ABSTRACT
Activating transcription factor 3 (ATF3) is a transcriptional repressor that is rapidly induced in cells exposed to a wide range of stress stimuli. To clarify the role of ATF3 in determining cell fate, we overexpressed it in human umbilical vein endothelial cells (HUVECs) by adenovirus-mediated gene transfer. ATF3 protected these cells from tumor necrosis factor (TNF)-alpha-induced apoptosis, as measured by flow cytometric analysis, trypan blue exclusion assay, and cleavage of procaspase 3 and poly(ADP-ribose) polymerase. Northern blot and nuclear run on assay showed that the transcription of tumor suppressor gene p53 was down-regulated in the ATF3-overexpressing cells. In the transient expression assay, ATF3 suppressed the p53 gene promoter activity through its specific binding to an atypical AP-1 element, PF-1 site, in the p53 gene promoter. Furthermore, the cell-protecting effect of ATF3 was remarkably reduced in p53-deficient cells. These results demonstrate that overexpression of ATF3 suppresses TNF-alpha-induced cell death of HUVECs, at least in part, through down-regulating the transcription of p53 gene. ATF3 may function as a cell survival factor of endothelial cells during vascular inflammation and atherogenesis.
Subject(s)
Apoptosis , Down-Regulation , Endothelium, Vascular/drug effects , Repressor Proteins/metabolism , Transcription Factors/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Tumor Suppressor Protein p53/metabolism , Activating Transcription Factor 3 , Caspase 3 , Caspases/metabolism , Cell Line , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Gene Transfer Techniques , Genes, Reporter , Humans , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases , Promoter Regions, Genetic , Proteins/metabolism , Repressor Proteins/genetics , Transcription Factors/genetics , Transcription, Genetic , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/physiology , Tumor Suppressor Protein p53/geneticsABSTRACT
Activating transcription factor 3 (ATF3) is a member of the ATF/CREB family of transcription factors and its expression is increased by various pathophysiological conditions and in several cancer cells. In this study, we describe two alternatively spliced ATF3DeltaZip mRNAs: ATF3DeltaZip2a and ATF3DeltaZip2b. Both variants encoded the same truncated protein of 135 amino acids, which lacked the leucine zipper domain and was incapable of binding to the ATF/CRE motif. The ATF3DeltaZip2 protein was shown to be localized in the nuclei and counteracted the transcriptional repression by the full-length ATF3. Western blot analysis showed that ATF3DeltaZip2 was expressed in cells exposed to A23187. Further study showed that, similar to the full-length ATF3, the expression of ATF3DeltaZip2 was induced by a wide range of stress stimuli. However, its expression was not detectable in cancer cells that constitutively over-expressed ATF3. Taken together, our results suggest that ATF3DeltaZip2, a protein derived from alternatively spliced mRNAs, is induced by various stress signals and may modulate the activity of the full-length ATF3 protein during stress response.
Subject(s)
Alternative Splicing/genetics , Oxidative Stress , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Transcription Factors/biosynthesis , Transcription Factors/genetics , Up-Regulation , Activating Transcription Factor 3 , Animals , Calcimycin/pharmacology , Cell Nucleus/metabolism , Cells, Cultured , Cloning, Molecular , DNA/genetics , DNA/metabolism , Homocysteine/pharmacology , Humans , Hydrogen Peroxide/pharmacology , Mice , Molecular Sequence Data , Oxidative Stress/drug effects , Protein Isoforms/biosynthesis , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Transport , RNA, Messenger/genetics , RNA, Messenger/metabolism , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Thapsigargin/pharmacology , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcription, Genetic/drug effects , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/pharmacology , Tunicamycin/pharmacology , Up-Regulation/drug effectsABSTRACT
Vascular endothelial cell death contributes to the progression of atherosclerotic lesion, and several transcriptional regulators are involved in the process. Activating transcription factor 3/liver regenerating factor-1 (ATF3/LRF-1), a stress-inducible transcriptional repressor, was shown to be highly expressed in vascular endothelial cells and macrophages of human atherosclerotic lesions by immunohistological assay. The expression was colocalized in these cells which were positive for TdT-mediated dUTP nick-end labeling (TUNEL) and annexin V. Treatment of human umbilical vein endothelial cells (HUVECs) by tumor necrosis factor (TNF)-alpha, oxidized low density lipoprotein (oxLDL), and lysophosphatidylcholine (LPC) rapidly induced ATF3/LRF-1, which showed an increased DNA binding to the consensus ATF/CRE sequence by supershift of gel shift assay. Flow cytometry analysis and immunostaining analysis with TUNEL assay showed that ATF3/LRF-1 was highly expressed in cell death induced by these agents. Moreover, antisense ATF3/LRF-1 cDNA partly suppressed the cell death induced by TNF-alpha, oxLDL, and LPC. From these results, it is indicated that ATF3/LRF-1 is one of the immediate early response genes in vascular endothelial cells in response to atherogenic stimuli, and may play a role in the endothelial cell death associated with atherogenesis.
