Mechanisms of KSR regulation in intestinal cell survival

描述（由申请人提供）：健康的肠上皮需要细胞增殖和凋亡的紧密协调，以维持完整的屏障和消化功能、环境采样和免疫调节控制。我们实验室以前的工作已经确定Ras激酶抑制因子（KSR）作为肿瘤坏死因子（TNF）启动的信号通路在这个组织中的一个重要介质。事实上，在没有KSR激酶活性的情况下，暴露于病理水平的TNF的肠上皮细胞（IEC）经历凋亡。此外，我们的研究结果表明，KSR是需要TNF刺激细胞外调节激酶1和2（ERK 1/ERK 2），核因子（NF）-κ B和Akt/蛋白激酶B。综上所述，这些数据将KSR定位为精氨酸介导的细胞存活的关键调节因子。本提案的目的是检验我们的假设，即KSR激酶活性通过激活抗凋亡信号转导途径调节炎症反应期间IEC的存活。支持KSR功能在体内损伤和修复中的作用，我们有初步数据表明KSR+/-IL - 10+/-小鼠自发发展炎性肠病（IBD），结肠上皮细胞凋亡增加。因此，目的1旨在通过诱变、胰蛋白酶磷酸肽图谱和体外神经酰胺激活研究来确定调节KSR激活的机制。为此，我们开发了一种新的KSR -/-小鼠结肠上皮细胞系，这将大大有助于结构-功能分析。目标2的重点是通过体外激酶试验、突变分析、共沉淀试验和cDNA表达文库的筛选来鉴定IEC中KSR的底物和下游靶标。在目标3中，我们将使用TNF诱导的肠病和炎症性肠病（IBD）的动物模型研究KSR在体内肠上皮损伤中的生物学作用。由于TNF与许多胃肠道疾病的发病机制有关，包括IBD、坏死性小肠结肠炎、乳糜泻和非甾体抗炎药肠病，因此这些研究对由细胞增殖、分化和凋亡程序改变引起的许多肠道疾病具有意义。此外，他们将确定KSR是否是胃肠道炎症的潜在治疗靶点。

DESCRIPTION (provided by applicant): A healthy intestinal epithelium requires tight coordination of cell proliferation and apoptosis to maintain intact barrier and digestive functions, environmental sampling and immunoregulatory control. Previous work from our laboratory has identified kinase suppressor of Ras (KSR) as an essential mediator of tumor necrosis factor (TNF)-initiated signaling pathways in this tissue. In fact, without KSR kinase activity, intestinal epithelial cells (IECs) exposed to pathological levels of TNF undergo apoptosis. Furthermore, our findings indicate that KSR is required for TNF stimulation of extracellular-regulated-kinases 1 and 2 (ERK1/ERK2), nuclear factor (NF)-kappaB and Akt/protein kinase B. Taken together, these data position KSR as a key regulator of cytokine-mediated cell survival. The goal of this proposal is to test our hypothesis that KSR kinase activity regulates IEC survival during the inflammatory response through activation of anti-apoptotic signal transduction pathways. Supporting a role for KSR function in injury and repair in vivo, we have preliminary data that the KSR+/-IL - 10+/- mouse spontaneously develops inflammatory bowel disease (IBD) with increased apoptosis of colon epithelial cells. Therefore, Aim 1 is designed to determine the mechanisms regulating KSR activation through mutagenesis, tryptic phosphopeptide mapping and in vitro ceramide activation studies. For this Aim, we have developed a novel KSR -/- mouse colon epithelial cell line which will greatly facilitate structure-function analyses. The focus of Aim 2 is to identify substrates and downstream targets of KSR in IECs through in vitro kinase assays, mutational analysis, co-precipitation assays and screening of Cdna expression libraries. In Aim 3 we will study the biological role of KSR in intestinal epithelial injury in vivo using animal models of TNF-induced enteropathy and inflammatory bowel disease (IBD). Because TNF has been implicated in the pathogenesis of a number of gastrointestinal diseases including IBD, necrotizing enterocolitis, celiac disease and non-steroidal anti-inflammatory drug enteropathy, these studies have implications for a number of intestinal conditions resulting from altered programs of cellular proliferation, differentiation and apoptosis. Further, they will determine if KSR is a potential therapeutic target for gastrointestinal inflammation.