Detection of bioavailable peroxisome proliferator-activated receptor gamma modulators by a cell-based luciferase reporter system

2012 ◽  
Vol 427 (2) ◽  
pp. 187-189 ◽  
Author(s):  
Josep M. del Bas ◽  
Sirle Laos ◽  
Antoni Caimari ◽  
Anna Crescenti ◽  
Lluís Arola
Keyword(s):  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Reporter System ◽  
Peroxisome Proliferator Activated Receptor ◽  
A Cell ◽  
Luciferase Reporter System

scholarly journals A Cell-Based High-Throughput Assay for the Screening of Small-Molecule Inhibitors of p53–MDM2 Interaction

2011 ◽  
Vol 16 (4) ◽  
pp. 450-456 ◽  
Author(s):  
Jing Li ◽  
Shuyong Zhang ◽  
Linghuan Gao ◽  
Ying Chen ◽  
Xin Xie
Keyword(s):  
High Throughput ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Direct Inhibition ◽  
A Cell ◽  
Luciferase Reporter System ◽  
Novel Strategy ◽  
High Throughput Assay ◽  
Two Hybrid ◽  
Rule Out

The p53 tumor suppressor is a potent transcription factor that regulates cell growth inhibition and apoptosis. The oncoprotein MDM2 suppresses p53 activity by direct inhibition of its transcriptional activity and enhances the degradation of p53 via the ubiquitin–proteosome pathway. Overexpression of MDM2, found in many human tumors, impairs p53-mediated cell death effectively. Inhibition of the p53–MDM2 interaction can stabilize p53 and may offer a novel strategy for cancer therapy. To search for new inhibitors of the p53–MDM2 interaction, the authors developed a cell-based high-throughput assay system based on mammalian two-hybrid technology. They also used a dual-luciferase reporter system to rule out false- positive hits due to the cytotoxic effect of compounds. Using this assay, they screened a library consisting of 3840 compounds and identified one compound that activates p53 pathway and induces growth arrest in tumor cells.

scholarly journals miR-299-3p suppresses cell progression and induces apoptosis by downregulating PAX3 in gastric cancer

2021 ◽  
Vol 16 (1) ◽  
pp. 266-276
Author(s):  
Zhenfen Wang ◽  
Qing Liu ◽  
Ping Huang ◽  
Guohao Cai
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Western Blot Assay ◽  
Normal Tissues ◽  
Cell Progression ◽  
Luciferase Reporter System ◽  
Proliferation And Invasion

Abstract Gastric cancer (GC) is ranked the fourth leading cause of cancer-related death, with an over 75% mortality rate worldwide. In recent years, miR-299-3p has been identified as a biomarker in multiple cancers, such as acute promyelocytic leukemia, thyroid cancer, and lung cancer. However, the regulatory mechanism of miR-299-3p in GC cell progression is still largely unclear. Cell viability and apoptosis tests were performed by CCK8 and flow cytometry assay, respectively. Transwell assay was recruited to examine cell invasion ability. The interaction between miR-299-3p and PAX3 was determined by the luciferase reporter system. PAX3 protein level was evaluated by western blot assay. The expression of miR-299-3p was downregulated in GC tissues and cell lines (MKN-45, AGS, and MGC-803) compared with the normal tissues and cells. Besides, overexpression of miR-299-3p significantly suppressed proliferation and invasion and promoted apoptosis in GC. Next, we clarified that PAX3 expression was regulated by miR-299-3p using a luciferase reporter system, qRT-PCR, and western blot assay. Additionally, downregulation of PAX3 repressed GC cell progression. The rescue experiments indicated that restoration of PAX3 inversed miR-299-3p-mediated inhibition on cell proliferation and invasion. miR-299-3p suppresses cell proliferation and invasion as well as induces apoptosis by regulating PAX3 expression in GC, representing desirable biomarkers for GC diagnosis and therapy.

scholarly journals TGFβ1 Controls PPARγExpression, Transcriptional Potential, and Activity, in Part, through Smad3 Signaling in Murine Lung Fibroblasts

PPAR Research ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Allan Ramirez ◽  
Erin N. Ballard ◽  
Jesse Roman
Keyword(s):  
Transforming Growth Factor ◽  
Gene Promoter ◽  
Negative Regulator ◽  
Lung Fibroblasts ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Reporter Expression ◽  
Peroxisome Proliferator Activated Receptor ◽  
3T3 Fibroblasts ◽  
Nih 3T3

Transforming growth factorβ1 (TGFβ1) promotes fibrosis by, among other mechanisms, activating quiescent fibroblasts into myofibroblasts and increasing the expression of extracellular matrices. Recent work suggests that peroxisome proliferator-activated receptorγ(PPARγ) is a negative regulator of TGFβ1-induced fibrotic events. We, however, hypothesized that antifibrotic pathways mediated by PPARγare influenced by TGFβ1, causing an imbalance towards fibrogenesis. Consistent with this, primary murine primary lung fibroblasts responded to TGFβ1 with a sustained downregulation of PPARγtranscripts. This effect was dampened in lung fibroblasts deficient in Smad3, a transcription factor that mediates many of the effects of TGFβ1. Paradoxically, TGFβ1 stimulated the activation of the PPARγgene promoter and induced the phosphorylation of PPARγin primary lung fibroblasts. The ability of TGFβ1 to modulate the transcriptional activity of PPARγwas then tested in NIH/3T3 fibroblasts containing a PPARγ-responsive luciferase reporter. In these cells, stimulation of TGFβ1 signals with a constitutively active TGFβ1 receptor transgene blunted PPARγ-dependent reporter expression induced by troglitazone, a PPARγactivator. Overexpression of PPARγprevented TGFβ1 repression of troglitazone-induced PPARγ-dependent gene transcription, whereas coexpression of PPARγand Smad3 transgenes recapitulated the TGFβ1 effects. We conclude that modulation of PPARγis controlled by TGFβ1, in part through Smad3 signals, involving regulation of PPARγexpression and transcriptional potential.

scholarly journals Autophagy-activating strategies to promote innate defense against mycobacteria

2019 ◽  
Vol 51 (12) ◽  
pp. 1-10 ◽  
Author(s):  
Yi Sak Kim ◽  
Prashanta Silwal ◽  
Soo Yeon Kim ◽  
Tamotsu Yoshimori ◽  
Eun-Kyeong Jo
Keyword(s):  
Host Defense ◽  
Defense Mechanism ◽  
Multidrug Resistant ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Innate Defense ◽  
Host Defense Mechanism ◽  
A Cell

AbstractMycobacterium tuberculosis (Mtb) is a major causal pathogen of human tuberculosis (TB), which is a serious health burden worldwide. The demand for the development of an innovative therapeutic strategy to treat TB is high due to drug-resistant forms of TB. Autophagy is a cell-autonomous host defense mechanism by which intracytoplasmic cargos can be delivered and then destroyed in lysosomes. Previous studies have reported that autophagy-activating agents and small molecules may be beneficial in restricting intracellular Mtb infection, even with multidrug-resistant Mtb strains. Recent studies have revealed the essential roles of host nuclear receptors (NRs) in the activation of the host defense through antibacterial autophagy against Mtb infection. In particular, we discuss the function of estrogen-related receptor (ERR) α and peroxisome proliferator-activated receptor (PPAR) α in autophagy regulation to improve host defenses against Mtb infection. Despite promising findings relating to the antitubercular effects of various agents, our understanding of the molecular mechanism by which autophagy-activating agents suppress intracellular Mtb in vitro and in vivo is lacking. An improved understanding of the antibacterial autophagic mechanisms in the innate host defense will eventually lead to the development of new therapeutic strategies for human TB.

scholarly journals Cyclin G2 Regulates Adipogenesis through PPARγ Coactivation

Endocrinology ◽  
2010 ◽  
Vol 151 (11) ◽  
pp. 5247-5254 ◽  
Author(s):  
Victor Aguilar ◽  
Jean-Sébastien Annicotte ◽  
Xavier Escote ◽  
Joan Vendrell ◽  
Dominique Langin ◽  
...  
Keyword(s):  
Adipocyte Differentiation ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Cell Cycle Regulators ◽  
Luciferase Reporter Gene ◽  
Peroxisome Proliferator Activated Receptor ◽  
Cyclin G2 ◽  
Regulatory Effects

Cell cycle regulators such as cyclins, cyclin-dependent kinases, or retinoblastoma protein play important roles in the differentiation of adipocytes. In the present paper, we investigated the role of cyclin G2 as a positive regulator of adipogenesis. Cyclin G2 is an unconventional cyclin which expression is up-regulated during growth inhibition or apoptosis. Using the 3T3-F442A cell line, we observed an up-regulation of cyclin G2 expression at protein and mRNA levels throughout the process of cell differentiation, with a further induction of adipogenesis when the protein is transiently overexpressed. We show here that the positive regulatory effects of cyclin G2 in adipocyte differentiation are mediated by direct binding of cyclin G2 to peroxisome proliferator-activated receptor γ (PPARγ), the key regulator of adipocyte differentiation. The role of cyclin G2 as a novel PPARγ coactivator was further demonstrated by chromatin immunoprecipitation assays, which showed that the protein is present in the PPARγ-responsive element of the promoter of aP2, which is a PPARγ target gene. Luciferase reporter gene assays, showed that cyclin G2 positively regulates the transcriptional activity of PPARγ. The role of cyclin G2 in adipogenesis is further underscored by its increased expression in mice fed a high-fat diet. Taken together, our results demonstrate a novel role for cyclin G2 in the regulation of adipogenesis.

scholarly journals Peroxisome Proliferator-Activated ReceptorγRegulates the Expression of Lipid Phosphate Phosphohydrolase 1 in Human Vascular Endothelial Cells

PPAR Research ◽  
2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Yazi Huang ◽  
Beilei Zhao ◽  
Yahan Liu ◽  
Nanping Wang
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Mrna Level ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Lipid Phosphate ◽  
Lipid Phosphate Phosphohydrolase

Lipid phosphate phosphohydrolase 1 (LPP1), a membrane ectophosphohydrolase regulating the availability of bioactive lipid phosphates, plays important roles in cellular signaling and physiological processes such as angiogenesis and endothelial migration. However, the regulated expression of LPP1 remains largely unknown. Here, we aimed to examine a role of peroxisome proliferator-activated receptorγ(PPARγ) in the transcriptional control ofLPP1gene expression. In human umbilical vein endothelial cells (HUVECs), quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) demonstrated that activation of PPARγincreased the mRNA level of LPP1. Chromatin immunoprecipitation assay showed that PPARγbinds to the putative PPAR-responsive elements (PPREs) within the 5′-flanking region of the humanLPP1gene. Genomic fragment containing 1.7-kilobase of the promoter region was cloned by using PCR. The luciferase reporter assays demonstrated that overexpression of PPARγand rosiglitazone, a specific ligand for PPARγ, could significantly upregulate the reporter activity. However, site-directed mutagenesis of the PPRE motif abolished the induction. In conclusion, our results demonstrated that PPARγtranscriptionally activated the expression ofLPP1gene in ECs, suggesting a potential role of PPARγin the metabolism of phospholipids.

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