SIGNAL TRANDUCTION IN CELL PROLIFERATION

本申请中提出的工作的目的是探索NF-κ B在溶血磷脂酸（LPA）和1-磷酸鞘氨醇（S1 P）信号转导中的活化机制和意义。NF-κ B是二聚体家族的原型，其成分是转录因子Rel家族的成员。响应于细胞因子，NF-κ B在炎症、凋亡/抗凋亡和细胞复制过程中通常起着强制性作用。LPA和S1 P激活NF-κ B的发现是特别重要的，因为这两种激动剂作用于细胞以实现广泛的即时和持久作用，包括细胞形状和张力、趋化性、增殖和分化的范围。LPA和S1 P被认为在伤口愈合、组织再生和发育中至关重要。该项目的第一个目标是评估LPA和S1 P用于实现NF-κ B激活的中间途径，重点关注ERK激活级联反应的组分，单体G蛋白的Rho家族，活性氧以及与促炎细胞因子所采用的途径会聚的作用。成纤维细胞将构成主要实验模型，其中显性负性分子将通过显微注射或转染引入。第二个目标是检验NF-κ B的激活是通过G蛋白偶联受体的Edg家族的一个或多个成员实现的假设。这将通过将受体重建到其中NF-κ B通常不被LPA或S1 P激活的细胞中来进行。将使用新的[35 S] GTP γ S/GDP交换测定来确定由这些受体激活的G蛋白的身份。第三个目标是使用突变激活的亚基和嵌合体来鉴定引起NF-κ B激活的G蛋白亚基。相关α和β γ亚基的鉴定将有助于建立GPCR与相关下游现象（包括活性氧物质产生）之间的联系。第四个目标是确定NF-kappaB在LPA和S1 P信号传导中的特定作用。将测试NF-κ B在正常或异常信号传导过程中抑制细胞凋亡的假设，以及在DNA合成重新启动中的作用。我们还将鉴定由LPA和S1 P激活的基因，以及其激活依赖于NF-κ B的子集。

The objective of the work proposed in this application is to explore the mechanisms and significance of the activation of NF-kappaB in lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) signaling. NF-kappaB is the prototype of a family of dimers whose constituents are members of the Rel family of transcription factors. Responsive to cytokines, NF-kappaB plays an often obligatory role in the processes of inflammation, apoptosis/anti-apoptosis, and cell replication. The finding that LPA and S1P activate NF-kappaB is particularly significant, as both agonists act on cells to achieve a broad range of immediate and long-lasting effects which include ranges in cell shape and tension, chemotaxis, proliferation, and differentiation. LPA and S1P are thought to be critical in wound healing, tissue regeneration and development. The first goal of the project is to evaluate intermediate pathways employed by LPA and S1P to achieve activation of NF-kappaB, with a concentration on roles played by components of the ERK activation cascade, the Rho family of monomeric G proteins, reactive oxygen species, and pathways that converge with those employed by pro-inflammatory cytokines. Fibroblasts will constitute the primary experimental model, wherein dominant negative molecules will be introduced by microinjection or transfection. The second goal is to test the hypothesis that the activation of NF-kappaB is achieved through one or more members of the Edg family of G protein-coupled receptors. This will be carried out by reconstitution of receptors into cells in which NF-kappaB is nor normally activated by LPA or S1P. The identity of the G proteins activated by these receptors will be ascertained using a novel [35S]GTPgammaS/GDP exchange assay. The third goal is to identify G protein subunits that cause activation of NF-kappaB, using mutationally activated subunits and chimeras. The identification of relevant alpha and betagamma subunits will help forge the link between GPCRs and relevant downstream phenomena including reactive oxygen species generation. The fourth goal is to determine specific roles for NF-kappaB in LPA and S1P signaling. The hypothesis that NF-kappaB suppresses apoptosis in the course of normal or aberrant signaling will be tested, as well a role in reinitiation of DNA synthesis. We will also identify genes activated by LPA and S1P, and the subset whose activation is dependent on NF-kappaB.