Yiu-Kay Lai

Localization of GRP78 to mitochondria under the unfolded protein response

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.

Differential Activation of p38 Mitogen-activated Protein Kinase and Extracellular Signal-regulated Protein Kinases Confers Cadmium-induced HSP70 Expression in 9L Rat Brain Tumor Cells

We have reported that treatment with CdCl2 at 40–100 μm induces the heat shock proteins (HSPs) in 9L rat brain tumor cells, during which the activation of heat shock factor (HSF) is essentially involved. By exploiting protein kinase inhibitors, we further analyzed the possible participation of specific protein kinases in the above processes. It was found that induction of HSP70 in cells treated with a high concentration of cadmium (i.e. 100 μm) is preceded by the phosphorylation and activation of p38 mitogen-activated protein kinase (p38MAPK), while that in cells treated with a low concentration (60 μm) is accompanied by the phosphorylation and activation of extracellular-regulated protein kinases 1 and 2 (ERK1/2). In 100 μm cadmium-treated cells, both HSP70 induction and HSF1 activation are eliminated in the presence of SB203580, a specific inhibitor of p38MAPK. By contrast, in 60 μm cadmium-treated cells, the processes are not affected by SB203580 but are significantly suppressed by PD98059, which indirectly inhibits ERK1/2 by acting on MAPK-ERK kinase. Taken together, we demonstrate that p38MAPK and ERK1/2 can be simultaneously or independently activated under different concentrations of cadmium and that the signaling pathways participate in the induction of HSP70 by acting on the inducible phosphorylation of HSF1. We thus provide the first evidence that both p38MAPKand ERK signaling pathways can differentially participate in the activation of HSF1, which leads to the induction of HSP70 by cadmium.

Dual cytotoxic mechanisms of submicromolar taxol on human leukemia HL-60 cells

Taxol-induced mitotic block and apoptosis were investigated using taxol-sensitive human leukemia HL-60 cells at submicromolar concentrations of the drug. Cells exposed to either 20 nM taxol for 1 hr or 10 nM taxol for 12 hr were able to resume normal growth, whereas cells exposed to 60 nM taxol for 1 hr or 10 nM taxol for 24 hr failed to proliferate after drug removal. Progressive changes in the percentage of mitotic block and apoptosis induced by these four treatment protocols were monitored continuously for 3-5 days after drug removal. Cells treated with 20 nM taxol for 1 hr showed a mitotic block without a subsequent increase in apoptosis, whereas cells treated with 10 nM taxol for 12 hr showed an increase in apoptotic ratio within several hours without an increase in mitotic block. These results indicate that apoptosis does not necessarily result from mitotic block and that these two phenomena can occur independently of each other. Drug sensitivity at progressive stages of the cell cycle was also investigated. The results showed that, in addition to the cells in G2/M phase, the cells in S phase were also sensitive to the drug, especially to a prolonged treatment. These results suggest that, in HL-60 cells, the apoptotic programs can be initiated in either the G2/M or S phase and represent two different cytotoxic mechanisms of taxol.

Combining Multiplex Reverse Transcription-PCR and a Diagnostic Microarray To Detect and Differentiate Enterovirus 71 and Coxsackievirus A16

ABSTRACT Cluster A enteroviruses, including enterovirus 71 (EV71) and coxsackievirus A16 (CA16), are known to cause hand-foot-and-mouth disease (HFMD). Despite the close genetic relationship between these two viruses, EV71 is generally known to be a more perpetuating pathogen involved in severe clinical manifestations and deaths. While the serotyping of enteroviruses is mostly done by conventional immunological methods, many clinical isolates remain unclassifiable due to the limited number of antibodies against enterovirus surface proteins. Array-based assays are able to detect several serotypes with high accuracy. We combined an enterovirus microarray with multiplex reverse transcription-PCR to try to develop a method of sensitively and accurately detecting and differentiating EV71 and CA16. In an effort to design serotype-specific probes for detection of the virus, we first did an elaborate bioinformatic analysis of the sequence database derived from different enterovirus serotypes. We then constructed a microarray using 60-mer degenerate oligonucleotide probes covalently bound to array slides. Using this enterovirus microarray to study 144 clinical specimens from patients infected with HFMD or suspected to have HFMD, we found that it had a diagnostic accuracy of 92.0% for EV71 and 95.8% for CA16. Diagnostic accuracy for other enteroviruses (non-EV71 or -CA16) was 92.0%. All specimens were analyzed in parallel by real-time PCR and subsequently confirmed by neutralization tests. This highly sensitive array-based assay may become a useful alternative in clinical diagnostics of EV71 and CA16.

Involvement of p38 Mitogen-activated Protein Kinase Signaling Pathway in the Rapid Induction of the 78-kDa Glucose-regulated Protein in 9L Rat Brain Tumor Cells

We have previously shown that treatment with okadaic acid (OA) followed by heat shock (HS) (termed OA → HS treatment) leads to rapid transactivation of the 78-kDa glucose-regulated protein gene (grp78) in 9L rat brain tumor cells. A cAMP-responsive element-like (CRE-like, TGACGTGA) promoter sequence and a protein kinase A signaling pathway are involved in this induction, and activation of both CRE binding protein (CREB) and activating transcription factor-2 (ATF-2) is required in the above process. Herein, we report that transactivation of grp78, as well as phosphorylation/activation of ATF-2, can be completely annihilated by SB203580, a highly specific inhibitor of p38 mitogen-activated protein kinase (p38MAPK). Activation of p38MAPK by OA → HS is also substantiated by its own phosphorylation as well as the phosphorylation and activation of MAPK activating protein kinase-2 in cells subjected to this treatment. The involvement of p38MAPK in the activation of ATF-2, which leads to the transactivation of rat grp78, is confirmed by electrophoretic mobility shift assay using a probe containing the CRE-like sequence as well as by transient transfection assays with a plasmid containing a 710-base pair stretch of the grp78promoter. Together with our previous studies, these results led us to conclude that phosphorylation/activation of CREB upon OA → HS treatment is mediated by cAMP-dependent protein kinase, whereas that of ATF-2 is mediated by p38MAPK. The transcription factors may bind to each other to form heterodimers that in turn transactivate grp78 by binding to the CRE-like element. This suggests that distinct signaling pathways converge on CREB-ATF-2, where each subunit is individually activated by a specific class of protein kinases. This may allow modulation ofgrp78 transactivation by diverse external stimuli.

GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis

The chaperone glucose‐regulated protein, 78/immunoglobulin binding protein (GRP78/Bip), protects cells from cytotoxicity induced by DNA damage or endoplasmic reticulum (ER) stress. In this study, we showed that GRP78 is a major inducible protein in human non‐small cell lung cancer H460 cells treated with ER stress inducers, including A23187 and thapsigargin. AEBSF, an inhibitor of serine protease, diminished GRP78 induction, enhanced mitochondrial permeability, and augmented apoptosis in H460 cells during ER stress. Simultaneously, AEBSF promoted Raf‐1 degradation and suppressed phosphorylation of Raf‐1 at Ser338 and/or Tyr340 during ER stress. Coimmunoprecipitation assays and subcellular fractionations showed that GRP78 associated and colocalized with Raf‐1 on the outer membrane of mitochondria, respectively. While treatment of cells with ER stress inducers inactivated BAD by phosphorylation at Ser75, a Raf‐1 phosphorylation site; AEBSF attenuated phosphorylation of BAD, leading to cytochrome c release from mitochondria. Additionally, overexpression of GRP78 and/or Raf‐1 protected cells from ER stress‐induced apoptosis. Taken together, our results indicate that GRP78 may stabilize Raf‐1 to maintain mitochondrial permeability and thus protect cells from ER stress‐induced apoptosis. J. Cell. Physiol. 215: 627–635, 2008.

Enterovirus 71 triggering of neuronal apoptosis through activation of Abl-Cdk5 signalling

The molecular mechanism behind what causes an infection of Enterovirus 71 (EV71) in young children to result in severe neurological diseases is unclear. Herein, we show that Cdk5, a critical signalling effector of various neurotoxic insults in the brain, is activated by EV71 infection of neuronal cells. EV71‐induced neuronal apoptosis could be effectively repressed by blocking either Cdk5 kinase activity or its protein expression. Moreover, EV71‐induced Cdk5 activation was modulated by c‐Abl. The suppression of c‐Abl kinase activity by STI571 notably repressed both the Cdk5 activation and neuronal apoptosis in cells infected with EV71. Although EV71 also induces apoptosis in non‐neuronal cells, it did not affect Abl and Cdk5 activities in several non‐neuronal cell lines. Intriguingly, coxsackievirus A16 (CA16), a genetically closely related serotype to EV71 that usually does not induce severe neurological disorders, could only weakly stimulate Abl, but not Cdk5 kinase activity. Taken together, our data suggest a serotype‐ and cell type‐specific mechanism, by which EV71 induces Abl kinase activity, which in turn triggers Cdk5‐signalling for neuronal apoptosis.

Induction of stress response and differential expression of 70 kDa stress proteins by sodium fluoride in HeLa and rat brain tumor 9L cells.

We herein demonstrate that sodium fluoride (NaF) acts as a stress response inducer on HeLa and 9L rat brain tumor cells. NaF is only slightly cytotoxic, and inhibitory to Ser/Thr‐phosphatases but not to Tyr‐phosphatases in both cell lines. After treatment with 5 mM NaF for 2 h, the phosphorylation levels of vimentin and an alkali‐resistant 65‐kDa phosphoprotein were enhanced, a common phenomenon detected in cells under a variety of stress conditions. Under an identical treatment protocol, in which the cells were treated with 5 mM NaF for 2 h and then allowed to recover under normal growing conditions for up to 12 h, NaF differentially induced the cytoplasmic/nuclear heat‐shock protein70s (including both the inducible and the constitutively expressed members of this protein family) in HeLa cells and the endoplasmic reticulum residing heat‐shock protein70 (the glucose‐regulated protein with an apparent molecular weight of 78 kDa) in 9L cells. Electrophoretic mobility shift assays (EMSA) using probes containing well‐characterized regulatory elements revealed the activation of the heat‐shock factor in HeLa but not in 9L cells; this is in good agreement with the stress protein induction pattern. Additional differential induction of binding activities toward EMSA probes individually containing NF‐κB, AP‐2, and CRE‐like elements were detected in NaF‐treated cells. The possible involvement of these binding sites as well as the corresponding factors in the stress response are discussed. J. Cell. Biochem. 69:221–231, 1998.

Identification of mitogen-activated protein kinase-activated protein kinase-2 as a vimentin kinase activated by okadaic acid in 9L rat brain tumor cells

Organization of intermediate filament, a major component of cytoskeleton, is regulated by protein phosphorylation/dephosphorylation, which is a dynamic process governed by a balance between the activities of involved protein kinases and phosphatases. Blocking dephosphorylation by protein phosphatase inhibitors such as okadaic acid (OA) leads to an apparent activation of protein kinase(s) and to genuine activation of phosphatase‐regulated protein kinase(s). Treatment of 9L rat brain tumor cells with OA results in a drastically increased phosphorylation of vimentin, an intermediate filament protein. In‐gel renaturing assays and in vitro kinase assays using vimentin as the exogenous substrate indicate that certain protein kinase(s) is activated in OA‐treated cells. With specific protein kinase inhibitors, we show the possible involvement of the cdc2 kinase‐ and p38 mitogen‐activated protein kinase (p38MAPK)‐mediated pathways in this process. Subsequent in vitro assays demonstrate that vimentin may serve as an excellent substrate for MAPK‐activated protein kinase‐2 (MAPKAPK‐2), the downstream effector of p38MAPK, and that MAPKAPK‐2 is activated with OA treatment. Comparative analysis of tryptic phosphopeptide maps also indicates that corresponding phosphopeptides emerged in vimentin from OA‐treated cells and were phosphorylated by MAPKAPK‐2. Taken together, the results clearly demonstrate that MAPKAPK‐2 may function as a vimentin kinase in vitro and in vivo. These findings shed new light on the possible involvement of the p38MAPK signaling cascade, via MAPKAPK‐2, in the maintenance of integrity and possible physiological regulation of intermediate filaments. J. Cell. Biochem. 71:169–181, 1998.

Involvement of calcium in the differential induction of heat shock protein 70 by heat shock protein 90 inhibitors, geldanamycin and radicicol, in human non-small cell lung cancer H460 cells

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. J. Cell. Biochem.