Binding of prostaglandins to human PPARgamma: tool assessment and new natural ligands.

The peroxisome proliferator-activated receptors (PPAR) form a family of nuclear receptors with a wide variety of biological roles from adipogenesis to carcinogenesis. More ligands (agonist and antagonist) are needed to explore the multiple functions of PPAR, particularly PPARgamma. In order to complete such ligand screening, a binding test should be assessed versus the classical transactivation reporter gene assay. In the present work, the full-length human PPARgamma protein as well as its ligand binding domain portion were expressed in Escherichia coli. Bacterial membrane preparations expressing those constructs were characterized using a classical binding competition assay [3H]rosiglitazone as the radioligand. When the receptor preparations were soluble, binding had to be measured with a new alternative method. The systems were assessed using a series of reference PPAR (alpha, beta and gamma) ligands. The full-length human PPARgamma fused to glutathione-S-transferase, expressed in E. coli and tested as a bacterial membrane-bound protein led to the most accurate results when compared to the literature. Furthermore, in an attempt to complete the panel of natural PPARgamma ligands, 29 commercially available prostaglandins were screened in the binding assay. Prostaglandins H(1) and H(2) were found to be modest ligands, however as potent as 15Delta(12-14 )prostaglandin J(2). These results were confirmed in the classical transactivation assay. The fact that these three prostaglandins were equally potent, suggests new pathways of PPARgamma-linked gene activation.