Mechanisms of Gastrointestinal Growth and Transformation

描述（由申请方提供）：肠嗜铬细胞（EC）隔室在急性损伤、微生物感染和结肠炎时扩张，产生5-羟色胺（5-羟色胺，5 HT）。血浆5-羟色胺水平升高刺激体液分泌和肠道运动，以排出感染性或毒性刺激物。虽然这些肠道功能是先天免疫应答的重要组成部分，但它们会导致患者不适，如果持续存在，则会成为显著发病率和可能死亡率的来源。尽管它的核心作用，它是不明白是什么调节EC细胞功能。在之前的资助周期中，我们发现锌指转录因子ZBP-89直接与肿瘤抑制蛋白共济失调毛细血管扩张突变（ATM）相互作用，以响应组蛋白脱乙酰酶抑制（HDACi），例如，丁酸盐或曲马斯他汀A（TSA）。丁酸盐诱导ZBP-89的表达，并在几个启动子中与富含GC的DNA元件结合，包括细胞周期蛋白依赖性激酶抑制剂（CDKI）p21。丁酸盐还触发ATM在丝氨酸1981（pATMS 1981）处的自磷酸化，其随后与ZBP-89复合以激活p21。在目前的建议中，pATMS 1981在肠道中的阳性表达被发现只发生在肠EC细胞的细胞质中，表明pATMS 1981参与这些细胞的基本功能。在微阵列上，结肠中ZBP-89条件无效的小鼠表现出EC细胞数量减少和色氨酸羟化酶1基因（TPH 1）转录物减少。事实上，我们发现富含GC的DNA元件在TPH 1基因的近端启动子中，该基因是5 HT生物合成的限速合成酶。此外，APCmin息肉中不存在pATMS 1981表达细胞，表明过量的Wnt信号传导阻止EC细胞分化。在本申请中，提出了三个目的来检验总体假设，即ZBP-89通过调节TPH 1基因表达刺激肠EC细胞中5-羟色胺产生，而细胞质中的pATM调节5-HT释放。此外，我们假设炎症扰乱了它们的功能，为肿瘤转化奠定了基础。在目标1中，我们将确定ZBP-89如何调节TPH 1基因表达。在目的2中，我们将剖析pATMS 1981在丁酸盐介导的5 HT分泌中的作用。在目的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5 HT生物合成中的作用。总的来说，这些研究将建立ZBP-89和pATMS 1981之间的联系，在生物合成和功能的5 HT响应管腔丁酸。 公共卫生相关性：肠嗜铬（EC）细胞分泌5-羟色胺进入循环发挥许多临床效果，包括增加肠道蠕动和腹泻。转录因子ZBP-89和抑癌蛋白ATM均受大肠杆菌的鲁米那丁酸的调节，在维持EC细胞5 HT的合成和分泌中起重要作用。

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. PUBLIC HEALTH RELEVANCE: Enterochromaffin (EC) cells secrete serotonin into the circulation to exert a number of clinical effects including increased gut motility and diarrhea. Both the transcription factor ZBP-89 and the tumor suppressor protein ATM are regulated by luminal butyrate from commensal bacteria and are important in maintaining 5HT biosynthesis and secretion from EC cells.