Abstract
The endoplasmic reticulum (ER) is a multifunctional organelle controlling important cellular processes, including Ca2+ homeostasis, protein synthesis, protein trafficking, and apoptosis. Under physiologically or pharmacologically adverse conditions that perturb the calcium homeostasis, accumulation of unfolded or malfolded proteins in the ER lumen occurs, referred to as ER stress, and the cells will activate a series of signal transduction cascades collectively termed the unfolded protein response (UPR). One characteristic of the UPR is the induction of the ER resident stress proteins referred to as the glucose-regulated proteins (GRPs). The best characterized GRP78, also known as the immunoglobulin heavy chain binding protein or BiP, is thought to function in Ca2+ sequestration or as a molecular chaperone in the folding and assembly of membrane or secreted proteins. Previous reports of GRP78 in different cellular compartments prompted us to examine and compare the changes of GRP78 in intracellular distribution patterns in response to ER stress, specifically under calcium disturbance. Treatment with calcium ionophore A23187 and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase inhibitor thapsigargin (TG) results in a decrease of [Ca2+]er with a concurrent increase of [Ca2+]c. The immunostaining of GRP78 coupled with confocal microscopy demonstrated the granular and perinuclear expression as normal ER distribution in cells under normal growing conditions. When the cells were exposed to A23187 or TG, the greater proportion of GRP78 displayed a diffused distribution throughout the cytoplasm at a slightly higher intensity. Overlapping of GRP78 and mitochondria marker apparently depicted worm-shaped strings, suggesting that there is an increase in the level of colocalization and that this might occur from an increase in the level of targeting. Cellular fractionation and protease digestion of isolated mitochondria from ER-stressed cells suggested that a significant portion of GRP78 is localized to the mitochondria and is protease-resistant. Localizations of GRP78 in ER and mitochondria were confirmed by immunoelectron microscopy. In ER-stressed cells, GRP78 mainly localized within the mitochondria and decorated the mitochondrial membrane compartment. Submitochondrial fractionation studies further indicated that the mitochondrial resided GRP78 is mainly located in the intermembrane space, inner membrane, and matrix. Furthermore, radioactive labeling followed by subcellular fractionation showed that a significant portion of the newly synthesized GRP78 is localized to the mitochondria in cells under UPR. The results in this study indicate that, at least under certain circumstances, the ER resided chaperone GRP78 can be retargeted to mitochondria and thereby may be involved in correlating UPR signaling between these two organelles. We also investigate the signaling pathway involved in the induction of GRP78 in cells under the treatment of geldanamycin (GA), which is a potent inducer of ER stress response. By using calcium monitoring, inhibitors screening and direct examination of the ROS contents rendered by GA, we show that GA exerts GRP78 inductive expression through a causative pathways connecting phospholipase C to intracellular calcium increase, PKC activation as well as ROS generation. Furthermore, the calcium influx from extracellular space, intracellular calcium store oscillations and mitochondrial calcium influx are important for the calcium mobilization and GRP78 expression induced by GA. The GA-induced GRP78 expression signaling cascades may represent regulation of the ER-stress response in cell survival or apoptotic program.