Mechanisms of tuft cell mediated regulation of the intestinal stem cell niche following injury

放射治疗的主要和毁灭性的不良反应之一是发展为放射性肠炎。 (Re)。在美国接受放射治疗的患者数量，作为其原发癌症的一个组成部分 预计未来十年治疗人数将增加20%以上，达到每年近60万人。Re是一种 放射治疗后发生的肠道炎症过程。这是一个主要的健康问题。 腹痛的以腹痛、腹泻和直肠出血为特征的肠道移位可使其复杂化。 肠道上皮细胞丢失，上皮内紧密连接中断，细菌进入循环， 再生能力丧失，导致肠道功能受损，甚至死亡。我们所知的相对较少 关于肠上皮损伤修复、干细胞存活和隐窝再生的机制 在RE.双重皮质醇样激酶1(Dclk1)标记肠道绒毛细胞和肠道内Dclk1的缺失 上皮细胞导致严重辐射损伤后小鼠的过早死亡，提示Dclk1是一种 隐窝上皮细胞对遗传毒性损伤反应的主要介体。我们报道了Dclk1与共济失调- 毛细血管扩张突变(ATM)和磷酸化ATM激活DNA损伤反应(DDR) 辐射损伤。此外，过表达Dclk1的肠上皮细胞比 控制细胞。这些数据表明，Dclk1+簇状细胞在上皮细胞存活过程中起关键作用。 严重的遗传毒性损伤。最近，对肠道中的单细胞分析发现，Dclk1+上皮丛细胞 是Cox1(Ptgs1)和COX2(Ptgs2)的主要来源。根据我们之前的研究，Cox1和Cox2是 肠道中PGE2的主要来源，在严重辐射损伤后保护肠道。然而，分子 严重创伤后调节肠绒毛细胞存活的机制尚不清楚，而 肠隐窝丛状细胞在上皮干细胞中的存活完全未知。我们的中心假设是 严重遗传毒性损伤后肠隐窝丛状细胞的存活是由Dclk1依赖性介导的 ATM/ATR DNA损伤反应的调节；此外，这些隐窝簇状细胞协调生存 通过PGE2依赖的机制对邻近的上皮干细胞进行诱导。我们将使用 目标1：阐明丛生细胞存活致死的分子机制 剂量辐射损伤。目的2：确定簇状细胞协调细胞存活的机制。 隐窝肠道干细胞(ISC)。目的3：确定丛状细胞特异性前列腺素E_2在肠道中的作用 严重辐射损伤后的隐窝再生和上皮重建。 这项拟议的研究将(1)确定存活的肠道上皮Dclk1+Tuft的机制 严重遗传毒性损伤后通过旁分泌调节隐窝干细胞存活 机制和(2)最终回答了RE如何导致肠道功能障碍的临床相关问题 并了解在严重的遗传毒性损伤后，上皮绒毛细胞在提高隐窝存活率方面的重要性。

One of the major and devastating adverse effects of radiation therapy is the development of radiation enteritis (RE). The number of patients receiving radiation therapy in the USA, as a component of their primary cancer treatment, is forecast to increase by more than 20% over the next decade to almost 600,000 per year. RE is an intestinal inflammatory process that occurs in response to radiotherapy. It is a major health concern characterized by abdominal pain, diarrhea, and rectal bleeding. It can be complicated by translocation of gut bacteria into the circulation due to the loss of intestinal epithelial cells, disruption of intraepithelial tight junctions, and loss of regenerative ability resulting in impairment of gut function and even death. Relatively little is known about the mechanisms underlying the intestinal epithelial injury repair, stem cell survival and crypt regeneration in RE. Doublecortin like kinase 1 (Dclk1) marks intestinal tuft cells and deletion of Dclk1 within intestinal epithelial cells resulted in premature death of mice following severe radiation injury, suggesting that Dclk1 is a major mediator of the crypt epithelial response to genotoxic injury. We reported that Dclk1 interacts with ataxia- telangiectasia mutated (ATM) and phosphorylates ATM activating the DNA damage response (DDR) following radiation injury . Furthermore, intestinal epithelial cells overexpressing Dclk1 are more resistant to radiation than the control cells. These data suggest that Dclk1+ tuft cell plays a critical role in epithelial cell survival following severe genotoxic injury. Recently, single cell analysis in the intestine has revealed that Dclk1+ epithelial tuft cell is the primary source of Cox1 (Ptgs1) and Cox2 (Ptgs2). Based on our previous studies, Cox1 and Cox2 are the major source of PGE2 in the intestine, that protects the gut after severe radiation injury. However, the molecular mechanism that regulates the survival of intestinal tuft cells following severe injury is unclear and the role of intestinal crypt tuft cells in epithelial stem cell survival is completely unknown. Our central hypothesis is that intestinal crypt tuft cell survival following severe genotoxic injury is mediated by Dclk1-dependent regulation of the ATM/ATR DNA damage response; further these crypt tuft cells coordinate the survival of neighboring epithelial stem cells via a PGE2-dependent mechanism. We will test our hypothesis with the following specific aims: Aim 1: To elucidate the molecular mechanism by which tuft cells survive lethal dose radiation injury. Aim 2: To determine the mechanisms by which tuft cells coordinate the survival of crypt intestinal stem cells (ISCs). Aim 3: To determine the role of tuft cell specific PGE2 on intestinal crypt regeneration and epithelial restitution following severe radiation injury. The proposed study will (1) determine the mechanisms by which surviving intestinal epithelial Dclk1+ tuft following severe genotoxic injury coordinately regulate the crypt stem cell survival via a paracrine mediated mechanism and (2) eventually answer the clinically-relevant question of how RE contribute to gut dysfunction and understand the importance of epithelial tuft cells in augmenting crypt survival, after severe genotoxic injury.