Stable reporter cell lines for peroxisome proliferator-activated receptor γ (PPARγ)-mediated modulation of gene expression

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• 作者：Linda Gijsbers^a ; ^{linda.gijsbers@wur.nl"" rel=""nofollow} ; Hai-Yen Man^b ; Samantha K. Kloet^a ; Laura H.J. de Haan^a ; Jaap Keijer^c ; Ivonne M.C.M. Rietjens^a ; Bart van der Burg^b ; Jac M.M.J.G. Aarts^a
• 关键词：Peroxisome proliferator-activated receptor &#x3b3 ; (PPAR&#x3b3 ; ) ; Peroxisome proliferator-responsive element (PPRE) ; Stable luciferase reporter cell line ; Human U2OS cells ; Thiazolidinediones
• 刊名：Analytical Biochemistry
• 出版年：2011
• 出版时间：1 July 2011
• 年：2011
• 卷：414
• 期：1
• 页码：77-83
• 全文大小：461 K

文摘

Activation of peroxisome proliferator-activated receptor γ (PPARγ) by ligands is associated with beneficial health effects, including anti-inflammatory and insulin-sensitizing effects. The aim of the current study was to develop luciferase reporter gene assays to enable fast and low-cost measurement of PPARγ agonist and antagonist activity. Two reporter gene assays, PPARγ1 CALUX and PPARγ2 CALUX, were developed by stable transfection of U2OS cells with an expression vector for PPARγ1 or PPARγ2 and a pGL3–3xPPRE–tata-luc or pGL4–3xPPRE–tata-luc reporter construct, respectively. PPARγ1 CALUX and PPARγ2 CALUX cells showed similar concentration-dependent luciferase induction upon exposure to the PPARγ agonists rosiglitazone, troglitazone, pioglitazone, ciglitazone, netoglitazone, and 15-deoxy-Δ^12,14-prostaglandin J₂. The potency to induce luciferase decreased in the following order: rosiglitazone > troglitazone = pioglitazone > netoglitazone > ciglitazone. A concentration-dependent decrease in the response to 50 nM rosiglitazone was observed on the addition of PPARγ antagonist GW9662 or T0070907 in both PPARγ1 CALUX and PPARγ2 CALUX cells. The PPARα agonists WY14643 and fenofibrate failed to induce luciferase activity, confirming the specificity of these cell lines for PPARγ agonists. In conclusion, PPARγ1 CALUX and PPARγ2 CALUX cells provide a reliable and useful tool to screen (bio)chemicals for PPARγ agonist or antagonist activity.