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: 8397529
• 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/8397529
• 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 injury; liver transplantation; mortality; mouse model; novel; novel therapeutic intervention; novel therapeutics; paracrine; population based; prevent; primary sclerosing cholangitis; promoter; public health relevance; 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.

描述（由申请人提供）： 胆管细胞增殖/损失是专门针对不同大小胆管细胞的胆汁淤积性肝病的典型标志。我们的研究是我们持续努力的直接产物，以了解调节大小肝内胆管细胞对胃肠道激素和肝损伤/毒素的功能异质反应的细胞内机制。虽然大胆管细胞的功能受CAMP依赖性信号传导的激活调节，但小胆管细胞的病理生理（假定由IP3/CA2？i依赖性信号传导调节）是不确定的。由于存在：（i）SR平行与增强的胆管细胞增生的SR表达，Synectin受体（SR）在调节胆管细胞生长/丧失的调节中发挥作用； （ii）与胆管损伤相关的病理状态下的SR表达和分泌素刺激的胆管细胞分泌。然而，缺乏直接证明Secritin及其受体（仅由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/损失中的作用。我们提出的关键假设是：（i）分泌蛋白是一种营养因子（由胆管细胞分泌），该因素激活了正常和胆汁淤积性的（在BDL）大型胆管细胞（在BDL）中的生长（唯一通过cAMP依赖性信号激活的自动内； （ii）Secralin是针对CCL4诱导的大胆管细胞损伤的自分泌保护因素； （iii）体内（在KO小鼠模型中）和体外（在大小和大的胆管细胞中）沉默的sectionin基因及其受体会减少大型胆管细胞的生长（例如，响应于BDL），并使对CCL4响应大型造成大型造成损害； （iv）小胆管细胞增殖并分泌IP3/Ca2的上调？ 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）。拟议的研究表明，Ca2+和CAMP依赖性表型的协调表达（通过小胆管细胞）对于在肝损伤/毒素损伤期间补充胆汁树可能很重要。为了检验这一假设，我们设计了三个特定的目的：（i）证明secralin是胆管细胞的营养因子，而Secritin在正常和病理条件下通过自分泌机制差异地调节了大小胆管细胞的生长/丧失； （ii）定义了分泌素受体基因的体内和体外分子操纵，从而消除了大小的大胆管细胞对胆汁淤积和肝损伤的增殖和凋亡反应； （iii）定义在大小胆管细胞对胆汁淤积和肝损伤的增殖/凋亡反应期间调节秘密蛋白和秘密蛋白受体表达的体外细胞内机制。我们将原位使用许多体内（sectin和sr KO鼠标模型）（例如，肝脏中的免疫组织化学），以及在体外分子（例如，沉默和实时PCR）和细胞（隔离和培养的小鼠类胆管造成的工具）和细胞（隔离和较大的杂物性工具）的体外分子化学（例如，沉默和实时PCR），并具有较大的含量胆管造成的工具。大小的胆管细胞的机制差异化或因肝损伤/损害而丢失。拟议的研究将介绍一个新的概念，即胆管细胞分泌激素秘书素，而胆管细胞中秘密素水平的操纵对于胆管疾病中胆管细胞生长/胆管疾病之间的平衡的管理可能很重要。