Nitric oxide regulation of CD55 and infection

一氧化氮对 CD55 和感染的调节

• 批准号: 7759623
• 负责人: CHANDRASEKHAR YALLAMPALLI
• 金额: $ 31.02万
• 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/7759623
• 关键词: 3' Untranslated Regions; ANXA2 gene; Actins; Affect; Animals; 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; Conceptions; Conceptus; 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; public health relevance; receptor; reproductive; trafficking; transcription factor

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.

描述（由申请人提供）：一氧化氮（NO）及其活性衍生物广泛用于调节血管张力。我们已经证明，NO是子宫感染由于大肠杆菌表达博士菌毛（博士+）的调制器。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，可保护细胞免受自体补体介导的损伤。NO浓度升高可显著降低子宫内膜石川细胞中CD 55蛋白和mRNA的表达，并呈时间和剂量依赖性，从而减少细菌的侵袭。因此，我们推测NO通过直接作用于转录水平（Aim 1），间接通过调节鞘脂代谢，从而改变P13 K/Akt通路（Aim 2）或通过改变脂筏组分及其分布（Aim 3）在膜上局部抑制CD 55表达和细胞分布。此外，NO调节大鼠子宫中的CD 55表达类似于细胞系。这些假设将在三个具体目标中得到检验。AIM 1.本研究旨在探讨NO对CD 55 DNA转录和CD 55 mRNA稳定性的影响。1.1.我们将通过定点突变来确定CD 55启动子-74至-43区域的CREB、AP 1、AP 2和SP 1结合位点在NO调节的CD 55转录中的作用。1.2.我们将通过放线菌素D测定来检测NO对CD 55 mRNA稳定性的影响。AIM 2.我们将确定鞘脂代谢和PI 3 K/Akt通路信号转导在NO诱导的CD 55表达下调中的参与。我们将评估如果没有2.1.通过减少鞘磷脂的水解抑制子宫内膜细胞中神经酰胺的产生，2.2.改变神经酰胺的细胞内分布。2.3.导致PI 3 K/Akt通路激活，下调CD 55表达。AIM 3.我们将研究NO对CD 55分子与膜中脂筏组分相互作用及其分布的局部影响。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锚定的CD 55和小窝蛋白-1，并增加CD 55在质膜中的横向迁移率，3.2。将改变CD 55分子与膜联蛋白II和肌动蛋白细胞骨架的解离。AIM 4.我们将证明在体内和离体NO操作对大鼠子宫中CD 55表达的影响，并评估其作用机制。4.1.我们将确定体内操作NO的影响及其对大鼠子宫中CD 55表达、PI 3 K/Akt通路活化和神经酰胺水平的影响。4.2.我们将研究NO调节剂对大鼠子宫CD 55表达的体外影响，以及PI 3 K/Akt通路和神经酰胺在此过程中的参与。4.3.我们将研究NO调节剂对人子宫内膜异位症中CD 55表达的影响以及PI 3 K/Akt通路和神经酰胺在此过程中的参与。这些研究将为NO诱导的CD 55表达下调以及对着床失败、妊娠丢失和感染严重程度的影响提供机制性见解。公共卫生相关性：我们推测NO通过直接在转录水平上作用，间接通过调节鞘脂代谢，从而改变P13 K/Akt通路，或通过改变脂筏组分及其分布在膜局部抑制CD 55表达和细胞分布。