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）和腹泻仍然是一个主要的健康负担，阻碍了旨在更好的治疗选择的研究。越来越多的证据表明，在人类和炎症或感染的实验模型中，肠道5-HT转运蛋白（SERT）的减少和随之而来的高管腔5-HT水平在结肠炎和肠道炎性疾病的病理生理学中。作为一个重要的例子，SERT KO小鼠表现出结肠炎表型、异常运动性和炎症反应的加剧。因此，了解SERT的调节是至关重要的，SERT是治疗GI疾病的新靶点。然而，很少有人知道有关的机制失调的肠道SERT的病理生理条件下。我们实验室和其他实验室的先前研究表明，肠道SERT活性受蛋白激酶、蛋白酪氨酸磷酸酶和基因表达水平改变的调节。先前已显示SERT通过不同的机制被促炎介质LPS和TNF抑制。例如，LPS降低了质膜上的SERT水平，而TNF降低了SERT mRNA的表达，尽管这些作用的详细机制尚不清楚。SERT上调是否可以证明有效抵消SERT抑制和减轻炎症和结肠炎表型也是未知的。在这方面，我们的初步研究表明，SERT功能由TGF-β1通过翻译后机制以及通过改变IEC中的基因表达水平来刺激。因此，为了确定肠SERT作为GI疾病的新靶点的作用，提出的研究将测试以下假设：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，这可以抵消病理生理状态下的SERT抑制以逆转腹泻或炎症。具体目标1中提出的研究将阐明响应于TGF-β1或LPS调节SERT的信号传导和膜运输事件。具体目标2将阐明调节SERT基因表达的分子机制，以响应长期TNF或TGF- β1，并研究其信号传导/分子途径的串扰。具体目标3将严格检查TGF-β1是否逆转SAMP/yitc小鼠（类似克罗恩病的回肠炎模型）中TNF或炎症引起的管腔液体积聚（腹泻的标志）。此外，将利用野生型、SERT敲除和DNIIR小鼠（缺乏TGF- β受体信号传导）在天然肠中检查响应于TNF调节SERT的机制以及TGF-β1对其的逆转。总体而言，这些研究旨在为病理生理条件下上调肠道SERT的机制提供有价值的见解，其中TGF-β1信号传导缺陷和/或SERT表达下调。因此，这些研究应该确立SERT作为IBD和相关腹泻干预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.