Transcriptional Inhibition of Type I Collagen Gene Expression in Scleroderma Fibroblasts by the Antineoplastic Drug Ecteinascidin 743*

We previously showed that COL1A1 expression is up-regulated at the transcriptional level in systemic sclerosis (SSc) fibroblasts and that the CCAAT-binding factor (CBF) is involved in this increased expression. Ecteinascidin 743 (ET-743) is a chemotherapeutic agent that binds with sequence specificity to the minor groove of DNA and inhibits CBF-mediated transcriptional activation of numerous genes. Therefore, we examined the effects of ET-743 on the increased COL1A1 expression in SSc fibroblasts. The drug caused a potent and dose-dependent inhibition of type I collagen biosynthesis, which reached 70-90% at 700 pm without affecting cell viability. The same drug concentration caused 60-80% reduction in COL1A1 mRNA levels. The stability of the corresponding transcripts was not affected. In vitro nuclear transcription assays demonstrated a 54% down-regulation of COL1A1 transcription. Transient transfections with COL1A1 promoter constructs containing the specific CBF binding sequence into SSc cells previously treated with 700 pm ET-743 failed to show an effect on COL1A1 promoter activity. Furthermore, ET-743 did not affect the binding of CBF or Sp1 transcription factors to their cognate COL1A1 elements. However, treatment with 700 pm ET-743 of stably transfected NIH 3T3 cells expressing a human type II procollagen gene under the control of the human COL1A1 promoter caused a greater than 50% reduction in the production of type II procollagen and a similar decrease in the corresponding type II procollagen transcripts. These results indicate that ET-743 is a potent inhibitor of COL1A1 transcription. However, this effect cannot be explained by a direct effect on CBF binding to the COL1A1 promoter. Although the exact mechanisms responsible for the transcriptional inhibition of COL1A1 by ET-743 are not apparent, our observations suggest that the drug may be an effective agent to decrease collagen overproduction in SSc and other fibrotic diseases.

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