activities of glutathione-S-transferase, glutamine synthetase, CYP3A as well as secretion of lactate and urea into the culture medium. Establishment of constant metabolic activities after an initial decrease compared with freshly isolated hepatocytes showed that the cultured hepatocytes achieve a new equilibrium state that was not affected by our starving conditions. To verify the highly reproducible dynamic activation of signalling pathways in the in vitro system, the authors examined the JAK-STAT, SMAD, PI3 kinase, MAP kinase, NF-kappaB and Wnt/beta-catenin signalling pathways. For the induction of gp130, JAK1 and STAT3 phosphorylation IL6 was used, whereas TGFbeta was applied to activate the phosphorylation of SMAD1, SMAD2 and SMAD3. Both Akt/PKB and ERK1/2 phosphorylation were stimulated by the addition of hepatocyte growth factor. The time-dependent induction of a pool of signalling competent beta-catenin was monitored in response to the inhibition of GSK3beta. To analyse whether phosphorylation is actually leading to transcriptional responses, luciferase reporter gene constructs driven by multiple copies of TGFbeta-responsive motives were applied, demonstrating a dose-dependent increase in luciferase activity. Moreover, the induction of apoptosis by the TNF-like cytokine Fas ligand was studied in the in vitro system. Thus, the mouse hepatocyte in vitro system provides an important basis for the generation of high-quality quantitative data under standardised cell culture conditions that is essential to elucidate critical hepatocellular functions by the systems biology approach.

An assay for the determination of biologically active bone morphogenetic proteins using cells transfected with an inhibitor of differentiation promoter-luciferase construct

Bone morphogenetic proteins (BMPs) control cell fate by regulating gene expression, especially inhibitor of differentiation (Id) genes. This property has been exploited to create a highly sensitive assay for quantification of active BMP. Embryonic mouse cells (C3H10T1/2) were stably transfected with an expression construct (BRE-Luc) containing a BMP-responsive element fused to the firefly luciferase reporter gene. BRE results from a multimerization of distinct sequences elements from a mouse Id1 promoter [15]. The addition of BMP-2 (0.5–100 ng/ml) to the transfectants resulted in a dose-dependent increase in luciferase activity in the cell lysates. This new assay was 100-fold more sensitive than the classical alkaline phosphatase (ALP) activity assay (0.5–1 vs. 50–100 ng/ml, respectively) as well as much more rapid (24 h vs. 3–6 days, respectively, of BMP treatment). This new assay is specific to BMPs (BMP-2, BMP-4, and BMP7) as evidenced by its relative insensitivity to TGFβ1, bFGF, and VEGF. Because of its BMP specificity, this rapid, sensitive, nonradioactive, and easily performed assay could be used in monitoring the biological activity of BMP and, eventually, as a cell-based screening assay to identify and evaluate molecules that modulate BMP signaling in cells.

Functional Relevance of the Conserved DNA-binding Domain of STAT2

Several distinct type I interferon (IFN)-inducible STAT2-containing complexes have been identified. For the IFN-stimulated gene factor 3 (ISGF3), STAT1 and IRF-9 mediate IFN-stimulated response element (ISRE) binding, whereas STAT2 provides a potent transactivational domain. ISGF3-independent STAT2-containing complexes, specifically STAT2:1 and STAT2:3, bind a gamma-activated sequence (GAS)-like element, yet the contribution of each STAT to DNA binding is unknown. Moreover, the contribution of these ISGF3-independent STAT2-containing complexes to IFN-inducible responses is not defined. Accordingly, we generated mutant cDNAs, targeting the DNA-binding domain in STAT2. These cDNAs were introduced by transfection into U6A cells lacking STAT2, resulting in a panel of cell lines expressing mutant STAT2 proteins. Studies assessed the sensitivity of U6A cells reconstituted with intact STAT2 (U6A-2) and cells expressing mutant STAT2s (U6A-2E426A,E427A (EE-AA), U6A-2V453I, U6A-2V454I, U6A-2V454A, U6A-2V453I,V454I(VV-II), U6A-2N458A) to IFN-inducible responses. Our data reveal that none of the mutations in the STAT2 DNA-binding domain affected IFN-inducible ISGF3 activation, and only the VV-II mutation restricted antiviral and growth inhibitory responses to IFN. Indeed, U6A-2VV-II cells are refractory to these IFN-inducible biological activities and also exhibit impaired IFN-inducible GAS-driven transcriptional activation and subsequent gene expression. Chromatin immunoprecipitation assays revealed that the VV-II mutation in STAT2 does not abrogate, but reduces the DNA binding activity of STAT2:1 heterodimers. Taken together, these data suggest a role for the conserved DNA-binding domain of STAT2 specific to the activity of ISGF3-independent STAT2-containing complexes.

A rapid assay for glial cell line-derived neurotrophic factor and neurturin based on transfection of cells with tyrosine hydroxylase promoter-luciferase construct

Glial cell line-derived neurotrophic factor (GDNF), a potent survival and trophic factor for various neuronal cells, has been measured by assaying its bioactivity based on neurite outgrowth or cell proliferation. We describe herein a sensitive and simple non-radioactive quantitative bioassay for GDNF family proteins based on their ability to induce tyrosine hydroxylase (TH) gene expression. Human neuroblastoma SK-N-MC cells were stably transfected with expression constructs of c-ret and with a luciferase reporter gene driven by 2 kb of the rat TH gene promoter region. In the presence of GDNF, luciferase activity increased with 20 h of incubation. A dose-dependent increase in luciferase activity was observed in the presence of GDNF between 1 and 300 ng/ml. This assay was also applicable for the quantification of the bioactivity of neurturin, another member of the GDNF family showing an even more sensitive profile of dose dependency than GDNF. Typical culture media were applicable in this assay. This method can be easily applied to numerous samples of conditioned medium in a short time.

Regulation of rat hepatic hydroxysteroid sulfotransferase (SULT2-40/41) gene expression by glucocorticoids: evidence for a dual mechanism of transcriptional control.

Glucocorticoid-inducible hydroxysteroid sulfotransferase (SULT2-40/41) gene transcription was investigated in primary cultured rat hepatocytes transiently transfected with a series of SULT2-40/41 5'-flanking region-luciferase reporter constructs, with emphasis on examining the functional role of an inverted repeat-0 nuclear receptor motif (IR0). Treatment of transfected cultures with any of four glucocorticoids activated luciferase expression from a construct containing 1938 base pairs (bp) of the SULT2-40/41 gene 5'-flanking sequence, whereas deletion of bp -227 to -158 (containing the IR0 motif) largely abolished the effect. On closer analysis, treatment of hepatocyte cultures with either of the potent glucocorticoids dexamethasone [strong cytochrome P-450 3A (CYP3A) inducer] or triamcinolone acetonide (weak CYP3A inducer) produced dose-dependent increases in luciferase activity when hepatocytes were transiently transfected with a construct containing as little as 158 bp of 5'-flanking sequence or containing a mutated IR0 motif. The dexamethasone dose-dependent increase in luciferase activity continued through a dose of 10(-6) M when the transfected construct contained the IR0 motif, but was maximal at 10(-7) M when the transfected construct lacked the IR0 motif. In contrast, triamcinolone acetonide-induced luciferase activity was maximal at a dose of 10(-7) M, irrespective of the presence or absence of the IR0 motif. Coincubation of transfected hepatocytes with 10(-8) M dexamethasone and the antiglucocorticoid RU486 inhibited luciferase expression. Luciferase induction by the prototypical CYP3A inducer pregnenolone 16alpha-carbonitrile was restricted to constructs containing the IR0 motif. These data suggest that glucocorticoid-inducible SULT2-40/41 gene expression occurs through a dual mechanism, whose components possibly involve the glucocorticoid receptor and the pregnane X receptor.

Evidence for transcriptional activation of ERalpha by IL-1beta in breast cancer cells.

Estrogen is mitogenic in breast cancer where IL-1beta also fulfils a role. The aim of this study was to determine any relationship between IL-1beta and ERalpha in breast cancer. By RT-PCR, 26/77 tumours expressed IL-1beta, and 57/77 expressed ERalpha. Samples which were IL-1beta positive were categorised against those which expressed ERalpha. Of the 26 tumours which expressed IL-1beta, all were ERalpha positive. We next examined whether IL-1beta could directly activate ERalpha. MCF-7 cells stably transfected with a plasmid reporter (ERE-TK-LUC) were incubated with either 17beta-estradiol (E2, 10-9-10-13 M), IL-1beta (10 ng/ml), the pure antiestrogen ZM 182780 (10 nM) or combinations of these substances. Transcriptional activity was measured in cell lysates 48 h later. E2 caused a dose-dependent increase in luciferase activity. With IL-1beta, transcriptional activity was typically half of the E2 response. To determine the role of the IL-1 receptor, parallel cultures were incubated with IL-1 receptor antagonist. This reduced, but did not completely block the effect of IL-1beta, suggesting that IL-1beta was affecting transcriptional activity via another pathway. Confirmation that the effect was via ERalpha was verified using the pure antiestrogen, ZM 182370, which completely abrogated the effects of E2, when added alone or in combination with IL-1beta. These results provide compelling evidence for direct transcriptional activation of ERalpha by IL-1beta. Interactions of these factors may thus modulate hormonal activity in human breast tumours.

α,β-Unsaturated Aldehydes Increase GlutathioneS-Transferase mRNA and Protein: Correlation with Activation of the Antioxidant Response Element

A series of α,β-unsaturated aldehydes was evaluated to determine if these compounds could mediate inducible expression of glutathioneS-transferase (GST) through the 5′-flanking antioxidant response element (ARE). The ARE from rGST A1 was subcloned into a luciferase reporter construct and used to transiently transfect rat Clone 9 hepatoma cells. Transfected cells were treated with 4-hydroxy-trans-2-nonenal (4-HNE),trans-2-hexenal (t-2-HE), 2-propenal (acrolein, 2-PE), and ethacrynic acid (EA), a control compound also containing an α,β-unsaturated carbonyl moiety. Each compound was evaluated for cytotoxicity to construct dosing regimens in transfection studies. IC50values for growth inhibition were measured using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. IC50values in Clone 9 cells were: 4-HNE, 6.3 ± 0.7 μM;t-2-HE, 16.0 ± 0.7 μM; 2-PE, 2.2 ± 0.4 μM; and EA, 38.0 ± 1.6 μM. A dose-dependent increase in luciferase activity was observed in transfected cells with all four compounds tested, indicating that α,β-unsaturated aldehydes function as direct activators of the ARE. To determine whether or not the observed promoter activation led to increased transcriptional and translational induction of GST, cells were treated with the various compounds and assayed for increases in GST mRNA, protein, and enzyme activity. Studies in Clone 9 cells revealed increased steady-state message for GST A1 and A4, increased GST A4-4 protein by Western blotting, and increased GST activity toward 1-chloro-2,4-dinitrobenzene in response to treatment with all four compounds evaluated. Collectively, these studies demonstrate that EA and certain α,β-unsaturated aldehydes produced as a result of cellular membrane lipid peroxidation are activators of the ARE and efficient inducers of GST A1-1 and A4-4.

A new chemiluminescent assay for the rapid detection of thyroid stimulating antibodies in Graves' disease.

Thyroid stimulating antibodies (TSAb) are the cause of Graves' disease (GD). At present, these antibodies can only be measured using bioassays, which are generally time consuming and difficult to apply to whole serum. Here we describe a new chemiluminescent assay for the detection of TSAb in serum samples. A Chinese hamster ovary (CHO) cell line, stably transformed with a reporter plasmid containing the firefly luciferase gene under the transcriptional control of multiple cAMP responsive elements (CRE), was transfected with the human thyroid stimulating hormone (TSH) receptor. A clonal cell line, (NA-4), was obtained which showed a dose-dependent increase in luciferase activity in response to bovine and human TSH stimulation. NA-4 was subsequently used to study TSAb activity in 42 GD sera. Of the GD sera 25 (60%) produced an increase in luciferase activity of 1.4-9 fold that obtained with control sera. Results were compared with an established bioassay for TSAb, based on the direct measurement of intracellular cAMP, and there was a significant correlation between the two assays (r = 0.8; P < 0.0001). IgG from GD patients, but not controls, also increased luciferase activity in NA-4 cells using concentrations as low as 3 mg/l in selected samples. The present report describes a simple, sensitive and rapid assay for the detection of TSAb, with application both in the clinical analysis of GD patient sera and as a potential research tool for use in the isolation of TSAb monoclonal antibodies.