Pathophysiology of Biliary Disease

描述（由申请人提供）：本次更新的总体目标是确定原发性硬化性胆管炎（PSC）综合征中胆管细胞衰老的机制、后果和病理结果。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。最近来自我们实验室的证据表明：(i)胆管细胞表达病原体识别受体（PRRs），包括toll样受体（TLRs）；（ii）在病原体识别后，胆管细胞被激活，启动涉及核因子κ B (NF？B)， p21 Ras亚型，丝裂原活化蛋白激酶（MAPK）和涉及microrna （miRNA）的转录后调控网络，包括let-7i；（iii）培养的人胆管细胞对潜在的有害微生物分子作出反应，产生具有应激诱导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），其特征是过度表达和释放促炎介质，包括IL6和IL8；(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央假设，即持续的外源性损伤诱导tlr依赖性的Ras/MAPK激活，促进let-7i miRNA依赖性的胆管细胞衰老和SASP，这是一种有助于PSC纤维炎症特征的表型。我们将通过使用新的体外生化和分子技术以及体内动物模型来验证这一假设，以解剖胆管细胞衰老和sasp诱导的促炎介质分泌的机制，后果和病理结果。我们的建议有三个综合的具体目标。首先，我们将验证胆汁中细菌来源的分子通过Ras/MAPK激活和E2F应答细胞周期基因的let-7i依赖性表观遗传沉默诱导胆管细胞衰老的假设。其次，我们将验证通过il - 6诱导的C/EBP激活胆管细胞向SASP过渡的假设，并通过促炎介质的高分泌促进旁分泌介导的胆管细胞衰老和星状细胞活化。最后，我们将验证以下假设，即在mdr2缺陷小鼠PSC模型中，胆管细胞衰老有助于特征性炎症，而遗传逆转或抑制胆管细胞衰老和SASP将改善胆道疾病。我们的创新实验结果将从本质上阐明潜在的有害胆道成分导致PSC中胆管细胞衰老和SASP的信号机制，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.