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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

基本信息

批准号：
10653257
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
项目类别：
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/10653257
关键词：
ATAC-seq Address Algorithms Apoptosis Apoptotic Autophagocytosis Autophagosome CRISPR/Cas technology Cell Separation Cell Survival Cells Cellular Stress Chromatin Cicatrix Cirrhosis Data Data Analyses 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 Phenotype Physiologic Ossification Prevalence Process Procollagen Production Protein Biosynthesis Protein Secretion Proteins Regulation Research Personnel Role Sampling Secretory Cell Signal Transduction Starvation Stimulus Stress Tissues Transforming Growth Factor beta Up-Regulation Work activating transcription factor cell type chromatin remodeling chronic liver injury 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 therapeutically effective transcription factor transcriptome sequencing

项目摘要

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.

肝硬化是因慢性肝损伤而引发的全球健康危机。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 产生和分泌大量的基质蛋白沉积到细胞外空间导致纤维化，如果不加以控制，肝硬化。虽然纤维化在去除有害刺激后是可逆的，但没有任何疗法可以有效促进纤维化回归。纤维化 HSC 产生基质蛋白导致内质中蛋白过多网状结构 (ER)，对 ER 施加压力。内质网应激启动未折叠蛋白反应 (UPR)，这是一种信号传导级联使 HSC 能够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 信号传导就会从适应性转变为促凋亡。我们建议瞄准促进 HSC 适应 ER 应激的机制将促进 HSC 凋亡并限制纤维发生，导致体内纤维化消退。初步数据显示，激活转录因子 6α (ATF6α) 是一种转录因子和 UPR 效应子，对于 HSC 的激活和存活至关重要。体外和体内纤维形成；然而，这种作用的机制尚不清楚。进行 RNA 测序注射 CCl4 4 周后，从小鼠体内分离出的 ATF6αΔ/Δ HSC 发现基因失调参与骨化、蛋白质降解、细胞凋亡信号传导、染色质重塑和细胞反应与从 WT 小鼠中分离的 HSC 相比，其对饥饿的抵抗力更强。我们假设 ATF6α 激活促进纤维化 HSC 适应 ER 应激和 HSC 的促纤维化转录程序生存。目标 1 将研究我们确定的 ATF6α 调节基因参与骨化的作用对从 CCl4 诱导的纤维化小鼠中分离出的 HSC 进行 RNAseq。我们还将使用 RNAseq/ATACseq 来了解 HSC 中 ATF6α 缺失的短期转录影响。这些分析将揭示 ATF6α 依赖性转录和染色质景观变化驱动纤维发生。目标2将研究 ATF6α 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 中的吞噬通量增加。此外，ER-吞噬受体在肝硬化肝脏中上调和激活的 HSC，这种上调是 ATF6α 依赖性的。目标 2 将研究 ER 吞噬如何维持 ER 功能并促进 HSC 存活以驱动纤维形成，ATF6α 如何促进活化的 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，拟议的研究将确立 ATF6α 作为关键的促纤维化转录 HSC 中的因子，深入了解纤维形成过程中 ATF6α 调节的纤维形成转录，并确定 ER 吞噬的关键促纤维化作用。这些研究将有助于为我的最初的 R01 申请，帮助我从 K01 接受者过渡到独立研究者。