ENTEROCYTE APOPTOSIS AFTER INTESTINAL RESECTION

描述（由申请人提供）：肠适应是对大面积小肠切除术（SBR）的一种关键性代偿反应，其特征是肠上皮细胞更新增加，通过增殖和凋亡的升高幅度来衡量。细胞凋亡的适应程度的重要性，揭示了在此赠款的前一个资助周期，作为放大的适应时，观察到细胞凋亡被积极抑制。虽然SBR后细胞凋亡增加的机制目前尚不清楚，但我们已经确定这种反应受表皮生长因子受体（EGFR）信号转导的调节，并需要促凋亡Bcl 02家族成员Bax和转录因子信号转导子和转录激活子（STAT）-1的表达。作为这些关键观察的延伸，我们提出了一个全球性的假设，即EGFR信号调节Bax的表达和活性，以调节切除诱导的细胞凋亡。为了验证这一假设，我们的目标是：1）确定STAT-1在肠道适应期间调节Bax表达中的作用。将在SBR后的回肠中以及在诱导细胞凋亡后的细胞培养物中测定STAT-1表达和活性。将测量STAT-1缺陷对Bax表达和细胞凋亡的影响，并研究Bax启动子上推定的STAT-1结合位点。2)确定p38 α丝裂原活化蛋白激酶（MAPK）在切除诱导适应过程中作为Bax活性调节剂的作用。在小鼠和细胞凋亡的补充体外模型中SBR后，将记录p38表达/活性和Bax活化的时间和空间分布。将在SBR后测定p38表达的条件性、精氨酸特异性缺失对Bax活性和细胞凋亡的影响。3)确定EGFR调节Bax表达和活性的机制。将记录增强或破坏的EGFR信号传导对STAT-1和p38活化和表达的影响。将确定在EGFR抑制的情况下减弱的STAT-1或p38表达对细胞凋亡和Bax活性和表达的影响。本申请中提出的研究将鉴定大规模SBR后直接细胞凋亡的最相关信号通路。对诱导细胞凋亡的精确机制的透彻理解对于旨在最大限度地刺激肠粘膜再生以响应大量肠损失的治疗靶点的从实验室到床边的转化是基础。公共卫生相关性：在大量肠道损失后，剩余的肠道试图重新生长以进行补偿。如果这种反应是不完全的，病人将受到终身的营养静脉和所有相关的并发症。本项目旨在了解粘膜细胞死亡对肠道再生过程的贡献。了解这一重要反应的确切分子调控可能会导致旨在改善肠道灾难性损失后肠道再生的创新疗法。

DESCRIPTION (provided by applicant): Intestinal adaptation is a critical, compensatory response to massive small bowel resection (SBR) and characterized by increased enterocyte turnover as gauged by elevated rages of both proliferation and apoptosis. The significance of apoptosis to the magnitude of adaptation was revealed during the previous funding cycle of this grant as amplified adaptation was observed when apoptosis was actively inhibited. While the mechanisms(s) for elevated apoptosis after SBR is presently unknown, we have established that this response is regulated by epidermal growth factor receptor (EGFR) signaling and requires expression of the proapototic Bcl02 family member Bax and the transcription factor signal transducer and activator of transcription (STAT)-1. As an extension of these key observations, we propose the global hypothesis that EGFR signaling modulates the expression and activity of Bax to regulate resection-induced apoptosis. To test this hypothesis our aims are: 1) Determine the role for STAT-1 in the regulation of Bax expression during intestinal adaptation. STAT-1 expression and activity will be determined in the ileum after SBR as well as in cell culture following induction of apoptosis. The effect of STAT-1 deficiency on Bax expression and apoptosis will be measured and putative STAT-1 binding sites on Bax promoter will be investigated. 2) Determine the role of p38alpha mitogen-activated protein kinase (MAPK) as modulator of Bax activity during resection induced adaptation. A temporal and spatial profile of p38 expression/activity and Bax activation will be recorded after SBR in mice and complementary in vitro models of apoptosis. The effect of conditional, intestine-specific deletion of p38 expression on Bax activity and apoptosis will be determined after SBR. 3) Determine the mechanism for EGFR regulation of Bax expression and activity. The effect of enhanced or disrupted EGFR signaling on STAT-1 and p38 activation and expression will be recorded. The effect of attenuated STAT-1 or p38 expression in the context of EGFR inhibition on apoptosis and Bax activity and expression will be determined. The proposed studies in this application will identify the most relevant signaling pathway to direct apoptosis after massive SBR. A thorough understanding of the precise mechanism for induction of apoptosis is fundamental for bench-to-bedside translation of therapeutic targets intended to maximally stimulate regrowth of the intestinal mucosa in response to massive intestinal loss. PUBLIC HEALTH RELEVANCE: Following massive intestinal loss, the remaining bowel attempts to grow back to compensate. If this response is incomplete, the patient will be subjected to a lifetime of nutrition by vein and all the associated complications. This project is designed to understand the contribution of mucosal cell death to the process of intestinal regrowth. Understanding the exact molecular regulation of this important response may lead to innovative therapy intended to improve intestinal regrowth following a catastrophic loss of the intestine.