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Transactivation
Kinds of Transactivation Terms modified by Transactivation Selected AbstractsContext-specific regulation of LINE-1GENES TO CELLS, Issue 10 2007Ivo Teneng The present study was conducted to evaluate the contextual specificity of long interspersed nuclear element-1 (LINE-1 or L1) activation by cellular stress and the role of the aryl hydrocarbon receptor (AHR) transcription factor and oxidative stress in the gene activation response. Activation of the AHR by the genotoxic carcinogen benzo(a)pyrene (BaP) increased L1 expression in human cervical carcinoma (HeLa) cells, human microvascular endothelial cells (HMEC), mouse vascular smooth muscle cells (mVSMC) and mouse embryonic kidney cells (mK4). In contrast, challenge with a different AHR ligand 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), or UV irradiation (10,20 J/m2), induced L1 only in HeLa cells. Transactivation of the mouse L1Md-A5 promoter was observed in all cell types challenged with BaP, while TCDD was without effect, and UV only activated L1 in HeLa cells. Genetic and pharmacological experiments implicated the AHR and oxidative stress as contextual determinants of L1 inducibility by cellular stress. [source] MEK/ERK Signaling Controls Osmoregulation of Nucleus Pulposus Cells of the Intervertebral Disc by Transactivation of TonEBP/OREBP,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2007Tsung-Ting Tsai Abstract Earlier studies have shown that intervertebral disc cells express TonEBP, a transcription factor that permits adaptation to osmotic stress and regulates aggrecan gene expression. However, the mechanism of hyperosmotic activation of TonEBP in disc cells is not known. Results of this study show that hypertonic activation of ERK signaling regulates transactivation activity of TonEBP, modulating its function. Introduction: In an earlier report, we showed that tonicity enhancer binding protein (TonEBP) positively regulates aggrecan gene expression in disc cells, thereby autoregulating its osmotic environment. Although these studies indicated that the cells of the nucleus pulposus were optimally adapted to a hyperosmotic state, the mechanism by which the cells transduce the osmotic stress was not delineated. The primary goal of this study was to test the hypothesis that, in a hyperosmotic medium, the extracellular signal-regulated kinase (ERK) signaling pathway regulated TonEBP activity. Materials and Methods: Nucleus pulposus cells were maintained in isotonic or hypertonic media, and MAPK activation and TonEBP expression were analyzed. To study the role of MAPK in regulation of TonEBP function, gel shift and luciferase reporter assays were performed. ERK expression in cells was modulated by using expression plasmids or siRNA, and transactivation domain (TAD)-TonEBP activity was studied. Results: We found that hypertonicity resulted in phosphorylation and activation of ERK1/2 proteins and concomitant activation of C terminus TAD activity of ELK-1, a downstream transcription factor. In hypertonic media, treatment with ERK and p38 inhibitors resulted in downregulation of TonE promoter activity of TauT and HSP-70 and decreased binding of TonEBP to TonE motif. Similarly, forced expression of DN-ERK and DN-p38 in nucleus pulposus cells suppressed TauT and HSP-70 reporter gene activity. Finally, we noted that ERK was needed for transactivation of TonEBP. Expression of DN-ERK significantly suppressed, whereas, WT-ERK and CA-MEK1 enhanced, TAD activity of TonEBP. Experiments performed with HeLa cells indicated that the ERK signaling pathway also served a major role in regulating the osmotic response in nondiscal cells. Conclusions: Together, these studies showed that adaptation of the nucleus pulposus cells to their hyperosmotic milieu is dependent on activation of the ERK and p38- MAPK pathways acting through TonEBP and its target genes. [source] Transactivation of Src, PDGF receptor, and Akt is involved in IL-1,-induced ICAM-1 expression in A549 cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Chih-Chung Lin In previous study, interleukin-1, (IL-1,) has been shown to induce ICAM-1 expression through MAPKs and NF-,B in A549 cells. In addition to these pathways, transactivation of non-receptor tyrosine kinase (Src), PDGF receptors (PDGFRs), and phosphatidylinositol 3-kinase (PI3K)/Akt has been implicated in the expression of inflammatory genes. Here, we further investigated whether these different mechanisms participating in IL-1,-induced ICAM-1 expression in A549 cells. We initially observed that IL-1,-induced ICAM-1 promoter activity was attenuated by the inhibitors of Src (PP1), PDGFR (AG1296), PI3-K (LY294002 and wortmannin), and Akt (SH-5), revealed by reporter gene assay, Western blotting, and RT-PCR analyses. The involvement of Src and PI3-K/Akt in IL-1,-induced ICAM-1 expression was significantly attenuated by transfection of A549 cells with dominant negative plasmids of Src, p85 and Akt, respectively. Src, PDGFR, and PI3K/Akt mediated the effects of IL-1, because pretreatment with PP1, AG1296, and wortmannin also abrogated IL-1,-stimulated Src, PDGFR, and Akt phosphorylation, respectively. Moreover, pretreatment with p300 inhibitor (curcumin) also blocked ICAM-1 expression. We further confirmed that p300 was associated with ICAM-1 promoter which was dynamically linked to histone H4 acetylation stimulated by IL-1,, determined by chromatin immunoprecipitation assay. Association of p300 and histone-H4 to ICAM-1 promoter was inhibited by LY294002. Up-regulation of ICAM-1 enhanced the adhesion of neutrophils onto A549 cell monolayer exposed to IL-1,, which was inhibited by PP1, AG1296, LY294002, wortmannin, and helenalin. These results suggested that Akt phosphorylation mediated through transactivation of Src/PDGFR promotes the transcriptional p300 activity and eventually leads to ICAM-1 expression induced by IL-1,. J. Cell. Physiol. 211: 771,780, 2007. © 2007 Wiley-Liss, Inc. [source] Transactivation of BARNASE under the AtLTP1 promoter affects the basal pole of the embryo and shoot development of the adult plant in ArabidopsisTHE PLANT JOURNAL, Issue 5 2001Célia Baroux Summary Genetically controlled expression of a toxin provides a tool to remove a specific structure and consequently study its role during a developmental process. The availability of many tissue-specific promoters is a good argument for the development of such a strategy in plants. We have developed a conditional system for targeted toxin expression and demonstrated its use for generating embryo phenotypes that can bring valuable information about signalling during embryogenesis. The BARNASE gene was expressed in the Arabidopsis embryo under the control of two promoters, one from the cyclin AtCYCB1 gene and one from the AtLTP1 gene (LipidTransferProtein 1). One-hundred percent seed abortion was obtained with the cyclin promoter. Surprisingly however, the embryos displayed a range of lethal phenotypes instead of a single arrested stage as expected from this promoter. We also show that BARNASE expression under the control of the AtLTP1 promoter affects the basal pole of the globular embryo. Together with reporter expression studies, this result suggests a role of the epidermis in controlling the development of the lower tier of the embryo. This defect was not embryo-lethal and we show that the seedlings displayed a severe shoot phenotype correlated to epidermal defects. Therefore, the epidermis does not play an active role during organogenesis in seedlings but is important for the postgermination development of a viable plant. [source] Nuclear autoantigen CENP-B transactivation of the epidermal growth factor receptor via chemokine receptor 3 in vascular smooth muscle cellsARTHRITIS & RHEUMATISM, Issue 9 2009Geneviève Robitaille Objective We have previously found that the CENP-B nuclear autoantigen, which is specifically targeted by autoantibodies in the limited cutaneous form of systemic sclerosis, behaved as a potent migratory factor for human pulmonary artery smooth muscle cells (PASMCs). Other recent studies have shown that several disease-associated autoantigens induced cell migration by interacting with various chemokine receptors. Prompted by this hypothesis, we undertook this study to determine whether CENP-B interacts with chemokine receptors on the surface of human PASMCs, to explore the relevant signaling pathways, and to characterize the effects of anti,CENP-B binding on SMC stimulation. Methods To demonstrate the expression of specific chemokine receptors by human PASMCs at both the messenger RNA and protein levels, reverse transcription,polymerase chain reaction, immunoblotting, and flow cytometry analyses were performed. Desensitization studies and specific inhibitors were used to further identify the CENP-B target on the surface of human PASMCs. Results Our data strongly suggested that CENP-B used chemokine receptor 3 (CCR3) to mediate human PASMCs signaling. Moreover, several lines of evidence indicated that CENP-B binding subsequently stimulated the cross-talk between CCR3 and epidermal growth factor receptor (EGFR) via a matrix metalloprotease,dependent mechanism that involved the processing of heparin-binding EGF-like growth factor. Transactivation of the EGFR through CCR3 was found to be a critical pathway that elicits MAP kinase activation and secretion of cytokines such as interleukin-8. Finally, anti,CENP-B autoantibodies were found to abolish this signaling pathway, thus preventing CENP-B from transactivating EGFR and exerting its cytokine-like activities toward vascular smooth muscle cells. Conclusion The identification of CENP-B as a CCR3 ligand opens up new perspectives for the study of the pathogenic role of anti,CENP-B autoantibodies. [source] Cardioprotection of bradykinin at reperfusion involves transactivation of the epidermal growth factor receptor via matrix metalloproteinase-8ACTA PHYSIOLOGICA, Issue 4 2009C. Methner Abstract Aim:, The endogenous autacoid bradykinin (BK) reportedly reduces myocardial infarct size when given exogenously at reperfusion. Muscarinic and opioid G-protein-coupled receptors are equally protective and have been shown to couple through a matrix metalloproteinase (MMP)-dependent transactivation of the epidermal growth factor receptor (EGFR). Here we test whether BK protects the rat heart through the EGFR by an MMP-dependent pathway. Methods:, Infarct size was measured in isolated perfused rat hearts undergoing 30 min regional ischaemia followed by 120 min reperfusion. In additional studies HL-1 cardiomyocytes were loaded with tetramethylrhodamine ethyl to measure their mitochondrial membrane potential (,m). Adding the calcium ionophore calcimycin, causes ,m-collapse presumably due to calcium-induced mitochondrial permeability transition. Results:, As expected, BK (100 nmol L,1) started 5 min prior to reperfusion reduced infarct size from 38.9 ± 2.0% of the ischaemic zone in control hearts to 22.2 ± 3.3% (P < 0.001). Co-infusing the EGFR inhibitor AG1478, the broad-spectrum MMP-inhibitor GM6001, or a highly selective MMP-8 inhibitor abolished BK's protection, thus suggesting an MMP-8-dependent EGFR transactivation in the signalling. Eighty minutes of exposure to calcimycin reduced the mean cell fluorescence to 37.4 ± 1.8% of untreated cells while BK could partly preserve the fluorescence and, hence, protect the cells (50.5 ± 2.3%, P < 0.001). The BK-induced mitochondrial protection could again be blocked by AG1478, GM6001 and MMP-8 inhibitor. Finally, Western blotting revealed that BK's protection was correlated with increased phosphorylation of EGFR and its downstream target Akt. Conclusion:, These results indicate that BK at reperfusion triggers its protective signalling pathway through MMP-8-dependent transactivation of the EGFR. [source] The taurine transporter: mechanisms of regulationACTA PHYSIOLOGICA, Issue 1-2 2006X. Han Abstract Taurine transport undergoes an adaptive response to changes in taurine availability. Unlike most amino acids, taurine is not metabolized or incorporated into protein but remains free in the intracellular water. Most amino acids are reabsorbed at rates of 98,99%, but reabsorption of taurine may range from 40% to 99.5%. Factors that influence taurine accumulation include ionic environment, electrochemical charge, and post-translational and transcriptional factors. Among these are protein kinase C (PKC) activation and transactivation or repression by proto-oncogenes such as WT1, c-Jun, c-Myb and p53. Renal adaptive regulation of the taurine transporter (TauT) was studied in vivo and in vitro. Site-directed mutagenesis and the oocyte expression system were used to study post-translational regulation of the TauT by PKC. Reporter genes and Northern and Western blots were used to study transcriptional regulation of the taurine transporter gene (TauT). We demonstrated that (i) the body pool of taurine is controlled through renal adaptive regulation of TauT in response to taurine availability; (ii) ionic environment, electrochemical charge, pH, and developmental ontogeny influence renal taurine accumulation; (iii) the fourth segment of TauT is involved in the gating of taurine across the cell membrane, which is controlled by PKC phosphorylation of serine 322 at the post-translational level; (iv) expression of TauT is repressed by the p53 tumour suppressor gene and is transactivated by proto-oncogenes such as WT1, c-Jun, and c-Myb; and (v) over-expression of TauT protects renal cells from cisplatin-induced nephrotoxicity. [source] Intracellular HMGB1 transactivates the human IL1B gene promoter through association with an Ets transcription factor PU.1EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2008Fumihiko Mouri Abstract High mobility group box 1 protein (HMGB1), originally described as a non-histone, DNA binding protein, was recently identified as a late mediator of inflammation via its extracellular release from activated macrophages/monocytes. In the present study, we report that intracellular HMGB1 synergizes with a macrophage/monocyte-specific E26 transformation-specific sequence (Ets) transcription factor PU.1 to transactivate the promoter of the IL1B gene coding a 31-kDa proIL-1, protein. The ,131 to +12 IL1B promoter, which possesses a PU.1 binding motif essential for its transactivation, was induced when HMGB1 expression vector was transfected into murine RAW264.7 macrophage cells. Our glutathione S -transferase-pulldown and coimmunoprecipitation assays demonstrated direct physical interaction of HMGB1 with PU.1. Deletion of the PU.1 winged helix-turn-helix DNA-binding domain inhibited the association of the two proteins. In electrophoretic mobility shift assay using recombinant PU.1 protein, a ternary complex of PU.1, HMGB1 and PU.1-binding element within the IL1B promoter was generated. The importance of PU.1 was further supported by our observation that induction of the IL1B promoter was obtained only after PU.1 expression in PU.1-deficient murine EL4 thymoma cells. Thus, our data raise the possibility of a novel mechanism which sustains and amplifies inflammatory reactions through physical interaction of PU.1 with intracellular HMGB1 in macrophages/monocytes. [source] PI3K limits TNF- , production in CD16-activated monocytesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2009Phillip R. Kramer Abstract IgG complexes bind to Fc receptor family members Fc,RI (CD64), Fc,RII (CD32) and Fc,RIII (CD16), activating cell MAPK and PI3K resulting in increased cytokine production from particular leukocytes. The signaling molecules involved in cytokine production after cross-linking CD16 have not been determined in monocytes. To address this question, TNF-,, IL-1, and IL-6 were measured in activated monocytes after inhibiting MEK1/2, PI3K and glycogen synthase kinase-, (GSK-3,). The roles of GSK-3, and NF-,B were then determined using reporter assays and siRNA treatment. The data suggested that an MAPK pathway stimulated TNF-, release but that active PI3K limited TNF-,, IL-1, and IL-6 cytokine production after cross-linking CD16. PI3K was also shown to limit nuclear translocation of NF-,B. The limiting effect of PI3K on TNF-, production from activated monocytes depended on the decrease of GSK-3, activity, which significantly reduced the transactivation of NF-,B. Moreover, the TNF-, production induced by CD16 cross-linking was reduced in monocytes after treatment with siRNA against NF-,B, implying that this transcription factor functioned in TNF-, production. The results suggest that CD16 cross-linking activated PI3K and that active PI3K limited TNF-, production by inhibiting GSK-3, activity, that blocked the action of NF-,B. [source] PU.1 is required for transcriptional activation of the Stat6 response element in the Ig, promoterEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2003Marko Pesu Abstract Signal transducer and activator of transcription 6 (Stat6) has a crucial role in regulation of IL-4-induced gene responses. Stat6-binding sites are present in the promoters of both ubiquitously and cell-type-specifically expressed genes. The promoter regions of IL-4-inducible genes contain cis -acting elements for several transcription factors that act in concert with Stat6 and are also likely to modulate lineage-specific gene expression. We have observed that the Stat6 response element from the B-cell-specific Ig, promoter is readily activated upon IL-4 stimulation in B cells but not in non-hematopoietic cells. A minimal low-affinity PU.1-core-binding sequence (5,-AGAA-3,) was identified within the Stat6 DNA-binding site in the Ig, promoter. Ectopic expression of the myeloid- and B-cell-specific transcription factor PU.1 restored the IL-4-inducibility of the Ig,-Stat6 response element in HepG2 cells, and the induction required an intact PU.1-binding sequence. Both the transactivation and the DNA-binding domains of PU.1 were required for induction of Stat6-mediated transcription. The co-operation between PU.1 and Stat6 in transactivation of the Ig, gene represents a molecular mechanism for the fine-tuning of cell-type-restricted expression of IL-4-induced gene responses. [source] Regulation of ,FosB transcriptional activity by Ser27 phosphorylationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2007Paula G. Ulery Abstract The transcription factor, ,FosB, is an important mediator of the long-term plasticity induced in brain by chronic exposure to drugs of abuse, stress, or several other psychoactive stimuli. We have previously demonstrated that the casein kinase 2 (CK2)-mediated phosphorylation of a highly conserved N-terminal serine (Ser27) plays a critical role in regulating ,FosB's unusual stability, while it does not affect that of the full-length FosB protein. In the present study, we analysed whether CK2 and Ser27 phosphorylation also play a role in regulating ,FosB's transcriptional activity. Our findings indicate that CK2 activation increases ,FosB's transactivation potential, while CK2 inhibition decreases it. Further, we show that preventing Ser27 phosphorylation by mutating the site to Ala results in a significant decrease in ,FosB transactivation, without affecting ,FosB's subcellular localization or DNA-binding affinity. In contrast, Ser27 does not seem to play a role in the transactivation potential of full-length FosB. These findings constitute the first evidence of a role for phosphorylation in ,FosB's transcriptional activity. [source] Modulation of glucocorticoid receptor-interacting protein 1 (GRIP1) transactivation and co-activation activities through its C-terminal repression and self-association domainsFEBS JOURNAL, Issue 10 2006Pei-Yao Liu Glucocorticoid receptor-interacting protein 1 (GRIP1), a p160 family nuclear receptor co-activator, possesses at least two autonomous activation domains (AD1 and AD2) in the C-terminal region. AD1 activity appears to be mediated by CBP/p300, whereas AD2 activity is apparently mediated through co-activator-associated arginine methyltransferase 1 (CARM1). The mechanisms responsible for regulating the activities of AD1 and AD2 are not well understood. We provide evidence that the GRIP1 C-terminal region may be involved in regulating its own transactivation and nuclear receptor co-activation activities through primary self-association and a repression domain. We also compared the effects of the GRIP1 C terminus with those of other factors that functionally interact with the GRIP1 C terminus, such as CARM1. Based on our results, we propose a regulatory mechanism involving conformational changes to GRIP1 mediated through its intramolecular and intermolecular interactions, and through modulation of the effects of co-repressors on its repression domains. These are the first results to indicate that the structural components of GRIP1, especially those of the C terminus, might functionally modulate its putative transactivation activities and nuclear receptor co-activator functions. [source] Two CCAAT/enhancer binding protein sites in the cytochrome P4503A1 locusFEBS JOURNAL, Issue 3 2003Potencial role in the glucocorticoid response Induction of CYP3A genes by the ligand-activated pregnane-X-receptor (PXR) involves the interaction of other as yet unidentified liver transcription factors. Here we show that the CYP3A1 promoter contains two active sites controlled by the CCAAT/enhancer-binding protein , (C/EBP,), previously shown to regulate a number of liver stress response genes. We have identified two functional C/EBP binding sites at the CYP3A1 promoter that confer luciferase activity to C/EBP, cotransfected CHO cells. When inserted upstream of a thymidine kinase promoter, oligonucleotides corresponding to these elements (,350/,311 and ,628/,608), increase reporter gene expression when cotransfected with a C/EBP, expression vector. Point mutations in the most conserved nucleotides in either element prevent binding of C/EBP, and abolish transactivation of the CYP3A1 promoter. Moreover, we demonstrate that C/EBP, accumulates in the rat liver nuclei in response to dexamethasone, and that under these conditions C/EBP, binds to the CYP3A1 promoter elements. Our results suggest a correlation between transcription of C/EBP,, nuclear protein function and induction of CYP3A1 by dexamethasone in the liver. They also support the notion that C/EBP, participates in the up-regulation of the CYP3A1 gene in response to synthetic glucocorticoids. [source] Transcriptional activity of interferon regulatory factor (IRF)-3 depends on multiple protein,protein interactionsFEBS JOURNAL, Issue 24 2002Hongmei Yang Virus infection results in the activation of a set of cellular genes involved in host antiviral defense. IRF-3 has been identified as a critical transcription factor in this process. The activation mechanism of IRF-3 is not fully elucidated, yet it involves a conformational change triggered by the virus-dependent phosphorylation of its C-terminus. This conformational change leads to nuclear accumulation, DNA binding and transcriptional transactivation. Here we show that two distinct sets of Ser/Thr residues of IRF-3, on phosphorylation, synergize functionally to achieve maximal activation. Remarkably, we find that activated IRF-3 lacks transcriptional activity, but activates transcription entirely through the recruitment of the p300/CBP coactivators. Moreover, we show that two separate domains of IRF-3 interact with several distinct regions of p300/CBP. Interference with any of these interactions leads to a complete loss of transcriptional activity, suggesting that a bivalent interaction is essential for coactivator recruitment by IRF-3. [source] Expression of immediate early gene pip92 during anisomycin-induced cell death is mediated by the JNK- and p38-dependent activation of Elk1FEBS JOURNAL, Issue 15 2000Kwang C. Chung We report here that immediate early gene pip92 is expressed during anisomycin-induced cell death in fibroblast NIH3T3 cells. To determine the mechanism by which this occurs and to identify downstream signaling pathways, we investigated the induction of the pip92 promoter. The activation of pip92 by anisomycin is mediated by the activation of MAP kinases, such as JNK and p38 kinase, but not ERK. Deletion analysis of the pip92 promoter indicated that pip92 activation occurs primarily within the region containing a serum response element (SRE). Further analysis of the SRE using a heterologous thymidine kinase promoter showed that both an Ets and CArG-like site are required for anisomycin-induced pip92 expression. Elk1, which binds to the Ets site, was phosphorylated by the JNK- and p38-dependent pathways and the phosphorylation of Elk1,GAL4 fusion proteins by these pathways was sufficient for the transactivation. Overall, this study suggested that different MAPK pathways are involved in the expression of immediate early gene pip92 by growth factors and environmental stresses. [source] Gq/11-induced intracellular calcium mobilization mediates Per2 acute induction in Rat-1 fibroblastsGENES TO CELLS, Issue 9 2006Naoyuki Takashima Phase resetting is one of the essential properties of circadian clocks that is required for the adjustment to a particular environment and the induction of Per1 and Per2 clock genes is believed to be a primary molecular event during this process. Although the intracellular signal transduction pathway underlying Per1 gene activation has been well characterized, the mechanisms that control Per2 up-regulation have not yet been elucidated. In our present study, we demonstrate that Gq/11 coupled receptors mediate serum-induced immediate rat Per2 (rPer2) transactivation in Rat-1 fibroblasts via intracellular Ca2+ mobilization. Stimulation of these cells with a high concentration of serum was found to rapidly increase the intracellular Ca2+ levels and strongly up-regulated rPer2 gene. rPer2 induction by serum stimulation was abrogated by intracellular Ca2+ chelation and depletion of intracellular Ca2+ store, which suggests that the calcium mobilization is necessary for the up-regulation of rPer2 gene. In addition, suppression of Gq/11 function was observed to inhibit both Ca2+ mobilization and rPer2 induction. Further, we demonstrated that endothelin-induced acute rPer2 transactivation via Gq/11-coupled endothelin receptors is also suppressed by a Gq/11 specific inhibitor. These findings together suggest that serum and endothelin utilize a common Gq/11-PLC mediated pathway for the transactivation of rPer2, which involves the mobilization of calcium from the intracellular calcium store. [source] In vivo potentiation of human oestrogen receptor , by Cdk7-mediated phosphorylationGENES TO CELLS, Issue 10 2004Saya Ito Phosphorylation of the Ser118 residue in the N-terminal A/B domain of the human oestrogen receptor , (hER,) by mitogen-activated protein kinase (MAPK), stimulated via growth factor signalling pathways, is known to potentiate ER, ligand-induced transactivation function. Besides MAPK, cyclin dependent kinase 7 (Cdk7) in the TFIIH complex has also been found to potentiate hER, transactivation in vitro through Ser118 phosphorylation. To investigate an impact of Cdk7 on hER, transactivation in vivo, we assessed activity of hER, in a wild-type and cdk7 inactive mutant Drosophila that ectopically expressed hER, in the eye disc. Ectopic expression of the wild-type or mutant receptors, together with a green fluorescent protein (GFP) reporter gene, allowed us to demonstrate that hER, expressed in the fly tissues was transcriptionally functional and adequately responded to hER, ligands in the patterns similar to those observed in mammalian cells. Replacement of Ser118 with alanine in hER, (S118A mutant) significantly reduced the ligand-induced hER, transactivation function. Importantly, while in cdk7 inactive mutant Drosophila the wild-type hER, exhibited reduced response to the ligand; levels of transactivation by the hER, S118A mutant were not affected in these inactive cdk7 mutant flies. Furthermore, phosphorylation of hER, at Ser118 has been observed in vitro by both human and Drosophila Cdk7. Our findings demonstrate that Cdk7 is involved in regulation of the ligand-induced transactivation function of hER,in vivo via Ser118 phosphorylation. [source] Enhanced Expression of Transcription Factor E2F in Helicobacter pylori -infected Gastric MucosaHELICOBACTER, Issue 3 2002Hajime Isomoto Abstract Objective.Helicobacter pylori is implicated in gastric carcinogenesis through increased gastric epithelial cell turnover. In fact, high proportions of proliferating and apoptotic epithelial cells are found in H. pylori -infected gastric mucosa. E2F, a transcription factor, induces coordinated transactivation of a set of genes involved in cell cycle progression. The aim of this study was to investigate the expression of E2F in H. pylori -infected gastric mucosa and examine the correlation between such expression and gastric epithelial cell proliferation and apoptosis. Methods. Twenty-five patients with H. pylori -associated gastritis (HAG) and 13 control subjects negative for H. pylori were examined. E2F expression was studied in situ by Southwestern histochemistry, a method used to localize transcription factors. Labeled double-stranded oligo-DNA with specific consensus sequence for E2F binding sites was reacted with frozen sections from antral biopsy specimens obtained at endoscopy. Gastric epithelial cell proliferation was assessed by immunostaining of proliferating cell nuclear antigen (PCNA), while apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). The percentages of epithelial cells with nuclear staining for PCNA and E2F were expressed as a positivity index (PI). The percentage of TUNEL-positive epithelial cells was defined as apoptotic index. Results. E2F was expressed in the nuclei of gastric epithelial cells within gastric pits. E2F PI in H. pylori -infected gastric mucosa was significantly higher than that in noninfected. Expression of E2F correlated well with PCNA-positive epithelial cells. We also demonstrated colocalization of PCNA with E2F expression in the same epithelial cells. Apoptotic index was also high in H. pylori -infected mucosa, and correlated with E2F PI. Conclusion. Our results demonstrated a significant increase in the expression of E2F in H. pylori -infected mucosa, which correlated with both the percentages of PCNA- and TUNEL-positive cells. Our results suggest that enhanced E2F expression in gastric mucosa may be involved in H. pylori -related gastric carcinogenesis through accelerated cell turnover. [source] MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells,HEPATOLOGY, Issue 1 2009Teng Xu Growing evidence indicates that deregulation of microRNAs (miRNAs) contributes to tumorigenesis. Down-regulation of miR-195 has been observed in various types of cancers. However, the biological function of miR-195 is still largely unknown. In this study we aimed to elucidate the pathophysiologic role of miR-195. Our results showed that miR-195 expression was significantly reduced in as high as 85.7% of hepatocellular carcinoma (HCC) tissues and in all of the five HCC cell lines examined. Moreover, introduction of miR-195 dramatically suppressed the ability of HCC and colorectal carcinoma cells to form colonies in vitro and to develop tumors in nude mice. Furthermore, ectopic expression of miR-195 blocked G1/S transition, whereas inhibition of miR-195 promoted cell cycle progression. Subsequent investigation characterized multiple G1/S transition-related molecules, including cyclin D1, CDK6, and E2F3, as direct targets of miR-195. Silencing of cyclin D1, CDK6, or E2F3 phenocopied the effect of miR-195, whereas overexpression of these proteins attenuated miR-195-induced G1 arrest. In addition, miR-195 significantly repressed the phosphorylation of Rb as well as the transactivation of downstream target genes of E2F. These results imply that miR-195 may block the G1/S transition by repressing Rb-E2F signaling through targeting multiple molecules, including cyclin D1, CDK6, and E2F3. Conclusion: Our data highlight an important role of miR-195 in cell cycle control and in the molecular etiology of HCC, and implicate the potential application of miR-195 in cancer therapy. (HEPATOLOGY 2009.) [source] Molecular characterization of the role of orphan receptor small heterodimer partner in development of fatty liver,HEPATOLOGY, Issue 1 2007Jiansheng Huang The orphan receptor Small Heterodimer Partner (SHP, NROB2) regulates metabolic pathways, including hepatic bile acid, lipid, and glucose homeostasis. We reported that SHP- deletion in leptin-deficient OB,/, mice increases insulin sensitivity, and prevents the development of fatty liver. The prevention of steatosis in OB,/,/SHP,/, double mutants is not due to decreased body weight but is associated with increased hepatic very-low-density lipoprotein (VLDL) secretion and elevated microsomal triglyceride transfer protein (MTP) mRNA and protein levels. SHP represses the transactivation of the MTP promoter and the induction of MTP mRNA by LRH-1 in hepatocytes. Adenoviral overexpression of SHP inhibits MTP activity as well as VLDL-apoB protein secretion, and RNAi knockdown of SHP exhibits opposite effects. The expression of SHP in induced in fatty livers of OB,/, mice and other genetic or dietary models of steatosis, and acute overexpression of SHP by adenovirus, result in rapid accumulation of neutral lipids in hepatocytes. In addition, the pathways for hepatic lipid uptake and lipogenic program are also downregulated in OB,/,/SHP,/, mice, which may contribute to the decreased hepatic lipid content. Conclusion: These studies demonstrate that SHP regulates the development of fatty liver by modulating hepatic lipid export, uptake, and synthesis, and that the improved peripheral insulin sensitivity in OB,/,/SHP,/, mice is associated with decreased hepatic steatosis. (HEPATOLOGY 2007.) [source] Nuclear translocation of UDCA by the glucocorticoid receptor is required to reduce TGF-,1,induced apoptosis in rat hepatocytes,HEPATOLOGY, Issue 4 2005Susana Solá Ursodeoxycholic acid (UDCA) inhibits classical mitochondrial pathways of apoptosis by either directly stabilizing mitochondrial membranes or modulating specific upstream targets. Furthermore, UDCA regulates apoptosis-related genes from transforming growth factor ,1 (TGF-,1),induced hepatocyte apoptosis by a nuclear steroid receptor (NSR),dependent mechanism. In this study, we further investigated the potential role of the glucocorticoid receptor (GR) in the antiapoptotic function of UDCA. Our results with short interference RNA (siRNA) technology confirmed that UDCA significantly reduces TGF-,1,induced apoptosis of primary rat hepatocytes through a GR-dependent effect. Immunoprecipitation assays and confocal microscopy showed that UDCA enhanced free GR levels with subsequent GR nuclear translocation. Interestingly, when a carboxy-terminus deleted form of GR was used, UDCA no longer increased free GR and/or GR translocation, nor did it protect against TGF-,1,induced apoptosis. In co-transfection experiments with GR response element reporter and overexpression constructs, UDCA did not enhance the transactivation of GR with TGF-,1. Finally, using a flourescently labeled UDCA molecule, the bile acid appeared diffuse in the cytosol but was aggregated in the nucleus of hepatocytes. Both siRNA assays and transfection experiments with either wild-type or mutant forms of GR showed that nuclear trafficking occurs through a GR-dependent mechanism. In conclusion, these results further clarify the antiapoptotic mechanism(s) of UDCA and suggest that GR is crucial for the nuclear translocation of this bile acid for reducing apoptosis. (HEPATOLOGY 2005;42:925,934.) [source] Mechanisms for sensitization to TNF-induced apoptosis by acute glutathione depletion in murine hepatocytesHEPATOLOGY, Issue 6 2003Katsuhiko Matsumaru We previously reported that depletion of glutathione in murine hepatocytes by diethylmaleate (DEM) or acetaminophen (APAP) leads to oxidative stress,dependent necrosis and sensitizes to tumor necrosis factor (TNF)-induced apoptosis in an oxidative stress,independent fashion, which could not be explained by interference with nuclear factor ,B (NF-,B) nuclear translocation. The present report explores the mechanisms of these effects. We observed that DEM led to necrosis when both mitochondrial and cytosol glutathione were depleted profoundly but sensitized to TNF-induced apoptosis when cytosol glutathione was depleted selectively. DEM and APAP lead to a significant decrease in reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio. Glutathione depletion by DEM or APAP was associated with inhibition of TNF-induced NF-,B transactivation of anti-apoptotic genes, including inducible nitric oxide synthase (i-NOS). Provision of exogenous NO partially abrogated the sensitization to TNF in response to glutathione depletion. Glutathione depletion alone led to sustained increase in phospho-jun levels and c-Jun-N-terminal kinase (JNK) activity. JNK inhibitor partially blocked the sensitization to TNF-induced apoptosis accompanying glutathione depletion. In conclusion, these findings suggest that extramitochondrial glutathione depletion alters the thiol-disulfide redox state, leading to inhibition of NF-,B transactivation of survival genes and to sustained activation of JNK, both of which contribute to the sensitization to TNF-induced apoptosis. [source] Heterozygous SOX9 Mutations Allowing for Residual DNA-binding and Transcriptional Activation Lead to the Acampomelic Variant of Campomelic Dysplasia,HUMAN MUTATION, Issue 6 2010Alex Staffler Abstract Campomelic dysplasia is a malformation syndrome with multiple symptoms including characteristic shortness and bowing of the long bones (campomelia). CD, often lethal due to airway malformations, is caused by heterozygous mutations in SOX9, an SRY-related gene regulating testis and chondrocyte development including expression of many cartilage genes such as type II collagen. Male to female sex reversal occurs in the majority of affected individuals with an XY karyotype. A mild form without campomelia exists, in which sex-reversal may be also absent. We report here two novel SOX9 missense mutations in a male (c.495C>G; p.His165Gln) and a female (c.337A>G; p.Met113Val) within the DNA-binding domain leading to non-lethal acampomelic CD. Functional analyses of mutant proteins demonstrate residual DNA-binding and transactivation of SOX9-regulated genes. Combining our data and reports from the literature we postulate a genotype-phenotype correlation: SOX9 mutations allowing for residual function lead to a mild form of CD in which campomelia and sex reversal may be absent. © 2010 Wiley-Liss, Inc. [source] Novel missense mutations in the FOXC2 gene alter transcriptional activity,HUMAN MUTATION, Issue 12 2009M.A.M. van Steensel Abstract Mutations in the FOXC2 gene that codes for a forkhead transcription factor are associated with primary lymphedema that usually develops around puberty. Associated abnormalities include distichiasis and, very frequently, superficial and deep venous insufficiency. Most mutations reported so far either truncate the protein or are missense mutations in the forkhead domain causing a loss of function. The haplo-insufficient state is associated with lymphatic hyperplasia in mice as well as in humans. We analyzed the FOXC2 gene in 288 patients with primary lymphedema and found 11 pathogenic mutations, of which 9 are novel. Of those, 5 were novel missense mutations of which 4 were located outside of the forkhead domain. To examine their pathogenic potential we performed a transactivation assay using a luciferase reporter construct driven by FOXC1 response elements. We found that the mutations outside the forkhead domain cause a gain of function as measured by luciferase activity. Patient characteristics conform to previous reports with the exception of distichiasis, which was found in only 2 patients out of 11. FOXC2 mutations causing lymphedema-distichiasis syndrome reported thus far result in haplo-insufficiency and lead to lymphatic hyperplasia. Our results suggest that gain-of-function mutations may also cause lymphedema. One would expect that in this case, lymphatic hypoplasia would be the underlying abnormality. Patients with activating mutations might present with Meige disease. © 2009 Wiley-Liss, Inc. [source] Differential functional effects of novel mutations of the transcription factor FOXL2 in BPES patients,HUMAN MUTATION, Issue 8 2008Jeyabalan Nallathambi Abstract Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development lead to the Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome (BPES) in human. Here, we describe nine mutations in the open reading frame of FOXL2. Six of them are novel: c.292T>A (p.Trp98Arg), c.323T>C (p.Leu108Pro), c.650C>G (p.Ser217Cys) and three frameshifts. We have performed localization and functional studies for three of them. We have observed a strong cytoplasmic mislocalization induced by the missense mutation p.Leu108Pro located in the forkhead (FKH) domain of FOXL2. In line with this, transcriptional activity assays confirmed the loss-of-function induced by this variant. Interestingly, the novel mutation p.Ser217Cys, mapping between the FKH and the polyalanine domain of FOXL2 and producing a mild eyelid phenotype, led to normal localization and transactivation. We have also modeled the structure of the FKH domain to explore the potential structural impact of the mutations reported here and other previously reported ones. This analysis shows that mutants can be sorted into two classes: those that potentially alter protein-protein interactions and those that might disrupt the interactions with DNA. Our findings reveal new insights into the molecular effects of FOXL2 mutations, especially those affecting the FKH binding domain. © 2008 Wiley-Liss, Inc. [source] The p38 mitogen-activated protein kinase regulates interleukin-1,-induced IL-8 expression via an effect on the IL-8 promoter in intestinal epithelial cellsIMMUNOLOGY, Issue 4 2003Kuljit Parhar Summary Several lines of evidence implicate the p38 mitogen-activated protein kinase (p38 MAPK) in the proinflammatory response to bacterial agents and cytokines. Equally, the transcription factor, nuclear factor (NF)-,B, is recognized to be a critical determinant of the inflammatory response in intestinal epithelial cells (IECs). However, the precise inter-relationship between the activation of p38 MAPK and activation of the transcription factor NF-,B in the intestinal epithelial cell (IEC) system, remains unknown. Here we show that interleukin (IL)-1, activates all three MAPKs in Caco-2 cells. The production of IL-8 and monocyte chemotactic protein 1 (MCP-1) was attenuated by 50% when these cells were preincubated with the p38 MAPK inhibitor, SB 203580. Further investigation of the NF-,B signalling system revealed that the inhibitory effect was independent of the phosphorylation and degradation of I,B,, the binding partner of NF-,B. This effect was also independent of the DNA binding of the p65 Rel A subunit, as well as transactivation, determined by an NF-,B luciferase construct, using both SB 203580 and dominant,negative p38 MAPK. Evaluation of IL-8 and MCP-1 RNA messages by reverse transcription,polymerase chain reaction (RT,PCR) revealed that the inhibitory effect of SB 203580 was associated with a reduction in this parameter. Using an IL-8,luciferase promoter construct, an effect of p38 upon its activation by both pharmacological and dominant,negative p38 construct co-transfection was demonstrated. It is concluded that p38 MAPK influences the expression of chemokines in intestinal epithelial cells, through an effect upon the activation of the chemokine promoter, and does not directly involve the activation of the transcription factor NF-,B. [source] Regulation of HER expression and transactivation in human prostate cancer cells by a targeted cytotoxic bombesin analog (AN-215) and a bombesin antagonist (RC-3095)INTERNATIONAL JOURNAL OF CANCER, Issue 8 2010Sandra Sotomayor Abstract Bombesin (BN) and gastrin-releasing peptide (GRP) have been shown to stimulate the growth of human prostate cancer in vivo and in vitro by mechanisms initiated by binding of the peptide to BN/GRP receptor (GRPR). GRPR is overexpressed in a variety of human cancers, including human prostatic carcinoma. This led us to evaluate the effectiveness of blocking GRPR and of chemotherapy targeted to GRPR in androgen-dependent (LNCaP) and androgen-independent (PC-3) prostate cancer cells, which exhibit different features of disease progression. Thus, we used a cytotoxic BN/GRP analog, AN-215, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to BN-like carrier peptide, and a BN/GRP receptor antagonist, RC-3095. Semiquantitative RT-PCR and Western blotting revealed that mRNA and protein levels for GRPR increased in prostate cancer cells as compared with nonneoplastic RWPE-1 cells. Immunofluorocytochemistry and Western blot assays revealed that AN-215 was the most effective analog decreasing both the expression of epidermal growth factor receptor family members and the activation of epidermal growth factor receptor and HER-2, which are associated to a poor prognosis. Furthermore, analogs targeted to BN/GRP receptors, AN-215 and RC-3095, blocked the effect of BN on cell growth in RWPE-1, LNCaP and PC-3 cells. These findings shed light on the mechanisms of action of these analogs and support the view that the use of AN-215 and RC-3095 for blocking BN/GRP receptors for targeted therapy may be of benefit for treatment of advanced prostate cancer. [source] Ochratoxin A impairs Nrf2-dependent gene expression in porcine kidney tubulus cellsJOURNAL OF ANIMAL PHYSIOLOGY AND NUTRITION, Issue 5 2009C. Boesch-Saadatmandi Summary The mycotoxin, ochratoxin A (OTA), which is produced by Aspergillus and Penicillium subspecies, is frequently present in feedstuffs. Ochratoxin A exhibits a wide range of toxic activities including nephrotoxicity. However, the underlying molecular mechanisms of OTA-induced cellular nephrotoxicity have yet not been fully elucidated. Nrf2 is a basic leucine zipper transcriptional activator essential for the coordinated transcriptional induction of antioxidant and xenobiotic metabolizing enzymes in the kidney. Therefore, in the present study, the effects of OTA on the nuclear translocation and transactivation of the transcription factor Nrf2 as well as mRNA levels of Nrf2 target genes including glutathione- S -transferase and ,-glutamylcysteinyl synthetase have been studied in cultured porcine kidney tubulus cells (LLC-PK1). Nrf2 was induced by sulforaphane, a well-known activator of this transcription factor. Ochratoxin A significantly decreased ,-glutamylcysteinyl synthetase and glutathione- S -transferase mRNA levels in LLC-PK1 cells. Decreased mRNA levels of ,-glutamylcysteinyl synthetase and glutathione- S -transferase were accompanied by a lowered nuclear translocation and transactivation of Nrf2. Furthermore, OTA also lowered Nrf2 mRNA levels. Current data indicate that OTA nephrotoxicity may be, at least partly, mediated by an Nrf2-dependent signal transduction pathway. [source] TCDD suppresses insulin-responsive glucose transporter (GLUT-4) gene expression through C/EBP nuclear transcription factors in 3T3-L1 adipocytesJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 2 2006Phillip Chin-Chen Liu Abstract TCDD is known to reduce significantly the level of the functionally active form of glucose transporter type 4 (GLUT4) in vivo in adipose tissue and muscles. To study the mechanistic basis of this phenomenon, we conducted transient transfection and DNA deletion analysis in 3T3-L1 cells using chloramphenicol acetyltransferase (CAT) reporter plasmids containing the GLUT4 promoter joined to the bacterial CAT. It was found that in transfected control samples, CAT activity was significantly higher in cells transfected with p469CAT and p273CAT than those with p78CAT, indicating that the region between ,78 and ,273 contained elements that play major roles in transactivation of this gene. Treatment with TCDD decreased CAT activity with p469CAT and p273CAT, but not with p78CAT, indicating the same region to contain the element(s) affected by TCDD. A gel-shift (EMSA) analysis result indicated that TCDD shows the profound effect only on the nuclear proteins binding to the [32P]-labeled probe containing C/EBP response element equivalent of the ,265 to ,242 stretch of the GLUT4 promoter. The results of supershift analysis showed that TCDD caused a decrease in the tier of C/EBP, and an increase in that of C/EBP, among the proteins bound to this C/EBP response element. We studied the effect of TCDD in cells overexpressing either C/EBP,, C/EBP,, or C/EBP, through transient transfection of p273CAT or p469CAT. The results clearly showed that the effect of TCDD to suppress the CAT activity of p273 or p469 disappeared in those cells overexpressing C/EBP, or C/EBP,. These results implicate the C/EBP proteins to be the main mediator of suppressive action of TCDD on GLUT4 gene expression in 3T3-L1 cells. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:79,87, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20120 [source] Compound Heterozygous Mutations in the Vitamin D Receptor in a Patient With Hereditary 1,25-Dihydroxyvitamin D-Resistant Rickets With Alopecia,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2009Yulin Zhou Abstract Hereditary vitamin D-resistant rickets (HVDRR) is a rare recessive genetic disorder caused by mutations in the vitamin D receptor (VDR). In this study, we examined the VDR in a young girl with clinical features of HVDRR including rickets, hypophosphatemia, and elevated serum 1,25(OH)2D. The girl also had total alopecia. Two mutations were found in the VDR gene: a nonsense mutation (R30X) in the DNA-binding domain and a unique 3-bp in-frame deletion in exon 6 that deleted the codon for lysine at amino acid 246 (,K246). The child and her mother were both heterozygous for the 3-bp deletion, whereas the child and her father were both heterozygous for the R30X mutation. Fibroblasts from the patient were unresponsive to 1,25(OH)2D3 as shown by their failure to induce CYP24A1 gene expression, a marker of 1,25(OH)2D3 responsiveness. [3H]1,25(OH)2D3 binding and immunoblot analysis showed that the patient's cells expressed the VDR,K246 mutant protein; however, the amount of VDR,K246 mutant protein was significantly reduced compared with wildtype controls. In transactivation assays, the recreated VDR,K246 mutant was unresponsive to 1,25(OH)2D3. The ,K246 mutation abolished heterodimerization of the mutant VDR with RXR, and binding to the coactivators DRIP205 and SRC-1. However, the ,K246 mutation did not affect the interaction of the mutant VDR with the corepressor Hairless (HR). In summary, we describe a patient with compound heterozygous mutations in the VDR that results in HVDRR with alopecia. The R30X mutation truncates the VDR, whereas the ,K246 mutation prevents heterodimerization with RXR and disrupts coactivator interactions. [source] | |||||||||||||||||||||||||||||||||||||