REGULATION OF NA,K ATPASE BY THE AH RECEPTOR

人类在怀孕期间暴露于持久性环境污染物，如2，3，7，8-四氯二苯并-对二恶英(TCDD)和相关化学品，会导致出生体重下降、神经缺陷、甲状腺激素变化、肺部听诊和色素沉着。TCDD介导发育毒性的机制尚未阐明。基本的螺旋-环-螺旋-PAS转录因子，芳烃受体(AhR)，是TCDD诱导的小鼠致畸所必需的，并可能介导其他物种的致畸。TCDD毒性可能是由AhR引起的基因转录改变所致。可能解释TCDD某些致畸作用的一个潜在的AhR基因靶点是Na+/K+ATPaseα1。二恶英应答元件在哺乳动物和禽类Na+/K+ATPaseα1基因的5‘增强子区域保守。在鸡胚中，TCDD降低了心肌Na+/K+ATPaseα1蛋白的表达，诱导了扩张型心肌病，改变了ECG，所有这些都与Na+/K+ATPase活性降低相一致。在缺乏AhR的小鼠中，胚胎会发展成肥厚性心肌病和心肌纤维化，并随着年龄的增长而恶化，这与Na+/K+ATPaseα1的潜在过度表达和高血压的发展是一致的。我将使用鸡胚胎和AhR基因缺失的小鼠来验证AhR调节心肌Na+/K+ATPaseα1基因表达，从而改变心血管发育的假设。本研究的目的是：(1)通过RT-PCR和体外禽类基因启动子分析，阐明心肌Na~+/K~+-ATPaseα1基因在AhR基因缺失小鼠和发育中的鸡胚中的调控作用；(2)通过对AhR缺失小鼠和TCDD暴露的鸡胚心肌哇巴因结合部位和Na~+/K~+-ATPase活性的测定，确定这种调控的组织意义；(3)通过检测AhR缺失小鼠的血压和TCDD暴露的鸡胚心肌对哇巴因的敏感性来确定这种调控的功能意义；以及(4)对亚利桑那大学Selmin博士鉴定的TCDD可改变其在大鼠胚胎心脏和肺中表达的候选基因的小鼠和禽类同源基因的表达进行分析，采用PCR选择消减杂交方法和基因芯片的筛选。

Human exposure during pregnancy to persistent environmental pollutants, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related chemicals, results in decreased birth weights, neurological deficits, thyroid hormone alterations, lung auscultation, and hyperpigmentation. The mechanism by which TCDD mediates developmental toxicity has not been elucidated. The basic helix-loop-helix-PAS transcription factor, aryl hydrocarbon receptor (AhR), is required for TCDD-induced teratogenicity in mice and likely mediates teratogenicity in other species. TCDD toxicity may result from alterations in gene transcription by the AhR. One potential AhR gene target that could account for some of TCDD's teratogenic effects is the Na+/K+ ATPase alpha1. Putative dioxin response elements are conserved in the 5' enhancer region of the mammalian and avian Na+/K+ ATPase alpha1 gene. In the chick embryo, TCDD reduces myocardial Na+/K+ ATPase alpha1 protein expression, induces a dilated cardiomyopathy, and alters ECGs, all consistent with reduced Na+/K+ ATPase activity. In mice lacking the AhR, embryos develop a hypertrophic cardiomyopathy and cardiac fibrosis which worsens with age, consistent with the potential overexpression of Na+/K+ ATPase alpha1 and development of hypertension. I will use the chick embryo and AhR null mice to test the hypothesis that the AhR regulates myocardial expression of the Na+/K+ ATPase alpha1 gene, altering cardiovascular development. The aims of this proposal are to (1) elucidate the regulation of myocardial Na+/K+ ATPase alpha1 gene in AhR null mice and in the developing chick embryo by TCDD using RT-PCR, and in vitro by promoter analysis of the avian gene; (2) determine the tissue significance of this regulation by quantitating myocardial ouabain binding sites and Na+/K+ ATPase enzyme activity in AhR null mice and TCDD-exposed chick embryos; (3) determine the functional significance by measuring blood pressure in AhR null mice and myocardial sensitivity to ouabain in TCDD-exposed chick embryos by ECG; and (4) analyze the expression of murine and avian homologues of candidates genes, whose expression is altered by TCDD in the rat embryo heart and lung as identified by Dr. Selmin, University of Arizona, by PCR-selected subtractive hybridization method and screening of gene microarrays.