Role of AP2 consensus sites in regulation of rat Npt2 (sodium-phosphate cotransporter) promoter

2000 ◽  
Vol 278 (3) ◽  
pp. F406-F416 ◽  
Author(s):  
C. Shachaf ◽  
K. L. Skorecki ◽  
M. Tzukerman
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Proximal Tubule ◽  
Cell Lines ◽  
Promoter Activity ◽  
Promoter Sequence ◽  
Luciferase Reporter ◽  
Inverted Repeats ◽  
Proximal Tubule Cell ◽  
Flanking Sequence

Expression of the Npt2 gene, encoding the type II sodium-dependent phosphate cotransporter, is restricted to renal proximal tubule epithelium. We have isolated a 4,740-bp fragment of the 5′-flanking sequence of the rat Npt2 gene, identified the transcription initiation site, and demonstrated that this 5′-flanking sequence drives luciferase-reporter gene expression, following transfection in the proximal tubule cell-derived opossum kidney (OK) cell line but not in unrelated cell lines. Analysis of the promoter sequence revealed the presence of 10 consensus binding motifs for the AP2 transcription factor. Transient transfection assays revealed an important effect of the number of tandemly repeated AP2 sites in enhancing promoter activity. The promoter sequence also revealed a pair of inverted repeats enclosing 1,324 bp of intervening sequence and containing 8 of the total 10 AP2 consensus sites in the promoter sequence. Deletion or reversal of orientation of the distal inverted repeat resulted in marked enhancement of promoter activity. Electrophoretic mobility shift analysis revealed a distinct pattern of transcription factor binding to oligonucleotides containing AP2 sites, using nuclear extracts from OK cells, compared with unrelated cell lines. Taken together, these results suggest an important role for AP2 consensus binding sites in regulating Npt2 gene expression and suggest a mechanism of regulation mediated by the interaction of inverted repeats enclosing these sites.

scholarly journals HCK Transcription Is Regulated By AP1, NF-Kb and STAT3 Transcription Factors in MYD88 Mutated WM and ABC-DLBCL Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2931-2931
Author(s):  
Xia Liu ◽  
Jiaji G Chen ◽  
Jie Chen ◽  
Lian Xu ◽  
Nicholas Tsakmaklis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cell Lines ◽  
Binding Sites ◽  
Research Funding ◽  
Promoter Activity ◽  
Promoter Sequence ◽  
Wild Type ◽  
Mutation Status ◽  
Bcr Signaling

Abstract Hematopoietic cell kinase (HCK) is a member of the SRC family of tyrosine kinases (SFKs). HCK transcription is aberrantly upregulated in Waldenström's Macroglobulinemia (WM) and Activated B-cell (ABC) subtype Diffuse Large B-cell Lymphoma (DLBCL) in response to activating mutations in MYD88 (Yang et al, Blood 2016). To clarify the mechanism responsible for the aberrant upregulation of HCK transcription inMYD88 mutated cells, we analyzed the promoter sequence of HCK using PROMO and identified consensus binding sites for transcription factors (AP1, NF-kB, STAT3, and IRF1) that are regulated by mutated MYD88 (Ngo et al, Nature 2011; Treon et al, NEJM 2012; Yang et al, Blood 2013; Juilland et al, Blood 2016; Yang et al, Blood 2016). We performed Chromatin Immuno-precipitation (ChIP) assays using ChIP grade antibodies to JunB, c-Jun, NF-kB-p65, STAT3 and IRF1 in MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8, HBL-1, OCI-Ly3) cells that highly express HCK transcripts, as well as wild type MYD88 expressing GCB DLBCL (OCI-Ly7, OCI-Ly19) cells that show low HCK transcription. Following ChIP, a HCK promoter specific quantitative PCR assay was used to detect HCK promoter sequences. These studies showed that JunB, NF-kB-p65 and STAT3 bound more robustly to the HCK promoter in MYD88 mutated WM and ABC-DLBCL cells versus MYD88 wild type GCB DLBCL cell lines, while c-Jun bound more abundantly to the HCK promoter sequence in all DLBCL cell lines, regardless of MYD88 mutation status. In contrast c-Jun binding was low in MYD88 mutated WM cells. IRF1 binding to the HCK promoter was similar in all cell lines, regardless of the MYD88 mutation status. To further investigate HCK regulation, we developed an HCK promoter driven luciferase reporter vector (WT) with mutated AP-1 binding (AP1-mu-1~6), NF-kB binding (NF-kB-mu-1~5), and STAT3 binding (STAT3-mu) sites and investigated their impact on HCK promoter activity in MYD88 mutated BCWM.1 cells. We observed that mutation of AP1-mu-1,4,5,6; NF-kB-mu-1,4,5, as well as STAT3-mu binding sites greatly reduced HCK promoter activity, thereby supporting a role for AP-1, NF-kB and STAT3 transcription factors in HCK gene expression in MYD88 mutated cells. To further clarify the importance of these transcription factors in aberrant HCK gene expression in MYD88 mutated cells, we treated BCWM.1, MWCL-1, TMD-8 and HBL-1 cells with the AP-1 inhibitor SR 11302; NF-kB inhibitor QNZ; and the STAT3 inhibitor STA-21. Treatment of cells for 2 hours with SR 11302, QNZ, and STA-21 at sub-EC50 concentrations resulted in decreased HCK expression in MYD88 mutated all cell lines. Lastly, we investigated the contribution of BCR signaling to HCK transcription. BCWM.1, MWCL-1, TMD-8, and HBL-1 cells were treated with the Syk kinase inhibitor R406, and HCK transcription levels were then assessed. Differences in HCK expression were observed between MYD88 mutated WM and ABC DLBCL cells following R406, supporting a contributing role for BCR signaling in ABC DLBCL but not WM cells to HCK expression. Our data provide critical new insights into HCK regulation, and a framework for targeting pro-survival HCK signaling in WM and ABC DLBCL cells dependent on activating MYD88 mutations. Disclosures Castillo: Biogen: Consultancy; Otsuka: Consultancy; Millennium: Research Funding; Janssen: Honoraria; Abbvie: Research Funding; Pharmacyclics: Honoraria. Treon:Janssen: Consultancy; Pharmacyclics: Consultancy, Research Funding.

scholarly journals Differential regulation of the TRH gene promoter by triiodothyronine and dexamethasone in pancreatic islets

2001 ◽  
Vol 170 (1) ◽  
pp. 91-98 ◽  
Author(s):  
P Fragner ◽  
SL Lee ◽  
S Aratan de Leon
Keyword(s):  
Gene Expression ◽  
Pancreatic Islets ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Flanking Sequence ◽  
Luciferase Reporter Gene ◽  
Neoplastic Cells

TRH was initially found in the hypothalamus and regulates TSH secretion. TRH is also produced by insulin-containing beta-cells. Endogenous TRH positively regulates glucagon secretion and attenuates pancreatic exocrine secretion. We have previously shown that triiodothyronine (T(3)) down-regulates pre-pro-TRH gene expression in vivo and in vitro. The present study was designed to determine the initial impact of T(3) on rat TRH gene promoter and to compare this effect with that of dexamethasone (Dex). Primary islet cells and neoplastic cells (HIT T-15 and RIN m5F) were transiently transfected with fragments of the 5'-flanking sequence of TRH fused to the luciferase reporter gene. The persistence of high TRH concentrations in fetal islets in culture, probably due to transactivating factors, allowed us to explore how T(3) and Dex regulate the TRH promoter activity in transfected cells and whether the hormone effect is dependent on the cell type considered. TRH gene promoter activity is inhibited by T(3) in primary but not neoplastic cells and stimulated by Dex in both primary and neoplastic cells of islets. These findings validate previous in vivo and in vitro studies and indicate the transcriptional impact of these hormones on TRH gene expression in the pancreatic islets.

TRAF3 Loss Drives Alternative NF-κB Pathway Activation in Diffuse Large B-Cell Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-23
Author(s):  
Michael Y. Li ◽  
Lauren C. Chong ◽  
Elizabeth Chavez ◽  
Bruce W Woolcock ◽  
Adele Telenius ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
B Cell ◽  
Cell Lines ◽  
Copy Number ◽  
Research Funding ◽  
B Cell Lymphoma ◽  
Luciferase Reporter ◽  
Published Data ◽  
Loss Of Function

Introduction: Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a transcription factor family that regulates gene expression programs contributing to inflammation and cell survival. NF-κB signaling occurs via two branches: classical and alternative, and is often enriched in somatic mutations of key pathway members in several lymphoid malignancies. Here, we reveal deregulation and constitutive activation of the alternative NF-κB pathway in a subset of DLBCL patients with recurrent genomic loss of the gene encoding tumor necrosis factor receptor-associated factor 3 (TRAF3), a regulator of the NF-κB signaling pathway. Methods and Results: To uncover novel driver mutations of DLBCL pathogenesis and tumor maintenance, we performed Affymetrix SNP6.0 copy number analysis on 347 de novo DLBCL samples from patients uniformly treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). We observed frequent, focal genomic loss of chr:14q32.31-32 which included TRAF3 and RCOR1 (7%, 22/313) in the minimally deleted region and an enrichment of activated B-cell-like (ABC) subtype cases over germinal center B-cell-like (GCB) subtype cases, confirming previously published data (Chan et al, Blood 2014). RNAseq of these DLBCL samples revealed a significant reduction of TRAF3 mRNA in chr:14q32.31-32 deleted cases compared to copy number neutral cases (p=0.002). Next, we focused on characterizing the phenotypic consequences of TRAF3 loss in DLBCL. We used CRISPR/Cas9 gene editing to knock out TRAF3 in 2 GCB-DLBCL (DOHH2, OCI-LY1) and 2 ABC-DLBCL (HBL1, OCI-LY3) cell lines. We performed immunoblotting analysis of NF-κB pathway members on cell fractionated samples of TRAF3 knockout cells and found increased levels of the NF-κB inducing kinase NIK (a direct target of TRAF3-mediated ubiquitin-proteasome degradation) and a concomitant increased nuclear translocation of NF-κB transcription factor complex subunits RelB and p52. Proteasome blockade restored RelB cytoplasmic localization and reduced processed p52 protein in TRAF3 knockout GCB-DLBCL lines only, indicating other factors may contribute to alternative NF-κB activation in ABC-DLBCL. Moreover, classical NF-κB activation remained unaffected, highlighting the specific role of TRAF3 regulation on the alternative NF-κB pathway in DLBCL. Consistent with these findings, TRAF3 knockout cells exhibited NF-κB-dependent transcriptional upregulation by luciferase reporter activity and elevated pro-inflammatory cytokine production (IL-6, TNF-β) by Luminex and ELISA. To study transcriptome changes as a result of TRAF3 loss-of-function, we performed RNAseq and differential gene expression analysis on wildtype and TRAF3 knockout DLBCL cell lines as well as primary DLBCL samples (N=347). We found enrichment of NIK and NF-κB associated pathways in TRAF3 deficient DLBCL and uncovered additional enriched gene sets including those involved in cell cycle regulation, cell division and metabolism, suggesting a potential proliferative and survival advantage. Conclusion: Our findings link TRAF3 loss-of-function to clinical and gene expression phenotypes in DLBCL and highlight alternative NF-κB activation as a pathogenically important pathway in both GCB and ABC subtypes. Future studies will be directed towards comprehensive evaluation of NF-κB inhibitors for effective blockade of constitutive alternative NF-κB activation in DLBCL. Disclosures Scott: NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding. Steidl:Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Consultancy; Curis Inc: Consultancy; Juno Therapeutics: Consultancy; Bayer: Consultancy; AbbVie: Consultancy.

Expression-Based Screen Identifies the Calcium Channel Antagonist Bepridil as a Notch1 Modulator in T-ALL.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 366-366 ◽  
Author(s):  
Giovanni Roti ◽  
Kenneth N. Ross ◽  
Adolfo A. Ferrando ◽  
Stephen C Blacklow ◽  
Jon Aster ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Calcium Channel ◽  
Cell Line ◽  
Cell Lines ◽  
Small Molecule ◽  
Lymphoblastic Leukemia ◽  
Receptor Activation ◽  
Luciferase Reporter ◽  
Small Molecule Library

Abstract Abstract 366 Gain of function mutations in Notch1, which encodes a signaling protein that is converted into a transcription factor upon activation, are the most common genetic abnormality in human T-cell acute lymphoblastic leukemia (T-ALL). Although inhibiting Notch1 activity represents a potential therapeutic opportunity, the discovery of new Notch1 pathway antagonists poses a difficult challenge. Traditional small molecule library screening approaches have not been amenable to modulating transcription factor abnormalities. In order to overcome this challenge, we applied Gene Expression-based High-throughput Screening (GE-HTS) to discover new Notch1 modulators. GE-HTS uses gene expression signatures as surrogates for different biological states. We derived a 32-gene Notch1 expression signature from genome-wide microarray expression profiling of 7 different Notch1 mutant T-ALL cell lines treated with vehicle (Notch1 on) versus a Notch1 inactivating γ-secretase inhibitor (GSI; Notch off) and screened a small molecule library for compounds inducing the Notch1 off state in DND41 mutant Notch1 T-ALL cells. Among numerous ion flux modulators validated to induce the Notch1 off signature, one of the top hits was the FDA-approved calcium channel blocker, bepridil, used to treat patients with cardiac disease. In multiple mutant Notch1 T-ALL cell lines, bepridil induced the Notch1 off signature. We next confirmed that bepridil indeed targets Notch signaling by demonstrating its inhibitory effects on a Notch-sensitive luciferase reporter gene in heterologous U2OS cells expressing a mutated form of Notch1. Similar to the phenotypic effects of GSI, bepridil induced a G0/G1 cell cycle arrest, inhibited cellular viability, and decreased cell size in multiple T-ALL cell lines, including the GSI-resistant cell line PF382. Next, in order to confirm dependency of the induced phenotype on inhibition of Notch, we utilized the 8946 T-ALL cell line. This murine line depends on a doxycycline-repressible human c-myc transgene for growth and can be rescued from transgene withdrawal with activated Notch1, which upregulates the endogenous c-myc gene. In these cells, the phenotypic effect of bepridil on viability is also dependent on Notch1 inhibition because cells rescued from transgene withdrawal with activated Notch1 were more sensitive to the effects of the drug than were those cells still dependent on the c-myc transgene. Finally, we asked whether bepridil altered the level of active Notch1 protein in T-ALL cell lines. As with GSI, bepridil treatment results in decreased levels of intracellular Notch (ICN1). In contrast to GSI, however, bepridil treatment decreased levels of the furin-processed extracellular and transmembrane forms of Notch1 while the full length Notch1 precursor form accumulated upon bepridil treatment. One hypothesis is that by altering ER/Golgi compartment calcium, bepridil prevents the folding of newly synthesized Notch1 polypeptides, leading to its retention in the ER/Golgi and a failure to traffic to cellular compartments where receptor activation occurs. Consistent with this hypothesis co-localization studies in U2OS cell lines expressing the L1601P mutant Notch1 suggest retention of Notch1 in the ER/Golgi. An alternative hypothesis under investigation is that bepridil affects the activity of furin, a calcium-dependent protease that is required for processing of Notch receptors. In summary, we have identified an FDA-approved drug with Notch1 modulating activity in T-ALL by a mechanism unique from GSI. These studies have potential for rapid translation to clinical testing. Disclosures: Ferrando: Merck, Pfizer: Research Funding.

scholarly journals Real-time imaging of gene promoter activity using an adenoviral reporter construct demonstrates transcriptional dynamics in normal anterior pituitary cells

2003 ◽  
Vol 178 (1) ◽  
pp. 61-69 ◽  
Author(s):  
JA Stirland ◽  
ZC Seymour ◽  
S Windeatt ◽  
AJ Norris ◽  
P Stanley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Cell Lines ◽  
Promoter Activity ◽  
Expression Patterns ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Pituitary Cells ◽  
Clonal Cell ◽  
Clonal Cell Lines

Although analysis of luciferase activity using luminescence imaging has provided new insights into the dynamic regulation of gene expression in living tIssues, studies in vitro have relied on stably transfected clonal cell lines, limiting the choice of cell type and species, or DNA microinjection, which is arduous and highly selective. We report here the first use of a recombinant adenovirus in which the firefly luciferase reporter gene was regulated by the prolactin gene promoter, to study temporal dynamics of promoter activity. This vector was used to infect the pituitary GH3 cell line, and also primary cultures of Syrian hamster pituitary cells. We show that adenovirally transduced cells retained normal regulation of the promoter-reporter transgene by appropriate signals. Furthermore, microscopic imaging studies indicated that both clonal and primary pituitary cells were transduced efficiently, giving readily detectable luminescence signals in real-time over long periods. Finally, analysis of single-cell expression patterns indicated that prolactin promoter activity was highly dynamic with pulses in gene expression, revealing that the transcriptional instability seen in clonal cells is a feature of normal pituitary cells. Adenoviral vectors offer a valuable tool for studies of gene regulation where conventional transgenesis and clonal cell lines are not available.

Inhibition of inducible TNF-α expression by oxaspirodion, a novel spiro-compound from the ascomycete Chaetomium subspirale

2004 ◽  
Vol 385 (9) ◽  
pp. 829-834 ◽  
Author(s):  
Jan Rether ◽  
Gerhard Erkel ◽  
Timm Anke ◽  
Olov Sterner
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Transcription Factor ◽  
Mode Of Action ◽  
Promoter Activity ◽  
Spiro Compound ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Jurkat T Cells ◽  
Tnf Α

Abstract In a search for compounds inhibiting the inducible TNF-αa promoter activity in T cells, a new spiro-compound, designated oxaspirodion, was isolated from fermentations of the ascomycete Chaetomium subspirale. Oxaspirodion inhibited TNF-α promoter-driven luciferase reporter gene expression with an IC50 value of 2.5 µg/ml (10 µM) in TPA/ionomycin-stimulated Jurkat T cells. Studies on the mode of action of the compound revealed that the inhibition of the TNF-α promoter activity is caused by an inhibition of the phosphorylation of the ERK1/2 kinases. In addition, oxaspirodion inhibited the activation of the transcription factor NF-κB, which is involved in the inducible expression of many proinflammatory genes.

scholarly journals Comparative activity of pulsed or continuous estradiol exposure on gene expression and proliferation of normal and tumoral human breast cells

2002 ◽  
Vol 28 (3) ◽  
pp. 165-175 ◽  
Author(s):  
V Cavailles ◽  
A Gompel ◽  
MC Portois ◽  
S Thenot ◽  
N Mabon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Hormone Replacement ◽  
Primary Cultures ◽  
Continuous Treatment ◽  
Luciferase Reporter ◽  
Breast Cancer Cell Lines ◽  
Human Breast ◽  
Kinetic Profile ◽  
Breast Cells

Intranasal administration of hormone replacement therapy presents an original plasma kinetic profile with transient estrogen levels giving rise to the concept of pulsed therapy. To further understand the molecular effects of this new therapy, we have compared the effects of pulsed and continuous estradiol treatments on two critical aspects of estradiol action: gene expression and cell proliferation. Cells were stimulated with estradiol as 1-h pulsed or 24-h continuous treatments at concentrations such that the 24-h exposure (concentration x time) was identical in both conditions. In MCF7 cells, the transcriptional activity of estrogen receptors (ER) on a transiently transfected responsive estrogen response element-luciferase reporter construct was shown to be drastically (approximately 10-fold) and similarly stimulated after both treatments. Moreover, the increased mRNA expression of three representative estradiol-sensitive genes (pS2, cathepsin D, progesterone receptor), evaluated by Northern blot, was identical after 1-h pulse with 7 nM estradiol or continuous treatment with 0.29 nM estradiol with the same kinetic profile over 48 h. Proliferation was quantified by a histomorphometric method on primary cultures of human normal breast cells from reduction mammoplasties and using a fluorescence DNA assay in six human breast cancer cell lines which were ER positive or negative. After a 7-day treatment period, estradiol had no effect on the proliferation of the three ER negative cell lines (BT20, MDA MB231, SK BR3) but significantly stimulated the proliferation of the normal cells and of the three tumoral hormone-sensitive cell lines (MCF7, T47D, ZR 75-1); both hormone treatments producing the same increases in cell growth. In conclusion, we have shown that the genomic or proliferative effects of estradiol were identical with pulsed or continuous treatments, thus indicating that estrogenic effects are not strictly related to concentrations but rather to total hormone exposure.

scholarly journals Sequence of the 5′-flanking region and promoter activity of the human mucin gene MUC5B in different phenotypes of colon cancer cells

2000 ◽  
Vol 348 (3) ◽  
pp. 675-686 ◽  
Author(s):  
Isabelle VAN SEUNINGEN ◽  
Michaël PERRAIS ◽  
Pascal PIGNY ◽  
Nicole PORCHET ◽  
Jean-Pierre AUBERT
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Promoter Activity ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Start Site ◽  
Transcription Start ◽  
Mucin Gene ◽  
Intron 1 ◽  
Flanking Region

Control of gene expression in intestinal cells is poorly understood. Molecular mechanisms that regulate transcription of cellular genes are the foundation for understanding developmental and differentiation events. Mucin gene expression has been shown to be altered in many intestinal diseases and especially cancers of the gastrointestinal tract. Towards understanding the transcriptional regulation of a member of the 11p15.5 human mucin gene cluster, we have characterized 3.55 kb of the 5ʹ-flanking region of the human mucin gene MUC5B, including the promoter, the first two exons and the first intron. We report here the promoter activity of successively 5ʹ-truncated sections of 956 bases of this region by fusing it to the coding region of a luciferase reporter gene. The transcription start site was determined by primer-extension analysis. The region upstream of the transcription start site is characterized by the presence of a TATA box at bases -32/-26, DNA-binding elements for transcription factors c-Myc, N-Myc, Sp1 and nuclear factor ĸB as well as putative activator protein (AP)-1-, cAMP-response-element-binding protein (CREB)-, hepatocyte nuclear factor (HNF)-1-, HNF-3-, TGT3-, gut-enriched Krüppel factor (GKLF)-, thyroid transcription factor (TTF)-1- and glucocorticoid receptor element (GRE)-binding sites. Intron 1 of MUC5B was also characterized, it is 2511 nucleotides long and contains a DNA segment of 259 bp in which are clustered eight tandemly repeated GA boxes and a CACCC box that bind Sp1. AP-2α and GATA-1 nuclear factors were also shown to bind to their respective cognate elements in intron 1. In transfection studies the MUC5B promoter showed a cell-specific activity as it is very active in mucus-secreting LS174T cells, whereas it is inactive in Caco-2 enterocytes and HT-29 STD (standard) undifferentiated cells. Within the promoter, maximal transcription activity was found in a segment covering the first 223 bp upstream of the transcription start site. Finally, in co-transfection experiments a transactivating effect of Sp1 on to MUC5B promoter was seen in LS174T and Caco-2 cells.

Phospholipase A 2 Metabolites Mediate Endothelin Stimulation of the Human Brain Natriuretic Peptide Promoter

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 721-721
Author(s):  
Quan He
Keyword(s):  
Gene Expression ◽  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Promoter Activity ◽  
Luciferase Activity ◽  
Phospholipase A ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
P450 Monooxygenase ◽  
Stimulation Of

P155 Brain natriuretic peptide (BNP) gene expression accompanies cardiac hypertrophy and heart failure. The vasoconstrictor endothelin-1 (ET)may be involved in the development of these diseases. ET has also been shown to activate phospholipase A 2 (PLA 2 ). Thus we studied whether ET and PLA 2 metabolites regulate BNP gene expression. The hBNP promoter (-1818 to + 100) coupled to a luciferase reporter gene was transferred into neonatal ventricular myocytes (NVM),and luciferase activity was measured as an index of promoter activity. ET (10 -7 M)induced BNP mRNA in NVM as assessed by Northern blot. It also stimulated the hBNP promoter 4-fold vs control, an effect completely inhibited by actinomycin D. To test the involvement of different PLA 2 isoforms, transfected cells were treated with the Ca ++ -independent PLA 2 (iPLA 2 )inhibitor bromoenol lactone (BEL), the cytosolic PLA 2 inhibitor methyl arachidonyl fluorophosphonate, or the secretory PLA 2 inhibitor ONO-RS-082 prior to stimulation with ET. Only the iPLA 2 inhibitor BEL prevented ET-stimulated hBNP promoter activity. The PLA 2 metabolite lysophosphatidic acid (LPA) also activated the hBNP promoter (2.2-fold; n = 3), but lysophosphatidylcholine did not. To test whether arachidonic acid metabolites are involved in ET’s effect, cells were pretreated with either a lipoxygenase (LO), cyclooxygenase, or p450 monooxygenase inhibitor. Only the LO inhibitor baicalein prevented ET stimulation of the hBNP promoter. Finally, we studied the involvement of cis elements in ET-stimulated hBNP promoter activity. Deletion of BNP promoter sequences from -1818 to -408 and from -408 to -40 reduced ET’s effect by 54% and 78%, respectively. Moreover, ET-stimulated luciferase activity was reduced by 53% when the GATA element (at position -85 relative to the start site of transcription) was mutated. These data suggest that: 1) ET activates the hBNP promoter through a transcriptional mechanism; 2) LPA, perhaps generated by a BEL-sensitive iPLA 2 , is involved in ET’s effect; 3) a LO pathway may also mediate ET signaling; and 4) ET regulation of the hBNP promoter targets both distal and proximal cis elements, including GATA.

Establishment of renal proximal tubule cell lines by targeted oncogenesis in transgenic mice using the L-pyruvate kinase-SV40 (T) antigen hybrid gene

1993 ◽  
Vol 104 (3) ◽  
pp. 695-704 ◽  
Author(s):  
N. Cartier ◽  
R. Lacave ◽  
V. Vallet ◽  
J. Hagege ◽  
R. Hellio ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Proximal Tubule ◽  
Pyruvate Kinase ◽  
Cell Lines ◽  
Cultured Cells ◽  
Simian Virus ◽  
T Antigen ◽  
Regulatory Sequence ◽  
Proximal Tubule Cell ◽  
Proximal Tubule Cells

Targeted oncogenesis allowed us to obtain two cell lines which have been derived from the proximal tubule of kidney from transgenic mice harbouring the simian virus (SV40) large T and small t antigens placed under the control of the 5′ regulatory sequence from the rat L-type pyruvate kinase (L-PK) gene. The cell lines (PKSV-PCT and PKSV-PR cells) were derived from early (PCT) and late (Pars Recta, PR) microdissected proximal tubules grown in D-glucose-enriched medium. In such conditions of culture, both cell lines exhibited L-PK transcripts, a stable expression of SV40-encoded nuclear large T antigen, a prolonged life span but failed to induce tumors when injected sub-cutaneously into athymic (nu-nu) mice. Confluent cells, grown on plastic support or porous filters, were organized as monolayers of polarized cuboid cells with well developed apical microvilli and formed domes. Both cell lines exhibited morphological features of proximal tubule cells with villin located in the apical brush-border and substantial amounts of hydrolase activity. By immunofluorescence studies using specific antibodies, aminopeptidase N appeared restricted to the apical microvillar domain, whereas the H2 histocompatibility antigen was distributed in the cytoplasm and lateral membranes. These results demonstrate that the proximal morphological phenotype has been fully preserved in these cultured cells derived from tissue-specific targeted oncogenesis in transgenic mice.

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