Elucidating the role of ATF6α as a critical pro-fibrogenic transcription factor in Hepatic Stellate Cells

阐明 ATF6α 作为肝星状细胞中关键的促纤维化转录因子的作用

• 批准号: 10526974
• 负责人: Jessica L Maiers
• 金额: $ 11.89万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10526974
• 关键词: ATAC-seq; Address; Algorithms; Apoptosis; Apoptotic; Autophagocytosis; Autophagosome; Bacteriophages; CRISPR/Cas technology; Cell Survival; Cells; Cellular Stress; Chromatin; Cicatrix; Cirrhosis; Data; Data Set; Degradation Pathway; Deposition; Endoplasmic Reticulum; Excision; Exhibits; Extracellular Matrix Proteins; Extracellular Space; Fibrosis; Gene Expression; Genes; Genetic Transcription; Goals; Health; Hepatic Stellate Cell; Hepatocyte; Impairment; In Vitro; Injections; Liver; Liver Failure; Liver Fibrosis; Liver diseases; Membrane; Mus; Myofibroblast; Olive oil preparation; Patients; Persons; Phage Receptors; Phenotype; Physiologic Ossification; Prevalence; Process; Procollagen; Production; Protein Biosynthesis; Protein Secretion; Proteins; Regulation; Research Personnel; Role; Sampling; Secretory Cell; Signal Transduction; Starvation; Stimulus; Stress; Therapeutic; Tissues; Transforming Growth Factor beta; Up-Regulation; Work; activating transcription factor; cell type; chromatin remodeling; chronic liver injury; delta opioid receptor; endoplasmic reticulum stress; fibrogenesis; global health; in vivo; insight; liver function; liver injury; mRNA sequencing; misfolded protein; novel; programs; protein degradation; protein folding; proteostasis; receptor; response; sensor; transcription factor; transcriptome sequencing

Cirrhosis is a global health crisis that develops in response to chronic liver injury. Liver injury activates Hepatic Stellate Cells (HSCs) which differentiate into fibrogenic myofibroblasts. Fibrogenic HSCs produce and secrete vast amounts of matrix proteins that deposit into the extracellular space leading to fibrosis, and if unchecked, cirrhosis. While fibrosis is reversible upon removal of injurious stimuli, no therapies effectively promote fibrosis regression. Production of matrix proteins by fibrogenic HSCs leads to excess proteins in the endoplasmic reticulum (ER), placing stress on the ER. ER stress initiates the Unfolded Protein Response (UPR), a signaling cascade allowing HSCs to adapt to increased protein load and facilitate efficient protein folding and secretion. If ER stress is unresolved, UPR signaling switches from adaptive to pro-apoptotic. We propose that targeting mechanisms facilitating HSC adaptation to ER stress would promote HSC apoptosis and limit fibrogenesis, leading to fibrosis regression in vivo. Preliminary data shows that Activating Transcription Factor 6α (ATF6α), a transcription factor and effector of the UPR, is crucial for HSC activation and survival in vitro and fibrogenesis in vivo; however, the mechanisms underlying this role are unknown. RNAseq performed on ATF6αΔ/Δ HSCs isolated from mice following 4 weeks of CCl4 injection revealed dysregulation of genes involved in ossificaiton, protein degradation, apoptotic signaling, chromatin remodeling, and cellular response to starvation compared to HSCs isolated from WT mice. We hypothesize that ATF6α activates profibrogenic transcriptional programs to promote adaption of fibrogenic HSCs to ER stress and HSC survival. Aim 1 will investigate the role of the ATF6α-regulated genes involved in ossification identified by our RNAseq on HSCs isolated from mice with CCl4-induced fibrosis. We will additionally use RNAseq/ATACseq to understand the short-term transcriptional impact of ATF6α deletion in HSCs. These analyses will reveal ATF6α-dependent changes in the transcriptional and chromatin landscapes that drive fibrogenesis. Aim 2 will study how ATF6α promotes HSC survival through ER-phagy: selective autophagic degradation of the ER. ER- phagy is critical for secretory cell survival but its role in HSCs and fibrogenesis is unknown. We show that ER- phagic flux increases in activated HSCs. Furthermore, ER-phagy receptors are upregulated in cirrhotic livers and activated HSCs, and this upregulation is ATF6α-dependent. Aim 2 will study how ER-phagy maintains ER function and promotes HSC survival to drive fibrogenesis, how ATF6α promotes ER-phagic flux in activated HSCs, and the mechanisms by which key ER-phagy receptors target unfolded and misfolded proteins for degradation. Together, the proposed studies will establish ATF6α as a key profibrotic transcription factor in HSCs, provide insight into fibrogenic transcription regulated by ATF6α during fibrogenesis, and identify a critical pro-fibrogenic role for ER-phagy. These studies will help lay the groundwork for my initial R01 application, facilitating my transition from K01 recipient to independent investigator.

肝硬化是一种全球性的健康危机，是由于慢性肝损伤而引起的。肝损伤激活肝脏 星状细胞（HSC）分化为纤维化肌成纤维细胞。纤维化HSC产生并分泌 大量的基质蛋白存款到细胞外间隙中，导致纤维化，如果不加抑制， 肝硬化虽然纤维化在去除有害刺激后是可逆的，但没有治疗有效地促进纤维化 回归分析由纤维化HSC产生的基质蛋白导致内质网中过量的蛋白质， 内质网（ER），强调ER。内质网应激启动未折叠蛋白反应（UPR），一种信号转导途径。 级联反应允许HSC适应增加的蛋白质负载并促进有效的蛋白质折叠和分泌。 如果ER应激未得到解决，UPR信号从适应性切换到促凋亡。我们建议， 促进HSC适应ER应激的机制将促进HSC凋亡并限制 纤维化，导致体内纤维化消退。初步数据显示， 转录因子6α（ATF 6 α）是UPR的一种转录因子和效应子，对HSC的活化和存活至关重要。 体外和体内纤维化;然而，这种作用的机制尚不清楚。进行RNAseq 在注射CCl 4 4 4周后从小鼠分离的ATF 6 αΔ/Δ HSC上显示基因调控异常， 参与骨化、蛋白质降解、凋亡信号传导、染色质重塑和细胞反应 与从WT小鼠分离的HSC相比，我们假设ATF 6 α激活 促进纤维化HSC适应ER应激的促纤维化转录程序和HSC 生存目的1：研究ATF 6 α调控基因在骨化过程中的作用。 RNAseq对从具有CCl 4诱导的纤维化的小鼠分离的HSC的作用。我们还将使用RNAseq/ATACseq 了解ATF 6 α缺失对HSC的短期转录影响。这些分析将揭示 转录和染色质景观中驱动纤维化的ATF 6 α依赖性变化。目标2将 研究ATF 6 α如何通过ER-吞噬促进HSC存活：ER的选择性自噬降解。ER- 吞噬对于分泌细胞的存活至关重要，但其在HSC和纤维形成中的作用尚不清楚。我们证明，ER- 活化的HSC中吞噬通量增加。此外，ER-吞噬受体在炎症性肝脏中上调， 并且这种上调是ATF 6 α依赖性的。目的2研究ER-吞噬是如何维持ER的 功能并促进HSC存活以驱动纤维化，ATF 6 α如何促进活化的HSC中的ER-吞噬通量， HSC，以及关键ER吞噬受体靶向未折叠和错误折叠蛋白质的机制 降解总之，拟议的研究将确定ATF 6 α作为关键的促纤维化转录 HSC中的因子，提供了对纤维形成过程中ATF 6 α调控的纤维形成转录的深入了解， 并确定ER吞噬的关键促纤维化作用。这些研究将有助于奠定基础， 最初的R 01申请，帮助我从K 01接收者过渡到独立调查员。