The ubiquitously expressed molecular chaperone GRP78 (78 kDa glucose-regulated protein) generally localizes to the ER (endoplasmic reticulum). GRP78 is specifically induced in cells under the UPR (unfolded protein response), which can be elicited by treatments with calcium ionophore A23187 and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor TG (thapsigargin). By using confocal microscopy, we have demonstrated that GRP78 was concentrated in the perinuclear region and co-localized with the ER marker proteins, calnexin and PDI (protein disulphide-isomerase), in cells under normal growth conditions. However, treatments with A23187 and TG led to diminish its ER targeting, resulting in redirection into a cytoplasmic vesicular pattern, and overlapping with the mitochondrial marker MitoTracker. 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 using immunoelectron microscopy. In ER-stressed cells, GRP78 mainly localized within the mitochondria and decorated the mitochondrial membrane compartment. Submitochondrial fractionation studies indicated further that the mitochondria-resided GRP78 is mainly located in the intermembrane space, inner membrane and matrix, but is not associated with the outer membrane. Furthermore, radioactive labelling followed by subcellular fractionation showed that a significant portion of the newly synthesized GRP78 is localized to the mitochondria in cells under UPR. Taken together, our results 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 signalling between these two organelles.

BackgroundEosinophilic granulocytes are important for the human immune system. Many cationic proteins with cytotoxic activities, such as eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), are released from activated eosinophils. ECP, with low RNase activity, is widely used as a biomarker for asthma. ECP inhibits cell viability and induces apoptosis to cells. However, the specific pathway underlying the mechanisms of ECP-induced cytotoxicity remains unclear. This study investigated ECP-induced apoptosis in bronchial epithelial BEAS-2B cells and elucidated the specific pathway during apoptosis.ResultsTo address the mechanisms involved in ECP-induced apoptosis in human BEAS-2B cells, investigation was carried out using chromatin condensation, cleavage of poly (ADP-ribose) polymerase (PARP), sub-G1 distribution in cell cycle, annexin V labeling, and general or specific caspase inhibitors. Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-α). Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-α antibody.ConclusionIn conclusion, our results have demonstrated that ECP increased TNF-α production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.

Both geldanamycin (GA) and radicicol (RA) are HSP90 binding agents that possess antitumour activities. Although the in vitro data indicated that the inhibitory constant of RA is much bigger than that of GA, the in vivo data on drug efficacy might reveal different results. We have recently shown that treatment with GA induces a heat-shock response and that calcium mobilization may be involved in the process. By using induction of HSP70 as the endpoint assay, we found changes in upstream signaling mediators, including HSF1 and calcium mobilization, as well as possible involvement of protein kinase in human non-small cell lung cancer H460 cells treated with GA and RA. Our results demonstrated that calcium mobilization, a calcium dependent and H7-sensitive protein kinase, along with HSF1 activation by phosphorylation, are all involved in the HSP70 induction process triggered by the drugs. However, only GA, but not RA, can provoke a rapid calcium mobilization and thereby result in an instant induction of HSP70. Furthermore, the rapid calcium influx, followed by instant HSP induction, could be achieved in GA- or RA-treated cells placed in a medium containing excessive calcium while the response was completely abolished in cells depleted of calcium. Taken together, our findings suggest that differential calcium signaling may account for the differential induction of HSP and the action of GA and RA.

HSP90 chaperones are transducer proteins of many signaling pathways in cells. Using a highly specific inhibitor, geldanamycin (GA), an increasing number of the HSP90 client proteins have been identified. Nevertheless, there is little information on the differential transactivation of the two isoforms of the hsp90 genes, hsp90alpha and beta, in cells under stress conditions. Here, we demonstrate the differential expression of the HSP90 isoforms, HSP90alpha and beta, in rat gliosarcoma 9L cells using a modified SDS-PAGE system that allowed us to distinguish the isoforms. We subsequently assessed the transcriptional controls involving the transcription elements located in the promoter regions of the hsp90 genes. At the protein level, HSP90alpha is more responsive to GA in terms of rate of de novo synthesis and amount of accumulation, as shown by metabolic-labeling and Western-blotting analyses. Upregulation of the hsp90 genes was demonstrated by real-time qPCR. The promoter elements hsp90alpha-HSE2 and hsp90beta-HSE1 were also identified to be the major transcription elements involved in GA-activated gene expression, as shown by EMSA, whereas the results of supershift showed that the transcription factor HSF1 is also involved. Moreover, EMSA results of analysis of the GC box showed differences in both the initial amounts and inductive response of hsp90s transcripts, whereas analysis of the TATA box showed GA responsiveness in hsp90alpha only. Collectively, these results indicate that GA exerts its regulatory effects through transcription elements including heat-shock elements (HSEs), GC boxes and TATA boxes, resulting in differential transactivation of hsp90alpha and hsp90beta in rat gliosarcoma 9L cells.

Although the differential expression of heat shcok proteins, Hsp90alpha and Hsp90beta was extensively studied in many kinds of cells, the post-transcriptional regulation of Hsp90 isoforms remains unclear. In control and GA-treated rat gliosarcoma cells, it has been reported that the translational efficiency of hsp90alpha is higher than hsp90beta. In this study, we present evidences identifying the roles for leaky scanning and 5'-UTR sequence in translational regulation of Hsp90beta. The result of in vitro transcription and translation (IVTT) experiment showed that hsp90alpha exhibited higher translation efficiency than hsp90beta. Sequence analysis revealed that there is an out-of-frame downstream AUG codon in hsp90beta gene. However, elimination of the downstream AUG by site-directly mutagenesis or introducing Kozak context sequence around the initiator AUG of hsp90beta open reading frame increased its translational efficiency, which indicated that leaky scanning might be a possible mechanism regulating hsp90beta. Furthermore, we also constructed a firefly luciferase reporter system to verify the effect of subsequent translation at the downstream out-of-frame AUG codon in 9L and A549 cells. Furthermore, it is believed that 5'-untranslated region (5'-UTR) also plays a significant role in translational control. We showed hsp90beta 5'-UTR gives rise to the reduction of the translation efficiency in IVTT experiment. Additionally, the reductive effect of hsp90beta 5'-UTR was further confirmed by luciferase reporter assay using truncated deletion analyses of 5'-UTR of hsp90beta. Our results support the hypothesis that ribosome leaky scanning mechanism and 5'-UTR sequence acts as negative regulators in hsp90beta mRNA.