The role of MKP-1/MAPK in hepatocytes and macrophages in alcohol-associated liver disease pathogenesis

项目摘要 酒精相关性肝病(ALD)占所有肝病死亡的近一半。尽管 鉴于这种疾病的严重性，目前还没有FDA批准的针对该疾病任何阶段的治疗方法， 强调迫切需要阐明ALD进展的潜在机制以开发新的 治疗。丝裂原活化蛋白激酶磷酸酶1(MKP-1)是丝裂原激活蛋白激酶-1(MKP-1)的原型成员。 特异性磷酸酶在器官炎症和损伤中起关键作用，包括在肝脏中。早期工作 研究表明，MKP-1水平的降低与c-Jun氨基末端激酶(JNK)的激活有关， 这会触发细胞凋亡级联反应，导致酒精所致的肝损伤。然而，其确切的机制是 在ALD中，MKP-1下调是如何导致肝细胞损伤和/或炎症的已知。此外， 在ALD中，MKP-1在巨噬细胞和MAPK驱动的炎症中的作用从未被研究过。我们的 初步研究表明，肝细胞MKP-1缺失增加酒精性肝损伤是一种NIAAA 酒精性肝病慢性暴饮症模型。数据还表明，肝细胞中MKP-1的缺失会使它们对 肿瘤坏死因子α具有毒性作用。作为一名有抱负的科学家，他的目标是从事与酒精相关的多器官研究 病理学，研究ALD发病机制中的新因素不仅有助于满足这一医学需求，而且还将 作为一个学习如何成为一名成功科学家的极好平台。在我们的帮助下 指导我的导师Gobejishvili博士(ALD基础研究专家)和McClain博士( 疾病的临床方面)以及几位知识渊博的合作者，我将调查 酒精相关MKP-1表达下调在酒精性肝病发病机制中的作用这项研究的第一个目的是进一步 肝细胞特异性MKP-1基因敲除小鼠在肝细胞中MKP-1缺失的作用 不同的ALD小鼠模型。我将从这些模型中分离出肝细胞和免疫细胞，检查 MKP-1缺失对促炎性趋化因子产生和免疫细胞表型的影响 改变肝细胞和免疫细胞的健康和功能。第二个目标将决定MKP-1的作用 特别是在使用RAW 264.7巨噬细胞系的巨噬细胞、原代库普弗细胞和巨噬细胞中- 特定的基因敲除小鼠。为了完成这项研究，我将遵循我的同事为我制定的详细培训计划。 指导和追求我的职业目标。其中包括，继续我在以下领域的教学培训 与我的研究相关，在新的实验室技术方面积累经验，培养有效的书面和口头表达能力 沟通能力，并建立一个专业的科学家网络。总体而言，培训和研究 在这里提出的建议不仅将满足ALD治疗发展的重大需求，而且还将促进我的 从一位年轻的科学家发展成为酒精诱导领域成功的独立调查者 器官疾病。

Project Summary Alcohol-associated liver disease (ALD) is responsible for nearly half of all deaths from liver disease. Despite the severity of this disease, there remain no FDA-approved treatments for any stage of the disease, highlighting the critical need to elucidate the underlying mechanisms of ALD progression to develop new therapies. Mitogen-activated protein kinase phosphatase 1 (MKP-1) is the archetypal member of the dual- specificity phosphatases with a pivotal role in organ inflammation and injury, including in the liver. Earlier work showed that decreased MKP-1 levels are associated with the activation of c-Jun N-terminal kinase (JNK), which triggers an apoptotic cascade leading to alcohol induced liver injury. However, the exact mechanisms of how MKP-1 downregulation leads to hepatocyte injury and/or inflammation in ALD are known. Moreover, the role of MKP-1 in macrophages and MAPK-driven inflammation in ALD has never been examined. Our preliminary studies show that MKP-1 deletion in hepatocytes augments alcohol-induced liver injury is a NIAAA chronic plus binge model of ALD. Data also suggest that MKP-1 deletion in hepatocytes sensitizes them to TNFα induced toxicity. As an aspiring scientist whose goal is to pursue a career in alcohol-related multi-organ pathology, investigating new players in ALD pathogenesis would not only help fill this medical need but also serve as an excellent platform from which to learn how to become a successful scientist. With the help and supervision of my mentors Dr. Gobejishvili (an expert in basic research on ALD) and Dr. McClain (an expert on clinical aspects of the disease) as well as several knowledgeable collaborators, I will investigate the role of alcohol-associated downregulation of MKP-1 in the pathogenesis of ALD. The first aim of this study is to further characterize the role of MKP-1 deletion in hepatocytes using hepatocyte specific MKP-1 knpockout mice in two different mouse models of ALD. I will isolate hepatocytes and immune cells from these models, examine the effect of MKP-1 deletion on pro-inflammatory chemokine production and immune cell phenotype and describe changes in hepatocyte and immune cell health and function. The second aim will determine the role of MKP-1 specifically in macrophages using RAW 264.7 macrophage cell line, primary Kupffer cells and macrophage- specific knockout mice. To complete this research, I will follow a detailed training plan laid out for me by my co- mentors and pursue my career goals. These include, among others, continuing my didactic training in areas relevant to my research, gaining experience in new laboratory techniques, developing effective written and oral communication skills, and building a professional network of scientists. Collectively, the training and research proposed here will not only fill a significant need for the development of ALD treatments but will also foster my development from a young scientist to a successful, independent investigator in the field of alcohol-induced organ disease.