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Responsive Element (responsive + element)
Terms modified by Responsive Element Selected AbstractsRegulatory mechanisms of intestinal iron absorption,Uncovering of a fast-response mechanism based on DMT1 and ferroportin endocytosisBIOFACTORS, Issue 2 2010Marco T. Núñez Abstract Knowledge on the intestinal iron transport process and the regulation of body iron stores has greatly increased during the last decade. The liver, through the sensing of circulating iron, is now recognized as the central organ in this regulation. High iron levels induce the synthesis of hepcidin, which in turn decreases circulating iron by inhibiting its recycling from macrophages and its absorption at the intestine. Another mechanism for the control of iron absorption by the enterocyte is an active Iron Responsive Element (IRE)/Iron Regulatory Protein (IRP) system. The IRE/IRP system regulates the expression of iron uptake and storage proteins thus regulating iron absorption. Similarly, increasing evidence points to the transcriptional regulation of both divalent metal transporter 1 (DMT1) and ferroportin expression. A new mechanism of regulation related to a phenomenon called the mucosal block is starting to be unveiled. The mucosal block describes the ability of an initial dose of ingested iron to block absorption of a second dose given 2,4 h later. Here, we review the mechanisms involved in the expression of DMT1 and ferroportin, and present recent evidence on the molecular components and cellular processes involved in the mucosal block response. Our studies indicate that mucosal block is a fast-response endocytic mechanism destined to decrease intestinal iron absorption during a high ingest of iron. [source] Granulocyte function in patients with L-ferritin iron-responsive element (IRE) 39C,T-positive hereditary hyperferritinaemia,cataract syndromeEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 10 2004R. Fritsche-Polanz Abstract Background, Hereditary hyperferritinaemia,cataract syndrome (HHCS) is an autosomal dominant trait associated with mutations in the iron responsive element (IRE) of the ferritin light-chain (L-ferritin) gene. Patients typically show elevated serum ferritin concentrations without iron overload and a bilateral cataract. Hyperferritinaemia can be associated with granulocyte dysfunction in patients with thalassemia beta and in haemodialysis patients. The effect of increased L-ferritin levels on granulocyte function in patients with HHCS is unknown. Material and methods, We examined glucose uptake, oxidative burst, chemotaxis, phagocytosis, apoptosis and intracellular calcium concentrations in polymorphonuclear leucocytes (PMNLs) of five affected members of a family with HHCS and in five healthy individuals matched for age and gender. Results, Mutation testing revealed a 39C,T transition in IRE in all five patients with HHCS. Serum ferritin levels of patients ranged between 907 and 2030 µg L,1, respectively. In comparison with healthy individuals, PMNLs of patients with HHCS showed a significant increase in PMA-mediated stimulation of the oxidative burst, as well as a significantly higher stimulation of glucose uptake but no difference with respect to chemotaxis, phagocytosis, apoptosis and intracellular calcium concentrations. Conclusion, In summary, our study suggests that hyperferritinaemia in patients with IRE 39C,T-positive HHCS is associated with activation of PMNLs but not with disturbance of fundamental PMNL function. [source] Impaired IL-4 production by CD8+ T,cells in NOD mice is related to a defect of c-Maf binding to the IL-4 promoterEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2005Xiao-Ping Chen Abstract CD8+ T,cells play an important role in the induction of the autoimmune response in non-obese diabetic (NOD) mice. Here we describe abnormalities in the control of cytokine production by NOD CD8+ T,cells. NOD CD8+ T,cells had an increased propensity to produce IFN-, upon TCR activation, in both adult and 2-week-old mice. NOD CD8+ T,cells had a reduced capacity to produce IL-4 in type,2 conditions compared to CD8+ T,cells from the diabetes-resistant strains BALB/c and C57BL/6. Both GATA-3 and c-Maf, two positive transactivators for IL-4 gene expression, were expressed in type,2 conditions at comparable levels in NOD CD8+ T,cells. The GATA-3 was functional since normal levels of IL-5 were produced and the IL-4 promoter was hyperacetylated in NOD CD8+ T,cells. In contrast, c-Maf failed to bind to its responsive element as determined by chromatin immunoprecipitation (ChIP) assay. These results suggest that NOD CD8+ T,cells possess an increased propensity to produce IFN-, and impaired c-Maf-dependent DNA binding activities in vivo that lead to reduced IL-4 production following TCR activation. These defects may facilitate the development of the autoimmune response by inducing an overall type,1-biased immune response in NOD mice. [source] Transcription of mammalian cytochrome c oxidase subunit IV-2 is controlled by a novel conserved oxygen responsive elementFEBS JOURNAL, Issue 21 2007Maik Hüttemann Subunit 4 of cytochrome c oxidase (CcO) is a nuclear-encoded regulatory subunit of the terminal complex of the mitochondrial electron transport chain. We have recently discovered an isoform of CcO 4 (CcO4-2) which is specific to lung and trachea, and is induced after birth. The role of CcO as the major cellular oxygen consumer, and the lung-specific expression of CcO4-2, led us to investigate CcO4-2 gene regulation. We cloned the CcO4-2 promoter regions of cow, rat and mouse and compared them with the human promoter. Promoter activity is localized within a 118-bp proximal region of the human promoter and is stimulated by hypoxia, reaching a maximum (threefold) under 4% oxygen compared with normoxia. CcO4-2 oxygen responsiveness was assigned by mutagenesis to a novel promoter element (5,-GGACGTTCCCACG-3,) that lies within a 24-bp region that is 79% conserved in all four species. This element is able to bind protein, and competition experiments revealed that, within the element, the four core bases 5,-TCNCA-3, are obligatory for transcription factor binding. CcO isolated from lung showed a 2.5-fold increased maximal turnover compared with liver CcO. We propose that CcO4-2 expression in highly oxygenated lung and trachea protects these tissues from oxidative damage by accelerating the last step in the electron transport chain, leading to a decrease in available electrons for free radical formation. [source] Identification of ERR, as a specific partner of PGC-1, for the activation of PDK4 gene expression in muscleFEBS JOURNAL, Issue 8 2006Makoto Araki Pyruvate dehydrogenase kinase 4 (PDK4) is a key regulatory enzyme involved in switching the energy source from glucose to fatty acids in response to physiological conditions. Transcription of the PDK4 gene is activated by fasting or by the administration of a PPAR, ligand in a tissue-specific manner. Here, we show that the two mechanisms are independent, and that ERR, is directly involved in PPAR,-independent transcriptional activation of the PDK4 gene with PGC-1, as a specific partner. This conclusion is based on the following evidence. First, detailed mutation analyses of the cloned PDK4 gene promoter sequence identified a possible ERR,-binding motif as the PGC-1, responsive element. Second, overexpression of ERR, by cotransfection enhanced, and the knockout of it by shRNAs diminished, PGC-1,-dependent activation. Third, specific binding of ERR, to the identified PGC-1, responsive sequence was confirmed by the electrophoresis mobility shift assay. Finally, cell-type-specific responsiveness to PGC-1, was observed and this could be explained by differences in the expression levels of ERR,, however, ectopic expression of ERR, in poorly responsive cells did not restore PGC-1, responsiveness, indicating that ERR, is necessary, but not sufficient for the response. [source] Hepatitis C virus core protein activates ERK and p38 MAPK in cooperation with ethanol in transgenic miceHEPATOLOGY, Issue 4 2003Takeya Tsutsumi In human chronic hepatitis C, alcohol intake is a synergistic factor for the acceleration of hepatocarcinogenesis. Recently, we showed a significant increase of reactive oxygen species (ROS) in hepatitis C virus (HCV) core-transgenic mice fed ethanol-containing diets. Because previous studies indicated that ROS is closely associated with mitogen-activated protein kinases (MAPK), we examined activities of c-Jun N-terminal kinase, p38 MAPK, and extracellular signal-regulated kinase (ERK) in the liver of core-transgenic and nontransgenic mice with short-term ethanol feeding. Activity of ERK and p38 MAPK was increased in core-transgenic mice compared with nontransgenic mice, whereas neither ERK nor p38 MAPK was activated in core-transgenic mice with normal diets. In addition, activity of cyclic-AMP and serum responsive element, downstream pathways of p38 MAPK and ERK, was also increased. Comparison of gene expression profiles by cDNA microarray and real-time PCR revealed that galectin-1, which is associated with cell transformation, was significantly increased in ethanol-fed core-transgenic mice. On the other hand, glutathione S-transferase (GST), which plays a key role in protecting cells from oxidative stress, was decreased. In conclusion, these results suggest that HCV core protein cooperates with ethanol for the activation of some MAPK pathways, and leads to the modulation of several genes, contributing to the pathogenesis of liver disease of HCV- infected patients with high ethanol consumption. (Hepatology 2003;38:820,828). [source] Microelectronic DNA chip for hereditary hyperferritinemia cataract syndrome, a model for large-scale analysis of disorders of iron metabolism,HUMAN MUTATION, Issue 2 2006Francesca Ferrari Abstract Hereditary hyperferritinemia cataract syndrome (HHCS) is caused by mutations in the regulatory iron responsive element (IRE) in the 5,UTR of the L-ferritin transcript that reduce binding affinity to the iron regulatory proteins (IRPs) and lead to a constitutive upregulation of the protein in tissue and serum. Twenty-nine mutations have been reported within the L-ferritin (FTL) IRE sequence, 21 of which were available to us. In addition, we included in this study three new mutations. Thus, we analyzed 24 mutations spanning over a DNA stretch of 48 nucleotides, including four deletions 2,29 nucleotides long and 20 substitutions, seven of which were conservative transversions. With this unique experimental model we developed a microchip diagnostic platform for identifying known molecular defects in the L-ferritin IRE structure with a microelectronic array approach, which we optimized after studying the effects of various parameters. The system enables electronic deposition of biotinylated amplicons to selected pads. Under optimized conditions, no cross-hybridization was found, even for mutations that affected the same or adjacent nucleotide positions. The same cartridge could be serially hybridized with all the 24 reporter probe sets, which allowed correct genotyping right up until the end of the analysis. Extensive validation on 200 samples in a blinded fashion gave total concordance of results. This pilot study represents a first step toward developing a diagnostic microchip for large-scale analyses for epidemiological studies and screening of mutations associated with iron disorders. Hum Mutat 27(2), 201,208, 2006. © 2006 Wiley-Liss, Inc. [source] Hepatocyte Growth Factor Contributes to Fracture Repair by Upregulating the Expression of BMP Receptors,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2005Yuuki Imai MD Abstract Hepatocyte growth factor (HGF) is activated and the expression of BMP receptors (BMPRs) is induced around the fracture site during the early phase of fracture repair. HGF facilitates the expression of BMPRs in mesenchymal cells. This study suggests that HGF contributes to fracture repair by inducing the expression of BMPRs. Introduction: The precise mechanisms that control the upregulation of BMP, BMPRs, and other molecules involved in bone repair are not completely understood. In this study, we hypothesized that HGF, activated through the action of thrombin on the HGF activator, may enhance BMP action through the local induction of BMP or BMPRs. Materials and Methods: Callus samples from tibial fractures in mice were harvested for immunohistochemical analysis of HGF and phosphorylated c-Met, for in situ hybridization of BMPRs, and for real-time RT-PCR analysis for the expression of HGF, c-Met, and BMPRs. To study the changes in gene expression of BMPRs in response to HGF, C3H10T1/2 cells were cultured with or without HGF and harvested for real-time RT-PCR and for Western blot analysis. To evaluate the contribution of HGF to the biological action of BMP2, C3H10T1/2 cells and primary muscle-derived mesenchymal cells were precultured with HGF and cultured with BMP2. In addition, the expression of the luciferase gene linked to the Id1 promoter containing the BMP responsive element and alkaline phosphatase (ALP) activity were assayed. Results: Positive immunostaining of HGF and phosphorylated c-Met was detected around the fracture site at 1 day after the fracture was made. mRNA expression of BMPRs was increased 1 day after fracture and localized in mesenchymal cells at the fracture site. From an in vitro study, the expression of mRNA for BMPRs was elevated by treatment with HGF, but the expression of BMP4 did not change. Western blot analysis also showed the upregulation of BMPR2 by HGF treatment. The results from the luciferase and ALP assays indicated increased responsiveness to BMPs by treating with HGF. Conclusions: This study indicates that HGF is activated and expressed at the fracture site and that HGF induces the upregulation of BMPRs in mesenchymal cells. Furthermore, HGF may facilitate BMP signaling without altering the expression of BMP molecules. [source] CREB Cooperates with BMP-stimulated Smad signaling to enhance transcription of the Smad6 promoterJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Andreia M. Ionescu Growth plate chondrocytes integrate a multitude of growth factor signals during maturation. PTHrP inhibits maturation through stimulation of PKA/CREB signaling while the bone morphogenetic proteins (BMPs) stimulate maturation through Smad mediated signaling. In this manuscript, we show that interactions between CREB and the BMP associated Smads are promoter specific, and demonstrate for the first time the requirement of CREB signaling for Smad mediated activation of a BMP responsive region of the Smad6 promoter. The 28 base pairs (bp) BMP responsive element of the Smad6 promoter contains an 11 bp Smad binding region and an adjacent 17 bp region in which we characterize a putative CRE site. PKA/CREB gain of function enhanced BMP stimulation of this reporter, while loss of CREB function diminished transcriptional activity. In contrast, ATF-2 and AP-1 transcription factors had minimal effects. Electrophoretic mobility shift assay (EMSA) confirmed CREB binding to the Smad6 promoter element. Mutations eliminating binding resulted in loss of transcriptional activity, while mutations that maintained CREB binding had continued reporter activation by CREB and BMP-2. The Smad6 gene was similarly regulated by CREB. Dominant negative CREB reduced BMP-2 stimulated Smad6 gene transcription by 50%, but markedly increased BMP-2 mediated stimulation of colX and Ihh expression. In contrast, PTHrP which activates CREB signaling, blocked the stimulatory effect of BMP-2 on colX and Ihh, but minimally inhibited the stimulatory effect of BMP on Smad6. These findings are the first to demonstrate a cooperative association between CREB and BMP regulated Smads in cells from vertebrates and demonstrate that promoter-specific rather than generalized interactions between PKA/CREB and BMP signaling regulate gene expression in chondrocytes. J. Cell. Physiol. 198: 428,440, 2004© 2003 Wiley-Liss, Inc. [source] Dual effects of hepatitis C virus Core protein on the transcription of cyclin-dependent kinase inhibitor p21 geneJOURNAL OF VIRAL HEPATITIS, Issue 4 2003H. J. Kwun Summary. Transcription of p21 was activated in hepatitis C virus (HCV) Core-expressing HepG2 cells where its upstream p53 was stabilized. However, this effect was not absolutely required for the activation of p21 by Core, as demonstrated in Hep3B cells. In addition, an opposite effect on the transcription of p21 was observed in NIH3T3 and primary hepatocytes, where p53 was not decreased by Core. To explain the p53-independent regulation of p21 by Core, we identified a Core-responsive element between positions ,74 and ,83 of the p21 promoter, exactly overlapped with a tumour growth factor , (TGF- ,)/butyrate responsive element. Furthermore, we demonstrated that Core could activate the p21 through the element by stimulating a butyrate pathway, whereas this was inhibited through a TGF- , pathway. The opposing effects of Core protein on the transcription of p21 might be important in understanding the progression of hepatic disease in HCV-positive patients. [source] Rev response elements (RRE) in lentiviruses: An RNAMotif algorithm-based strategy for RRE predictionMEDICINAL RESEARCH REVIEWS, Issue 6 2002Elena A. Lesnik Abstract Lentiviruses (a sub-family of the retroviridae family) include primate and non-primate viruses associated with chronic diseases of the immune system and the central nervous system. All lentiviruses encode a regulatory protein Rev that is essential for post-transcriptional transport of the unspliced and incompletely spliced viral mRNAs from nuclei to cytoplasm. The Rev protein acts via binding to an RNA structural element known as the Rev responsive element (RRE). The RRE location and structure and the mechanism of the Rev-RRE interaction in primate and non-primate lentiviruses have been analyzed and compared. Based on structural data available for RRE of HIV-1, a two step computational strategy for prediction of putative RRE regions in lentivirus genomes has been developed. First, the RNAMotif algorithm was used to search genomic sequence for highly structured regions (HSR). Then the program RNAstructure, version 3.6 was used to calculate the structure and thermodynamic stability of the region of , 350 nucleotides encompassing the HSR. Our strategy correctly predicted the locations of all previously reported lentivirus RREs. We were able also to predict the locations and structures of potential RREs in four additional lentiviruses. © 2002 Wiley Periodicals, Inc. Med Res Rev, 22, No. 6, 617,636, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/med.10027 [source] Identification of the core element responsive to runt-related transcription factor 2 in the promoter of human type x collagen geneARTHRITIS & RHEUMATISM, Issue 1 2009Akiro Higashikawa Objective Type X collagen and runt-related transcription factor 2 (RUNX-2) are known to be important for chondrocyte hypertrophy during skeletal growth and repair and development of osteoarthritis (OA) in mice. Aiming at clinical application, this study was undertaken to investigate transcriptional regulation of human type X collagen by RUNX-2 in human cells. Methods Localization of type X collagen and RUNX-2 was determined by immunohistochemistry, and their functional interaction was examined in cultured mouse chondrogenic ATDC-5 cells. Promoter activity of the human type X collagen gene (COL10A1) was examined in human HeLa, HuH7, and OUMS27 cells transfected with a luciferase gene containing a 4.5-kb promoter and fragments. Binding to RUNX-2 was examined by electrophoretic mobility shift assay and chromatin immunoprecipitation. Results RUNX-2 and type X collagen were co-localized in mouse limb cartilage and bone fracture callus. Gain and loss of function of RUNX-2 revealed that RUNX-2 is essential for type X collagen expression and terminal differentiation of chondrocytes. Human COL10A1 promoter activity was enhanced by RUNX-2 alone and more potently by RUNX-2 in combination with the coactivator core-binding factor , in all 3 human cell lines examined. Deletion, mutagenesis, and tandem repeat analyses identified the core responsive element as the region between ,89 and ,60 bp (termed the hypertrophy box [HY box]), which showed specific binding to RUNX-2. Other putative RUNX-2 binding motifs in the human COL10A1 promoter did not respond to RUNX-2 in human cells. Conclusion Our findings indicate that the HY box is the core element responsive to RUNX-2 in human COL10A1 promoter. Studies on molecular networks related to RUNX-2 and the HY box will lead to treatments of skeletal growth retardation, bone fracture, and OA. [source] Mediation of interleukin-1,,induced transforming growth factor ,1 expression by activator protein 4 transcription factor in primary cultures of bovine articular chondrocytes: Possible cooperation with activator protein 1ARTHRITIS & RHEUMATISM, Issue 6 2003R. Andriamanalijaona Objective Interleukin-1 (IL-1) and transforming growth factor ,1 (TGF,1) play major roles in osteoarticular diseases, exerting opposite effects on both the catabolism and anabolism of cartilage matrix. Previous findings suggest that IL-1 and TGF,1 could function in a feedback interaction. However, the effect exerted by IL-1 on expression of TGF, by articular chondrocytes is, so far, poorly understood. The present study was carried out to determine the influence of IL-1, on the expression of TGF,1 by bovine articular chondrocytes (BACs) in primary culture. Methods BAC primary cultures were treated with IL-1,, and TGF,1 messenger RNA (mRNA) steady-state levels and protein expression were measured by real-time reverse transcription,polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Transient transfection of TGF,1 gene promoter constructs was performed to delineate the DNA sequences that mediate the IL-1, effect. Electrophoretic mobility shift assays (EMSAs) and supershift analysis were used to characterize the transcription factors binding to these sequences. Results Cultured BACs responded to IL-1, exposure by exhibiting an increase of TGF,1 expression at both the mRNA and protein levels. The effect was found to be mediated by a major 80-bp sequence located between ,732 and ,652 upstream of the transcription initiation site. EMSA and supershift analysis revealed that the transcription factors activator protein 4 (AP-4) and AP-1 specifically bound to the ,720/,696 part of this sequence under IL-1, treatment. Overexpression of AP-4 in the BAC cultures resulted in stimulation of the transcriptional activity of the ,732/+11 TGF,1 promoter construct through the same IL-1,,responsive element. Conclusion IL-1, induces an increase of TGF,1 in articular chondrocytes through activation of AP-4 and AP-1 binding to the TGF,1 gene promoter. These findings may help us understand the role of IL-1, in the disease process. Notwithstanding its deleterious effect on cartilage, IL-1 could initiate the repair response displayed by injured cartilage in the early stages of osteoarthritis through its ability to enhance TGF,1 expression by local chondrocytes. [source] Paradoxical early glucocorticoid induction of stem cell factor (SCF) expression in inflammatory conditionsBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2004Carla Alexandra Da Silva Stem cell factor (SCF) is a major growth factor for mast cells, promoting their differentiation and chemotaxis. Its expression is regulated by glucocorticoids in inflammatory conditions, showing an early increased protein expression, before the expected anti-inflammatory decrease (Da Silva et al., Br. J. Pharmacol. 2002:135,1634). We here evaluated the early kinetic of SCF expression regulated by interleukin (IL)-1,, budesonide and the combination of both in human lung fibroblasts in culture. Budesonide potentiated the IL-1, -enhanced expression of SCF mRNA (+103%) and protein (+98%) very shortly after treatment (at 30 min and 1 h, respectively). A gentle downregulation followed. This potentiating effect of budesonide was related to increased SCF mRNA stability and SCF gene transcription. Deletion of a ,B-like site that we identified in the first intron of the SCF gene, in a luciferase reporter system, abolished the potentiation by budesonide, as well as the effect of IL-1, alone, as compared to the wild-type construction activity. All budesonide-induced effects were glucocorticoid-receptor dependent, since they were reproduced by dexamethasone and blocked by RU486. IL-1,+budesonide did not affect the relative expression of the soluble and membrane-bound forms of SCF. In conclusion, our results clearly show that glucocorticoids act very early to adversely increase the expression of SCF mRNA and protein in the inflammatory conditions created by IL-1,, and that this effect involves increased mRNA stability and increased gene expression through activation of the NF- ,B-like responsive element. British Journal of Pharmacology (2004) 141, 75,84. doi:10.1038/sj.bjp.0705598 [source] Subcellular distribution of S100A4 and its transcriptional regulation under hypoxic conditions in gastric cancer cell line BGC823CANCER SCIENCE, Issue 5 2010Ruixiu Zhang It is well known that S100A4 is overexpressed in many tumors and involved in tumor invasion and metastasis. But the regualtion of it is ill understood. We previously found that hypoxia mimicking cobalt chloride (CoCl2) enhanced the mRNA and protein expressions of the S100A4 gene in the gastric cancer cell line BGC823. In this study we found that S100A4 also displayed increased expression in BGC823 cells after exposure to real hypoxia (2.5% O2) as that by CoCl2 treatment. Moreover, S100A4 protein showed different subcellular distribution under real hypoxia compared with that by CoCl2 treatment or in normoxic conditions. To investigate the underlying molecular mechanism by which hypoxia regulates the expression of S100A4, we analyzed the regulatory sequences of the genes by bioinformatics and found a putative hypoxia responsive element (HRE) motif in the first intron of S1004. Furthermore, luciferase reporter assay showed that it is responsive to hypoxia. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrated that hypoxia-inducible factor 1 (HIF-1) binds to the functional HRE in vitro and in vivo. The results provide evidence that S100A4 is a hypoxia-inducible gene, whose transcription is stimulated at least partly through the interaction of HIF-1 and HRE located at +329 to +334 of S100A4. (Cancer Sci 2010; 101: 1141,1146) [source] The hepatitis E virus ORF3 protein stabilizes HIF-1, and enhances HIF-1-mediated transcriptional activity through p300/CBPCELLULAR MICROBIOLOGY, Issue 9 2009Syed M. Moin Summary The hepatitis E virus (HEV) causes hepatitis E and is an important human pathogen. We have previously shown that the HEV open reading frame 3 (ORF3) protein promotes survival of the host cell. Here we report finding increased expression of glycolytic pathway enzymes in ORF3-expressing cells. Promoter analysis of these genes revealed the ubiquitous presence of hypoxia inducible factor (HIF) responsive element (HRE). Dominant-negative and siRNA studies showed increased expression of glycolytic pathway genes by the ORF3 to be mediated by the HIF-1 transcription factor. Our results showed that HIF-1,, a highly unstable subunit of the HIF-1, was stabilized in ORF3-expressing cells. This was through phosphatidylinositol-3-kinase (PI3K) mediated activation of Akt/protein kinase B. Enhanced binding to the consensus HRE and increased transactivation activity of HIF-1 were also observed in ORF3-expressing cells. The HIF complex recruits the transcriptional adapter/histone acetyltransferase protein p300/CBP to target gene promoters and p300/CBP phosphorylation is required for this interaction. We show that ORF3-mediated extracellularly regulated kinase (Erk) activation was responsible for the observed increase in phosphorylation and transactivation activity of p300/CBP. Our results reveal a two-pronged strategy through which the ORF3 protein might modulate the energy homeostasis in HEV infected cells and thus contribute to pathogenesis. [source] FGF-2, IL-1, and TGF-, regulate fibroblast expression of S100A8FEBS JOURNAL, Issue 11 2005Farid Rahimi Growth factors, including fibroblast growth factor-2 (FGF-2) and transforming growth factor-, (TGF-,) regulate fibroblast function, differentiation and proliferation. S100A8 and S100A9 are members of the S100 family of Ca2+ -binding proteins and are now accepted as markers of inflammation. They are expressed by keratinocytes and inflammatory cells in human/murine wounds and by appropriately activated macrophages, endothelial cells, epithelial cells and keratinocytes in vitro. In this study, regulation and expression of S100A8 and S100A9 were examined in fibroblasts. Endotoxin (LPS), interferon , (IFN,), tumour-necrosis factor (TNF) and TGF-, did not induce the S100A8 gene in murine fibroblasts whereas FGF-2 induced mRNA maximally after 12 h. The FGF-2 response was strongly enhanced and prolonged by heparin. Interleukin-1, (IL-1,) alone, or in synergy with FGF-2/heparin strongly induced the gene in 3T3 fibroblasts. S100A9 mRNA was not induced under any condition. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblasts. S100A8 mRNA induction by FGF-2 and IL-1, was partially dependent on the mitogen-activated-protein-kinase pathway and dependent on new protein synthesis. FGF-2-responsive elements were distinct from the IL-1,-responsive elements in the S100A8 gene promoter. FGF-2-/heparin-induced, but not IL-1,-induced responses were significantly suppressed by TGF-,, possibly mediated by decreased mRNA stability. S100A8 in activated fibroblasts was mainly intracytoplasmic. Rat dermal wounds contained numerous S100A8-positive fibroblast-like cells 2 and 4 days post injury; numbers declined by 7 days. Up-regulation of S100A8 by FGF-2/IL-1,, down-regulation by TGF-,, and its time-dependent expression in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair. [source] Oxygen Tension Regulates the Expression of ANK (Progressive Ankylosis) in an HIF-1-Dependent Manner in Growth Plate Chondrocytes,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2009Raihana Zaka Abstract The proximal promoter region of ANK, a gene that codes for a protein that regulates the transport of inorganic pyrophosphate, contains two hypoxia responsive elements (HREs); therefore, we studied the expression and function of ANK at different oxygen tensions. ATDC5 and N1511 clonal chondrocytic cells were cultured in either hypoxia (2% O2) or normoxia (21% O2). Transcript and protein levels of ANK were depressed in hypoxic conditions, as were levels of extracellular pyrophosphate (ePPi). To determine whether HIF-1 was involved in the oxemic response, Hif-1, knockdown cells were exposed to varying oxygen conditions and ANK expression was assessed. Knockdown of Hif-1, resulted in low levels of expression of ANK in hypoxia and normoxia. Chromatin immunoprecipitation (ChIP) assays explored the binding of Hif-1, to ANK HREs and showed that Hif-1, is able to bind to the HREs of ANK more avidly in normoxia than in hypoxia. Furthermore, functional studies of Hif-1, activity using luciferase reporter assays of wildtype and mutagenized HREs showed that only HRE-1 binds Hif-1, in normoxia. Expression of ANK in growth plate and articular cartilage was low in hypoxic regions of the tissues, and higher levels of ANK expression were observed in the synovium and meniscus in regions that have a normally higher oxygen tension. The data suggest that ANK expression and function in vitro and in vivo are repressed in hypoxic environments and that the effect is regulated by HIF-1. [source] Vitamin D Receptor: Key Roles in Bone Mineral Pathophysiology, Molecular Mechanism of Action, and Novel Nutritional Ligands,JOURNAL OF BONE AND MINERAL RESEARCH, Issue S2 2007Peter W Jurutka Abstract The vitamin D hormone, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], binds with high affinity to the nuclear vitamin D receptor (VDR), which recruits its retinoid X receptor (RXR) heterodimeric partner to recognize vitamin D responsive elements (VDREs) in target genes. 1,25(OH)2D3 is known primarily as a regulator of calcium, but it also controls phosphate (re)absorption at the intestine and kidney. Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone produced in osteoblasts that, like PTH, lowers serum phosphate by inhibiting renal reabsorption through Npt2a/Npt2c. Real-time PCR and reporter gene transfection assays were used to probe VDR-mediated transcriptional control by 1,25(OH)2D3. Reporter gene and mammalian two-hybrid transfections, plus competitive receptor binding assays, were used to discover novel VDR ligands. 1,25(OH)2D3 induces FGF23 78-fold in osteoblasts, and because FGF23 in turn represses 1,25(OH)2D3 synthesis, a reciprocal relationship is established, with FGF23 indirectly curtailing 1,25(OH)2D3 -mediated intestinal absorption and counterbalancing renal reabsorption of phosphate, thereby reversing hyperphosphatemia and preventing ectopic calcification. Therefore, a 1,25(OH)2D3,FGF23 axis regulating phosphate is comparable in importance to the 1,25(OH)2D3,PTH axis that regulates calcium. 1,25(OH)2D3 also elicits regulation of LRP5, Runx2, PHEX, TRPV6, and Npt2c, all anabolic toward bone, and RANKL, which is catabolic. Regulation of mouse RANKL by 1,25(OH)2D3 supports a cloverleaf model, whereby VDR-RXR heterodimers bound to multiple VDREs are juxtapositioned through chromatin looping to form a supercomplex, potentially allowing simultaneous interactions with multiple co-modulators and chromatin remodeling enzymes. VDR also selectively binds certain ,3/,6 polyunsaturated fatty acids (PUFAs) with low affinity, leading to transcriptionally active VDR-RXR complexes. Moreover, the turmeric-derived polyphenol, curcumin, activates transcription of a VDRE reporter construct in human colon cancer cells. Activation of VDR by PUFAs and curcumin may elicit unique, 1,25(OH)2D3 -independent signaling pathways to orchestrate the bioeffects of these lipids in intestine, bone, skin/hair follicle, and other VDR-containing tissues. [source] Cloning and molecular dissection of the 8.8 kb pig uroplakin II promoter using transgenic mice and RT4 cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Deug-Nam Kwon Abstract Uroplakin II (UPII) gene expression is highly tissue and cell specific, with mRNA present in the suprabasal cell layers of the bladder and urethra. Previous reports described the mouse UPII (mUPII) promoter as primarily urothelium selective. However, ectopic expression of a transgene under the 3.6 kb mUPII promoter was also detected in brain, kidney, and testis in some transgenic mouse lines. Here, we have cloned an 8.8 kb pig UPII (pUPII) promoter region and investigated which cells within the bladder and urethra express a transgene consisting of the pUPII promoter fused to human erythropoietin (hEPO) or a luciferase gene. pUPII-luciferase expression vectors with various deletions of the promoter region were introduced into mouse fibroblast (NIH3T3), Chinese hamster ovary (CHO), and human bladder transitional carcinoma (RT4). A 2.1 kb pUPII promoter fragment displayed high levels of luciferase activity in transiently transfected RT4 cells, whereas the 8.8 kb pUPII promoter region displayed only low levels of activity. The pUPII-hEPO expression vector was injected into the pronucleus of zygotes to make transgenic mice. To elucidate the in vivo molecular mechanisms controlling the tissue- and cell-specific expression of the pUPII promoter gene, transgenic mice containing 2.1 and 8.8 kb pUPII promoter fragments linked to the genomic hEPO gene were generated. An erythropoietin (EPO) assay showed that all nine transgenic lines carrying the 8.8 kb construct expressed recombinant human erythropoietin (rhEPO) only in their urethra and bladder, whereas two transgenic lines carrying the 2.1 kb pUPII promoter displayed hEPO expression in several organs including bladder, kidney, spleen, heart, and brain. These studies demonstrate that the 2.1 kb promoter contains the DNA elements necessary for high levels of expression, but lacks critical sequences necessary for tissue-specific expression. We compared binding sites in the 2.1 and 8.8 kb promoter sequences and found five peroxisome proliferator responsive elements (PPREs) in the 8.8 kb promoter. Our data demonstrated that proliferator-activated receptor (PPAR)-, activator treatment in RT4 cells induced the elevated expression of hEPO mRNA under the control of the 8.8 kb pUPII promoter, but not the 2.1 kb promoter. Collectively, our data suggested that all the major trans-regulatory elements required for bladder- and urethra-specific transcription are located in the 8.8 kb upstream region and that it may enhance tissue-specific protein production and be of interest to clinicians who are searching for therapeutic modalities with high efficacy and low toxicity. J. Cell. Biochem. 99: 462,477, 2006. © 2006 Wiley-Liss, Inc. [source] How to overcome (and exploit) tumor hypoxia for targeted gene therapyJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2003Olga Greco Tumor hypoxia has long been recognized as a critical issue in oncology. Resistance of hypoxic areas has been shown to affect treatment outcome after radiation, chemotherapy, and surgery in a number of tumor sites. Two main strategies to overcome tumor hypoxia are to increase the delivery of oxygen (or oxygen-mimetic drugs), and exploiting this unique environmental condition of solid tumors for targeted therapy. The first strategy includes hyperbaric oxygen breathing, the administration of carbogen and nicotinamide, and the delivery of chemical radiosensitizers. In contrast, bioreductive drugs and hypoxia-targeted suicide gene therapy aim at activating cytotoxic agents at the tumor site, while sparing normal tissue from damage. The cellular machinery responds to hypoxia by activating the expression of genes involved in angiogenesis, anaerobic metabolism, vascular permeability, and inflammation. In most cases, transcription is initiated by the binding of the transcription factor hypoxia-inducible factor (HIF) to hypoxia responsive elements (HREs). Hypoxia-targeting for gene therapy has been achieved by utilizing promoters containing HREs, to induce selective and efficient transgene activation at the tumor site. Hypoxia-targeted delivery and prodrug activation may add additional levels of selectivity to the treatment. In this article, the latest developments of cancer gene therapy of the hypoxic environment are discussed, with particular attention to combined protocols with ionizing radiation. Ultimately, it is proposed that by adopting specific transgene activation and molecular amplification systems, resistant hypoxic tumor tissues may be effectively targeted with gene therapy. J. Cell. Physiol. 197: 312,325, 2003© 2003 Wiley-Liss, Inc. [source] Sp proteins play a critical role in histone deacetylase inhibitor-mediated derepression of CYP46A1 gene transcriptionJOURNAL OF NEUROCHEMISTRY, Issue 2 2010Maria João Nunes J. Neurochem. (2010) 113, 418,431. Abstract We investigated whether the CYP46A1 gene, a neuronal-specific cytochrome P450, responsible for the majority of brain cholesterol turnover, is subject to transcriptional modulation through modifications in histone acetylation. We demonstrated that inhibition of histone deacetylase activity by trichostatin A (TSA), valproic acid and sodium butyrate caused a potent induction of both CYP46A1 promoter activity and endogenous expression. Silencing of Sp transcription factors through specific small interfering RNAs, or impairing Sp binding to the proximal promoter, by site-directed mutagenesis, led to a significant decrease in TSA-mediated induction of CYP46A1 expression/promoter activity. Electrophoretic mobility shift assay, DNA affinity precipitation assays and chromatin immunoprecipitation assays were used to determine the multiprotein complex recruited to the CYP46A1 promoter, upon TSA treatment. Our data showed that a decrease in Sp3 binding at particular responsive elements, can shift the Sp1/Sp3/Sp4 ratio, and favor the detachment of histone deacetylase (HDAC) 1 and HDAC2 and the recruitment of p300/CBP. Moreover, we observed a dynamic change in the chromatin structure upon TSA treatment, characterized by an increase in the local recruitment of euchromatic markers and RNA polymerase II. Our results show the critical participation of an epigenetic program in the control of CYP46A1 gene transcription, and suggest that brain cholesterol catabolism may be affected upon treatment with HDAC inhibitors. [source] Interleukin-4 activates androgen receptor through CBP/p300THE PROSTATE, Issue 2 2009Soo Ok Lee Abstract BACKGROUND Aberrant activation of androgen receptor (AR) plays an important role in the progression of castration resistant prostate cancer. Interleukin-4 (IL-4) enhances AR activation in the absence of androgen and stimulates castration resistant growth of androgen-sensitive prostate cancer cells. However, the mechanism of IL-4 mediated AR activation has not yet been revealed. METHODS The effect of IL-4 on CBP/p300 expression was examined by Western blot analysis. The effect of IL-4 on the interactions of AR and CBP/p300 was examined by co-immunoprecipitation and ChIP assays. CBP/p300 siRNA was used to knockdown CBP/p300 expression to examine the role of CBP/p300 expression on IL-4 mediated AR activation. RESULTS We found that IL-4 increases CBP/p300 protein expression and enhances interaction of AR with CBP/p300 proteins through an increase in the recruitment of CBP/p300 protein to the androgen responsive elements in the promoters of androgen responsive genes. Down regulation of CBP/p300 expression using CBP/p300 specific siRNA abolished IL-4 mediated AR activation, suggesting that CBP/p300 is responsible for AR activation induced by IL-4. Furthermore, AR activation can be enhanced by AR acetylation induced by IL-4 in prostate cancer cells. The IL-4 mediated AR acetylation can be blocked by knocking down CBP/p300 expression using CBP/p300 specific siRNA. CONCLUSION These results suggest that IL-4 activates AR through enhanced expression of CBP/p300 and its histone acetyltransferase activity. Prostate 69: 126,132, 2009. © 2008 Wiley,Liss, Inc. [source] Crystallization and preliminary X-ray analyses of catabolite control protein A, free and in complex with its DNA-binding siteACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2000Jan Tebbe The catabolite control protein (CcpA) from Bacillus megaterium is a member of the bacterial repressor protein family GalR/LacI. CcpA with an N-terminal His-tag was used for crystallization. Crystals of free CcpA and of CcpA in complex with the putative operator sequence (catabolite responsive elements, CRE) were obtained by vapour-diffusion techniques at 291,K using the hanging-drop method. CcpA crystals grown in the presence of polyethylene glycol 8000 belong to the hexagonal space group P6122 or P6522, with unit-cell parameters a = 74.4, c = 238.8,Å. These crystals diffract X-rays to 2.55,Å resolution and contain one monomer of the homodimeric protein per asymmetric unit. Crystals of the CcpA,CRE complex were obtained with ammonium sulfate as precipitant and belong to the tetragonal space group I4122, with unit-cell parameters a = 125, c = 400,Å and one complex per asymmetric unit. Although these co-crystals grew to a sufficient size, X-ray diffraction was limited to 8,Å resolution. [source] Hybrid promoters directed tBid gene expression to breast cancer cells by transcriptional targetingBIOTECHNOLOGY PROGRESS, Issue 2 2010Samila Farokhimanesh Abstract Developing cancer gene therapy constructs based on transcriptional targeting of genes to cancer cells is a new and promising modality for treatment of cancer. Introducing truncated Bid (tBid), a recently known member of the Bcl-2 family, eradicates cancer cells efficiently. For transcriptional targeting of tBid, two dual-specificity promoters, combining cancer specific core promoters and response modules, were designed. These two core promoter modules contained cancer specific promoters of MUC1 and Survivin genes accompanied by hypoxia-responsive elements and estrogen responsive elements (microenvironment condition of breast cancer cells) which were employed to achieve a higher and more specific level of tBid expression in breast cancer cells. Correlation of the level of tBid expression in normal and cancer cell lines with promoter activity was measured by RT-PCR after treatment with hypoxia and estrogen. The level of tBid expression under control of new hybrid promoters was compared with its expression under control of cytomegalovirus (CMV) promoter as a control. Our data revealed that the level of tBid expression in breast cancer cells were nearly 11 times more than normal cells because of the cancer specific promoters, although tBid expression under control of CMV promoter was almost the same in normal and cancer cell lines. Increased apoptosis was detected in the transfected breast cancer cell lines by the Caspase-3 activity assay. The application of these promoters may prove to have the advantage of tumor selective gene therapy in breast cancer cells and low-potential toxicity for normal tissues. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Gene transfer of disease regulated promoters during experimental autoimmune uveitisACTA OPHTHALMOLOGICA, Issue 2009V ELMALEH Purpose Adeno-associated virus (AAV) vectors have been successfully used to transfer immunosuppressive genes into the retina to prevent experimental uveitis development. Transgene expression is classically regulated by constitutive or tetracycline inducible promoters. It might be more advantageous that the control of transgene expression depends on the pathological process itself. Inflammation activates transcription factors acting on promoters containing short responsive sequences, responding, for example to nuclear factor kappa B (NF,B-RE). These responsive elements can be used to generate disease regulated promoters. Methods An AAV vector with the GFP gene under the control of a NF-kB-RE containing promoter will be injected subretinally in C57Bl6 mice. Autoimmune uveitis will be induced by adoptive transfer of IRBP specific lymphocytes. Animals will be sacrificed at different time points. GFP expression will be analysed by immunofluorescence. VCAM1, MHC II and CD45 will be analysed by immunofluorescence and used to monitor the level of retinal inflammation. Results One week after disease induction, GFP expression was found in eyes injected with this new vector. Milder GFP expression was also found in mice who did not received adoptive transfer. This background was increased a J14. Conclusion Our preliminary results suggest that disease driven GFP expression can be obtained by the use of AAV vectors containing disease regulated promoters. We still need some more times to improve our model. In the future, we plan to replace the GFP gene by an immunosuppressive gene and test if the system can be use to treat experimental uveitis. [source] | ||||||||||||||||