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.

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.

scholarly journals Transcriptional regulation of the human GD3 synthase gene expression in Fas-induced Jurkat T cells: a critical role of transcription factor NF-κB in regulated expression

Glycobiology ◽  
2006 ◽  
Vol 16 (5) ◽  
pp. 375-389 ◽  
Author(s):  
Young Kang ◽  
Sung-Koo Kang ◽  
Young-Choon Lee ◽  
Hee-Jeong Choi ◽  
Young-Seek Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Critical Role ◽  
Jurkat T Cells ◽  
Regulated Expression ◽  
Gd3 Synthase

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 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.

scholarly journals Calcium Dynamics and Resting Transcriptional Activity Regulates Prolactin Gene Expression

Endocrinology ◽  
2002 ◽  
Vol 143 (9) ◽  
pp. 3548-3554 ◽  
Author(s):  
Carlos Villalobos ◽  
Lucía Núñez ◽  
William J. Faught ◽  
David C. Leaumont ◽  
Fredric R. Boockfor ◽  
...  
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Pituitary Cells ◽  
Luciferase Reporter Gene ◽  
Transcriptional Responses ◽  
Prolactin Gene ◽  
Before And After ◽  
Single Living

Abstract Research on the regulation of hormone gene expression by calcium signaling is hampered by the difficulty of monitoring both parameters within the same individual, living cells. Here we achieved concurrent, dynamic measurements of both intracellular Ca2+ concentration ([Ca2+]i) and prolactin (PRL) gene promoter activity in single, living pituitary cells. Cells were transfected with the luciferase reporter gene under control of the PRL promoter and subjected to bioluminescence and fluorescence imaging before and after presentation of TSH-releasing hormone (TRH), a prototypic regulator of PRL secretion and gene expression that induces a transient Ca2+ release, followed by sustained Ca2+ influx. We found that cells displaying specific photonic emissions (i.e. mammotropes) showed heterogeneous calcium and transcriptional responses to TRH. Transcriptionally responsive cells always exhibited a TRH-induced [Ca2+]i increase. In addition, transcriptional responses were related to the rate of Ca2+ entry but not Ca2+ release. Finally, cells lacking transcriptional responses (but showing [Ca2+]i rises) exhibited larger levels of resting PRL promoter activity than transcriptionally responsive cells. Thus, our results suggest that the sustained entry of Ca2+ induced by TRH (but not the Ca2+ release) regulates transcriptional responsiveness. Superimposed on this regulation, the previous, resting PRL promoter activity also controls transcriptional responses.

scholarly journals Potentiation of TNF-α–Stimulated Group IIA Phospholipase A2 Expression by Peroxisome Proliferator–Activated Receptor α Activators in Rat Mesangial Cells

2002 ◽  
Vol 13 (3) ◽  
pp. 611-620 ◽  
Author(s):  
Kirsten Scholz-Pedretti ◽  
Annette Gans ◽  
Karl-Friedrich Beck ◽  
Josef Pfeilschifter ◽  
Marietta Kaszkin
Keyword(s):  
Promoter Activity ◽  
Mesangial Cells ◽  
Site Directed Mutagenesis ◽  
Lipid Lowering ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Glomerular Mesangial Cells ◽  
Luciferase Reporter Gene ◽  
Peroxisome Proliferator Activated Receptor ◽  
Tnf Α

ABSTRACT. Natural activators of peroxisome proliferator–activated receptors (PPAR) are lipid metabolites, including those produced by phospholipases A2 (PLA2). In glomerular mesangial cells, the secreted group IIA PLA2 (sPLA2-IIA), which is thought to be a crucial factor in pathologic processes in the kidney, may provide free fatty acids and eicosanoids directly or indirectly, by activating a cytosolic PLA2. The scope of this study was to investigate whether synthetic PPARα activators have an effect on sPLA2-IIA mRNA expression in rat mesangial cells, thus constituting a feedback modulation of sPLA2-IIA transcription. In the presence of tumor necrosis factor–α (TNF-α), the PPARα agonists WY14643 and LY171883 as well as the lipid-lowering compound clofibrate potentiated expression, secretion, and activity of group IIA sPLA2 in mesangial cells. MK886, known as a noncompetitive inhibitor of PPARα, completely abolished the potentiation of sPLA2-IIA secretion and activity by WY14643, thus indicating that the effect of WY14643 is specifically mediated by PPARα. When cells were transfected with different constructs of the rat sPLA2-IIA promoter fused to a luciferase reporter gene, a stimulation with TNF-α in the presence of the PPARα activators caused an enhanced promoter activity compared with that induced by TNF-α alone. Site-directed mutagenesis of a putative PPRE site in the sPLA2-IIA promoter abolished the potentiating effect of PPARα agonists, thus strongly indicating its contribution to the enhanced promoter activity. In summary, this study shows that the rat sPLA2-IIA promoter is sensitive to PPARα agonists, which act synergistically with cytokines, resulting in an enhanced expression of sPLA2-IIA in rat mesangial cells.

scholarly journals Nuclear Factor of Activated T Cells Transcription Factor Nfatp Controls Superantigen-Induced Lethal Shock

2000 ◽  
Vol 192 (4) ◽  
pp. 581-586 ◽  
Author(s):  
Alla V. Tsytsykova ◽  
Anne E. Goldfeld
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Serum Levels ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Tnf Α ◽  
Lethal Shock

Tumor necrosis factor α (TNF-α) is the key mediator of superantigen-induced T cell lethal shock. Here, we show that nuclear factor of activated T cells transcription factor, NFATp, controls susceptibility to superantigen-induced lethal shock in mice through its activation of TNF-α gene transcription. In NFATp-deficient mice, T cell stimulation leads to delayed induction and attenuation of TNF-α mRNA levels, decreased TNF-α serum levels, and resistance to superantigen-induced lethal shock. By contrast, after lipopolysaccharide (LPS) challenge, serum levels of TNF-α and susceptibility to shock are unaffected. These results demonstrate that NFATp is an essential activator of immediate early TNF-α gene expression in T cells and they present in vivo evidence of the inducer- and cell type–specific regulation of TNF-α gene expression. Furthermore, they suggest NFATp as a potential selective target in the treatment of superantigen-induced lethal shock.

Ceramide decreases surfactant protein B gene expression via downregulation of TTF-1 DNA binding activity

2006 ◽  
Vol 290 (2) ◽  
pp. L351-L358 ◽  
Author(s):  
Loretta Sparkman ◽  
Hemakumar Chandru ◽  
Vijayakumar Boggaram
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Dna Binding ◽  
Promoter Activity ◽  
Surfactant Protein ◽  
Binding Activity ◽  
Mrna Levels ◽  
Dna Binding Activity ◽  
Surfactant Protein B ◽  
Tnf Α

Ceramide, a sphingolipid, is an important signaling molecule in the inflammatory response. Mediators of acute lung injury such as TNF-α, platelet-activating factor, and Fas/Apo ligand stimulate sphingomyelin hydrolysis to increase intracellular ceramide levels. Surfactant protein B (SP-B), a hydrophobic protein of pulmonary surfactant, is essential for surfactant function and lung stability. In this study we investigated the effects of ceramide on SP-B gene expression in H441 lung epithelial cells. Ceramide decreased SP-B mRNA levels in control and dexamethasone-treated cells after 24-h incubation and inhibition of SP-B mRNA was associated with inhibition of immunoreactive SP-B. In transient transfections assays, ceramide inhibited SP-B promoter activity, indicating that the inhibitory effects are exerted at the transcriptional level. Deletion mapping experiments showed that the ceramide-responsive region is located within the −233/−80-bp region of human SP-B promoter. Electrophoretic mobility shift and reporter assays showed that ceramide reduced the DNA binding activity and transactivation capability of thyroid transcription factor 1 (TTF-1/Nkx2.1), a key factor for SP-B promoter activity. Collectively these data showed that ceramide inhibits SP-B gene expression by reducing the DNA biding activity of TTF-1/Nkx2.1 transcription factor. Protein kinase C inhibitor bisindolylmaleimide and the protein tyrosine kinase inhibitor genistein partially reversed ceramide inhibition, indicating that protein kinases play important roles in the ceramide inhibition of SP-B gene expression. Chemical inhibitors of de novo ceramide synthesis and sphingomyelin hydrolysis had no effect on TNF-α inhibition of SP-B promoter activity and mRNA levels, suggesting that ceramide does not play a role in the inhibition.

scholarly journals Modulation of PTPN2/22 Function by Spermidine in CRISPR-Cas9-Edited T-Cells Associated with Crohn’s Disease and Rheumatoid Arthritis

2021 ◽  
Vol 22 (16) ◽  
pp. 8883
Author(s):  
Ameera M. Shaw ◽  
Ahmad Qasem ◽  
Saleh A. Naser
Keyword(s):  
Rheumatoid Arthritis ◽  
Gene Expression ◽  
Cell Proliferation ◽  
T Cells ◽  
Cell Response ◽  
Cell Activation ◽  
Clinical Samples ◽  
Jurkat T Cells ◽  
Tnf Α ◽  
Ifn Γ

Crohn’s Disease (CD) and Rheumatoid Arthritis (RA) share some single nucleotide polymorphisms (SNPs) in protein tyrosine phosphatase non-receptor types 2 and 22 (PTPN2/22). Recently, we reported that clinical samples from CD and RA patients associated with PTPN2:rs478582 or PTPN22:rs2476601 genotypes were linked to overactive immune response and exacerbation of inflammation. Here, we investigated in vitro the effects of these SNPs in Jurkat T-cells using CRISPR-Cas9. All cells were evaluated for PTPN22/22 loss of function and effects on cell response. We measured gene expression via RT-qPCR and cytokines by ELISA. We also measured cell proliferation using a BrdU labeling proliferation ELISA, and T-cell activation using CD-25 fluorescent immunostaining. In PTPN2 SNP-edited cells, PTPN2 expression decreased by 3.2-fold, and proliferation increased by 10.2-fold compared to control. Likewise, expression of PTPN22 decreased by 2.4-fold and proliferation increased by 8.4-fold in PTPN22 SNP-edited cells. IFN-γ and TNF-α secretions increased in both edited cell lines. CD25 expression (cell activation) was 80.32% in PTPN2 SNP-edited cells and 85.82% in PTPN22 SNP-edited cells compared to 70.48% in unedited Jurkat T-cells. Treatment of PTPN2 and PTPN22-edited cells with a maximum 20 μM spermidine restored PTPN2/22 expression and cell response including cell proliferation, activation, and cytokines secretion. Most importantly, the effect of spermidine on edited cells restored normal expression and secretion of IFN-γ and TNF-α. The data clearly demonstrated that edited SNPs in PTPN2 or PTPN22 were associated with reduced gene expression, which resulted in an increase in cell proliferation and activation and overactive immune response. The data validated our earlier observations in CD and RA clinical samples. Surprisingly, spermidine restored PTPN2/22 expression in edited Jurkat T-cells and the consequent beneficial effect on cell response and inflammation. The study supports the use of polyamines dietary supplements for management of CD and in RA patients.

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