Nitric oxide regulation of CD55 and infection

一氧化氮对 CD55 和感染的调节

• 批准号: 8206846
• 负责人: CHANDRASEKHAR YALLAMPALLI
• 金额: $ 29.78万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 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 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8206846
• 关键词: 3' Untranslated Regions; ANXA2 gene; Actins; Affect; Antiphospholipid Antibodies; Antiphospholipid Syndrome; Autologous; Bacteremia; Binding; Binding Sites; Biological Assay; Biological Availability; Blood Vessels; CD55 Antigens; CREB1 gene; Cell Line; Cell membrane; Cells; Ceramides; Cholesterol; Complement; Cyclic GMP; Cytoskeleton; DNA; Dactinomycin; Data; Disease; Dissociation; Dose; Down-Regulation; Endometrial; Endometrium; Epithelial Cells; Escherichia coli; Generations; Genetic Transcription; Human; Hydrolysis; Immune Tolerance; Implant; In Vitro; Infection; Inositol; Lateral; Lipids; Maternal Mortality; Mediating; Membrane; Membrane Microdomains; Messenger RNA; Metabolism; Modeling; Mutation; Names; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nuclear; Pathology; Pathway interactions; Patient observation; Phase; Phosphatidylinositols; Phosphotransferases; Play; Pre-Eclampsia; Pregnancy; Pregnancy loss; Premature Labor; Process; Production; Proteins; Publishing; Rat Cell Line; Rattus; Recurrence; Regulation; Reporter; Reporting; Role; Running; SP1 gene; Severities; Signal Pathway; Site; Sphingolipids; Sphingomyelins; Spontaneous abortion; Testing; Time; Tissues; Transcript; Transcription Factor AP-1; Transcription Factor AP-2 Alpha; Transfection; Up-Regulation; Uterus; caveolin 1; endometriosis; failure Implantation; fimbria; genetic regulatory protein; implantation; in vivo; inhibitor/antagonist; insight; mRNA Stability; pregnant; promoter; receptor; reproductive; transcription factor

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.

项目摘要/摘要 一氧化氮（NO）及其活性衍生物以调节血管张力而闻名。我们有 证明NO是由于表达Fimbriae博士（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+结合与衰减加速因子（CD55）的结合，这是一种补体调节蛋白，可保护 自体补体介导的损伤的细胞。无明显降低的CD55蛋白质和 子宫内膜Ishikawa细胞中的mRNA以剂量依赖性方式，因此减少了 细菌入侵。因此，我们假设NO抑制CD55表达和细胞分布 通过转录级别的直接动作（AIM 1）间接通过鞘脂的调节 代谢，从而改变p13k/akt路径（AIM 2）或通过改变在膜上的局部 脂筏的成分及其分布（AIM 3）。此外，没有调节CD55的表达 大鼠子宫类似于细胞系。这些假设将以三个特定目的进行检验。目标1。我们将 研究NO对CD55 DNA转录和CD55 mRNA的稳定性的影响。 1.1。 我们将根据位点定向突变确定-74至-43的CREB，AP1，AP2和SP1结合位点的作用 CD55的无调制转录中CD55启动子的区域。 1.2。我们将检查NO的效果 CD55 mRNA稳定性通过使用放线菌素D分析。目标2。我们将确定 鞘脂代谢和PI3K/AKT途径在无诱导的CD55下调中的信号传导 表达。我们将评估是否2.1。通过减少来抑制子宫内膜细胞中神经酰胺的产生 鞘磷脂的水解，2.2。改变陶瓷的细胞内分布。 2.3。引起PI3K/AKT的激活 下调CD55表达的途径。 AIM 3。我们将检查NO的局部影响 CD55分子与膜中脂质筏的成分的相互作用及其 分配。我们将确定是否没有3.1。增加脂质筏相关分子之间的距离：GPI 锚定CD55和Caveolin-1并增加了质膜中CD55的横向迁移率，3.2。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。我们将证明 在体内和ex-Vivo中无操纵对大鼠子宫中CD55表达的影响，并评估 作用机理。 4.1。我们将确定对NO的体内操纵及其对该的影响的影响 CD55表达，PI3K/AKT途径激活和大鼠子宫中的神经酰胺水平。 4.2。我们将 研究无修饰符对大鼠子宫中CD55表达的体外影响，并参与 在此过程中，PI3K/AKT途径和神经酰胺。 4.3。我们将研究没有修饰符对CD55的影响 人子宫内膜异位症的表达以及PI3K/AKT途径和神经酰胺在此过程中的参与。 这些研究将提供有关CD55无引起的下调的机械见解 对植入失败，妊娠丧失和感染严重程度的表达和影响。