PPARs are known widely for their roles in lipid metabolism and inflammation. Agonist ligands for PPARα and PPARγ have been used clinically for the management of dyslipidemia and control of glycemia in patients with type 2 diabetes [30
]. More recently, attention to pleiotropic effects of these agents has grown with evidence that PPARγ is a negative regulator of cardiomyocyte hypertrophy through its interaction with NFATc4, an important transcription factor that is both necessary and sufficient for development of cardiomyocyte hypertrophy [13
]. Proof of PPARα participation in the pathophysiology of hypertensive heart diseases [35
], suggested that PPARα ligands could be useful beyond their hypolipidaemic effects, in the management of disorders associated with hypertrophy and myocardial remodeling.
Multiple signaling systems function as downstream effectors of ET-1 [39
], including the calcineurin/NFAT pathway, which is important for cardiomyocyte hypertrophy [13
]. In a current model for the calcineurin/NFATc4 pathway in cardiomyocyte hypertrophy, NFATc4 is usually hyperphosphorylated and sequestered in the cytoplasm. Actions of ET-1, angiotenin II (AngII), and possibly other hypertrophic stimuli lead to elevation of intracellular Ca2+
and activation of cytoplasmic calcineurin. Activated calcineurin dephosphorylates NFATc4, resulting in its translocation to the nucleus, where it interacts with molecules such as GATA-4 to bind to a target sequence in the promoter of hypertrophic genes and trigger gene transcription [13
Here, we identified by co-IP a interaction of PPARα and NFATc4 in nuclei of cardiomyocytes. This interaction was enhanced by Fen, an activator of PPARα, or by overexpression of PPARα-EGFP. Association of NFATc4 with PPARα was significantly increased by Fen treatment of ET-1-stimulated cardiomyocytes, suggesting a novel mechanism of action of PPARα in cardiomyocyte hypertrophy induced by ET-1 through the NFATc4 pathway. To understand better how PPARα regulated NFATc4 function, we investigated two important actions of NFATc4 on binding to its target gene and its interaction with cofactors.
It had been reported that the binding of NF-AT3 (analogous to NFATc4) at a position −927 nucleotides upstream of the human BNP gene, which is a marker of cardiac hypertrophy and heart failure, was involved in the activation of the promoter [13
]. Promoter analysis based on transcription factor-binding sites, revealed a putative binding site for NFATc4 in region −330 to −351bp of the rat BNP promoter; EMSA confirmed this. Although a supershifted band was not observed with anti-NFATc4 antibodies, the shifted band was markedly diminished by the antibodies. One possible reason for the lack of a supershifted band was shielding the DNA binding site after NFATc4 binding with the antibody. These data are consistent with the findings of Zhu et al. [45
]. We showed that binding of NFATc4 to this site was increased in a time-dependent manner by ET-1 treatment. The enhanced binding was clearly decreased by activation or overexpression of PPARα, suggesting that interaction of NFATc4 with PPARα interfered with its binding to the BNP promoter.
GATA-4, a zinc-finger transcription factor was an important role in cardiac hypertrophy [46
]. GATA-4 also acted synergistically with NF-AT3 to activate the BNP promoter in cardio myocytes [13
]. Consistent with the report of Kakita et al. that ET-1 translocated NFATc into nuclei and enhanced its interaction with GATA-4 [24
], the association of NFATc4 and GATA-4 was significantly increased in nuclei of cardiomyocytes after ET-1 stimulation. The interaction between NFATc4 and GATA-4 was markedly decreased by PPARα activation or overexpression, indicating the interaction of NFATc4 with PPARα decreased its association with GATA-4. Our data also showed that Fen or overexpression of PPARα significantly attenuated increases in BNP mRNA and cardiomyocyte size induced by ET-1, consistent with a significant contribution of PPARα to ET-1-induced BNP transcription and cardiomyocyte hypertrophy.
In addition, using siRNA to deplete cells of PPARα (without affecting PPARβ/δ or PPARγ), we showed that PPARα siRNA failed to block the enhanced interaction of NFATc4 with BNP promoter or GATA-4, resulting from Fen and ET-1 co-stimulation. Quantitative RT-PCR and confocal microscopy similarly confirmed the effects of PPARα siRNA on elevation of BNP mRNA content and cardiomyocyte hypertrophy.
Although the mechanism by which NFATc4 binding to PPARα decreased its binding to GATA-4 remains to be determined, it is possible that the two molecules compete for the same binding site. Molkentin et al. [13
] reported that NF-AT3 interacted with GATA-4 through its Rel-homology domain, which also mediated DNA binding. In our study, Fen activation of PPARα apparently enhanced its interaction with NFATc4 while, in a concentration-dependent manner, decreasing NFATc4 interactions with GATA-4 and the BNP promoter (), consistent with the notion that PPARα and GATA-4 competed for binding to the Rel-homology domain of NFATc4.
Overall, our data fit a model () in which PPARα can participate in transcription complexes with NFATc4 that interfere with an NFATc4–GATA-4 interaction in cardiomyocytes, thereby decreasing its transactivation potential and preventing induction of cardiomyocyte hypertrophy by ET-1. These findings provide novel mechanistic insight into a role for PPARα in cardiac hypertrophy, and suggest that interference with interactions of nuclear transcription factors could be a useful therapeutic approach to prevent cardiac hypertrophy.
Fig. 7 A model for the function of PPARα activation on cardiomyocyte hypertrophy induced by ET-1. Activated PPARα associates with activated NFATc4 induced by ET-1 in the nucleus to prevent the interaction of NFATc4 with GATA-4, and further reduce (more ...)