Mechanisms of Glucocorticoid Resistance in Prenatal Alcohol Exposure

项目摘要 许多受产前酒精暴露（PAE）影响的生理过程受到以下因素的调节： 糖皮质激素（GC）。GC抗性（即，对GC作用的敏感性降低），这可能是由于 子宫内糖皮质激素编程异常与多种慢性疾病有关， 与免疫功能相关的蛋白质和PAE。我们的目标是确定酒精介导的 发育中胎儿糖皮质激素敏感性编程的改变，并跟踪这些变化 长大成人我们将利用这些信息制定有针对性的干预措施，以扭转或减少影响 PAE对GC敏感性和随后的免疫信号分子反应的影响。这项提案将考验 假设PAE修饰了长的非编码RNA，生长停滞特异性5（Gas 5），它作为一种非编码RNA， 糖皮质激素受体（GR）诱饵调节GR介导的基因表达。 我们的假设是双重的：1）。PAE产生糖皮质激素抗性，其表示为：a.） 下丘脑-垂体（HPA）轴失调，促炎性细胞因子增加， 应激条件下的细胞因子比率，B.）正常压力和免疫系统的关键发育转变 出生后早期低反应期和c.）GRα/GRβ比值降低。2.）这种适应不良 由于胎儿脑Gas 5升高，糖皮质激素抵抗在胎儿脑中被编程， 在成年期通过FK 506-结合蛋白-51（FKBP 51）水平的增加来维持。 我们将使用我们建立的PAE小鼠模型和三种特定的研究设计来测试这些假设： 目标1.）PAE产生可测量的糖皮质激素抵抗增加：我们将从生理学上证实 通过评估HPA在成年雄性和雌性PAE和糖精（SAC）对照小鼠中的相关GC抗性 响应、促炎和抗炎细胞因子/趋化因子蛋白水平和特定GR调节的水平 基因转录物（包括细胞因子/趋化因子）响应于应激激活。GC抗性也将是 通过GRα/GRβ比值以及FKPB 5甲基化水平的测量进行评估。 目标2）PAE影响GC反应的发育编程：我们将确定 使用母体分离对免疫和应激低反应期进行PAE。HPA轴 反应性、额叶皮质细胞因子/趋化因子水平、FKBP 51蛋白和Gas 5 RNA表达以及 将确定它们与GR的关联。我们还将测量GR亚型GRα的相对表达， GRβ。 目标3）产前Gas 5的抑制或减少应恢复PAE小鼠的正常GC敏感性。 靶向Gas-5或shRNA的GRE结合区的LNA-寡核苷酸靶位点阻断剂的递送 在胚胎期介导的gas 5敲低将恢复正常的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）在成年PAE小鼠。

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.