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

肝硬变是一种全球性的健康危机，是对慢性肝损伤的反应。肝损伤激活肝脏 分化为纤维性肌成纤维细胞的星状细胞(HSCs)。致纤维化的HSCs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， 肝硬变。虽然肝纤维化在去除有害刺激后是可逆的，但没有任何治疗方法有效地促进肝纤维化。 回归。致纤维化的肝星状细胞产生基质蛋白导致内质中蛋白质过剩 网状结构(ER)，对内质网施加压力。内质网应激启动未折叠蛋白反应(UPR)，这是一种信号 级联使HSCs能够适应增加的蛋白质负荷，并促进有效的蛋白质折叠和分泌。 如果内质网应激得不到解决，UPR信号就会从适应性转变为促凋亡。我们建议将目标定为 促进HSC对内质网应激适应的机制促进HSC凋亡并限制 纤维化形成，导致体内纤维化消退。初步数据显示，激活转录 转录因子6α(atf6α)是UPR的转录因子和效应因子，对HSC的激活和存活起着至关重要的作用。 体外和体内的纤维化形成；然而，这一作用背后的机制尚不清楚。已执行RNAseq 注射四氯化碳4周后分离的小鼠肝星状细胞在ATF6ATF6HSCs上显示基因调控异常 参与骨化、蛋白质降解、凋亡信号、染色质重塑和细胞反应 与从WT小鼠分离的HSCs相比，饥饿的可能性更大。我们假设ATF6α激活了 促纤维化HSCs对内质网应激和HSC适应的促纤维化转录程序 生死存亡。目的1研究由ATF6α调控的基因在成骨过程中的作用 从CCl4诱导的纤维化小鼠分离的HSCs上的RNAseq。我们还将使用RNAseq/ATACseq来 了解ATF6α缺失对造血干细胞转录的短期影响。这些分析将揭示 ATF6α依赖的转录和染色质环境的变化，推动了纤维化的发生。目标2将 研究atf6α如何通过内质网吞噬：选择性自噬降解内质网促进肝星状细胞存活。呃- 吞噬对于分泌细胞的生存至关重要，但它在肝干细胞和纤维化形成中的作用尚不清楚。我们证明了ER- 激活的肝星状细胞的吞噬通量增加。此外，吞噬ER受体在肝硬变中表达上调。 和激活的HSC，这种上调是ATF6α依赖的。目标2将研究内质网吞噬如何维持内质网 ATF6α如何促进活化的内质网吞噬通量 HSCs，以及关键的ER-吞噬受体靶向未折叠和错误折叠的蛋白质用于 退化。总之，拟议的研究将建立atf6α作为关键的促纤维化转录 HSCs中的因子，提供了在纤维化形成过程中由ATF6α调节的纤维化转录的洞察力， 并确定内质网吞噬在促纤维化中的关键作用。这些研究将有助于为我的 最初的R01申请，帮助我从K01接受者转变为独立调查员。

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.