Inhibition of basal and transforming growth factor-beta-induced stimulation of COL1A1 transcription by the DNA intercalators, mitoxantrone and WP631, in cultured human dermal fibroblasts.
The Sp1 transcription factor plays a crucial role in COL1A1 transcriptional regulation under normal and pathologic conditions and under the effects of transforming growth factor-beta (TGF-beta). Sp1 activity is elevated in numerous diseases characterized by tissue fibrosis. Therefore, inhibition of Sp1 binding to COL1A1 regulatory elements may represent an effective treatment for these diseases. Here we examined the effect of two DNA intercalators that prevent Sp1 binding on the expression of COL1A1 in human dermal fibroblasts. Cultured human adult dermal fibroblasts were treated with WP631 (50 pm/ml to 500 nm/ml) or mitoxantrone (5-500 nm/ml). Cytotoxicity, cellular apoptosis, and collagen deposition were examined by fluorescence microscopy. Collagen production was examined by enzyme-linked immunosorbent assay and metabolic labeling, COL1A1 steady-state mRNA levels, and stability were assessed by Northern hybridizations, and COL1A1 transcription by in vitro nuclear transcription assays and transient transfections. Competition of the drugs for Sp1 binding and their effect on TGF-beta-induced stimulation of COL1A1 transcription was also examined. Both drugs caused a dose-related inhibition of COL1A1 production and mRNA levels without cytotoxicity or apoptosis. COL1A1 transcriptional activity showed a profound reduction mediated by a short proximal promoter region containing an Sp1-binding element at -87 to -82 bp. Furthermore, both drugs inhibited Sp1 DNA complex formation and abrogated the stimulation of COL1A1 transcription induced by TGF-beta. WP631 showed 10-fold higher potency than mitoxantrone. These data indicate that mitoxantrone and WP631 are very potent inhibitors of basal and TGF-beta-stimulated COL1A1 expression and suggest that Sp1-DNA intercalators may be an effective and novel approach for the treatment of fibrotic diseases and modulation of profibrogenic effects of TGF-beta.
Division of Rheumatology, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Svetlana
S
Gaidarova
Sergio A
SA
Jiménez
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002470:Cell Survival; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics / Q000378:metabolism*; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D003630:Daunorubicin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D020405:Dermis / Q000166:cytology; D005347:Fibroblasts / Q000187:drug effects* / Q000502:physiology; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007364:Intercalating Agents / Q000494:pharmacology*; D008942:Mitoxantrone / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020871:RNA Stability; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D000970:Antineoplastic Agents; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D005353:Fibronectins; D007364:Intercalating Agents; D016329:Sp1 Transcription Factor; D016212:Transforming Growth Factor beta; C104426:WP 631; D003094:Collagen; D008942:Mitoxantrone; D003630:Daunorubicin