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

The peroxisome proliferator-activated receptor alpha (PPAR alpha) plays a k ey role in lipid and lipoprotein metabolism. However, important inter- and intraspecies differences exist in the response to PPAR alpha activators. Th is incited us to screen for PPAR alpha variants with different signaling fu nctions. In the present study, using a RT-PCR approach a variant human PPAR alpha mRNA species was identified, which lacks the entire exon 6 due to al ternative splicing. This deletion leads to the introduction of a premature stop codon, resulting in the formation of a truncated PPAR alpha protein (P PAR alpha(tr)) lacking part of the hinge region and the entire ligand-bindi ng domain. RNase protection analysis demonstrated that PPAR alpha(tr) mRNA is expressed in several human tissues and cells, representing between 20-50 % of total PPAR alpha mRNA. By contrast, PPAR alpha(tr) mRNA could not be d etected in rodent tissues. Western blot analysis using PPAR alpha-specific antibodies demonstrated the presence of an immunoreactive protein migrating at the size of in vitro produced PPAR alpha(tr) protein both in human hepa toma HepG2 cells and in human hepatocytes. Both in the presence or absence of 9-cis-retinoic acid receptor, PPAR alpha(tr) did not bind to DNA in gel shift assays. Immunocytochemical analysis of transfected CV-1 cells indicat ed that, whereas transfected PPAR alpha(wt) was mainly nuclear localized, t he majority of PPAR alpha(tr) resided in the cytoplasm, with presence in th e nucleus depending on cell culture conditions. Whereas a chimeric PPAR alp ha(tr) protein containing a nuclear localization signal cloned at its N-ter minal localized into the nucleus and exhibited strong negative activity on PPAR alpha(wt) transactivation function, PPAR alpha(tr) interfered with PPA R alpha(wt) transactivation function only under culture conditions inducing its nuclear localization. Cotransfection of the coactivator CREB-binding p rotein relieved the transcriptional repression of PPAR alpha(wt) by PPAR al pha(tr), suggesting that the dominant negative effect of PPAR alpha(tr) mig ht occur through competition for essential coactivators. In addition, PPAR alpha(tr) interfered with transcriptional activity of other nuclear recepto rs such as PPAR gamma hepatic nuclear factor-4, and glucocorticoid receptor -alpha which share CREB-binding protein/p300 as a coactivator. Thus, we hav e identified a human PPAR alpha splice variant that may negatively interfer e with PPAR alpha(wt) function. Factors regulating either the ratio of PPAR alpha(wt) vs. PPAR alpha(tr) mRNA or the nuclear entry of PPAR alpha(tr) p rotein should therefore lead to altered signaling via the PPAR alpha and, p ossibly also, other nuclear receptor pathways.