Nitric oxide regulation of CD55 and infection

一氧化氮对 CD55 和感染的调节

• 批准号: 8794626
• 负责人: CHANDRASEKHAR YALLAMPALLI
• 金额: $ 11.67万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-15 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8794626
• 关键词: Nitric; oxide; regulation; CD55; infection

PROJECT SUMMARY/ABSTRACT Nitric oxide (NO) and its reactive derivatives are widely known for regulating vascular tone. We have demonstrated that NO is a modulator of uterine infections due to Escherichia coli expressing Dr fimbriae (Dr+). E. coli through its Dr+ binds to decay-accelerating factor (CD55), a complement regulatory protein that protects cells from autologous complement-mediated damage. Elevated NO significantly decreased CD55 protein and mRNA in endometrial Ishikawa cells in a time and dose-dependent manner and consequently reduced bacterial invasion. We, therefore, hypothesize that NO inhibits CD55 expression and cellular distribution through direct actions at transcriptional level (Aim 1), indirectly through the modulation of sphingolipid metabolism and, thus altering P13K/Akt path-way (Aim 2) or locally at the membrane through altering the components of lipid rafts and their distribution (Aim 3). Further, NO modulates CD55 expression in rat uterus similar to the cell lines. These hypotheses will be tested in three specific aims. AIM 1. We will investigate the effect of NO on the transcription of CD55 DNA and on the stability of CD55 mRNA. 1.1. We will determine, by site directed mutations, the role of CREB, AP1, AP2, and SP1 binding sites at -74 to -43 region of CD55 promoter in the NO modulated transcription of CD55. 1.2. We will examine the effect of NO on CD55 mRNA stability by using actinomycin D assay. AIM 2. We will determine the involvement of sphingolipid metabolism and PI3K/Akt pathway signaling in NO-induced downregulation of CD55 expression. We will assess if NO 2.1. inhibits generation of ceramide in endometrial cells through decreasing hydrolysis of sphingomyelin, 2.2. alters intracellular distribution of ceramiide. 2.3. causes activation of PI3K/Akt pathway in down-regulating CD55 expression. AIM 3. We will examine the local effects of NO on the interaction of CD55 molecules with the components of lipid rafts in the membrane and their distribution. We will determine if NO 3.1. increases distance between lipid raft-associated molecules: GPI anchored CD55 and caveolin-1 and increase CD55 lateral mobility in the plasma membrane, 3.2. will alter dissociation of CD55 molecule from annexin II and actin cytoskeleton. AIM 4. We will demonstrate the effects of NO manipulations in vivo and ex-vivo on CD55 expression in the rat uterus, and assess the mechanisms of action. 4.1. We will determine the effects of in vivo manipulation of NO and its effects on the CD55 expression, PI3K/Akt pathway activation and ceramide levels in the uterus of rats. 4.2. We will investigate the in vitro effects of NO modifiers on CD55 expression in the rat uterus, and the involvement of PI3K/Akt pathway and ceramide in this process. 4.3. We will investigate the effects of NO modifiers on CD55 expression in human endometriosis and the involvement of PI3K/Akt pathway and ceramide in this process. These studies will provide mechanistic insights into the NO-induced down-regulation of CD55 expression and implications for implantation failure, pregnancy loss and severity of infection.

项目概要/摘要 一氧化氮 (NO) 及其活性衍生物因调节血管张力而广为人知。我们有 证明 NO 是表达 Dr fimbriae (Dr+) 的大肠杆菌引起的子宫感染的调节剂。 大肠杆菌通过其 Dr+ 与腐烂加速因子 (CD55) 结合，这是一种补体调节蛋白，可保护 细胞免受自体补体介导的损伤。 NO 升高显着降低 CD55 蛋白和 子宫内膜石川细胞中的 mRNA 以时间和剂量依赖性方式减少 细菌入侵。因此，我们假设 NO 抑制 CD55 表达和细胞分布 通过转录水平的直接作用（目标 1），通过鞘脂的调节间接作用 代谢，从而改变 P13K/Akt 途径（目标 2）或通过改变局部在膜上 脂筏的组成及其分布（目标 3）。此外，NO 调节 CD55 的表达 大鼠子宫细胞系相似。这些假设将在三个具体目标中得到检验。目标 1. 我们将 研究 NO 对 CD55 DNA 转录和 CD55 mRNA 稳定性的影响。 1.1. 我们将通过定点突变确定 -74 至 -43 处 CREB、AP1、AP2 和 SP1 结合位点的作用 NO 调节 CD55 转录中的 CD55 启动子区域。 1.2.我们将研究 NO 对 使用放线菌素 D 测定 CD55 mRNA 稳定性。目标 2. 我们将确定以下人员的参与 NO 诱导的 CD55 下调中的鞘脂代谢和 PI3K/Akt 通路信号传导 表达。我们将评估是否为“否”2.1。通过减少子宫内膜细胞中神经酰胺的生成 鞘磷脂的水解，2.2。改变神经酰胺的细胞内分布。 2.3.导致 PI3K/Akt 激活 下调 CD55 表达的途径。目标 3. 我们将研究 NO 对局部的影响 CD55分子与膜中脂筏成分的相互作用及其 分配。我们将确定是否为NO 3.1。增加脂筏相关分子之间的距离：GPI 锚定 CD55 和 Caveolin-1 并增加 CD55 在质膜中的横向移动性，3.2。会改变 CD55 分子从膜联蛋白 II 和肌动蛋白细胞骨架上解离。目标 4. 我们将展示 体内和离体 NO 操作对大鼠子宫中 CD55 表达的影响，并评估 行动机制。 4.1.我们将确定 NO 体内操作的效果及其对 大鼠子宫中 CD55 表达、PI3K/Akt 通路激活和神经酰胺水平。 4.2.我们将 研究NO修饰剂对大鼠子宫CD55表达的体外影响，以及参与 此过程中的 PI3K/Akt 途径和神经酰胺。 4.3.我们将研究 NO 修饰剂对 CD55 的影响 人类子宫内膜异位症中的表达以及 PI3K/Akt 途径和神经酰胺在此过程中的参与。 这些研究将为 NO 诱导的 CD55 下调提供机制见解 表达及其对着床失败、妊娠丢失和感染严重程度的影响。

项目成果

Dra/AfaE adhesin of uropathogenic Dr/Afa+ Escherichia coli mediates mortality in pregnant rats.

尿路致病性 Dr/Afa 大肠杆菌的 Dra/AfaE 粘附素介导妊娠大鼠的死亡率。

• DOI: 10.1128/iai.73.11.7597-7601.2005
• 发表时间: 2005
• 期刊: Infection and immunity
• 影响因子: 3.1
• 作者: Wroblewska-Seniuk,K;Selvarangan,R;Hart,A;Pladzyk,R;Goluszko,P;Jafari,A;duMerle,L;Nowicki,S;Yallampalli,C;LeBouguénec,C;Nowicki,B
• 通讯作者: Nowicki,B