EPIMORPHIN DELETION ALTERS STEM CELL NICHE MYOFIBROBLAST SECRETION

描述（由申请人提供）：作为华盛顿大学医学院胃肠病学系的医学助理教授，我专注于探索肌成纤维细胞蛋白缺失导致保护免受结肠炎和结肠炎相关癌症（CAC）的机制。近期和长期职业目标：该奖项将允许时间获得教学培训和技术专业知识，并发表更多的第一作者手稿，详细说明基质和结肠肌成纤维细胞在CAC发展中的作用，目标是在该奖项的第4年以R 01的形式申请独立资助。从长远来看，我希望过渡到一个独立的调查事业，作为一个胃肠病学的医生，科学家，调查炎症相关癌症发病机制中的基质。职业发展：住院临床职责仅占我时间的25%。除了完成我的研究计划的目标，我的职业发展计划包括个人和小组会议与导师博士鲁宾和纽贝里，会议与我的咨询委员会，会议在GI部门和医学院，参加全国会议，包括DDW，FASEB，和AAI，并在骨髓移植，流式细胞术和激光捕获显微切割的指令。我计划在第一年年底完成目标1的研究，并在手稿中详细说明我的研究结果;在第二至第三年完成目标2的大部分内容，沿着一份手稿;在第四至第五年完成目标2的研究和一份手稿。工作环境：华盛顿大学医学院和胃肠病学分部的研究和教育资源为成功的医生科学家的产生做出了贡献。我的导师Rubin博士和纽贝里博士是基质/上皮和免疫细胞生物学方面的知名专家，并致力于为拟议的研究项目提供培训。研究：上皮-基质相互作用的破坏与结肠炎和CAC的发病机制有关。表吗啡肽（epim）是一种基质和肌成纤维细胞蛋白，调节肠上皮形态发生。Epim-/-小鼠被部分保护免于与隐窝细胞增殖增加相关的急性结肠炎。我们已经在CAC的鼠模型中显示Epim-/-小鼠显著降低了发育异常肿瘤负荷。我们还证明，表吗啡肽缺失增加了结肠肌成纤维细胞的脊索蛋白分泌，并减少了肌成纤维细胞和巨噬细胞的白细胞介素-6（IL-6）分泌。我们认为这些调节事件是重要的，因为Chordin抑制骨形态发生蛋白（Bmp），一种已知抑制隐窝细胞增殖的蛋白质。此外，IL-6是一种与CAC有关的促炎细胞因子。这些观察结果构成了我们两个主要假设的基础：假设1：表吗啡肽缺失改变了结肠肌成纤维细胞分泌调节隐窝细胞增殖（Bmp和Bmp抑制剂）和调节炎症（细胞因子）的关键信号分子。假设二：表吗啡肽缺失改变了骨髓源性肌成纤维细胞和基质细胞的分泌功能，这些细胞在保护结肠炎和CAC中起重要作用。这些相关的假设将在以下具体目标进行追求：1。确定表吗啡肽缺失导致上皮隐窝细胞增殖增加和炎症减少的机制。这些研究将解决假设1。我们将使用Epim-/-和WT肌成纤维细胞和肌成纤维细胞-上皮细胞共培养物来检查表吗啡缺失对结肠肌成纤维细胞分泌参与调节隐窝细胞增殖（Bmps）和炎症（细胞因子）的信号分子的影响。我们将这些观察扩展到葡聚糖硫酸钠（DSS）结肠炎和氧化偶氮甲烷（AOM）/DSS CAC模型。我们预测，表吗啡肽缺失将改变肌成纤维细胞分泌的骨形成蛋白和细胞因子，以增加上皮细胞增殖和减少炎症。2.确定负责保护Epim-/-小鼠免受DSS结肠炎和AOM/DSS CAC的细胞的身份和来源。这些研究将解决假设2。肌成纤维细胞驻留在肠基质中，但在损伤或炎症的情况下，“新的”肌成纤维细胞和其他骨髓（BM）来源的基质细胞迁移到肠中。为了确定在Epim-/-小鼠中观察到的表型是否是BM衍生细胞的结果，我们将对致死性照射的Epim-/-和WT受体进行GFP标记的Epim-/-和WT BM拯救，并用DSS和AOM/DSS处理BM嵌合体。我们将确定移植的骨髓细胞在受体的细胞鉴定，并确定其细胞因子分泌谱。我们预测，Epim-/- BM供体来源的细胞将需要实现对DSS结肠炎和AOM/DSS发育不良的最大保护。这些研究可能为炎症性肠病和CAC的新治疗方法提供基础。

DESCRIPTION (provided by applicant): As an Assistant Professor of Medicine in the Division of Gastroenterology at Washington University School of Medicine, I am focused on exploring mechanisms whereby deletion of a myofibroblast protein results in protection from colitis and colitis associated cancer (CAC). Immediate and long-term career goals: This award will allow time to obtain didactic training and technical expertise, and publish additional first-authored manuscripts detailing the role of the stroma and colonic myofibroblast in the development of CAC, with the goal of applying for independent funding in the form of an R01 in year 4 of this award. In the long-term I hope to transition to an independent investigative career as a physician-scientist in gastroenterology, investigating the stroma in the pathogenesis of inflammation associated cancer. Career development: Inpatient clinical duties are limited to < 25% of my time. In addition to completing the aims of my research proposal, my career development plan consists of individual and group meetings with mentors Drs. Rubin and Newberry, meetings with my Advisory Committee, conferences in the GI Division and the School of Medicine, participation in national conferences including DDW, FASEB, and AAI, and instruction in bone marrow transfer, flow cytometry, and laser capture microdissection. I plan on completing Aim 1 studies and detailing my findings in a manuscript by the end of year 1; completing a substantial portion of Aim 2 along with a manuscript by years 2-3; and completing Aim 2 studies and a manuscript in years 4-5. Environment: The research and educational resources of Washington University School of Medicine and the Gastroenterology Division have contributed to the generation of successful physician-scientists. My mentors Drs. Rubin and Newberry are well-established experts in stromal/epithelial and immune cellular biology and have committed to providing training for the proposed research project. Research: Disrupted epithelial-stromal interactions are implicated in the pathogenesis of colitis and CAC. Epimorphin (epim) is a stromal and myofibroblast protein that regulates intestinal epithelial morphogenesis. Epim-/- mice are partially protected from acute colitis in association with an increase in crypt cell proliferation. We have shown in a murine model of CAC that Epim-/- mice have markedly reduced dysplastic tumor burden. We have also demonstrated that epimorphin deletion increases secretion of Chordin from colonic myofibroblasts and decreases secretion of Interleukin-6 (IL-6) from myofibroblasts and macrophages. We propose that these regulatory events are important because Chordin inhibits bone morphogenetic protein (Bmp), a protein known to inhibit crypt cell proliferation. In addition, IL-6 is a pro-inflammatory cytokine that has been implicated in CAC. These observations underlie our two major hypotheses: Hypothesis 1: Epimorphin deletion alters colonic myofibroblast secretion of key signaling molecules that regulate crypt cell proliferation (Bmps and Bmp inhibitors) and modulate inflammation (cytokines). Hypothesis 2: Epimorphin deletion alters the secretory function of bone marrow derived myofibroblasts and stromal cells which play a significant role in conferring protection from colitis and CAC. These related hypotheses will be pursued in the following specific aims: 1. Determine the mechanisms by which epimorphin deletion results in an increase in epithelial crypt cell proliferation and reduced inflammation. These studies will address hypothesis 1. We will use Epim-/- and WT myofibroblasts and myofibroblast-epithelial co-cultures to examine the effect of epimorphin deletion on colonic myofibroblast secretion of signaling molecules involved in the regulation of crypt cell proliferation (Bmps) and inflammation (cytokines). We will extend these observations to the dextran sodium sulfate (DSS) colitis and azoxymethane (AOM)/DSS CAC models. We predict epimorphin deletion will alter myofibroblast secretion of Bmps and cytokines to increase epithelial cell proliferation and reduce inflammation. 2. Define the identity and origin of the cells that are responsible for protection from DSS colitis and AOM/DSS CAC in Epim-/- mice. These studies will address hypothesis 2. Myofibroblasts are resident in the intestinal stroma but in the setting of injury or inflammation, "new" myofibroblasts and other bone marrow (BM) derived stromal cells migrate into the intestine. To determine whether the observed phenotype in Epim-/- mice is a consequence of BM derived cells, we will perform GFP labeled Epim-/- and WT BM rescue of lethally irradiated Epim-/- and WT recipients and treat BM chimeras with DSS and AOM/DSS. We will determine the cellular identify of the transplanted BM cells in the recipient and define their cytokine secretory profile. We predict that Epim-/- BM donor derived cells will be required to achieve maximal protection from DSS colitis and AOM/DSS dysplasia. These studies may provide the basis for novel therapeutic approaches to inflammatory bowel disease and CAC.