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

描述（由申请人提供）：我们的申请的主旨是基于我们报告的数据，该数据证明了促纤维化细胞因子在由于原纤维蛋白-1基因突变而发展成硬皮病样综合征的TSK小鼠中发生皮肤纤维化中的体内作用。这些小鼠在IL-4、IL-4 R和TGF-α基因破坏后没有发生纤维化。IL-4基因缺失小鼠TGF-β对胶原合成的代偿作用缺乏，提示IL-4可能影响成纤维细胞TGF-β基因的表达。关于IL-4和IL-13上调成纤维细胞中胶原基因的分子机制、IL-4和TGF-β基因之间的上位相互作用的分子基础以及突变的Fbn-1基因与TSK/小鼠中胶原过度合成之间的联系知之甚少。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.表征导致成纤维细胞中胶原基因上调的IL-4和IL-13信号通路。这将通过测量胶原启动子活性、胶原基因转录水平和成纤维细胞胶原合成水平来研究Stat 6和三种主要哺乳动物MAPK家族在IL-4/IL-13刺激的成纤维细胞中的参与来研究 2.阐明IL-4和TGF-β相互作用调控胶原基因表达的分子机制。我们建议克隆TGF-β启动子I和2，并使用TGF-β/CAT以及TGF-β启动子/CAT融合基因的缺失突变体来研究促纤维化细胞因子对TGF-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、GATA 3、Stat 6、c-Maf和NF-1在TGF-α启动子缺失突变体转染的成纤维细胞中与TGF-α启动子的DNA结合活性。 3.确定突变的Fbn-1与TSK/+小鼠皮肤增生之间的联系。由于我们没有发现用TSK-Fbn-1基因转染的成纤维细胞中胶原合成增加，我们假设突变的Fbn-1基因和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。将通过产生Fbn-1特异性T细胞克隆来测试该假设。在过继转移实验中选择致病性克隆，然后在体外用于测量与产生促纤维化细胞因子的T细胞克隆共培养的成纤维细胞的胶原合成。

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.