Hsp72 functions as a natural inhibitory protein of c-Jun N-terminal kinase

Hsp72, a major inducible member of the heat shock protein family, can protect cells against many cellular stresses including heat shock. In our present study, we observed that pretreatment of NIH 3T3 cells with mild heat shock (43 degrees C for 20 min) suppressed UV-stimulated c-Jun N-terminal kinase 1 (JNK1) activity. Constitutively overexpressed Hsp72 also inhibited JNK1 activation in NIH 3T3 cells, whereas it did not affect either SEK1 or MEKK1 activity. Both in vitro binding and kinase studies indicated that Hsp72 bound to JNK1 and that the peptide binding domain of Hsp72 was important to the binding and inhibition of JNK1. In vivo binding between endogenous Hsp72 and JNK1 in NIH 3T3 cells was confirmed by co-immunoprecipitation. Hsp72 also inhibited JNK-dependent apoptosis. Hsp72 antisense oligonucleotides blocked Hsp72 production in NIH 3T3 cells in response to mild heat shock and concomitantly abolished the suppressive effect of mild heat shock on UV-induced JNK activation and apoptosis. Collectively, our data suggest strongly that Hsp72 can modulate stress-activated signaling by directly inhibiting JNK.     

Positive regulation of ASK1-mediated c-Jun NH(2)-terminal kinase signaling pathway by the WD-repeat protein Gemin5

Gemin5 is a 170-kDa WD-repeat-containing protein that was initially identified as a component of the survival of motor neurons (SMN) complex. We now show that Gemin5 facilitates the activation of apoptosis signal-regulating kinase 1 (ASK1) and downstream signaling. Gemin5 physically interacted with ASK1 as well as with the downstream kinases SEK1 and c-Jun NH(2)-terminal kinase (JNK1), and it potentiated the H(2)O(2)-induced activation of each of these kinases in intact cells. Moreover, Gemin5 promoted the binding of ASK1 to SEK1 and to JNK1, as well as the ASK1-induced activation of JNK1. In comparison, Gemin5 did not physically associate with MKK7, MKK3, MKK6, or p38. Furthermore, depletion of endogenous Gemin5 by RNA interference (RNAi) revealed that Gemin5 contributes to the activation of ASK1 and JNK1, and to apoptosis induced by H(2)O(2) and tumor necrosis factor-alpha (TNFalpha) in HeLa cells. Together, our results suggest that Gemin5 functions as a scaffold protein for the ASK1-JNK1 signaling module and thereby potentiates ASK1-mediated signaling events.     

Mammalian 105 kDa heat shock family proteins suppress hydrogen peroxide-induced apoptosis through a p38 MAPK-dependent mitochondrial pathway in HeLa cells

Hsp105alpha and Hsp105beta are major heat shock proteins in mammalian cells that belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105alpha has opposite effects on stress-induced apoptosis depending on the cell type. However, it is not fully understood how Hsp105 regulates stress-induced apoptosis. In this study, we examined how Hsp105alpha and Hsp105beta regulate H2O2-induced apoptosis by using HeLa cells in which expression of Hsp105alpha or Hsp105beta was regulated using doxycycline. Overexpression of Hsp105alpha and Hsp105beta suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria in H2O2-treated cells. Furthermore, both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) were activated by treatment with H2O2, and the activation of both kinases was suppressed by overexpression of Hsp105alpha and Hsp105beta. However, H2O2-induced apoptosis was suppressed by treatment with a potent inhibitor of p38 MAPK, SB202190, but not a JNK inhibitor, SP600125. These findings suggest that Hsp105alpha and Hsp105beta suppress H2O2-induced apoptosis by suppression of p38 MAPK signaling, one of the essential pathways for apoptosis.     

Protein kinase D specifically mediates apoptosis signal-regulating kinase 1-JNK signaling induced by H2O2 but not tumor necrosis factor

Although both tumor necrosis factor (TNF) and H2O2 induce activation of c-Jun N-terminal kinase (JNK) kinase cascades, it is not known whether they utilize distinct intracellular signaling pathways. In this study, we first examined a variety of pharmacological inhibitors on TNF and H2O2-induced JNK activation. Go6983 or staurosporine, which inhibits protein kinase C isoforms had no effects on TNF or H2O2-induced JNK activation. However, Go6976 and calphostin, which can inhibit protein kinase C as well as protein kinase D (PKD), blocked H2O2- but not TNF-induced JNK activation, suggesting that PKD may be specifically involved in H2O2-induced JNK activation. Consistently, H2O2, but not TNF, induced phosphorylation of PKD and translocation of PKD from endothelial cell membrane to cytoplasm where it associates with the JNK upstream activator, apoptosis signal-regulating kinase 1 (ASK1). The association is mediated through the pleckstrin homology domain of PKD and the C-terminal domain of ASK1. Inhibition of PKD by Go6976 or by small interfering RNA of PKD blocked H2O2-induced ASK1-JNK activation and endothelial cell apoptosis. Interestingly, H2O2 induced 14-3-3 binding to PKD via the phospho-Ser-205/208 and phospho-Ser-219/223 and H2O2-induced 14-3-3 binding of PKD was specifically blocked by Go6976 but not by Go6983. More significantly, the 14-3-3-binding defective forms of PKD failed to associate with ASK1 and to activate JNK signaling, highlighting the importance of 14-3-3 binding of PKD in H2O2-induced activation of ASK1-JNK cascade. Thus, our data have identified PKD as a critical mediator in H2O2- but not TNF-induced ASK1-JNK signaling.     

Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2Cepsilon

ASK1 (apoptosis signal-regulating kinase 1), a MKKK (mitogen-activated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H2O2 and TNFalpha (tumour necrosis factor alpha). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H2O2, ASK1 is transiently activated by autophosphorylation at Thr845. The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr845. Although this feedback regulation mechanism has been elucidated, it remains unclear how ASK1 is maintained in the dephosphorylated state under non-stressed conditions. In the present study, we have examined the possible role of PP2Cepsilon (protein phosphatase 2Cepsilon), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cepsilon inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cepsilon mutant enhanced AP-1 activity. Exogenous PP2Cepsilon associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr845 phosphorylation. The association of endogenous PP2Cepsilon and ASK1 was also observed in mouse brain extracts. PP2Cepsilon directly dephosphorylated ASK1 at Thr845 in vitro. In contrast with PP5, PP2Cepsilon transiently dissociated from ASK1 within cells upon H2O2 treatment. These results suggest that PP2Cepsilon maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cepsilon and PP5 play different roles in H2O2-induced regulation of ASK1 activity.     

Hsp72 and stress kinase c-jun N-terminal kinase regulate the bid-dependent pathway in tumor necrosis factor-induced apoptosis

The major inducible heat shock protein Hsp72 has been shown to protect cells from certain apoptotic stimuli. Here we investigated the mechanism of Hsp72-mediated protection from tumor necrosis factor (TNF)-induced apoptosis of primary culture of IMR90 human fibroblasts. Hsp72 temporarily blocked apoptosis in response to TNF and permanently protected cells from heat shock. An Hsp72 mutant (Hsp72 Delta EEVD) with a deletion of the four C-terminal amino acids, which are essential for the chaperone function, blocked TNF-induced apoptosis in a manner similar to that of normal Hsp72 but did not inhibit heat shock-induced death. Therefore, the chaperone activity of Hsp72 is dispensable for suppression of TNF-induced apoptosis but is required for protection from heat shock. In fibroblasts derived from Bid knockout mice, similar temporal inhibition of TNF-induced apoptosis was seen. In these cells neither normal Hsp72 nor Hsp72 Delta EEVD conferred additional protection from apoptosis, suggesting that Hsp72 specifically affects Bid-dependent but not Bid-independent apoptotic pathways. Furthermore, both normal Hsp72 and Delta Hsp72EEVD inhibited Bid activation and downstream events, including release of cytochrome c, activation of caspase 3, and cleavage of poly-ADP-ribose polymerase. Both Hsp72 and Delta Hsp72EEVD blocked activation of the stress kinase c-jun N-terminal kinase (JNK) by TNF, and specific inhibition of JNK similarly temporarily blocked Bid activation and the downstream apoptotic events. These data strongly suggest that in TNF-induced apoptosis, Hsp72 specifically interferes with the Bid-dependent apoptotic pathway via inhibition of JNK.     

Heat shock pretreatment inhibited the release of Smac/DIABLO from mitochondria and apoptosis induced by hydrogen peroxide in cardiomyocytes and C2C12 myogenic cells

Oxidative stress may cause apoptosis of cardiomyocytes in ischemia-reperfused myocardium, and heat shock pretreatment is thought to be protective against ischemic injury when cardiac myocytes are subjected to ischemia or simulated ischemia. However, the detailed mechanisms responsible for the protective effect of heat shock pretreatment are currently unclear. The aim of this study was to determine whether heat shock pretreatment exerts a protective effect against hydrogen peroxide(H2O2)-induced apoptotic cell death in neonatal rat cardiomyocytes and C2C12 myogenic cells and whether such protection is associated with decreased release of second mitochondria-derived activator of caspase-direct IAP binding protein with low pl (where IAP is inhibitor of apoptosis protein) (Smac/DIABLO) from mitochondria and the activation of caspase-9 and caspase-3. After heat shock pretreatment (42 +/- 0.3 degrees C for 1 hour, recovery for 12 hours), cardiomyocytes and C2C12 myogenic cells were exposed to H2O2 (0.5 mmol/L) for 6, 12, 24, and 36 hours. Apoptosis was evaluated by Hoechst 33258 staining and DNA laddering. Caspase-9 and caspase-3 activities were assayed by caspase colorimetric assay kit and Western analysis. Inducible heat shock proteins (Hsp) were detected using Western analysis. The release of Smac/DIABLO from mitochondria to cytoplasm was observed by Western blot and indirect immunofluorescence analysis. (1) H2O2 (0.5 mmol/L) exposure induced apoptosis in neonatal rat cardiomyocytes and C2C12 myogenic cells, with a marked release of Smac/DIABLO from mitochondria into cytoplasm and activation of caspase-9 and caspase-3, (2) heat shock pretreatment induced expression of Hsp70, Hsp90, and alphaB-crystallin and inhibited H2O2-mediated Smac/DIABLO release from mitochondria, the activation of caspase-9, caspase-3, and subsequent apoptosis. H2O2 can induce the release of Smac/DIABLO from mitochondria and apoptosis in cardiomyocytes and C2C12 myogenic cells. Heat shock pretreatment protects the cells against H2O2-induced apoptosis, and its mechanism appears to involve the inhibition of Smac release from mitochondria.     

Identification of a novel antiapoptotic protein that antagonizes ASK1 and CAD activities

Diverse stimuli initiate the activation of apoptotic signaling pathways that often causes nuclear DNA fragmentation. Here, we report a new antiapoptotic protein, a caspase-activated DNase (CAD) inhibitor that interacts with ASK1 (CIIA). CIIA, by binding to apoptosis signal-regulating kinase 1 (ASK1), inhibits oligomerization-induced ASK1 activation. CIIA also associates with CAD and inhibits the nuclease activity of CAD without affecting caspase-3-mediated ICAD cleavage. Overexpressed CIIA reduces H2O2- and tumor necrosis factor-alpha-induced apoptosis. CIIA antisense oligonucleotides, which abolish expression of endogenous CIIA in murine L929 cells, block the inhibitory effect of CIIA on ASK1 activation, deoxyribonucleic acid fragmentation, and apoptosis. These findings suggest that CIIA is an endogenous antagonist of both ASK1- and CAD-mediated signaling.     

Induction of heat shock proteins (HSPs) by sodium arsenite in cultured astrocytes and reduction of hydrogen peroxide-induced cell death

Induction of heat shock proteins (HSPs) protects cells from oxidative injury. Here Hsp72, Hsp27 and heme oxygenase-1 (HO-1) were induced in cultured rat astrocytes, and protection against oxidative stress was investigated. Astrocytes were treated with sodium arsenite (20-50 micro m) for 1 h, which was non-toxic to cells, 24 h later they were exposed to 400 micro m H2O2 for 1 h, and cell death was evaluated at different time points. Arsenite triggered strong induction of HSPs, which was prevented by 1 micro g/mL cycloheximide (CXH). H2O2 caused cell loss and increased cell death with features of apoptosis, i.e. TdT-mediated dUTP nick-end labelling (TUNEL) reaction and caspase-3 activation. These features were abrogated by pre-treatment with arsenite, which prevented cell loss and significantly reduced the number of dead cells. The protective effect of arsenite was not detected in the presence of CHX. Pre-treatment with arsenite increased protein kinase B (Akt) and extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation after H2O2. However, while Akt phosphorylation was prevented by CHX, Erk1/2 phosphorylation was further enhanced by CHX. The results show that transient arsenite pre-treatment induces Hsp72, HO-1 and, to a lesser extent, Hsp27; it reduces H2O2-induced astrocyte death; and it causes selective activation of Akt following H2O2. It is suggested that HSP expression at the time of H2O2 exposure protects astrocytes from oxidative injury and apoptotic cell death by means of pro-survival Akt.     

The function of HSP72 in suppression of c-Jun N-terminal kinase activation can be dissociated from its role in prevention of protein damage.

Activation of the c-Jun N-terminal kinase (JNK) by a variety of stimuli is critical for regulation of many cellular processes including apoptosis. The major inducible heat shock protein Hsp72 has previously been demonstrated to inhibit activation of JNK in cells exposed to heat shock and other protein-damaging agents, thus suppressing apoptosis. Hsp72 can protect proteins from stress-induced damage. To test if this protective function of Hsp72 is involved in JNK suppression, we investigated whether Hsp72 can avert activation of JNK by stimuli that do not cause protein damage. We show that Hsp72 suppresses activation of JNK induced by non-protein-damaging stimuli, interleukin-1 and UV irradiation, as well as by constitutively active components of the JNK signaling cascade Cdc42 and MEKK1. Furthermore, Hsp72 strongly reduced activation of JNK by phosphatase inhibitors. We also demonstrate that an Hsp72 mutant that lacks the ATPase domain is still capable of JNK suppression, thus indicating that the protein refolding activity of Hsp72 is not critical for inhibition of JNK activation. Taken together these data suggest that Hsp72 plays a regulatory role in JNK signaling and that the function of Hsp72 in protein protection or refolding is not involved in JNK regulation.     

Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress.

Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that activates the JNK and p38 MAP kinase cascades and is activated in response to oxidative stress such as hydrogen peroxide (H(2)O(2)). A yeast two-hybrid screening identified a serine/threonine protein phosphatase 5 (PP5) as a binding partner of ASK1. PP5 directly dephosphorylated an essential phospho-threonine residue within the kinase domain of ASK1 and thereby inactivated ASK1 activity in vitro and in vivo. The interaction between PP5 and ASK1 was induced by H(2)O(2) treatment and was followed by the decrease in ASK1 activity. PP5 inhibited not only H(2)O(2)-induced sustained activation of ASK1 but also ASK1-dependent apoptosis. Thus, PP5 appears to act as a physiological inhibitor of ASK1-JNK/p38 pathways by negative feedback.     

A novel function of peroxiredoxin 1 (Prx-1) in apoptosis signal-regulating kinase 1 (ASK1)-mediated signaling pathway

We report a novel function of peroxiredoxin-1 (Prx-1) in the ASK1-mediated signaling pathway. Prx-1 interacts with ASK1 via the thioredoxin-binding domain of ASK1 and this interaction is highly inducible by H2O2. However, catalytic mutants of Prx1, C52A, C173A, and C52A/C173A, could not undergo H2O2 inducible interactions, indicating that the redox-sensitive catalytic activity of Prx-1 is required for the interaction with ASK1. Prx-1 overexpression inhibited the activation of ASK1, and resulted in the inhibition of downstream signaling cascades such as the MKK3/6 and p38 pathway. In Prx-1 knockdown cells, ASK1, p38, and JNK were quickly activated, leading to apoptosis in response to H2O2. These findings suggest a negative role of Prx-1 in ASK1-induced apoptosis.     

Analysis of properties of small heat shock protein Hsp25 in MAPK-activated protein kinase 2 (MK2)-deficient cells: MK2-dependent insolubilization of Hsp25 oligomers correlates with susceptibility to stress

Small heat shock proteins (sHsps) exist in dynamic oligomeric complexes and display diverse biological functions ranging from chaperone properties to modulator of apoptosis. So far, the role of stress-dependent phosphorylation of mammalian sHsps for its structure and function has been analyzed by using various phosphorylation site mutants overexpressed in different cell types as well as by non-exclusive inhibitors of the p38 MAPK cascade. Here we investigate the role of phosphorylation of endogenous sHsp in a genetic model lacking the major Hsp25 kinase, the MAP kinase-activated protein kinase MK2. We demonstrate that in MK2-deficient fibroblasts, where no stress-dependent phosphorylation of Hsp25 at Ser86 and no in vitro binding to 14-3-3 was detectable, stress-dependent disaggregation of endogenous Hsp25 complexes is impared and kinetics of arsenite-dependent, H2O2-dependent, and sublethal heat shock-induced insolubilization of Hsp25 is delayed. Similarly, green fluorescent protein-tagged Hsp25 shows retarded subcellular accumulation into stress granules in MK2-deficient cells after arsenite treatment. Decreased insolubilization of Hsp25 in MK2-deficient cells correlates with increased resistance against arsenite, H2O2, and sublethal heat shock treatment and with decreased apoptosis. In contrast, after severe, lethal heat shock MK2-deficient embryonic fibroblasts cells show fast and complete insolubilization of Hsp25 independent of MK2 and no increased stress resistance. Hence, MK2-dependent formation of insoluble stress granules and irreversible cell damage by oxidative stresses and sublethal heat shock correlate and only upon severe, lethal heat shock MK2-independent processes could determine insolubilization of Hsp25 and are more relevant for cellular stress damage.     

C-terminus of heat shock protein 70-interacting protein facilitates degradation of apoptosis signal-regulating kinase 1 and inhibits apoptosis signal-regulating kinase 1-dependent apoptosis

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that is regulated under conditions of cellular stress. ASK1 phosphorylates c-Jun N-terminal kinase (JNK) and elicits an apoptotic response. ASK1 activity is regulated at multiple levels, 1 of which is through inhibition by cytosolic chaperones of the heat shock protein (Hsp) 70 family. Among the proteins that determine Hsp70 function, CHIP (C-terminus of Hsp70-interacting protein) is a cochaperone and ubiquitin ligase that interacts with Hsp70 through an amino-terminal tetratricopeptide repeat (TPR) domain. Prominent among the cellular functions mediated by CHIP is protection against physiologic stress. Because ASK1 is known to contain a TPR-acceptor site, we examined the role of CHIP in regulating ASK1 function. CHIP interacted with ASK1 in a TPR-dependent fashion and induced ubiquitylation and proteasome-dependent degradation of ASK1. Targeting of ASK1 by CHIP inhibited JNK activation in response to oxidative challenge and reduced ASK1-dependent apoptosis, whereas short interfering RNA (siRNA)-dependent depletion of CHIP enhanced JNK activation. Consistent with its ability to reduce cytoplasmic ASK1 levels, CHIP triggered the translocation of ASK1 partner protein death-associated protein (Daxx) into the nucleus, where it is known to activate an antiapoptotic response. These results indicate that CHIP regulates ASK1 activity by inducing its ubiquitylation and degradation, which, together with its effects on Daxx localization, provides a mechanism for the antiapoptotic effects of CHIP observed in the face of cellular and physiologic stress.     

Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival

The serine/threonine kinase Akt (also known as protein kinase B) is activated in response to various stimuli by a mechanism involving phosphoinositide 3-kinase (PI3-K). Akt provides a survival signal that protects cells from apoptosis induced by growth factor withdrawal, but its function in other forms of stress is less clear. Here we investigated the role of PI3-K/Akt during the cellular response to oxidant injury. H(2)O(2) treatment elevated Akt activity in multiple cell types in a time- (5-30 min) and dose (400 microM-2 mm)-dependent manner. Expression of a dominant negative mutant of p85 (regulatory component of PI3-K) and treatment with inhibitors of PI3-K (wortmannin and LY294002) prevented H(2)O(2)-induced Akt activation. Akt activation by H(2)O(2) also depended on epidermal growth factor receptor (EGFR) signaling; H(2)O(2) treatment led to EGFR phosphorylation, and inhibition of EGFR activation prevented Akt activation by H(2)O(2). As H(2)O(2) causes apoptosis of HeLa cells, we investigated whether alterations of PI3-K/Akt signaling would affect this response. Wortmannin and LY294002 treatment significantly enhanced H(2)O(2)-induced apoptosis, whereas expression of exogenous myristoylated Akt (an activated form) inhibited cell death. Constitutive expression of v-Akt likewise enhanced survival of H(2)O(2)-treated NIH3T3 cells. These results suggest that H(2)O(2) activates Akt via an EGFR/PI3-K-dependent pathway and that elevated Akt activity confers protection against oxidative stress-induced apoptosis.     

Apoptosis signal-regulating kinase 1 is involved not only in apoptosis but also in non-apoptotic cardiomyocyte death

The molecular basis of myocardial cell death in the ischemia-reperfused heart still remains to be clarified. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays an important role in stress-induced apoptosis. We studied ASK1(-/-) mice to examine the role of ASK1 in ischemia-reperfusion injury. In the wild-type heart, ischemia-reperfusion resulted in necrotic injury, whereas infarct size was drastically reduced in the ASK1(-/-) heart. The necrotic injury was not accompanied with any evidence of apoptosis such as an increase in TUNEL-positive cells, DNA fragmentation or the activation of caspase-3. ASK1(-/-) cardiomyocytes were more resistant to H(2)O(2)- or Ca(2+)-induced apoptotic and non-apoptotic cell death compared with wild-type cells. These data suggest that ASK1 is involved in necrosis as well as apoptosis and that ASK1-dependent necrosis is likely to contribute to myocardial cell death in the ischemia-reperfused heart.     

Catalase, but not MnSOD, inhibits glucose deprivation-activated ASK1-MEK-MAPK signal transduction pathway and prevents relocalization of Daxx: hydrogen peroxide as a major second messenger of metabolic oxidative stress

Overexpression of catalase, but not manganese superoxide dismutase (MnSOD), inhibited glucose deprivation-induced cytotoxicity and c-Jun N-terminal kinase 1 (JNK1) activation in human prostate adenocarcinoma DU-145 cells. Suppression of JNK1 activation by catalase overexpression resulted from inhibition of apoptosis signal-regulating kinase 1 (ASK1) activation by preventing dissociation of thioredoxin (TRX) from ASK1. Overexpression of catalase also inhibited relocalization of Daxx from the nucleus to the cytoplasm as well as association of Daxx with ASK1 during glucose deprivation. Taken together, hydrogen peroxide (H(2)O(2)) rather than superoxide anion (O(2) (*-)) acts as a second messenger of metabolic oxidative stress to activate the ASK1-MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-mitogen-activated protein kinase (MAPK) signal transduction pathway.