Role of E4BP4 in Glucocorticoid-Evoked Lymphocyte Apoptosis

描述(申请人提供)：细胞凋亡是一种精心安排的生理性细胞死亡途径，通过改变生长调节/死亡诱导基因的表达而激活。这种PI的长期目标是了解细胞凋亡的分子途径，重点是基因调控的变化。在一对姐妹人类白血病T细胞系CEM-C7-14和CEM-C1-15中，我们观察到转录抑制因子E4BP4的表达和去磷酸化与细胞凋亡之间的相关性。CEM-C7-14和CEM-C1-15分别对糖皮质激素(GC)诱导的凋亡敏感或无效。虽然其他几种基因产物也与GC诱导的白血病T细胞凋亡有关，但在本提案中，我们将重点评估E4BP4在调节这一过程中的相对贡献，其作用模式，以及它与GC引发的其他变化的关系。根据新的初步数据和审查员的意见，对重新提交的具体目标进行了修订。具体目的1将通过报告基因和磷酸酶抑制剂处理的细胞的凋亡检测，分析E4BP4去磷酸化与其抑制转录和调控细胞凋亡的能力的相关性。在特定目标2中，E4BP4调节下游进化保守的凋亡调节因子(Slug/Snail，Bim/Puma和Bcl2/Bcl2/Bclxl)的能力将通过E4BP4在CEM-C1-15细胞中的异位表达或通过siRNA介导的E4BP4在CEM-C7-14细胞中的下调来评估。异位表达小鼠E4BP4的CE-C1-15衍生克隆已被开发出来，并对GC诱导的细胞凋亡表现出敏感性。具体目标3将分别使用GRE驱动的荧光素酶报告PHH-Luc和c-myc启动子-荧光素酶报告HBMLuc来研究E4BP4调节GR依赖的转录激活和抑制的能力。将在CEM细胞中测试E4BP4与其他已知的T细胞凋亡调节因子的表达的关系，包括Cyclin D3、p27kip1、c-jun、c-Myc和BTG1，这些细胞被操纵过表达或关闭E4BP4的表达。 与公共卫生相关：细胞凋亡异常或不足会导致自身免疫、免疫缺陷、神经变性和癌症等疾病。这里提出的研究将促进促进或防止细胞凋亡的策略的发展，并管理这些情况。

DESCRIPTION (provided by applicant): Apoptosis is a well-orchestrated pathway of physiological cell death, activated through alterations in the expression of growth regulatory/death-inducing genes. The long-term goal of this PI is to understand the molecular pathway for apoptosis, with emphasis on gene regulatory changes. In a pair of sister human leukemic T cell lines, CEM-C7-14 and CEM-C1-15, that are susceptible or refractory, respectively, to glucocorticoid (GC)-evoked apoptosis, we have observed a correlation between expression and dephosphorylation of the transcriptional repressor E4BP4, a homolog of the pro-apoptotic C. elegans ces-2 gene product, and apoptosis. While several other gene products are also implicated in GC-evoked apoptosis of leukemic T cells, in this proposal we will focus on evaluating the relative contribution of E4BP4 in modulating the process, its mode of action, and its relationship to other changes triggered by GCs. Based on new preliminary data and comments from reviewers, the specific aims of this resubmission have been revised. Specific aim 1 will analyze the relevance of E4BP4 dephosphorylation on its ability to repress transcription and modulate apoptosis through reporter gene and apoptosis assays of cells treated with kinase and phosphatase inhibitors. In specific aim 2, the ability of E4BP4 to regulate downstream evolutionary conserved modulators of apoptosis (Slug/Snail, Bim/Puma, and Bcl2/Bcl-xL) will be evaluated through ectopic E4BP4 expression in CEM-C1-15 cells, or siRNA-mediated E4BP4 knock-down in CEM-C7-14 cells. A CE-C1-15 derived clone expressing ectopic mouse E4BP4 has been developed and exhibits sensitivity to GC-evoked apoptosis. Specific aim 3 will study the ability of E4BP4 to modulate GR-dependent transcriptional activation and repression using the GRE-driven luciferase reporter pHH-Luc, and the c-myc promoter-luciferase reporter HBMLuc, respectively. The relationship between E4BP4 and expression of other known modulators of T cell apoptosis, including cyclin D3, p27kip1, c-Jun, c-Myc and BTG1, will be tested in CEM cells manipulated to either over-express or shut off E4BP4 expression. Relevance to Public Health: Aberrant or inadequate apoptosis contributes to diseases such as autoimmunity, immunodeficiency, neurodegeneration, and cancer. Studies proposed here will promote development of strategies to enforce or prevent apoptosis and manage these conditions.