The long-term objectives of our research focus on elucidation of the mechanisms that mediate controlled regulation of the extracellular matrix (ECM) turnover in healthy tissues and uncontrolled ECM turnover under pathological conditions. Excessive deposition of the ECM occurs in fibrotic diseases and is the main pathological manifestation of scleroderma (SSc), while accelerated ECM degradation occurs in the rheumatoid arthritis and during cancer progression and metastasis. During the previous funding period we proposed to test the hypothesis that constitutive upregulation of collagen and other ECM genes in SSc is caused by different expression levels and modification patterns of transcription factors involved in collagen gene expression by SSc fibroblasts. Our recent findings significantly strengthen this hypothesis. We demonstrated that Ets family transcription factors, particularly Fli-1, are important regulators of collagen homeostasis in healthy skin fibroblasts and among the key contributors to the excessive collagen production by SSc fibroblasts. We demonstrated that Fli-1 is a specific repressor of collagen gene transcription in cultured fibroblasts and in healthy skin in vivo. In addition, we made a novel observation that Fli-1 and Ets-1 are targeted by the TGF-beta signaling pathway, which induces rapid hyperacetylation of these proteins in healthy fibroblasts. In contrast, Fli-1 is constitutively hyperacetylated in SSc fibroblasts. Fli-l is also underexpressed in SSc fibroblasts in vitro. Significantly, during active stage of disease, Fli-1 protein is undetectable in fibroblasts in SSc lesions, correlated with increased collagen production. Based on these observations we hypothesize that in human dermal fibroblasts, Ets factors are the critical mediators of the fibrogenic TGF-beta signaling pathway and that alterations of the levels and acetylation status of Fli-1 contribute to dysregulation of ECM deposition in SSc. To test this hypothesis and to establish a direct linkage between Fli-1 and the development of fibrosis in SSc we propose five Specific Aims.
In Specific Aim 1 we will further delineate the role of Fli-1 in the formation of the multiprotein complex that regulates transcription of the COL 1A2 gene, focusing on the TGF-beta induced post-translational modifications of Fli-1, including acetylation and phosphorylation.
In Specific Aim 2 we will employ several approaches, including yeast two-hybrid screens to identify fibroblast-specific proteins that physically interact with Fli-1.
In Specific Aim 3 we will determine whether Fli-1 contributes to phenotypic alterations of SSc fibroblasts.
In Specific Aim 4 we will analyze in vivo expression of Fli-1 protein and its partners, as well as Fli-1 target genes in SSc and healthy skin biopsies.
In Specific Aim 5 we will use established animal models of SSc to determine the contribution of Fli-1 expression to fibrosis development in vivo.
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