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.
