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，这是动脉粥样硬化和心血管疾病风险增加的基础。表现为老年人血浆主要急性期蛋白如C反应蛋白(CRP)、血清淀粉样蛋白A(SAA)和α1酸性糖蛋白(AGP)水平的慢性低幅度升高。这项拟议研究的长期目标是加深我们对衰老、慢性炎症和急性时相蛋白调节之间的关系的理解。我们最近发现，在炎症过程中介导CRP、AGP和SAA急性刺激的第二信使分子神经酰胺在老年大鼠和小鼠的肝脏中慢性增加。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C/EBP转录因子的慢性激活和C/EBP转录因子的慢性激活，以及C/EBP转录因子、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增加(S)APP表达中的作用，并确定这在多大程度上类似于调节炎症诱导的APP分泌变化的机制。我们提出了以下特定的目标：(I)确定神经酰胺产生的哪条途径导致急性时相蛋白(APP)mRNA的增加。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度表达酸性和中性SMase，并将测试哪种酶足以上调小鼠SAP、AGP和SAA三种主要APP的mRNA水平。为了确定参与信号转导的神经酰胺亚细胞池，将研究过表达的ASMase和NSMase的亚细胞定位，并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)在体外测试衰老和炎症诱导APP mRNA是否需要有活性的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ASMase(-/-)小鼠的肝细胞和NSMase的特异性抑制剂，我们将确定哪些SMase是APP上调所必需的。(3)破译酸性和中性sMase在体内APP调控中的作用。利用酸性鞘磷脂酶基因敲除小鼠，我们将验证从Sp.生理学背景下的目标1和目标2。 这些研究的结果将阐明在炎症和衰老过程中调节神经酰胺水平的因素。了解这种调控的细胞和分子机制将有助于确定神经酰胺是如何发挥作用的，以及它在心血管疾病、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.