Functionally Novel Tumor Necrosis Factor-&agr;–Modulated CHR-Binding Protein Mediates Cyclin A Transcriptional Repression in Vascular Endothelial Cells

Abstract

Abstract— Local expression of tumor necrosis factor-&agr; (TNF-&agr;) at the sites of arterial injury after balloon angioplasty, suppresses endothelial cell (EC) proliferation and negatively affects reendothelialization of the injured vessel. We have previously reported that in vitro exposure of ECs to TNF-&agr; induced EC growth arrest and apoptosis. These effects were mediated, at least in part, by downregulation of cell cycle regulatory proteins. In the present study, we report potential mechanism(s) for TNF-&agr;–mediated suppression of cyclin A in ECs. TNF-&agr; exposure to ECs completely abrogated cyclin A mRNA expression via mechanisms involving both transcriptional and posttranscriptional modifications. TNF-&agr; inhibited de novo cyclin A mRNA synthesis and suppressed cyclin A promoter activity. Utilizing deletion mutants of human cyclin A promoter, we have identified CDE-CHR (C ell cycle–D ependent E lements–C ell cycle genes H omology R egion) region of cyclin A promoter as a target for TNF-&agr; suppressive action. Experiments to investigate CDE-CHR binding proteins/factors revealed a TNF-&agr;–mediated increase in specific DNA binding activity to the CHR elements. This increase in binding activity by TNF-&agr; was mediated via the induction of a functionally novel 84-kDa protein that binds specifically to CHR in Southwestern assays. UV cross-linking and SDS-PAGE analysis of proteins eluted from specific complex confirmed the presence of this 84-kDa protein. Moreover, induction of this protein by TNF-&agr; was protein synthesis dependent. Additionally, exposure of ECs to TNF-&agr; markedly reduced cyclin A mRNA stability. Targeted disruption of this protein could potentially be a therapeutic strategy to rescue EC proliferation in vivo.