Role and regulation of beta-catenin in cholestatic liver disease

慢性胆汁淤积症是由胆汁分泌缺陷或胆汁流动障碍引起的，目前的治疗方法很少。 可用的医疗疗法。在过去的筹资期间，我们在确定 Wnt/β-Catenin通路在胆汁淤积性肝病中的多因素作用我们展示β-连锁素 抑制减少胆汁酸(BA)的合成，限制胆管结扎后的CLD和纤维化。然而， β-连环蛋白在MDR2基因敲除(KO)小鼠中的抑制作用 (PSC)，加重而不是减轻伤害。事实上，mdr2-β-catenin-KO(DKO1)小鼠 提示这可能是一种临床上相关的模型，用于研究生物学和 PSC的治疗学。β-连环蛋白也是黏附连接(AJ)的关键成分，其丢失是 通过β-连环蛋白的自发增加来补偿。我们证明了两个β-&γ-连环蛋白从 DKO2小鼠的肝细胞(HCS)和胆管细胞(CCS)可导致CLD，生长受阻，BA升高， 纤维化和死亡率，类似于进行性家族性肝内胆汁淤积症(PFIC)，这是一种儿科CLD。 尽管DKO1和DKO2有不同的基础，但它们有表型上的共同点，包括 肝细胞分化丧失和获得间充质特征；细胞极性丧失； 细胞-细胞连接中的微扰。有趣的是，这些事件中没有一个在CLD中得到了深入研究 和胆管病，使我们的研究具有创新性和重要意义。基于这些发现，我们假设 我们的DKO模型代表了PSC和PFIC病例的子集，这些病例将通过转录本进行验证 分析。无论疾病亚型如何，这些小鼠都可以作为深入研究的强大临床前模型 对细胞成熟/分化、细胞极性和/或细胞-细胞等过程的机制研究 在疾病发病机制中研究和了解较少的连接。我们进一步假设 调节关键过程的决定因素，如转化生长因子β信号和β-连环蛋白在HCs和CCs中都不会 不仅为β-连环蛋白在细胞极性和胆管细胞生物学中的作用提供了新的见解，而且还可能 在胆管病的一般情况下具有重要的治疗意义。我们建议有3个 独立但有凝聚力的目标是检验我们的假设。在目标1中，我们将验证DKO1和 DKO2模型到人类CLD亚类。在AIM2中，我们将探讨转化生长因子β在进展性慢性肝病中的作用。 与β-连环蛋白丢失相关的模型，因为我们的两个临床前模型的特征都是转化生长因子β升高 信号转导和随之而来的细胞极性丧失与上皮向间充质转化表达增强 (EMT)标记。在目标3中，我们将通过删除CCβ-catenin在mdr2KO小鼠中的作用来确定它的作用 单元类型。我们还将研究β-γ-连环蛋白双重缺失对CC的影响。因此，我们建议的研究将 全面解决3个鲜为人知的上皮到间充质重编程、丢失的过程 细胞极性和细胞间连接的扰动在CLD的发病机制中的作用。

Chronic cholestasis results from bile secretory defects or impairment of bile flow, and there are few effective medical therapies available. During the past funding period, we have made significant progress in determining the multifactorial role of the Wnt/β-catenin pathway in cholestatic liver disease (CLD). We show β-catenin inhibition decreases bile acid (BA) synthesis, limiting CLD and fibrosis after bile duct ligation. However, inhibition of β-catenin in the Mdr2 knockout (KO) mice, a model mimicking Primary Sclerosing Cholangitis (PSC), aggravated rather than alleviated injury. In fact, Mdr2-β-catenin-KO (DKO1) mice more closely resembled PSC cases than Mdr2 KO, suggesting this might be a clinically relevant model to study biology and therapeutics for PSC. β-catenin is also a critical component of adherens junctions (AJs), where its loss is compensated by spontaneous increase in β-catenin. We show that deletion of both β- & γ-catenin from hepatocytes (HCs) & cholangiocytes (CCs) in DKO2 mice resulted in CLD, failure to thrive, increased BA, fibrosis, and mortality, and resembled progressive familial intrahepatic cholestasis (PFIC), a pediatric CLD. Although DKO1 and DKO2 had a disparate underlying basis, they share phenotypic commonalities including loss of hepatocyte differentiation & acquisition of mesenchymal characteristics; loss of cell polarity; and pertubations in cell-cell junctions. Intriguingly, none of these events have been studied in great depth in CLD and cholangiopathies, making our study innovative and significant. Based on these findings, we hypothesize that our DKO models represent a subset of PSC and PFIC cases which will be validated by transcriptomic analyses. Regardless of the disease subtype, these mice serve as powerful pre-clinical models for in-depth mechanistic studies of processes such as cell maturation/differentiation, cell polarity and/or cell-cell junctions, that are less well investigated and understood in disease pathogenesis. We further posit that modulating the determinants of key processes such as TGFβ signaling & β-catenin in both HCs & CCs, will not only provide novel insights into the role of β-catenin in cell polarity and cholangiocyte biology, but might also have major therapeutic implications in the general context of cholangiopathies. We are proposing 3 independent yet cohesive aims to test our hypothesis. In Aim 1, we will validate the relevance of the DKO1 and DKO2 models to human CLD subclasses. In Aim2, we will investigate the role of TGFβ in progressive CLD models associated with β-catenin loss, since both our preclinical models are characterized by increased TGFβ signaling and ensuing loss of cell polarity with enhanced expression of epithelial-to-mesenchymal transition (EMT) markers. In aim 3, we will determine the role of CC β-catenin in Mdr2 KO mice by its deletion from this cell type. We will also study the impact of dual deletion of β-γ-catenin in CC. Thus our proposed studies will comprehensively address the 3 poorly understood processes of epithelial to mesenchymal reprogramming, loss of cell polarity, and perturbations in cell-cell junctions, in the pathogenesis of CLD.