Systemic sclerosis, or scleroderma (SD), is a disease with vascular, autoimmune, and fibrotic components. The fibrotic component is characterized by an excess synthesis of extracellular matrix by fibroblasts, including overproduction of collagen. Collagen overproduction continues in ex-vivo cultured SD fibroblasts. This study is designed examine the transcriptional component of collagen overproduction. The promoter region of the alpha1(I) collagen gene has been characterized by several groups. Among the transcription factors that bind to the mouse collagen promoter is cKROX (mcKROX). This factor belongs to a group of Zn-finger containing early growth response genes, which play important roles in embryonic development. The mouse cKROX gene has been clones, but nothing is known about the human gene (hcKROX). In preliminary studies, a partial cDNA and a genomic clone for hcKROX have been isolated. In order to examine the possible importance of hcKROX in collagen dysregulation in scleroderma, the full length human cKROX cDNA and genomic sequences will be cloned. After determining the nucleotide sequence of hcKROX, its role in collagen regulation will be addressed. The expression of hcKROX will be compared in cultured fibroblasts from normal human skin, as well as from scleroderma patients, looking for a correlation between hcKROX and collagen expression. Cells will be treated with various proinflammatory cytokines to determine whether cKROX levels can be altered by these substances implicated in autoimmune disease. Initial characterization of the cKROX promoter will be carried out to delineate DNA sequences responsible for expression. Direct effects of hcKROX on collagen gene expression will be measured using a transient transfection assay in cultured fibroblasts. Collagen promoter fragments fused to the chloramphenicol acetyltransferase gene will be transfected into cultured human and mouse fibroblasts, along with a vector expressing the hcKROX protein. The initial description of mcKROX indicated that it may be a repressor of collagen gene transcription. If indeed hcKROX can repress collagen gene transcription, it could be an important target for possible therapeutic intervention for treating fibrosis associated with scleroderma.
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