EFFECTS OF AGING ON ACUTE PHASE RESPONSE REGULATION

老化对急性期反应调节的影响

• 批准号: 5204813
• 负责人: JOHN PAPACONSTANTINOU
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5204813
• 关键词: DNA binding protein; DNA footprinting; RNase protection assay; acute phase protein; aging; alpha 1 acid glycoprotein; animal genetic material tag; animal old age; animal tissue; gel mobility shift assay; gene expression; gene induction /repression; genetic models; genetic promoter element; genetic transcription; glucocorticoids; high performance liquid chromatography; hormone regulation /control mechanism; interleukin 6; juvenile animal; laboratory mouse; laboratory rat; lipopolysaccharides; liver cells; messenger RNA; nuclear runoff assay; phosphorylation; posttranslational modifications; protein purification; protein structure function; transcription factor

Current theories suggest that progressive age-associated declines in tissue function are caused by changes in intrinsic processes, and that these can occur in the absence of disease. We hypothesize that components of eukaryotic gene regulatory processes may be intrinsically altered by aging to cause increased or decreased gene expression, in turn producing the observed declines in tissue functions. Following such systemic injuries as bacterial lipopolysaccharide (LPS) mediated acute inflammation, the liver responds with a striking increase in the synthesis of a subset of serum proteins, the acute phase reactants (APR). Our preliminary studies indicate that aging affects the regulation of one of the APR genes, the alpha1-acid glycoprotein (AGP) gene. Changes include (a) an increase in the constitutive level of the AGP MRNA pool, and (b) slowed induction of AGP by LPS. In this project we will use the AGP gene as a model to determine whether the structure and function of regulatory factors are affected by aging. AGP MRNA levels will be determined to establish complete LPS-stimulated induction curves in 2, 12, and 24 month Balb/cNNia mice, and nuclear run-on analyses will be done to ask if aging alters both constitutive and LPS-inducible levels of AGP gene transcription in aged mice. Preliminary studies have shown that the mouse AGP promoter has two trans-acting factor binding sites, namely region B (-104 to -91) and region C (-125 to -104), and that the binding activity of these factors may be altered in aged animals. In addition the proteins that binding activity of these factors may be altered in aged animals. In addition the proteins that bind to region C have been identified as C/EBPalpha (constitutive) and C/EBPbeta (LPS-inducible). To determine if trans-acting factors of the AGP promoter are altered in aged animals, activity of these factors will be determined by Dnase I (in vitro) and in vivo footprinting, and gel- shift/Scatchard plot analysis. Experiments will be done to ask whether age-associated structural changes in these trans-acting factors is the basis for their altered binding activity. These experiments include protein modification by phosphorylation and homodimer/heterodimer function. Trans-acting factor MRNA and protein levels will be analyzed to determine if aging affects the expression of their genes. An in vitro transcription assay system will also be developed in order to conduct functional assays of the trans-acting factors. Our long range goal is to determine how gene regulation at the transcriptional or post transcriptional level is intrinsically affected by aging.

目前的理论表明，随着年龄的增长，组织的进行性下降 函数是由内在过程中的变化引起的，这些过程可以 在没有疾病的情况下发生。我们假设其中的组件 真核基因调控过程可能会因衰老而发生本质变化 导致基因表达增加或减少，进而产生 观察到组织功能的下降。在发生系统性损伤后，如 细菌脂多糖(LPS)介导的急性炎症，肝脏 作为回应，血清的一个子集的合成显著增加 蛋白质，急性相反应物(APR)。我们的初步研究 表明衰老影响APR基因之一的调节，即 α1酸性糖蛋白(AGP)基因。变化包括：(A)增加 AGP信使核糖核酸池的组成水平，以及(B)减缓了对 脂多糖诱导的AGP。在这个项目中，我们将使用AGP基因作为模型 确定调控因子的结构和功能是否 受衰老的影响。AGP mRNA水平将被测定以建立 2个月、12个月和24个月Balb/cNNia完整的内毒素刺激诱导曲线 将进行小鼠和核连续分析，以询问衰老是否会改变两者 老年人AGP基因转录的组成性和脂多糖诱导水平 老鼠。初步研究表明，小鼠AGP启动子有两个 反式作用因子结合位点，即B区(-104至-91)和区 C(-125到-104)，并且这些因子的结合活性可能是 在年老的动物身上发生改变。此外，结合活性的蛋白质 这些因素可能会在老年动物身上发生变化。此外，这些蛋白质 与C区结合的已被鉴定为C/EBPalpha(构成)和 C/EBPbeta(脂多糖诱导)。确定AGP的反式作用因子 启动子在老年动物中发生改变，这些因子的活性将被 通过DNase I(体外)和体内足迹测定，以及凝胶- Shift/Scatchard图分析。将进行实验，以询问是否 这些反式作用因子中与年龄相关的结构变化是 它们的结合活性改变的基础。这些实验包括 蛋白质的磷酸化修饰和同源/异源二聚体功能。 将分析反式作用因子的mRNA和蛋白质水平以确定 如果衰老影响了他们基因的表达。体外转录 还将开发检测系统，以便进行功能检测 反式作用因子。我们的长期目标是确定基因如何 转录或转录后水平的调控是 本质上受衰老的影响。