Biological significance of Mallory-Denk bodies in mouse and human liver diseases

描述（由申请方提供）：马洛里登克小体（MDB）是肝细胞胞质包涵体，是脂肪性肝炎（酒精性和非酒精性）患者的组织学标志，但也可在其他肝脏疾病（包括肝细胞癌和铜代谢疾病）中发现。最近的证据表明，MDB的存在与加速肝脏疾病进展有关。MDB的主要成分是细胞骨架中间丝（IF）蛋白，角蛋白多肽8和18（K8/K18），它们形成专性非共价K8和K18异二聚体，聚结为四聚体和更高级的寡聚体。MDB的形成是通过给小鼠喂食卟啉原药物诱导的，并且需要K8的优先过表达（以建立K8>K18状态）和通过转氨酶的K8交联。在铁螯合酶基因点突变的小鼠（fch/fch小鼠）中描述了自发性MDB，由于肝脏中的原卟啉沉积，这些小鼠发生红细胞生成性原卟啉症（EPP）。此外，与雌性小鼠相比，一种测试品系的雄性小鼠更容易积聚MDB。 本提案中将测试的中心假设是，卟啉沉积、慢性酒精暴露和遗传性别相关差异创造了一个促进MDB形成的环境，MDB的形成有助于肝细胞最终的细胞命运。本提案涵盖的主题是一个新的和未充分研究的领域，因为MDB形成的生物学意义以及MDB对肝细胞是有益还是有害，尽管Frank马洛里博士在近100年前首次描述，但仍然不清楚。我们的假设将利用三个具体目标进行测试：（i）表征携带铁螯合酶基因突变的EPP小鼠模型中MDB形成的遗传易感性和机制，（ii）开发小鼠MDB酒精模型，（iii）表征小鼠和人类肝脏中含MDB肝细胞的细胞命运。 拟议的研究结合了分子和生物化学方法以及最先进的技术，包括小鼠MDB模型以及人类肝脏MDB分子分析。我们希望建立的基本发现包括迄今缺乏的酒精小鼠MDB模型的产生，识别酒精性肝病和EPP模型中MDB形成的潜在性别关联，以及确定单个肝细胞中MDB形成的后果。拟议研究的结果将有助于澄清MDB的药理学操作是否有助于治疗肝脏疾病。我们预期，我们的发现也将有助于了解其他与包涵体相关的肝外疾病，如肌病和神经病，这些疾病含有胞质含IF包涵体。 公共卫生相关性：患有各种引起炎症的肝病的患者在肝细胞中产生不溶性沉积物，称为Mallory-Denk Bodies（MDB），临床医生使用它来帮助他们建立肝病诊断。MDBs的重要性知之甚少，但促进其消退或形成的药物可能提供治疗益处。我的建议将开发小鼠模型来研究MDB的形成，并帮助我们了解人类和小鼠肝脏中的MDB是否对肝细胞具有保护作用或有害作用。

DESCRIPTION (provided by applicant): Mallory Denk Bodies (MDBs) are hepatocyte cytoplasmic inclusions that serve as histological hallmark in patients with steatohepatitis (alcoholic and nonalcoholic), but can also be found in other liver disorders including hepatocellular carcinoma and copper metabolism disorders. Recent evidence suggests that the presence of MDBs associates with accelerated liver disease progression. The major constituents of MDBs are the cytoskeletal intermediate filament (IF) proteins, keratin polypeptides 8 and 18 (K8/K18), which form obligate non-covalent K8 and K18 heterodimers that coalesce as tetramers and higher order oligomers. MDB formation is induced by feeding mice with porphyrinogenic drugs, and requires the preferential overexpression of K8 (to establish a K8>K18 state) and K8 crosslinking by transglutaminase. Spontaneous MDBs have been described in mice with a point mutation in the ferrochelatase gene (fch/fch mice), which develop erythropoietic protoporphyria (EPP) due to protoporphyrin deposition in the liver. Also, male mice in one tested strain are more predisposed to MDB accumulation as compared with female mice. The central hypothesis that will be tested in this proposal is that porphyrin deposition, chronic alcohol exposure and genetic gender-associated differences create an environment that promotes MDB formation, and that MDB formation contributes to the hepatocyte ultimate cell fate. The topic covered by this proposal is a novel and understudied area given that the biological significance of MDB formation, and whether MDBs are beneficial or detrimental to hepatocytes, is still not clear despite being first described nearly 100 years ago by Dr. Frank Mallory. Our hypothesis will be tested utilizing three specific aims: (i) Characterize the genetic susceptibility and mechanism of MDB formation in a mouse model of EPP that carries a ferrochelatase gene mutation, (ii) Develop a mouse MDB alcohol model, (iii) Characterize the cell fate of MDB-containing hepatocytes in mouse and human livers. The proposed study combines molecular and biochemical approaches, and state-of-the-art technologies and includes mouse MDB models, as well as human liver MDB molecular analysis. Fundamental findings that we hope to establish include the generation of an alcohol mouse MDB model that is hitherto lacking, the identification of potential gender association with MDB formation in alcoholic liver disease and EPP models, and determining the consequences of MDB formation in individual hepatocytes. Findings from the proposed studies will help clarify whether the pharmacological manipulation of MDBs might be helpful in the treatment of liver diseases. We anticipate that our findings will also contribute to the understanding of other inclusion-associated extra-hepatic disorders, such as myopathies and neuropathies that harbor cytoplasmic IF- containing inclusions. PUBLIC HEALTH RELEVANCE: Patients with a variety of cirrhosis-causing liver diseases develop insoluble deposits in liver cells, termed Mallory-Denk Bodies (MDBs) that clinicians use to help them establish a liver disease diagnosis. The significance of MDBs is poorly understood, but agents that promote their resolution or formation may provide a therapeutic benefit. My proposal will develop mouse models to study MDB formation, and should help us understand in human and mouse livers whether MDBs are protective or detrimental to liver cells.