Chimeric transcriptional control units for improved liver-specific transgene expression

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Peroxisome proliferator-activated receptor ␤/␦ (PPAR␤/␦) is a ligand-regulated nuclear receptor with essential functions in metabolism and inflammation. We have synthesized a new derivative [methyl 3-(N-(4-(hexylamino)-2-methoxyphenyl)sulfamoyl)thiophene-2-carboxylate (ST247) structurally related to the published PPAR␤/␦ inhibitory ligand methyl 3-(N-(2-methoxy-4-(phenylamino)phenyl)sulfamoyl)thiophene-2-carboxylate (GSK0660). ST247 has a higher affinity to PPAR␤/␦ than GSK0660, and at equimolar concentrations, it more efficiently 1) induces the interaction with corepressors both in vitro and in vivo, 2) inhibits the agonist-induced transcriptional activity of PPAR␤/␦, and 3) downregulates basal level expression of the peroxisome proliferator responsive element-driven PPAR␤/␦ target gene ANGPTL4. Methyl 3-(N-(4-(tert-butylamino)-2-methoxyphenyl)sulfamoyl)thiophene-2-carboxylate (PT-S58), another high-affinity derivative from our series, also efficiently inhibits agonist-induced transcriptional activation, but in contrast to ST247, it does not enhance the interaction of PPAR␤/␦ with corepressors. PT-S58 rather prevents corepressor recruitment triggered by the inverse agonist ST247. These findings classify ST247 as an inverse agonist, whereas PT-S58 is the first pure PPAR␤/␦ antagonist described to date. It is noteworthy that ST247 and PT-S58 are also effective on PPREindependent functions of PPAR␤/␦: in monocytic cells, both ligands modulate expression of the activation marker CCL2 in the opposite direction as an established PPAR␤/␦ agonist. The possibility to differentially modulate specific functions of PPAR␤/␦ makes these novel compounds invaluable tools to advance our understanding of PPAR␤/␦ biology.

Section: Methodsmentioning

confidence: 99%

High-Affinity Peroxisome Proliferator-Activated Receptor β/δ-Specific Ligands with Pure Antagonistic or Inverse Agonistic Properties

Naruhn

Toth²,

Adhikary³

et al. 2011

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“…Cells were transfected on 12-well plates at 70 to 80% confluence in DMEM plus 2% FCS with 2.5 g of plasmid DNA and 2.5 l of polyethylenimine (1:1000 dilution, adjusted to pH 7.0 and preincubated for 15 min in 100 l of phosphate-buffered saline for complex formation). Four hours after transfection, the medium was changed and cells were incubated in normal growth medium for 24 h. Luciferase assays were performed as described previously (Gehrke et al, 2003). Values from three independent experiments were combined to calculate averages and standard deviations.…”

Section: Methodsmentioning

confidence: 99%

15-Hydroxyeicosatetraenoic Acid Is a Preferential Peroxisome Proliferator-Activated Receptor β/δ Agonist

Naruhn¹,

Meißner²,

Adhikary³

et al. 2009

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Peroxisome proliferator-activated receptor (PPARs) modulate target gene expression in response to unsaturated fatty acid ligands, such as arachidonic acid (AA). Here, we report that the AA metabolite 15-hydroxyeicosatetraenoic acid (15-HETE) activates the ligand-dependent activation domain (AF2) of PPAR␤/␦ in vivo, competes with synthetic agonists in a PPAR␤/␦ ligand binding assay in vitro, and triggers the interaction of PPAR␤/␦ with coactivator peptides. These agonistic effects were also seen with PPAR␣ and PPAR␥, but to a significantly weaker extent. We further show that 15-HETE strongly induces the expression of the bona fide PPAR target

“…Cells were transfected on six-well plates at 70 to 80% confluence in Dulbecco's minimal essential medium plus 2% fetal calf serum with 5 g of plasmid DNA and 10 l of polyethylenimine (1:1000 dilution, adjusted to pH 7.0 and preincubated for 15 min in 200 l of phosphate-buffered saline for complex formation). Four hours after transfection, the medium was changed, and cells were incubated in normal growth medium for 24 h. Luciferase assays were performed as described previously (Gehrke et al, 2003). Values from three independent experiments were combined to calculate averages and standard deviations.…”

Section: Methodsmentioning

confidence: 99%

Ligand-Mediated Regulation of Peroxisome Proliferator-Activated Receptor (PPAR) β/δ: A Comparative Analysis of PPAR-Selective Agonists and All-trans Retinoic Acid

Rieck

Meißner²,

Ries³

et al. 2008

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