Molecular studies on the role of IL4 & IL13 in fibrosis

描述（由申请人提供）：我们的申请的主旨是基于两组调查结果。我们先前的数据表明促纤维化细胞因子在发生硬皮病样综合征的TSK小鼠皮肤纤维化中的作用。TSK小鼠在IL-4、IL-4 R基因或TGF-基因破坏后不发生皮肤硬化。IL-4受体基因缺失的小鼠TGF基因对胶原合成缺乏代偿作用，提示IL-4可能影响TGF基因在成纤维细胞中的表达。此外，报道的数据表明，促纤维化细胞因子的水平在人硬皮病患者的血清中升高。IL-4和IL-13上调胶原蛋白表达的分子机制知之甚少。具体而言，导致胶原过度合成和纤维化形成的信号传导途径以及IL-4和TGF-基因之间上位相互作用的分子基础尚不清楚。本应用程序的总体目标是解决这些问题。具体目标如下： 1.表征IL-4和IL-13在导致正常和TSK成纤维细胞中胶原表达上调的STAT-6依赖性途径中的作用。这将通过研究IL-4和IL-13刺激的成纤维细胞中STAT-6活化、启动子活性、胶原转录和胶原合成来研究。 2.阐明IL-4和TGF-基因相互作用调节胶原基因表达的分子机制。这将使用一组缺失构建体进行研究，所述缺失构建体含有TGF基因启动子和随后的CAT基因;将评估IL-4和IL-13对启动子活性的影响。我们还将研究STAT-6与TGF-基因启动子的DNA结合。还将检查AP-1、SP-1、NF-1、GATA 3和c-Maf转录因子，因为这些转录因子的DNA结合位点已被鉴定。 3.研究IL-4和IL-13激活MAPK模块对人皮肤成纤维细胞株胶原（I）基因表达的影响。将检查ERK、p38和c-Jun MAPK模块的各种分子的活化动力学。将在存在或不存在促纤维化细胞因子的情况下研究显性阳性和显性阴性构建体对胶原基因表达的影响。

DESCRIPTION (provided by applicant): The thrust of our application is based on two groups of findings. Our previous data demonstrate a role for profibrogenic cytokines in the occurrence of skin fibrosis in TSK mice that develop a scleroderma-like syndrome. TSK mice do not develop skin sclerosis following disruption of IL-4, IL-4R gene, or TGF-gene. The lack of a compensatory effect on collagen synthesis by TGF gein mice with a disrupted IL-4R gene suggests that IL-4 may influence the expression of TGF gene in fibroblasts. Additionally, reported data demonstrate that levels of profibrogenic cytokines are elevated in the serum of human scleroderma patients. Little is known of the molecular mechanisms by which IL-4 and IL-13 up-regulate collagen expression. Specifically, the signaling pathways that lead to over-synthesis of collagen and formation of fibrosis as well as the molecular basis of the epistatic interaction between IL-4 and TGF-genes are not known. The overall goal of this application is to address these questions. The specific aims are as follow: 1. To characterize the role of IL-4 and IL-13 in STAT-6-dependent pathways leading to up-regulation of collagen expression in normal and TSK fibroblasts. This wilt be studied by investigating STAT-6 activation, promoter activity, collagen transcription, and collagen synthesis in fibroblasts stimulated with IL-4 and IL-13. 2. To delineate the molecular mechanisms of the IL-4 and TGF-gene interaction that regulate the expression of collagen genes. This will be studied using a set of deletion constructs containing the TGF-gene promoter followed by the CAT gene; the effects of IL-4 and IL-13 on promoter activity will be assessed. We will also study the DNA binding of STAT-6 to the TGF-gene promoter. AP-1, SP-1, NF-1, GATA3, and c-Maf transcription factors will also be examined since the DNA binding sites for these transcription factors have been identified. 3. To study the effect of the activation of MAPK modules by IL-4 and IL-13 on the expression of the collagen (I) gene in a human skin fibroblast line. The kinetics of activation of various molecules of ERK, p38, and c- Jun MAPK modules will be examined. The effect of dominant-positive and dominant-negative constructs on collagen gene expression will be studied in the presence or absence of profibrogenic cytokines.