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

作者：Gij**ers Linda^*; Man Hai Yen; Kloet Samantha K; de Haan Laura H J; Keijer Jaap; Rietjens Ivonne M C M; van der Burg Bart; Aarts Jac M M J G

来源：Analytical Biochemistry, 2011, 414(1): 77-83.

DOI：10.1016/j.ab.2011.02.032

• Peroxisome proliferator-activated receptor gamma (PPAR gamma)
• Peroxisome proliferator-responsive element (PPRE)
• Stable luciferase reporter cell line
• Human U2OS cells
• Thiazolidinediones

Activation of peroxisome proliferator-activated receptor gamma (PPAR gamma) 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 PPAR gamma agonist and antagonist activity. Two reporter gene assays, PPAR gamma 1 CALUX and PPAR gamma 2 CALUX, were developed by stable transfection of U2OS cells with an expression vector for PPAR gamma 1 or PPAR gamma 2 and a pGL3-3xPPRE-tata-luc or pGL4-3xPPRE-tata-luc reporter construct, respectively. PPAR gamma 1 CALUX and PPAR gamma 2 CALUX cells showed similar concentration-dependent luciferase induction upon exposure to the PPAR gamma agonists rosiglitazone, troglitazone, pioglitazone, ciglitazone, netoglitazone, and 15-deoxy-Delta(12,14)-prostaglandin J(2). 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 PPAR gamma antagonist GW9662 or T0070907 in both PPAR gamma 1 CALUX and PPAR gamma 2 CALUX cells. The PPAR alpha. agonists WY14643 and fenofibrate failed to induce luciferase activity, confirming the specificity of these cell lines for PPAR gamma agonists. In conclusion, PPAR gamma 1 CALUX and PPAR gamma 2 CALUX cells provide a reliable and useful tool to screen (bio)chemicals for PPAR gamma agonist or antagonist activity.

• 出版日期2011-7-1