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

转化生长因子-β通过激活肝星状细胞促进肝纤维化。转化生长因子-β也有助于急性损伤后的肝再生，很可能是通过终止肝细胞的增殖。控制转化生长因子-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)是肝脏病理生理学中的关键因子。ECM1在健康的人和小鼠肝脏中含量丰富。它是由肝细胞合成的，一旦肝脏受损，ECM1水平就会迅速下降。ECM1KO小鼠自发地发展为肝纤维化，并在大约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的Smad2/3，而没有显示出转化生长因子-β的表达上调。AAV8介导的可溶性转化生长因子βR2在ECM1KO小鼠肝脏中的异位表达和经CCL4处理的6周小鼠的ECM1异位表达延长了小鼠的存活时间，并防止了纤维化的发生。初步的机制数据表明，ECM1参与了肝基质中潜伏的转化生长因子-β的沉积和稳定性。我们的目标是从以下几个方面加深对这一关键因素在肝脏中的相关性的认识：(1)利用电子显微镜和体外启动子分析以及刺激/抑制实验，我们将描述ECM1在肝细胞中的调控。这些发现将在健康小鼠身上得到验证，并在CCl4模型的肝脏损伤期间得到验证。这将通过调节ECM1的表达来提供潜在的纤维化治疗方法的知识。(2)初步数据显示在CCl_4和肝部分切除肝再生模型中ECM1下调的模式明显。通过AAV8介导的再表达，我们将描述ECM1对再生效果的影响，并评价其与转化生长因子-β激活的关系。(3)我们将产生可诱导的ECM1KO小鼠，以在慢性肝病(CLD)动态过程中的特定疾病阶段耗尽ECM1，例如在CCl4模型中。(4)通过研究蛋白质之间的相互作用，使用转化生长因子-β/Smad报告系统，以及通过检测潜在的转化生长因子-β结合伙伴和激活剂，将获得对ECM1在基质沉积和潜伏的转化生长因子-β激活中的作用的机械性见解。在人类患者样本和表达番茄-ECM1的小鼠中进行的共定位研究将补充数据。这一小鼠品系将被生成，并随后被CCl4长期挑战。因此，我们将获得关于ECM1如何在CLD和再生中调节转化生长因子-β激活的机制知识。(5)诊断：在多家医院同事的广泛支持下，我们将测试ECM1水平对CLD分期的预测价值。建立了一个酶联免疫吸附试验，初步数据显示了相关性的证据。为了建立ECM1作为生物标记物，我们将分析大量患者队列中的丰度。总之，这一研究项目的发现将为研究肝纤维化和再生的机制提供重要的见解，这些机制可能有可能用于临床翻译。

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.