Ceramide and acute phase proteins elevation during aging

描述（由申请人提供）：衰老与慢性亚临床炎症相关，这是动脉粥样硬化和心血管疾病风险增加的基础。表现为老年人血浆中C反应蛋白（CRP）、血清淀粉样蛋白A （SAA）、α - 1酸性糖蛋白（AGP）等主要急性期蛋白水平的慢性低幅度升高。这项研究的长期目标是加深我们对衰老、慢性炎症和急性期蛋白调控之间关系的理解。我们最近发现，神经酰胺是一种第二信使分子，在炎症期间介导CRP， AGP和SAA的急性刺激，在老年大鼠和小鼠肝脏中慢性增加。我们推测，衰老过程中神经酰胺水平的升高导致C/EBP转录因子的慢性激活以及CRP、AGP和SAA的表达增加。我们的目标是破译神经酰胺增加在衰老相关的APP表达增加中的作用，并确定这与调节炎症诱导的APP分泌变化的机制在多大程度上相似。我们提出以下具体目标：(I)确定神经酰胺生成的哪个途径导致诱导急性期蛋白（APP） mRNA增加。利用腺病毒介导的基因转移，我们将在小鼠原代肝细胞中过表达酸性和中性SMases，并将测试哪一种酶足以上调小鼠三种主要APP， SAP， AGP和SAA的mRNA水平。为了确定参与信号传导的神经酰胺的亚细胞池，我们将研究过表达的ASMase和NSMase的亚细胞定位，并将其与衰老和炎症过程中产生的过量神经酰胺的亚细胞定位进行比较(2)在体外测试衰老和炎症诱导APP mRNA是否需要激活ASMase或NSMase。在这个特定的目标中，我们将确定炎症和衰老过程中神经酰胺的产生和APP表达的诱导机制。利用来自ASMase（-/-）小鼠的肝细胞和NSMase的特异性抑制剂，我们将确定哪些SMases对于APP的上调是必不可少的。(3)揭示酸性和中性SMase在体内APP调控中的作用。使用酸性鞘磷脂酶敲除小鼠，我们将在生理背景下验证Sp. Aim 1和2得出的结论。

DESCRIPTION (provided by applicant): Aging is associated with chronic sub-clinical inflammation that underlies the increased risk of atherosclerosis and cardiovascular disease. It is manifested by chronic low-amplitude increases in the plasma levels of the major acute phase proteins, such as C reactive protein (CRP), serum amyloid A (SAA) and alpha1 acid glycoprotein (AGP) in the elderly. The long term objective of the proposed research is to deepen our understanding for the relationship between aging, chronic inflammation and regulation of the acute phase proteins. We recently found that ceramide, a second messenger molecule that mediates the acute stimulation of CRP, AGP and SAA during inflammation is chronically increased in liver from old rats and mice. We hypothesize that increased level of ceramide in aging cause chronic activation of C/EBP transcription factor and increased expression of CRP, AGP and SAA. Our objectives are to decipher the role of ceramide increases in aging - associated increase(s) in the expression of APP and to determine to what extent this is similar to the mechanisms that regulate the inflammation-induced changes in APP secretion. We propose the following specific aims: (I) To identify which pathway for ceramide generation leads to induction of acute phase proteins (APP) mRNA increases. Using adenovirus-mediated gene transfer we will overexpress acid and neutral SMases in primary mouse hepatocytes and will test which of the enzymes is sufficient to up-regulate the level of mRNA for three of the major APP in mouse, SAP, AGP and SAA. To identify the subcellular pool of ceramide involved in signaling, subcellular localization of overexpressed ASMase and NSMase will be studied and compared to the localization of the excess ceramide generated during aging and inflammation (2) To test in vitro whether active ASMase or NSMase is required for the induction of APP mRNA by aging and inflammation. In this specific aim we will identify the mechanism for generation of ceramide and induction of APP expression during inflammation and aging. Using hepatocytes from ASMase(-/-) mice and specific inhibitors of NSMase, we will identify which of SMases is indispensable for APP up-regulation. (3) To decipher the role of acid and neutral SMase in APP regulation in vivo. Using acid sphingomyelinase knockout mice, we will validate the conclusions drawn from Sp. Aim 1 and 2 in a physiological context. The results of these studies will elucidate the factors that regulate ceramide level in inflammation and during aging. An understanding of the cellular and molecular mechanisms responsible for this regulation will help to define how ceramide exerts its effects and its role in cardiovascular disease, atherosclerosis and, perhaps other diseases of the elderly, such as stroke and Alzheimer disease.