Covalent binding of 15-deoxy-delta 12,14 -prostaglandin J 2 to PPARc A.F. Soares a , O. Nosjean b , D. Cozzone a , D. DÕOrazio c , M. Becchi d , M. Guichardant a , G. Ferry b , J.A. Boutin b , M. Lagarde a , A. Ge ´loe ¨n a, * a UMR 585 INSERM/INSA-Lyon, IMBL, 69621 Villeurbanne, France b Institut de Recherches Servier, Croissy-sur-Seine, France c DSM Nutritional Products Ltd., Basel, Switzerland d UMR 5086 CNRS/UCBL, IBCP, Lyon, France Received 9 September 2005 Available online 22 September 2005 Abstract Since 15-deoxy-delta 12,14 -prostaglandin J 2 (15dPGJ 2 ) has been identified as an endogenous ligand of PPARc thus inducing adipogen- esis, it has been reported to play active parts in numerous cellular regulatory mechanisms. As 15dPGJ 2 has been shown to covalently bind several peptides and proteins, we investigated whether it also covalently binds PPARc. We first observed that after incubation of 15dPGJ 2 with recombinant PPARc, the quantity of free 15dPGJ 2 measured was always lower than the initial amount. We then measured the ability of the labeled agonist rosiglitazone to displace the complex PPARc 2 /15dPGJ 2 obtained after pre-incubation. We observed that the binding of rosiglitazone was dependent on the initial concentration of 15dPGJ 2 . Finally using MALDI-TOF mass spectrometry anal- ysis, after trypsinolysis of an incubate of the PPARc 2 ligand binding domain (GST-LBD) with 15dPGJ2, we found a fragment (m/ z = 1314.699) corresponding to the addition of 15dPGJ 2 (m/z = 316.203) to the GST-LBD peptide (m/z = 998.481). All these observa- tions demonstrate the existence of a covalent binding of 15dPGJ 2 to PPARc, which opens up new perspectives to study the molecular basis for selective activities of PPARs. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Peroxisome proliferator-activated receptor gamma (PPARc); 15-deoxy-delta 12,14 -prostaglandin J 2 ; Covalent binding; Mass spectrometry Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily. PPARc heterodimerizes with the 9-cis RXR to bind PPAR response element, present in the promoters of target genes involved in lipid metabolism, glucose homeostasis, adipose cell proliferation and differ- entiation, and modulated inflammatory responses [1]. To heterodimerize, PPARs require to be activated by ligands. Various ligands have been described for PPARs amongst which are natural fatty acids and their metabolites such as arachidonate derivatives. Arachidonic acid can be con- verted into prostaglandin D 2 (PGD 2 ), which, in turn, can be converted into PGJ 2 , then into 15dPGJ 2 by successive dehydration and isomerization steps. Several studies have shown that 15dPGJ 2 is a natural ligand of PPARc that is involved in the terminal differentiation of adipocytes [2,3]. Prostaglandins are formed from arachidonic acid through the action of cyclooxygenases which are expressed in numerous tissues including adipose tissues. Prostaglan- din D 2 synthase enzymes are expressed in mouse 3T3-L1 adipocytes and human tissues [4]. Although numerous evi- dences have suggested the biological activities of the PGD 2 metabolite 15dPGJ 2 , the question of its formation in vivo remains open. Using liquid chomatography–tandem mass spectrometry Bell-Parikh et al. [5] have measured a small amount of 15dPGJ 2 in 3T3-L1 and concluded that it is not the endogenous mediator of PPARc-dependent adipo- cyte activation. Despite the fact that these authors found non-physiological relevant levels of 15dPGJ 2 , numerous observations are in favor of a physiological role of 15dPGJ 2 . The question of the efficient concentration of 15dPGJ 2 has been recently summarized by Nosjean and Boutin [6]. 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.09.085 * Corresponding author. Fax: +33 4 72 43 85 24. E-mail address: alain.geloen@insa-lyon.fr (A. Ge ´loe ¨n). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 337 (2005) 521–525 BBRC