Xenobiotic Receptors

PPARalpha：为了在小鼠中概括人类PPARalpha的调控和组织特异性表达，制备了一种新的PPARalpha人源化小鼠细胞系，该细胞系由人类原生基因表达人类受体。利用P1噬菌体人工染色体（PAC）基因组克隆，在pparalpha缺失的小鼠背景上培育pparalpha人源化转基因小鼠，命名为pparalpha （PAC）。在hpparα (PAC)小鼠中，人pparα基因在脂肪酸高分解代谢的组织中表达，并在禁食时被诱导表达，类似于野生型小鼠的小鼠pparα。经过氧化物酶体增殖剂非诺贝特治疗后，hppar （PAC）小鼠表现出与野生型小鼠相似的反应，包括过氧化物酶体增殖、降低血清甘油三酯和诱导pparα靶基因编码参与肝、肾和心脏脂肪酸代谢的酶，这表明人类pparα在调节脂肪酸代谢和降低血清甘油三酯方面的功能与小鼠pparα相同。然而，与野生型小鼠相比，非诺贝特治疗hparα (PAC)小鼠并未引起明显的肝脏肥大和肝细胞增殖，这表明pparα影响脂质代谢的机制与肝细胞增殖反应不同，后者仅由小鼠pparα诱导。PPARalpha（PAC）小鼠模型为研究PPARalpha介导的物种差异提供了一个体内平台，也是评估人类过氧化物酶体增殖物暴露风险的理想模型。为了确定物种对过氧化物酶体增殖剂的反应差异机制以及过氧化物酶体增殖剂诱导肝癌发生的机制，我们对野生型和hppar α (TetOFF)小鼠进行了微阵列分析，分别对肝细胞增殖和肝癌敏感和耐药。揭示了pparα调控基因表达和肝细胞增殖的新机制。MicroRNA （miRNA）表达谱显示，活化的pparα是肝脏miRNA表达的主要调节因子。特别有趣的是，在野生型小鼠中，强效PPAR_激动剂wey - 14643治疗4小时、2周和11个月后，let-7C（一种对细胞生长很重要的miRNA）被抑制。let-7C通过与c-myc的3个非翻译区直接相互作用而靶向c-myc。ppar_介导的通过let-7C诱导的c-myc随后增加了致癌mir-17-92簇的表达；这些事件在pparα缺失的小鼠中没有发生。过表达let-7C可降低c-myc和mir-17，抑制Hepa-1细胞的生长。此外，利用表达人类ppar -的小鼠模型，该模型对wy - 14643对β -氧化和血清甘油三酯的影响有反应，但对肝细胞增殖和肿瘤发生有抗性，我们证明了let-7C在肝脏肿瘤发生中的关键作用。wy - 14643处理没有抑制let-7C或诱导c-myc和mir-17的表达。这些观察结果揭示了let-7C信号级联对PPAR_激动剂诱导的肝脏增殖和肿瘤发生至关重要。PXR：人类PXR激活最常见的临床意义是发生由上调的细胞色素P450 3A （CYP3A）同工酶介导的药物-药物相互作用。典型的啮齿动物模型不能预测人类PXR介导的药物-药物相互作用，因为物种对PXR配体的反应存在差异。本研究采用细菌人工染色体（BAC）转基因技术，在PXR缺失的小鼠中建立了PXR人源化小鼠模型，BAC克隆含有完整的人类PXR基因和5′-和3′-侧翼序列。在这个PXR人源化小鼠模型中，PXR在肝脏和肠道中选择性表达，与CYP3A的组织表达模式相同。用PXR配体处理PXR人源化小鼠模拟了人的反应，因为肝脏和肠道CYP3As都被利福平（人类特异性PXR配体）强烈诱导，但不被孕烯醇酮16 α -碳腈（啮齿动物特异性PXR配体）诱导。在利福平预处理的pxr人源化小鼠中，由于咪达唑仑1′-羟基化增加了3倍，咪达唑仑最大血清浓度（Cmax）和浓度-时间曲线下面积均下降了约60%。这些结果说明了PXR人源化小鼠在CYP3A介导的药物-药物相互作用研究中的潜在效用，并表明PXR人源化小鼠模型将是评估药物激活人类PXR的整体药代动力学后果的适当体内工具。利福昔明是一种被批准用于治疗旅行者腹泻的利福霉素类似物，也有益于治疗多种慢性胃肠道疾病。然而，利福昔明对慢性胃肠疾病的作用机制尚不完全清楚。在目前的研究中，利福昔明被研究其在PXR激活中的作用，PXR是一种核受体，调节参与外源性和有限内源性沉积和解毒的基因。将PXR人源化（hPXR）、PXR零型和野生型小鼠口服利福昔明和利福平（一种表征良好的人PXR配体）。与利福平相比，利福昔明在肠道中浓度较高。利福昔明在hPXR小鼠肠道中显著诱导PXR靶基因，而在野生型和无PXR小鼠肠道中无明显诱导作用。然而，利福昔明治疗对野生型、无PXR和hPXR小鼠肝脏PXR靶基因没有显著影响。与体内数据一致，基于细胞的报告基因分析显示利福昔明介导的人类PXR活化，但不包括其他外源核受体CAR， (PPAR) α， PPAR γ和FXR。利福昔明预处理不影响CYP3A底物咪达唑仑的药代动力学，但增加了1′-羟咪达唑仑的Cmax，降低了其Tmax。总的来说，目前的研究确定了利福昔明是一种肠道特异性的人类PXR配体，并为hPXR小鼠作为研究人类PXR激活剂的关键工具提供了进一步的证据。建议进一步的人体研究来评估利福昔明介导的肠道PXR激活在慢性胃肠道疾病治疗中的潜在作用。AHR: CYP1A1是最重要的解毒酶之一，因为它具有广泛的底物特异性和广泛的分布在全身。另一方面，CYP1A1也可以通过氧化多环芳烃产生高致癌的中间代谢物。我们描述了我们认为是一种新的调节系统，通过一种源于肠道的因子来调节全身CYP1A1的表达。产生了一种突变小鼠，其中Arnt基因主要在肠上皮中被破坏。令人惊讶的是，CYP1A1 mRNA的表达和酶活性在几乎所有的非肠道组织中都显著升高。这种诱导甚至在怀孕突变雌性的早期胚胎中也观察到。有趣的是，这种上调是CYP1A1选择性的，并且在使用合成纯化饮食后消失。M[截断为7800个字符的摘要]

PPARalpha: A new PPARalpha humanized mouse line was produced in which the human receptor is expressed from the native human gene in order to recapitulate in the mouse the regulation and tissue-specific expression of the human PPARalpha. PPARalpha-humanized transgenic mice were generated using a P1 phage artificial chromosome (PAC) genomic clone bred onto a PPARalpha-null mouse background, designated hPPARalpha(PAC). In hPPARalpha(PAC) mice, the human PPARalpha gene is expressed in tissues with high fatty acid catabolism and induced upon fasting, similar to mouse PPARalpha in wild-type mice. Upon treatment with the peroxisome proliferator fenofibrate, hPPARalpha(PAC) mice exhibited responses similar to wild-type mice, including peroxisome proliferation, lowering of serum triglycerides and induction of PPARalpha target genes encoding enzymes involved in fatty acid metabolism in liver, kidney and heart, suggesting that human PPARalpha functions in the same manner as mouse PPARalpha in regulating fatty acid metabolism and lowering serum triglycerides. However, in contrast to wild-type mice, treatment of hPPARalpha(PAC) mice with fenofibrate did not cause significant hepatomegaly and hepatocyte proliferation, thus indicating that the mechanisms by which PPARalpha affects lipid metabolism are distinct from the hepatocyte proliferation response, the latter of which is only induced by mouse PPARalpha. The hPPARalpha(PAC) mouse model provides an in vivo platform to investigate the species difference mediated by PPARalpha and an ideal model for human risk assessment peroxisome proliferators exposure. To determine the mechanism of species differences in responses to peroxisome proliferators and the mechanism of peroxisome proliferator-induced hepatocarcinogenesis, microarray analysis was done on Wy-14,643 (a PPARalpha activator) treated wild-type and hPPARalpha (TetOFF) mice, sensitive and resistant, respectively to hepatocyte proliferation and liver cancer. A novel mechanism by which PPARalpha regulates gene expression and hepatocellular proliferation was uncovered. MicroRNA (miRNA) expression profiling demonstrated that activated PPARalpha was a major regulator of hepatic miRNA expression. Of particular interest, let-7C, an miRNA important in cell growth, was inhibited following 4-h treatment and 2-week and 11-month sustained treatment with the potent PPAR_ agonist Wy-14,643 in wild-type mice. let-7C was shown to target c-myc via direct interaction with the 3 untranslated region of c-myc. The PPAR_-mediated induction of c-myc via let-7C subsequently increased expression of the oncogenic mir-17-92 cluster; these events did not occur in PPARalpha-null mice. Overexpression of let-7C decreased c-myc and mir-17 and suppressed the growth of Hepa-1 cells. Furthermore, using the human PPARalpha-expressing mouse model, which is responsive to Wy-14,643 effects on beta-oxidation and serum triglycerides but resistant to hepatocellular proliferation and tumorigenesis, we demonstrated a critical role for let-7C in liver oncogenesis. Wy-14,643 treatment did not inhibit let-7C or induce c-myc and mir-17 expression. These observations reveal a let-7C signaling cascade critical for PPAR_ agonist-induced liver proliferation and tumorigenesis. PXR: The most common clinical implication for the activation of the human PXR is the occurrence of drug-drug interactions mediated by up-regulated cytochromes P450 3A (CYP3A) isozymes. Typical rodent models do not predict drug-drug interactions mediated by human PXR because of species differences in response to PXR ligands. In the current study, a PXR-humanized mouse model was generated by bacterial artificial chromosome (BAC) transgenesis in PXR-null mice using a BAC clone containing the complete human PXR gene and 5'- and 3'-flanking sequences. In this PXR-humanized mouse model, PXR is selectively expressed in the liver and intestine, the same tissue expression pattern as CYP3A. Treatment of PXR-humanized mice with the PXR ligands mimicked the human response, since both hepatic and intestinal CYP3As were strongly induced by rifampicin, a human-specific PXR ligand, but not by pregnenolone 16alpha-carbonitrile, a rodent-specific PXR ligand. In rifampicin-pretreated PXR-humanized mice, an approximately 60% decrease was observed for both the maximal midazolam serum concentration (Cmax) and the area under the concentration-time curve, as a result of a 3-fold increase in midazolam 1'-hydroxylation. These results illustrate the potential utility of the PXR-humanized mice in the investigation of drug-drug interactions mediated by CYP3A and suggest that the PXR-humanized mouse model would be an appropriate in vivo tool for evaluation of the overall pharmacokinetic consequences of human PXR activation by drugs. Rifaximin, a rifamycin analog approved for the treatment of travelers' diarrhea, is also beneficial in the treatment of multiple chronic gastrointestinal disorders. However, the mechanisms contributing to the effects of rifaximin on chronic gastrointestinal disorders are not fully understood. In the current study, rifaximin was investigated for its role in activation of the PXR, a nuclear receptor that regulates genes involved in xenobiotic and limited endobiotic deposition and detoxication. PXR-humanized (hPXR), PXR-null, and wild-type mice were treated orally with rifaximin, and rifampicin, a well characterized human PXR ligand. Rifaximin was highly concentrated in the intestinal tract compared with rifampicin. Rifaximin treatment resulted in significant induction of PXR target genes in the intestine of hPXR mice, but not in wild-type and PXR-null mice. However, rifaximin treatment demonstrated no significant effect on hepatic PXR target genes in wild-type, PXR-null, and hPXR mice. Consistent with the in vivo data, cell-based reporter gene assay revealed rifaximin-mediated activation of human PXR, but not the other xenobiotic nuclear receptors CAR, (PPAR)alpha, PPARgamma, and FXR. Pretreatment with rifaximin did not affect the pharmacokinetics of the CYP3A substrate midazolam, but it increased the Cmax and decreased Tmax of 1'-hydroxymidazolam. Collectively, the current study identified rifaximin as a gut-specific human PXR ligand, and it provided further evidence for the utility of hPXR mice as a critical tool for the study of human PXR activators. Further human studies are suggested to assess the potential role of rifaximin-mediated gut PXR activation in therapeutics of chronic gastrointestinal disorders. AHR: CYP1A1 is one of the most important detoxification enzymes due to its broad substrate specificity and wide distribution throughout the body. On the other hand, CYP1A1 can also produce highly carcinogenic intermediate metabolites through oxidation of polycyclic aromatic hydrocarbons. We describe what we believe to be a novel regulatory system for whole-body CYP1A1 expression by a factor originating in the gut. A mutant mouse was generated in which the Arnt gene is disrupted predominantly in the gut epithelium. Surprisingly, CYP1A1 mRNA expression and enzymatic activities were markedly elevated in almost all non-gut tissues in this mouse line. The induction was even observed in early-stage embryos in pregnant mutant females. Interestingly, the upregulation was CYP1A1 selective and lost upon administration of a synthetic purified diet. M [summary truncated at 7800 characters]