SIGNALLING MECHANISMS DURING AIRWAY SMOOTH MUSCLE PROLIFERATION

呼吸道平滑肌质量增加被认为是导致呼吸道疾病的原因 哮喘患者的高反应性观察。潜力 呼吸道平滑肌细胞异常增殖的重要性凸显 需要了解与呼吸道有关的早期事件 平滑肌有丝分裂。为了达到这一总体目标，我们建议 以下具体目标：具体目标1：确定 细胞周期蛋白D(1)在牛气管肌细胞G(1)中的表达。我们将评估 细胞周期蛋白D(1)的表达及细胞周期蛋白D(1)依赖的激酶活性 视网膜母细胞瘤的免疫印迹促有丝分裂刺激及检测 蛋白质磷酸化。细胞周期蛋白D的必要性和充分性(1) 对于牛气管肌细胞周期进程将通过以下方法评估 Cyclin D(1)反义或正义基因稳定转染细胞 使用可诱导的Tet阻遏物系统，然后进行流式细胞术 和细胞计数。转化生长因子β和血管紧张素转换酶激活剂Forsklin的作用 腺苷环化酶对细胞周期蛋白D(1)合成、相关激酶活性的影响 并测定CDK抑制因子的表达。特定目标2： 确定牛气管肌细胞中MAPK的下游靶点。我们会 瞬时评估细胞周期蛋白D(1)启动子转录活性 转染细胞周期蛋白D(1)启动子，亚克隆到荧光素酶 记者。荧光素酶活性将在刺激后进行测量 有丝分裂原和非有丝分裂原，以及与 编码显性-阴性或结构性活性形式的MEK的质粒- 1，MAPK激活所需的和足够的双功能激酶 这些细胞。MAPK诱导所需的顺式作用DNA序列 Cyclin D(1)启动子活性将通过一系列Cyclin进行评估 D(1)5‘侧翼区缺失突变体。我们将确定准确的 参与转录的核转录因子(S) 通过执行凝胶迁移率变化分析、DNA酶足迹和 西南印迹。具体目标3：确定替代方案，RAF-1- 牛气管MAPK的独立上游激活途径 肌细胞。MEK-1的激酶活性将从Forsklin和 用Mono-Q色谱柱检测PDGF对细胞的影响 磷酸化试验。具有激酶活性的级分将被探测到 抗MEK激酶(Mekk)、Raf-1、A-Raf、B-Raf和MOS抗体。如果 经免疫印迹检测，MEK激活剂(S)的作用将为 通过顺序免疫沉淀确认，然后重新测试 MEK激酶活性。如果MEK激活剂是新的，它将被提纯 通过柱层析，通过活性部位标记来可视化， 进行了微测序和克隆。这项工作可能会对并行研究有所帮助 可能在哮喘中起作用并导致治疗的机制 干预措施。

Increased airway smooth muscle mass is thought to contribute to the airway hyperresponsiveness observed in patients with asthma. The potential importance of abnormal airway smooth muscle cell proliferation highlights the need for an understanding of the early events involved in airway smooth muscle mitogenesis. To achieve this overall goal, we propose the following Specific Aims: SPECIFIC AIM 1: Determine the precise role of cyclin D(1) in bovine tracheal myocyte G(1) traversal. We will assess cyclin D(1) expression and cyclin D(1)-dependent kinase activity following mitogenic stimulation by immunoblotting and measurement of retinoblastoma protein phosphorylation. The requirement and sufficiency of cyclin D(1) for bovine tracheal myocyte cell cycle progression will be assessed by stable transfection of cells with either cyclin D(1) antisense or sense cDNA using the inducible TET repressor system, followed by flow cytometry and cell counting. The effects of TGFbeta and forskolin, an activator of adenylate cyclase, on cyclin D(1) synthesis, associated kinase activity and cdk inhibitor expression will be determined. SPECIFIC AIM 2: Identify downstream targets of MAPK in bovine tracheal myocytes. We will assess cyclin D(1) promoter transcriptional activity in cells transiently- transfected with the cyclin D(1) promoter, subcloned into a luciferase reporter. Luciferase activity will be measured after stimulation with mitogens and non-mitogens, as well as following co-transfection with plasmids encoding dominant-negative or constitutively-active forms of MEK- 1, a dual-function kinase required and sufficient for MAPK activation in these cells. The cis-acting DNA sequences required for MAPK-induced cyclin D(1) promoter activity will be assessed with a series of cyclin D(1) 5' flanking region deletion mutants. We will identify the precise nuclear transcription factor(s) involved in this transcriptional regulation by performing gel mobility shift assays, DNase footprinting and Southwestern blotting. SPECIFIC AIM 3: Identify alternative, Raf-1- independent upstream activation pathways of MAPK in bovine tracheal myocytes. Kinase activity for MEK-1 will be isolated from forskolin and PDGF-treated cells using a Mono-Q column and assessed by in vitro phosphorylation assay. Fractions with kinase activity will be probed with antibodies against MEK kinase (MEKK), Raf-1, A-Raf, B-Raf and Mos. If detected by immunoblotting, the role of the MEK activator(s) will be confirmed by sequential immunoprecipitation, followed by re-testing for MEK kinase activity. If the MEK activator is novel, it will be purified by column chromatography, visualized by active-site labeling, microsequenced and cloned. This work may shed light on parallel mechanisms that may operate in asthma, and lead to therapeutic interventions.