Tseng Pei-Chi, Hsu Hsing-Chih, Janmanchi Damodar, Lin Chih-Hsiu, Kuo Yueh-Hsiung, Chou Chen-Kung, Yeh Sheau-Farn
Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei City 112, Taiwan, ROC.
Biochem Pharmacol. 2008 Oct 30;76(9):1121-33. doi: 10.1016/j.bcp.2008.08.023. Epub 2008 Aug 27.
An elevated level of macrophage inflammatory protein-1beta (MIP-1beta) induced by IL-1beta has been correlated with chronic hepatic inflammatory disease. However, molecular mechanism of IL-1beta-induced MIP-1beta expression in hepatic cells is obscure. Previously, we reported the mechanism of the anti-hepatitis B virus (HBV) activity of helioxanthin (HE-145). Here, we demonstrated that HE-145 inhibited IL-1beta-induced MIP-1beta expression in a dose-dependent manner in Huh7 cells. To understand the mode of action of HE-145, we first examined how IL-1beta induced MIP-1beta expression at the molecular level. Using selective inhibitors, we found that JNK and p38 pathways participated in IL-1beta-induced MIP-1beta expression. HE-145 specifically suppressed IL-1beta-induced c-jun mRNA and protein expression and prevented c-jun-mediated AP-1 DNA-binding activity, whereas it had no effect on IL-1beta-induced activation of JNK, p38 and ATF2. Further studies indicated that HE-145 may downregulate c-jun mRNA expression directly at transcriptional level without requirement of de novo protein synthesis. Mutational analysis and supershift assays indicated that IL-1beta stimulated c-jun and CREB1 binding to the essential AP-1/CRE site of the MIP-1beta promoter. The inhibitory effect of HE-145 on IL-1beta-induced MIP-1beta promoter activity was completely reversed by overexpressing c-jun. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay consistently revealed that HE-145 reduced c-jun binding to the AP-1/CRE site in vitro and in vivo. Our results established a major role for c-jun in IL-1beta-induced MIP-1beta expression in hepatic cells. The reduction in IL-1beta-induced c-jun expression and subsequent binding of the c-jun/CREB1 complex to AP-1/CRE site mainly contributed to the inhibitory action of HE-145 on IL-1beta-induced MIP-1beta production.
白细胞介素-1β（IL-1β）诱导的巨噬细胞炎性蛋白-1β（MIP-1β）水平升高与慢性肝脏炎性疾病相关。然而，IL-1β诱导肝细胞中MIP-1β表达的分子机制尚不清楚。此前，我们报道了海葵黄质（HE-145）抗乙型肝炎病毒（HBV）活性的机制。在此，我们证明HE-145在Huh7细胞中以剂量依赖的方式抑制IL-1β诱导的MIP-1β表达。为了解HE-145的作用模式，我们首先在分子水平上研究了IL-1β如何诱导MIP-1β表达。使用选择性抑制剂，我们发现JNK和p38信号通路参与了IL-1β诱导的MIP-1β表达。HE-145特异性抑制IL-1β诱导的c-jun mRNA和蛋白表达，并阻止c-jun介导的AP-1 DNA结合活性，而对IL-1β诱导的JNK、p38和ATF2激活没有影响。进一步研究表明，HE-145可能在转录水平直接下调c-jun mRNA表达，而无需从头合成蛋白质。突变分析和超迁移实验表明，IL-1β刺激c-jun和CREB1与MIP-1β启动子的必需AP-1/CRE位点结合。过表达c-jun完全逆转了HE-145对IL-1β诱导的MIP-1β启动子活性的抑制作用。电泳迁移率变动分析（EMSA）和染色质免疫沉淀（ChIP）实验一致表明，HE-145在体外和体内均减少了c-jun与AP-1/CRE位点的结合。我们的结果确定了c-jun在IL-1β诱导的肝细胞MIP-1β表达中起主要作用。IL-1β诱导的c-jun表达减少以及随后c-jun/CREB1复合物与AP-1/CRE位点的结合减少主要促成了HE-145对IL-1β诱导的MIP-1β产生的抑制作用。
