B-Myb acts as a repressor of human COL1A1 collagen gene expression by interacting with Sp1 and CBF factors in scleroderma fibroblasts.
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.
Cicchillitti
Lucia
L
Division of Rheumatology, Department of Medicine, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.
Jimenez
Sergio A
SA
Sala
Arturo
A
Saitta
Biagio
B
D001665:Binding Sites; D023081:CCAAT-Binding Factor / Q000378:metabolism; D018797:Cell Cycle Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D024042:Collagen Type I / Q000235:genetics* / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D020405:Dermis / Q000166:cytology; D015536:Down-Regulation; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D020868:Gene Silencing; D006801:Humans; D046911:Macromolecular Substances; D008297:Male; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000378:metabolism*; D012595:Scleroderma, Systemic / Q000235:genetics* / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*
D023081:CCAAT-Binding Factor; D018797:Cell Cycle Proteins; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D004268:DNA-Binding Proteins; C064352:MYBL2 protein, human; D046911:Macromolecular Substances; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor; D015534:Trans-Activators