Tumor necrosis factor α-dependent up-regulation of Lrh-1 and Mrp3(Abcc3) reduces liver injury in obstructive cholestasis
Tumor necrosis factor α-dependent up-regulation of Lrh-1 and Mrp3(Abcc3) reduces liver injury in obstructive cholestasis
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DOI：
10.1074/jbc.m304011200
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发表时间：
2003-09-19
影响因子：
4.8
通讯作者：
Boyer, JL
中科院分区：
文献类型：
--
作者：
Bohan, A;Chen, WS;Boyer, JL
Mrp 3（Abcc 3）在大鼠和一些人胆汁淤积性肝病中在胆管结扎（BDL）后被显著诱导，并且被认为在这种情况下改善肝损伤。最近，孤儿核受体甲胎蛋白转录因子/胆固醇-7 α-羟化酶启动子因子（CPF/FTF/Lrh-1）已被证明激活Mrp 3的表达。然而，是否炎性细胞因子或胆汁酸水平升高增加Lrh 1/Mrp 3的表达在梗阻性胆汁淤积是未知的。我们假设Mrp 3的诱导与Lrh-1的上调相关，并且需要完整的细胞因子信号传导。对雄性肿瘤坏死因子（Tnf）受体I（Tnfr-/-）小鼠和C57 BLJ野生型（WT）对照进行假手术或胆管结扎。用胆汁酸或细胞因子处理HepG 2细胞。免疫印迹法和真实的实时RT-PCR检测MRP 3/Mrp 3、CPF/Lrh-1、Mrp 2和Bsep的表达。CPF/Lrh-1 DNA与MRP 3/Mrp 3启动子的结合使用电泳迁移率变动分析来评估，并且启动子活性通过荧光素酶分析来确定。采用比色法测定总胆汁酸和乳酸脱氢酶，酶联免疫吸附试验测定细胞因子丰度。WT小鼠BDL后Lrh-1和Mrp 3显著诱导，但Tnfr-/-小鼠未诱导。这与Tnfr-/-小鼠中更严重的肝细胞坏死相关。WT小鼠BDL后Lrh-1与Mrp 3启动子的结合增加，但Tnfr-/-小鼠中没有。TNF α处理HepG 2细胞还上调CPF和MRP 3，增加CPF与MRP 3启动子的结合，并上调MRP 3启动子活性。这些结果表明BDL后Mrp 3的诱导是由于Lrh-1的TNF α依赖性上调。他们提供了强有力的证据，Mrp 3的诱导在阻塞性胆汁淤积期间的肝细胞保护中起着重要作用。
Mrp3(Abcc3) is markedly induced following bile duct ligation (BDL) in the rat and in some human cholestatic liver diseases and is believed to ameliorate liver injury in this setting. Recently, the orphan nuclear receptor fetoprotein transcription factor/cholesterol-7alpha-hydroxylase promoter factor (CPF/FTF/Lrh-1) has been shown to activate Mrp3 expression. However, whether inflammatory cytokines or elevated bile acid levels increased Lrh1/Mrp3 expression in obstructive cholestasis was not known. We hypothesized that induction of Mrp3 would be associated with Lrh-1 up-regulation and would require intact cytokine signaling. Male tumor necrosis factor (Tnf) receptor I (Tnfr-/-) mice and C57BLJ wild type (WT) controls were subjected to sham surgery or bile duct ligation. HepG2 cells were treated with bile acids or cytokines. Immunoblot assay and real time reverse transcriptase-PCR were used to determine expression of MRP3/Mrp3, CPF/Lrh-1, Mrp2, and Bsep. CPF/Lrh-1 DNA binding to the MRP3/Mrp3 promoter was assessed using electrophoretic mobility shift assay, and promoter activity was determined by luciferase assay. Total bile acids and lactate dehydrogenase were measured using colorimetric assays, and cytokine abundance was determined by enzyme-linked immunosorbent assay. Lrh-1 and Mrp3 were significantly induced after BDL in WT but not Tnfr-/- mice. This was associated with more severe hepatocellular necrosis in Tnfr-/- mice. Lrh-1 binding to the Mrp3 promoter increased after BDL in WT but not in Tnfr-/- mice. Tnfalpha treatment of HepG2 cells also up-regulated CPF and MRP3, increased CPF binding to the MRP3 promoter, and up-regulated MRP3 promoter activity. These results indicate that induction of Mrp3 after BDL is due to Tnfalpha-dependent up-regulation of Lrh-1. They provide strong evidence that induction of Mrp3 plays a significant role in hepatocyte protection during obstructive cholestasis.