SOX9, Enteroendocrine & Intestinal Epithelial Stem Cells in the intestinal crypt

描述(由申请人提供)： 肠上皮的更新是由位于隐窝底部的多潜能肠上皮干细胞(IESCs)池驱动的。我们的初步研究表明，小肠隐窝上皮中的有丝分裂后细胞亚群表达高水平的转录因子Sox9；这些表达Sox9的细胞与肠内分泌细胞一致，可能通过分泌多肽来调节IESCs的增殖和分化能力。初步数据显示，部分表达Sox9的P物质阳性细胞在成年IESC的“生态位”中具有相对“较长的寿命”。我们提供了进一步的证据，证明高表达Sox9的细胞存在于发育早期的肠道中，是在发育中观察到的第一种分化的细胞类型，并且存在于辐射后再生的上皮微克隆中。这些观察表明，以高水平的Sox9为标志的细胞是增殖的肠上皮的保守特征。SOX9在EE群体中的作用机制尚不清楚。EE细胞既能分泌促进生长的多肽，又能分泌抑制生长的多肽，因此随着NIDDK-K01研究的进展，我们现在准备研究EE细胞之间的关系及其对IESC增殖和分化能力的影响。我们的中心假设是，基于隐窝的肠内分泌细胞(CBEE)对于调节IESCs的增殖/自我更新和分化以及指导隐窝的形态也是至关重要的。我们将这一假设扩展到预测CBEE中的Sox9控制着下游的靶基因，这些基因外在地影响IESC和隐窝生物学。为了开始检验这些假设，我们提出了以下具体目标：1)表征CBEE细胞，并确定其基因表达谱；1)鉴定EE细胞中依赖Sox9的基因。2)应用新的共培养方法建立EE细胞对IESC增殖和隐窝发育的影响。3)建立EE细胞消融转基因小鼠模型。这项研究的成功完成将：1)确定CBEE细胞的作用，2)识别与干细胞维持和分化有关的新的EE衍生蛋白，3)为进一步的外部资金提供理论基础、初步数据和试剂。这项研究的潜在临床意义是识别可用于治疗的多肽，以通过减缓IESCs的分裂速率在化疗或放射治疗期间保护IESC；或者相反，识别可用于通过加快感染、辐射或药物治疗引起的上皮枯竭后的再生过程来促进IESC增殖的促生长多肽。 公共卫生相关性： 在正常和受损状态下，上皮干细胞池驱动肠上皮的再生。了解调节和影响肠上皮干细胞增殖和分化的机制对于确定在疾病期间或损伤后促进组织再生的治疗方法至关重要。有证据表明，肠上皮干细胞区周围的一小部分细胞可能既为更大的肠上皮结构解剖提供了指导队列，也提供了控制增殖的信号。我们假设这群细胞是在肠道干细胞生物学中起关键作用的“长寿”荷尔蒙分泌细胞的子集，称为肠内分泌细胞。这项研究将调查这些细胞的作用和肠道内分泌细胞亚群中的活跃基因通路，以提供关于它们在干细胞区的作用的信息。这项研究收集的数据可能会为这些细胞类型提供新的作用机制，并确定再生治疗的新靶点。

DESCRIPTION (provided by applicant): The renewal of the intestinal epithelium is driven by a pool of multipotential intestinal epithelial stem cells (IESCs) residing in the base of the crypts. Our preliminary studies indicate that a sub-set of post-mitotic cells in the small intestine crypt epithelium express high levels of the transcription factor, Sox9; these Sox9-expressing cells are consistent with enteroendocrine cells, and may modulate proliferative and differentiation capacity of IESCs through secreted peptides. Preliminary data demonstrates that a subset of Sox9-expressing Substance-P positive cells have a relatively 'long-lived' tenure in the adult IESC 'niche'. We provide further evidence that high Sox9-expressing cells populate the early developing gut, are the first differentiated cell type observed in developing 'mini-guts' (crypt-villus units derived from a single IESC in vitro), and are present in regenerating epithelial micro-colonies following irradiation. These observations suggest that cells marked by high levels of Sox9 are a conserved feature of the proliferating intestinal epithelium. The mechanism by which SOX9 functions in the EE population is unknown. EE cells are known to secrete both growth promoting and inhibitory peptides, so with advances made with NIDDK-K01 funding, we are now poised to study the relationships between EE cells and their influence on IESC proliferation and differentiation capacity. Our central hypothesis for this proposal is that crypt-based enteroendocrine cells (CBEE) are critical for modulating proliferation/self-renewal and differentiation of IESCs, and also for instructing crypt morphology. We extend this hypothesis to predict that Sox9 in the CBEE controls downstream target genes that extrinsically influence IESC and crypt biology. To begin to test these hypotheses we propose the following Specific Aims: 1a) To characterize CBEE cells, and determine their gene expression profile, and 1b) to indentify Sox9-dependent genes in EE cells. 2) To establish the effect of EE cells on IESC proliferation and crypt development using novel co-culture assays. 3) To generate an EE cell ablation transgenic mouse model. Successful completion of this study will: 1) establish a role for the CBEE cells, 2) identify novel EE-derived proteins involved in stem cell maintenance and differentiation, and 3) provide rationale, preliminary data and reagents for further extramural funding. The potential clinical implications of this study are identification of peptides that could be used as therapeutics to protect the IESC during chemotherapeutic or radiation treatments by slowing division rates of IESCs; or conversely identification of pro-growth peptides that could be used to enhance IESC proliferation by speeding the regenerative process following epithelium depletion cause by infection, irradiation or drug treatments. PUBLIC HEALTH RELEVANCE: A pool of epithelial stem cells drives regeneration of the intestinal epithelium during normal and injured states. Understanding the mechanisms that regulate and influence the proliferation and differentiation of the intestinal epithelial stem cell is critical for identifying therapies to enhance tissue regeneration during disease or following injury. There is evidence that a small population of cells that surround the stem cell zone in the intestinal epithelium may be providing both instructional queues for the larger structural anatomy of the intestinal epithelium, and also signals that control proliferation. We hypothesize that this population of cells is a subset of 'long-lived' hormone producing cells called enteroendocrine cells that have a critical role in intestinal stem cell biology. This study will investigate the role of these cells and active gene pathways within this subset of enteroendocrine cells to provide information regarding their role in the stem cell zone. Data gathered from this study will likely provide new mechanisms of action for these cell types and indentify novel targets for regenerative therapies.