Akt Isoforms In the Pathogenesis of Alcoholic Liver Disease

酒精性肝病发病机制中的 Akt 亚型

• 批准号: 9314661
• 负责人: Karina Reyes-Gordillo
• 金额: $ 13.68万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9314661
• 关键词: 1-Phosphatidylinositol 3-Kinase; AKT1 gene; AKT2 gene; Acetaldehyde; Address; Advisory Committees; Alcohol dehydrogenase; Alcoholic Liver Diseases; Applications Grants; Area; B Cell Proliferation; Biochemical; Biological; C57BL/6 Mouse; CYP2E1 gene; Cell Culture Techniques; Cell Proliferation; Cells; Chemicals; Chronic; Collagen; Committee Members; Complex; Development; Endotoxins; Environment; Epigenetic Process; Ethanol; Extracellular Matrix Proteins; FRAP1 gene; Faculty; Fibronectins; Fibrosis; Generations; Genes; Goals; Grant; Hepatic; Hepatic Stellate Cell; Hepatocyte; Human; In Vitro; Individual; Inflammation; Inflammatory; Interleukin-1 beta; Investigation; Knockout Mice; Knowledge; Kupffer Cells; Lead; Lipids; Lipopolysaccharides; Liver; Mediating; Mentored Research Scientist Development Award; Mentors; Mentorship; Methyl-CpG-Binding Protein 2; Modeling; Molecular; NF-kappa B; Oxidative Stress; Oxides; PDPK1 gene; PPAR gamma; Pathogenesis; Pathogenicity; Permeability; Peroxisome Proliferator-Activated Receptors; Pharmacology; Phenotype; Phosphotransferases; Platelet-Derived Growth Factor beta Receptor; Play; Prevention; Process; Production; Protein Isoforms; Protein-Serine-Threonine Kinases; Reactive Oxygen Species; Research; Research Assistant; Research Personnel; Role; Scientist; Signal Transduction; Small Interfering RNA; Smooth Muscle Actin Staining Method; TNF gene; Training; Up-Regulation; Vitamin A; Wild Type Mouse; base; career development; cell motility; cytokine; experience; fibrogenesis; in vivo; inflammatory marker; inhibitor/antagonist; innovation; liver injury; migration; mouse model; new therapeutic target; next generation; phosphoinositide-dependent kinase 1; prevent; professor; skills; stellate cell; tenure track; transdifferentiation

The goal of this revised K01 application is to promote the development of the applicant to become a multi- disciplinarily trained, and independent academic investigator. The collective strengths of this application are defined in these 3 major areas: 1) Credentials: Reyes-Gordillo’s application builds upon her productive track- record establishing her scientific independence in the field of Alcoholic Liver Disease (ALD) based on which GWU appointed her as Assistant Research Professor since 07/01/2015. The applicant’s goals include achieving professional skills, and maturing as an independent tenure-track academic researcher. 2) Training Environment: Drs. Lakshman (mentor) and Bin Gao (co-mentor), the two world class hepatologists, are fully committed to implement and complete the training necessary to advance the PI as an outstanding junior faculty. Drs. Kumar, Szabo, Schrum, Diehl, and Casey with credentials in developing the next generation of successful academic scientists will serve as Advisory Committee members. The knowledge and experience gained during this K01 award period will facilitate the candidate to successfully earn a R01 grant to become an independent tenured investigator. 3) Innovative Models and Research: PI’s main hypothesis is that each Akt isoform has a specific regulatory function in chronic Ethanol(EtOH)/Binge/LPS (EBL)-mediated liver injury, which she plans to prove utilizing both in vitro and in vivo approaches with the following encouraging preliminary results: In vitro: In the acetaldehyde (ACE)/LPS and/or EtOH/LPS human culture models, siRNA- directed silencing of (A) Akt2, but not Akt1, significantly suppressed cell inflammatory markers in KC and HSC; (B) Akt1, Akt2 inhibited cell proliferation in HSC; (C) Akt2 alone inhibited cell migration in HSC; (D) Akt1, Akt2, but not Akt3 inhibited the fibrogenic markers in VL17A hepatocytes and HSC. In vivo: EBL mouse model (a) stimulated all Akt isoforms with concomitant increases in phosphorylated PDK1 and mTORC-2, and PI3K, thereby up regulating inflammatory, proliferative, and fibrogenic genes; (b) caused no inflammation when Akt2, but not Akt1 was pharmacologically blocked, whereas blocking of both Akt1 and Akt2 inhibited fibrosis. PI will use Akt-isoform-specific silenced HSC/KC/hepatocyte cultures in vitro and hepatocyte/HSC-specific Akt1/2- KO, and wild type mice in the EBL model, and biochemical, molecular biological, immuno- & histo-chemical approaches to accomplish the following specific aims: Specific Aim 1. What are the specific respective roles of the three Akt isoforms in EtOH/LPS or ACE/LPS-mediated (A) NFκB signaling cascade, TNFα and IL1β, (B) cell proliferation and migration & (C) fibrogenic and adipogenic gene cascades in individual human HSC, KC and hepatocyte cultures? Specific Aim 2. (2A) What are the action/s of EBL on Akt isoforms and consequent effects on inflammatory, proliferative, fibrogenic and adipogenic genes? (2B) Could specific pharmacological inhibition of Akt1 and/or Akt2 protect against EBL liver damage? & (2C) Could EBL-induced liver damage be prevented by the deletion of Akt1 and/or Akt2 gene using hepatocyte-specific or HSC-specific knockout mice?

修订后的K 01申请的目标是促进申请人的发展，使其成为一个多 受过纪律训练，独立的学术调查员。该应用程序的集体优势是 定义在这3个主要领域：1）证书：雷耶斯戈迪略的应用程序建立在她的生产轨道- 在酒精性肝病（ALD）领域建立科学独立性的记录， GWU任命她为助理研究教授自07/01/2015。申请人的目标包括 实现专业技能，并作为一个独立的终身制学术研究成熟。2)培训 环境：两位世界级肝病学家Lakshman（导师）和Bin Gao（共同导师）博士完全 致力于实施和完成必要的培训，以促进PI作为一个优秀的初级 教师。Kumar博士、Szabo博士、Schrum博士、Diehl博士和凯西博士在开发下一代 成功的学术科学家将担任咨询委员会成员。的知识和经验 在此K 01奖励期间获得的奖励将有助于候选人成功获得R 01补助金， 独立终身研究员3)创新模型和研究：PI的主要假设是，每个Akt 同种型在慢性乙醇（EtOH）/酗酒/LPS（EBL）介导的肝损伤中具有特异性调节功能， 她计划利用体外和体内方法证明这一点， 初步结果：体外：在乙醛（ACE）/LPS和/或EtOH/LPS人培养模型中，siRNA- （A）Akt 2而非Akt 1的定向沉默显著抑制KC和HSC中的细胞炎症标志物; (B)Akt 1、Akt 2抑制HSC中的细胞增殖;（C）Akt 2单独抑制HSC中的细胞迁移;（D）Akt 1、Akt 2、 而Akt 3不能抑制VL 17 A肝细胞和HSC中的纤维化标志物。体内：EBL小鼠模型（a） 刺激所有Akt同种型，伴随磷酸化PDK 1和mTORC-2以及PI 3 K的增加， 从而上调炎性、增殖性和纤维化基因;（B）当Akt 2， 但Akt 1未被阻断，而阻断Akt 1和Akt 2均抑制纤维化。PI将 使用Akt同种型特异性沉默的HSC/KC/肝细胞体外培养物和肝细胞/HSC特异性Akt 1/2- KO和野生型小鼠的EBL模型，以及生化，分子生物学，免疫和组织化学 实现以下具体目标的方法：具体目标1。它们各自的具体作用是什么？ EtOH/LPS或ACE/LPS介导的（A）NFκB信号级联、TNFα和IL 1 β（B）细胞中的三种Akt亚型 增殖和迁移&（C）单个人HSC、KC和HSC中的纤维形成和脂肪形成基因级联 肝细胞培养具体目标2。(2A)EBL对Akt亚型的作用及其后果是什么 炎症、增生、纤维化和脂肪形成基因的影响(2B)特异性药物抑制 Akt 1和/或Akt 2是否能保护EBL肝损伤？&（2C）是否可以防止EBL诱导的肝损伤， 使用肝细胞特异性或HSC特异性敲除小鼠缺失Akt 1和/或Akt 2基因？