Extracellular Matrix Protein 1 in liver homeostasis, chronic disease and regeneration

转化生长因子(TGF)-β通过激活肝星状细胞促进肝纤维化。TGF-β也有助于急性损伤后的肝脏再生，最可能是通过终止肝细胞增殖。控制TGF-β激活的潜在机制尚不完全清楚。我们已经确定细胞外基质蛋白1 （ECM1）是肝脏病理生理的关键因素。ECM1在健康的人和小鼠肝脏中含量丰富。它由肝细胞合成，在肝损伤时，ECM1水平迅速下降。ECM1KO小鼠自发发生肝纤维化，并在2个月大时死亡。这些小鼠的肝组织的特点是高水平的磷酸化Smad2/3，而没有表现出TGF-β表达上调。AAV8介导的可溶性tgf - β r2在ECM1KO小鼠和ECM1肝脏中的异位表达在ccl4处理小鼠6周后延长了生存期并阻止了纤维化的发生。初步机制数据表明，ECM1参与肝基质中潜伏TGF-β的沉积和稳定。我们的目标是在几个方面加深对这一关键因素在肝脏中的相关性的见解：(1)使用硅和体外启动子分析和刺激/抑制实验，我们将描述肝细胞中的ECM1调控。研究结果将在健康小鼠和CCl4肝损伤模型中得到验证。这将提供通过调节ECM1表达的潜在纤维化治疗方法的知识。(2)初步数据显示，在肝再生的CCl4和部分肝切除模型中，ECM1下调的模式不同。通过AAV8介导的再表达，我们将描述ECM1对再生效果的影响，并评估其与TGF-β激活的关系。(3)我们将产生可诱导的ECM1KO小鼠，在慢性肝病（CLD）动力学的选定疾病阶段消耗ECM1，例如在CCl4模型中。(4)通过研究蛋白-蛋白相互作用，使用TGF-β/Smad报告系统，以及测试潜在的TGF-β结合伙伴和激活剂，将获得ECM1在基质沉积和潜在TGF-β激活中的功能机制。数据将通过在人类患者样本和表达Tomato-ECM1的小鼠中进行共定位研究来补充。这一小鼠系将被生成并随后被CCl4慢性激发。因此，我们将获得关于ECM1如何调节TGF-β在CLD和再生中的激活的机制知识。(5)诊断：在多家医院合作同事的广泛支持下，我们将测试ECM1血药浓度对CLD分期诊断的预测价值。建立了酶联免疫吸附试验，初步数据显示相关性。为了确定ECM1作为生物标志物，我们将分析大型患者队列中的丰度。总之，这项研究项目的发现将为肝纤维化和再生的机制提供重要的见解，可能具有临床转化的潜力。

The transforming growth factor (TGF)-β promotes liver fibrosis by activating hepatic stellate cells. TGF-β also contributes to liver regeneration after acute damage, most probably by terminating hepatocyte proliferation. The underlying mechanisms controlling TGF-β activation are not entirely clear. We have identified extracellular matrix protein 1 (ECM1) as crucial factor in liver pathophysiology. ECM1 is abundant in healthy human and mouse livers. It is synthesized by hepatocytes and upon liver damage, ECM1 levels are decreasing quickly. ECM1KO mice spontaneously develop liver fibrosis and die at an age of ~2 months. Liver tissue from these mice is characterized by high levels of phosphorylated Smad2/3 without showing upregulation of TGF-β expression. AAV8 mediated ectopic expression of soluble TGFβR2 in livers of ECM1KO mice and of ECM1 in 6 weeks CCl4-treated mice prolonged survival and prevented fibrogenesis. Preliminary mechanistic data indicate that ECM1 is involved in deposition and stability of latent TGF-β in the liver matrix. We aim to deepen insights on the relevance of this critical factor in liver for several aspects: (1) Using in silico and in vitro promoter analyses and stimulation/inhibition experiments, we will delineate ECM1 regulation in hepatocytes. Findings will be validated in healthy mice and during liver damage in the CCl4 model. This will provide knowledge on potential fibrosis treatment approaches by modulating ECM1 expression. (2) Preliminary data show distinct patterns of ECM1 downregulation in CCl4 and partial hepatectomy models of liver regeneration. By AAV8 mediated re-expression, we will delineate the impact of ECM1 on efficacy of regeneration, and evaluate the relation to TGF-β activation. (3) We will generate inducible ECM1KO mice to deplete ECM1 at selected disease stages during chronic liver disease (CLD) dynamics, e.g. in the CCl4 model. (4) Mechanistic insights on ECM1 functions in matrix deposition and activation of latent TGF-β will be obtained by studying protein-protein interactions and using a TGF-β/Smad reporter system, as well as by testing latent TGF-β binding partners and activators. Data will be complemented by colocalization studies in human patient samples and in mice expressing Tomato-ECM1. This mouse line will be generated and subsequently chronically challenged with CCl4. Thus, we will gain mechanistic knowledge on how ECM1 modulates TGF-β activation in CLD and regeneration. (5) Diagnosis: With extensive support from collaborating colleagues from several hospitals, we will test the predictive value of ECM1 blood levels for diagnosis of CLD stages. An ELISA is established and preliminary data show evidence of correlation. To establish ECM1 as biomarker, we will analyze abundance in large patient cohorts. In summary, the discoveries of this research project will provide important insights into mechanisms of liver fibrosis and regeneration that may have potential for clinical translation.