Elucidating the Role and Regulation of Proteostasis in Hepatic Fibrogenesis

阐明蛋白质稳态在肝纤维形成中的作用和调节

• 批准号: 10718882
• 负责人: Jessica L Maiers
• 金额: $ 42.65万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10718882
• 关键词: Apoptosis; Autophagocytosis; Binding; Cell Separation; Cell Survival; Cells; Cellular Stress; Chemicals; Cicatrix; Cirrhosis; Data; Deposition; Development; Endoplasmic Reticulum; Excision; Extracellular Space; Failure; Fibrosis; GADD45A gene; Genes; Genetic Transcription; Genomics; Goals; Golgi Apparatus; Health; Hepatic Fibrogenesis; Hepatic Stellate Cell; Hepatocyte; Impairment; In Vitro; Injury; Intervention; Liver; Liver Cirrhosis; Liver Failure; Liver Fibrosis; Lysosomes; Mediating; Mus; Pathway interactions; Patients; Phosphotransferases; Post-Translational Regulation; Process; Procollagen; Production; Protein Secretion; Proteins; Proteome; Proteomics; Regulation; Research; Role; Secretory Cell; Signal Transduction; Stress; Testing; Therapeutic; Tissues; Transforming Growth Factor beta; Universities; Validation; antifibrotic treatment; arm; chronic liver injury; end stage liver disease; endoplasmic reticulum stress; fibrogenesis; gene induction; global health; in vivo; insight; liver function; liver injury; lysosomal proteins; misfolded protein; new therapeutic target; non-alcoholic fatty liver disease; posttranscriptional; protein degradation; protein folding; protein transport; proteostasis; receptor; response; therapeutically effective; trafficking; transcriptome sequencing

Liver cirrhosis is a major health crisis caused by chronic liver injury. Liver injury leads to fibrosis which impairs liver function and can progress to cirrhosis and end-stage liver disease if unchecked. Fibrosis can reverse with removal of the injury; however, no therapeutics effectively targeted fibrosis. The major fibrogenic cells in the liver are hepatic stellate cells (HSCs), which produce and secrete matrix proteins into the extracellular space to drive fibrogenesis. Production of matrix proteins in fibrogenic HSCs exceeds the folding capacity of the endoplasmic reticulum (ER), leading to ER stress and induction of the Unfolded Protein Response (UPR). The UPR propa- gates signaling cascades to increase protein folding demands, restore proteostasis, and promote cell survival (adaptive UPR); however prolonged ER stress leads to apoptosis. We propose that targeting the adaptive UPR in HSCs is an effective anti-fibrotic strategy, through disrupting proteostasis, promting apoptosis, and limiting fibrogenesis. A major effector of the adaptive UPR is ATF6α. We found that ATF6α is necessary and sufficient to promote HSC activation in vitro, and HSC-specific loss of ATF6α limited fibrogenesis in vivo; however, the pro-fibrotic mechanisms downstream of ATF6α are unclear. To gain insight into these mechanisms, we performed RNAseq on HSCs isolated from fibrotic Atf6aHSCΔ/Δ mice, revealing disruption of several pathways compared to Atf6afl/fl HSCs, including pathways crucial for maintaining proteostasis through autophagy, protein secretion, and cell survival. We hypothesize that ATF6α drives fibrogenesis through regulating proteosta- sis in fibrogenic HSCs. Based on RNAseq as well as proteomics data from patient derived HSCs, this proposal will focus on how ATF6α regulates proteostasis to drive fibrogenesis through three distinct mechanisms: protein degradation, regulation of pro-survival signals, and protein trafficking. Degrading misfolded proteins is crucial for relieving ER stress. Misfolded proteins in the ER can undergo ER-to-lysosomal associated degradation (ER- LAD), with ERLAD receptors targeting misfolded proteins for lysosomal degradation. ERLAD activity and ERLAD receptors increased in fibrogenic HSCs in an ATF6α-dependent manner. Aim 1 will study how ATF6α regulates ERLAD to reduce ER stress and promote HSC survival and fibrogenesis. Second, ATF6α is crucial for survival of secretory cells, and we show that ATF6α induces expression of GADD45A, a protein which binds to and regulates several kinases. We show that GADD45A loss limits HSC activation in vitro, thus Aim 2 will study how ATF6α regulates GADD45A expression and association with pro-survival kinases to promote HSC survival and fibrogenesis in vitro and in vivo. Finally, we identified a potential role for ATF6α in procollagen I secretion through SORT1. Aim 3 will utilize a combination of in vitro mechanistic studies, proteomics, and in vivo studies to reveal how ATF6α regulation of SORT1 promotes procollagen I trafficking and secretion to drive fibrogenesis. Together these studies will elucidate how ATF6α coordinates signaling mechanisms to restore proteostasis in activated HSCs, and identify novel therapeutic targets to limit liver fibrosis in vivo.

肝硬化是由慢性肝损伤引起的主要健康危机。肝损伤导致纤维化， 肝功能，如果不加检查，可能进展为肝硬化和终末期肝病。纤维化可以逆转， 去除损伤;然而，没有有效靶向纤维化的治疗剂。肝脏中的主要纤维化细胞 是肝星状细胞（HSC），其产生并分泌基质蛋白到细胞外空间中以驱动 纤维化纤维化HSC中基质蛋白的产生超过了内质网的折叠能力 内质网（ER），导致ER应激和未折叠蛋白反应（UPR）的诱导。普遍定期审议的建议 门信号级联增加蛋白质折叠需求，恢复蛋白质稳态，促进细胞存活 （适应性UPR）;然而，延长的ER应激导致细胞凋亡。我们建议，针对适应性 UPR是一种有效的抗肝纤维化策略，通过破坏蛋白质稳态，促进细胞凋亡， 和限制纤维化。适应性UPR的主要效应子是ATF 6 α。我们发现ATF 6 α是必需的 在体外足以促进HSC活化，而在体内HSC特异性的ATF 6 α缺失限制了纤维化的发生; 然而，ATF 6 α下游的促纤维化机制尚不清楚。为了深入了解这些机制， 我们对从纤维化Atf 6aHSC Δ/Δ小鼠分离的HSC进行了RNAseq，揭示了几种途径的破坏， 与Atf 6afl/fl HSC相比，包括通过自噬维持蛋白质稳态的关键途径， 分泌和细胞存活。我们假设ATF 6 α通过调节蛋白质骨架来驱动纤维化。 在纤维化HSC中。基于RNAseq以及来自患者来源的HSC的蛋白质组学数据， 将重点关注ATF 6 α如何通过三种不同的机制调节蛋白稳态以驱动纤维化：蛋白 降解、促生存信号的调节和蛋白质运输。降解错误折叠的蛋白质对于 缓解ER应力。ER中的错误折叠蛋白质可以经历ER-至-溶酶体相关降解（ER-至-溶酶体相关降解）。 LAD），其中ERLAD受体靶向错误折叠的蛋白质用于溶酶体降解。ERLAD活性和ERLAD 受体以ATF 6 α依赖的方式在纤维化HSC中增加。目的1研究ATF 6 α如何调节 ERLAD减少ER应激并促进HSC存活和纤维化。其次，ATF 6 α对生存至关重要 我们发现，ATF 6 α诱导GADD 45 A的表达，GADD 45 A是一种与分泌细胞结合并 调节几种激酶。我们发现GADD 45 A的缺失限制了体外HSC的激活，因此Aim 2将研究GADD 45 A的缺失如何限制HSC的激活。 ATF 6 α调节GADD 45 A表达并与促生存激酶相关以促进HSC存活和 体外和体内纤维化。最后，我们确定了ATF 6 α在I型前胶原分泌中的潜在作用， SORT 1.目标3将利用体外机制研究，蛋白质组学和体内研究的组合来揭示 ATF 6 α调节SORT 1如何促进I型前胶原运输和分泌以驱动纤维化。一起 这些研究将阐明ATF 6 α如何协调信号传导机制，恢复蛋白质稳态， 激活的HSC，并确定新的治疗靶点，以限制体内肝纤维化。