REGULATION OF KERATINOCYTE SURVIVAL

角质细胞存活的调节

• 批准号: 2896780
• 负责人: ULRICH RODECK
• 金额: $ 24.37万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2896780
• 关键词: RNase protection assay; apoptosis; biological signal transduction; cellular oncology; cysteine endopeptidases; epidermal growth factor; epithelium; gene expression; growth factor receptors; human tissue; immunoprecipitation; keratinocyte; northern blottings; polymerase chain reaction; protein tyrosine kinase; tissue /cell culture; western blottings

Homeostasis of multicellular organisms depends in part on the ability of cells to undergo controlled cell death or apoptosis. Conversely, disruption of pathways that induce apoptosis is often associated with tumor development. At present, the molecular control of epithelial cell survival is poorly understood except that activation of a tyrosine kinase (focal adhesion kinase) through substrate adhesion is known to be required. Our recent studies indicate that the epidermal growth factor-receptor (EGF-R) tyrosine kinase similarly supports survival of human normal keratinocytes but not of non-epithelial normal human cells, including melanocytes and fibroblasts. The proposed experiments are designed to define molecular pathways linking EGF-R activation to regulation of epithelial cell survival. In preliminary studies we observed that (i) EGF-R-dependent apoptosis is accelerated by detachment of keratinocytes from extracellular matrix; (ii) activation of the tyrosine kinase moiety of the EGF-R and activation of the Ras/Raf/MEK signaling cascade is required to protect keratinocytes from apoptosis; and (iii) blocking EGF-R is associated with down-regulation of the anti- apoptotic Bcl-xL molecule. Based on these results we hypothesize that signaling pathways triggered by tyrosine phosphorylation of the EGF-R are critical to keratinocyte survival and that EGF-R activation protects normal keratinocytes from apoptosis via regulation of members of the Bcl-2 protein family. To test these hypotheses we will: 1) investigate the effect of EGF-R blockade on mRNA and protein expression of members of the Bcl-2 protein family (Bcl-2, Bad, Bak, Bax) and death genes (ICE; CPP32); 2) determine by forced overexpression the functional contribution of Bcl-xL and other relevant Bcl-2 family members to keratinocyte apoptosis and, 3) examine EGF-R-dependent signal transduction pathways responsible for survival and up-regulation of Bcl- xL in keratinocytes. EGF-R dependent expression of relevant molecules will be assessed using established immunoprecipitation, Western and Northern blotting, RNAse protection, and RT-PCR methods. Forced gene expression is accomplished using advanced gene expression systems which enable regulatable gene expression in the immortalized keratinocyte cell line HaCaT and in normal keratinocytes. The long-term goal of these studies is to elucidate molecular pathways relevant to aberrant cell survival in epithelial malignancies.

多细胞生物体的动态平衡在一定程度上取决于 细胞发生受控的细胞死亡或凋亡。相反， 诱导细胞凋亡的通路中断通常与 肿瘤的发展。目前，上皮细胞的分子调控 除了酪氨酸的激活外，人们对存活的了解很少 已知通过底物黏附的蛋白激酶(焦点黏附激酶) 是必需的。我们最近的研究表明，表皮的生长 因子受体(EGF-R)酪氨酸激酶类似地支持 人类正常角质形成细胞，但不是非上皮性正常人类细胞， 包括黑素细胞和成纤维细胞。建议的实验是 旨在定义将EGF-R激活连接到 上皮细胞存活的调控。在初步研究中，我们 观察到(I)脱离可加速EGF-R依赖的细胞凋亡 来自细胞外基质的角质形成细胞；(Ii)激活 EGF-R酪氨酸激酶部分与Ras/Raf/MEK的激活 角质形成细胞的凋亡需要信号级联反应来保护； 和(Iii)阻断EGF-R与下调抗- 凋亡的Bclxl分子。根据这些结果，我们假设 EGF-R酪氨酸磷酸化触发的信号转导通路 对角质形成细胞的存活至关重要，并且EGF-R的激活保护 正常角质形成细胞通过调节 BCL-2蛋白家族。为了检验这些假设，我们将：1)调查 EGF-R受体拮抗剂对成员基因和蛋白表达的影响 Bcl2蛋白家族(Bcl2、Bad、Bak、Bax)和死亡基因(ICE； CPP32)；2)通过强制过度表达来确定功能 Bc l-xl等相关Bc l-2家族成员在肿瘤发病中的作用 角质形成细胞凋亡和，3)检测EGF-R依赖的信号 Bc l-2存活和上调的信号转导途径 角质形成细胞中的XL。EGF-R依赖的相关分子表达 将使用已建立的免疫沉淀、Western和 Northern印迹、核糖核酸酶保护和RT-PCR方法。强迫基因 表达是使用先进的基因表达系统完成的，该系统 在永生化角质形成细胞中实现可调控的基因表达 并在正常角质形成细胞中表达。这些项目的长期目标是 研究旨在阐明与异常细胞相关的分子途径 上皮性恶性肿瘤的存活率。