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

Natalia Louneva,Biagio Saitta,David J Herrick,Sergio A Jimenez

doi:10.1074/jbc.m301964200

Natalia Louneva, Biagio Saitta + Show 2 more

Open Access

PDF Available

https://doi.org/10.1074/jbc.m301964200

Copy DOI

Export

Save

Cite

Abstract
Highlights/Summary
Full-Text PDF
Similar Papers

Abstract

Listen

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.

Highlights

Fibroblast activation and increased extracellular matrix deposition are central to normal processes such as wound healing
We recently demonstrated that CCAAT-binding factor (CBF) binds the high affinity inverted CCAAT box of the human COL1A1 promoter and found a marked increase in CBF binding activity in systemic sclerosis (SSc) fibroblasts when compared with normal cells [9]
Effects of Ecteinascidin 743 (ET-743) on Normal and SSc Fibroblast Viability and Morphology—In order to examine cytotoxic effects of ET743 on human adult dermal fibroblasts, confluent cultures of normal and SSc cells were treated with various ET-743 concentrations for 24, 48, and 72 h, and cytotoxicity was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Fig. 1A)

Summary

Introduction

Fibroblast activation and increased extracellular matrix deposition are central to normal processes such as wound healing. 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.

Results

Conclusion