Oxidative stress is the main cause of cardiac injury during ischemia/reperfusion but the molecular mechanism for this process is unclear. In this study, it was found that hypoxia induces apoptosis in rat embryonic heart-derived H9c2 cells leading to the induction of GADD153, which is an apoptosis-related gene. Therefore, this study addressed the molecular role of GADD153 in hypoxia-induced apoptosis. The stable or inducible overexpression of GADD153 sensitized the H9c2 cells to apoptotic cell death. The results suggest that the transactivation domain of the GADD153 might be responsible for this cell execution and play a role in the nucleoplasmic localization of GADD153. The cells transiently transfected with the antisense GADD153 were more resistant to hypoxia-induced apoptosis than the vector control cells. Furthermore, GADD153 transcriptionally down-regulated the expression of the cardiac ankyrin repeat protein gene (CARP), which is a nuclear transcriptional co-factor that negatively regulates the expression of the cardiac gene. The ectopic expression of CARP in H9c2 cells increased the resistance to hypoxia-induced apoptosis. These results suggest that GADD153 overexpression and the concomitant down-regulation of CARP might have a causative role in the apoptotic cell injury of hypoxic H9c2 cells.
Apoptosis , CCAAT-Enhancer-Binding Proteins/physiology , Cell Hypoxia/physiology , Myocytes, Cardiac/physiology , Animals , CCAAT-Enhancer-Binding Proteins/chemistry , CCAAT-Enhancer-Binding Proteins/metabolism , Cell Line , Gene Expression Regulation , Humans , Muscle Proteins , Myocytes, Cardiac/metabolism , Nuclear Proteins , Rats , Reperfusion Injury/metabolism , Reperfusion Injury/physiopathology , Repressor Proteins , Transcription Factor CHOP , Transcriptional Activation
The transcription factor CHOP/GADD153 is reportedly induced by cellular stresses such as UV light, genotoxic agents, and protein misfolding in the endoplasmic reticulum. However, the mechanism whereby induction of the GADD153 gene is linked to a downstream pathway is still unclear. Previously, we observed that a synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) effectively impaired cell growth and survival (induction of growth arrest and apoptosis) in human hepatoma cells, which was accompanied by over expression of GADD153. Furthermore, GADD153-transfected Hep 3B cells were growth arrested and were sensitized to drug-induced apoptosis. Thus, in this study, we used suppression subtractive hybridization (SSH) to identify GADD153 target genes that were up-regulated or down-regulated in the GADD153 transfectants. We screened 614 sequence-verified clones by Northern blotting, of which 42 genes were scored as over expressed and 17 genes as under expressed in GADD153 transfectants compared with control vector transfectants. Of those genes, 49 corresponded to known genes in public databases. Among them, we further verified that the expression of transferrin (Tf), which is a negative acute-phase protein and is essential to cell survival as a growth factor, was highly modulated by drug-induced GADD153 over expression or by in vitro transfection. GADD153 significantly antagonized the C/EBP (C/EBP-alpha, -beta, and -delta)-mediated transcriptional activation of the Tf gene. In conclusion, Tf and other target genes identified may play a functional role in the downstream pathway of GADD153.
CCAAT-Enhancer-Binding Proteins/metabolism , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Transferrin/genetics , Antineoplastic Agents/pharmacology , CCAAT-Enhancer-Binding Proteins/pharmacology , Down-Regulation , Fenretinide/pharmacology , Gene Expression/drug effects , Humans , Transcription Factor CHOP , Transcription Factors/pharmacology , Transcription, Genetic/drug effects , Transcriptional Activation/drug effects , Tumor Cells, Cultured
