Pathophysiology of Biliary Disease

描述（由申请方提供）：本次更新的总体目的是明确原发性硬化性胆管炎（PSC）综合征中胆管细胞衰老的机制、后果和病理结局。虽然胆管细胞在包括PSC在内的胆管疾病中起着不可或缺的作用，但胆管细胞信号传导如何促进PSC的发生和进展尚不清楚。最近的证据表明，从我们的实验室支持这项拨款表明：（i）胆管细胞表达病原体识别受体（PRRs），包括toll样受体（TLR）;（ii）病原体识别后，胆管细胞被激活，启动下游信号通路，涉及核因子κ B（NF？B）、p21 Ras同种型、促分裂原活化蛋白激酶（MAPK）和涉及微小RNA（miRNA）（包括let-7 i）的转录后调节网络;（iii）培养的人胆管细胞，响应于潜在有害的微生物分子，发展具有应激诱导的衰老特征的衰老表型;（iv）这些衰老的胆管细胞转变为衰老相关的分泌表型（SASP），其特征在于促炎介质（包括IL 6和IL 8）的过度表达和释放;和（v）与正常和疾病对照组织相比，PSC患者的肝组织显示出具有衰老和SASP表型的胆管细胞比例增加。 因此，我们的数据支持最初的中枢假设，即持续的外源性损伤诱导Ras/MAPK的TLR依赖性激活，促进let-7i miRNA依赖性胆管细胞衰老和SASP，这是一种有助于PSC纤维炎性特征的表型。我们将测试这一假设，通过使用新的体外生物化学和分子技术和体内动物模型，解剖的机制，后果和病理结果的胆管细胞衰老和SASP诱导的促炎介质的分泌。我们的建议有三个综合的具体目标。 首先，我们将检验胆汁中细菌衍生的分子通过Ras/MAPK激活和E2 F反应性细胞周期基因的let-7i依赖性表观遗传沉默诱导胆管细胞衰老的假设。第二，我们将检验以下假设：胆管细胞通过IL-6诱导的C/EBP激活转变为SASP，并通过促炎介质的过度分泌促进旁分泌介导的胆管细胞衰老和星状细胞激活。最后，我们将检验以下假设：在PSC的Mdr 2缺陷小鼠模型中，胆管细胞衰老有助于特征性炎症，而胆管细胞衰老的遗传逆转或抑制和SASP将改善胆道疾病。我们创新实验的结果将基本上阐明潜在有害的胆汁成分导致PSC中胆管细胞衰老和SASP的信号传导机制，并将提供新的信息，可能影响我们减缓这种不治之症进展的能力。

DESCRIPTION (provided by applicant): The OVERALL OBJECTIVE of this renewal is to define the mechanisms, consequences and pathologic outcomes of cholangiocyte senescence in the syndrome of primary sclerosing cholangitis (PSC). While the cholangiocyte plays an integral role in the cholangiopathies, including PSC, how cholangiocyte signaling contributes to the initiation and progression of PSC is unknown. Recent evidence from our lab supported by this grant indicates that: (i) cholangiocytes express pathogen recognition receptors (PRRs) including toll-like receptors (TLRs); (ii) upon pathogen recognition, cholangiocytes are activated to initiate downstream signaling pathways involving Nuclear Factor Kappa B (NF?B), the p21 Ras isoform, Mitogen Activated Protein Kinase (MAPK) and post-transcriptional regulatory networks involving microRNAs (miRNA), including let-7i; (iii) cultured human cholangiocytes, in response to potentially injurious microbial molecules, develop a senescent phenotype with characteristics of stress-induced senescence; (iv) these senescent cholangiocytes transition to a senescence-associated secretory phenotype (SASP) characterized by excessive expression and release of proinflammatory mediators, including IL6 and IL8; and (v) liver tissue from patients with PSC exhibits an increased proportion of cholangiocytes with the senescent and SASP phenotype compared to normal and disease control tissue. Thus, our data support the original CENTRAL HYPOTHESIS that persistent exogenous insult induces TLR-dependent activation of Ras/MAPK promoting let-7i miRNA - dependent cholangiocyte senescence and SASP, a phenotype that contributes to the fibroinflammatory features of PSC. We will test this hypothesis by using novel in vitro biochemical and molecular techniques and in vivo animal models to dissect the mechanisms, consequences and pathologic outcomes of cholangiocyte senescence and SASP-induced secretion of proinflammatory mediators. Our proposal has three integrated SPECIFIC AIMS. FIRST, we will test the hypothesis that bacterial derived molecules in bile induce cholangiocyte senescence through Ras/MAPK activation and let-7i dependent epigenetic silencing of E2F- responsive cell cycle genes. SECOND, we will test the hypothesis that cholangiocytes transition to SASP by IL6- induced activation of C/EBP¿ and promote paracrine-mediated cholangiocyte senescence and stellate cell activation via hypersecretion of proinflammatory mediators. FINALLY, we will test the hypothesis that, in the Mdr2-deficient mouse model of PSC, cholangiocyte senescence contributes to the characteristic inflammation while genetic reversal or inhibition of cholangiocyte senescence and SASP will ameliorate the biliary disease. Results from our innovative experiments will substantially clarify the signaling mechanisms by which potentially injurious biliary components cause cholangiocyte senescence and SASP in PSC, and will provide novel information that could impact our ability to slow progression of this incurable disease.