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有关，慢性亚临床炎症是动脉粥样硬化和心血管疾病风险增加的基础。其表现为老年人血浆中主要急性时相蛋白水平慢性低幅度升高，如C反应蛋白（CRP）、血清淀粉样蛋白A（SAA）和α1酸性糖蛋白（AGP）。该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节之间关系的理解。我们最近发现，神经酰胺（一种在炎症过程中介导 CRP、AGP 和 SAA 急性刺激的第二信使分子）在老年大鼠和小鼠的肝脏中长期增加。我们假设衰老过程中神经酰胺水平的增加导致 C/EBP 转录因子的慢性激活以及 CRP、AGP 和 SAA 表达的增加。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，并确定这在多大程度上与调节炎症诱导的 APP 分泌变化的机制相似。我们提出以下具体目标： (I) 确定神经酰胺生成的哪种途径导致诱导急性期蛋白 (APP) mRNA 增加。使用腺病毒介导的基因转移，我们将在原代小鼠肝细胞中过表达酸性和中性 SMase，并测试哪种酶足以上调小鼠中三种主要 APP（SAP、AGP 和 SAA）的 mRNA 水平。为了确定参与信号传导的神经酰胺亚细胞库，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定位进行比较 (2) 体外测试衰老和炎症诱导 APP mRNA 是否需要活性 ASMase 或 NSMase。在这个具体目标中，我们将确定炎症和衰老过程中神经酰胺生成和诱导 APP 表达的机制。使用 ASMase(-/-) 小鼠的肝细胞和 NSMase 的特异性抑制剂，我们将确定哪些 SMase 对于 APP 上调是必不可少的。 (3)破译酸性和中性SMase在体内APP调节中的作用。使用酸性鞘磷脂酶敲除小鼠，我们将验证 Sp 得出的结论。在生理背景下实现目标 1 和 2。 这些研究的结果将阐明炎症和衰老过程中调节神经酰胺水平的因素。了解负责这种调节的细胞和分子机制将有助于确定神经酰胺如何发挥其作用及其在心血管疾病、动脉粥样硬化以及其他老年人疾病（如中风和阿尔茨海默病）中的作用。

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.