A truncated human peroxisome proliferator-activated receptor alpha splice variant with dominant negative activity.
The peroxisome proliferator-activated receptor alpha (PPARalpha) plays a key role in lipid and lipoprotein metabolism. However, important inter- and intraspecies differences exist in the response to PPARalpha activators. This incited us to screen for PPARalpha variants with different signaling functions. In the present study, using a RT-PCR approach a variant human PPARalpha 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 PPARalpha protein (PPARalphatr) lacking part of the hinge region and the entire ligand-binding domain. RNase protection analysis demonstrated that PPARalphatr mRNA is expressed in several human tissues and cells, representing between 20-50% of total PPARalpha mRNA. By contrast, PPARalphatr mRNA could not be detected in rodent tissues. Western blot analysis using PPARalpha-specific antibodies demonstrated the presence of an immunoreactive protein migrating at the size of in vitro produced PPARalphatr protein both in human hepatoma HepG2 cells and in human hepatocytes. Both in the presence or absence of 9-cis-retinoic acid receptor, PPARalphatr did not bind to DNA in gel shift assays. Immunocytochemical analysis of transfected CV-1 cells indicated that, whereas transfected PPARalphawt was mainly nuclear localized, the majority of PPARalphatr resided in the cytoplasm, with presence in the nucleus depending on cell culture conditions. Whereas a chimeric PPARalphatr protein containing a nuclear localization signal cloned at its N-terminal localized into the nucleus and exhibited strong negative activity on PPARalphawt transactivation function, PPARalphatr interfered with PPARalphatr transactivation function only under culture conditions inducing its nuclear localization. Cotransfection of the coactivator CREB-binding protein relieved the transcriptional repression of PPARalphawt by PPARalphatr, suggesting that the dominant negative effect of PPARalphatr might occur through competition for essential coactivators. In addition, PPARalphatr interfered with transcriptional activity of other nuclear receptors such as PPARgamma, hepatic nuclear factor-4, and glucocorticoid receptor-alpha, which share CREB-binding protein/p300 as a coactivator. Thus, we have identified a human PPARalpha splice variant that may negatively interfere with PPARalphawt function. Factors regulating either the ratio of PPARalphawt vs. PPARalphatr mRNA or the nuclear entry of PPARalphatr protein should therefore lead to altered signaling via the PPARalpha and, possibly also, other nuclear receptor pathways.
Gervois
P
P
U.325 INSERM, Département d'Athérosclérose, Institut Pasteur de Lille, France.
Torra
I P
IP
Chinetti
G
G
Grötzinger
T
T
Dubois
G
G
Fruchart
J C
JC
Fruchart-Najib
J
J
Leitersdorf
E
E
Staels
B
B
D000818:Animals; D016633:Apolipoprotein A-II / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014018:Tissue Distribution; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D016633:Apolipoprotein A-II; D017362:Cyclic AMP Response Element-Binding Protein; D009687:Nuclear Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D012097:Repressor Proteins; D014157:Transcription Factors