Molecular Mechanisms of Stellate Cell Activation in Liver Fibrosis

肝纤维化星状细胞激活的分子机制

• 批准号: 9559537
• 负责人: HIDEKAZU TSUKAMOTO
• 金额: --
• 依托单位: VA GREATER LOS ANGELES HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-10-01 至 2022-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9559537
• 关键词: 3' Untranslated Regions; ADD-1 protein; Ablation; Address; Attenuated; Binding; Binding Proteins; Biological; Cell Fate Control; Cells; Cholesterol; Cholesterol Homeostasis; Cirrhosis; Development; Down-Regulation; Eicosanoids; Encapsulated; Enhancers; Enzymes; Epigenetic Process; Fatty Liver; Gene Expression Profiling; Generations; Genes; Genetic; Genetic Transcription; Goals; Guanosine Triphosphate Phosphohydrolases; Hepatic; Hepatic Fibrogenesis; Hepatic Stellate Cell; Hepatocyte; Heterochromatin; HuR protein; Karyopherins; Lipids; Liver Fibrosis; Liver neoplasms; Malignant neoplasm of liver; Mediating; Messenger RNA; Metabolism; Methyl-CpG-Binding Protein 2; Modality; Modeling; Molecular; Monounsaturated Fatty Acids; Mus; Nitrosamines; Nuclear Import; Nude Mice; Oleic Acids; PPAR gamma; Pathway interactions; Patients; Protein Isoforms; Published Comment; Regulation; Research; Research Support; Role; Saturated Fatty Acids; Series; Stearoyl-CoA Desaturase; Testing; Therapeutic; Tissues; Transcriptional Regulation; Tumor Initiators; Tumor Promotion; Tumor Suppression; Veterans; Xenograft Model; base; beta catenin; cell type; chronic liver disease; desaturase; design; eicosanoid metabolism; feeding; hepatocellular carcinoma cell line; innovation; insight; lipid metabolism; liver development; mRNA Stability; morphogens; mouse model; neoplastic cell; new therapeutic target; novel; palmitoleic acid; prevent; recruit; response; screening; self-renewal; stellate cell; stem-like cell; stemness; transcriptome; transcriptomics; tumor; tumor growth; tumor initiation; tumor microenvironment; tumorigenic; wound

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.

活化的HSC（aHSC）参与肝纤维化的发生，促进肝脏的发育 肿瘤，慢性肝病的两个最具破坏性的后遗症。我们对Wnt的严格筛选- aHSC中的β-连环蛋白靶基因，鉴定了编码ER的Scd（硬脂酰-CoA去饱和酶） 一种负责合成单不饱和脂肪酸（MUFA）的酶， 脂肪肝、癌症和干性。Scd以Wnt依赖性方式被β- catenin结合SREBP-1c并增强近10倍的SREBP-1c诱导的转录。感应 在HSC中主要表达的同种型是HSC活化所需的，并提供了一种 正向环通过Lrp 5/6 mRNA稳定性扩增经典Wnt途径。这个新的循环是 通过SCD产生的MUFA干扰mRNA结合蛋白HuR的核输入， 转运蛋白（TNPO）和Ran 1 GT3，导致HuR细胞质积累，HuR结合到 Lrp 5/6 mRNA 3 'UTR，及其稳定化。SCD抑制或Scd 2沉默消除LRP 5/6 在aHSC中的β-连环蛋白的表达和稳定，并减弱小鼠中的肝纤维化。有趣的是， Wnt-SCD 2-LRP 5/6环也存在于肝肿瘤起始干细胞样细胞（TIC）和Huh 7中， 细胞，并需要他们的自我更新和肿瘤启动活动，建立这种新的 在aHSC和TIC/HCC细胞两者中作为功能核心的信号通路。我们进一步证明， aHSC中的条件性Scd 2 KO（Scd 2 cKO）使aHSC不能促进TIC引发的肿瘤 在裸鼠中生长并抑制肿瘤细胞Scd 2表达和肝脏的自然发育 二乙基亚硝胺（DEN）诱发小鼠肿瘤。我们最新的脂质组学和转录组学 对Scd 2 cKO小鼠的非肿瘤肝组织的分析显示， 胆固醇合成和类花生酸代谢中的脂质重编程。 基于这些结果，我们假设aHSC建立的SCD 2-HuR-Wnt阳性环可能与aHSC的表达有关。 通过靶向相关基因诱导微环境中促进肿瘤的脂质重编程 胆固醇合成和类花生酸的产生和代谢。为了验证这一假设， 为了研究HSC SCD 2的肿瘤增强剂作用的机制，我们将致力于以下目标： 目标1.为了确定肝细胞中的Scd 2条件性KO是否阻止DEN诱导的肝细胞凋亡， 使用Scd 2ff的肿瘤发展;Alb-Cre与Scd 2ff小鼠。 目标2.确定HSC SCD 2是否通过时间选择增强肿瘤起始与肿瘤促进 使用Scd 2ff进行Scd 2消融; Col 1a 1-Cre-ERT 2。 目标三确定aHSC中的SCD 2如何支持肿瘤促进脂质微环境。 瞄准3-1探讨β-catenin依赖性调控HMGCR转录的机制。 目标3-2确定β-catenin是否通过TCF转录刺激FADS 1/2和LTA 4 H。 瞄准3-3确定HuR是否通过SCD上调LTA 4 H mRNA。 瞄准3-4在HCC-PDX（患者源性异种移植物）中验证SCD介导的脂质重编程 模型 总的来说，这些努力将确定新的肿瘤增强作用的HSC的机制 SCD 2和帮助确定新的治疗肝癌的目标。