Autocrine/Paracrine Regulation of Intrahepatic Bile Duct Growth

描述（由申请人提供）： 胆管细胞增殖/丧失是胆汁淤积性肝病的典型标志，其特异性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由cAMP依赖性信号的激活调节，小胆管细胞的病理生理学（已被假定为由IP 3/Ca 2？I-依赖性信号传导）是未定义的。分泌素受体（SR）已被认为在胆管细胞生长/损失的调节中起作用，因为存在：（i）SR的功能性表达增加，与增强的胆管细胞增生平行;和（ii）在与胆管损伤相关的病理状态中SR表达和分泌素刺激的胆管细胞分泌减少。然而，胰泌素及其受体（仅在正常啮齿动物肝脏中由大胆管细胞表达）在胆管细胞异质性生长/丧失的调节中的作用的直接证据是缺乏的。我们提出的主要假设是：（i）分泌素是一种营养因子（由胆管细胞分泌），激活正常和胆汁淤积的生长（BDL期间）大胆管细胞（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（在小和大胆管细胞中）分泌素基因及其受体的沉默减少大胆管细胞生长（例如，响应于BDL），并且响应于CCl 4而加剧大导管的损伤;以及（iv）小胆管细胞增殖并通过两者分泌[a.] IP3/Ca2？I信号传导（由正常小胆管细胞组成性表达），和[B.]大胆管细胞表型的从头获得，例如分泌素的表达和合成（通过NeuroD 1和SP1的激活），和分泌素受体（通过CaMK I和腺苷酸环化酶AC 8的激活，以及随后CREB和SP1/3的激活）。拟议的研究表明，Ca 2+和cAMP依赖性表型（通过小胆管细胞）的协调表达可能对肝损伤/毒素导致的大胆管损伤期间的胆道系统补充很重要。为了验证这一假设，我们设计了三个具体的目标：（i）证明胰泌素是胆管细胞的营养因子，并且在正常和病理条件下，胰泌素通过自分泌机制差异地调节小胆管细胞和大胆管细胞的生长/损失;（二）定义了促胰液素受体基因的体内和体外分子操作消除了小的，大胆管细胞胆汁淤积和肝损伤;和（iii）定义在体外细胞内机制，调节促胰液素和促胰液素受体的表达过程中的增殖/凋亡反应的小和大胆管细胞胆汁淤积和肝损伤。我们将使用许多体内（分泌素和SR KO小鼠模型）、原位（例如，肝切片中的免疫组织化学），和体外分子（例如，沉默和实时PCR）和细胞（分离和培养的小和大鼠胆管细胞）工具结合生物化学和免疫学方法来查明小和大胆管细胞差异增殖或响应于肝损伤/损害而丢失的细胞内机制。拟议的研究将引入新的概念，即胆管细胞分泌激素促胰液素，并在胆管细胞中的促胰液素水平的操纵可能是重要的，在胆管疾病的胆管细胞生长/损失之间的平衡管理。 公共卫生相关性： 肝病的管理是退伍军人管理局的主要挑战之一。由于酒精和肝炎病毒，退伍军人中胆汁淤积性肝病的风险和发病率很高，这是美国退伍军人住院和死亡的最常见原因之一。胆管损伤是一种病理过程，几乎在所有胆管病（如药物诱导的胆管减少症、原发性胆汁性肝硬化和原发性硬化性胆管炎）中均可观察到。了解胆管在正常和疾病状态下增殖的机制（拟议的研究直接涉及）可能会导致新的治疗方法，并降低美国退伍军人肝病的发病率和死亡率。

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.