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; 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.

肝硬化是一种全球健康危机，响应慢性肝损伤而发展。肝损伤激活肝 星状细胞（HSC），分化为纤维化肌纤维细胞。纤维化HSC产生和秘密 大量沉积在细胞外空间中的基质蛋白会导致纤维化，如果未检查， 虽然纤维化在去除有害刺激后是可逆的，但没有疗法有效地促进纤维化 回归。通过纤维化HSC生产基质蛋白会导致内质中过量蛋白质 网状（ER），在ER上施加压力。 ER应力启动未折叠的蛋白质反应（UPR），一种信号传导 级联允许HSC适应增加的蛋白质负荷并促进有效的蛋白质折叠和分泌。 如果尚未解决ER应力，UPR信号传导从自适应转向促凋亡。我们提出了目标 支持HSC适应ER应力的机制将促进HSC凋亡并限制 纤维发生，导致体内纤维化回归。初步数据表明激活转录 因子6α（ATF6α）是UPR的转录因子和效应子，对于HSC激活和存活至关重要 体内体外和纤维化；但是，该作用的基础机制尚不清楚。 RNASEQ执行 在CCL4注射4周后，在从小鼠中分离的ATF6αΔ/δHSC上显示基因失调失调 涉及蛋白质降解，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，饥饿。我们假设ATF6α激活 纤维化转录程序，以促进纤维化HSC对ER应力和HSC的适应 生存。 AIM 1将研究我们的ATF6α调节基因与我们所识别的骨化基因的作用 从CCL4诱导的纤维化的小鼠中分离出的HSC上的RNASEQ。我们还将使用rnaseq/atacseq 了解ATF6α缺失在HSC中的短期转录影响。这些分析将揭示 驱动纤维发生的转录和染色质景观的ATF6α依赖性变化。 AIM 2意志 研究ATF6α如何通过ER-Phagy促进HSC存活：ER的选择性自噬降解。 er- Phagy对于分泌细胞存活至关重要，但其在HSC中的作用，纤维发生尚不清楚。我们证明了er- 活化的HSC中的吞噬通量增加。此外，Er-Phagy受体在cirrhotic Lives中也更新 和活化的HSC，该上调是ATF6α依赖性的。 AIM 2将研究ER-Phagy如何保持ER 功能并促进HSC存活以驱动纤维发生，ATF6α如何促进活化的ER-PHAGIC通量 HSC，以及关键的ER-Phagy接收器的靶向蛋白质和错误折叠的蛋白质的机制 降解。拟议的研究将共同​​确定ATF6α作为关键的专业转录 HSC中的因素，提供对纤维化调节期间ATF6α调控的纤维化转录的见解， 并确定ER-Phagy的关键促纤维化作用。这些研究将有助于为我奠定基础 最初的R01应用程序，支持我从K01接收者到独立研究者的过渡。