PPARγ regulators: Cyclic phosphatidic acid acts down

Lipidomics Gateway (26 August 2010) [doi:10.1038/lipidmaps.2010.24]

Cyclic phosphatidic acid is a second messenger and a competitive antagonist of PPARγ.

The transcriptional regulator PPARγ plays a key role in lipid and glucose homeostasis and is also involved in cell death decisions and inflammation. Gabor Tigyi and colleagues have identified an endogenous antagonist that adds to the growing list of small molecule PPARγ regulators that already include modified fatty acids, lysophosphatidic acid, and the insulin-sensitizing drug rosiglitazone.

Most known PPARγ ligands act as agonists, though it is clear that excess PPARγ activity can be detrimental to cells and tissues. In the search for antagonists of PPARγ, Tigyi and colleagues tested various naturally occurring lysophospholipids, based on the ability of lysophosphatidic acid (LPA) to modulate PPARγ. They found that whereas LPA 18:1 and its analog alkyl glycerol phosphate (AGP) strongly activated PPARγ, species of cyclic phosphatidic acid (cPA) inhibited PPARγ-dependent gene expression in a cell-based reporter assay. Competition experiments suggest that cPA binds to the same domain of PPARγ that other regulators bind, the ligand binding domain (LBD), and it does so at an affinity comparable to the highly active rosiglitazone.

On the basis of a published crystal structure of the PPARγ homolog PPARα bound to a small molecule antagonist, the authors generated a homology model of PPARγ-LBD and then computationally docked various PPARγ antagonists and antagonists, including cPA, to it. Although the docked positions of rosiglitazone mostly differ from those of cPA, consistent with the agonist activity of the former and the antagonist activity of the latter, the LBD residue R288 was found to be important for cPA binding in silico and in vitro, and this residue was previously found to bind to the phosphates of LPA and AGP 18:1. This and other common interactions preclude simultaneous binding of cPA and agonists, a further explanation of the competitive antagonistic effect of cPA.

Agonists induce the dissociation of PPARγ from co-repressor proteins such as SMRT. By contrast, the authors show here that cPA stabilizes this interaction, while antagonizing rosiglitazone-induced complex dissociation. cPA inhibits all other PPARγ-mediated effects tested, including uptake of acetylated low-density lipoprotein in macrophages, transcription of target genes such as Cd36 and Hadh, adipocyte differentiation of 3T3-L1 cells, and agonist-induced neointima formation in a rodent model of vascular remodeling.

An in vivo radiolabeling assay for phospholipase D (PLD) activity showed that cPA was an enzymatic product. On the basis of several PLD mutations, and RNAi experiments, the authors implicated the mammalian PLD2 isoform as responsible for cPA formation in vivo. Physiological stimulation of PLD2 with insulin, mastoparan, PMA, or lipopolysaccharide caused production of cPA 18:1 and 16:0 in primary and cultured cells, whereas PLD inhibition abolished cPA production. The insulin-stimulated production of cPA (via PLD2) inhibited PPARγ-dependent reporter gene expression as well as the resulting adipogenesis and neointima formation.

These results are striking evidence that cPA is a second messenger and a negative regulator of PPARγ, and point towards a scaffold for designing drugs that act on this important transcriptional activator.

Mirella Bucci