Study On The Mechanism Of ATF6in Autophagy And Apoptosis Induced By ER Stress In HepG2Cells

ATF6, as one of the three ER transmembrane proteins, is an important regulator of apoptosis and autophagy which induced by ER stress. There have been many researches about its regulatory mechanism, while the mechanism of ATF6in the apoptosis and autophagy of tumor cells induced by ER stress remains unclear. In present study, HepG2cells with overexpression of p50ATF6(active form of ATF6) were constructed to detect the roles of ATF6in apoptosis and autophagy that induced by ER stress and to elucidate the possible signal pathways, which may help to supply some data to provide an experimental basis for further elucidate the mechanism of ATF6in apoptosis and autophagy.In the present study, HepG2cells were treated with different doses of Thapsigargin (TG) for24h or48h, the cytotoxic effects were detected by MTT assay, morphological changes of apoptotic cells were observed by Hoechst33342, mitochondrial transmembrance potentials was detected through staining of Rhodamine (Rh123), the changes of three Caspases activities were observed by Multifunctional microplate reader. HepG2cells were treated with different doses of TG for24h or with1μM TG for different periods, or first treated with Caspase3inhibitor AC-DEVD-CHO and Caspase9inhibitor Z-LEDH-FMK and then treated with1μM TG for48h, apoptosis rate was determined by flow cytometry. The protein levels of apoptosis-related proteins were detected by Western blot. The data showed that, the HepG2cells’ viabilities were inhibited by thapsigargin in a time-dependently and dose-dependently mode after exposure to TQ the cells exhibited typical morphological changes of apoptotic cell, the mitochondrial membrane potentials were decreased significantly in a dose-dependent manner and the apoptosis rates were increased in a dose-and time-dependent manner in experiment range. The data showed that the TG induced a dose-dependent increase in the activities of Caspase3and Caspase9, but not Caspase8. After inhibited the activities of Caspase3and Caspase9, the apoptosis rates were also significantly decreased. Western blot results indicated that cleavage and activation of Caspase3,9, releasing of Cytochrome C from mitochondria, upregulation of Caspase12expression at the endoplasmic reticulum stress pathway. Expression of Bax Bcl-2were upregulated and downregulated respectively, while the expression of Caspase8,Fas and FADD were not changed. These results suggest that the ER stress which induced by TG can induce apoptosis in HepG2cells, its mechanism may be related to mitochondrial pathway.In order to explore the autophagy induction effect of thapsigargin-induced ER stress on liver cell line HepG2and its possible mechanism, HepG2cells were treated with different doses of TG for24h or with1μM TG for different periods, the MDC was used to evaluate the autophagic vacuoles and autophagy rate was determined by flow cytometry. By transiently transfected with pEGFP-LC3plasmid in HepG2cells and treated with1μM TG for24h, photomicrographs of GFP-LC3were obtained by fluorescence microscopy. The protein levels of ER stress-related and autophagy-related proteins were detected by Western blot. Fluorescent results revealed that MDC was accumulating in the cells with an autophagic response and after treated with Tg the autophagic rate of HepG2cells were higher than that of control group. After treated with TG, the number and intensity of punctate LC3fluorescence was increased in a time-dependently manner. The western blotting analysis revealed that the expression of Bip, p50ATF6, IRE-1and CHOP were increased apparently. The LC3II: LC3I ratio prominently increased in a time-dependently and dose-dependently mode, the expression of Beclinl and DAPK1were up-regulated and p62was down-regulated as time tends and dose increases. These results suggest the ER stress which induced by TG can induce autophagy in HepG2cells and DAPK1may play an important role in this process.To define the relationship between ER stress-induced apoptosis and autophagy, we used3-MA to inhibit autophagy, then apoptosis ratio was detected by flow cytometry and the expression of apoptosis-related proteins was detected by Western blot. FACS analysis indicated that the apoptotic ratio induced by TG was prominently increased after pretreatment with3-MA. Western blot results showed that the expression of Bcl-2was downregulated and Bax was increased. It suggests that inhibition autophagy accelerates the apoptosis induced by ER stress.In order to determine whether up-regulation of ATF6played a role in apoptosis and autophagy that induced by ER stress, the ATF6(p50ATF6) was transfected to HepG2cells and induced ER stress by using TG The morphological changes of apoptosis and autophagy were observed by fluorescence microscopy, the apoptosis (autophagy) rate and mitochondrial transmembrance potentials were detected by flow cytometry, the formation of autophagic vesicles was observed transmission electron microscopy (TEM), the changes of Caspase activities were detected by multifunctional microplate reader, the mRNA and protein levels of apoptosis and autophagy-related proteins were detected by QRT-PCR and Westen bolt. In ATF6overexpressed cells, marked morphologic apoptosis alterations were observed; autophagic vesicle (green fluorescence) formation and the number of MDC-labeled vesicles were significantly increased; extensive acidic vesicular organelles were seen and in high magnification image the autophagic vacuoles contained variously degraded cytoplasmic organelles; the percentage of apoptosis and autophagy were also prominently increased. In addition, the Caspase3and Caspase9activities were also increased apparently. The expression of Caspase9, Cyt C and Bax were significantly increased while the Bcl-2was significant decreased in the over-active ATF6cells. With regard to the extrinsic signaling molecules, the expression of Caspase8and Fas were not obviously different from control. And the markers of autophagy, the ratio of LC3II/LC3I was also increased, the expression of Beclinl and DAPK1were prominently increased while the p62was decreased. These results show that the over-expression of ATF6enhanced the apoptosis and autophagy induced by ER stress, the ATF6pathway in apoptosis was accompanied by reduced the ratio of Bcl-2/Bax to mediate the mitochondrial pathway and it may enhance autophagy by activating the DAPK-Beclinl pathway.To further investigate the molecular mechanism of ATF6in apoptosis and autophagy that induced by ER stress, we screened the differentially expressed proteins through iTRAQ. The identified proteins were further analyzed by bioinformatic software Gene Ontology and KEGG The apoptosis and autophagy-related mRNA and protein levels were validated by QRT-PCR and Western blot. And the Blebbistatin, SB203580and Dynasore were used as the inhibitor of MYH9, HSPBI and Dynamin Ⅱ, the protein levels of apoptosis and autophagy-related proteins were detected by Westen bolt. iTRAQ data showed that106differentially expressed proteins were identified, including52upregulated proteins and33downregulated proteins. Bioinformatics analysis revealed that a lot of the differentially expression proteins were related with the growth and development of tumor.24of the upregulated and downregulated proteins were connected with apoptosis and autophagy. After used Blebbistatin and Dynasore, the expression of Bcl-2was increased while Bax was significantly decreased; the expression of autophagy-related proteins LC3Ⅱ and Beclinl were prominently downregulated while the p62was upregulated. After used SB203580, the expression of Bcl-2, LC3Ⅱ and Beclinl were obviously decreased while Bax and p62were significantly increased. These results indicate MYH9, HSPBI and Dynamin Ⅱ may be involved in the apiptosis and autophagy which mediated by ATF6under the conditions of ER stress.In summary, ER stress can induce apoptosis and autophgay in HepG2cells. The ATF6can enhance the apoptosis and autophagy that induced by ER stress, the mechanism of mediation apoptosis may be connected with the mitochondrial pathway and the activation of DAPK1-Beclinl pathway may be involved in the process of regulation autophagy. MYH9, HSPB1and Dynamin Ⅱ were played an important role in the ATF6-induced apoptosis and autophagy, and could be used as molecular targets to study the mechanism of ATF6in apoptosis and autophagy.