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B-Myb acts as a repressor of human COL1A1 collagen gene expression by interacting with Sp1 and CBF factors in scleroderma fibroblasts

By Lucia Cicchillitti, Sergio A. Jimenez, Arturo Sala and Biaggio Saitta

Abstract

We investigated the role of B-Myb, a cell-cycle-regulated transcription factor, in the expression of the alpha1 (I) pro-collagen gene (COL1A1) in scleroderma fibroblasts. Scleroderma or SSc (systemic sclerosis) is a fibrotic disease characterized by excessive production of extracellular matrix components, especially type I collagen. Northern-blot analysis showed an inverse relationship between COL1A1 mRNA expression and that of B-Myb during exponential cell growth and during quiescence in human SSc fibroblasts. Overexpression of B-Myb in SSc fibroblasts was correlated with decreased COL1A1 mRNA expression. Transient transfections localized the down-regulatory effect of B-Myb to a region containing the proximal 174 bp of the COL1A1 promoter that does not contain B-Myb consensus binding sites. Gel-shift analysis, using nuclear extracts from normal and SSc fibroblasts transfected with B-Myb, showed no differences in DNA-protein complex formation when compared with the nuclear extracts from mock-transfected cells. However, we found that B-Myb decreases Sp1 (specificity protein 1) and CBF (CCAAT-binding factor) binding for their specific sites localized in the 174 bp COL1A1 proximal promoter. These results were also confirmed using B-Myb-immunodepleted nuclear extracts. Furthermore, immunoprecipitation assays using SSc nuclear extracts demonstrated a physical interaction of B-Myb with Sp1 and CBF transcription factors, and also an interaction between Sp1 and CBF. In addition, by employing full-length or deleted B-Myb cDNA construct, we found that B-Myb down-regulates the COL1A1 proximal promoter through its C-terminal domain. Thus these results suggest that B-Myb may be an important factor in the pathway(s) regulating collagen production in SSc fibroblasts

Topics: MyB, binding sites, CCAAT-Binding Factor, cell cycle proteins, cell line, cell nucleus, collagen type 1, DNA-Binding Proteins, dermis, Down-Regulation, fibroblasts, Gene Silencing, macromolecular substances, Promoter Regions, tertiary protein structure, Repressor Proteins, systemic scleroderma, Sp1 Transcription Factor, trans-activators, chemistry, metabolism, physiology, genetics, cytology, Rheumatology
Publisher: Jefferson Digital Commons
Year: 2004
OAI identifier: oai:jdc.jefferson.edu:sergio_jimenez-1000

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