Characterization of cis-elements required for osmotic response of rat Na+/H+exchanger-2 (NHE-2) gene

1999 ◽  
Vol 277 (4) ◽  
pp. R1112-R1119 ◽  
Author(s):  
Liqun Bai ◽  
James F. Collins ◽  
Yunhua L. Muller ◽  
Hua Xu ◽  
Pawel R. Kiela ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Dna Sequences ◽  
Collecting Duct ◽  
Specific Interaction ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Response Elements ◽  
Osmotic Response ◽  
Responsive Element

The Na+/H+exchanger ( NHE-2) has been implicated in osmoregulation in the kidney, because it transports Na+ across the cell membrane and efficiently alters intracellular osmolarity. On hyperosmotic stress, NHE-2 mRNA increases in abundance in mouse inner medullary collecting duct (mIMCD-3) cells, suggesting possible transcriptional regulation. To investigate the molecular mechanism of potential transcriptional regulation of NHE-2 by hyperosmolarity, we have functionally characterized the 5′-flanking region of the gene in mIMCD-3 cells. Transient transfection of luciferase reporter gene constructs revealed a novel cis-acting element, which we call OsmoE (osmotic-responsive element, bp −808 to −791, GGGCCAGTTGGCGCTGGG), and a TonE-like element (tonicity-responsive element, bp −1201 to −1189, GCTGGAAAACCGA), which together are shown to be responsible for hyperosmotic induction of the NHE-2gene. Electrophoretic mobility shift assays suggest that different DNA-protein interactions occur between these two osmotic response elements. However, both DNA sequences were shown to specifically bind nuclear proteins that dramatically increase in abundance under hyperosmotic conditions. Isolation of trans-acting factors and characterization of their specific interaction with these osmotic response elements will further elucidate the transcriptional mechanisms controlling NHE-2 gene expression under hyperosmolar conditions.

scholarly journals Characterization of a Viral Phosphoprotein Binding Site on the Surface of the Respiratory Syncytial Nucleoprotein

2012 ◽  
Vol 86 (16) ◽  
pp. 8375-8387 ◽  
Author(s):  
Marie Galloux ◽  
Bogdan Tarus ◽  
Ilfad Blazevic ◽  
Jenna Fix ◽  
Stéphane Duquerroy ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Specific Interaction ◽  
Viral Rna ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Potential Candidate ◽  
Luciferase Reporter Gene ◽  
Interaction Domain ◽  
Rna Complex

The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (PCTD) and N. However, the P binding region on N remains to be identified. In this study, glutathioneS-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (NNTD) as a P binding domain. A biochemical characterization of the PCTDand molecular modeling of the NNTDallowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the PCTDinteraction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolishedin vitroandin vivoP-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.

Potency of isothiocyanates to induce luciferase reporter gene expression via the electrophile-responsive element from murine glutathione S-transferase Ya

2009 ◽  
Vol 23 (4) ◽  
pp. 617-621 ◽  
Author(s):  
Martijn Vermeulen ◽  
Anne-Marie M.J.F. Boerboom ◽  
Barry M.G. Blankvoort ◽  
Jac M.M.J.G. Aarts ◽  
Ivonne M.C.M. Rietjens ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Reporter Gene Expression ◽  
Luciferase Reporter ◽  
Glutathione S Transferase ◽  
Luciferase Reporter Gene ◽  
Responsive Element

scholarly journals Phorbol 12-myristate 13-acetate (PMA) responsive sequence in Gαq promoter during megakaryocytic differentiation

2008 ◽  
Vol 100 (05) ◽  
pp. 821-828 ◽  
Author(s):  
Gauthami Jalagadugula ◽  
Danny N. Dhanasekaran ◽  
A. Koneti Rao
Keyword(s):  
Promoter Sequence ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Luciferase Reporter Gene ◽  
Nuclear Extracts ◽  
Upstream Promoter ◽  
Hel Cells ◽  
The Difference ◽  
Responsive Element ◽  
Increased Activity

SummaryGαq plays a major role in platelet signal transduction, but little is known regarding its transcriptional regulation. We have reported that Gαq is upregulated during phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic transformation of human erythroleukemia (HEL) cells and regulated by EGR-1, an early growth transcription factor. These studies focused on the initial 238 bp of the 5’ upstream region of the Gαq gene. In the present studies we characterize a minimal region -1042/-1037 bp from ATG in the 5’ upstream of the Gαq promoter that is associated with PMA responsiveness. In luciferase reporter gene studies in HEL cells, Gαq 5’ upstream promoter sequence -1042/-1 showed an about four-fold increased activity in PMA-treated compared to untreated cells. Deletion of 6-nt-1042/-1037 eliminated the difference. Gel-shift studies on Gαq probe (-1042/-1012 bp) revealed binding of EGR-1 with PMA-treated but not untreated nuclear extracts, and this was dependent on the sequence –1042/-1037.Silencing of endogenous EGR-1 inhibited Gαq induction by PMA. MEK/ERK inhibitor U0126 blocked PMA effect on promoter activity of the -1042/-1 construct. In conclusion, EGR-1 binding to sequence –1042/-1037 bp in Gαq promoter mediates the induction of Gαq gene by PMA via the MEK/ERK signaling pathway. These studies provide the first evidence of a PMA-responsive element in Gαq promoter, and new insights into regulation of Gαq gene by EGR-1.

scholarly journals Albuminuria induces a proinflammatory and profibrotic response in cortical collecting ducts via the 24p3 receptor

2013 ◽  
Vol 305 (7) ◽  
pp. F1053-F1063 ◽  
Author(s):  
Eva Dizin ◽  
Udo Hasler ◽  
Stellor Nlandu-Khodo ◽  
Marc Fila ◽  
Isabelle Roth ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
Collecting Duct ◽  
Transforming Growth Factor Β ◽  
Luciferase Reporter ◽  
Lipocalin 2 ◽  
Luciferase Reporter Gene ◽  
Puromycin Aminonucleoside ◽  
Gene Assay ◽  
Albumin Endocytosis

Albuminuria is strongly associated with progressive kidney tubulo-interstitial damage and chronic kidney disease (CKD) progression. In proteinuric nephropathies, albumin reabsorption by the proximal tubule is saturated and the distal nephron is exposed to high concentrations of luminal albumin that may produce adverse effects. Since proximal tubular cells exposed to albuminuria exhibit a proinflammatory and profibrotic response, we assessed the effect of albuminuria in the collecting duct (CD). With the use of kidney sections and isolated cortical CDs (CCDs) from puromycin-aminonucleoside-induced nephrotic rats (PAN rats) exhibiting proteinuria, immunofluorescence microscopy revealed internalized albumin in CD cells. In these proteinuric rats, increased expression levels of cytokines and profibrotic signaling markers were detected in isolated CCDs and bands of inflammatory fibrosis could be observed around CDs. Albumin endocytosis was confirmed by FITC-albumin uptake in cultured murine CCD (mCCDcl1) cells. Exposure of mCCDcl1 cells to albumin induced NF-κB activation as assessed by luciferase reporter gene assay, nuclear translocation of NF-κB p65 subunit, and increased NF-κB target gene expression. Moreover, albuminuria-like condition results in transforming growth factor-β1 (TGF-β1) overexpression and the upregulation of profibrotic signaling markers such as Snail or vimentin via an autocrine mechanism. In mCCDcl1 cells, neutrophil gelatinase-associated lipocalin (NGAL)/lipocalin-2/24p3 receptor (24p3R) mediates albumin endocytosis as well as activation of NF-κB and TGF-β1 signaling pathways. Therefore, CD may play a key role in initiation and/or progression of inflammation and fibrosis in response to proteinuria.

scholarly journals MicroRNA-194 (miR-194) regulates ROMK channel activity by targeting intersectin 1

2014 ◽  
Vol 306 (1) ◽  
pp. F53-F60 ◽  
Author(s):  
Dao-Hong Lin ◽  
Peng Yue ◽  
Chengbiao Zhang ◽  
Wen-Hui Wang
Keyword(s):  
Collecting Duct ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Seed Sequence ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Luciferase Reporter Gene ◽  
High K ◽  
Low K

The aim of the study is to explore the role of miR-194 in mediating the effect of high-K (HK) intake on ROMK channel. Northern blot analysis showed that miR-194 was expressed in kidney and that HK intake increased while low-K intake decreased the expression of miR-194. Real-time PCR analysis further demonstrated that HK intake increased the miR-194 expression in the cortical collecting duct. HK intake decreased the expression of intersectin 1 (ITSN1) which enhanced With-No-Lysine Kinase (WNK)-induced endocytosis of ROMK. Expression of miR-194 mimic decreased luciferase reporter gene activity in HEK293 T cells transfected with ITSN-1–3′UTR containing the complementary seed sequence for miR-194. In contrast, transfection of miR-194 inhibitor increased the luciferase activity. This effect was absent in the cells transfected with mutated 3′UTR of ITSN1 in which the complimentary seed sequence was deleted. Moreover, the inhibition of miR-194 expression increased the protein level of endogenous ITSN1 in HEK293T cells. Expression of miR-194 mimic also decreased the translation of exogenous ITSN1 in the cells transfected with the ITSN1 containing 3′UTR but not with 3′UTR-free ITSN1. Expression of pre-miR-194 increased K currents and ROMK expression in the plasma membrane in ROMK-transfected cells. Coexpression of ITSN1 reversed the stimulatory effect of miR-194 on ROMK channels. This effect was reversed by coexpression of ITSN1. We conclude that miR-194 regulates ROMK channel activity by modulating ITSN1 expression thereby enhancing ITSN1/WNK-dependent endocytosis. It is possible that miR-194 is involved in mediating the effect of a HK intake on ROMK channel activity.

Characterization of the rat intestinal Fc receptor (FcRn) promoter: transcriptional regulation of FcRn gene by the Sp family of transcription factors

2004 ◽  
Vol 286 (6) ◽  
pp. G922-G931 ◽  
Author(s):  
Lingling Jiang ◽  
Jiafang Wang ◽  
R. Sergio Solorzano-Vargas ◽  
Hugh V. Tsai ◽  
Edgar M Gutierrez ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Cell Lines ◽  
Promoter Region ◽  
Core Promoter ◽  
Dnase I ◽  
Fc Receptor ◽  
Minimal Promoter ◽  
Luciferase Reporter ◽  
Core Promoter Region

The regulatory elements that control the transcriptional regulation of the intestinal Fc receptor ( FcRn) have not been elucidated. The objective of this study was to characterize the core promoter region of the rat FcRn gene. Chimeric clones that contained various regions of the promoter located upstream of the luciferase reporter were transiently transfected into either IEC-6 or Caco-2 cell lines and nuclear extracts were used to perform DNase I footprint and DNA binding assays (EMSA). Transfection of chimeric upstream nested deletions-luciferase reporter clones into either of these cell lines supported robust reporter activity and identified the location of the minimal promoter at −157/+135. DNase I footprint analysis revealed two complexes located within the gene's core promoter region, and site-directed mutagenesis identified two regions that were critical to maintain basal expression. EMSA identified the presence of five Sp elements within the immediate promoter region that are capable of binding members of the Sp family of proteins. Among the five Sp elements, one element appears to not bind Sp1, Sp2, or Sp3 while influencing the interaction of Sp proteins with an adjacent Sp site. Overexpression of either Sp1 or Sp3 augments activity of the minimal promoter in Sp-deficient Drosophila SL2 cells. In summary, we report on the characterization of the rat FcRn minimal promoter, including the characterization of five Sp elements within this region that interact with members of the Sp family of transcriptional factors and drive promoter activity in intestinal cell lines.

scholarly journals Hormonal regulation of the human sterol 27-hydroxylase gene CYP27A1

2003 ◽  
Vol 372 (2) ◽  
pp. 529-534 ◽  
Author(s):  
Zufan ARAYA ◽  
Wanjin TANG ◽  
Kjell WIKVALL
Keyword(s):  
Reporter Gene ◽  
Hormonal Regulation ◽  
Promoter Activity ◽  
Luciferase Activity ◽  
Luciferase Reporter ◽  
Signal Transduction Pathways ◽  
Cytochrome P450 Enzyme ◽  
Luciferase Reporter Gene ◽  
Response Elements ◽  
P450 Enzyme

The mitochondrial sterol 27-hydroxylase (CYP27A1) is a multifunctional cytochrome P450 enzyme that catalyses important hydroxylations in the biosynthesis of bile acids and bioactivation of vitamin D3. Previous results [Babiker, Andersson, Lund, Xiu, Deeb, Reshef, Leitersdorf, Diczfalusy and Björkhem (1997) J. Biol. Chem. 272, 26253–26261] suggest that CYP27A1 plays an important role in cholesterol homoeostasis and affects atherogenesis. In the present study, the regulation of the human CYP27A1 gene by growth hormone (GH), insulin-like growth factor-1 (IGF-1), dexamethasone, thyroid hormones and PMA was studied. HepG2 cells were transfected transiently with luciferase reporter gene constructs containing DNA fragments flanking the 5′-region of the human CYP27A1 gene. GH, IGF-1 and dexamethasone increased the promoter activity by 2–3-fold, whereas thyroxine (T4) and PMA repressed the activity significantly when measured with luciferase activity expressed in the cells. The endogenous CYP27A1 enzyme activity in the cells was stimulated by GH, IGF-1 and dexamethasone, whereas T4 and PMA inhibited the activity. Experiments with progressive deletion/luciferase reporter gene constructs indicated that the response elements for GH may be localized in a region upstream to position −1094 bp. The putative response elements for dexamethasone were mapped to positions between −792 and −1095 bp. The −451 bp fragment of the human CYP27A1 gene was found to confer the activation by IGF-1, and the inhibition by T4 and PMA. Results of the present study suggest that CYP27A1 is regulated in human cells by hormones and signal-transduction pathways.

Abstract 09: Mtor Inhibition Decreases Ligand-induced Mineralocorticoid Receptor Activation

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Yusuf Ali ◽  
Elise P Gomez-sanchez ◽  
Celso E Gomez-sanchez
Keyword(s):  
Reporter Gene ◽  
Transcriptional Activity ◽  
Collecting Duct ◽  
Receptor Activation ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Mtor Inhibition ◽  
Positive Effects ◽  
Mtor Phosphorylation

Introduction: ULK1 phosphorylates the MR at S843, decreasing its ligand binding and transcriptional activity. Angiotensin II-induced mTOR phosphorylation of ULK1 inactivates ULK1, preventing its phosphorylation of MR. Aim: Further elucidate the role of mTOR in the regulation of MR transcriptional activity. Methods: M1 mouse cortical collecting duct cells stably transduced with the rat MR cDNA and a MMTV- Gaussia luciferase reporter gene, were incubated with an mTOR activator and several inhibitors, +/- aldosterone or corticosterone. Similar studies were done after lentiviral transduction of CRISPR/gRNA for raptor and rictor genes or mutated MR (mu/S843A) cDNA. Results: mTOR inhibition significantly decreased ligand activation of the MR reporter gene, while the mTOR activator MHY1485 had no effect suggesting that mTOR is tonically active. MR activation induced by aldosterone and corticosterone was also decreased by CRISPR/gRNA gene knockdown of raptor and rictor, the adaptors of mTOR complex 1 and 2, respectively, supporting a role for mTOR. The mTOR inhibitor AZD8055 (AZD) reduced phospho-ULK1 and attenuated ligand-mediated MR transactivation in a dose-dependent manner. The ULK1 inhibitor MRT68921 increased MR transactivation. We speculated that mTOR decreased ULK1 activity by phosphorylating it, thereby preventing ULK1 phosphorylation of the MR at Serine (S843). However, when M1 cells were transduced with an MR cDNA in which S843 was replaced with Alanine that cannot be phosphorylated, ligand-induced activation of the mu/S843A MR was still decreased by AZD, but unchanged by MRT68921. This suggests that mTOR has an additional effect on MR activity unrelated to ULK1 activity. AZD also decreased P70S6K and AKT phosphorylation in these cells. Conclusions: mTOR phosphorylation of ULK1 prevents its phosphorylation of the MR and reduction of MR transcriptional activity. mTOR inhibitors and deletion of raptor and rictor decreased MR transcriptional activity. mTOR has additional positive effects on MR activity possibly related to its phosphorylation of AKT and P70S6K. Inhibition of mTOR action may be a useful target for mitigating excessive MR activation.

scholarly journals Cloning and Functional Analysis of Rat Tweety-Homolog 1 Gene Promoter

2021 ◽  
Author(s):  
Malgorzata Gorniak-Walas ◽  
Karolina Nizinska ◽  
Katarzyna Lukasiuk
Keyword(s):  
Transcriptional Regulation ◽  
Reporter Gene ◽  
Hippocampal Neurons ◽  
Gene Promoter ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay

AbstractTweety-homolog 1 protein (Ttyh1) is abundantly expressed in neurons in the healthy brain, and its expression is induced under pathological conditions. In hippocampal neurons in vitro, Ttyh1 was implicated in the regulation of primary neuron morphology. However, the mechanisms that underlie transcriptional regulation of the Ttyh1 gene in neurons remain elusive. The present study sought to identify the promoter of the Ttyh1 gene and functionally characterize cis-regulatory elements that are potentially involved in the transcriptional regulation of Ttyh1 expression in rat dissociated hippocampal neurons in vitro. We cloned a 592 bp rat Ttyh1 promoter sequence and designed deletion constructs of the transcription factors specificity protein 1 (Sp1), E2F transcription factor 3 (E2f3), and achaete-scute homolog 1 (Ascl1) that were fused upstream of a luciferase reporter gene in pGL4.10[luc2]. The luciferase reporter gene assay showed the possible involvement of Ascl1, Sp1, and responsive cis-regulatory elements in Ttyh1 expression. These findings provide novel information about Ttyh1 gene regulation in neurons.

scholarly journals Genomic structure and transcriptional regulation of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene

2003 ◽  
Vol 31 (1) ◽  
pp. 105-121 ◽  
Author(s):  
A Nandy ◽  
S Jenatschke ◽  
B Hartung ◽  
K Milde-Langosch ◽  
AM Bamberger ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Binding Sites ◽  
Biological Activities ◽  
Genomic Structure ◽  
Start Codon ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Flanking Sequence ◽  
Luciferase Reporter Gene ◽  
Mobility Shift

The NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is a catabolic enzyme that controls the biological activities of prostaglandins by converting them into inactive keto-metabolites. Here we report the genomic organisation of the complete human PGDH gene and characterise its transcriptional regulation. The PGDH gene spans about 31 kb on chromosome 4 and contains 7 exons. Within 2.4 kb of the 5'-flanking sequence we identified two regions with clustered putative transcription factor binding sites. The distal promoter element PGDH-DE (positions-2152/-1944 relative to the start codon) contains binding sites for Ets and activating protein-1 (AP-1) flanked by two cAMP-responsive element-binding protein binding sites (CREB1, CREB2), whereas the proximal element PGDH-PE (-235/-153) includes an Ets and an AP-1 binding sequence. By electrophoretic mobility shift assay, no high affinity binding of Ets or AP-1 factors was observed with PGDH-PE, whereas we confirmed interaction of members of the Ets, AP-1 and CREB families of transcription factors with PGDH-DE. Transcriptional control of the PGDH promoter was assessed by transiently transfecting JEG-3 choriocarcinoma cells. A luciferase reporter gene construct containing the PGDH-PE was not induced by c-jun/c-fos in the absence or presence of co-expressed Ets-1. A construct carrying the PGDH-DE in front of the minimal homologous promoter was activated by co-transfection of expression vectors for AP-1 proteins. Mutation of the AP-1 or CREB2 site reduced the response to c-jun/c-fos, whereas mutation of the Ets site of the distal element reduced basal promoter activity. CREB activated the PGDH-DE construct through the CREB1 site. These results defined the distal element as an integrator of transcriptional regulation by AP-1, Ets and CREB proteins.

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