• Journal of Life Science
• /
• v.19 no.8
• /
• pp.1159-1163
• /
• 2009

It has been postulated that endoplasmic (ER) stress is involved in the development of several diseases. However, the detailed molecular mechanisms have not been fully understood. Therefore, we characterized a genetic network of genes induced by ER stress using cDNA microarray and gene set expression coherence analysis (GSECA), and identified gene function as well as several transcription regulators associated with ER stress. We analyzed time-dependent gene expression profiles in thapsigargin-treated Sk-Hep1 using an oligonucleotide expression chip, and then selected functional gene sets with significantly high expression coherence which was processed into functional clusters according to the expression similarities. The functions related to sugar binding, lysosome, ribosomal protein, ER lumen, and ER to golgi transport increased, whereas the functions with mRNA processing, DNA replication, DNA repair, cell cycle, electron transport chain and helicase activity decreased. Furthermore, functional clusters were investigated for the enrichment of regulatory motifs using GSECA, and several transcriptional regulators associated with regulation of ER-induced gene expression were found.

• Development and Reproduction
• /
• v.10 no.2
• /
• pp.105-113
• /
• 2006

Reactive oxygen species(ROS) generated in cellular metabolism have an effect on cell maturation and development. In human reproductive tract, oxidative injury by ROS may induce female infertility. Also, oxidative injury may be responsible for developmental retardation and arrest of mammalian preimplantation embryos. Activating transcription factor 4(ATF4) is a member of the cyclic-AMP response element-binding(CREB) familiy of basic region- leucine zipper(bZip). ATF4 is known to regulate stress response to protect cell from various stress factors and inducer of apoptisis. The purpose of this study was to investigate whether ATF4 is involved in the defensive mechanism in oxidative stress condition during the development of mouse preimplantation embryos. To verify the expression of ATF4 in oxidative stress condition, 2-cell stage embryos were cultured in HTF media containing 0.1mM, 0.5mM or 1mM hydrogen peroxide($H_2O_2$) for 1hr(2-cell), 8hr(4-cell), 17hr(8-cell), 24hr(morula), 48hr(early blastocyst) or 64hr(late blastocyst). The developmental rate decreased in the 0.1mM $H_2O_2$ treated group compared with control group. In embryos treated with 0.5mM and 1mM $H_2O_2$ showed 2-cell block. As a results of the semi-quantitative RT-PCR analysis of SOD1, ATF4 and Bax gene expression, SOD1, ATF4 and Bax genes were increased in 0.1mM, 0.5mM, 1mM $H_2O_2$ treated groups compared with control group. In 2-cell embryos, expression of SOD1, ATF4 and Bax genes were notably increased in 0.1mM, 0.5mM, 1mM $H_2O_2$ treated groups compared with control group. Immunofluorescence analysis showed that ATF4 protein was localized at the cytoplasm of preimplantation embryos. The increase in ATF4 immunoreactivety was observed in the 0.1mM, 0.5mM, 1mM $H_2O_2$ treated groups compared with control group. It suggests that oxidative stress by $H_2O_2$ induces expression of ATF4 and may be involved in protection mechanism in preimplantation embryos from oxidative injury.

• Tuberculosis and Respiratory Diseases
• /
• v.50 no.1
• /
• pp.52-66
• /
• 2001

Background : NF-${\kappa}B$ is the most important transcriptional factor in IL-8 gene expression. Triptolide is a new compound that recently has been shown to inhibit NF-${\kappa}B$ activation. The purpose of this study is to investigate how triptolide inhibits NF-${\kappa}B$-dependent IL-8 gene transcription in lung epithelial cells and to pilot the potential for the clinical application of triptolide in inflammatory lung diseases. Methods : A549 cells were used and triptolide was provided from Pharmagenesis Company (Palo Alto, CA). In order to examine NF-${\kappa}B$-dependent IL-8 transcriptional activity, we established stable A549 IL-8-NF-${\kappa}B$-luc. cells and performed luciferase assays. IL-8 gene expression was measured by RT-PCR and ELISA. A Western blot was done for the study of $I{\kappa}B{\alpha}$ degradation and an electromobility shift assay was done to analyze NF-${\kappa}B$ DNA binding. p65 specific transactivation was analyzed by a cotransfection study using a Gal4-p65 fusion protein expression system. To investigate the involvement of transcriptional coactivators, we perfomed a transfection study with CBP and SRC-1 expression vectors. Results : We observed that triptolide significantly suppresses NF-${\kappa}B$-dependent IL-8 transcriptional activity induced by IL-$1{\beta}$ and PMA. RT-PCR showed that triptolide represses both IL-$1{\beta}$ and PMA-induced IL-8 mRNA expression and ELISA confirmed this triptolide-mediated IL-8 suppression at the protein level. However, triptolide did not affect $I{\kappa}B{\alpha}$ degradation and NF-$_{\kappa}B$ DNA binding. In a p65-specific transactivation study, triptolide significantly suppressed Gal4-p65T Al and Gal4-p65T A2 activity suggesting that triptolide inhibits NF-${\kappa}B$ activation by inhibiting p65 transactivation. However, this triptolide-mediated inhibition of p65 transactivation was not rescued by the overexpression of CBP or SRC-1, thereby excluding the role of transcriptional coactivators. Conclusions : Triptolide is a new compound that inhibits NF-${\kappa}B$-dependent IL-8 transcriptional activation by inhibiting p65 transactivation, but not by an $I{\kappa}B{\alpha}$-dependent mechanism. This suggests that triptolide may have a therapeutic potential for inflammatory lung diseases.