Intestinal 5-HT Transporter: A novel therapeutic target for GI disorders

描述（由申请人提供）：胃肠道疾病，如炎症性肠病（IBD）和腹泻仍然是主要的健康负担，有必要进行研究，以寻求更好的治疗方案。越来越多的证据表明，肠道5-羟色胺转运体（SERT）的减少以及由此导致的肠道5-羟色胺高水平在人类和炎症或感染的实验模型中腹泻和肠道炎症疾病的病理生理中起着重要作用。作为一个重要的例子，SERT KO小鼠表现出腹泻表型、运动异常和炎症反应加剧。因此，了解SERT的调控是至关重要的，SERT是治疗胃肠道疾病的新靶点。然而，关于病理生理条件下肠道SERT失调的机制知之甚少。我们实验室和其他人之前的研究表明，肠道SERT活性受到蛋白激酶、蛋白酪氨酸磷酸酶和基因表达水平改变的调节。先前已经证明SERT通过不同的机制被促炎介质、LPS和TNF抑制。例如，LPS降低了质膜上SERT的水平，而TNF降低了SERT mRNA的表达，尽管这些影响的详细机制尚不清楚。SERT上调是否能有效抵消SERT抑制并减轻炎症和腹泻表型也尚不清楚。因此，我们的初步研究表明，TGF-β1通过翻译后机制以及IECs基因表达水平的改变来刺激SERT功能。因此，为了确定肠道SERT作为胃肠道疾病的新靶点的作用，拟议的研究将验证SERT功能和/或表达通过不同的信号传导、运输和分子机制增加的假设，这些机制可以抵消SERT在病理生理状态下的抑制作用，从而逆转腹泻或炎症。在Specific Aim 1中提出的研究将阐明在TGF-β1或LPS的作用下调节SERT的信号和膜转运事件。Specific Aim 2将阐明SERT基因在TNF或TGF- β1长期作用下表达调控的分子机制，并研究其信号/分子通路的串扰。特异性目的3将严格检查SAMP/yitc小鼠（类似克罗恩病的回肠炎模型）中由TNF或炎症引起的腔内积液（腹泻的标志）是否被TGF-β1逆转。此外，将利用野生型、SERT敲除和DNIIR小鼠（缺乏TGF-β受体信号）在天然肠道中研究SERT响应TNF及其被TGF-β1逆转的调节机制。总的来说，这些研究旨在为在TGF-β1信号缺陷和/或SERT表达下调的病理生理条件下上调肠道SERT的机制提供有价值的见解。因此，这些研究应该确立SERT作为干预IBD和相关腹泻的新治疗靶点的作用。

DESCRIPTION (provided by applicant): Gastrointestinal disorders such as inflammatory bowel diseases (IBD) and diarrhea remain a major health burden, warranting investigations aimed at better treatment options. Increasing evidence implicates a decrease in intestinal 5-HT transporter (SERT) and the consequent high luminal 5-HT levels in the pathophysiology of diarrheal and inflammatory disorders of the intestine both in humans and experimental models of inflammation or infection. As an important example, SERT KO mice exhibit diarrheal phenotype, abnormal motility and exacerbation of inflammatory responses. Therefore, it is critical to understand the regulation of SERT, a novel target for the treatment of GI disorders. However, very little is known regarding mechanisms underlying dysregulation of intestinal SERT under pathophysiological conditions. Previous studies from our laboratory and others have demonstrated that intestinal SERT activity is subject to regulation by protein kinases, protein tyrosine phosphatases and alterations at the level of gene expression. SERT has been previously shown to be inhibited by the pro-inflammatory mediators, LPS and TNF via distinct mechanisms. For example, LPS decreased SERT levels at the plasma membrane, whereas, TNF reduced SERT mRNA expression, albeit the detailed mechanisms underlying these effects are not known. Whether SERT upregulation can prove effective in counteracting SERT inhibition and alleviation of inflammatory and diarrheal phenotype is also not known. In this regard, our preliminary studies showed that SERT function is stimulated by TGF-β1 by post- translational mechanisms as well as via alterations at the level of gene expression in IECs. Thus, to establish the role of intestinal SERT as a novel target of GI disorders, proposed studies will test the hypothesis that SERT function and/or expression is increased by distinct signaling, trafficking and molecular mechanisms, which can counteract the inhibition of SERT in pathophysiological states to reverse diarrhea or inflammation. Studies proposed in Specific Aim 1 will elucidate the signaling and membrane trafficking events modulating SERT in response to TGF-β1 or LPS. Specific Aim 2 will elucidate the molecular mechanisms modulating SERT gene expression in response to long-term TNF or TGF- β1 and investigate the cross-talk of their signaling/molecular pathways. Specific Aim 3 will critically examine whether luminal fluid accumulation (hallmark of diarrhea) caused by TNF or inflammation in SAMP/yitc mice (model of ileitis resembling Crohn's disease) is reversed by TGF-β1. Furthermore, mechanisms modulating SERT in response to TNF and their reversal by TGF-β1 will be examined in the native intestine utilizing wild type, SERT knock out and DNIIR mice, (lacking TGF- β receptor signaling). Overall, these studies are designed to provide valuable insights into the mechanisms upregulating intestinal SERT in pathophysiological conditions, where TGF-β1 signaling is defective and/or SERT expression is downregulated. Thus, these studies should establish the role of SERT as a novel therapeutic target for intervention in IBD and associated diarrhea.