Inhibition of Collagen Gene Expression in Systemic Sclerosis Dermal Fibroblasts by Mithramycin
Nora Sandorfi, Thomas Jefferson University; Natalia Louneva, Thomas Jefferson University; Elena Hitraya, Thomas Jefferson University; Gyorgy Hajnoczky, Thomas Jefferson University; Biagio Saitta, Thomas Jefferson University; and Sergio A. Jimenez, Thomas Jefferson University

DATE: December 2005
SOURCE: Annals of the Rheumatic Diseases, vol64, iss12, pp.1685-1691

267 Sandorfi et al.doc

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This article has been peer reviewed. It is the authors' final version prior to publication in Annals of the Rheumatic Diseases, Volume 64, Issue 12, December 2005, Pages 1685-1691. The published version is available at DOI: 10.1136/ard.2005.037515. Copyright © BMJ Publishing Group Ltd..



Introduction. The antitumor antibiotic mithramycin has also been shown to be a potent and effective inhibitor of fibrosis following glaucoma surgery. This DNA interacting drug displays high affinity binding to GC rich sequences in DNA, including those present in the promoter of the gene encoding the Α1 chain of type I collagen (COL1A1).

Objective. To evaluate the effects of mithramycin on the production of type I collagen in fibroblast cultures from patients with systemic sclerosis. Methods. Confluent cultures of dermal fibroblasts from patients with recent onset of the diffuse form of systemic sclerosis were treated with varius concentrations of mithramycin in vitro. Cell viability and protein expression were examined using fluorescence and confocal imaging. Type I collagen production was analyzed by confocal imaging and metabolic labeling with radiolabeled precursors. Steady-state messenger RNA (mRNA) levels and mRNA stability of Α1(I) collagen mRNA were assessed by Northern hybridizations. The transcription of COL1A1 was examined by transient transfections with reporter constructs containing various deletions of the gene promoter. Results. Treatment of systemic sclerosis fibroblasts with 10 nM to 100 nM did not cause any significant cytotoxicity. Type I collagen biosynthesis examined by metabolic labeling was decreased by 33-40% in cells cultured with 10 nM and by 50-70% in cells treated with 100 nM mithramycin. Similar results were obtained when type I collagen production by individual cells was assessed by confocal microscopy following 48 h treatment. Mithramycin at 50 nM also decreased the steady-state mRNA level of Α1(I) collagen by 40-60%. The effects of mithramycin on collagen gene expression were mediated by transcriptional and post-transcriptional mechanisms as shown by a reduction of COL1A1 promoter activity and by a decrease in the stability of these transcripts, respectively. Conclusions. The data indicate that mithramycin causes potent inhibition of collagen production and gene expression by systemic sclerosis cells in the absence of cytotoxic effects. In addition to potent transcriptional inhibition, the effects of mythramycin on COL1A1 expression appear to be due to a decrease in COL1A1 mRNA stability. The results suggest that mithramycin may be an effective treatment for the fibrotic process which is the hallmark of systemic sclerosis.