REGULATION OF KERATINOCYTE SURVIVAL

多细胞生物的体内平衡部分取决于 使细胞经历受控的细胞死亡或凋亡。相反地， 诱导细胞凋亡的途径的中断通常与 肿瘤发展 目前，上皮细胞的分子调控 除了酪氨酸激活外，对存活了解很少 已知通过底物粘附激酶（粘着斑激酶） 被要求。 我们最近的研究表明， 因子受体（EGF-R）酪氨酸激酶同样支持 人正常角质形成细胞而不是非上皮正常人细胞， 包括黑素细胞和成纤维细胞。 建议的实验是 旨在确定连接EGF-R激活与 调节上皮细胞存活。 在初步研究中， 观察到（i）EGF-R依赖性凋亡通过脱离而加速 角质形成细胞的细胞外基质;（ii）激活的角质形成细胞， EGF-R的酪氨酸激酶部分和Ras/Raf/MEK的活化 需要信号级联以保护角质形成细胞免于凋亡; 和（iii）阻断EGF-R与抗EGF-R的下调有关。 凋亡Bcl-xL分子。 基于这些结果，我们假设， EGF-R酪氨酸磷酸化引发的信号通路 对角质形成细胞的存活至关重要，EGF-R的激活可以保护 正常角质形成细胞凋亡通过调节成员的 Bcl-2蛋白家族。 为了验证这些假设，我们将：1）调查 EGF-R阻断对成员mRNA和蛋白表达的影响 Bcl-2蛋白家族（Bcl-2、Bad、巴克、Bax）和死亡基因（ICE; CPP 32）; 2）通过强制过表达确定功能性 Bcl-xL和其他相关的Bcl-2家族成员对 角质形成细胞凋亡和，3）检查EGF-R依赖性信号 负责存活和上调Bcl-2的转导途径， 角质形成细胞中的xL。 EGF-R相关分子的依赖性表达 将使用已建立的免疫沉淀、Western和 北方印迹、RNA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， 使永生化角质形成细胞中的基因表达可调节， 细胞系HaCaT和正常角质形成细胞中。 这些长期目标 研究的目的是阐明与异常细胞相关的分子途径， 上皮恶性肿瘤的存活率。

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.