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

L. Gijsbers; H.Y. Man; S.K. Kloet; L.H.J. de Haan; J. Keijer; I. Rietjens; B.J. van der Burg; J.M.M.J.G. Aarts

doi:10.1016/j.ab.2011.02.032

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

L. Gijsbers, H.Y. Man, S.K. Kloet, L.H.J. de Haan, J. Keijer, I. Rietjens, B.J. van der Burg, J.M.M.J.G. Aarts

Research output: Contribution to journal › Article › Academic › peer-review

34 Citations (Scopus)

Abstract

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

Original language	English
Pages (from-to)	77-83
Journal	Analytical Biochemistry
Volume	414
Issue number	1
DOIs	https://doi.org/10.1016/j.ab.2011.02.032
Publication status	Published - 2011

Keywords

ppar-gamma
antidiabetic thiazolidinedione
insulin-resistance
binding-sites
fatty-acids
agonists
ligand
alpha
inflammation
obesity

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Gijsbers, L., Man, H. Y., Kloet, S. K., de Haan, L. H. J., Keijer, J., Rietjens, I., van der Burg, B. J., & Aarts, J. M. M. J. G. (2011). Stable reporter cell lines for peroxisome proliferator-activated receptor y (PPARy)-mediated modulation of gene expression. Analytical Biochemistry, 414(1), 77-83. https://doi.org/10.1016/j.ab.2011.02.032

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title = "Stable reporter cell lines for peroxisome proliferator-activated receptor y (PPARy)-mediated modulation of gene expression",

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

keywords = "ppar-gamma, antidiabetic thiazolidinedione, insulin-resistance, binding-sites, fatty-acids, agonists, ligand, alpha, inflammation, obesity",

author = "L. Gijsbers and H.Y. Man and S.K. Kloet and {de Haan}, L.H.J. and J. Keijer and I. Rietjens and {van der Burg}, B.J. and J.M.M.J.G. Aarts",

year = "2011",

doi = "10.1016/j.ab.2011.02.032",

language = "English",

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journal = "Analytical Biochemistry",

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Stable reporter cell lines for peroxisome proliferator-activated receptor y (PPARy)-mediated modulation of gene expression. / Gijsbers, L.; Man, H.Y.; Kloet, S.K.; de Haan, L.H.J.; Keijer, J.; Rietjens, I.; van der Burg, B.J.; Aarts, J.M.M.J.G.

In: Analytical Biochemistry, Vol. 414, No. 1, 2011, p. 77-83.

Research output: Contribution to journal › Article › Academic › peer-review

TY - JOUR

T1 - Stable reporter cell lines for peroxisome proliferator-activated receptor y (PPARy)-mediated modulation of gene expression

AU - Gijsbers, L.

AU - Man, H.Y.

AU - Kloet, S.K.

AU - de Haan, L.H.J.

AU - Keijer, J.

AU - Rietjens, I.

AU - van der Burg, B.J.

AU - Aarts, J.M.M.J.G.

PY - 2011

Y1 - 2011

N2 - Activation of peroxisome proliferator-activated receptor ¿ (PPAR¿) 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¿ agonist and antagonist activity. Two reporter gene assays, PPAR¿1 CALUX and PPAR¿2 CALUX, were developed by stable transfection of U2OS cells with an expression vector for PPAR¿1 or PPAR¿2 and a pGL3–3xPPRE–tata-luc or pGL4–3xPPRE–tata-luc reporter construct, respectively. PPAR¿1 CALUX and PPAR¿2 CALUX cells showed similar concentration-dependent luciferase induction upon exposure to the PPAR¿ agonists rosiglitazone, troglitazone, pioglitazone, ciglitazone, netoglitazone, and 15-deoxy-¿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 PPAR¿ antagonist GW9662 or T0070907 in both PPAR¿1 CALUX and PPAR¿2 CALUX cells. The PPARa agonists WY14643 and fenofibrate failed to induce luciferase activity, confirming the specificity of these cell lines for PPAR¿ agonists. In conclusion, PPAR¿1 CALUX and PPAR¿2 CALUX cells provide a reliable and useful tool to screen (bio)chemicals for PPAR¿ agonist or antagonist activity.

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

KW - ppar-gamma

KW - antidiabetic thiazolidinedione

KW - insulin-resistance

KW - binding-sites

KW - fatty-acids

KW - agonists

KW - ligand

KW - alpha

KW - inflammation

KW - obesity

U2 - 10.1016/j.ab.2011.02.032

DO - 10.1016/j.ab.2011.02.032

M3 - Article

VL - 414

SP - 77

EP - 83

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 1

ER -