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

 描述(由申请人提供)： 腹泻是炎症性肠病(IBD)或由食源性病原体引起的肠道感染的主要症状。由于年龄和许多并存疾病，我们的资深患者群体特别容易患腹泻疾病。尽管医学取得了重大进展，但腹泻性疾病的治疗仍然具有挑战性。因此，了解与这些疾病相关的腹泻的病理生理机制是很重要的。腹泻是由于肠道分泌物增加和/或水和电解质吸收减少所致。人体肠道电解质吸收的一个主要途径是Na+/H+(NHE)和Cl-/HCO3-交换器的耦合作用。研究表明，当NHE3基因敲除的小鼠表现出腹泻表型时，NHE3在调节肠道钠吸收方面发挥了关键作用。此外，研究还表明，NHE3 KO小鼠更容易发生炎症，因为这些小鼠缺乏NHE3会导致与结肠炎相关的腹泻。然而，到目前为止，关于腹泻疾病中NHE3表达减少的分子机制知之甚少。为了阐明IBD相关性腹泻中NHE3表达下调的机制，我们目前的研究集中在DNA甲基化和组蛋白修饰等表观遗传机制在IBD发病机制中的作用。我们大量的初步数据提供了强有力的证据，证明了DNA甲基化和组蛋白修饰的变化对NHE3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在调节NHE3基因表达方面发挥重要作用，而NHE3基因表达是腹泻的病理生理基础。因此，目前的应用旨在通过DNA甲基化和组蛋白修饰来研究体外(AIMS 1和2)和体内模型(AIMS 3)对NHE3基因表达的调控：目的1.详细阐明DNA甲基化对NHE3启动子活性的影响，确定参与NHE3启动子调控的特异性CpG二核苷酸，并确定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)亚型在NHE3表达调控中的作用、NHE3基因上组蛋白乙酰化标记的变化和染色质重塑；目的3：利用野生型、DSS诱导的结肠炎和NHE3基因敲除小鼠(腹泻表型)，研究正常和炎症条件下表观遗传学机制对NHE3表达的调节。这些研究的结果将为NHE3的表观遗传调控提供新的见解，并确定NHE3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在腹泻疾病中涉及NHE3失调的特定DNMT或HDAC亚型的身份。

 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.