Nitric oxide regulation of CD55 and infection

一氧化氮对 CD55 和感染的调节

• 批准号: 8004069
• 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/8004069
• 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; Health; 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

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）及其活性衍生物被广泛认为具有调节血管张力的作用。我们已经证明NO是大肠杆菌表达Dr菌膜（Dr+）引起的子宫感染的调节剂。大肠杆菌通过Dr+结合衰变加速因子（CD55）， 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。NO升高可显著降低子宫内膜石川细胞CD55蛋白和mRNA表达，并呈时间和剂量依赖性，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（Aim 1），间接通过调节鞘脂代谢，从而改变P13K/Akt通路（Aim 2）或通过改变脂筏成分及其分布在膜上的局部作用（Aim 3）来抑制CD55的表达和细胞分布。此外，NO调节CD55在大鼠子宫中的表达类似于细胞系。这些假设将在三个具体目标中得到检验。目的1。我们将研究NO对CD55 DNA转录和CD55 mRNA稳定性的影响。1.1. 我们将通过位点定向突变来确定CD55启动子-74至-43区域的CREB、AP1、AP2和SP1结合位点在NO调节CD55转录中的作用。1.2. 我们将通过放线菌素D法检测NO对CD55 mRNA稳定性的影响。目标2。我们将确定鞘脂代谢和PI3K/Akt通路信号在no诱导的CD55表达下调中的作用。我们将评估第2.1条。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。改变神经酰胺的细胞内分布。2.3. 激活PI3K/Akt通路下调CD55表达。目标3。我们将研究NO对CD55分子与膜中脂筏组分相互作用及其分布的局部影响。我们将决定是否NO . 3.1。增加脂筏相关分子之间的距离：GPI锚定CD55和caveolin-1，增加CD55在质膜中的横向迁移，3.2。会改变CD55分子与膜联蛋白II和肌动蛋白细胞骨架的分离。目标4。我们将展示NO在体内和离体处理对大鼠子宫CD55表达的影响，并评估其作用机制。4.1. 我们将研究NO在体内对大鼠子宫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. 我们将探讨NO修饰剂对人子宫内膜异位症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。这些研究将提供no诱导的CD55表达下调的机制，以及对着床失败、妊娠丢失和感染严重程度的影响。公共卫生相关性：我们假设NO通过直接作用在转录水平上抑制CD55表达和细胞分布，间接通过调节鞘脂代谢，从而改变P13K/Akt通路，或通过改变脂筏成分及其分布在膜上局部抑制CD55表达和细胞分布。