Cell proliferation in models of fibrosis

在真核细胞周期中，进入G1期和进入S期的过程受到仔细的调控。细胞增殖的诱导包括以下几个步骤：1)丝裂原活化蛋白激酶的激活，2)c-fos和c-jun等早期反应基因的表达，3)转录许可和S时相进入。石棉、活性氧和活性氮物种(ROS/RNS)通过激活或扰动MAPK级联、改变G1期细胞周期蛋白的活性或改变E2F的活性来影响细胞周期通过G1并进入S期。在这里，我们提出了特定的目标来研究变态反应性气道高反应/纤维化和石棉肺模型中通过G1和S阶段进入的进展情况。首先，我们将记录同步的小鼠肺泡II型(C1)细胞对石棉、RO2和阳离子蛋白的反应中，细胞周期进展与MAPK激活模式、细胞周期蛋白D1的表达以及CDC6来源许可的关系。其次，我们将使用细菌人工染色体(BAC)的同源重组来产生Cyclin D1和cfdc6的等位基因，这些等位基因在基因的3‘非翻译区包含内部核糖体进入位点(IRES)增强型绿色荧光蛋白(EGFP)表达盒。利用一种新的基因转移技术，这些等位基因将被转移到培养的细胞中，并将评估BAC等位基因在细胞周期中的调控表达。第三，这些BAC等位基因正确表达编码细胞周期蛋白D1-IRES-EGFP和CDC6-IRES-EGFP的mRNAs，将被用来产生转基因报告小鼠。携带这些转基因BAC等位基因的小鼠将被用于研究项目1-3中使用的吸入模型中上皮细胞通过G1期和S期的进展。最后，BAC转基因小鼠与表达显性阴性MEK1的小鼠的回交将被用来确定ERKs在G1期和S期进入进展中的调控作用，以及它们与上皮细胞增殖和纤维化发展的关系。

Progression through G1 and entry into the S phase are carefully regulated during the eukaryotic cell cycle. Induction of cell proliferation involves several sequential steps that include: 1) activation of mitogen-activated protein kinases (MAPK), 2) expression of early response genes such as c- fos and c-jun, 3) transcriptional licensing and S phase entry. Asbestos, reactive oxygen, and reactive nitrogen species (ROS/RNS) influence progression through G1 and entry into the S phase by activating or perturbing MAPK cascades, altering the activity of G1 cyclins or alt4ering the activation of E2F. Here, we propose specific aims to examine progression through G1 and S phase entry in models of allergic airway hyperresponsiveness/fibrosis and asbestosis. First, we will document the relationship between cell cycle progression and patterns of MAPK activation, expression of cyclin D1, and origin licensing by Cdc6 in synchronized murine alveolar type II (C1) cells in response to asbestos, RO2 and cationic proteins. Second, we will use homologous recombination of bacterial artificial chromosomes (BACs) to generate alleles of cyclin D1 and cfdc6 that contain internal ribosome entry sites (IRES)-enhanced green fluorescent protein (EGFP) expression cassettes in the 3' untranslated regions of the genes. Using a novel gene transfer technique, these alleles will be transferred into cells in cultured, and regulated expression during the cell cycle of dicistonic mRNAs from the BAC alleles will be assessed. Third, those BAC alleles displaying proper expression of dicistonic mRNAs encoding cyclin D1-IRES-EGFP and Cdc6-IRES-EGFP will be used to generate transgenic reporter mice. Mice bearing these transgenic BAC alleles will be used to study progression through G1 and commitment to S phase in epithelial cells in inhalation models used in projects 1-3. Finally, backcrossing of BAC transgenic mice with mice expressing dominant negative MEK1 will be used to define the role of ERKs in governing progression through G1 and S phase entry, as well as their relationship to the development of epithelial cell proliferation and fibrosis.