Mechanisms of KSR regulation in intestinal cell survival

描述(申请人提供)：健康的肠道上皮需要细胞增殖和凋亡的紧密协调，以保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的激酶抑制因子(KSR)是该组织中肿瘤坏死因子(TNF)启动的信号通路的重要中介。事实上，在没有KSR激酶活性的情况下，暴露于病理水平的肿瘤坏死因子的肠上皮细胞(IECs)会发生凋亡。此外，我们的发现表明，KSR是肿瘤坏死因子刺激细胞外调节激酶1和2(ERK1/ERK2)、核因子kappaB和Akt/蛋白激酶B所必需的。综上所述，KSR是细胞因子介导的细胞生存的关键调节因子。这项提议的目的是验证我们的假设，即KSR激酶活性通过激活抗凋亡信号转导通路来调节炎症反应期间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+/-小鼠自发发生炎症性肠病(IBD)，结肠上皮细胞凋亡增加。因此，Aim 1旨在通过诱变、胰酶磷酸肽定位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，它将极大地促进结构-功能分析。目标2的重点是通过体外激酶分析、突变分析、共沉淀法和筛选cDNA表达文库来确定IECS中KSR的底物和下游靶点。在目的3中，我们将利用肿瘤坏死因子诱导的肠病和炎症性肠病(IBD)的动物模型，研究KSR在活体肠道上皮损伤中的生物学作用。由于肿瘤坏死因子参与了许多胃肠道疾病的发病机制，包括IBD、坏死性小肠结肠炎、乳糜泻和非类固醇抗炎药物肠病，这些研究对细胞增殖、分化和凋亡程序改变引起的一些肠道疾病有一定的意义。此外，他们将确定KSR是否为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.