PHPT1 knockout for investigation of ethanol-induced hepatic steatosis

PHPT1 敲除用于研究乙醇诱导的肝脂肪变性

• 批准号: 9759732
• 负责人: BRANT Roger BURKHARDT
• 金额: $ 7.48万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9759732
• 关键词: Address; Adenoviruses; Affect; Alcoholic Liver Diseases; Animal Model; Area; Biological; Cause of Death; Cell physiology; Cessation of life; Chronic; Clinical; Complement; Data; Diet; Disease; Down-Regulation; Embryo; Ethanol; Eukaryotic Cell; Exposure to; Fatty Liver; Functional disorder; Funding Mechanisms; Gene Expression Regulation; Genetic Transcription; Goals; Grant; Hepatic; Hepatocyte; Heterozygote; Histidine; Investigation; Ion Transport; Knock-out; Knockout Mice; Liver; Liver diseases; Mammalian Cell; Mass Spectrum Analysis; Measures; Metabolic; Mission; Modeling; Molecular; Mus; National Institute on Alcohol Abuse and Alcoholism; Pathway interactions; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Predisposition; Prevention; Process; Proteins; Research; Research Project Grants; Role; Signal Transduction; Therapeutic Intervention; Time; Transgenic Organisms; Triglycerides; United States; Validation; alcohol exposure; base; differential expression; early onset; experimental study; fatty acid biosynthesis; in vivo; knockout animal; liver function; liver injury; mouse model; mutant; new therapeutic target; novel; overexpression; phosphohistidine; programs; protein function; targeted treatment; tool

PROJECT SUMMARY Histidine phosphorylation in mammalian cell signaling is largely unexplored despite the biological importance where few phosphohistidine (pHis) targets have been identified and validated. These critical biological targets include proteins involved in fatty acid biosynthesis, cell signaling, regulation of gene transcription and ion transport. Given the importance of these fundamental cellular processes and related impact of histidine phosphorylation on protein function, dysregulation of pHis levels has been implicated as a mechanism underlying the pathophysiology of disease including liver disease, which is among the top 15 leading causes of death in the United States. Alcoholic liver disease (ALD) accounts for approximately 50% of these deaths. Elucidation of the mechanisms underlying the pathophysiology of ALD is critical to ultimately developing novel clinical interventional therapy, particularly at early onset. Our research group has been actively investigating the role of a mammalian phosphohistidine phosphatase, PHPT1, in ethanol-induced hepatocyte dysfunction and our data has demonstrated the following critical results. First, we have identified that PHPT1 is significantly downregulated and cytoplasmic pHis levels are increased in the livers of chronic ethanol-exposed mice. Second, our in vivo data has demonstrated that transgenic delivery of PHPT1 inhibits hepatic steatosis in ethanol-fed mice. Taken together, we hypothesize that chronic ethanol exposure induces downregulation of PHPT1 and subsequent changes in hepatic pHis signaling, altering expression and activity of critical hepatic lipogenic targets contributing to the onset of hepatic steatosis. To address this, we have generated a novel PHPT1 knockout mouse. Our initial evidence indicates disruption of PHPT1 is embryonically lethal. Our heterozygote PHPT1 mice have an approximate 50% reduction of PHPT1, particularly in the liver but further characterization is needed. Therefore, the purpose of this R03 is to fully examine the steatotic phenotype of the PHPT1 knockout in relation to PHPT1 inhibition of ethanol-induced hepatic steatosis. Central to addressing our hypothesis, the PHPT1 KO will be exposed to the NIAAA chronic binge model with 2 specific aims: (1) Determine if the reduction of PHPT1 promotes ethanol-induced hepatic steatosis in the PHPT1 Knockout (PHPT1KO) mouse and (2) Determine if liver-specific expression of PHPT1 can rescue hepatic steatosis in PHPT1+/-. The proposed studies will significantly contribute to the understanding of ALD pathophysiology. Our primary scientific goals of this proposal are strongly suited to the mission of NIAAA and that of the R03 small grant program. Our self-contained research project already has both an established essential animal model (PHPT1KO) and additional tools (Ad-PHPT1) to rapidly employ to directly address the role of PHPT1 in ethanol-induced hepatic steatosis.

项目摘要 尽管生物学重要性 在很少鉴定和验证的磷酸组织（PHI）靶标。这些关键的生物学靶标 包括参与脂肪酸生物合成的蛋白质，细胞信号传导，基因转录和离子的调节 运输。鉴于这些基本细胞过程的重要性以及组氨酸的相关影响 蛋白质功能的磷酸化，PHIS水平的失调已被视为一种机制 包括肝病在内的疾病的病理生理学，这是15个主要的死亡原因之一 美国。酒精性肝病（ALD）约占这些死亡的50％。阐明 ALD病理生理学基础的机制对于最终发展新型临床至关重要 介入疗法，特别是在早期发作时。 我们的研究小组一直在积极研究哺乳动物磷酸组织磷酸酶的作用，即 PHPT1，在乙醇诱导的肝细胞功能障碍中，我们的数据证明了以下关键结果。 首先，我们已经确定PHPT1显着下调，并且细胞质PHIS水平增加 慢性乙醇暴露小鼠的肝脏。其次，我们的体内数据证明了转基因递送 PHPT1的抑制乙醇喂养小鼠的肝脂肪变性。综上所述，我们假设慢性乙醇 暴露会引起PHPT1的下调以及随后的肝PHIS信号变化，改变 关键的肝脂肪生成靶标的表达和活性导致肝脂肪变性的发作。到 解决这个问题，我们生成了一种新型的PHPT1敲除鼠标。我们的最初证据表明中断 PHPT1是胚胎致死的。我们的杂合子PHPT1小鼠的PHPT1降低了约50％， 特别是在肝脏中，但需要进一步的表征。因此，此R03的目的是完全 检查与PHPT1抑制乙醇诱导的PHPT1基因敲除的脂肪分裂表型 肝脂肪变性。核心解决我们的假设，PHPT1 KO将暴露于NIAAA慢性 具有2个特定目的的暴饮暴食模型：（1）确定PHPT1的减少是否促进乙醇诱导的肝 PHPT1敲除（PHPT1KO）小鼠中的脂肪变性和（2）确定PHPT1的肝脏特异性表达是否表达 可以在PHPT1 +/-中营救肝脂肪变性。拟议的研究将有助于理解 ALD病理生理学。我们该提案的主要科学目标非常适合NIAAA的使命 以及R03小型赠款计划的。我们独立的研究项目已经建立了 基本动物模型（PHPT1KO）和其他工具（AD-PHPT1）迅速采用直接解决 PHPT1在乙醇引起的肝脂肪变性中的作用。