Nitric oxide regulation of CD55 and infection

说明书(申请人提供)：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。我们已经证明，由于大肠杆菌表达DR菌毛(DR+)，NO是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，可以保护细胞免受自体补体介导的损伤。升高NO可明显降低子宫内膜Ishikawa细胞CD55蛋白和mRNA的表达，并呈时间和剂量依赖关系，从而减少细菌侵袭。因此，我们假设，NO通过在转录水平上的直接作用(目标1)、间接地通过调节鞘脂代谢从而改变P13K/Akt通路(目标2)或通过改变脂筏的成分及其分布而局部地在膜上抑制CD55的表达和细胞分布(目标3)。此外，与细胞系相似，NO对大鼠子宫中CD55的表达也有调节作用。这些假设将在三个具体目标上得到检验。目的1.探讨一氧化氮对CD55DNA转录和CD55mRNA稳定性的影响。1.1.我们将通过定点突变来确定CD55启动子-74到-43区域的CREB、AP1、AP2和SP1结合位点在CD55的NO调控转录中的作用。1.2.用放线菌素D法检测NO对CD55mRNA稳定性的影响。目的2.探讨鞘磷脂代谢和PI3K/Akt信号通路在NO诱导的CD55表达下调中的作用。我们将评估是否没有2.1。通过减少鞘磷脂的水解抑制子宫内膜细胞神经酰胺的产生，2.2。改变神经酰胺在细胞内的分布。2.3.通过激活PI3K/Akt途径下调CD55的表达。目的3.研究NO对CD55分子与膜上脂筏成分相互作用及其分布的局部影响。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在质膜中的横向迁移率，3.2。将改变CD55分子与膜联蛋白II和肌动蛋白细胞骨架的解离。目的4.研究体内、外一氧化氮对大鼠子宫CD55表达的影响，并探讨其作用机制。4.1.我们将确定体内操纵NO的效果及其对大鼠子宫CD55表达、PI3K/Akt通路激活和神经酰胺水平的影响。4.2.我们将在体外研究NO修饰剂对大鼠子宫CD55表达的影响，以及PI3K/Akt通路和神经酰胺在这一过程中的作用。4.3.我们将研究NO修饰物对人子宫内膜异位症中CD55表达的影响以及PI3K/Akt通路和神经酰胺在这一过程中的作用。这些研究将为NO诱导的CD55表达下调以及与植入失败、妊娠丢失和感染严重程度的关系提供机械性见解。与公共卫生相关：我们假设，NO通过在转录水平上的直接作用，间接地通过调节Spingolipid代谢从而改变P13K/Akt途径来抑制CD55的表达和细胞分布，或者局部地通过改变脂筏的组成和分布来抑制CD55的表达和细胞分布。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: We hypothesize that NO inhibits CD55 expression and cellular distribution through direct actions at the transcriptional level, indirectly through the modulation of spingolipid metabolism and, thus altering P13K/Akt pathway, or locally at the membrane through altering the components of lipid rafts and their distribution.