Mechanisms of Gastrointestinal Growth and Transformation

描述（由申请人提供）：肠肠嗜铬细胞（EC）室在急性损伤、微生物感染和结肠炎时扩张，产生5-羟色胺（5-羟色胺，5HT）。血浆血清素水平升高会刺激体液分泌和肠道运动以排出感染性或毒性刺激物。虽然是先天免疫反应的重要组成部分，但这些肠道功能会导致患者不适，如果持续下去，就会成为显著发病率和可能死亡率的来源。尽管它具有中心作用，但调控EC细胞功能的机制尚不清楚。在之前的资助周期中，我们发现锌指转录因子ZBP-89与肿瘤抑制蛋白ataxia毛细血管扩张突变（ATM）直接相互作用，以响应组蛋白去乙酰化酶抑制（HDACi），例如丁酸盐或trichostatin A （TSA）。丁酸盐诱导ZBP-89在包括细胞周期蛋白依赖性激酶抑制剂（CDKI） p21在内的多种启动子中表达并与富含gc的DNA元件结合。丁酸盐还能触发ATM丝氨酸1981 （pATMS1981）的自磷酸化，随后与ZBP-89复合物激活p21。在目前的研究中，发现肠道中pATMS1981的阳性表达仅发生在肠EC细胞的细胞质中，这表明pATMS1981参与了这些细胞的基本功能。在微阵列上显示，结肠条件阴性ZBP-89的小鼠EC细胞数量减少，色氨酸羟化酶1基因（TPH1）转录物减少。事实上，我们在TPH1基因的近端启动子中发现了富含gc的DNA元素，TPH1基因是5HT生物合成的限速合成酶。此外，在APCmin息肉中没有表达patms1981的细胞，这表明过量的Wnt信号传导阻止了EC细胞的分化。在目前的应用中，提出了三个目的来验证ZBP-89通过调节TPH1基因表达刺激肠EC细胞血清素产生，而细胞质中的pATM调节5HT释放的总体假设。此外，我们假设炎症干扰了它们的功能，为肿瘤转化奠定了基础。在Aim 1中，我们将确定ZBP-89-如何调节TPH1基因表达。在Aim 2中，我们将剖析pATMS1981在丁酸盐介导的5HT分泌中的作用。在Aim 3中，我们将研究这两种蛋白在慢性炎症和结肠转化过程中5HT生物合成中的作用。总的来说，这些研究将建立ZBP-89和pATMS1981之间的联系，在5HT的生物合成和功能响应于luminal butyrate。

DESCRIPTION (provided by applicant): The intestinal enterochromaffin cell (EC) compartment expands in response to acute injury, microbial infection and colitis to produce serotonin (5-hydroxytryptamine, 5HT). Elevated levels of plasma serotonin stimulate fluid secretion and gut motility to expel the infectious or toxic irritants. Although an essential component of the innate immune response, these gut functions contribute to patient discomfort and if sustained become a source of significant morbidity and possible mortality. Despite its central role, it is not understood what regulates EC cell function. During the prior funding cycle, we found that the zinc finger transcription factor ZBP-89 interacts directly with the tumor suppressor protein ataxia telangiectasia mutated (ATM) in response to histone deacetylase inhibition (HDACi), e.g., butyrate or trichostatin A (TSA). Butyrate induces ZBP-89 expression and binding to GC-rich DNA elements in several promoters including the cyclin-dependent kinase inhibitor (CDKI) p21. Butyrate also triggers auto-phosphorylation of ATM at serine 1981 (pATMS1981) that subsequently complexes with ZBP-89 to activate p21. In the current proposal, pATMS1981 positive expression in the gut was found to occur exclusively in the cytoplasm of intestinal EC cells suggesting that pATMS1981 participates in an essential function of these cells. Mice conditionally null for ZBP-89 in the colon exhibited reduced numbers of EC cells and reduced tryptophan hydroxylase 1 gene (TPH1) transcripts on a microarray. Indeed, we found GC-rich DNA elements in the proximal promoter of the TPH1 gene, the rate-limiting synthetic enzyme for 5HT biosynthesis. In addition, pATMS1981-expressing cells in APCmin polyps were absent suggesting that excess Wnt signaling prevents EC cell differentiation. In the current application, three aims are proposed to test the overarching hypothesis that ZBP-89 stimulates serotonin production in gut EC cells by regulating TPH1 gene expression while pATM in the cytoplasm regulates 5HT release. Moreover, we hypothesize that inflammation perturbs their function setting the stage for neoplastic transformation. In Aim 1, we will determine how ZBP-89- regulates TPH1 gene expression. In Aim 2, we will dissect the role of pATMS1981 in butyrate-mediated secretion of 5HT. In Aim 3, we will study the role of these two proteins in 5HT biosynthesis during chronic inflammation and colonic transformation. Collectively, these studies will establish a link between ZBP-89 and pATMS1981, in the biosynthesis and function of 5HT in response to luminal butyrate.