Molecular characterisation of peroxisome proliferator-activated receptor (PPAR) genes in mammals / Molekylær karakterisering av peroxisom proliferator-aktivert reseptor (PPAR) gener hos pattedyr
Peroxisome proliferator activated receptors (PPARs) are members of the nuclear hormone receptor superfamily and comprise a class of ligand-activated transcription factors that play a pivotal role in regulating lipid homeostasis. PPARs are activated by a broad range of compounds including various fatty acids. Three distinct PPARs, termed -α, -β and -γ, each encoded by a separate gene and showing a distinct tissue distribution pattern according to their respective functions, have been identified in mouse and humans.
Here we report the isolation of the cDNAs encoding the three PPAR subtypes in swine and cattle. The putative amino acid sequences of porcine and bovine PPARs were highly conserved (90-95%) to other species. However, whereas PPARγ showed an overall extensive sequence conservation among species, the -α and -β subtypes were evolutionary less conserved when comparing cattle, swine and humans to the corresponding rat and mouse sequences. This evolutionary divergence could reflect the observed species differences among humans and rat/mouse in the physiological response to PPARα activators. The expression patterns of the porcine and bovine PPARs were determined by Northern blot hybridisations in various tissues. Two transcripts of PPARγ (-γ1 and –γ2), that were highly expressed in adipose tissue, were detected in both species. Whereas both –γ1 and –γ2 mRNA were found in several additional bovine tissues, porcine PPARγ2 expression was confined to adipose tissue. PPARγ mRNA was also abundantly expressed in the spleen, while lower levels were detected in lung, ovary, mammary gland, intestine and muscle. PPARα was the predominant subtype expressed in liver and kidney, while lower levels were detected in spleen, ovary, adipose tissue, mammary gland, muscle, lung and heart. By genetic linkage analysis, the α- and γ- subtypes of PPAR were mapped to bovine chromosome 5 and 22, respectively. These chromosomal locations are consistent with comparative maps for human and mouse.
The expression profiles of porcine PPAR -α and -γ during maturation of the animals were analysed in two breeds of pigs (Norwegian Landrace and Duroc) that differ considerably in fat deposition. Opposite expression levels of PPARα in heart and liver were detected in piglets of the two breeds, which may reflect a physiological difference between the two breeds with respect to metabolising fatty acids. On the contrary, the expression profile in mature individuals revealed a similar expression profile for the two breeds with predominant lever of PPARα mRNA in liver and kidney. Interestingly, the PPARγ mRNA level increased with age and paralleled the level of subcutaneous adipose tissue depots in the two breeds of pigs. These results indicate a role for PPARγ in adipocyte differentiation in swine as reported in other species.
The generation of several transcripts from a single gene, by different promoter usage and/or alternative splicing, is frequently observed for members of the nuclear hormone receptor superfamily. An alternative spliced mRNA of porcine PPARα that lacked exon 5 was identified by RT-PCR and sequencing. Due to introduction of a premature stop codon, the putative protein lacks part of the DNA-binding domain and the entire ligang-binding domain. The truncated protein shares similarity to a variant present in man that results from alternative splicing and skipping of exon 6. In man, the truncated protein was demonstrated to inhibit the normal acitivity of PPARα by sequestering common cofactors. The function of the similar PPARα protein in swine remains to be established.
Sequence analysis of the human PPARγ gene revealed a putative regulatory region in intron B. The fragment was inserted into a reporter plasmid from which it was shown to promote transcription in several cell lines in transient transfection assays. Primer extension analysis on human white adipose tissue (WAT) indicated the presence of a novel mRNA, designated PPARγ4, with transcription initiation site located at the 5` boundary of exon 1. The upstream promoter region contained a responsive element (RORE) for the nuclear receptor RORα (retinoic acid receptor-related orphan receptor). By EMSA, we showed that the RORα1 isoform specifically bound to this RORE. Over-expression of RORα1 induced the transcription of the reporter construct containing RORE in several cell lines in transient transfection assays. In conclusion, this study demonstrated the presence of a fourth promoter within the human PPARγ gene from which the transcription of a novel mRNA was driven. The finding that RORα1 was shown to induce transcription from the new promoter in vitro indicates that RORα1 might form part of the complex transcription machinery regulating PPARγ gene expression in humans.
