SOX9, Enteroendocrine and Intestinal Epithelial Stem Cells: A Study of Gene Netwo

描述（由申请人提供）： 肠上皮的更新是由位于隐窝底部的多能肠上皮干细胞（IESC）库驱动的。我们的初步研究表明，一个子集的有丝分裂后的细胞在小肠隐窝上皮表达高水平的转录因子，Sox 9，这些Sox 9表达细胞与肠内分泌细胞一致，并可能通过分泌肽调节IESC的增殖和分化能力。初步数据表明，一个子集的Sox 9表达物质P阳性细胞有一个相对'长寿'的任期在成人IESC的'利基'。我们提供了进一步的证据表明，高Sox 9表达细胞填充早期发育的肠道，是在发育中的“小肠道”（来自体外单个IESC的隐窝绒毛单位）中观察到的第一种分化的细胞类型，并且存在于照射后再生的上皮微菌落中。这些观察结果表明，由高水平的Sox 9标记的细胞是增殖的肠上皮的保守特征。SOX 9在EE人群中发挥作用的机制尚不清楚。已知EE细胞分泌生长促进和抑制肽，因此随着NIDDK-K 01资助的进展，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。我们对这一提议的中心假设是，基于隐窝的肠内分泌细胞（CBEE）对于调节IESC的增殖/自我更新和分化以及指导隐窝形态至关重要。我们扩展了这一假设，预测CBEE中的Sox 9控制下游靶基因，这些靶基因对IESC和隐窝生物学产生显著影响。为了开始检验这些假设，我们提出了以下具体目标：1a）表征CBEE细胞，并确定其基因表达谱，和1b）鉴定EE细胞中的Sox 9依赖性基因。2)使用新型共培养试验确定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）确定参与干细胞维持和分化的新型EE衍生蛋白，3）为进一步的校外资助提供理论依据，初步数据和试剂。本研究的潜在临床意义是鉴定可用作治疗剂的肽，以通过减慢IESC的分裂速率在化学治疗或放射治疗期间保护IESC;或者相反地鉴定促生长肽，其可用于通过在由感染、辐射或药物治疗引起的上皮耗竭后加速再生过程来增强IESC增殖。 公共卫生相关性： 上皮干细胞库在正常和损伤状态下驱动肠上皮的再生。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增强组织再生的疗法至关重要。有证据表明，在肠上皮中围绕干细胞区的一小群细胞可能为肠上皮的较大结构解剖提供指导队列，并且还提供控制增殖的信号。我们假设这群细胞是一个“长寿”的激素产生细胞的子集，称为肠内分泌细胞，在肠道干细胞生物学中起着关键作用。本研究将探讨这些细胞的作用和活性基因通路内的肠内分泌细胞的这一子集，以提供有关其在干细胞区的作用的信息。从这项研究中收集的数据可能会为这些细胞类型提供新的作用机制，并确定再生疗法的新靶点。

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.