A selective inhibitor of p38 MAP kinase, SB202190, induced apoptotic cell death of a lipopolysaccharide-treated macrophage-like cell line, J774.1

A selective p38 MAP kinase (p38 MAPK) inhibitor, SB202190, induced apoptotic cell death of a macrophage-like cell line, J774.1, in the presence of lipopolysaccharide (LPS), as judged by DNA nicks revealed by terminal deoxy transferase (TdT)-mediated dUTP nick end labeling (TUNEL), activation of caspase-3, and subsequent release of lactate dehydrogenase. This cytotoxicity was dependent on both LPS and SB202190, and such inhibitors of the upstream LPS-signaling cascade as polymyxin B and TPCK blocked this macrophage cell death. SB202190 suppressed the kinase activity of p38, leading to inhibition of activation of MAPKAPK2 and then the subsequent phosphorylation of hsp27 in LPS-treated macrophages both in vitro and in vivo, but an inactive analog of SB202190, SB202474, did not. There was a threshold of the time of addition of SB202190 to LPS-treated macrophages to induce apoptosis, which was before full transmission of p38 activity to a direct downstream kinase, MAPKAPK2. Besides, localization of phosphorylated hsp27 in Golgi area of the LPS-treated macrophages was suppressed by SB202190, while it was not by SB202474. These results suggest that selective inhibition of p38 MAPK activity in LPS-induced MAP kinase cascade leads to apoptosis of macrophages.     

Short-chain fatty acids induced autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death

Short-chain fatty acids (SCFAs) are the major by-products of bacterial fermentation of undigested dietary fibers in the large intestine. SCFAs, mostly propionate and butyrate, inhibit proliferation and induce apoptosis in colon cancer cells, but clinical trials had mixed results regarding the anti-tumor activities of SCFAs. Herein we demonstrate that propionate and butyrate induced autophagy in human colon cancer cells to dampen apoptosis whereas inhibition of autophagy potentiated SCFA induced apoptosis. Colon cancer cells, after propionate treatment, exhibited extensive characteristics of autophagic proteolysis: increased LC3-I to LC3-II conversion, acidic vesicular organelle development, and reduced p62/SQSTM1 expression. Propionate-induced autophagy was associated with decreased mTOR activity and enhanced AMP kinase activity. The elevated AMPKα phosphorylation was associated with cellular ATP depletion and overproduction of reactive oxygen species due to mitochondrial dysfunction involving the induction of MPT and loss of Δψ. In this context, mitochondria biogenesis was initiated to recover cellular energy homeostasis. Importantly, when autophagy was prevented either pharmacologically (3-MA or chloroquine) or genetically (knockdown of ATG5 or ATG7), the colon cancer cells became sensitized toward propionate-induced apoptosis through activation of caspase-7 and caspase-3. The observations indicate that propionate-triggered autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death, whereas application of an autophagy inhibitor (Chloroquine) is expected to enhance the therapeutic efficacy of SCFAs in inducing colon tumor cell 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.     

Stress protein flux during recovery from simulated ischemia: induced heat shock protein 70 confers cytoprotection by suppressing JNK activation and inhibiting apoptotic cell death

Multiple stress proteins are recruited in response to stress in living cells. There are limited reports in the literature analyzing multiple stress protein shifts and their functional consequences on stress response. Using two-dimensional electrophoresis we have analyzed shifts in stress protein profiles in response to energy deprivation as a model of ischemic injury to kidneys. A group of chaperones and stress-induced mitogen activated protein (MAP) kinases were analyzed. In addition to examining stress protein induction and phosphorylation we have also examined the mechanism of cytoprotection by heat shock protein 70 (Hsp70). Our results show that, of the different stress proteins examined, only binding protein (BiP) and Hsp70 were significantly induced upon energy deprivation. Other stress proteins, including Hsp27, calnexin, Hsp90 and ERp57 showed alterations in their phosphorylation profiles. Three different MAP kinases, namely p38, extracellular signal regulated kisase and c-jun N-terminal kinase (JNK) were activated in response to energy deprivation. While JNK activation was linked to apoptosis, activated-p38 was involved in phosphorylation of Hsp27. Study of inhibitors of Hsp70 induction or pre-induction of Hsp70 indicated that induced Hsp70 was involved in the suppression of JNK activation thereby inhibiting apoptotic cell death. Our results provide important insights into the flux in stress protein profiles in response to simulated ischemia and highlight the antiapoptotic, cytoprotective mechanism of Hsp70 action.     

p38 mitogen-activated protein kinase is a key regulator of 5-phenylselenyl- and 5-methylselenyl-methyl-2'-deoxyuridine-induced apoptosis in human HL-60 cells

Two novel, modified thymidine nucleosides, 5-phenylselenyl-methyl-2'-deoxyuridine (PhSe-T) and 5-methylselenyl-methyl-2'-deoxyuridine (MeSe-T), trigger reactive oxygen species (ROS) generation and DNA damage and thereby induce caspase-mediated apoptosis in human HL-60 cells; however, the mechanism leading to caspase activation and apoptotic cell death remains unclear. Therefore, we investigated the signaling molecules involved in nucleoside derivative-induced caspase activation and apoptosis in HL-60 cells. PhSe-T/MeSe-T treatment activated two mitogen-activated protein kinases (MAPKs), extracellular-receptor kinase (ERK) and p38, and induced the phosphorylation of two downstream targets of p38, ATF-2 and MAPKAPK2. In addition, the selective p38 inhibitor SB203580 suppressed PhSe-T/MeSe-T-induced apoptosis and activation of caspase-3, -9, -8, and -2, whereas the jun amino-terminal kinase (JNK) inhibitor SP600125 and the ERK inhibitor PD98059 had no effect. SB203580 and an ROS scavenger, tiron, inhibited PhSe-T/MeSe-T-induced histone H2AX phosphorylation, which is a DNA damage marker. Moreover, tiron inhibited PhSe-T/MeSe-T-induced phosphorylation of p38 and enhanced p38 MAP kinase activity, indicating a role for ROS in PhSe-T/MeSe-T-induced p38 activation. Taken together, our results suggest that PhSe-T/MeSe-T-induced apoptosis is mediated by the p38 pathway and that p38 serves as a link between ROS generation and DNA damage/caspase activation in HL-60 cells.     

Mitogen Activated Protein Kinase Activated Protein Kinase 2 Regulates Actin Polymerization and Vascular Leak in Ventilator Associated Lung Injury

Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2^−/− mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2^−/− mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV_T MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.     

The protein kinase inhibitor SB203580 uncouples PMA-induced differentiation of HL-60 cells from phosphorylation of Hsp27

HL-60 cells are an attractive model for studies of human myeloid cell differentiation. Among the well-examined parameters correlated to differentiation of HL-60 cells are the expression and phosphorylation of the small heat shock protein Hsp27. Here we demonstrate that PMA treatment of HL-60 cells stimulates different MAP kinase cascades, leading to significant activation of ERK2 and p38 reactivating kinase (p38RK). Using the protein kinase inhibitor SB 203580, we specifically inhibited p38RK and, thereby, activation of its target MAP kinase-activated protein kinase 2(MAPKAP kinase 2), which is the major enzyme responsible for small Hsp phosphorylation. As a result, PMA-induced Hsp27 phosphorylation is inhibited in SB 203580-treated HL-60 cells indicating that p38RK and MAPKAP kinase 2 are components of the PMA-induced signal transduction pathway leading to Hsp27 phosphorylation. We further demonstrate that, although PMA-induced phosphorylation is inhibited, SB 203580-treated HL-60 cells are still able to differentiate to the macrophage-like phenotype as judged by decrease in cell proliferation, induction of expression of the cell surface antigen CD11b and changes in cell morphology. These results indicate that, although correlated, Hsp27 phosphorylation is not required for HL-60 cell differentiation. However, the results do not exclude that increased Hsp27 expression is involved in HL-60 cell differentiation.     

p38 mitogen-activated protein kinase and mitogen-activated protein kinase-activated protein kinase 2 (MK2) signaling in atrophic and hypertrophic denervated mouse skeletal muscle

Background

p38 mitogen-activated protein kinase has been implicated in both skeletal muscle atrophy and hypertrophy. T317 phosphorylation of the p38 substrate mitogen-activated protein kinase-activated protein kinase 2 (MK2) correlates with muscle weight in atrophic and hypertrophic denervated muscle and may influence the nuclear and cytoplasmic distribution of p38 and/or MK2. The present study investigates expression and phosphorylation of p38, MK2 and related proteins in cytosolic and nuclear fractions from atrophic and hypertrophic 6-days denervated skeletal muscles compared to innervated controls.

Methods

Expression and phosphorylation of p38, MK2, Hsp25 (heat shock protein25_rodent/27_human, Hsp25/27) and Hsp70 protein expression were studied semi-quantitatively using Western blots with separated nuclear and cytosolic fractions from innervated and denervated hypertrophic hemidiaphragm and atrophic anterior tibial muscles. Unfractionated innervated and denervated atrophic pooled gastrocnemius and soleus muscles were also studied.

Results

No support was obtained for a differential nuclear/cytosolic localization of p38 or MK2 in denervated hypertrophic and atrophic muscle. The differential effect of denervation on T317 phosphorylation of MK2 in denervated hypertrophic and atrophic muscle was not reflected in p38 phosphorylation nor in the phosphorylation of the MK2 substrate Hsp25. Hsp25 phosphorylation increased 3-30-fold in all denervated muscles studied. The expression of Hsp70 increased 3-5-fold only in denervated hypertrophic muscles.

Conclusions

The study confirms a differential response of MK2 T317 phosphorylation in denervated hypertrophic and atrophic muscles and suggests that Hsp70 may be important for this. Increased Hsp25 phosphorylation in all denervated muscles studied indicates a role for factors other than MK2 in the regulation of this phosphorylation.

     

Short-chain fatty acids induce acute phosphorylation of the p38 mitogen-activated protein kinase/heat shock protein 27 pathway via GPR43 in the MCF-7 human breast cancer cell line

The expression of GPR41 and 43, which have recently been identified as G-protein-coupled cell-surface receptors for short-chain fatty acids (SCFAs), was detected in a human breast cancer cell line (MCF-7) by RT-PCR. Acetate, propionate and butyrate induced an increase in intracellular Ca2+ in these cells that was not blocked by treatment with pertussis toxin (PTX). SCFAs significantly reduced forskolin-induced cAMP levels in these cells. The phosphorylation of mitogen-activated protein kinase (MAPK) p38 was selectively increased by SCFAs. The downstream substrate heat shock protein 27 (HSP27) was also phosphorylated by SCFAs at Ser-78 and-82, but not-15. Propionate induced elevations in intracellular Ca2+ and the phosphorylation of p38 were inhibited by the silencing of GPR43 using a specific siRNA. These results suggest that GPR41 and 43 mediate SCFA signaling in mammary epithelial cells and thereby play an important role in their stress management.     

JlpA of Campylobacter jejuni interacts with surface-exposed heat shock protein 90alpha and triggers signalling pathways leading to the activation of NF-kappaB and p38 MAP kinase in epithelial cells

Campylobacter jejuni is a leading cause of acute bacterial gastroenteritis in humans. The mechanism by which C. jejuni interacts with host cells, however, is still poorly understood. Our previous study has shown that the C. jejuni surface lipoprotein JlpA mediates adherence of the bacterium to epithelial cells. In this report, we demonstrated that JlpA interacts with HEp-2 cell surface heat shock protein (Hsp) 90alpha and initiates signalling pathways leading to activation of NF-kappaB and p38 MAP kinase. Gel overlay and GST pull down assays showed that JlpA interacts with Hsp90alpha. Geldanamycin, a specific inhibitor of Hsp90, and anti-human Hsp90alpha antibody significantly blocked the interaction between JlpA and Hsp90alpha, suggesting a direct interaction between JlpA and HEp-2 cell surface-exposed Hsp90alpha. The treatment of HEp-2 cells with GST-JlpA initiated two signalling pathways: one leading to the phosphorylation and degradation of IkappaB and nuclear translocation of NF-kappaB; and another one to the phosphorylation of p38 MAP kinase. The activation of NF-kappaB and p38 MAP kinase in HEp-2 cells suggest that JlpA triggers inflammatory/immune responses in host cells following C. jejuni infection.     

Arachidonic acid activates mitogen-activated protein (MAP) kinase-activated protein kinase 2 and mediates adhesion of a human breast carcinoma cell line to collagen type IV through a p38 MAP kinase-dependent pathway

Adhesion of metastatic human mammary carcinoma MDA-MB-435 cells to the basement membrane protein collagen type IV can be activated by treatment with arachidonic acid. We initially observed that this arachidonic acid-mediated adhesion was inhibited by the tyrosine kinase inhibitor genistein. Therefore, we examined the role of the mitogen-activated protein (MAP) kinase family tyrosine phosphorylation-regulated pathways in arachidonic acid-stimulated cell adhesion. Arachidonic acid stimulated the phosphorylation of p38, the activation of MAP kinase-activated protein kinase 2 (MAPKAPK2, a downstream substrate of p38), and the phosphorylation of heat shock protein 27 (a downstream substrate of MAP kinase-activated protein kinase 2). Treatment with the p38 inhibitor PD169316 completely and specifically inhibited arachidonic acid-mediated cell adhesion to collagen type IV. p38 activity was specifically associated with arachidonic acid-stimulated adhesion; this was demonstrated by the observation that 12-O-tetradecanoylphorbol 13-acetate-activated cell adhesion was not blocked by inhibiting p38 activity. Extracellular signal-regulated protein kinases (ERKs) 1 and 2 were also activated by arachidonic acid; however, cell adhesion to collagen type IV was not highly sensitive to PD98059, an inhibitor of MAP kinase kinase/ERK kinase 1 (MEK1) that blocks activation of the ERKs. c-Jun NH(2)-terminal kinase was not activated by arachidonic acid treatment of these cells. Together, these data suggest a novel role for p38 MAP kinase in regulating adhesion of breast cancer cells to collagen type IV.     

Proteomic identification of 14-3-3zeta as a mitogen-activated protein kinase-activated protein kinase 2 substrate: role in dimer formation and ligand binding

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK2) mediates multiple p38 MAPK-dependent inflammatory responses. To define the signal transduction pathways activated by MAPKAPK2, we identified potential MAPKAPK2 substrates by using a functional proteomic approach consisting of in vitro phosphorylation of neutrophil lysate by active recombinant MAPKAPK2, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and phosphoprotein identification by peptide mass fingerprinting with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and protein database analysis. One of the eight candidate MAPKAPK2 substrates identified was the adaptor protein, 14-3-3zeta. We confirmed that MAPKAPK2 interacted with and phosphorylated 14-3-3zeta in vitro and in HEK293 cells. The chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated p38-MAPK-dependent phosphorylation of 14-3-3 proteins in human neutrophils. Mutation analysis showed that MAPKAPK2 phosphorylated 14-3-3zeta at Ser-58. Computational modeling and calculation of theoretical binding energies predicted that both phosphorylation at Ser-58 and mutation of Ser-58 to Asp (S58D) compromised the ability of 14-3-3zeta to dimerize. Experimentally, S58D mutation significantly impaired both 14-3-3zeta dimerization and binding to Raf-1. These data suggest that MAPKAPK2-mediated phosphorylation regulates 14-3-3zeta functions, and this MAPKAPK2 activity may represent a novel pathway mediating p38 MAPK-dependent inflammation.     

Progressive left ventricular remodeling, myocyte apoptosis, and protein signaling cascades after myocardial infarction in rabbits

To determine the temporal changes in oxidative stress, mitogen-activated protein (MAP) kinases and mitochondrial apoptotic proteins, and their relationship to myocyte apoptosis in the remote noninfarcted myocardium after myocardial infarction (MI), rabbits were randomly assigned to either coronary artery ligation to produce MI or sham operation. The animals were sacrificed at 1, 4, 8, or 12 weeks after coronary artery occlusion. Sham rabbits were sacrificed at 12 weeks after surgery. MI rabbits exhibited progressive increases of left ventricular (LV) end-diastolic pressure and end-diastolic dimension, and progressive decreases of LV fractional shortening and dP/dt over 12 weeks. The LV remodeling with LV chamber dilation and LV systolic dysfunction was temporally associated with progressive increases of cardiac oxidative stress as evidenced by decreased myocardial reduced-to-oxidized-glutathione ratio and increased myocardial 8-hydroxydeoxyguanosine and myocyte apoptosis. The ERK and JNK activities were decreased while p38 MAP kinase activity was increased with age of MI. The extent of p38 MAP kinase activation correlated with Bcl-2 phosphorylation. Bcl-2 protein was decreased in both mitochondrial and cytosolic fractions with age of MI. Bax protein was increased in both mitochondrial and cytosolic fractions. Cytochrome c was reduced in mitochondrial fraction and increased in cytosolic fraction in a time-dependent manner after MI. Cleaved caspase 9 and caspase 3 proteins were time-dependently increased after MI. These data suggest that p38 MAP kinase activation is not only time-dependent after MI, but also correlates with oxidative stress, Bcl-2 phosphorylation, and myocyte apoptosis. These changes in the remote noninfarcted myocardium may contribute to LV remodeling and dysfunction after MI.     

Apoptosis induced by domoic acid in mouse cerebellar granule neurons involves activation of p38 and JNK MAP kinases

In mouse cerebellar granule neurons (CGNs) the marine neurotoxin domoic acid (DomA) induces neuronal cell death, either by apoptosis or by necrosis, depending on its concentration, with apoptotic damage predominating in response to low concentrations (100 nM). DomA-induced apoptosis is due to selective activation of AMPA/kainate receptors, and is mediated by DomA-induced oxidative stress, leading to mitochondrial dysfunction and activation of caspase-3. The p38 MAP kinase and the c-Jun NH2-terminal protein kinase (JNK) have been shown to be preferentially activated by oxidative stress. Here we report that DomA increases p38 MAP kinase and JNK phosphorylation, and that this effect is more pronounced in CGNs from Gclm (-/-) mice, which lack the modifier subunit of glutamate-cysteine ligase, have very low glutathione (GSH) levels, and are more sensitive to DomA-induced apoptosis than CGNs from wild-type mice. The increased phosphorylation of JNK and p38 kinase was paralleled by a decreased phosphorylation of Erk 1/2. The AMPA/kainate receptor antagonist NBQX, but not the NMDA receptor antagonist MK-801, prevents DomA-induced activation of p38 and JNK kinases. Several antioxidants (GSH ethyl ester, catalase and phenylbutylnitrone) also prevent DomA-induced phosphorylation of JNK and p38 MAP kinases. Inhibitors of p38 (SB203580) and of JNK (SP600125) antagonize DomA-induced apoptosis. These results indicate the importance of oxidative stress-activated JNK and p38 MAP kinase pathways in DomA-induced apoptosis in CGNs.     

Protein kinase inhibitors can suppress stress-induced dissociation of Hsp27

We previously showed that the aggregated form of Hsp27 in cultured cells becomes dissociated as a result of phosphorylation with various types of stress. In order to clarify the signal transduction cascade involved, the effects of various inhibitors of protein kinases and dithiothreitol on the dissociation of Hsp27 were here examined by means of an immunoassay after fractionation of cell extracts by sucrose density gradient centrifugation. The dissociation of Hsp27 induced by exposure of U251 MG human glioma cells to metals (NaAsO₂ and CdCl₂), hypertonic stress (sorbitol and NaCl), or anisomycin, an activator of p38 mitogen–activated protein (MAP) kinase, was completely suppressed by the presence of SB 203580 or PD 169316, inhibitors of p38 MAP kinase, but not by PD 98059 and Uo 126, inhibitors of MAP kinase kinase (MEK), nor by staurosporine, Go 6983, and bisindolylmaleimide I, inhibitors of protein kinase C. Phorbol ester (PMA)–induced dissociation of Hsp27 was completely suppressed by staurosporine, Go 6983, or bisindolylmaleimide I and partially suppressed by SB 203580, or PD 169316 but not by PD 98059 or Uo 126, indicating mediation by 2 cascades. The presence of 1 mM dithiothreitol in the culture medium during exposure to chemicals suppressed the dissociation of Hsp27 induced by arsenite and CdCl₂ but not by other chemicals. These results suggest that the phosphorylation of Hsp27 is catalyzed by 2 protein kinases, p38 MAP kinase–activated protein (MAPKAP) kinase- 2/3 and protein kinase C. In addition, metal-induced signals are sensitive to reducing power.     

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.