Autocrine/Paracrine Regulation of Intrahepatic Bile Duct Growth

肝内胆管生长的自分泌/旁分泌调节

• 批准号: 8195932
• 负责人: Gianfranco D Alpini
• 金额: --
• 依托单位: OLIN TEAGUE VETERANS CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8195932
• 关键词: Acute; Address; Adenylate Cyclase; Alcoholic Liver Cirrhosis; Alcohols; American; Animals; Apoptosis; Apoptotic; Bicarbonates; Bile fluid; Biliary; Biochemical; CREB1 gene; Calcium/calmodulin-dependent protein kinase; Carbon Tetrachloride; Cells; Cessation of life; Cholangiocarcinoma; Cholestasis; Chronic; Cirrhosis; Clinic; Complex; Coupled; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Diffuse; Down-Regulation; Duct (organ) structure; Ductal; Duodenum; ELK1 gene; Equilibrium; Evaluation; Extrahepatic Cholestasis; Functional disorder; Gastrointestinal Hormones; Gene Expression; Gene-Modified; Genes; Growth; Health; Hepatitis Viruses; Hepatocyte; Hormones; Hospitalization; Human; Human Cell Line; Hyperplasia; Immunohistochemistry; In Situ; In Vitro; Incidence; Inflammation; Inflammatory; Injury; Injury to Liver; Intrahepatic bile duct; Italy; Laboratories; Lead; Letters; Ligation; Liver; Liver Cirrhosis; Liver diseases; MAPK3 gene; Measures; Modeling; Molecular; Morbidity - disease rate; Mus; Nature; Pathologic Processes; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Play; Primary biliary cirrhosis; Primary carcinoma of the liver cells; Process; Proliferating; Protein Isoforms; Proteins; Reaction; Receptor Activation; Receptor Up-Regulation; Regulation; Resistance; Rodent; Role; Rome; S Cells (Intestine); SCTR gene; SP1 gene; Sampling; Secretin; Serum; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Staging; Structure; Testing; Time; Toxin; Transcriptional Activation; United States Department of Veterans Affairs; Universities; Up-Regulation; Vascular Endothelial Cell; Veterans; Wild Type Mouse; Work; autocrine; bile duct; biliary tract; cell type; cholangiocyte; design; gastrointestinal; high risk; in vivo; indexing; inhibitor/antagonist; intrahepatic; liver transplantation; mortality; mouse model; novel; novel therapeutic intervention; novel therapeutics; paracrine; population based; prevent; primary sclerosing cholangitis; promoter; receptor; receptor expression; research study; response; secretin receptor; small hairpin RNA; therapeutic development; tool; trait; treatment strategy

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.

描述（由申请人提供）： 胆管细胞增殖/丢失是胆汁淤积性肝病的典型标志，特别针对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小型和大型肝内胆管细胞对胃肠激素和肝损伤/毒素的功能异质反应的细胞内机制的直接成果。虽然大胆管细胞的功能是通过 cAMP 依赖性信号传导的激活来调节的，但小胆管细胞的病理生理学（假设其受到 IP3/Ca2?I 依赖性信号传导的调节）尚不清楚。促胰液素受体 (SR) 被认为在胆管细胞生长/损失的调节中发挥作用，因为存在： (i) SR 的功能性表达增加与胆管细胞增生增强平行； (ii) 在与胆管损伤相关的病理状态下，SR 表达和促胰液素刺激的胆管细胞分泌减少。然而，缺乏促胰液素及其受体（仅由正常啮齿动物肝脏中的大胆管细胞表达）在调节胆管细胞异质生长/损失中的作用的直接证据。我们提出的关键假设是：（i）促胰液素是一种营养因子（由胆管细胞分泌），通过激活 cAMP 依赖性信号传导，通过自分泌机制激活正常和胆汁淤积（BDL 期间）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，可抵抗 CCl4 引起的大胆管细胞损伤； (iii)体内（在KO小鼠模型中）和体外（在小型和大型胆管细胞中）沉默促胰液素基因及其受体会减少大型胆管细胞的生长（例如，响应BDL），并加剧响应CCl4的大型胆管的损伤； (iv) 小胆管细胞通过 [a.] IP3/Ca2? 的上调而增殖和分泌？ I 信号传导（由正常小胆管细胞组成型表达），以及 [b.] 大胆管细胞表型的从头获得，例如促胰液素的表达和合成（通过激活 NeuroD1 和 SP1）和促胰液素受体（通过激活 CaMK I 和腺苷酸环化酶 AC8，以及随后激活 CREB ​​和 SP1/3）。拟议的研究表明，Ca2+ 和 cAMP 依赖性表型（通过小胆管细胞）的协调表达可能对于在肝损伤/毒素导致大胆管损伤期间补充胆管树很重要。为了检验这一假设，我们设计了三个具体目标：（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，促胰液素通过自分泌机制差异调节小胆管细胞和大胆管细胞的生长/损失； (ii) 确定促胰液素受体基因的体内和体外分子操作可消除小胆管细胞和大胆管细胞对胆汁淤积和肝损伤的增殖和凋亡反应； (iii) 确定在小胆管细胞和大胆管细胞对胆汁淤积和肝损伤的增殖/凋亡反应期间调节促胰液素和促胰液素受体表达的体外细胞内机制。我们将使用多种体内（促胰液素和 SR KO 小鼠模型）、原位（例如肝脏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（分离和培养的小型和大型小鼠胆管细胞）工具，结合生化和免疫学方法来查明细胞内的 小胆管细胞和大胆管细胞因肝损伤/损害而不同地增殖或丢失的机制。拟议的研究将引入新概念，即胆管细胞分泌激素促胰液素，并且控制胆管细胞中的促胰液素水平对于管理胆管病中胆管细胞生长/损失之间的平衡可能很重要。 公共卫生相关性： 肝脏疾病的管理是退伍军人管理局面临的主要挑战之一。退伍军人因酒精和肝炎病毒而罹患胆汁淤积性肝病的风险和发病率较高，这是美国退伍军人住院和死亡的最常见原因之一。胆管损伤是几乎所有胆管病（例如药物引起的胆管减少症、原发性胆汁性肝硬化和原发性硬化性胆管炎）中都观察到的病理过程。了解胆管在正常和患病状态下增殖的机制（拟议的研究直接解决）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。