Abstract
Previously, our group found that geldanamycin (GA) with sublethal dose provokes the ER stress in rat brain tumour 9L (9L RBT) cells, which followed by the generation of reactive oxygen species (ROS). However, the effect of GA on the unfolded protein response (UPR) signaling pathway remains unclear. Recently, activation of JAK/STAT pathway has been observed in response to generation of intracellular ROS and exogenous hydrogen peroxide (H2O2). Here, we investigated the effect of AG490, the specific inhibitor of Janus kinase-2 (JAK2), on the expression of grp78 coding for ER stress protein and the mechanistic relationship of GA signaling to ER stress. The mRNA and protein level of grp78 and grp94 were examined by real-time quantitative RT-PCR, Western blotting analysis and metabolic labeling experiment in 9L RBT cells treated with GA or AG490. In this study, we firstly discovered that AG490 only could specifically transactivate the ER-resident molecular chaperones GRP78 and GRP94 in 9L RBT cells. In addition, calcium chelator BAPTA-AM, mitochondrial uniporter inhibitor ruthenium red (RR), antioxidant N-acetylcysteine (NAC), and the inhibitor of mitochondrial PT pore, cyclosporin A (CyA), abolished the grp78 and grp94 induction by AG490. Therefore, it suggests that intracellular calcium disturbances and oxidative stress are involved in AG490-induced ER stress. Furthermore, serine/threonine kinase inhibitor H7, PKA inhibitor KT5720, and additional PKC isozyme-selective inhibitors including Gö6983 and Gö6976 could diminished the AG490-induced upregulation of grp78 and grp94 genes. However, the similar suppression effect of Gö6983 and Gö6976 was not observed on the GA-mediated induction of grp78 and grp94 mRNA. Thus, we suggest that the signaling pathway of AG490-induced ER stress response might different from GA. In conclusion, AG490, as an ER stress inducer, might evoke intracellular calcium disturbances and generation of ROS, which lead to activation of cPKC and PKA for upregulation of grp78 and grp94 genes in 9L RBT cells.