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

描述(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，需要进行旨在更好的治疗方案的研究。越来越多的证据表明，在人类腹泻和炎症性疾病的病理生理过程中，肠道5-羟色胺转运体(SERT)减少，从而导致肠腔内5-羟色胺水平升高，无论是在人类还是在炎症或感染的实验模型中都是如此。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，对于肠道SERT在病理生理条件下调节失调的机制知之甚少。我们实验室和其他实验室以前的研究表明，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变化的调节。以往的研究表明，SERT可通过不同的机制被促炎介质--内毒素和肿瘤坏死因子所抑制。例如，内毒素降低了质膜上的SERT水平，而肿瘤坏死因子则降低了SERT mRNA的表达，尽管这些影响的详细机制尚不清楚。SERT上调是否在对抗SERT抑制和减轻炎症和腹泻表型方面是否有效也尚不清楚。在这方面，我们的初步研究表明，转化生长因子-β1通过翻译后机制以及通过改变内皮细胞基因表达水平来刺激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，拟议的研究将检验这样一种假设，即SERT的功能和/或表达通过不同的信号、运输和分子机制而增加，这些机制可以抵消SERT在病理生理状态下对SERT的抑制，从而逆转腹泻或炎症。在特定目标1中提出的研究将阐明调节转化生长因子-β1或脂多糖反应的SERT的信号和膜转运事件。特异性目标2将阐明肿瘤坏死因子和转化生长因子-β-1长期作用下调节SERT基因表达的分子机制，并研究它们之间的信号/分子通路的相互作用。具体目标3将关键地检查在SAMP/YITC小鼠(类似克罗恩病的回肠炎模型)中由肿瘤坏死因子或炎症引起的腔液堆积(腹泻的特征)是否被转化生长因子-β1逆转。此外，将利用野生型、SERT基因敲除和DNIIR小鼠(缺乏转化生长因子-β受体信号)在自然肠道中研究调节肿瘤坏死因子反应和转化生长因子-β逆转的机制。总体而言，这些研究旨在为在转化生长因子-SERT 1信号缺陷和/或β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和相关腹泻的新的治疗靶点的作用。

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.