Mitochondrial Protein Phosphatase 2A Regulates Cell Death Induced by Simulated Ischemia in Kidney NRK-52E Cells

Acute renal failure can occur after an ischemic injury and results in significant mortality. The stress-signaling pathways that are activated during renal ischemia are unknown. PP2A has emerged as an important regulator of cell death. To study the role of PP2A in ischemia-induced cell death, we used an in vitro model of simulated ischemia. In the present study, simulated ischemia in rat renal tubule epithelial NRK-52E cells (a) results in cell death that involves both necrosis and apoptosis, (b) activates PP2A, and (c) up-regulates the PP2A B56 α regulatory subunit. Previous data have shown that PKC α negatively regulates B56 α protein expression. Consistent with this finding, simulated ischemia suppressed PKC α and up-regulated B56 α. Treatment of NRK-52E cells with ceramide suppressed PKC α and activated PP2A in a manner that mimicked simulated ischemia. A role for PP2A in simulated ischemia-induced cell death is likely since inhibition of PP2A protected NRK-52E cells. In addition, overexpression of exogenous B56 α but not B55 in NRK-52E cells enhanced simulated ischemia-induced cell death. These findings suggest that activation of a PP2A isoform that contains the B56 α regulatory subunit is required for ischemia-induced cell death in kidney epithelial proximal tubule cells.     

Effect of single and repeated heat stress on chemical signals of heat shock response cascade in the rat’s heart

Exposure to sublethal heat stress activates a complex cascade of signaling events, such as activators (NO), signal molecules (PKCε), and mediators (HSP70 and COX-2), leading to implementation of heat preconditioning, an adaptive mechanism which makes the organism more tolerant to additional stress. We investigated the time frame in which these chemical signals are triggered after heat stress (41?±?0.5°С/45 min), single or repeated (24 or 72 h after the first one) in heart tissue of male Wistar rats. The animals were allowed to recover 24, 48 or 72 h at room temperature. Single heat stress caused a significant increase of the concentration of HSP70, NO, and PKC level and decrease of COX-2 level 24 h after the heat stress, which in the next course of recovery gradually normalized. The second heat stress, 24 h after the first one, caused a significant reduction of the HSP70 levels, concentration of NO and PKC?, and significant increase of COX-2 concentration. The second exposure, 72 h after the first heat stress, caused more expressive changes of HSP70 and NO in the 24 h-recovery groups. The level of PKC? was not significantly changed, but there was significantly increased COX-2 concentration during recovery. Serum activity of AST, ALT, and CK was reduced after single exposure and increased after repeated exposure to heat stress, in both time intervals. In conclusion, a longer period of recovery (72 h) between two consecutive sessions of heat stress is necessary to achieve more expressive changes in mediators (HSP70) and triggers (NO) of heat preconditioning.     

Altered ischemic induction of immediate early gene and heat shock protein 70 mRNAs after preconditioning in rat hearts

Immediate early genes and heat shock protein (HSP) 70s, which may play a role in adaptation and cellular protection, respectively, are induced by ischemia in hearts. We examined if the induction of immediate early gene (c-fos, c-myc, c-jun, and junB) and HSP70 mRNAs by ischemia is affected by ischemic preconditioning. Transient ischemia (5 or 10 minute) was applied to Wistar rat (n=75) hearts, by tightening a snare placed around left coronary arterial branches 7 days before applying ischemia. Rats without earlier ischemia (control group, C) and rats with 5-minute ischemia 12 or 24 hours earlier (EI12 or 24 group) were given 10-minute ischemia and sacrificed at 0, 0.5, 1, 2, and 4 hour. RNA was extracted from the ischemic region and Northern blot analysis was performed. The induction of c-fos and c-myc mRNAs was significantly increased in EI12 but not in EI24 compared with that in C. The induction of c-jun and junB mRNAs showed no change in both EI12 and EI24 compared with that in C. The induction of HSP72 and 73 mRNAs was decreased in EI12 and decreased further in EI24. Thus, ischemic preconditioning altered the induction of immediate early gene and HSP70 mRNAs by ischemia. The effect of preconditioning differed among genes studied and changed with time after preconditioning. Ischemic preconditioning alters protective and adaptive responses to ischemia at the gene level.     

The stress protein response in cultured neurons: characterization and evidence for a protective role in excitotoxicity.

We used purified cultures of cerebellar granule cells to investigate the possible protective role of stress proteins in an in vitro model of excitotoxicity. Initial experiments used one- and two-dimensional polyacrylamide gel electrophoresis to confirm the induction of typical stress protein size classes by heat shock, sodium arsenite, and the calcium ionophore A23187. Immunoblot analysis and immunocytochemistry verified the expression of the highly inducible 72 kd heat shock protein (HSP72). Granule cell cultures exposed to glutamate showed evidence of cellular injury that was prevented by the noncompetitive NMDA antagonist MK-801, yet glutamate did not induce a detectable stress protein response. Nonetheless, preinduction of heat shock proteins was associated with protection from toxic concentrations of glutamate. These results imply that the HSP72 expression observed in in vivo models of excitotoxicity may not be directly related to the effects of excitatory amino acids. However, the ability of stress protein induction to protect against injury from glutamate may offer a novel approach toward ameliorating damage from excitotoxins.     

Exercise-heat acclimation in humans alters baseline levels and ex vivo heat inducibility of HSP72 and HSP90 in peripheral blood mononuclear cells

The induction of cellular acquired thermal tolerance (ATT) during heat acclimation (HA) in humans is not well described. This study determined whether exercise-HA modifies the human heat shock protein (HSP)72 and HSP90 responses and whether changes are correlated with physiological adaptations to HA. Using a 10-day HA protocol comprising daily exercise (treadmill walking) in a hot environment (T(a) = 49 degrees C, 20% RH), we analyzed baseline and ex vivo heat-induced expression of HSP72 and HSP90 in peripheral blood mononuclear cells (PBMCs) isolated prior to exercise from eight subjects on day 1 and 10 of the HA protocol. Classical physiological responses to HA were observed, including significantly reduced heart rate and core body temperature, and significantly increased sweating rate. Baseline levels of HSP72 and HSP90 were significantly increased following acclimation by 17.7 +/- 6.1% and 21.1 +/- 6.5%, respectively. Ex vivo induction of HSP72 in PBMCs exposed to heat shock (43 degrees C) was blunted on day 10 compared with day 1. A correlation was identified (r(2) = 0.89) between changes in core temperature elevation and ex vivo HSP90 responses to heat shock between days 1 and 10, indicating that volunteers demonstrating the greatest physiological HA tended to exhibit the greatest blunting of ex vivo HSP induction in response to heat shock. In summary, 1) exercise-HA resulted in increased baseline levels of HSP72 and HSP90, 2) ex vivo heat inducibility of HSP72 was blunted after HA, and 3) volunteers demonstrating the greatest physiological HA tended to exhibit the greatest blunting of ex vivo HSP induction in response to heat shock. These data demonstrate that physiological adaptations in humans undergoing HA are accompanied by both increases in baseline levels and changes in regulation of cytoprotective HSPs.     

Sexual dimorphism of the intracellular heat shock protein 72 response.

The majority of previous work examining stress responses has been done in males. Recently, it has become clear that the impact of stressor exposure is modulated by sex. One stress response that may be affected by sex is the induction of intracellular heat shock protein (HSP) 72, which is a stress- responsive molecular chaperone that refolds denatured proteins and promotes cellular survival. The following study compared HSP72 in males and females and also examined whether the estrous cycle altered HSP72 induction in females. We hypothesized that females compared with males would have a constrained HSP72 response after an acute stressor and that the stress-induced HSP72 response in females would fluctuate with the estrous cycle. Male and female F344 rats were either left in their home cage or exposed to acute tail-shock stress (8-10/group). Immediately following stressor, trunk blood was collected and tissues were flash frozen. Vaginal smear and estrogen enzyme immunoassay were used to categorize the phase of estrous. Results show that female rats had a greater corticosterone response than males, that both males and females exhibit a stress-induced release of progesterone, and that males and females had equal levels of stress-induced circulating norepinephrine. Sexual dimorphism of the HSP72 (ELISA) response existed in pituitary gland, mesenteric lymph nodes, and liver such that female rats had an attenuated HSP72 response compared with males after stress. The adrenal glands, spleen, and heart did not exhibit sexual dimorphism of the HSP72 response. The estrous cycle did not have a significant effect on basal or stress-induced HSP72 in females.     

Increased stability of Bcl-2 in HSP70-mediated protection against apoptosis induced by oxidative stress

We have previously shown that heat shock protein 70 (HSP70) markedly inhibits H₂O₂-induced apoptosis in mouse C2C12 myogenic cells by reducing the release of Smac. However, the molecular mechanism by which HSP70 interferes with Smac release during oxidative stress-induced apoptosis is not understood. In the current study, we showed that HSP70 increased the stability of Bcl-2 during oxidative stress. An antisense phosphorothioate oligonucleotide against Bcl-2 caused selective inhibition of Bcl-2 protein expression induced by HSP70 and significantly attenuated HSP70-mediated cell protection against H₂O₂-induced release of Smac and apoptosis. Taken together, our results indicate that there are important relationships among HSP70, Bcl-2, release of Smac, and induction of apoptosis by oxidative stress.     

Heat stress preconditioning does not protect renal epithelial Na(+),K(+),Cl(-) and Na(+),P(i) cotransporters from their modulation by severe heat stress

This study compares the effects of heat and osmotic stress on heat stress protein (HSP) production while examining the putative protective action of HSPs on modulation of Na(+),K(+),Cl(-) and Na(+),P(i) cotransporters in Madin-Darby canine kidney (MDCK) epithelial cells by severe heat stress (46 degrees C, 15 min). Preconditioning heat stress (43 degrees C, 20 min) followed by 4 h recovery at 37 degrees C led to a 35-fold increase of HSP70 mRNA expression measured by Northern blot analysis. The protein content of HSP70 and HSP27, assessed by Western blots, was augmented by 5- and 2-fold, respectively, after 6 h of recovery. In contrast to preconditioning heat stress, hyperosmotic stress (520 vs. 320 mosm) elevated HSP70 mRNA content only by 7-fold and did not significantly affect the protein content of HSP70 or HSP27. Neither cell survival, assessed as lactate dehydrogenase (LDH) release, nor the basal activities of the ion transporters and their modulation by protein kinase C, P(2)-purinoceptor and cell volume were altered by preconditioning heat stress. Severe heat stress increased extracellular LDH content from 3+/-2 to 23+/-5% and enhanced Na(+),K(+),Cl(-) and Na(+),P(i) cotransport activity by 2-3-fold. The volume- and protein kinase C-dependent regulation of these carriers was abolished by severe heat stress while regulation by P(2)-purinoceptors was preserved. Preconditioning heat stress diminished severe heat stress-induced LDH release to 11+/-4% but did not protect Na(+),K(+),Cl(-) and Na(+),P(i) cotransporters from activation by severe heat stress and did not prevent severe heat stress-induced inactivation of protein kinase C- and volume-dependent signaling pathways. These results show that in MDCK cells, preconditioning heat stress-induced HSPs are not involved in the regulation of Na(+),K(+),Cl(-) and Na(+),P(i) cotransporters and do not protect them from modulation by severe heat stress.     

Stressful preconditioning and HSP70 overexpression attenuate proteotoxicity of cellular ATP depletion

Rat H9c2 myoblasts were preconditioned by heat or metabolic stress followed by recovery under normal conditions. Cells were then subjected to severe ATP depletion, and stress-associated proteotoxicity was assessed on 1) the increase in a Triton X-100-insoluble component of total cellular protein and 2) the rate of inactivation and insolubilization of transfected luciferase with cytoplasmic or nuclear localization. Both heat and metabolic preconditioning elevated the intracellular heat shock protein 70 (HSP70) level and reduced cell death after sustained ATP depletion without affecting the rate and extent of ATP decrease. Each preconditioning attenuated the stress-induced insolubility among total cellular protein as well as the inactivation and insolubilization of cytoplasmic and nuclear luciferase. Transient overexpression of human HSP70 in cells also attenuated both the cytotoxic and proteotoxic effects of ATP depletion. Quercetin, a blocker of stress-responsive HSP expression, abolished the effects of stressful preconditioning but did not influence the effects of overexpressed HSP70. Analyses of the cellular fractions revealed that both the stress-preconditioned and HSP70-overexpressing cells retain the soluble pool of HSP70 longer during ATP depletion. Larger amounts of other proteins coimmunoprecipitated with excess HSP70 compared with control cells deprived of ATP. This is the first demonstration of positive correlation between chaperone activity within cells and their viability in the context of ischemia-like stress.     

Cerebral ischemic preconditioning reduces glutamate excitotoxicity by up-regulating the uptake activity of GLT-1 in rats

Our previous study has shown that cerebral ischemic preconditioning (CIP) can up-regulate the expression of glial glutamate transporter-1 (GLT-1) during the induction of brain ischemic tolerance in rats. The present study was undertaken to further explore the uptake activity of GLT-1 in the process by observing the changes in the concentration of extracellular glutamate with cerebral microdialysis and high-performance liquid chromatography. The results showed that a significant pulse of glutamate concentration reached the peak value of sevenfold of the basal level after lethal ischemic insult, which was associated with delayed neuronal death in the CA1 hippocampus. When the rats were pretreated 2 days before the lethal ischemic insult with CIP which protected the pyramidal neurons against delayed neuronal death, the peak value of glutamate concentration decreased to 3.9 fold of the basal level. Furthermore, pre-administration of dihydrokainate, an inhibitor of GLT-1, prevented the protective effect of CIP on ischemia-induced CA1 cell death. At the same time, compared with the CIP + Ischemia group, the peak value of glutamate concentration significantly increased and reached sixfold of the basal level. These results indicate that CIP induced brain ischemic tolerance via up-regulating GLT-1 uptake activity for glutamate and then decreasing the excitotoxicity of glutamate.     

Higher induction of heat shock protein 72 by heat stress in cisplatin-resistant than in cisplatin-sensitive cancer cells

Induction of the heat shock proteins (HSPs) is involved in the increased resistance to cancer therapies such as chemotherapy and hyperthermia. We used two human ovarian cancer cell lines; a cisplatin (CDDP)-sensitive line A2780 and its CDDP-resistant derivative, A2780CP. The concentration of intracellular glutathione (GSH) is higher (2.7-fold increase) in A2780CP cells than in A2780 cells. A mild treatment with a heat stress (42 degrees C for 30 min) induced synthesis of both the heat shock protein 72 (Hsp72) mRNA and the HSP72 protein in A2780CP cells, but not in A2780 cells. In contrast, a severe heat stress (45 degrees C for 30 min) increased synthesis of the HSP72 protein in the two cell lines. The induced level of the HSP72 protein by the severe treatment was higher in A2780CP than in A2780 cells. The gel mobility shift assay showed that DNA binding activities of the heat shock factor (HSF) in the two cell lines were induced similarly and significantly by the mild heat stress. Immunocytochemistry using an anti HSF1 antibody also indicated that mild heat stress activated the HSF1 translocation from the cytosol to the nucleus similarly in the both cell lines. Pretreatment of CDDP-sensitive A2780 cells with N-acetyl-L-cysteine, a precursor of GSH, effectively enhanced induction of the Hsp72 mRNA by the mild heat stress. The present findings demonstrate that induction of the Hsp72 mRNA by the mild heat stress was more extensive in CDDP-resistant A2780CP cells. It is likely that the higher GSH concentration in A2780CP cells plays an important role in promoting Hsp72 gene expression induced by the mild heat stress probably through processes downstream of activation of HSF-DNA binding.     

Invited review: Interplay between molecular chaperones and signaling pathways in survival of heat shock.

Heat shock of mammalian cells causes protein damage and activates a number of signaling pathways. Some of these pathways enhance the ability of cells to survive heat shock, e.g., induction of molecular chaperones [heat shock protein (HSP) HSP72 and HSP27], activation of the protein kinases extracellular signal-regulated kinase and Akt, and phosphorylation of HSP27. On the other hand, heat shock can activate a stress kinase, c-Jun NH2-terminal kinase, thus triggering both apoptotic and nonapoptotic cell death programs. Recent data indicate that kinases activated by heat shock can regulate synthesis and functioning of the molecular chaperones, and these chaperones modulate activity of the cell death and survival pathways. Therefore, the overall balance of the pathways and their interplay determine whether a cell exposed to heat shock will die or survive and become stress tolerant.