Inhibition of collagen gene expression in systemic sclerosis dermal fibroblasts by mithramycin

Abstract

Background: The anti-tumour antibiotic mithramycin is also a potent inhibitor of fibrosis after glaucoma surgery. This 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 COL1A1 expression in systemic sclerosis fibroblasts.

Methods: Confluent cultures of dermal fibroblasts from patients with recent onset diffuse systemic sclerosis were treated with mithramycin in vitro. Cell viability and protein expression were examined by fluorescence and confocal imaging. Type I collagen production was analysed by confocal imaging and metabolic labelling. COL1A1 messenger RNA levels and stability were assessed by northern hybridisation, and COL1A1 transcription was examined by transient transfections.

Results: Treatment of systemic sclerosis fibroblasts with mithramycin (10–100 nmol/l) did not cause significant cytotoxicity. Type I collagen biosynthesis decreased by 33–40% and 50–70% in cells cultured with mithramycin at 10 nmol/l and 100 nmol/l, respectively. Mithramycin at 50 nmol/l decreased COL1A1 mRNA levels by 40–60%. The effects of mithramycin on collagen gene expression were mediated by transcriptional and post-transcriptional mechanisms as shown by the reduction of COL1A1 promoter activity and by a decrease in the stability of these transcripts, respectively.

Conclusions: Mithramycin causes potent inhibition of collagen production and gene expression in systemic sclerosis dermal fibroblasts in vitro in the absence of cytotoxic effects. These results suggest that this drug may be an effective treatment for the fibrotic process which is the hallmark of systemic sclerosis.

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Figure 1.

 Effects of mithramycin (MM) on cell viability. Cell viability was examined as described in "Materials and methods" employing various concentrations of mithramycin (10 nmol/l to 1 µmol/l) applied for 48 hours to confluent systemic sclerosis fibroblast cultures in three experiments using three different cell lines, each in duplicate. The results showed that mithramycin at 1 µmol/l decreased the number of cells by over 80%, whereas 10 nmol/l (or 50 nmol/l; not shown) did not cause any significant change in cell viability. The phase contrast image shows that treatment with mithramycin at 1 µmol/l altered the shape of the cells which shrunk and lost their cytoplasm, although the nuclear staining with Hoechst did not indicate apoptotic process. No change in cell shape was seen using mithramycin at 10 nmol/l.

Figure 2.

 Evaluation of type I collagen production in systemic sclerosis dermal fibroblast monolayer cultures by confocal imaging after mithramycin treatment. Type I collagen present in individual cells of systemic sclerosis fibroblast cultures was examined by immunomicroscopy and confocal microscopy imaging as described in "Materials and methods" in five separate experiments using four cell lines. (A) Fluorescence of the FITC conjugated antirabbit IgG labelling type I collagen is shown in green and that of rhodamine labelled phalloidin labelling actin in red in one illustrative experiment. (B) Quantitative evaluation of the mithramycin effect on type I collagen production at the individual cell level showed that 48 hours of treatment with mithramycin at 10 nmol/l and 100 nmol/l decreased the protein level by about 33% and 50%, respectively. The results show the averages (SD) from five separate experiments with four different cell lines.

Figure 3.

 Evaluation of type I collagen biosynthesis by metabolic labelling after mithramycin treatment. Confluent monolayer cultures of systemic sclerosis fibroblasts were labelled with [¹⁴C]proline under control conditions or under treatment with various concentrations of mithramycin, and collagen biosynthesis was determined employing a specific collagenase assay in pooled media plus cell lysate samples as described in "Materials and methods". (A) Collagen biosynthesis was decreased by 30% using mithramycin at 10 nmol/l and by 53% using mithramycin at 100 nmol/l. The results of one experiment are shown. (B) SDS gel electrophoresis of labelled proteins from the medium showed similar results with a 30% decrease in [¹⁴C]collagen after 10 nmol/l mithramycin treatment and about a 50% decrease after 100 nmol/l mithramycin administration. Two separate experiments, each in duplicate, were performed with similar results.

Figure 4.

 Analysis of steady state mRNA levels by northern blot hybridisation. Confluent monolayer cultures of two systemic sclerosis dermal fibroblast cell lines were treated with various concentrations of mithramycin and total RNA extracted from the cultures was examined by northern hybridisation with cDNA for COL1A1 and GAPDH as described in "Materials and methods". Inset: northern analysis of cell line 1. Bar graph: open circles, cell line 1; closed circles, cell line 2. Mithramycin (50 nmol/l) decreased the steady state mRNA level of α1(I) by 40–60%.

Figure 5.

 COL1A1 promoter activity after mithramycin treatment. Transient transfection of systemic sclerosis fibroblasts with COL1A1 promoter-CAT constructs followed by mithramycin (50–100 nmol/l) was performed as described in "Materials and methods". (A) The results with –84 bp, –174 bp, and –804 bp constructs are shown. The efficiency of transfection was corrected by assays of ß-galactosidase activity measured by an enzymatic assay. These experiments were repeated three times, each in duplicate. (B) Densitometric analysis of three separate experiments, each performed in duplicate. p Values obtained comparing the transcriptional activity of each of the constructs in cells treated with mithramycin compared with the transcriptional activity of the same constructs in the untreated cells were significant (p<0.001 for each of the constructs).

Figure 6.

 mRNA stability after mithramycin treatment. The effects of mithramycin on COL1A1 mRNA stability were examined as described in "Materials and methods". (A) Confluent cultures of systemic sclerosis dermal fibroblasts were either kept in culture medium containing 10% FBS and ascorbic acid (50 µg/ml) (lanes 1–7) or were incubated for 4 hours with mithramycin (50 nmol/l; lanes 8–14) before addition of α-amanitin (1 µg/ml). Total RNA was prepared from cells at various intervals after α-amanitin addition and examined by northern analysis. Northern analysis was performed on samples prepared at the time of α-amanitin addition (time point 0): lanes 1, 2, 8, 9; at 6 hours after α-amanitin administration: lanes 3, 4, 10, 11; at 18 hours after α-amanitin treatment: lanes 5, 6, 12, 13; and at 24 hours after α-amanitin: lanes 7 and 14. (B) COL1A1 mRNA stability after mithramycin treatment assessed by densitometric analysis. Values shown were corrected to GAPDH and represent the average of duplicate cultures of different cell lines. The 24 hour value was not included in the graph because it was not performed in duplicate.