scholarly journals CREB-1α Is Recruited to and Mediates Upregulation of the Cytochrome c Promoter during Enhanced Mitochondrial Biogenesis Accompanying Skeletal Muscle Differentiation

2008 ◽  
Vol 28 (7) ◽  
pp. 2446-2459 ◽  
Author(s):  
Andras Franko ◽  
Sabine Mayer ◽  
Gerald Thiel ◽  
Ludovic Mercy ◽  
Thierry Arnould ◽  
...  
Keyword(s):  
Mitochondrial Biogenesis ◽  
Critical Role ◽  
Dominant Negative ◽  
Mitochondrial Proteins ◽  
Luciferase Reporter ◽  
Mitochondrial Activity ◽  
Mobility Shift ◽  
Mitochondrial Dna Copy Number ◽  
Genes Encoding ◽  
Phosphorylated Creb

ABSTRACT To further understand pathways coordinating the expression of nuclear genes encoding mitochondrial proteins, we studied mitochondrial biogenesis during differentiation of myoblasts to myotubes. This energy-demanding process was accompanied by a fivefold increase of ATP turnover, covered by an eightfold increase of mitochondrial activity. While no change in mitochondrial DNA copy number was observed, mRNAs as well as proteins for nucleus-encoded cytochrome c, cytochrome c oxidase subunit IV, and mitochondrial transcription factor A (TFAM) increased, together with total cellular RNA and protein levels. Detailed analysis of the cytochrome c promoter by luciferase reporter, binding affinity, and electrophoretic mobility shift assays as well as mutagenesis studies revealed a critical role for cyclic AMP responsive element binding protein 1 (CREB-1) for promoter activation. Expression of two CREB-1 isoforms was observed by using specific antibodies and quantitative reverse transcription-PCR, and a shift from phosphorylated CREB-1Δ in myoblasts to phosphorylated CREB-1α protein in myotubes was shown, while mRNA ratios remained unchanged. Chromatin immunoprecipitation assays confirmed preferential binding of CREB-1α in situ to the cytochrome c promoter in myotubes. Overexpression of constitutively active and dominant-negative forms supported the key role of CREB-1 in regulating the expression of genes encoding mitochondrial proteins during myogenesis and probably also in other situations of enhanced mitochondrial biogenesis.

scholarly journals High glucose upregulates osteopontin expression by FoxO1 activation in macrophages

2019 ◽  
Vol 242 (2) ◽  
pp. 51-64 ◽  
Author(s):  
Qiongge Zhang ◽  
Chaoqun Wang ◽  
Yehua Tang ◽  
Qiangqiang Zhu ◽  
Yongcheng Li ◽  
...  
Keyword(s):  
High Glucose ◽  
Glycated Albumin ◽  
Critical Role ◽  
Dominant Negative ◽  
Raw 264.7 Cells ◽  
Luciferase Reporter ◽  
Dominant Negative Mutant ◽  
Diabetic Patients ◽  
Mobility Shift ◽  
Expression Levels

Hyperglycemia plays a major role in the development of diabetic macrovascular complications, including atherosclerosis and restenosis, which are responsible for the most of disability and mortality in diabetic patients. Osteopontin (OPN) is an important factor involved in atherogenesis, and hyperglycemia enhances the transcriptional activity of FoxO1 which is closely association with insulin resistance and diabetes. Here, we showed that plasma OPN levels were significantly elevated in type 2 diabetic patients and positively correlated with glycated albumin (GA). The more atherosclerotic lesions were observed in the aorta of diabetic ApoE−/− mice analyzed by Sudan IV staining. High glucose increased both the mRNA and protein expression levels of OPN and inhibited the phosphorylation of FoxO1 in RAW 264.7 cells. Overexpression of WT or constitutively active mutant FoxO1 promoted the expression levels of OPN, while the dominant-negative mutant FoxO1 decreased slightly the expression of OPN. Conversely, knockdown of FoxO1 reduced the expression of OPN. Luciferase reporter assay revealed that high glucose and overexpression of FoxO1 enhanced the activities of the OPN promoter region nt −1918 ~ −713. Furthermore, the interactions between FoxO1 and the OPN promoter were confirmed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). Our results suggest that high glucose upregulates OPN expression via FoxO1 activation, which would play a critical role in the development of diabetic atherogenesis.

scholarly journals A distal activation domain is critical in the regulation of the rat androgen receptor gene promoter

1993 ◽  
Vol 294 (3) ◽  
pp. 779-784 ◽  
Author(s):  
C S Song ◽  
S Her ◽  
M Slomczynska ◽  
S J Choi ◽  
M H Jung ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Nucleotide Sequence ◽  
Receptor Gene ◽  
Critical Role ◽  
Androgen Receptor Gene ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Cis Element

The far upstream region of the rat androgen receptor (AR) gene has been cloned, and the nucleotide sequence up to -2656 bp established. Nested deletion mutants of rat AR 5′ flanking sequences were ligated to the luciferase reporter gene, and their promoter activities were examined in transfected COS1 cells. Results show a critical cis-acting domain located between positions -960 and -940. Deletion of this cis element resulted in a greater than 90% decrease in the promoter activity. A nuclear protein that specifically binds to this 21-nucleotide sequence was identified by gel mobility shift analysis. The -960/-940 cis element has no identify to the binding sequence of any known transcription factor. Furthermore, the cognate binding protein is present in both rat and human (HeLa) cell nuclear extracts. We conclude that a novel trans-activator interacting at the -960/-940 region plays a critical role in the regulation of AR gene expression.

scholarly journals Divergent homeobox gene Hex regulates promoter of the Na+-dependent bile acid cotransporter

2000 ◽  
Vol 279 (2) ◽  
pp. G347-G355 ◽  
Author(s):  
Lee A. Denson ◽  
Saul J. Karpen ◽  
Clifford W. Bogue ◽  
Harris C. Jacobs
Keyword(s):  
Bile Acid ◽  
Homeobox Gene ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Specific Factor ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Mobility Shift ◽  
Luciferase Reporter Construct ◽  
G2 Cells

The divergent homeobox gene Hex is expressed in both developing and mature liver. A putative Hex binding site was identified in the promoter region of the liver-specific Na+-bile acid cotransporter gene ( ntcp), and we hypothesized that Hex regulates the ntcp promoter through this site. Successive 5′-deletions of the ntcp promoter in a luciferase reporter construct transfected into Hep G2 cells confirmed a Hex response element (HRE) within the ntcppromoter (nt −733/−714). Moreover, p-CMHex transactivated a heterologous promoter construct containing HRE multimers (p4xHRELUC), whereas a 5-bp mutation of the core HRE eliminated transactivation. A dominant negative form of Hex (p- Hex-DN) suppressed basal luciferase activity of p-4xHRELUC and inhibited activation of this construct by p-CMHex. Interestingly, p-CMHex transactivated the HRE in Hep G2 cells but not in fibroblast-derived COS cells, suggesting the possibility that Hex protein requires an additional liver cell-specific factor(s) for full activity. Electrophoretic mobility shift assays confirmed that liver and Hep G2 cells contain a specific nuclear protein that binds the native HRE. We have demonstrated that the liver-specific ntcp gene promoter is the first known target of Hex and is a useful tool for evaluating function of the Hex protein.

scholarly journals Hypoxia regulates the mitochondrial activity of hepatocellular carcinoma cells through HIF/HEY1/PINK1 pathway

2019 ◽  
Vol 10 (12) ◽  
Author(s):  
David Kung-Chun Chiu ◽  
Aki Pui-Wah Tse ◽  
Cheuk-Ting Law ◽  
Iris Ming-Jing Xu ◽  
Derek Lee ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Mitochondrial Biogenesis ◽  
Molecular Mechanisms ◽  
Hypoxia Inducible Factor ◽  
Notch Pathway ◽  
Redox Balance ◽  
Therapeutic Interventions ◽  
Luciferase Reporter ◽  
Mitochondrial Activity ◽  
Chip Sequencing

AbstractHypoxia is commonly found in cancers. Hypoxia, due to the lack of oxygen (O2) as the electron recipient, causes inefficient electron transfer through the electron transport chain at the mitochondria leading to accumulation of reactive oxygen species (ROS) which could create irreversible cellular damages. Through hypoxia-inducible factor 1 (HIF-1) which elicits various molecular events, cells are able to overcome low O2. Knowledge about the new molecular mechanisms governed by HIF-1 is important for new therapeutic interventions targeting hypoxic tumors. Using hepatocellular carcinoma (HCC) as a model, we revealed that the HIF-1 and the Notch signaling pathways cross-talk to control mitochondrial biogenesis of cancer cells to maintain REDOX balance. From transcriptome sequencing, we found that HEY1, a transcriptional repressor, in the NOTCH pathway was consistently induced by hypoxia in HCC cell lines. We identified a strong hypoxia response element (HRE) in HEY1 by chromatin immunoprecipitation (ChIP) and luciferase reporter assays. Transcriptome and ChIP sequencing further identified PINK1, a gene essential for mitochondrial biogenesis, as a novel transcriptional target of HEY1. HCC cells with HEY1 knockdown re-expressed PINK1. HEY1 and PINK1 expressions inversely correlated in human HCC samples. Overexpression of HEY1 and under-expression of PINK1 were detected in human HCC and associated with poor clinical outcomes. Functionally, we found that overexpression of HEY1 or knockdown of PINK1 consistently reduced mitochondrial cristae, mitochondrial mass, oxidative stress level, and increased HCC growth.

scholarly journals Regulation of Interleukin-6 Promoter Activation in Gastric Epithelial Cells Infected withHelicobacter pylori

2005 ◽  
Vol 16 (10) ◽  
pp. 4954-4966 ◽  
Author(s):  
Hong Lu ◽  
Jeng Yih Wu ◽  
Takahiko Kudo ◽  
Tomoyuki Ohno ◽  
David Y. Graham ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Signal Transduction Pathway ◽  
Dominant Negative ◽  
Gastric Epithelium ◽  
Luciferase Reporter ◽  
Gastric Epithelial Cells ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
H Pylori ◽  
Cag Pai

The regulation of Helicobacter pylori induced interleukin (IL)-6 in the gastric epithelium remains unclear. Primary gastric epithelial cells and MKN28 cells were cocultured with H. pylori and its isogenic cag pathogenicity island (PAI) mutant and/or oipA mutants. H. pylori infection-induced IL-6 mRNA expression and IL-6 protein production, which was further enhanced by the cag PAI and OipA. Luciferase reporter gene assays and electrophoretic mobility shift assays showed that full IL-6 transcription required binding sites for nuclear factor-κB (NF-κB), cAMP response element (CRE), CCAAT/enhancer binding protein (C/EBP), and activator protein (AP)-1. The cag PAI and OipA were involved in binding to NF-κB, AP-1, CRE, and C/EBP sites. The cag PAI activated the extracellular signal-regulated kinase (ERK) and Jun N-terminal kinase (JNK) pathways; OipA activated the p38 pathway. Transfection of dominant negative G-protein confirmed roles for Raf, Rac1, and RhoA in IL-6 induction. Overall, the cag PAI-related IL-6 signal transduction pathway involved the Ras/Raf/MEK1/2/ERK/AP-1/CRE pathway and the JNK/AP-1/CRE pathway; the OipA-related pathway is p38/AP-1/CRE and both the cag PAI and OipA appear to be involved in the RhoA/Rac1/NF-κB pathway. Combination of different pathways by the cag PAI and OipA will lead to the maximum IL-6 induction.

scholarly journals CLUH regulates mitochondrial biogenesis by binding mRNAs of nuclear-encoded mitochondrial proteins

2014 ◽  
Vol 207 (2) ◽  
pp. 213-223 ◽  
Author(s):  
Jie Gao ◽  
Désirée Schatton ◽  
Paola Martinelli ◽  
Henriette Hansen ◽  
David Pla-Martin ◽  
...  
Keyword(s):  
Mitochondrial Biogenesis ◽  
Mitochondrial Proteins ◽  
Control Mechanisms ◽  
Messenger Ribonucleic Acid ◽  
Mrna Binding Protein ◽  
Protein Biogenesis ◽  
Genes Encoding ◽  
Rna Immunoprecipitation ◽  
Tyrosinated Tubulin ◽  
Mrna Binding

Mitochondrial function requires coordination of two genomes for protein biogenesis, efficient quality control mechanisms, and appropriate distribution of the organelles within the cell. How these mechanisms are integrated is currently not understood. Loss of the Clu1/CluA homologue (CLUH) gene led to clustering of the mitochondrial network by an unknown mechanism. We find that CLUH is coregulated both with genes encoding mitochondrial proteins and with genes involved in ribosomal biogenesis and translation. Our functional analysis identifies CLUH as a cytosolic messenger ribonucleic acid (RNA; mRNA)–binding protein. RNA immunoprecipitation experiments followed by next-generation sequencing demonstrated that CLUH specifically binds a subset of mRNAs encoding mitochondrial proteins. CLUH depletion decreased the levels of proteins translated by target transcripts and caused mitochondrial clustering. A fraction of CLUH colocalizes with tyrosinated tubulin and can be detected close to mitochondria, suggesting a role in regulating transport or translation of target transcripts close to mitochondria. Our data unravel a novel mechanism linking mitochondrial biogenesis and distribution.

scholarly journals c-Myb Contributes to G2/M Cell Cycle Transition in Human Hematopoietic Cells by Direct Regulation of Cyclin B1 Expression

2007 ◽  
Vol 27 (6) ◽  
pp. 2048-2058 ◽  
Author(s):  
Yuji Nakata ◽  
Susan Shetzline ◽  
Chizuko Sakashita ◽  
Anna Kalota ◽  
Ravikumar Rallapalli ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Critical Role ◽  
Hematopoietic Cells ◽  
Biological Significance ◽  
Cyclin B1 ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
M Cell ◽  
Cell Cycle Transition

ABSTRACTMyb family proteins are ubiquitously expressed transcription factors. In mammalian cells, they play a critical role in regulating the G1/S cell cycle transition but their role in regulating other cell cycle checkpoints is incompletely defined. Herein, we report experiments which demonstrate that c-Myb upregulates cyclin B1 expression in normal and malignant human hematopoietic cells. As a result, it contributes directly to G2/M cell cycle progression. In cell lines and primary cells, cyclin B1 levels varied directly with c-Myb expression. Chromatin immunoprecipitation assays, mutation analysis, and luciferase reporter assays revealed that c-Myb bound the cyclin B1 promoter preferentially at a site just downstream of the transcriptional start site. The biological significance of c-Myb, versus B-Myb, binding the cyclin B1 promoter was demonstrated by the fact that expression of inducible dominant negative c-Myb in K562 cells accelerated their exit from M phase. In addition, expression of c-Myb in HCT116 cells rescued cyclin B1 expression after B-mybexpression was silenced with small interfering RNA. These results suggest that c-Myb protein plays a previously unappreciated role in the G2/M cell cycle transition of normal and malignant human hematopoietic cells and expands the known repertoire of c-mybfunctions in regulating human hematopoiesis.

scholarly journals RORα Augments Thyroid Hormone Receptor-Mediated Transcriptional Activation*

Endocrinology ◽  
1999 ◽  
Vol 140 (3) ◽  
pp. 1356-1364 ◽  
Author(s):  
Noriyuki Koibuchi ◽  
Ying Liu ◽  
Harumi Fukuda ◽  
Akira Takeshita ◽  
Paul M. Yen ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Transcriptional Activation ◽  
Hormone Receptor ◽  
Hormone Receptors ◽  
Thyroid Hormone Receptor ◽  
Critical Role ◽  
Nuclear Hormone Receptor ◽  
Dominant Negative ◽  
Mobility Shift ◽  
Response Element

Abstract This study is designed to clarify the role of an orphan nuclear hormone receptor, RORα, on thyroid hormone (TH) receptor (TR)-mediated transcription on a TH-response element (TRE). A transient transfection study using various TREs [i.e., F2 (chick lysozyme TRE), DR4 (direct repeat), and palindrome TRE] and TR and RORα1 was performed. When RORα1 and TR were cotransfected into CV1 cells, RORα1 enhanced the transactivation by liganded-TR on all TREs tested without an effect on basal repression by unliganded TR. By electrophoretic mobility shift assay, on the other hand, although RORα bound to all TREs tested as a monomer, no (or weak) TR and RORα1 heterodimer formation was observed on various TREs except when a putative ROR-response element was present. The transactivation by RORα1 on a ROR-response element, which does not contain a TRE, was not enhanced by TR. The effect of RORα1 on the TREs is unique, because, whereas other nuclear hormone receptors (such as vitamin D receptor) may competitively bind to TRE to exert dominant negative function, RORα1 augmented TR action. These results indicate that RORα1 may modify the effect of liganded TR on TH-responsive genes. Because TR and RORα are coexpressed in cerebellar Purkinje cells, and perinatal hypothyroid animals and RORα-disrupted animals show similar abnormalities of this cell type, cross-talk between these two receptors may play a critical role in Purkinje cell differentiation.

scholarly journals Nicotine Modulates CTSS (Cathepsin S) Synthesis and Secretion Through Regulating the Autophagy-Lysosomal Machinery in Atherosclerosis

2020 ◽  
Vol 40 (9) ◽  
pp. 2054-2069 ◽  
Author(s):  
Huaner Ni ◽  
Shuang Xu ◽  
Hangwei Chen ◽  
Qiuyan Dai
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Nuclear Translocation ◽  
Critical Role ◽  
Mammalian Target Of Rapamycin ◽  
Immunofluorescent Staining ◽  
Luciferase Reporter ◽  
Cathepsin S ◽  
Mobility Shift

Objective: Increased CTSS (cathepsin S) has been reported to play a critical role in atherosclerosis progression. Both CTSS synthesis and secretion are essential for exerting its functions. However, the underlying mechanisms contributing to CTSS synthesis and secretion in atherosclerosis remain unclear. Approach and Results: In this study, we showed that nicotine activated autophagy and upregulated CTSS expression in vascular smooth muscle cells and in atherosclerotic plaques. Western blotting and immunofluorescent staining showed that nicotine inhibited the mTORC1 (mammalian target of rapamycin complex 1) activity, promoted the nuclear translocation of TFEB (transcription factor EB), and upregulated the expression of CTSS. Chromatin immunoprecipitation-qualificative polymerase chain reaction, electrophoretic mobility shift assay, and luciferase reporter assay further demonstrated that TFEB directly bound to the CTSS promoter. mTORC1 inhibition by nicotine or rapamycin promoted lysosomal exocytosis and CTSS secretion. Live cell assays and IP-MS (immunoprecipitation-mass spectrometry) identified that the interactions involving Rab10 (Rab10, member RAS oncogene family) and mTORC1 control CTSS secretion. Nicotine promoted vascular smooth muscle cell migration by upregulating CTSS, and CTSS inhibition suppressed nicotine-induced atherosclerosis in vivo. Conclusions: We concluded that nicotine mediates CTSS synthesis and secretion through regulating the autophagy-lysosomal machinery, which offers a potential therapeutic target for atherosclerosis treatment.

scholarly journals Prolactin regulates ZNT2 expression through the JAK2/STAT5 signaling pathway in mammary cells

2009 ◽  
Vol 297 (2) ◽  
pp. C369-C377 ◽  
Author(s):  
Linxi Qian ◽  
Veronica Lopez ◽  
Young Ah Seo ◽  
Shannon L. Kelleher
Keyword(s):  
Signaling Pathway ◽  
Promoter Activity ◽  
Critical Role ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Luciferase Reporter ◽  
Primary Role ◽  
Jak2 Inhibitor ◽  
Hc11 Cells

The zinc transporter ZnT2 ( SLC30A2) plays an important role in zinc secretion into milk during lactation. The physiological process of mammary gland secretion is regulated through complex integration of multiple lactogenic hormones. Prolactin plays a primary role in this regulation through the activation of various signaling cascades including Jak2/STAT5, mitogen-activated protein kinase (MAPK), p38, and phosphatidylinositol 3-kinase (PI3K). The precise mechanisms that regulate the transfer of specific nutrients such as zinc into milk are not well understood. Herein we report that prolactin increased ZnT2 abundance transcriptionally in cultured mammary epithelial (HC11) cells. To delineate the responsible mechanisms, we first determined that prolactin-mediated ZnT2 induction was inhibited by pretreatment with the Jak2 inhibitor AG490 but not by the MAPK inhibitor PD-98059. Using a luciferase reporter assay, we demonstrated that ZnT2 promoter activity was increased by prolactin treatment, which was subsequently abolished by expression of a dominant-negative STAT5 construct, implicating the Jak2/STAT5 signaling pathway in the transcriptional regulation of ZnT2. Two putative consensus STAT5 binding sequences in the ZnT2 promoter were identified (GAS1:−674 to −665 and GAS2:−377 to −368). Mutagenesis of the proximal GAS2 element resulted in complete abrogation of PRL-induced ZnT2 promoter activity. The promoter incorporating the distal GAS1 mutation was only able to respond to very high PRL concentrations. Results from both the mutagenesis and gel shift assays indicated that a cooperative relationship exists between GAS1 and GAS2 for PRL-induced activation; however, the proximal GAS2 plays a more critical role in STAT5-mediated signal transduction compared with the GAS1 element. Finally, chromosome immunoprecipition assay further confirmed that prolactin activates STAT5 binding to the ZnT2 promoter in vivo. Taken together, these results illustrate that prolactin regulates the transcription of ZnT2 through activation of the Jak2/STAT5 signaling pathway to assist in providing optimal zinc for secretion into milk during lactation.

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