Molecular studies on the role of IL-4 & IL-13 in fibrosi

描述(申请人提供)：我们申请的主旨是基于我们报告的数据，在体内证明促纤维化细胞因子在TSK小鼠由于纤维蛋白-1基因突变而发展为硬皮病样综合征的皮肤纤维化发生中所起的作用。这些小鼠在IL-4、IL-4R和转化生长因子-α基因中断后没有出现纤维化。IL-4基因缺失的小鼠缺乏对转化生长因子-β合成胶原的代偿作用，提示1L-4可能影响成纤维细胞转化生长因子-β基因的表达。IL-4和IL-13上调成纤维细胞胶原基因的分子机制、IL-4和转化生长因子-β基因上位性相互作用的分子基础以及突变的FBN-1基因与TSK/小鼠胶原过度合成之间的联系尚不清楚。本应用程序的总体目标是解决这些特定问题。具体目标是： 1.研究导致成纤维细胞胶原基因上调的IL-4和1L-13信号通路。这将通过检测成纤维细胞的胶原启动子活性、胶原基因转录水平和胶原合成水平来研究Stat6和三个主要哺乳动物MAPK家族在IL-4/IL-13刺激的成纤维细胞中的作用 2.探讨IL-4和转化生长因子-β相互作用调控胶原基因表达的分子机制。我们建议克隆转化生长因子-β启动子I和2，并利用转化生长因子-β/猫以及转化生长因子-β启动子/猫融合基因的缺失突变体来研究促纤维化细胞因子对转化生长因子-α启动子活性的影响。此外，我们还将研究转录因子SP-1、AP-1、GATA3、Stat6、c-Maf和NF-1在1L-4和IL-13刺激下转染成纤维细胞后与转化生长因子-α启动子的DNA结合活性。 3.探讨Fbn-1基因突变与TSK/小鼠皮肤增生的关系。由于我们没有发现转导TSK-FBN-1基因的成纤维细胞胶原合成增加，我们推测突变的FBN-1基因与纤维化之间的联系是由产生促纤维化细胞因子的FBN-1特异性T细胞介导的。这一假设将通过产生FBN-1特异性T细胞克隆来验证。这些致病克隆将在过继转移实验中筛选出来，然后用于体外测定成纤维细胞与产生促纤维化细胞因子的T细胞克隆共培养时胶原的合成。

DESCRIPTION (provided by applicant): The thrust of our application is based on our reported data demonstrating in vivo the role of profibrogenic cytokines in the occurrence of skin fibrosis in TSK mice developing a scleroderma-like syndrome due to a mutation of Fibrillin-1 gene. These mice did not develop fibrosis subsequent to disruption of IL-4, IL-4R and TGF-a genes. The lack of compensatory effect on collagen synthesis by TGF-beta in mice with disrupted IL-4 gene suggested that 1L-4 may influence the expression of TGF-beta gene in fibroblasts. Little is known about the molecular mechanisms of upregulation of collagen genes in fibroblasts by 1L-4 and IL-13, on molecular basis of epistatic interaction between IL-4 and TGF-beta genes and on the link between mutated Fbn-1 gene and over synthesis of collagen in TSK/mice. The overall goal of this application is to address these specific questions. The specific aims are: 1. Characterize the IL-4 and 1L-13 signaling pathways leading to upregulation of collagen genes in fibroblasts. This will be studied by investigating the involvement of Stat6 and three major mammalian MAPK families in IL-4/IL-13 stimulated fibroblasts by measuring collagen promoter activity, the levels of the transcription of collagen genes and the level of synthesis of collagen by fibroblasts 2. To delinate the molecular mechanisms of IL-4 and TGF-beta interactions regulating the expression of collagen genes. We propose to clone TGF-beta promoter I and 2 and to study the effect of profibrogenic cytokines on TGF-a promoter activity using TGF-beta /CAT as well as deletion mutants of TGF-beta promoter/CAT fusion genes. In addition we will study the DNA binding activity to TGF-a promoter of transcription factors such as SP-, AP-1 GATA3, Stat6, c-Maf and NF-1 in fibroblasts transfected with deletion mutants of promoter and stimulated with 1L-4and IL-13. 3. To identify the link between mutated Fbn-1 and cutaneous hyperplasia in TSK/+ mice. Since we did not find an increased synthesis of collagen in fibroblasts transfected with TSK-Fbn-1 gene, we hypothesized that the link between mutated Fbn-1 gene and fibrosis is mediated by Fbn-1 specific T cells producing profibrogenic cytokines. This hypotheses will be tested by generating Fbn-1 specific T cell clones. The pathogenic clones will be selected in adoptive transfer experiments and then used in vitro to measure the synthesis of collagen by fibroblasts cocultured with T cell clones producing profibrogenic cytokines.