Mechanisms of Glucocorticoid Resistance in Prenatal Alcohol Exposure

项目摘要 胎儿期酒精暴露(PAE)影响的许多生理过程受 糖皮质激素(GC)。GC抗性(即对GC作用的敏感度降低)，可能是由于 宫内糖皮质激素编程异常，与多种慢性病有关，许多 它们与免疫功能有关，还有PAE。我们的目标是确定酒精介导的分子机制。 发育中胎儿对糖皮质激素敏感性编程的改变，并追踪这些变化 进入成年期。我们将利用这些信息制定有针对性的干预措施，逆转或减少影响 PAE对免疫信号分子GC敏感性和后续反应的影响。这项提案将考验 假设PAE修改了长的非编码RNA，生长停滞特异的5(Gas5)，它作为一个 糖皮质激素受体(GR)诱饵调节GR介导的基因表达。 我们的假设有两个：1)PAE产生糖皮质激素抵抗，表现为：a.) 下丘脑垂体(HPA)轴调节失调和促炎与抗炎的增加 应激条件下的细胞因子比率，b。正常应激和免疫的关键发育转变 出生后早期反应迟钝和c.)GRα/GRβ比率下降。2.)这是不适应的 糖皮质激素抵抗是由于胎脑Gas5和Gas5升高而在胎脑中编程产生的 成年期通过增加FK506结合蛋白-51(FKBP51)水平维持。 我们将使用我们建立的PAE小鼠模型和三个特定的研究设计来验证这些假设： 目标1。)PAE产生可测量的糖皮质激素抵抗增加：我们将从生理上证实 通过评估HPA对成年雄性和雌性PAE和糖精(SAC)对照组小鼠的相关GC抵抗 反应性、促炎性和抗炎性细胞因子/趋化因子蛋白水平和特异性GR调节水平 应激激活反应的基因转录产物(包括细胞因子/趋化因子)。GC耐药性也将是 通过GRα/GRβ比值以及FKPB5甲基化水平的测量进行评估。 目标2。)PAE影响GC反应的发育规划：我们将确定 使用母体分离对免疫期和应激低反应期的PAE。百帕轴线 反应性、额叶皮质细胞因子/趋化因子水平、FKBP51蛋白和Gas5 RNA表达以及 它们与GR的关联将被确定。我们还将测量GR亚型GRα的相对表达 和GRβ。 目标3。)抑制或减少胎儿期Gas5应能恢复PAE小鼠正常的GC敏感性。 针对Gas-5或shRNA的GRE结合区的LNA-寡核苷酸靶点阻断剂的传递 胚胎期介导的Gas5基因敲除将恢复正常的GC反应和敏感性 (包括应激反应、免疫信号分子和核GR调节基因)。

Project Summary Many of the physiological processes affected by prenatal alcohol exposure (PAE) are regulated by glucocorticoids (GCs). GC resistance (i.e., reduced sensitivity to the actions of GCs), which may result from aberrant in utero glucocorticoid programming, is associated with both a variety of chronic diseases, many of which are immune function related, and PAE. Our goal is to identify molecular mechanisms of alcohol-mediated alterations in the programming of glucocorticoid sensitivity in the developing fetus, and to track these changes into adulthood. We will use this information to develop targeted interventions that reverse or reduce the effects of PAE on GC sensitivity and consequent responses of immune signaling molecules. This proposal will test the hypothesis that PAE modifies the long noncoding RNA, Growth arrest-specific 5 (Gas5), which acts as a glucocorticoid receptor (GR) decoy to regulate GR-mediated gene expression. Our hypotheses are two-fold: 1.) PAE produces glucocorticoid resistance that is expressed as: a.) dysregulation of the hypothalamic pituitary (HPA) axis and an increase in pro-inflammatory to anti-inflammatory cytokine ratio under stressful conditions, b.) a critical developmental shift in the normal stress and immune hyporesponsive early postnatal periods and c.) a decrease in the GRα/GRβ ratio. 2.) This maladaptive glucocorticoid resistance is programmed in the fetal brain as a result of an elevation in fetal brain Gas5 and maintained in adulthood by increases in FK506-bindinig protein-51 (FKBP51) levels. We will test these hypotheses using our established mouse model of PAE and three specific study designs: Aim 1.) PAE produces a measurable increase in glucocorticoid resistance: We will confirm physiologically relevant GC resistance in adult male and female PAE and saccharin (SAC) control mice by assessing HPA responding, pro- and anti-inflammatory cytokine/chemokine protein levels and levels of specific GR-regulated gene transcripts (including cytokines/chemokines) in response to stress activation. GC resistance will also be assessed by the ratio of GRα/GRβ, as well as measures of the levels of FKPB5 methylation. Aim 2.) PAE affects the developmental programming of GC responding: We will determine the impact of PAE on both the immune and stress hyporesponsive periods using maternal separation. HPA axis responsiveness, levels of frontal cortical cytokines/chemokines, FKBP51 protein and Gas5 RNA expression and their associations with GR will be determined. We will also measure the relative expression of GR isoforms GRα and GRβ. Aim 3.) Inhibition or reduction of prenatal Gas5 should restore normal GC sensitivity in the PAE mice. Delivery of an LNA-oligonucleotide target site blocker directed at the GRE binding region of Gas-5 or shRNA mediated gas5 knockdown during the embryonic period will restore normal GC responding and sensitivity (inclusive of stress responses, immune signaling molecules and nuclear GR-regulated genes) in adult PAE mice.