C/EBP alpha in Aging Liver

描述(申请人提供)：老年肝脏在部分肝切除后失去增殖能力，导致老年人死亡率较高。我们的实验室研究控制肝脏增殖的分子机制。肝脏特异性蛋白CCAAT/增强子结合蛋白α(C/EBPalpha)是一种强大的肝脏增殖抑制因子。C/EBPalpha通过与细胞周期蛋白依赖性激酶2和4直接相互作用抑制年轻肝脏的增殖。在脂肪组织中，C/EBPalpha通过抑制E2F转录而导致生长停滞。我们最近发现，衰老将肝脏中的C/EBPalpha从对CDKs的抑制转变为对E2F的抑制，E2F是通常在脂肪组织中运行的途径。在老年肝脏中，C/EBPalpha存在于高相对分子质量的复合体C/EBPalpha-RB-E2F4-BRM中。这种复合体占据E2F依赖的启动子，并阻止肝脏增殖所需表达的基因的激活。部分肝切除后未能减少这种复合体似乎是老肝脏增殖反应减弱的主要原因。该复合体的三个组分，RB，C/EBPalpha和E2F4，在老年肝脏中被差异磷酸化。C/EBPalpha在Ser193位的磷酸化是形成年龄特异性复合体的关键事件。我们最近发现CDK4和PP2A是调节C/EBPalpha的Ser193的磷酸化-去磷酸化的酶。在这个应用中，我们建议研究衰老对这些信号转导途径的影响，并确定它们在C/EBPalpha-Rb-E2F4-BRM复合体的出现和减少增殖反应中的作用。我们的工作假设是：1)C/EBPalpha、E2F4和Rb的磷酸化促进了AGE相关复合体的形成，2)肝脏中C/EBPalpha的Serl93的磷酸化是由CDK4介导的，并被PI3K-Akt-PP2A途径逆转，3)衰老的肝脏通过减少PI3K-Akt-PP2A途径和CDK4的激活来增加AGE专一性复合体的形成。在特定的目标1中，我们将研究衰老激活CDK4和下调PP2A活性的机制。特定目的2检查RB和E2F4的磷酸化是否影响它们形成年龄特异性C/EBPalpha复合体的能力。在具体目标3中，将鉴定该复合体的其他蛋白质，并研究它们在肝脏增殖中的作用。基于这个项目中获得的数据，我们计划开始制定一项纠正老年人肝脏增殖的策略。

DESCRIPTION (provided by applicant): An aging liver loses the ability to proliferate after partial hepatic resections leading to a higher mortality in the elderly. Our laboratory' investigates molecular mechanisms that control liver proliferation. Liver specific protein, CCAAT/Enhancer Binding Protein alpha (C/EBPalpha), is a strong inhibitor of liver proliferation. C/EBPalpha inhibits proliferation of young livers through direct interactions with cyclin dependent kinases 2 and 4. In adipose tissues, C/EBPalpha causes growth arrest via repression of E2F transcription. We have recently found that aging switches C/EBPalpha in liver from inhibition of cdks to repression of E2F, the pathway that normally operates in adipose tissues. In old livers, C/EBPalpha is observed in a high MW complex C/EBPalpha-Rb-E2F4-Brm. This complex occupies E2F-dependent promoters and blocks activation of genes whose expression is required for liver proliferation. A failure to diminish this complex after partial hepatectomy seems to be a major cause for the reduced proliferative response in old livers. Three components of the complex, Rb, C/EBPalpha and E2F4, are differentially phosphorylated in old livers. Phosphorylation of C/EBPalpha at Serl93 is a key event in the formation of the age-specific complex. We have recently identified cdk4 and PP2A as enzymes that regulate phosphorylation-dephosphorylation of Serl93 of C/EBPalpha. In this application, we propose to examine the effects of aging on these signal transduction pathways and determine their roles -in the appearance of the C/EBPalpha-Rb-E2F4-Brm complex and in the reduction of the proliferative response. Our working hypotheses are that: 1) Phosphorylation of C/EBPalpha, E2F4, and Rb promotes the formation of the age related complex, 2) Phosphorylation of Serl93 of C/EBPalpha in the liver is mediated by cdk4 and is reversed by the action of the PI3K-Akt-PP2A pathway, 3) Aging liver increases the age-specific complex by diminishing the PI3K-Akt-PP2A pathway and by activation of cdk4. In Specific Aim 1, we will examine mechanisms by which aging activates cdk4 and down-regulates activity of PP2A. Specific Aim 2 examines whether phosphorylation of Rb and E2F4 affects their ability to form the age-specific C/EBPalpha complex. In Specific Aim 3, additional proteins of the complex will be identified and their role in liver proliferation will be examined. Based on data obtained in this project, we are planning to start developing a strategy to correct liver proliferation in elderly.