Functionally novel tumor necrosis factor-alpha-modulated CHR-binding protein mediates cyclin A transcriptional repression in vascular endothelial cells.
Local expression of tumor necrosis factor-alpha (TNF-alpha) 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-alpha 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-alpha-mediated suppression of cyclin A in ECs. TNF-alpha exposure to ECs completely abrogated cyclin A mRNA expression via mechanisms involving both transcriptional and posttranscriptional modifications. TNF-alpha 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 (Cell cycle-Dependent Elements-Cell cycle genes Homology Region) region of cyclin A promoter as a target for TNF-alpha suppressive action. Experiments to investigate CDE-CHR binding proteins/factors revealed a TNF-alpha-mediated increase in specific DNA binding activity to the CHR elements. This increase in binding activity by TNF-alpha 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-alpha was protein synthesis dependent. Additionally, exposure of ECs to TNF-alpha markedly reduced cyclin A mRNA stability. Targeted disruption of this protein could potentially be a therapeutic strategy to rescue EC proliferation in vivo.
Kishore
Raj
R
Division of Cardiovascular Research, St Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Mass 02135, USA.
Spyridopoulos
Ioakim
I
Luedemann
Corinne
C
Losordo
Douglas W
DW
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D019925:Cyclin A / Q000235:genetics / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D020868:Gene Silencing / Q000187:drug effects / Q000502:physiology*; D005809:Genes, Regulator / Q000502:physiology*; D017930:Genes, Reporter; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D020871:RNA Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000502:physiology*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D019925:Cyclin A; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D012097:Repressor Proteins; D014409:Tumor Necrosis Factor-alpha