Mithramycin selectively inhibits collagen-alpha 1(I) gene expression in human fibroblast.
The products of the collagen-alpha 1(I) and -alpha 2(I) genes form the triple helical molecule collagen type I, which constitutes the major ECM protein in tissue fibrosis. The collagen-alpha 1(I) gene is mainly transcriptionally regulated, and its promoter activity depends on the interaction of the transcription factors NF-I and Sp1 with a tandem repeat of evolutionary conserved NF-I/Sp1 switch elements. An increased affinity of Sp1 to these elements has been observed in experimental liver fibrosis. Here, we demonstrate that the DNA binding drug mithramycin displays a high affinity binding to the GC-rich elements in the collagen-alpha 1(I) promoter as measured by DNAse I protection and gel retardation assays. Mithramycin interferes with Sp1 but not with NF-I binding to these sites. At a concentration of 100 nM, mithramycin efficiently reduces basal and TGF-beta-stimulated alpha 1(I) gene expression in human primary fibroblasts. The transcriptional activity and mRNA steady state levels of other genes, including the collagenase gene, as well as the growth rate of fibroblasts remained unchanged on exposure to this drug. Taken together, our results indicate that the transcriptional activity of the type I collagen gene highly depends on its GC-rich regulatory elements, and further, that these elements can be differentially blocked, thereby changing the balance between ECM structural and degrading gene activities in human fibroblasts.
Nehls
M C
MC
Max-Delbrück Center for Molecular Medicine, Berlin, Germany.
Brenner
D A
DA
Gruss
H J
HJ
Dierbach
H
H
Mertelsmann
R
R
Herrmann
F
F
D015152:Blotting, Northern; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003094:Collagen / Q000096:biosynthesis*; D015342:DNA Probes; D018076:DNA, Complementary / Q000032:analysis; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects*; D016758:Genes, jun; D006801:Humans; D007700:Kinetics; D008168:Lung / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D008926:Plicamycin / Q000633:toxicity*; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism; D012313:RNA / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D015342:DNA Probes; D018076:DNA, Complementary; D009687:Nuclear Proteins; D014157:Transcription Factors; D012313:RNA; D003094:Collagen; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D008926:Plicamycin