Genetic analysis of four novel peroxisome proliferator activated receptor- [formula omitted] splice variants in monkey macrophages

Abstract

Peroxisome proliferator activated receptor-γ (PPAR-γ) is abundantly expressed in atherosclerotic lesions and is implicated in atherogenesis. The existence of three splice variants, PPAR-γ1, PPAR-γ2, and PPAR-γ3 has been established. Using monocyte-derived macrophages from cynomolgus monkeys, we demonstrate here the identification of two new PPAR-γ exons, exon C and exon D, which splice together with already established exons A1, A2, and B in the 5 ′ terminal region to generate four novel PPAR-γ subtypes, PPAR-γ4, -γ5, -γ6, and -γ7. PPAR-γ4 and γ5 were detected only in macrophages whereas γ6 and γ7 were expressed both in macrophages and adipose tissues. None of these novel isoforms were detected in muscle, kidney, and spleen from monkeys. We found sequences identical to exons C and D in the human genome database. These and all PPAR-γ exons known to date are encoded by a single gene, located from region 10498 K to 10384 K on human chromosome 3. We cloned and expressed PPAR-γ1, PPAR-γ4, and PPAR-γ5 proteins in yeast using the expression vector pPICZB. As expected, all recombinant proteins showed a molecular weight of approximately 50 kDa. We also investigated the effect of a high-fat diet on the level of macrophage PPAR-γ expression in monkeys. RT-PCR showed a significant increase in total PPAR-γ and ABCA1 mRNA levels in macrophages of fat-fed monkeys ( n=7) compared to those maintained on a normal diet ( n=2). However, none of the novel isoforms seemed to be induced by fat-feeding. We used tetracycline-responsive expression vectors to obtain moderate expression of PPAR-γ4 and -γ5 in CHO cells. In these cells, expression of PPAR-γ5 but not -γ4 repressed the expression of ABCA1. Neither isoform modulated the expression of lipoprotein lipase. Our results suggest that individual PPAR-γ isoforms may be responsible for unique tissue-specific biological effects and that PPAR-γ4 and -γ5 may modulate macrophage function and atherogenesis.