Nitric oxide regulation of CD55 and infection

项目摘要/摘要 一氧化氮(NO)及其活性衍生物是众所周知的调节血管张力的物质。我们有 证明NO是由表达DR菌毛(DR)的大肠杆菌引起的子宫感染的调节因子。 大肠杆菌通过其DR与衰变加速因子(CD55)结合，CD55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。升高的NO显著降低CD55蛋白和 子宫内膜Ishikawa细胞的mRNA以时间和剂量依赖的方式表达，并因此减少 细菌入侵。因此，我们假设NO抑制CD55的表达和细胞分布 通过转录水平的直接作用(目标1)，间接通过鞘糖脂的调节 代谢，从而改变P13K/Akt途径(目标2)或通过改变局部在膜上 脂筏的成分及其分布(目标3)。此外，NO对CD55的表达具有调节作用。 大鼠子宫与细胞系相似。这些假设将在三个具体目标上得到检验。目标1.我们将 探讨NO对CD55DNA转录及CD55mRNA稳定性的影响。1.1. 我们将通过定点突变来确定CREB、AP1、AP2和SP1结合位点在-74到-43处的作用 CD55启动子区域在NO调控CD55转录中的作用。1.2.我们将研究NO对人类健康的影响 放线菌素D法检测CD55mRNA的稳定性。目标2.我们将确定 鞘磷脂代谢和PI3K/Akt信号通路在NO诱导的CD55下调中的作用 表情。我们将评估是否没有2.1。抑制子宫内膜细胞神经酰胺的生成 神经鞘磷脂的水解度，2.2。改变神经酰胺在细胞内的分布。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在局部地区对 CD55分子与膜上脂筏成分的相互作用及其机制 分发。我们将确定是否3.1。增加脂筏相关分子之间的距离：GPI 锚定CD55和小窝蛋白-1并增加CD55在质膜的横向迁移率，3.2。将会改变 CD55分子与膜联蛋白II和肌动蛋白细胞骨架的解离。目标4。我们将演示 体内、外NO处理对大鼠子宫CD55表达的影响 行动机制。4.1.我们将确定体内操纵一氧化氮的效果及其对血管生成的影响。 大鼠子宫CD55表达、PI3K/Akt通路激活及神经酰胺水平。4.2.我们会 探讨一氧化氮修饰剂对大鼠子宫CD55表达的影响及其在其中的作用 PI3K/Akt通路和神经酰胺在此过程中起重要作用。4.3.我们将研究NO修饰物对CD55的影响 PI3K/Akt通路和神经酰胺在子宫内膜异位症中的表达及意义 这些研究将为NO诱导的CD55下调提供机械性的见解 表达和对植入失败、妊娠丢失和感染严重程度的影响。

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.