Biological Clock Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan.
Mol Pharmacol. 2010 Jul;78(1):135-41. doi: 10.1124/mol.110.064402. Epub 2010 Apr 16.
A functional interaction between peroxisome proliferator-activated receptor alpha (PPARalpha) and components of the circadian clock has been suggested, but whether these transcriptional factors interact to regulate the expression of their target genes remains obscure. Here we used a PPARalpha ligand, bezafibrate, to search for PPARalpha-regulated genes that are expressed in a CLOCK-dependent circadian manner. Microarray analyses using hepatic RNA isolated from bezafibrate treated-wild type, Clock mutant (Clk/Clk), and PPARalpha-null mice revealed that 136 genes are transcriptionally regulated by PPARalpha in a CLOCK-dependent manner. Among them, we focused on the plasminogen activator inhibitor-1 (PAI-1) gene, because its expression typically shows circadian variation, and it has transcriptional response elements for both PPAR and CLOCK. The bezafibrate-induced expression of PAI-1 mRNA was attenuated in Clk/Clk mice and in PPARalpha-null mice. The protein levels of PPARalpha were reduced in Clk/Clk hepatocytes. However, the overexpression of PPARalpha could not rescue bezafibrate-induced PAI-1 expression in Clk/Clk hepatocytes, suggesting that impaired bezafibrate-induced PAI-1 expression in Clk/Clk mice is not due to reduced PPARalpha expression. Luciferase reporter and chromatin immunoprecipitation analyses using primary hepatocytes demonstrated that DNA binding of both PPARalpha and CLOCK is essential for bezafibrate-induced PAI-1 gene expression. Pull-down assays in vitro showed that both PPARalpha and its heterodimerized partner retinoic acid receptor-alpha can serve as potential interaction targets of CLOCK. The present findings revealed that molecular interaction between the circadian clock and the lipid metabolism regulator affects the bezafibrate-induced gene expression.
过氧化物酶体增殖物激活受体α（PPARα）与生物钟成分之间存在功能相互作用，但这些转录因子是否相互作用以调节其靶基因的表达尚不清楚。在这里，我们使用 PPARα 配体 bezafibrate 来搜索以 CLOCK 依赖性节律方式表达的 PPARα 调节的基因。使用来自 bezafibrate 处理的野生型、Clock 突变（Clk/Clk）和 PPARα 缺失小鼠的肝 RNA 进行微阵列分析，揭示了 136 个基因受 PPARα 以 CLOCK 依赖性方式转录调节。其中，我们重点关注纤溶酶原激活物抑制剂-1（PAI-1）基因，因为其表达通常表现出昼夜节律变化，并且具有 PPAR 和 CLOCK 的转录反应元件。在 Clk/Clk 小鼠和 PPARα 缺失小鼠中，bezafibrate 诱导的 PAI-1 mRNA 表达减弱。Clk/Clk 肝细胞中的 PPARα 蛋白水平降低。然而，PPARα 的过表达不能挽救 Clk/Clk 肝细胞中 bezafibrate 诱导的 PAI-1 表达，表明 Clk/Clk 小鼠中受损的 bezafibrate 诱导的 PAI-1 表达不是由于 PPARα 表达减少所致。使用原代肝细胞进行的荧光素酶报告基因和染色质免疫沉淀分析表明，PPARα 和 CLOCK 的 DNA 结合对于 bezafibrate 诱导的 PAI-1 基因表达都是必需的。体外下拉测定表明，PPARα 及其异二聚体伙伴视黄酸受体-α都可以作为 CLOCK 的潜在相互作用靶标。本研究结果表明，生物钟和脂质代谢调节剂之间的分子相互作用会影响 bezafibrate 诱导的基因表达。
