Functions and Regulatory Mechanisms of Peroxisome Proliferator-activated Receptor (PPAR)

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The following four themes were studied on PRAR.1. Transcriptional regulation of PEX11α gene : To clarify the mechanism of peroxisome proliferation by the peroxisome proliferators, the mechanism of transcriptional regulation was investigated on the PEX11α gene, which is possibly implicated in this process. An enhancer element of the mouse PEX11αgene was searched for by reporter assays, using cultured mammalian cells. An effective enhancer sequence was found in the downstream region of the gene, ca. 8 kb apart from the transcriptional start site. This sequence matched the consensus PPAR-binding site, and indeed bound to PPARα/RXR heterodimer. The physiological significance of this element is further studied.2. Stabilization of PPARα by the ligand : When PPARα was forcedly expressed in IIeLa cells, the efficiency of expression was poor, but it was significantly improved by a ligand. A pulse-chase radiolabeling experiment showed that the expressed PPARα was quite unstable in the cells, and … More the half-life was markedly extended by the ligand. When RXR was co-expressed the stability of PPARα was improved, and not affected any more by the ligand. Even if the rat hepatoma H4IIEC3 was cultured in the presence of the ligand, the intracellular level of PPARα was not changed. Hence, PPARα is probably stabilized by the heterodimerization with RXR in the cells, and the excess PPARα is rapidly degraded. The ligand seems to suppress this dgradative process.3. Coativators interacting with the PPARα AF-1 : Coactivators that cooperate with the N-terminalligand-independent transactivating domain of PPARα were sought. Typical coactivators, such as CBP, SRC-1, and TAFII31, known to interact with the AF-1 of other nuclear receptors, did not exhibit any interaction with the AF-1 of PPARα, on the yeast and mammalian two-hybrid assay. Screening for a novel coactivator by conventional yeast two-hybrid method was unsuccessful, because of the strong transactivating activity of the AF-1 in the yeast. Further screening is now attempted using a new two-hybrid strategy.4.Transcriprional regulation of PPARγ gene : PPARγ, a critical regulator of adipocite differentiation, is itself induced significantly in the differentiation process. The enhancer element of the mouse PPARγ2 gene was searched for, by the transfection assay employing the 3T3-L1 preadipocites. No enhancer activity responding to the differentiation was found, in the 15 kb upstream region as well as the first intron region 7.5 jb downstream from the start site. Further screening of the enhancer sequence is continued for the PPAEγ2 as well as PPARγ1. Less