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.

肝硬变是由慢性肝损伤引起的主要健康危机。肝损伤导致肝纤维化，肝纤维化损害 如果不加以控制，可能会发展为肝硬变和终末期肝病。纤维化可以通过以下方式逆转 去除损伤；然而，没有有效针对纤维化的治疗药物。肝脏中的主要纤维化细胞 是肝星状细胞(HSCs)，它产生并分泌基质蛋白到细胞外空间以驱动 纤维化。纤维化HSCs产生的基质蛋白超过内质的折叠能力 内质网(ER)，导致内质网应激和诱导未折叠蛋白反应(UPR)。普遍定期审议提案-- 盖茨信号级联增加蛋白质折叠需求，恢复蛋白质平衡，促进细胞存活 (适应性UPR)；然而，长期的内质网应激会导致细胞凋亡。我们建议将目标对准适应性强的 在HSCs中，UPR是一种有效的抗纤维化策略，通过破坏蛋白平衡，促进细胞凋亡， 和限制纤维化的发生。自适应UPR的一个主要效应器是ATF6α。我们发现atf6α是必要的。 在体外足以促进肝星状细胞活化，体内肝星状细胞特异性缺失ATF6α可限制肝纤维化的发生； 然而，ATF6α下游的促纤维化机制尚不清楚。为了深入了解这些机制， 我们对从纤维化的atf6aHSCΔ/Δ小鼠分离的HSC进行了RNAseq，揭示了几条途径的中断 与Atf6afl/fl HSCs相比，包括通过自噬维持蛋白稳定至关重要的途径，蛋白质 分泌和细胞存活。我们假设ATF6α通过调节蛋白水解酶来促进纤维化的发生。 SIS在纤维化HSCs中的表达。基于RNAseq和来自患者来源的HSCs的蛋白质组学数据，这一建议 将侧重于atf6α如何通过三种不同的机制调节蛋白平衡以推动纤维化形成：蛋白质 降解、有利于生存的信号的调节以及蛋白质的贩运。降解错误折叠的蛋白质对 缓解内质网压力。内质网中的错误折叠蛋白可以经历内质网到溶酶体的相关降解(ER- LAD)，ERLAD受体靶向错误折叠的蛋白进行溶酶体降解。ERLAD活动与ERLAD 成纤维细胞的受体以ATF6α依赖的方式增加。目标1将研究atf6α如何调节 ERLAD可减轻内质网应激，促进HSC存活和纤维化形成。其次，atf6α对生存至关重要。 我们发现ATF6α可以诱导GADD45A的表达，GADD45A是一种结合和 调节着几种激活酶。我们发现GADD45A丢失在体外限制了HSC的激活，因此Aim 2将研究如何 血管内皮生长因子6α调节GADD45A的表达及其与促生存蛋白激酶的关联以促进造血干细胞的存活和 体外和体内的纤维化形成。最后，我们确定了atf6α在I型前胶原分泌中的潜在作用。 SORT1.AIM 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 血管紧张素转运蛋白6α调控SORT1如何促进I型前胶原的运输和分泌，从而推动纤维化的发生。同舟共济 这些研究将阐明atf6α如何协调信号机制以恢复蛋白平衡。 激活的HSCs，并确定新的治疗靶点，以限制体内的肝纤维化。