Nitric oxide regulation of CD55 and infection

项目概要/摘要 一氧化氮（NO）及其活性衍生物被广泛认为是调节血管张力的物质。我们有 证明NO是由于表达Dr菌毛（Dr+）的大肠杆菌引起的子宫感染的调节剂。 E.大肠杆菌通过其Dr+与衰变加速因子（CD 55）结合，这是一种补体调节蛋白， 自体补体介导的损伤。NO升高可显著降低CD 55蛋白表达， mRNA在子宫内膜石川细胞中呈时间和剂量依赖性， 细菌入侵因此，我们假设NO抑制CD 55的表达和细胞分布， 通过在转录水平上的直接作用（Aim 1），间接通过调节鞘脂 P13 K/Akt通路（Aim 2）或通过改变P13 K/Akt通路在细胞膜上的表达而局部改变P13 K/Akt通路。 脂筏的组成及其分布（目的3）。此外，NO调节CD 55表达， 类似于大鼠子宫的细胞系。这些假设将在三个具体目标中得到检验。AIM 1.我们将 探讨NO对CD 55 DNA转录及CD 55 mRNA稳定性的影响。1.1. 我们将通过定点突变确定CREB、AP 1、AP 2和SP 1结合位点在-74至-43位点的作用。 CD 55启动子区域在NO调节的CD 55转录中的作用。1.2.我们将研究NO对 使用放线菌素D测定的CD 55 mRNA稳定性。AIM 2.我们将确定 NO诱导CD 55下调中鞘脂代谢和PI 3 K/Akt信号通路 表情我们将评估如果没有2.1.抑制子宫内膜细胞神经酰胺的产生， 鞘磷脂的水解，2.2.改变神经酰胺的细胞内分布。2.3.导致PI 3 K/Akt激活 下调CD 55表达。AIM 3.我们将研究NO对细胞的局部影响。 CD 55分子与细胞膜脂筏组分的相互作用及其 分布我们将确定是否没有3.1.增加脂筏相关分子之间的距离：GPI 锚定CD 55和小窝蛋白-1，并增加CD 55在质膜中的横向移动性，3.2.将改变 CD 55分子从膜联蛋白II和肌动蛋白细胞骨架上解离。AIM 4.我们将展示 体内和离体NO操作对大鼠子宫中CD 55表达的影响，并评估 行动机制。4.1.我们将确定体内操作NO的效果及其对细胞增殖的影响。 大鼠子宫中CD 55表达、PI 3 K/Akt通路活化和神经酰胺水平。4.2.我们将 研究NO修饰剂对大鼠子宫中CD 55表达的体外作用，以及 PI 3 K/Akt通路和神经酰胺参与了这一过程。4.3.我们将研究NO修饰剂对CD 55的影响， PI 3 K/Akt通路和神经酰胺在子宫内膜异位症中的表达。 这些研究将为NO诱导的CD 55下调提供机制上的见解 表达和对着床失败、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.