Autocrine/Paracrine Regulation of Intrahepatic Bile Duct Growth

描述(由申请人提供)： 胆管细胞增殖/缺失是胆汁淤积性肝病的典型特征，其靶向不同大小的胆管细胞。我们的研究是我们不断努力了解调节大小肝内胆管细胞对胃肠激素和肝脏损伤/毒素的功能异质性反应的细胞内机制的直接结果。虽然大胆管细胞的功能是通过激活cAMP依赖的信号来调节的，但小胆管细胞的病理生理(被认为是受IP3/Ca2？依赖于i的信号)是未定义的。促胰液素受体(SR)被认为在胆管细胞的生长/丧失中起着调节作用，其原因在于：(1)与胆管损伤相关的病理状态下，促肾上腺皮质激素释放激素受体(SR)表达增加，同时伴有胆管细胞增生；(2)与胆管损伤相关的病理状态下，SR表达减少，促胆管细胞分泌分泌素。然而，促胰液素及其受体(仅在正常啮齿动物肝脏中的大胆管细胞表达)在胆管细胞异质性生长/缺失调节中的作用缺乏直接证据。我们提出的关键假设是：(I)分泌素是一种营养因子(由胆管细胞分泌)，通过激活cAMP依赖的信号通路，通过自分泌机制激活正常和胆汁淤积期(BDL)大胆管细胞(唯一表达SR的肝细胞类型)的生长；(Ii)促胰液素是一种自分泌保护因子，可对抗CCl_4诱导的大胆管细胞损伤；(Iii)体内(在KO小鼠模型中)和体外(在大小胆管细胞中)促胰液素基因及其受体的沉默减少了大胆管细胞的生长(例如，对BDL的反应)，并加剧了对CCl4的反应的大胆管的损伤；以及(Iv)小胆管细胞的增殖和分泌[a.]IP3/Ca2+上调？I信号(由正常小胆管细胞组成性表达)，和[b.]大的胆管细胞表型的从头获得，如分泌素的表达和合成(通过激活NeuroD1和SP1)，以及分泌素受体(通过激活CaMK I和腺苷环化酶AC8，以及随后激活CREB和SP1/3)。这些研究表明，钙离子和cAMP依赖的表型的协调表达(由小的胆管细胞)可能在肝损伤/毒素损伤大胆管时对胆道树的补充起重要作用。为了验证这一假说，我们设计了三个特定的目标：(I)证明促胰液素是胆管细胞的营养因子，并且在正常和病理条件下，促胰液素以自分泌机制差异地调节大小胆管细胞的生长/丧失；(Ii)确定在体内和体外对促胰液素受体基因的分子操作抑制大小胆管细胞对胆汁淤积和肝损伤的增殖和凋亡反应；以及(Iii)确定在大小胆管细胞对胆汁淤积和肝损伤的增殖/凋亡反应过程中，促胰液素及其受体表达的细胞内机制。我们将使用一些体内(促胰液素和SR KO小鼠模型)、原位(例如肝脏切片的免疫组织化学)和体外分子(例如沉默和实时荧光聚合酶链式反应)和细胞(分离和培养的小鼠和大鼠胆管细胞)工具，结合生化和免疫学方法来精确定位小鼠和大鼠胆管细胞因肝脏损伤/损伤而分化增殖或丢失的细胞内机制。这些研究将引入一个新的概念，即胆管细胞分泌分泌素，并且控制胆管细胞中的分泌素水平可能在胆管疾病中胆管细胞生长/缺失之间的平衡中起重要作用。 公共卫生相关性： 肝病管理是退伍军人管理局面临的主要挑战之一。退伍军人中因酒精和肝炎病毒引起的胆汁淤积性肝病的风险和发病率都很高，这是美国退伍军人住院和死亡最常见的原因之一。胆管损伤是几乎所有胆管病(如药物性胆管减少症、原发性胆汁性肝硬化症和原发性硬化性胆管炎)都可观察到的病理过程。了解胆管在正常和患病状态下增殖的机制(建议的研究直接针对这些机制)可能会导致新的治疗方法，并降低患有肝病的美国退伍军人的发病率和死亡率。

DESCRIPTION (provided by applicant): Cholangiocyte proliferation/loss is a typical hallmark of cholestatic liver diseases specifically targeting different sized cholangiocytes. Our studies are a direct outgrowth of our continuing efforts to understand the intracellular mechanisms regulating the functional heterogeneous responses of small and large intrahepatic cholangiocytes to gastrointestinal hormones and liver injury/toxins. While the function of large cholangiocytes is regulated by activation of cAMP-dependent signaling, the pathophysiology of small cholangiocytes (which has been postulated to be regulated by the IP3/Ca2? I-dependent signaling) is undefined. Secretin receptor (SR) has been suggested to play a role in the regulation of cholangiocyte growth/loss since there is: (i) functional increased expression of SR parallel to enhanced cholangiocyte hyperplasia; and (ii) decreased SR expression and secretin-stimulated cholangiocyte secretion in pathological states associated with damage of bile ducts. However, direct evidence for the role of secretin and its receptor (expressed only by large cholangiocytes in normal rodent liver) in the regulation of cholangiocyte heterogeneous growth/loss is lacking. We propose the key hypotheses that: (i) secretin is a trophic factor (secreted by cholangiocytes) that activates the growth of normal and cholestatic (during BDL) large cholangiocytes (the only hepatic cell type expressing SR) by an autocrine mechanism via activation of cAMP-dependent signaling; (ii) secretin is an autocrine protective factor against CCl4-induced damage of large cholangiocytes; (iii) in vivo (in KO mouse models) and in vitro (in small and large cholangiocytes) silencing of the secretin gene and its receptor reduces large cholangiocyte growth (e.g., in response to BDL), and exacerbates the damage of large ducts in response to CCl4; and (iv) small cholangiocytes proliferate and secrete by both [a.] the upregulation of IP3/Ca2? I signaling (that is constitutively expressed by normal small cholangiocytes), and [b.] the de novo acquisition of large cholangiocyte phenotypes such as the expression and synthesis of secretin (through activation of NeuroD1 and SP1), and secretin receptor (by activation of CaMK I and the adenylyl cyclase, AC8, and the subsequent activation of CREB and SP1/3). The proposed studies suggest that the coordinated expression of Ca2+ and cAMP-dependent phenotypes (by small cholangiocytes) may be important to replenish the biliary tree during damage of large ducts by liver injury/toxins. To test this hypothesis, we have designed three specific aims to: (i) demonstrate that secretin is a trophic factor for cholangiocytes, and that secretin differentially regulates the growth/loss of small and large cholangiocytes by an autocrine mechanism in normal and pathological conditions; (ii) define that in vivo and in vitro molecular manipulation of the secretin receptor gene ablates the proliferative and apoptotic responses of small and large cholangiocytes to cholestasis and liver injury; and (iii) To define the in vitro intracellular mechanisms regulating secretin and secretin receptor expression during the proliferative/apoptotic response of small and large cholangiocytes to cholestasis and liver injury. We will use a number of in vivo (secretin and SR KO mouse models), in situ (e.g., immunohistochemistry in liver sections), and in vitro molecular (e.g., silencing, and real-time PCR) and cellular (isolated and cultured small and large murine cholangiocytes) tools in conjunction with biochemical and immunological approaches to pinpoint the intracellular mechanisms by small and large cholangiocytes differentially proliferate or are lost in response to liver injury/damage. The proposed studies will introduce the novel concept that cholangiocytes secrete the hormone secretin, and that manipulation of secretin levels in cholangiocytes may be important in the management of the balance between cholangiocyte growth/loss in cholangiopathies. PUBLIC HEALTH RELEVANCE: Management of liver diseases represents one of the major challenges of the Veterans Administration. There is a high risk and incidence of cholestatic liver diseases due to alcohol and hepatitis viruses in Veterans, which is one of the most common reasons for hospitalization and mortality in American Veterans. Damage of bile ducts is a pathological process that is observed in virtually all cholangiopathies (such as, drug induced ductopenia, primary biliary cirrhosis and primary sclerosing cholangitis). Understanding the mechanisms by which bile ducts proliferate in normal and diseased states (which the proposed studies directly address) will likely lead to new therapeutic approaches and a reduction of morbidity and mortality in American Veterans with liver diseases.