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

描述(申请人提供)：Mallory Denk小体(MDB)是脂肪性肝炎(酒精性和非酒精性)患者的肝细胞胞浆内含物，但也可在其他肝脏疾病中发现，包括肝细胞癌和铜代谢障碍。最近的证据表明，MDBS的存在与肝病进展加速有关。MDBS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)，它们形成专有的非共价K8和K18杂二聚体，以四聚体和更高阶齐聚物的形式结合。MDB的形成是通过给小鼠喂食产卟啉药物诱导的，需要K8的优先过表达(以建立K8和GT；K18状态)和K8通过转谷氨酰胺酶进行交联。在铁螯合酶基因点突变的小鼠(FCH/FCH小鼠)中已发现自发性MDB，这些小鼠由于原卟啉沉积在肝脏中而患上红细胞生成性原卟啉(EPP)。此外，与雌性小鼠相比，同一测试品系中的雄性小鼠更容易积累MDB。这项提议将检验的中心假设是，卟啉沉积、长期酒精暴露和遗传性别相关的差异创造了促进MDB形成的环境，MDB的形成有助于肝细胞的最终细胞命运。由于MDB形成的生物学意义，以及MDB对肝细胞是有益的还是有害的，尽管弗兰克·马洛里博士在近100年前就首次描述了MDB，但这一提议所涵盖的主题是一个新的且研究不足的领域。我们的假设将利用三个特定目标进行验证：(I)在携带铁络合酶基因突变的EPP小鼠模型中表征MDB形成的遗传易感性和机制，(Ii)建立小鼠MDB酒精模型，(Iii)表征含有MDB的肝细胞在小鼠和人肝脏中的细胞命运。这项拟议的研究结合了分子和生化方法，以及最先进的技术，并包括小鼠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建立迄今缺乏的酒精小鼠MDB模型，在酒精性肝病和EPP模型中确定潜在的性别与MDB形成的关联，以及确定MDB在个体肝细胞中形成的后果。拟议研究的结果将有助于澄清MDBS的药理操作是否有助于肝病的治疗。我们预计，我们的发现也将有助于理解其他与包涵体相关的肝外疾病，如肌病和含有胞浆IF包涵体的神经疾病。 公共卫生相关性：各种引起肝硬变的肝病患者在肝细胞中形成不溶性沉积，称为Mallory-Denk小体(MDB)，临床医生用它来帮助他们建立肝病诊断。MDB的意义尚不清楚，但促进其分解或形成的药物可能提供治疗益处。我的建议将建立小鼠模型来研究MDB的形成，并应有助于我们了解MDB在人类和小鼠肝脏中对肝细胞是保护还是有害。

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.