EPIGENETIC REGULATION OF INTESTINAL Na+/H+ EXCHANGER-3

 描述（由申请人提供）： 腹泻是由食源性病原体引起的炎症性肠病（IBD）或肠道感染的主要症状。我们的退伍军人患者群体由于年龄和许多合并症而特别容易受到创伤性疾病的影响。尽管医学取得了重大进展，但牙周病的治疗仍然具有挑战性。因此，重要的是要了解与这些疾病相关的腹泻的病理生理机制。腹泻是由于肠分泌增加和/或水和电解质吸收减少所致。人体肠道中电解质吸收的主要途径涉及Na+/H+（NHE）和Cl-/HCO 3-交换剂的耦合操作。研究表明，NHE 3在介导肠钠吸收中起关键作用，因为NHE 3敲除小鼠表现出结肠炎表型。此外，已显示NHE 3 KO小鼠更易受炎症影响，因为这些小鼠中的NHE 3缺乏导致与结肠炎相关的腹泻。然而，迄今为止，很少有人知道的分子机制参与减少NHE 3的表达在牙周炎疾病。为了阐明IBD相关腹泻中NHE 3表达下调的机制，我们目前的研究集中在表观遗传机制的作用，如DNA甲基化和组蛋白修饰，这些机制与IBD的发病机制有关。我们广泛的初步数据提供了强有力的证据，通过DNA甲基化和组蛋白修饰的变化NHE 3表达的表观遗传调控。基于这些数据，我们假设DNA甲基化、组蛋白修饰和染色质重塑的变化在调节腹泻病理生理学基础的NHE 3基因表达中起重要作用。因此，本申请被设计为利用体外（目的1和2）和体内模型（目的3）来研究通过DNA甲基化和组蛋白修饰对NHE 3基因表达的调节，如下所述：详细阐明DNA甲基化对NHE 3启动子活性的影响，鉴定参与NHE 3启动子调控的特异性CpG二核苷酸，确定DNA甲基转移酶（DNMT）亚型的作用;目的2：研究组蛋白乙酰转移酶、p300和组蛋白脱乙酰酶（HDAC）同种型在NHE 3表达调节中的作用，NHE 3基因上组蛋白乙酰化标记的变化和刺激NHE 3表达中的染色质重塑;以及目的3：利用野生型、DSS诱导的结肠炎和NHE 3敲除小鼠（结肠炎表型），在正常和炎症条件下检查表观遗传机制对NHE 3表达的调节。这些研究的结果将为NHE 3的表观遗传调控提供新的见解，并确定NHE 3基因的DNA甲基化/组蛋白修饰改变与IBD相关腹泻之间的重要机制联系。此外，这些研究将首次确定参与神经系统疾病中NHE 3失调的特定DNMT或HDAC同种型的身份。

 DESCRIPTION (provided by applicant): Diarrhea is a predominant symptom of inflammatory bowel diseases (IBD) or intestinal infections caused by food-borne pathogens. Our veteran patient population is particularly more vulnerable to diarrheal illnesses due to their age and numerous co-morbidities. In spite of significant medical advances, the treatment of diarrheal disorders still remains challenging. Therefore, it is important to understand the mechanisms involved in the pathophysiology of diarrhea associated with these diseases. Diarrhea results from increased intestinal secretion and/or decreased absorption of water and electrolytes. A major route of electrolyte absorption in the human intestine involves coupled operation of Na+/H+ (NHE) and Cl-/HCO3- exchangers. Studies have shown NHE3 to play a critical role in mediating intestinal sodium absorption as NHE3 knockout mice exhibit diarrheal phenotype. Also, it has been shown that NHE3 KO mice are more susceptible to inflammation as NHE3 deficiency in these mice resulted in diarrhea associated with colitis. To date, however, very little is known about the molecular mechanisms involved in decreasing NHE3 expression in diarrheal disorders. In order to elucidate the mechanisms underlying the down-regulation of NHE3 expression in IBD-related diarrhea, our current studies are focused on the role of epigenetic mechanisms such as DNA methylation and histone modifications shown to be implicated in the pathogenesis of IBD. Our extensive preliminary data provides strong evidence for the epigenetic regulation of NHE3 expression by changes in DNA methylation and histone modifications. Based on these data, we hypothesize that changes in DNA methylation, histone modifications and chromatin remodeling play important roles in modulating NHE3 gene expression that underlie the pathophysiology of diarrhea. The current application is, therefore, designed to investigate the regulation of NHE3 gene expression by DNA methylation and histone modifications utilizing both in vitro (Aims 1 & 2) and in vivo models (Aim 3) as follows: Aim 1. Elucidate in detail the effect of DNA methylation on NHE3 promoter activity, identify the specific CpG dinucleotide involved in the modulation of NHE3 promoter and determine the roles of DNA methyltransferase (DNMT) isoforms; Aim 2: Investigate the role of histone acetyl transferase, p300 & histone deacetylase (HDAC) isoforms in the modulation of NHE3 expression, changes in histone acetylation marks on NHE3 gene and chromatin remodeling in the stimulation of NHE3 expression; and Aim 3: Examine the regulation of NHE3 expression by epigenetic mechanisms under normal and inflammatory conditions utilizing wild type, DSS-induced colitis and NHE3 knockout mice (diarrheal phenotype). The outcome of these studies should provide novel insights into the epigenetic regulation of NHE3 and define important mechanistic link between alterations in DNA methylation/histone modifications of NHE3 gene and diarrhea associated with IBD. Also, these studies will establish for the first time the identity of a particular DNMT or HDAC isoforms involved in the dysregulation of NHE3 in diarrheal disorders.