A truncated human peroxisome proliferator-activated receptor α splice variant with dominant negative activity

Abstract

The peroxisome proliferator-activated receptor α (PPARα) plays a key role in lipid and lipoprotein metabolism. However, important inter- and intraspecies differences exist in the response to PPARα activators. This incited us to screen for PPARα variants with different signaling functions. In the present study, using a RT-PCR approach a variant human PPARα mRNA species was identified, which lacks the entire exon 6 due to alternative splicing. This deletion leads to the introduction of a premature stop codon, resulting in the formation of a truncated PPARα protein (PPARα(tr)) lacking part of the hinge region and the entire ligand-binding domain. RNase protection analysis demonstrated that PPARα(tr) mRNA is expressed in several human tissues and cells, representing between 20-50% of total PPARα mRNA. By contrast, PPARα(tr) mRNA could not be detected in rodent tissues. Western blot analysis using PPARα-specific antibodies demonstrated the presence of an immunoreactive protein migrating at the size of in vitro produced PPARα(tr) protein both in human hepatoma HepG2 cells and in human hepatocytes. Both in the presence or absence of 9-cis-retinoic acid receptor, PPARα(tr) did not bind to DNA in gel shift assays. Immunocytochemical analysis of transfected CV-1 cells indicated that, whereas transfected PPARα(wt) was mainly nuclear localized, the majority of PPARα(tr) resided in the cytoplasm, with presence in the nucleus depending on cell culture conditions. Whereas a chimeric PPARα(tr) protein containing a nuclear localization signal cloned at its N-terminal localized into the nucleus and exhibited strong negative activity on PPARoα(wt) transactivation function, PPARα(tr) interfered with PPARα(wt) transactivation function only under culture conditions inducing its nuclear localization. Cotransfection of the coactivator CREB-binding protein relieved the transcriptional repression of PPARoα(wt) by PPARα(tr), suggesting that the dominant negative effect of PPARα(tr) might occur through competition for essential coactivators. In addition, PPARα(tr) interfered with transcriptional activity of other nuclear receptors such as PPARγ, hepatic nuclear factor-4, and glucocorticoid receptor-α, which share CREB-binding protein/p300 as a coactivator. Thus, we have identified a human PPARα splice variant that may negatively interfere with PPARα(wt) function. Factors regulating either the ratio of PPARα(wt) vs. PPARα(tr) mRNA or the nuclear entry of PPARα(tr) protein should therefore lead to altered signaling via the PPARα and, possibly also, other nuclear receptor pathways.