Molecular Mechanisms of Stellate Cell Activation in Liver Fibrosis

活化的肝星状细胞(AHSCs)参与肝纤维化，促进肝脏发育 肿瘤是慢性肝病最具破坏性的两种后遗症。我们为筛选WNT所做的严格努力- AHSCs中的β-catenin靶基因，发现了编码ER的SCD(硬脂酰辅酶A去饱和酶) 负责合成单不饱和脂肪酸(MUFA)的酶，并与 脂肪肝、癌症和干性痴呆。β能够以依赖于Wnt的方式上调SCD- 连接素与SREBP-1c结合并增强近10倍的SREBP-1c-诱导转录。归纳 在Scd2中，主要在HSC中表达的异构体是HSC激活所必需的，并提供了 正向环通过LRP5/6mRNA的稳定性来扩增典型的Wnt通路。这个新奇的环路是 SCD产生的多不饱和脂肪酸通过干扰mRNA结合蛋白HUR的核输入而启动 转运蛋白(TNPO)和Ran1 GTP酶导致HUR细胞质聚集，HUR结合到 LRP5/6mRNA3‘非编码区及其稳定性。SCD抑制或Scd2沉默取消LRP5/6 β-连环蛋白在急性肝星状细胞中的表达和稳定，并减轻小鼠肝纤维化。有趣的是， WNT-SCD2-LRP5/6环也存在于肝肿瘤起始干细胞样细胞(TICS)和HuH7中 细胞，是它们自我更新和启动肿瘤活动所必需的，建立了这一新的 通路在AHSCs和TICS/HCC细胞中都是一个功能核心。我们进一步证明了 AHSCs中的条件性Scd2 KO(Scd2 CKO)使AHSCs失能促进TIC启动的肿瘤 抑制肿瘤细胞Scd2的表达和肝脏的自然发育 二乙基亚硝胺(DEN)致小鼠肿瘤。我们最新的脂肪组学和转录组 Scd2 CKO小鼠非肿瘤肝组织分析揭示促肿瘤的整体抑制作用 胆固醇合成和二十烷类代谢中的脂质重编程。 根据这些结果，我们假设AHSCs建立了SCD2-Hur-Wnt正环 通过靶向相关基因在微环境中诱导促肿瘤脂质重编程 胆固醇合成和二十烷类化合物的生成和代谢。为了检验这一假设和 关于HSC SCD2的肿瘤增强作用的机制，我们将解决以下目标： 目的1.确定肝细胞中Scd2条件性KO是否能阻止DEN诱导的肝脏 Scd2ff；Alb-Cre小鼠与Scd2ff小鼠的肿瘤形成。 目的2.确定HSC SCD2是否通过时机促进肿瘤的启动与促进 用Scd2ff；Col1a1-Cre-ERT2消融Scd2。 AIM-3。探讨SCD2在AHSCs中对促肿瘤脂质微环境的支持作用。 目标3-1。目的：探讨β-连环蛋白依赖的HMGCRs转录调控机制。 目标3-2。确定β-连环蛋白是否通过Tcf在转录水平上刺激FADS1/2和LTA4H。 目标3-3。确定HUR是否通过SCD上调LTA4H基因的表达。 目标3-4。验证SCD介导的脂质重编程在人肝癌-PDX(患者来源的异种移植)中的作用 模特。 总而言之，这些努力将确定HSC新的肿瘤促进作用的机制。 SCD2，并帮助确定肝癌的新治疗靶点。

Activated HSCs (aHSCs) participate in liver fibrogenesis and promote the development of liver tumor, the two most devastating sequela of chronic liver disease. Our rigorous efforts to screen Wnt- β-catenin target genes in aHSCs, identified Scd (stearoyl-CoA desaturase) which encodes an ER enzyme responsible for the synthesis of mono-unsaturated fatty acids (MUFA) and is implicated in fatty liver, cancer and stemness. Scd is upregulated in a Wnt-dependent manner by the ability of β- catenin to bind to SREBP-1c and potentiate nearly 10-fold SREBP-1c-induce transcription. Induction of Scd2, an isoform dominantly expressed in HSCs is required for HSC activation and provides a positive forward loop to amplify canonical Wnt pathway via Lrp5/6 mRNA stability. This novel loop is initiated by SCD-generated MUFA interfering nuclear import of the mRNA binding protein HuR via Transportin (TNPO) and Ran1 GTPase, resulting in HuR cytoplasmic accumulation, HuR binding to Lrp5/6 mRNA 3'UTR, and their stabilization. SCD inhibition or Scd2 silencing abrogates LRP5/6 expression and stabilization of β-catenin in aHSCs, and attenuates liver fibrosis in mice. Intriguingly, the Wnt-SCD2-LRP5/6 loop also exists in liver tumor-initiating stem-cell like cells (TICs) and Huh7 cells and is required for their self-renewal and tumor-initiating activity, establishing this novel pathway as a functional core in both aHSCs and TICs/HCC cells. We further demonstrate that conditional Scd2 KO in aHSCs (Scd2 cKO) incapacitates aHSCs to promote TIC-initiated tumor growth in nude mice and suppresses tumor cell Scd2 expression and natural development of liver tumors induced by diethyl nitrosamine (DEN) in mice. Our most recent lipidomic and transcriptomic analyses of non-tumor liver tissues of Scd2 cKO mice, reveal global suppression of tumor-promoting lipid reprogramming in cholesterol synthesis and eicosanoid metabolism. Based on these results, we hypothesize that SCD2-HuR-Wnt positive loop established by aHSCs induces tumor-promoting lipid reprogramming in microenvironment by targeting genes involved in cholesterol synthesis and eicosanoid generation and metabolism. To test this hypothesis and the mechanisms of the tumor enhancer role of HSC SCD2, we will address the following aims: Aim 1. To determine whether Scd2 conditional KO in hepatocytes prevents DEN-induced liver tumor development by using Scd2ff;Alb-Cre vs. Scd2ff mice. Aim 2. To determine whether HSC SCD2 enhances tumor initiation vs. tumor promotion by timing Scd2 ablation using Scd2ff;Col1a1-Cre-ERT2. Aim-3. To determine how SCD2 in aHSCs supports tumor promoting lipid microenvironment. Aim 3-1. To determine the mechanism of β-catenin dependent regulation of HMGCR transcription. Aim 3-2. To determine whether β-catenin transcriptionally stimulates FADS1/2 and LTA4H via TCF. Aim 3-3. To determine if LTA4H mRNA is up-regulated by HuR via SCD. Aim 3-4. To validate SCD-mediated lipid reprogramming in HCC-PDX (patient-derived xenograft) model. Collectively, these efforts will define the mechanisms of the novel tumor enhancer role of HSC SCD2 and help identify new therapeutic targets for HCC.