The Role And Molecular Mechanism Of JWA Involved In Cadmium-induced Apoptosis In HEK-293T Cells

As the Human Genome Project(HGP) was completed, the Human Epigenome Project which focuses on the relationship of the genome structure and function, such as the Environment Genome Project(EGP), has been becoming a hot spot of life science. At present, it is hard to prevent and cure health lesion casued by many environmental pollutants, the main reason for this is that a little is known about the nature of 'environment-organism response system'. Studying the interaction of lesion induced by environmental adverse factors and organism defensive system on gene level is an important foundation to know the 'environment-organism response system'. In 1998, using the differential display high-throughput technique, Zhou et al cloned JWA, a novel environmental responsive gene from human bronchial epithelial(HBE) cells. In this study, we investigated the activity of JWA promoter region in NIH-3T3 cells. And we also studied the role and molecular mechanism of JWA involved in cadmium-induced apoptosis in HEK-293T cells.1. Study on the activity of JWA promoter region in NII-I-3T3 cells.Using constructed a series of reporter plasmids of JWA promoter region, transient transfection of these constructs into NIH-3T3 cells and followed by reporter gene assay, we found that the transcriptional activity of -194/+107 and -107/+107 cnstructs was significantly higher than other constructs. And the transcriptional activity of -194/+107 cnstruct was also higher than -107/+107 cnstruct.In order to evaluate the core regulation fragment of JWA promoter, we inserted -194/-107 fragment of JWA promoter into the Luciferace repoter vector PGL3-Basic. The reporter gene assay showed that the transcriptional activity of -194/-107 fragment was even higher than -194/+107 fragment. We also employed Southwestern assay to determine whether there was certain transcription factor binds to -194/-107 fragment. As a result, we identified an about 32kD of transcription factor binds to -194/-107 fragment.In order to locate more exactly the core regulation fragment, we divided -194/-107 fragment into two fragments(-194/-147 and -156/-107). Then we employed both Southwestern and EMSA assays to confirm the 32kD of transcription factor. It reminded that -194/-147 fragment might be the core regulation fragment of JWA promoter.2. Study on the role and molecular mechanism of JWA involved in cadmium-induced apoptosis in HEK-293T cells.To evaluate the effect of CdCl₂ on the growth of HEK-293T cells in vitro, both time- and concentration-dependent studies were designed. Cells were treated with varying concentrations of CdCl₂(0-80μM) for different hours(0-48 h) and cell viability was determined by the MTT assay. The results showed that CdCl₂ induced a significant cytotoxic effect on HEK-293T cells. A concentration- and time-dependent inhibitory effect of CdCl₂ on cell growth was observed. The cell apoptosis induced by CdCl₂ was detected by Hoechst33258 staining. Cells were treated with 5μM,10μM,20μM and 40/aM CdCl₂ for 12 h and stained, respectively. The results indicated that every treated concentration of CdC12 could induce apoptosis in HEK-293T cells. The apotosis ratio and the concentrations of CdCI2 were showed direct correlation in some way.In order to evaluate whether JWA was involved in Cd-induced apoptosis, we employed Western blot to detect the expression of JWA protein. 12 h following exposure of cells to 5, 10, 20 or 40μM CdCl₂ resulted in a significant up-regulation of JWA protein. The number of pyknotic cells in the JWA knock-down cells was markedly greater than in vector control cells following CdCl₂ exposure (20μM for 12 h).In order to evaluate whether the CdCl₂ exposure produced oxidative stress was involved in the over-expression of JWA during Cd-induced apoptosis, Dihydroethidium and 2, 7-dichlorofluorescin diacetate (DCFH-DA), dyes specific for intracellular O₂^- and H₂O₂, respectively, were used to detect the intracellular levels of the O₂^- and H₂O₂. Exposure of cells to 20μM CdCl₂ for 12 h produced elevated intracellular levels of both O₂^- and H₂O₂, indicating that CdCl₂ might induce JWA over-expression through activating ROS generation. To understand whether Cd alters activity of MAPKs in HEK-293T cells, the three major MAPKs, JNK, ERK and p38, were examined by Western blots. Results showed that 10, 20, 40μM CdCl₂ activated JNK and ERK but p38. To determine whether the up-regulation of JWA by Cd is regulated by transcriptional mechanisms, transient tranfection and repoter gene assay were employed. The results revealed significant increase in promoter activity with the -1680/+107 and -257/+107 promoter constructs in HEK-293T cells after treatment with 20μM CdCl₂ for 12 h. These data suggest that a putative enhancing element that mediates the effect of CdCl₂ resides between -257 and - 194 of the JWA promoter.