Activity In Reporter Gene Assays Research Articles

GnRH-II Antagonists Induce Apoptosis in Human Endometrial, Ovarian, and Breast Cancer Cells via Activation of Stress-Induced MAPKs p38 and JNK and Proapoptotic Protein Bax

Recently, we could show that gonadotropin-releasing hormone (GnRH)-II antagonists induce apoptosis in human endometrial, ovarian, and breast cancer cells in vitro and in vivo. In the present study, we have ascertained receptor binding and effects of GnRH-II antagonists on mitogenic signal transduction and on activation of proapoptotic protein Bax. The GnRH-II antagonists tested showed EC50 values for GnRH-I receptor binding in the range of 1 to 2 nmol/L. The GnRH-II agonist [D-Lys6]GnRH-II showed an EC50 value for GnRH-I receptor binding of approximately 1,000 nmol/L. Agonistic activity on GnRH-I receptor function with an EC50 of 13 nmol/L has been determined for [D-Lys6]GnRH-II. Antagonistic activities with EC50 values in the range of 1 nmol/L were determined for the GnRH-II antagonists. Treatment of human endometrial, ovarian, and breast cancer cells with GnRH-II antagonists resulted in time-dependent activation of stress-induced mitogen-activated protein kinases p38 and c-Jun NH2-terminal kinase. In addition, treatment with GnRH-II antagonists induced time-dependent activation of proapoptotic protein Bax. GnRH-II antagonists are not involved in activation of protein kinase B/Akt or extracellular signal-regulated kinase 1/2. The GnRH-II antagonists tested had similar binding affinities to the GnRH-I receptor comparable with that of GnRH-I antagonist Cetrorelix. Referring to the cyclic AMP response element reporter gene activation assay, the GnRH-II agonist [D-Lys6]GnRH-II has to be classified as an agonist at the GnRH-I receptor, whereas the GnRH-II antagonists tested are clear antagonists at the GnRH-I receptor. GnRH-II antagonists induce apoptotic cell death in human endometrial, ovarian, and breast cancer cells via activation of stress-induced mitogen-activated protein kinases p38 and c-Jun NH2-terminal kinase followed by activation of proapoptotic protein Bax.

Complementation of diverse HIV-1 Env defects through cooperative subunit interactions: a general property of the functional trimer.

BackgroundThe HIV-1 Env glycoprotein mediates virus entry by catalyzing direct fusion between the virion membrane and the target cell plasma membrane. Env is composed of two subunits: gp120, which binds to CD4 and the coreceptor, and gp41, which is triggered upon coreceptor binding to promote the membrane fusion reaction. Env on the surface of infected cells is a trimer consisting of three gp120/gp41 homo-dimeric protomers. An emerging question concerns cooperative interactions between the protomers in the trimer, and possible implications for Env function.ResultsWe extended studies on cooperative subunit interactions within the HIV-1 Env trimer, using analysis of functional complementation between coexpressed inactive variants harboring different functional deficiencies. In assays of Env-mediated cell fusion, complementation was observed between variants with a wide range of defects in both the gp120 and gp41 subunits. The former included gp120 subunits mutated in the CD4 binding site or incapable of coreceptor interaction due either to mismatched specificity or V3 loop mutation. Defective gp41 variants included point mutations at different residues within the fusion peptide or heptad repeat regions, as well as constructs with modifications or deletions of the membrane proximal tryptophan-rich region or the transmembrane domain. Complementation required the defective variants to be coexpressed in the same cell. The observed complementation activities were highly dependent on the assay system. The most robust activities were obtained with a vaccinia virus-based expression and reporter gene activation assay for cell fusion. In an alternative system involving Env expression from integrated provirus, complementation was detected in cell fusion assays, but not in virus particle entry assays.ConclusionOur results indicate that Env function does not require every subunit in the trimer to be competent for all essential activities. Through cross-talk between subunits, the functional determinants on one defective protomer can cooperatively interact to trigger the functional determinants on an adjacent protomer(s) harboring a different defect, leading to fusion. Cooperative subunit interaction is a general feature of the Env trimer, based on complementation activities observed for a highly diverse range of functional defects.

Lack of association of TIM3polymorphisms and allergic phenotypes

BackgroundT-cell immunoglobulin mucin-3 (TIM3) is a TH1-specific type 1 membrane protein that regulates TH1 proliferation and the development of immunological tolerance. TIM3 and its genetic variants have been suggested to play a role in regulating allergic diseases. Polymorphisms in the TIM3 promoter region have been reported to be associated with allergic phenotypes in several populations. The aims of this study were to examine whether genetic variation in the promoter region of TIM3 influenced transcription of the gene and risk for allergic phenotypes.MethodsWe performed 5' rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction. We screened for polymorphisms in the promoter region. Deletion analysis was used to localize the promoter region of TIM3. Genotyping was performed by TaqMan assays in three asthma/allergy population samples.ResultsWe found two regions with promoter activity in TIM3. One region was from -214 bp to +58 bp and the other from -1.6 kb to -914 bp relative to the transcription start site. None of the single nucleotide polymorphisms (SNPs) or haplotypes affected the transcriptional activity in reporter gene assays. No association between the SNPs and any phenotype was observed in the study cohorts.ConclusionOur findings indicate that SNPs and haplotypes in the TIM3 promoter region do not have a functional effect in vitro and are not associated with allergic diseases. These data suggest that polymorphisms in the TIM3 promoter region are unlikely to play an important role in susceptibility to allergic diseases.

Lineage-specific DNA methylation in T cells correlates with histone methylation and enhancer activity

DNA methylation participates in establishing and maintaining chromatin structures and regulates gene transcription during mammalian development and cellular differentiation. With few exceptions, research thus far has focused on gene promoters, and little is known about the extent, functional relevance, and regulation of cell type-specific DNA methylation at promoter-distal sites. Here, we present a comprehensive analysis of differential DNA methylation in human conventional CD4(+) T cells (Tconv) and CD4(+)CD25(+) regulatory T cells (Treg), cell types whose differentiation and function are known to be controlled by epigenetic mechanisms. Using a novel approach that is based on the separation of a genome into methylated and unmethylated fractions, we examined the extent of lineage-specific DNA methylation across whole gene loci. More than 100 differentially methylated regions (DMRs) were identified that are present mainly in cell type-specific genes (e.g., FOXP3, IL2RA, CTLA4, CD40LG, and IFNG) and show differential patterns of histone H3 lysine 4 methylation. Interestingly, the majority of DMRs were located at promoter-distal sites, and many of these areas harbor DNA methylation-dependent enhancer activity in reporter gene assays. Thus, our study provides a comprehensive, locus-wide analysis of lineage-specific methylation patterns in Treg and Tconv cells, links cell type-specific DNA methylation with histone methylation and regulatory function, and identifies a number of cell type-specific, CpG methylation-sensitive enhancers in immunologically relevant genes.

Liver X receptor is a regulator of orphan nuclear receptor NOR-1 gene transcription in adipocytes

The liver X receptors (LXRs) are ligand-activated nuclear transcription factors that have been shown to play major roles in lipid, glucose and cholesterol metabolism. Recently, members of the NR4A orphan nuclear receptor family have also been shown to regulate the expression of important genes in metabolically active tissues such as liver, adipose and skeletal muscle. Here, we investigated the role of LXRs to regulate the expression of the nuclear receptor NOR-1 (neuron-derived orphan receptor-1) in adipocytes. White and brown adipose tissues from wild-type, LXRalpha-/-- and LXRalpha:beta-deficient mice were collected from animals at room temperature or following cold exposure to measure NOR-1 mRNA. The expression of NOR-1 and its promoter activity in response to LXR ligands were determined in cultured primary brown adipocytes or mouse embryo fibroblasts derived from wild-type or LXRalpha-/- mice differentiated into adipocytes. In LXRalpha-/-- and LXRalpha:beta-deficient adipocytes, basal levels of NOR-1 were significantly reduced while retaining an equivalent proportional induction by beta-adrenergic agonists. This reduced basal expression of NOR-1 in adipose tissue from LXR-deficient mice is a cell-autonomous event as it was also preserved in adipocytes differentiated from mouse embryo fibroblasts derived from these mice. In cultured primary brown adipocytes or cell lines, the expression of NOR-1 increased in response to an LXR agonist. A DNA sequence element (DR-4) capable of binding LXRs was found at -997 bp of the NOR-1 promoter, which was shown to be functional by promoter reporter gene activity, gel shift and chromatin immunoprecipitation assays. These data describe a new role for LXR to regulate NOR-1 gene expression in adipocytes and demonstrate that these two nuclear receptors have an interdependent regulatory relationship, in addition to each being involved in the control of metabolic fuel usage.

An insulator with barrier-element activity promotes α-spectrin gene expression in erythroid cells

AbstractUnderstanding mechanisms controlling expression of the α-spectrin gene is important for understanding erythropoiesis, membrane biogenesis, and spectrin-linked hemolytic anemia. We showed previously that a minimal α-spectrin promoter directed low levels of expression only in early erythroid development, indicating elements outside the promoter are required for expression in adult erythrocytes. Addition of noncoding exon 1′ and intron 1′ conferred a 10-fold increase in activity in reporter gene assays. In this report, we used a transgenic mouse model to show that addition of exon 1′ and intron 1′ to the α-spectrin promoter conferred tissue-specific expression of a linked Aγ-globin gene in erythroid cells at all developmental stages. Expression was nearly position-independent, as 21 of 23 lines expressed the transgene, and γ-globin protein was present in 100% of erythrocytes, indicating uniform expression. Additional in vivo studies revealed that exon 1′ functions as an insulator with barrier-element activity. Chromatin immunoprecipitation assays demonstrated that this region was occupied by the upstream stimulatory factors 1/2 (USF1/USF2), similar to the well-characterized chicken HS4 insulator. These data identify the first barrier element described in an erythrocyte membrane protein gene and indicate that exon 1′ and intron 1′ are excellent candidate regions for mutations in patients with spectrin-linked hemolytic anemia.

A quantitative high-throughput screen for modulators of IL-6 signaling: a model for interrogating biological networks using chemical libraries

Small molecule modulators are critical for dissecting and understanding signaling pathways at the molecular level. Interleukin 6 (IL-6) is a cytokine that signals via the JAK-STAT pathway and is implicated in cancer and inflammation. To identify modulators of this pathway, we screened a chemical collection against an IL-6 responsive cell line stably expressing a beta-lactamase reporter gene fused to a sis-inducible element (SIE-bla cells). This assay was optimized for a 1536-well microplate format and screened against 11 693 small molecules using quantitative high-throughput screening (qHTS), a method that assays a chemical library at multiple concentrations to generate titration-response profiles for each compound. The qHTS recovered 564 actives with well-fit curves that clustered into 32 distinct chemical series of 13 activators and 19 inhibitors. A retrospective analysis of the qHTS data indicated that single concentration data at 1.5 and 7.7 microM scored 35 and 71% of qHTS actives, respectively, as inactive and were therefore false negatives. Following counter screens to identify fluorescent and non-selective series, we found four activator and one inhibitor series that modulated SIE-bla cells but did not show similar activity in reporter gene assays induced by EGF and hypoxia. Small molecules within these series will make useful tool compounds to investigate IL-6 signaling mediated by JAK-STAT activation.

Real-time Monitoring of Non-specific Toxicity Using a Saccharomyces cerevisiae Reporter System.

Baker's yeast, Saccharomyces cerevisiae, is the simplest and most well-known representative of eukaryotic cells and thus a convenient model organism for evaluating toxic effects in human cells and tissues. Yeast cell sensors are easy to maintain with short generation times, which makes the analytical method of assessing antifungal toxicity cheap and less-time consuming. In this work, the toxicity of test compounds was assessed in bioassays based on bioluminescence inhibition and on traditional growth inhibition on agar plates. The model organism in both tests was a modified S. cerevisiae sensor strain that produces light when provided with D-luciferin in an insect luciferase reporter gene activity assay. The bioluminescence assay showed toxic effects for yeast cell sensor of 5,6-benzo-flavone, rapamycin, nystatin and cycloheximide at concentrations of nM to μM. In addition, arsenic compounds, cadmium chloride, copper sulfate and lead acetate were shown to be potent non-specific inhibitors of the reporter organism described here. The results from a yeast agar diffusion assay correlated with the bioluminescence assay results.

The Epstein-Barr Virus LF2 Protein Inhibits Viral Replication

The switch from Epstein-Barr virus (EBV) latent infection to lytic replication is governed by two transcriptional regulators, Zta and Rta. We previously reported that the EBV protein encoded by the LF2 gene binds to Rta and can inhibit Rta activity in reporter gene assays. We now report that LF2 associates with Rta in the context of EBV-infected cells induced for lytic replication. LF2 inhibition of Rta occurs in both epithelial and B cells, and this downregulation is promoter specific: LF2 decreases Rta activation of the BALF2, BMLF1, and BMRF1 promoters by 60 to 90% but does not significantly decrease Rta activation of its own promoter (Rp). LF2 decreases Rta activation by at least two mechanisms: decreased DNA binding and interference with transcriptional activation by the Rta acidic activation domain. Coexpression of LF2 also specifically induces modification of Rta by the small ubiquitin-like modifiers SUMO2 and SUMO3. We further demonstrate that LF2 overexpression blocks lytic activation in EBV-infected cells induced with Rta or Zta. Our results demonstrate that LF2, a gene deleted from the EBV reference strain B95-8, encodes a potent inhibitor of EBV replication, and they suggest that future studies of EBV replication need to account for the potential effects of LF2 on Rta activity.

Protein Phosphatase-2A Is a Target of Epigallocatechin-3-Gallate and Modulates p53-Bak Apoptotic Pathway

(-)-Epigallocatechin-3-gallate (EGCG) is a well-known chemoprevention factor. Recent studies have revealed that EGCG triggers cancer cells undergoing apoptosis through p53-dependent pathway. How EGCG activates p53-dependent apoptosis is not fully understood. In the present study using JB6 cell as a model system, we have shown that EGCG can negatively regulate protein serine/threonine phosphatase-2A (PP-2A) to positively regulate p53-dependent apoptosis. First, EGCG at physiologic levels down-regulates PP-2A at the protein and enzyme activity levels. Second, EGCG induces apoptosis of JB6 cells, which is associated with hyperphosphorylation of p53 and up-regulation of the proapoptotic gene, Bak. DNA sequence analysis, gel mobility shifting, chromatin immunoprecipitation, and reporter gene activity assays revealed that p53 directly controls Bak in JB6 cells. Knockdown of p53 and Bak expression with RNAi substantially inhibits EGCG-induced apoptosis. Third, PP-2A directly interacts with p53 and dephosphorylates p53 at Ser-15 in vitro and in vivo. Fourth, overexpression of the catalytic subunit for PP-2A down-regulates p53 phosphorylation at Ser15, attenuates expression of the downstream proapoptotic gene, Bak, and antagonizes EGCG-induced apoptosis. Inhibition of PP-2A activity enhances p53 phosphorylation at Ser-15 and up-regulates Bak expression to promote EGCG-induced apoptosis. Finally, in the p53(-/-) H1299 and p53(+/+) H1080 cells, EGCG down-regulates PP-2A similarly but induces differential apoptosis. In summary, our results show that (a) PP-2A directly dephosphorylates p53 at Ser-15; (b) P53 directly controls Bak expression; and (c) EGCG negatively regulates PP-2A. Together, our results show that EGCG-mediated negative regulation of PP-2A is an important molecular event for the activation of p53-dependent apoptosis during its chemoprevention.

Characterization of the Inosine Triphosphatase (ITPA) Gene: Haplotype Structure, Haplotype-Phenotype Correlation and Promoter Function

Inosine triphosphatase (ITPA) cleaves phosphate residues from inosine triphosphate (ITP) and deoxy ITP (dITP), thereby recovering inosine monophosphate, which is a substrate for further purine nucleotide pathways. Deficient ITPA activity leads to intracellular accumulation of ITP/dITP and would, under thiopurine therapy, lead to accumulation of unusual thio-inosine metabolites (thio-ITP) with the potential for adverse metabolic effects. ITPA is a promising candidate for a more comprehensive understanding of thiopurine pharmacogenetics. We therefore studied the haplotype structure, haplotype-phenotype association, and promoter function of ITPA in a Western European population.ITPA haplotyping was performed based on haplotype tagging SNPs (selected from HapMap data) in healthy 130 controls. Haplotypes were reconstructed, and the haplotype-phenotype association was assessed by haplotype trend regression. A 1.5 kb upstream region and stepwise deletions thereof were tested for promoter activity in reporter gene assays in HepG2 and CCRF-CEM cells. Transcription factor binding (Sp1, Sp3) to the proximal promoter region was studied by chromatin immunoprecipitation. Among haplotypes with a frequency greater than 0.01, we did not find any new low-activity haplotypes besides those carrying 94C>A or IVS2 + 21A>C variants. Two promoter SNPs had no influence on promoter activity. An approximately 200 bp sized GC-rich proximal promoter region was sufficient to fully drive reporter gene activity. Chromatin immunoprecipitation showed binding of Sp1 and Sp3 transcription factors to this region. Only the two haplotypes carrying 94C>A or IVS2 + 21A>C were associated with reduced enzyme activity. The gene promoter is associated with a CpG island and driven by Sp-family transcription factors. There was no evidence for functional promoter SNPs, and it is suggested that only SNPs within the very proximal promoter region (approximately 200 bp) have the potential to be functionally significant.

HADA2a and hADA3 are required for acetylation, transcriptional activity and proliferative effects of beta-catenin

Beta-catenin is the key transcriptional activator of the Wnt pathway important for development and tissue homeostasis of multicellular organisms. Its deregulation contributes to many human cancers. The beta-catenin transcriptional activator complex continues to be defined, but already contains several proteins with chromatin remodeling activity. Here we show that two members of histone acetyltransferase complexes without enzymatic activity, hADA2a and hADA3, are required for full activity of beta-catenin. hADA2a and hADA3 physically interact with beta-catenin, and the interaction is mediated through Armadillo repeats 6 through 12 and the C-terminal transactivation domain of beta-catenin. Both hADA2a and hADA3 reside with beta-catenin at the enhancer for the Wnt target gene c-Myc. RNA interference-mediated reduction of hADA2a and hADA3 results in reduced beta-catenin acetylation, reduced activity in reporter gene assays and reduced activation of endogenous beta-catenin target genes. Overall, loss of hADA2a and hADA3 negatively impacts beta-catenin-mediated proliferation. Our studies identify hADA2a and hADA3 as crucial cofactors of beta-catenin that are likely involved in the assembly of transactivation-competent beta-catenin complexes at Wnt target genes.

The histone deacetylase inhibitor trichostatin A mediates upregulation of 5-lipoxygenase promoter activity by recruitment of Sp1 to distinct GC-boxes

The histone deacetylase inhibitor trichostatin A (TsA) potently induces 5-lipoxygenase (5-LO) promoter activity in reporter gene assays as well as 5-LO mRNA expression. We identified two proximal Sp1/Sp3 binding sites in the 5-LO gene promoter mediating the TsA effect in both 5-LO-negative HeLa cells and in 5-LO expressing Mono Mac 6 (MM6) cells, the tandem GC-boxes, by contrast, were not important for the TsA effect. TsA neither altered the protein expression levels of Sp1/Sp3 nor of the histone deacetylases HDAC1/2, nor did it apparently change the protein complex formation by these factors. Also, treatment of cells with TsA did not change the binding affinity of Sp1/Sp3 in cell extracts, as tested by DAPA analysis using probes containing the proximal GC boxes. However, in the living cell TsA induced Sp1, Sp3 and RNA polymerase II recruitment to the 5-LO promoter without changing the acetylation status of histone protein H4. Cotransfection studies suggest that both Sp1 and Sp3 can mediate the TsA effect. This is the first report demonstrating that Sp3 is involved in the regulation of 5-LO promoter activity. In summary, we show that TsA increases 5-LO promoter activity by the enhanced recruitment of Sp1 and Sp3 to the 5-LO promoter.

Identification of five human novel genes associated with cell proliferation by cell-based screening from an expressed cDNA ORF library

The development of functional profiling technologies provides opportunity for high-throughput functional genomics studies. We describe a cell-based screening system to identify novel human genes associated with cell proliferation. The method integrates luciferase reporter gene activity, fluorescence stain, automated microscopy and cellular phenotype assays. We successfully used the system to screen 409 novel human genes cloned by our lab and found that 27 genes significantly up-regulated promoter-Renilla luciferase reporter plasmid (pRL) activity. Among them, five genes, TRAF3IP3, ZNF306, ZNF250, SGOL1, and ZNF434, were determined through morphological observation, calcein AM fluorescence stain, MTT assay and cell cycle analysis to be associated with cell proliferation. Furthermore, we showed that the gene TRAF3IP3, which initially was identified to specifically interact with TRAF3, stimulated cell growth by modulating the c-Jun N-terminal kinase (JNK) pathway, and RNAi of TRAF3IP3 confirmed that the effect was physiological and necessary. In summary, we integrated a rapid and efficient system for screening novel growth regulatory genes. Using the new screening system we identified five genes associated with cell proliferation for the first time.

Incorporation of an Internal Ribosome Entry Site–Dependent Mechanism in Arsenic-Induced GADD45α Expression

We have previously shown that trivalent arsenic (arsenite, As(3+)) is able to induce GADD45 alpha expression in human bronchial epithelial cells through activation of c-Jun NH(2)-terminal kinase and nucleolin-dependent mRNA stabilization. In the present report, we show that As(3+) is capable of inducing translation of the GADD45 alpha protein through a cap-independent, or rather, an internal ribosome entry site (IRES)-dependent mechanism. In growth-arrested cells, As(3+) elevated the GADD45 alpha protein level in a dose- and time-dependent manner which did not correlate with the GADD45 alpha mRNA expression. Pretreatment of the cells with rapamycin, an inhibitor for the cap-dependent translation machinery through the suppression of mTOR and p70S6 kinase, failed to affect the induction of the GADD45 alpha protein induced by As(3+). Sequence analysis revealed a potential IRES element in the 5'-untranslated region of the GADD45 alpha mRNA. This IRES element in the 5'-untranslated region of the GADD45 alpha mRNA is functional in mediating As(3+)-induced translation of the GADD45 alpha protein in a dicistronic reporter gene activity assay. Immunoprecipitation and proteomic studies suggest that As(3+) impairs the assembly of the cap-dependent initiating complex for general protein translation but increases the association of human elongation factor 2 and human heterogeneous nuclear ribonucleoprotin with this complex. Thus, these results suggest that in growth-arrested cells, As(3+) is still capable of inducing GADD45 alpha expression through an IRES-dependent translational regulation.

Novel Estrogen Receptor-α Binding Sites and Estradiol Target Genes Identified by Chromatin Immunoprecipitation Cloning in Breast Cancer

Estrogen receptor-alpha (ERalpha) and its ligand estradiol play critical roles in breast cancer growth and are important therapeutic targets for this disease. Using chromatin immunoprecipitation (ChIP)-on-chip, ligand-bound ERalpha was recently found to function as a master transcriptional regulator via binding to many cis-acting sites genome-wide. Here, we used an alternative technology (ChIP cloning) and identified 94 ERalpha target loci in breast cancer cells. The ERalpha-binding sites contained both classic estrogen response elements and nonclassic binding sequences, showed specific transcriptional activity in reporter gene assay, and interacted with the key transcriptional regulators, including RNA polymerase II and nuclear receptor coactivator-3. The great majority of the binding sites were located in either introns or far distant to coding regions of genes. Forty-three percent of the genes that lie within 50 kb to an ERalpha-binding site were regulated by estradiol. Most of these genes are novel estradiol targets encoding receptors, signaling messengers, and ion binders/transporters. mRNA profiling in estradiol-treated breast cancer cell lines and tissues revealed that these genes are highly ERalpha responsive both in vitro and in vivo. Among estradiol-induced genes, Wnt11 was found to increase cell survival by significantly reducing apoptosis in breast cancer cells. Taken together, we showed novel genomic binding sites of ERalpha that regulate a novel set of genes in response to estradiol in breast cancer. Our findings suggest that at least a subset of these genes, including Wnt11, may play important in vivo and in vitro biological roles in breast cancer.

Sodium butyrate modifies the stabilizing complexes of tyrosine hydroxylase mRNA

Multiple mechanisms regulate the expression of the tyrosine hydroxylase ( Th) gene, which encodes the rate-limiting enzyme in the biosynthesis of catecholamines. Sodium butyrate (SOB), a physiological histone deacetylase (HDAC) inhibitor, was reported to stimulate the Th gene promoter activity in reporter gene assays. However, the expression of the endogenous Th gene in PC12 cells was reported to be either stimulated or inhibited by SOB. Here, we report that SOB and other HDAC inhibitors drastically (up to 90%) and reversibly decrease the level of TH mRNA in PC12 cells. We also show that SOB does not influence the transcription initiation rate of the Th gene but perturbs the formation of protein–RNA complexes at the 3′UTR of the gene. Our results suggest that SOB inhibits the expression of the Th gene by destabilizing TH mRNAs.

A Functional Enhancer of Keratin14 Is a Direct Transcriptional Target of ΔNp63

Keratin14 (K14) is a prototypic marker of dividing basal keratinocytes where its gene is transcribed at high levels. Transcriptional regulation of K14 is governed by an evolutionarily conserved functional enhancer marked by DNase 1 hypersensitive sites present upstream of the gene. This enhancer is sufficient to confer epidermal-specific gene expression, which is mediated in part by binding of members of activator protein-2 (AP)-2, AP-1, Ets, and Sp1 families of transcription factors. Here we provide evidence that a keratinocyte-specific nuclear protein identified as deltaNp63 binds to a conserved motif within this enhancer. Interestingly, the selective expression profile of deltaNp63 in various cell lines correlates with both the nuclear complex and the expression of K14. Biochemical studies reveal that deltaNp63 can bind to a specific DNA sequence present in the K14 enhancer and this binding leads to transactivation. In addition, chromatin immunoprecipitation experiments with deltaNp63-specific antibodies demonstrate that the enhancer is occupied by deltaNp63 in cultured keratinocytes and in mouse skin epidermal cells in vivo. Finally, we show that ectopic expression of either p63 isoform (deltaN or TA) can induce de novo expression of K14. These studies provide a potential mechanism by which deltaNp63 directly governs the expression of K14 in a keratinocyte-specific manner.

Association between the T-381C polymorphism of the brain natriuretic peptide gene and risk of type 2 diabetes in human populations

Brain natriuretic peptide (BNP/NPPB) is a member of the natriuretic family involved in the regulation of blood pressure and blood volume as well as lipolysis control in human fat cells. Thus BNP may play a role in energy metabolism and metabolic diseases. We therefore assessed the association between the BNP promoter T-381C polymorphism and risk of type 2 diabetes and metabolic and BNP expression traits in several population samples. In French population-based samples (n = 3216), we found that individuals bearing the -381CC genotype had lower (P = 0.005) fasting glucose levels than -381TC or -381TT individuals. Moreover, the -381CC genotype was less frequent in individuals with type 2 diabetes (n = 280, 13.6%) or with impaired fasting glucose (n = 248, 12.9%) compared with normoglycaemic individuals (n = 2485, 17.8%). The adjusted odds ratio (OR) (95% CI) of type 2 diabetes for -381CC individuals was 0.69 (0.47-1.00), P = 0.05, when compared with -381T allele bearers. We replicated this association in four additional case-control studies for type 2 diabetes. The overall OR (95% CI) of type 2 diabetes was 0.85 (0.76-0.96), P = 0.008, (under a recessive model) (3593 cases and 6646 controls in total). We also found that the -381C allele was associated with higher plasma BNP concentrations (P = 0.015, n = 634) and higher BNP promoter activity in reporter gene assays. Collectively, these data suggest that relatively high BNP expression may protect against type 2 diabetes in humans.

Angiotensin II Type 1 Receptor Gene Regulation

Angiotensin II (Ang II), the major bioactive peptide of the renin–angiotensin system, was originally described as a potent vasoconstrictor. It is now recognized as a multifunctional hormone influencing many cellular processes, including cell growth, apoptosis, migration, inflammation, and fibrosis.1,2 Under pathological conditions, the vasoconstrictor, mitogenic, proinflammatory, and profibrotic actions of Ang II contribute to altered vascular tone, structural remodeling, and endothelial dysfunction.3–6 Thus, Ang II plays a key role in the pathogenesis of cardiovascular diseases. The biological responses of Ang II are mediated by its interaction with 2 distinct high-affinity G protein–coupled receptors now designated Ang II type 1 receptor (AT1R) and Ang II type 2 receptor.7 Most of the known physiological and pathophysiological effects of Ang II are mediated via the AT1R.1–7 This receptor subtype is expressed in cardiovascular-relevant cell types including vascular smooth muscle cells (VSMCs), endothelial cells, cardiac myocytes, cardiac fibroblasts, and renal mesangial cells.7 The multiple actions of Ang II, mediated through the AT1R, are a result of complex intracellular signaling pathways including stimulation of the phospholipase C/inositol 1,4,5-trisphosphate/diacylglycerol cascade, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases, tyrosine kinases, and RhoA/Rho kinase.1,2,4,6,7 In addition, AT1Rs mediate many of their pathophysiological effects by stimulating reactive oxygen species generation via an reduced nicotinamide-adenine dinucleotide/reduced nicotinamide-adenine dinucleotide phosphate oxidase-dependent mechanism.2,8 Reactive oxygen species, in turn, influences downstream signaling molecules, including transcription factors, tyrosine kinases/phosphatases, Ca2+ channels, and MAPKs.2,8 The expression level of the AT1R defines the biological efficacy of Ang II; hence, overexpression of the AT1R is one potential mechanism by which Ang II can contribute to cardiovascular disease. Importantly, aberrant expression of the AT1R has been shown to have pathophysiological relevance in cell culture, animal studies, …