Induced Reporter Gene Activity Research Articles

Evaluation of the Biological Effects of Novel Tobacco Product Vapor Using Two- or Three-Dimensional Culture Systems of Human Bronchial Epithelia

Introduction: Because of the rising popularity of novel tobacco products, there has been an increasing interest in the biological effects of these products. This study was performed to investigate the biological effects of vapor from a novel tobacco vapor product (NTV) in comparison with cigarette smoke (CS) from a 3R4F reference cigarette by using two types of in vitro model. Materials and Methods: A two-dimensional (2D) culture system composed of a submerged monolayer of BEAS-2B cells was used to detect cellular oxidative stress responses. A three-dimensional (3D) culture system composed of a pseudostratified air–liquid interface culture of normal human bronchial epithelial cells was used to detect tissue inflammation. In both culture systems, exposures were conducted with an aqueous extract (AqE) of NTV vapor or CS. Results: In the 2D culture system, exposure to CS AqE induced glutathione oxidization and antioxidant response element reporter gene activation. NTV vapor AqE also induced reporter gene activity, but the concentration required for activation was higher than that of CS AqE. In the 3D culture system, exposure to CS AqE increased the secretion of various inflammatory mediators. In contrast, the effect of NTV vapor AqE on tissue inflammation was limited, and only interferon-γ showed a slight increase in expression. Conclusions: Overall, these results demonstrate that NTV vapor has fewer effects on cellular oxidative stress and tissue inflammatory responses as compared with CS in in vitro models using human cells.

Oleocanthal Modulates Estradiol-Induced Gene Expression Involving Estrogen Receptor α.

Oleocanthal is a bioactive compound from olive oil. It has attracted considerable attention as it is anti-inflammatory, antiproliferative, and has been shown to possess neuroprotective properties in vitro and in vivo. Delineated from its polyphenolic structure, the aim of this study was to characterize oleocanthal towards estrogenic properties. This might contribute to partly explain the beneficial effects described for the Mediterranean diet. Estrogenic properties of oleocanthal were assessed by different methods: a) stimulation of reporter gene activity in MVLN or RNDA cells either expressing estrogen receptor α or β, b) stimulation of luciferase reporter gene activity in U2OS osteosarcoma cells expressing estrogen receptor α or β, and c) elucidation of the impact on estradiol-induced gene expression in U2OS cells transduced with both estrogen receptors. Depending on the cell line origin, oleocanthal inhibited luciferase activity (MVLN, U2OS-estrogen receptor β) or weakly induced reporter gene activity at 10 µM in U2OS-estrogen receptor α cells. However, oleocanthal inhibited stimulation of luciferase activity by estradiol from both estrogen receptors. Oleocanthal, if given alone, did not stimulate gene expression in U2OS cells, but it significantly modulated the response of estradiol. Oleocanthal enhanced the effect of estradiol on the regulation of those genes, which are believed to be regulated through heterodimeric estrogen receptors. As the estrogenic response pattern of oleocanthal is rather unique, we compared the results obtained with oleacein. Oleocanthal binds to both estrogen receptors inducing estradiol-agonistic or antiagonistic effects depending on the cell line. Regarding regulation of gene expression in U2OS-estrogen receptor α/β cells, oleocanthal and oleacein enhanced estradiol-mediated regulation of heterodimer-regulated genes.

Enteric bacterial metabolites propionic and butyric acid modulate gene expression, including CREB-dependent catecholaminergic neurotransmission, in PC12 cells--possible relevance to autism spectrum disorders.

Alterations in gut microbiome composition have an emerging role in health and disease including brain function and behavior. Short chain fatty acids (SCFA) like propionic (PPA), and butyric acid (BA), which are present in diet and are fermentation products of many gastrointestinal bacteria, are showing increasing importance in host health, but also may be environmental contributors in neurodevelopmental disorders including autism spectrum disorders (ASD). Further to this we have shown SCFA administration to rodents over a variety of routes (intracerebroventricular, subcutaneous, intraperitoneal) or developmental time periods can elicit behavioral, electrophysiological, neuropathological and biochemical effects consistent with findings in ASD patients. SCFA are capable of altering host gene expression, partly due to their histone deacetylase inhibitor activity. We have previously shown BA can regulate tyrosine hydroxylase (TH) mRNA levels in a PC12 cell model. Since monoamine concentration is known to be elevated in the brain and blood of ASD patients and in many ASD animal models, we hypothesized that SCFA may directly influence brain monoaminergic pathways. When PC12 cells were transiently transfected with plasmids having a luciferase reporter gene under the control of the TH promoter, PPA was found to induce reporter gene activity over a wide concentration range. CREB transcription factor(s) was necessary for the transcriptional activation of TH gene by PPA. At lower concentrations PPA also caused accumulation of TH mRNA and protein, indicative of increased cell capacity to produce catecholamines. PPA and BA induced broad alterations in gene expression including neurotransmitter systems, neuronal cell adhesion molecules, inflammation, oxidative stress, lipid metabolism and mitochondrial function, all of which have been implicated in ASD. In conclusion, our data are consistent with a molecular mechanism through which gut related environmental signals such as increased levels of SCFA's can epigenetically modulate cell function further supporting their role as environmental contributors to ASD.

Caffeine and rolipram affect Smad signalling and TGF-β1 stimulated CTGF and transgelin expression in lung epithelial cells.

Caffeine administration is an important part of the therapeutic treatment of bronchopulmonary dysplasia (BPD) in preterm infants. However, caffeine mediated effects on airway remodelling are still undefined. The TGF-β/Smad signalling pathway is one of the key pathways involved in airway remodelling. Connective tissue growth factor (CTGF), a downstream mediator of TGF-β, and transgelin, a binding and stabilising protein of the cytoskeleton, are both regulated by TGF-β1 and play an important role in airway remodelling. Both have also been implicated in the pathogenesis of BPD. The aim of the present study was to clarify whether caffeine, an unspecific phosphodiesterase (PDE) inhibitor, and rolipram, a prototypical PDE-4 selective inhibitor, were both able to affect TGF-β1-induced Smad signalling and CTGF/transgelin expression in lung epithelial cells. Furthermore, the effect of transgelin knock-down on Smad signalling was studied. The pharmacological effect of caffeine and rolipram on Smad signalling was investigated by means of a luciferase assay via transfection of a TGF-β1-inducible reporter plasmid in A549 cells. The regulation of CTGF and transgelin expression by caffeine and rolipram were studied by promoter analysis, real-time PCR and Western blot. Endogenous transgelin expression was down-regulated by lentiviral transduction mediating transgelin-specific shRNA expression. The addition of caffeine and rolipram inhibited TGF-β1 induced reporter gene activity in a concentration-related manner. They also antagonized the TGF-β1 induced up-regulation of CTGF and transgelin on the promoter-, the mRNA-, and the protein-level. Functional analysis showed that transgelin silencing reduced TGF-β1 induced Smad-signalling and CTGF induction in lung epithelial cells. The present study highlights possible new molecular mechanisms of caffeine and rolipram including an inhibition of Smad signalling and of TGF-β1 regulated genes involved in airway remodelling. An understanding of these mechanisms might help to explain the protective effects of caffeine in prevention of BPD and suggests rolipram to be a potent replacement for caffeine.

Regulation of CDKN2B expression by interaction of Arnt with Miz-1 - a basis for functional integration between the HIF and Myc gene regulatory pathways

BackgroundHypoxia- and Myc-dependent transcriptional regulatory pathways are frequently deregulated in cancer cells. These pathways converge in many cellular responses, but the underlying molecular mechanisms are unclear.MethodsThe ability of Miz-1 and Arnt to interact was identified in a yeast two-hybrid screen. The mode of interaction and the functional consequences of complex formation were analyzed by diverse molecular biology methods, in vitro. Statistical analyses were performed by Student’s t-test and ANOVA.ResultsIn the present study we demonstrate that the aryl hydrocarbon receptor nuclear translocator (Arnt), which is central in hypoxia-induced signaling, forms a complex with Miz-1, an important transcriptional regulator in Myc-mediated transcriptional repression. Overexpression of Arnt induced reporter gene activity driven by the proximal promoter of the cyclin-dependent kinase inhibitor 2B gene (CDKN2B), which is an established target for the Myc/Miz-1 complex. In contrast, mutated forms of Arnt, that were unable to interact with Miz-1, had reduced capability to activate transcription. Moreover, repression of Arnt reduced endogenous CDKN2B expression, and chromatin immunoprecipitation demonstrated that Arnt interacts with the CDKN2B promoter. The transcriptional activity of Arnt was counteracted by Myc, but not by a mutated variant of Myc that is unable to interact with Miz-1, suggesting mutually exclusive interaction of Arnt and Myc with Miz-1. Our results also establish CDKN2B as a hypoxia regulated gene, as endogenous CDKN2B mRNA and protein levels were reduced by hypoxic treatment of U2OS cells.ConclusionsOur data reveal a novel mode of regulation by protein-protein interaction that directly ties together, at the transcriptional level, the Myc- and hypoxia-dependent signaling pathways and expands our understanding of the roles of hypoxia and cell cycle alterations during tumorigenesis.

Aromatase activity induction in human adipose fibroblasts by retinoic acids via retinoic acid receptor α

Estrogen synthesis in adipose tissue is associated with the development of breast cancer. Tumors are preferentially found in breast quadrants with strongest expression of the cytochrome P450 aromatase (encoded by the gene CYP19A1). Several promoters regulated by various hormonal factors drive aromatase expression in human breast adipose fibroblasts (BAFs). As adipose tissue is a major source of retinoids, in this study, we investigated their role in the regulation of aromatase expression. The retinoids all-trans-retinoic acid (at-RA) and 9-cis-RA induce aromatase activity in human BAFs. In BAFs, at-RA induces aromatase gene expression via promoter I.4. In 3T3-L1 cells, both retinoids specifically drive luciferase reporter gene expression under the control of aromatase promoter I.4, whereas other promoters active in human adipose tissue are insensitive. Activation by retinoids depends on a 467 bp fragment (-256/+211) of promoter I.4 containing four putative retinoic acid response elements (RAREs). Site-directed mutagenesis revealed that only RARE2 (+91/+105) mediates the retinoid-dependent induction of reporter gene activity. In 3T3-L1 preadipocytes and human BAFs, RA receptor α (RARα (RARA)) expression is predominant, whereas RARβ (RARB) or RARγ (RARG) expression is low. Electrophoretic mobility shift assays with nuclear extracts obtained from human BAFs and 3T3-L1 cells identified a specific RARE2-binding complex. Retinoids enhanced complex formation, whereas pre-incubation with anti-RARα antibodies prohibited the binding of RARα to RARE2. Chromatin immunoprecipitation showed RA-dependent binding of RARα to the RARE2-containing promoter region in vivo. Furthermore, we provide evidence that RARE2 is also necessary for the basal activation of promoter I.4 in these cells. Taken together, these findings indicate a novel retinoid-dependent mechanism of aromatase activity induction in adipose tissue.

Synthesis and evaluation of small libraries of triazolylmethoxy chalcones, flavanones and 2-aminopyrimidines as inhibitors of mycobacterial FAS-II and PknG

A synthetic strategy to access small libraries of triazolylmethoxy chalcones 4{1–20}, triazolylmethoxy flavanones 5{1–10} and triazolylmethoxy aminopyrimidines 6{1–17} from a common substrate 4-propargyloxy-2-hydroxy acetophenone using a set of different reactions has been developed. The chalcones and flavanones were screened against mycobacterial FAS-II pathway using a recombinant mycobacterial strain, against which the most potent compound showed ∼88% inhibition in bacterial growth and substantially induction of reporter gene activity at 100μM concentration. The triazolylmethoxy aminopyrimdines were screened against PknG of Mycobaceterium tuberculosis displaying moderate to good activity (23–53% inhibition at 100μM), comparable to the action of a standard inhibitor.

RORγ directly regulates the circadian expression of clock genes and downstream targets in vivo

In this study, we demonstrate that the lack of retinoic acid-related orphan receptor (ROR) γ or α expression in mice significantly reduced the peak expression level of Cry1, Bmal1, E4bp4, Rev-Erbα and Per2 in an ROR isotype- and tissue-selective manner without affecting the phase of their rhythmic expression. Analysis of RORγ/RORα double knockout mice indicated that in certain tissues RORγ and RORα exhibited a certain degree of redundancy in regulating clock gene expression. Reporter gene analysis showed that RORγ was able to induce reporter gene activity through the RORE-containing regulatory regions of Cry1, Bmal1, Rev-Erbα and E4bp4. Co-expression of Rev-Erbα or addition of a novel ROR antagonist repressed this activation. ChIP-Seq and ChIP–Quantitative real-time polymerase chain reaction (QPCR) analysis demonstrated that in vivo RORγ regulate these genes directly and in a Zeitgeber time (ZT)-dependent manner through these ROREs. This transcriptional activation by RORs was associated with changes in histone acetylation and chromatin accessibility. The rhythmic expression of RORγ1 by clock proteins may lead to the rhythmic expression of RORγ1 target genes. The presence of RORγ binding sites and its down-regulation in RORγ−/− liver suggest that the rhythmic expression of Avpr1a depends on RORγ consistent with the concept that RORγ1 provides a link between the clock machinery and its regulation of metabolic genes.

Functional analysis of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) mutations associated with common variable immunodeficiency

Some, but not all, TACI mutations found in CVID impair TACI function and may potentially contribute to B cell dysfunction in heterozygotes via haploinsufficiency.

Constituents fromCistus salvifolius(Cistaceae) Activate Peroxisome Proliferator-Activated Receptor-γbut Not -δand Stimulate Glucose Uptake by Adipocytes

A number of medicinal/culinary herbs have been reported to improve glucose metabolism and to yield hypoglycemic effects in patients with diabetes. Since stimulation of insulin sensitivity appears to be a potential mechanism, peroxisome proliferator-activated receptor (PPAR) γ is a likely target molecule for small lipophilic compounds derived from endogenous metabolism and nutrition. Functionally, PPAR γ integrates the control of energy, lipid, and glucose homeostasis. In addition, PPAR δ activity is involved in energy expenditure. Therefore the aim of this study was to investigate whether PPAR γ and PPAR δ as well as the stimulation of glucose uptake is activated by botanical products. CISTUS SALVIFOLIUS (Cistaceae) has been identified as a candidate botanical in a preliminary screening of extracts from medicinal plants of Greek flora. In a bioguided approach, crude extracts, fractions and in the end purified compounds have been evaluated for PPAR γ and PPAR δ specific activities using cell-based transactivation assays. Glucose uptake was measured by nonradioactive 2-[ N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) uptake. Concerning PPAR γ several extracts induced reporter gene activity, and clear dose-response patterns (0.1-100 µg/mL) could be established in the case of the cyclohexane and dichloromethane extracts. Isolation of individual compounds from the cyclohexane extract revealed that at least 6 out of 7 compounds isolated were active with TRANS-cinnamic acid showing a clear dose-response pattern. In contrast, they were found to be inactive on PPAR δ. The same compounds, however, were also active in stimulating glucose uptake into 3T3-L1 adipocytes. In summary, the bioguided fractionation of CISTUS SALVIFOLIUS yields PPAR γ stimulating metabolites with differing chemical natures. In conclusion, PPAR γ represents a candidate molecule for the mediation of improvement of glucose metabolism by botanical/nutritional products.

Endothelin-1 enhances the expression of the androgen receptor via activation of the c-myc pathway in prostate cancer cells.

Increasing evidence suggests that androgen independent prostate cancer (PC) maintains a functional androgen receptor (AR) pathway despite the low levels of circulating androgen following androgen withdrawal, the molecular mechanisms of which are not well defined yet. To address this question, we investigated the effects of endothelin-1 (ET-1) on AR expression. Western analysis and RT-PCR revealed that in the presence of ET-1, levels of AR significantly increased in a time- and dose-dependent manner in LNCaP cells. Pretreatments with inhibitors of Src and phosphoinositide kinase 3 (PI-3K) suppressed ET-1-induced AR expression. As ET-1 was reported to cause a transient increase in c-myc mRNA levels, we examined the involvement of c-myc in ET-1-mediated AR expression. Transient transfection of c-myc siRNA neutralized ET-1-induced AR expression, suggesting that AR induction by ET-1 is c-myc dependent. AR can regulate the transcription of its own gene via a mechanism in which c-myc plays a crucial role. Therefore, we assessed if ET-1-induced-c-myc leads to the enhancement of AR transcription. Reporter gene assays using the previously identified AR gene enhancer containing a c-myc binding site were conducted in LNCaP cells. We found that ET-1 induced reporter gene activity from the construct containing the wild-type but not mutant c-myc binding site. Chromatin immunoprecipitation assays confirmed that ET-1 increased interaction between c-myc and c-myc binding sites in AR enhancer, suggesting that ET-1-induced AR transcription occurs via c-myc-mediated AR transcription. Together, these data support the notion that ET-1, via Src/PI-3K signaling, augments c-myc expression leading to enhanced AR expression in PC.

A chimeric fusion of the hASH1 and EZH2 promoters mediates high and specific reporter and suicide gene expression and cytotoxicity in small cell lung cancer cells

Transcriptionally targeted gene therapy is a promising experimental modality for treatment of systemic malignancies such as small cell lung cancer (SCLC). We have identified the human achaete-scute homolog 1 (hASH1) and enhancer of zeste homolog 2 (EZH2) genes as highly upregulated in SCLC compared to a panel of representative normal tissues. Here, we evaluate the use of regulatory regions from the hASH1- and EZH2-promoter regions alone and in combination for suicide gene therapy of SCLC. Two hASH1-promoter regions comprising 0.3 and 0.7 kb immediately upstream of (and including) the transcription start site were tested. Both constructs induced reporter gene activity (up to sevenfold SV40-promoter activity) in all tested classic (hASH1 positive) SCLC and in two hASH1-negative SCLC cell lines, whereas gene activity was low or absent (<4% of SV40 activity) in one hASH1-negative SCLC and in all control cell lines tested. To evaluate its therapeutic potential, the 0.7 kb hASH1 proximal-promoter region was evaluated for cytotoxicity in a suicide gene assay. The construct induced SCLC cytotoxicity at levels equivalent to those observed with the SV40 promoter, while control cells remained unaffected by the treatment. Analogously, a 1.1 kb EZH2-promoter region was evaluated by reporter and suicide gene assays. The EZH2 promoter potently induced reporter gene activity in SCLC (up to 25-fold of SV40 activity) while moderate reporter activity (up to 12% of SV40 activity), was detected in the control cells. However, in the suicide gene assay both control and SCLC cells demonstrated sensitivity indicating lack of promoter specificity. Finally, we fused the 0.7 kb hASH1 promoter to the EZH2 promoter generating a chimeric hASH1EZH2 regulatory construct. The chimeric promoter demonstrated increased activity in SCLC cells compared to the hASH1 promoter alone while retaining specificity in control cells. The hASH1EZH2 promoter thus constitutes a promising transcriptional regulator for SCLC gene therapy.

The cell‐cycle promoter cdc2aAt from Arabidopsis thaliana is induced in the lateral roots of the actinorhizal tree Allocasuarina verticillata during the early stages of the symbiotic interaction with Frankia

The symbiosis between the actinorhizal tree Allocasuarina verticillata and the actinomycete Frankia leads to the formation of root nodules inside which bacteria fix atmospheric nitrogen. Actinorhizal nodule organogenesis starts with the induction of cell divisions in the root cortex and in the pericycle cells opposite protoxylem poles near Frankia‐infected root hairs. To study the ability of Frankia to induce progression through the cell cycle, we monitored the expression of the β‐glucuronidase (gus) gene driven by the promoter from cdc2aAt, an Arabidopsis cyclin‐dependent kinase gene that displays competence for cell division, during plant growth and nodule ontogenesis. In non‐symbiotic tissues, the gus gene was mainly expressed in primary and secondary meristems of roots and shoots. Auxins and cytokinins were found to induce reporter gene activity in the root system of whole plants, showing that the promoter cdc2aAt displayed the same regulation by hormones in Allocasuarina as that reported in Arabidopsis. In transgenic nodules, gus expression was found to be restricted to the phellogen. During the early stages of the interaction between Frankia and the plant root system, cdc2aAt was strongly induced in the lateral roots surrounded by hyphae of the actinomycete. Histochemical analysis of β‐glucuronidase activity revealed that cells from the pericycle opposite protoxylem poles were very deeply stained. These data indicate that upon Frankia infection, cells from the lateral roots, and notably pericycle cells that can give rise to a nodule or a root primordium, prepare to re‐enter the cell cycle.

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.

Functional analysis of the polymorphism − 211C>T in the regulatory region of the human ABCC3 gene

The multidrug resistance protein 3 (MRP3/gene symbol: ABCC3) is an ATP-dependent efflux pump mediating the transport of endogenous glucuronides and conjugated drug metabolites across cell membranes. In humans the hepatic expression of ABCC3 mRNA seems to be influenced by the polymorphism C>T at the position − 211 in the promoter of the ABCC3 gene. The aim of this study was to investigate the possible mechanisms of how this SNP influences the MRP3 expression. Promoter luciferase reporter gene constructs representing 0.5, 1.1, 4.4, and 8.1 kb upstream of the translational start site were cloned with cytosine or thymine at position − 211 and transfected into HepG2, Caco-2, and LS174T cells. Reporter gene activity was dependent on the length of the promoter sequence but interestingly not on the nucleotide at position − 211. Cotransfection with FTF cDNA (Fetoprotein Transcription Factor) binding to elements near the − 211 polymorphism increased promoter activity in all constructs except the 0.5 kb fragment also independently of the − 211 SNP. Taken together, we did not find any influence of the − 211C>T ABCC3 promoter polymorphism on either the basal or the FTF induced reporter gene activity. Whether other tissue specific mechanisms reveal an impact of this SNP on the in vivo regulation of MRP3 remains to be determined.

Peroxisome Proliferator-Activated Receptor-γ (PPARγ) activity from Cistus salvifolius – Cistaceae

A number of medicinal/culinary herbs have been reported to improve glucose metabolism and to yield hypoglycemic effects in patients with diabetes. Since stimulation of insulin sensitivity appears to be a potential mechanism, PPARγ is a likely target molecule for small lipophilic compounds derived from metabolism and nutrition. As a member of the nuclear receptor superfamily, functionally PPARγ integrates the control of energy, lipid, and glucose homeostasis. The aim of this study was to investigate the activation of PPARγ by botanical products. Cistus salvifolius (Cistaceae), a wide spread evergreen shrub was selected as an example. It has revealed significant antihyperglycaemic activity in a preliminary screening of extracts from medicinal plants of Greek flora. The whole plant was extracted using organic solvents and water (cHex, CH2Cl2, MeOH, MeOH-H2O, and H2O). The MeOH-extract was re-extracted with EtOAc and ButOH to afford four additional sub-extracts. PPARγ specific activities were assessed in HEC-1B endometrial adenocarcinoma cells following co-transfection with a PPARγ expression and a PPRE containing J3-tk-Luc reporter plasmid. Extracts (doses 10 and 100µg/ml) were tested in comparison to a solvent control and a positive control (10µM troglitazone). Several extracts induced reporter gene activity with cHex-extract being most potent. For those fractions a clear dose response pattern (0,1–100µg/ml) could be established. For this reason cHex-extract was fractionated using VLC and tested again. PPARγ activity thereby resided in several fractions. In summary, extraction and fractionation of Cistus salvifolius yields PPARγ stimulating activities with differing chemical nature. In conclusion, PPARγ represents a candidate molecule for the mediation of improvement of glucose metabolism by botanical/nutritional products.

Analysis of the 5-lipoxygenase promoter and characterization of a vitamin D receptor binding site

1,25-Dihydroxyvitamin D 3 (1,25(OH) 2D 3) and transforming growth factor beta (TGFβ) potently induce 5-lipoxygenase (5-LO) in myeloid cells. We analyzed vitamin D receptor (VDR) binding to putative vitamin D response elements within the 5-LO promoter and analyzed its function by reporter gene analysis. Binding of VDR and retinoid X receptor to the promoter region was shown in DNase I footprinting, electrophoretic mobility shift and chromatin immunoprecipitation assays. However, the identified VDR binding region did not mediate induction of reporter gene activity by 1,25(OH) 2D 3/TGFβ, neither in the 5-LO promoter context nor with the thymidine kinase (tk) promoter. Insertion of the rat atrial natriuretic factor VDRE in reporter plasmids containing the 5-LO promoter diminished induction by 1,25(OH) 2D 3/TGFβ as compared with the tk promoter. Similarly, low inductions were obtained when cells were transiently or stably transfected with constructs containing various 5-LO promoter regions. Concerning basal promoter activity, we identified a positive regulatory region (−779 to −229), which includes the VDR binding region, in 5-LO-positive MonoMac6 cells. In summary, the VDR/RXR complex binds to putative VDREs in the 5-LO promoter, but other sequences outside the 5-LO promoter seem to be responsible or additionally required for the prominent induction of 5-LO mRNA expression by 1,25(OH) 2D 3 and TGFβ.

Pseudomonas fluorescens Tn5-B20 mutant RA92 responds to carbon limitation in soil

Tn5-B20 (lacZ as reporter gene) transcriptional fusion mutants of Pseudomonas fluorescens R2f Rpr were screened for their response to carbon limitation. Reporter gene expression was specifically induced by this stress factor in one mutant, designated RA92, and to a lower extent by phosphorus and nitrogen starvation. Expression was not detected during exponential growth. Reporter gene activity in RA92 cells increased shortly after exposure to carbon limitation and reached a plateau level within 48 h. Survival of RA92 in mineral medium without carbon source, as determined by CFU enumerations, was impaired compared to that of the parent strain, R2f Rpr. Survival and induction of reporter gene activity in exponentially grown as well as in carbon-starved RA92 cells were measured after introduction into Ede loamy sand soil. Both the exponentially grown and carbon-starved RA92 cells responded to carbon limitation in Ede loamy sand soil by induction of reporter gene expression. RA92 cells introduced into soil developed enhanced resistance to different stress factors in a fashion similar to that of the parent strain R2f Rpr. RA92 CFU numbers dropped progressively in Ede loamy sand soil portions at a rate greater than that of its parent strain. The addition of glucose to Ede loamy sand inoculated with RA92 cells gave no reduction in the fitness of RA92. Also in soil microcosms planted with wheat, the fitness of RA92 was enhanced in the layer 0–3 mm from the roots as compared to that in the deeper soil layer (3–8 mm from the roots).

Identification of the cAMP-Responsive Enhancer of the Murine ABCA1 Gene

To determine the mechanism by which expression of the murine ABCA1 gene is highly induced by cAMP analogues. ABCA1 mRNA turnover cannot account for its induction by cAMP. Thus cAMP induction of ABCA1 mRNA occurs at a transcriptional level. Shotgun cloning DNA fragments from the murine ABCA1 locus identified a strong cAMP responsive enhancer located in the first intron, which led to 25- to 100-fold cAMP-mediated induction of reporter gene activity. Deletions and mutations of this enhancer led to the identification a cAMP-responsive element (CRE) that was essential for the cAMP induction. Furthermore, the capacity of this CRE site to mediate the cAMP induction required the presence of a STAT3/4 element located 81 bp away. A dominant-negative CREB expression vector inhibited the cAMP induction of ABCA1, demonstrating that CREB was required for cAMP induction of ABCA1 expression in RAW264.7 cells. Phospho-CREB1 controls the cAMP-mediated induction of murine ABCA1 gene expression through a CRE site acting in cooperation with a nearby STAT element. This CRE site is not conserved in the human ABCA1 gene, explaining why human ABCA1 is not strongly stimulated by cAMP analogs.

The androgen receptor pathway is by‐passed in prostate cancer cells generated after prolonged treatment with bicalutamide

Experimental work in various prostate cancer models revealed that the androgen receptor is frequently upregulated and implicated in tumor progression. However, little attention has been paid to the androgen receptor-signaling pathway in the development of therapy resistance in patients who receive chronic treatment with a non-steroidal anti-androgen. We have generated a novel subline, LNCaP-Bic, after prolonged treatment with androgen and bicalutamide in vitro. Proliferation of LNCaP-Bic cells in the absence or presence of androgen, tocopherol succinate, and/or bicalutamide was assessed by cell counting. Androgen receptor expression was determined by Western blot. Luciferase activity was measured in cells transfected with an androgen-responsive reporter. In basal conditions, proliferation of LNCaP-Bic cells increased more than threefold over that of control LNCaP cells. Neither synthetic androgen R1881 nor bicalutamide showed any effect on LNCaP-Bic growth in vitro. Androgen receptor expression did not differ between the cell subline generated in the presence of bicalutamide and parental LNCaP cells. The ability of R1881 to induce reporter gene activity in LNCaP-Bic cells was reduced by 56%. Tocopherol succinate caused inhibition of proliferation only in the parental cell line although the androgen receptor and prostate-specific antigen were down regulated by the vitamin E derivative in both parental LNCaP and LNCaP-Bic cells. Androgen receptor-mediated signal transaction is not enhanced in cells selected in the presence of bicalutamide. Our data may suggest that a more differentiated approach in targeting the androgen receptor is needed in prostate cancers that become resistant to classic endocrine treatment.