Regulation of PTEN in chronic alcohol mediated liver disease

描述（由申请人提供）：本提案的目的是确定脂质磷酸酶PTEN在酒精性肝病（ALD）期间脂肪变性和肝损伤形成中的作用。虽然有关于PTEN在ALD中的作用的出版物，但这些报告使用的是流体模型中的静态时间点，并没有解决PTEN通过醛修饰失活和PTEN调节/表达在酒精暴露一段时间内发生变化的可能性。我们的工作假设是，慢性乙醇处理小鼠肝脏中产生的活性醛4-HNE共价加合PTEN，从而降低酶活性，导致PTEN下游信号通路失调。4-HNE已经与肝脏慢性乙醇暴露期间蛋白质的失活有关。也有假设认为PTEN表达的增加会导致下游通路的改变，最终导致ALD中脂肪变性的增加。为了实现本研究计划的目标，实验将使用质谱法鉴定PTEN上4-HNE修饰的位点，这些加合物抑制酶活性、膜结合和Trx1结合的能力。此外，通过western blotting、免疫组织化学和mRNA表达，研究慢性乙醇暴露9周后PTEN表达对下游信号通路的影响。

DESCRIPTION (provided by applicant): The objective of this proposal is to determine the involvement of the lipid phosphatase PTEN in the formation of steatosis and liver damage during alcoholic liver disease (ALD). Although there are publications concerning the role of PTEN in ALD, these reports are using a static time point in a fluid model and do not address the possibility of both PTEN inactivation via aldehyde modification and changes in PTEN regulation/expression over a period of alcohol exposure. Our working hypothesis states the reactive aldehyde 4-HNE, produced in the liver of chronic ethanol treated mice covalently adducts PTEN thereby reducing enzymatic activity leading to disregulation in PTEN downstream signaling. 4-HNE has already been implicated in the inactivation of proteins during chronic ethanol exposure in the liver. It is also hypothesized that increased PTEN expression leads to changes in downstream pathways ultimately leading to increased steatosis seen in ALD. In order to fulfill the objectives of this research proposal, experiments will be performed using mass spectrometry to identify the sites of 4-HNE modification on PTEN, the ability of these adducts to inhibit enzymatic activity, membrane association and Trx1 association. In addition, using western blotting, immunohistochemistry and mRNA expression, the effects of variable PTEN expression on downstream signaling pathways will be examined following 9-weeks of chronic ethanol exposure. PUBLIC HEALTH RELEVANCE: In the United States today, alcohol induced liver disease is a major cause of morbidity and mortality. Current statistics indicate that among chronic alcohol users, 15% will eventually be diagnosed with alcoholic liver disease. These symptoms include steatosis, alcoholic hepatitis and cirrhosis. The 5 and 10 year survival rates for alcohol induced cirrhosis are 23% and 7% respectively. Completion of the proposed experiments will provide greater insight into the mechanisms of chronic alcohol toxicity especially with respect to steatosis and damage caused by increased oxidative stress and altered PTEN signaling.