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

在prar - 1上研究了以下四个主题。PEX11α基因的转录调控：为了阐明过氧化物酶体增殖因子对过氧化物酶体增殖的调控机制，我们对可能参与过氧化物酶体增殖的PEX11α基因的转录调控机制进行了研究。利用培养的哺乳动物细胞，通过报告基因检测寻找小鼠pex11 α基因的增强子元件。在该基因的下游区域发现了一个有效的增强子序列，距离转录起始位点约8 kb。该序列与PPARα/RXR异源二聚体相匹配。该元素的生理意义有待进一步研究。配体对PPARα的稳定作用：在IIeLa细胞中强制表达时，PPARα的表达效率较差，但配体可显著提高其表达效率。脉冲追踪放射性标记实验表明，表达的PPARα在细胞内非常不稳定，而且配体明显延长了半衰期。当RXR共表达时，PPARα的稳定性提高，不受配体的影响。即使在有配体存在的情况下培养大鼠肝癌H4IIEC3，细胞内PPARα水平也未发生变化。因此，PPARα可能通过与RXR在细胞内的异源二聚化而稳定，并且过量的PPARα被迅速降解。配体似乎抑制了这种降解过程。与PPARα AF-1相互作用的共激活因子：寻找与PPARα的n端非依赖性反激活结构域合作的共激活因子。典型的共激活因子，如CBP、SRC-1和TAFII31，已知与其他核受体的AF-1相互作用，在酵母和哺乳动物的双杂交实验中，没有表现出与PPARα的AF-1相互作用。由于AF-1在酵母中具有很强的反激活活性，用传统的酵母双杂交方法筛选新的辅激活剂是不成功的。现在尝试使用新的双混合策略进行进一步筛选。PPARγ基因的转录调控：PPARγ是脂肪分化的关键调控因子，其本身在脂肪分化过程中受到显著诱导。采用3T3-L1前脂肪细胞转染法，寻找小鼠ppar γ - 2基因的增强子。在上游15kb区域和起始位点下游7.5 jb的第一个内含子区域，没有发现与分化相关的增强子活性。进一步筛选PPAEγ2和PPARγ1的增强子序列。少

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