5-HT/cAMP/Egr-1级联介导咖啡因抑制胎海马11β-HSD-2表达的转录调控机制

。A newly published review in Front Neuroendocrinol (2011) make clear that 11β-hydroxysteroid dehydrogenase 2 (11β-HSD-2) in fetal brain, which is responsible for glucocorticoid (GC) inactivation, plays an important role in mediating the GC programmed hypothalamic-pituitary-adrenal (HPA) axis related adult disease in the early life. However, no enough attention has been paid to studying the regulation and function of 11β-HSD-2 in the fetal brain. Our previous studies indicated that prenatal caffeine exposure can significantly inhibit the expression of hippocampal 11β-HSD-2 in fetal rats, which further stimulate the expressions of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD-1) and glucocorticoid receptor (GR) via a positive feedback loop regulation and cause GC metabolic activation in fetal hippocampus, all of these have been a big cause for the caffeine induced HPA axis related neuroendocrine metabolic programming alteration and adult disease (eg. fatty liver). The expression of 11β-HSD-2 is regulated at transcriptional stage. Some references indicated that the specificity protein 1 (Sp1) as well as early growth response protein 1 (Egr-1) are both important transcription factors of 11β-HSD-2; Sp1 can upregulate the expression of 11β-HSD-2 by recruiting the transcriptional coactivator p300 and altering the histone modifications after binding to the promoter region of 11β-HSD-2; while Egr-1 can occupy the binding sites of Sp1 and antagonism its effect; the expression of Egr-1 in hippocampus is regulated by serotonin (5-HT) induced ayclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway; caffeine can increase the level of 5-HT in brains and decrease the hydrolysis of cAMP by inbibiting the phosphodiesterase. Here from we put forward the research hypothesis, that prenatal caffeine exposure can reduce the binding of Sp1 as well as recruiting p300 to the promoter of 11β-HSD-2 in fetal hippocampus by activating the 5-HT/5-HT receptor and cAMP/PKA pathway and increasing Egr-1 expression, which in turn alters histone modifications of 11β-HSD-2 and inhibits its expression. All of these will further increase the level of active GC and promote the GC metabolic activity and GR expression in fetal hippocampus, which ultimately causes HPA axis related neuroendocrine metabolic programming alteration. Our goals for this project were to explore the direct links with respect to "caffeine - 5-HT/5-HT7 receptor - cAMP/PKA - Egr-1 - interactions of Egr-1-11β-HSD-2 and Sp1-11β-HSD-2 promoter - histone modifications of 11β-HSD-2 - expression of 11β-HSD-2" both in vivo and in vitro studies, and further to clarify the transcriptional regulation mechanism for the caffeine induced inhibition of 11β-HSD-2 mediated by the activated 5-HT/cAMP/Egr-1 cascade in fetal hippocampus. This project will be beneficial in expanded understanding the mechanism of caffeine induced developmental toxicity as well as elucidating the fetal origin of HPA axis related adult disease.

新近文献指出，胎脑组织2型11β羟类固醇脱氢酶(11β-HSD-2)在介导生命早期糖皮质激素编程下丘脑-垂体-肾上腺(HPA)轴相关成年疾病中发挥了重要作用。但目前对胎脑11β-HSD-2的表达调控机制及功能并不清楚。申请人前期发现，咖啡因可抑制胎海马11β-HSD-2表达，进一步通过高表达糖皮质激素受体引起HPA轴宫内编程改变。综合文献我们推测：咖啡因通过增加胎海马5-羟色胺(5-HT)水平，促进cAMP/PKA通路活化和早期生长反应因子1(Egr-1)表达，引起11β-HSD-2的组蛋白修饰改变和表达抑制。本项目拟在整体动物水平证实孕期咖啡因暴露引起胎海马5-HT/5-HT7R、cAMP/PKA/Egr-1通路、11β-HSD-2表达变化的基础上，进一步在细胞和分子水平确证5-HT/cAMP/Egr-1级联介导咖啡因抑制11β-HSD-2表达的转录调控机制，阐明咖啡因的发育毒性机制。

胎脑组织2型11β羟类固醇脱氢酶(11β-HSD-2)在介导生命早期糖皮质激素（GC）编程下丘脑-垂体-肾上腺轴相关成年疾病中发挥了重要作用，但对胎脑11β-HSD-2的表达调控及相关功能研究并不清楚。本项目在建立的孕期咖啡因暴露致子代大鼠宫内发育迟缓模型上，发现孕期咖啡因暴露可抑制胎盘11β-HSD-2表达，引起胎儿“母源性GC”过暴露；同时胎海马11β-HSD-2表达抑制并伴随启动子区组蛋白H3K4me3、H3K9me2和H3K27me3修饰改变；这些可激活海马局部GC代谢系统（11βHSDs/GR cEBPs）引起海马GC代谢活化改变，并通过抑制胰岛素样生长因子1（IGF1）信号通路引起海马发育损伤，且可遗传到出生后至成年。进一步，在体外培养的原代胎海马神经元和/或胎海马H19-7细胞系中，我们发现咖啡因及皮质酮均可直接抑制胎海马11β-HSD-2表达，同时伴随海马11β-HSD-2启动子区-358~77bp高甲基化改变，且糖皮质激素受体（GR）拮抗剂米非司酮可完全逆转皮质酮所致的胎海马11β-HSD-2表达抑制、GC代谢系统（11βHSDs/GR cEBPs）活化、IGF1信号通路抑制及海马发育损伤。我们认为，孕期咖啡因暴露所致胎儿“母源性高GC”可通过激活“11βHSDs/GR cEBPs”系统介导海马局部GC代谢活化和功能损伤，咖啡因可进一步通过表观遗传修饰机制（包括DNA甲基化和组蛋白修饰改变）直接抑制胎海马11β-HSD-2表达，由此加重海马局部GC代谢活化及相关损伤改变。本研究为解析孕期咖啡因暴露调控胎海马11β-HSD-2表达的分子机制及相关功能改变，阐明咖啡因发育毒性机制提供了实验依据。本项目在国际药理与毒理学权威杂志Toxicology、Toxicol Appl Pharmacol、Plos One、Arch Med Res上发表SCI论文4篇，发表中文核心2篇，待发表SCI论文2篇。