Mechanisms of Gastrointestinal Growth and Transformation

描述(由申请人提供)：肠道嗜铬细胞(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，5HT)。血浆5-羟色胺水平升高会刺激体液分泌和肠道运动，以排出感染性或有毒刺激物。虽然这些肠道功能是先天免疫反应的重要组成部分，但会导致患者不适，如果持续下去，会导致严重的发病率和可能的死亡。尽管它起着核心作用，但目前还不清楚是什么调节了EC细胞的功能。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)，例如丁酸盐或曲古菌素A(TSA)。丁酸盐在几个启动子中诱导ZBP-89的表达并与富含GC的DNA元件结合，其中包括细胞周期蛋白依赖性激酶抑制因子(CDKI)p21。丁酸还触发ATM在1981年丝氨酸(PATMS1981)的自动磷酸化，随后与ZBP-89络合激活p21。在目前的建议中，pATMS1981在肠道中的阳性表达仅出现在肠道EC细胞的胞浆中，这表明pATMS1981参与了这些细胞的一项重要功能。在基因芯片上，ZBP-89条件性缺失的小鼠表现出EC细胞数量减少和色氨酸羟化酶1基因(TPH1)转录本减少。事实上，我们在TPH1基因的近端启动子中发现了富含GC的DNA元件，TPH1基因是5HT生物合成的限速合成酶。此外，在APCmin息肉中没有pATMS1981表达的细胞，这表明过多的Wnt信号阻止了EC细胞的分化。在目前的应用中，有三个目标被提出来检验压倒一切的假设，即ZBP-89通过调节TPH1基因的表达来刺激肠内皮细胞产生5-羟色胺，而细胞质中的pATM调节5-羟色胺的释放。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-是如何调控Tph1基因表达的。在目标2中，我们将剖析pATMS1981在丁酸介导的5-羟色胺分泌中的作用。在目标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-羟色胺生物合成中的作用。总的来说，这些研究将建立ZBP-89和PATMS1981之间的联系，在5-羟色胺的生物合成和功能对鲁米那丁酸盐的反应中。

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.