Báo cáo khoa học: Mammalian 105 kDa heat shock family proteins suppress hydrogen peroxide-induced apoptosis through a p38 MAPK-dependent mitochondrial pathway in HeLa cells potx

Mammalian 105 kDa heat shock family proteins suppress hydrogen peroxide-induced apoptosis through a p38 MAPK-dependent mitochondrial pathway in HeLa cells Nobuyuki Yamagishi, Youhei Saito and Takumi Hatayama Department of Biochemistry, Division of Biological Sciences, Kyoto Pharmaceutical University, Japan Keywords apoptosis; H2O2; Hsp105; JNK; p38 MAPK Correspondence T Hatayama, Department of Biochemistry, Division of Biological Sciences, Kyoto Pharmaceutical University, Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan Fax: +81 75 595 4758 Tel: +81 75 595 4653 E-mail: hatayama@mb.kyoto-phu.ac.jp (Received 28 April 2008, revised 23 June 2008, accepted 15 July 2008) doi:10.1111/j.1742-4658.2008.06598.x Hsp105a and Hsp105b are major heat shock proteins in mammalian cells that belong to a subgroup of the HSP70 family, HSP105 ⁄ 110 Previously, we have shown that Hsp105a 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 Hsp105a and Hsp105b regulate H2O2-induced apoptosis by using HeLa cells in which expression of Hsp105a or Hsp105b was regulated using doxycycline Overexpression of Hsp105a and Hsp105b 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 Hsp105a and Hsp105b 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 Hsp105a and Hsp105b suppress H2O2-induced apoptosis by suppression of p38 MAPK signaling, one of the essential pathways for apoptosis Heat shock proteins (Hsps) are a set of highly conserved proteins produced in response to physiological and environmental stress that serve to protect cells from stress-induced damage by preventing protein denaturation and ⁄ or repairing such damage [1] Mammalian Hsps are classified into several families on the basis of their apparent molecular weight and function, such as the HSP105 ⁄ 110, HSP90, HSP70, HSP60, HSP40, and HSP27 families The HSP70 family is a major and well-characterized group of Hsps Several species of HSP70 family proteins are present in the various compartments of eukaryotic cells These proteins play important roles as molecular chaperones that prevent the irreversible aggregation of denatured proteins and assist the folding, assembly and translocation across membranes of cellular proteins [2,3] In addition, Hsp70 protects against apoptosis caused by a variety of stressors such as heat shock, oxidative stress and chemotherapeutic agents [4–6], and recent studies have demonstrated that Hsp70 can modulate the functions of several major components in the apoptotic process, including the caspase cascade and the c-Jun N-terminal kinase (JNK) signaling pathway [7–12] Hsp105a and Hsp105b are mammalian members of the HSP105 ⁄ 110 family, a subgroup of the HSP70 family Hsp105a is expressed constitutively and in response to various forms of stress, while Hsp105b is an alternatively spliced form of Hsp105a that is Abbreviations DOX, doxycycline; G3DPH, glyceraldehyde 3-phosphate dehydrogenase; Hsps, Heat shock proteins; JNK, c-Jun N-terminal kinase; p38 MAPK, p38 mitogen-activated protein kinase; PARP, poly(ADP-ribose)polymerase; PBS, phosphate-buffered saline; SDS-PAGE, SDSpolyacrylamide gel electrophoresis 4558 FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS N Yamagishi et al expressed specifically during mild heat shock [13–15] These proteins suppress the aggregation of denatured proteins caused by heat shock in vitro, as does Hsp70, but have not been found to have refolding activity [16] We have demonstrated previously that Hsp105a protects neuronal PC12 cells against apoptosis induced by various stresses such as H2O2, heat shock and anticancer drugs [17] In addition, Hsp105a suppresses protein aggregation and apoptosis caused by the expression of proteins with expanded polyQ tracts in a cellular model of spinal and bulbar muscular atrophy [18] Recently, we also showed that Hsp105a and Hsp105b suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria [19] In contrast, Hsp105a has the opposite effects on apoptosis induced by H2O2 in mouse embryonal F9 cells, suggesting that Hsp105a has opposing effects on stress-induced apoptosis depending on the cell type [20] However, it is not fully understood how Hsp105 regulates apoptosis caused by a variety of stresses Here, we examined the mechanisms by which Hsp105 regulates H2O2-induced apoptosis by using HeLa cells in which expression of Hsp105a or Hsp105b was regulated using doxycycline Our results suggest Hsp105a and Hsp105b suppress H2O2-induced apoptosis by inhibition of the p38 mitogen-activated protein kinase (p38 MAPK) pathway but not the JNK pathway Results Hsp105a and Hsp105b suppress apoptosis induced by H2O2 but not by heat shock in HeLa cells Previously, we have established human HeLa cell lines (HeLa-tet ⁄ Hsp105a and HeLa-tet ⁄ Hsp105b) in which overexpression of mouse Hsp105a or Hsp105b was induced by removing DOX from the culture medium In HeLa-tet ⁄ Hsp105a cells, the level of Hsp105a was increased approximately twofold by removal of DOX from the culture medium, and was comparable to that of Hsp105 (Hsp105a plus Hsp105b) after heat exposure at 42 °C for h The inducible level of mouse Hsp105b in HeLa-tet ⁄ Hsp105b cells after DOX removal was comparable to that of Hsp105b induced after heat exposure [19] Using these cells, we examined whether overexpression of Hsp105a or Hsp105b suppresses cell death induced by treatment with H2O2 and heat shock As shown in Fig 1A, these cells were more resistant to H2O2 as a result of overexpression of Hsp105a or Hsp105b As cell death is classified into two morphologically and biochemical distinct modes, apoptosis and necrosis, we next examined whether Suppression of H2O2-induced apoptosis by Hsp105 apoptotic features such as the fragmentation of chromatin and the externalization of phosphatidylserine on the plasma membrane were induced by treatment with H2O2 Treatment with H2O2 did induce the development of fragmented and ⁄ or condensed chromatin (Fig 1B,C) In addition, externalized phosphatidylserine in the plasma membrane, which is detected by annexin V binding, was observed on the plasma membrane of H2O2-treated cells but not on that of untreated control cells (Fig 1B) Next, we examined whether Hsp105a and Hsp105b suppress H2O2-induced apoptosis As shown in Fig 1C, overexpression of Hsp105a or Hsp105b reduced the number of apoptotic cells with fragmented and ⁄ or condensed chromatin In contrast, overexpression of Hsp105a or Hsp105b did not significantly affect cell death induced by heat shock, although apoptotic cell death was induced by heat shock treatment (Fig 2) Hsp105a and Hsp105b suppress H2O2-induced apoptosis upstream of cytochrome c release from the mitochondria Caspases are a family of cysteine aspartic acid protease that are central regulators of apoptosis and form a proteolytic cascade that results in the cleavage of distinct and vital proteins [21–23] In particular, caspase-3 is one of the key executioners of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins such as poly(ADP-ribose)polymerase (PARP) [24] Therefore, we next examined whether caspase-3 is activated in HeLa cells treated with H2O2 As shown in Fig 3A, active forms of caspase-3 and a cleaved fragment of PARP were detected in cells treated with H2O2 Furthermore, overexpression of Hsp105a or Hsp105b markedly reduced both the amount of the active forms of caspase-3 and PARP cleavage by treatment with H2O2 In contrast, although caspase-3 was markedly activated after heat shock, activation of caspase-3 was not affected by overexpression of Hsp105a or Hsp105b (Fig 3B) These results suggested that overexpression of Hsp105a and Hsp105b prevents the activation of caspase-3, resulting in suppression of H2O2-induced apoptosis However, Hsp105a and Hsp105b seem to suppress H2O2-induced apoptosis upstream of rather than by direct inhibition of caspase-3 activation, as they not suppress the activation of caspase-3 mediated by heat shock In mammalian cells, one of the main pathways that activates procaspase-3 is via mitochondria When the mitochondria receive appropriate signals from a variety of stresses, pro-apoptotic molecules such as cytochrome c are released from mitochondria into the FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS 4559 Suppression of H2O2-induced apoptosis by Hsp105 N Yamagishi et al A B C Fig Hsp105 suppresses apoptosis induced by H2O2 in HeLa cells (A) HeLa-tet ⁄ Hsp105 cells that overexpressed Hsp105a or Hsp105b ()DOX) or not (+DOX) were exposed to 0.2–0.6 mM H2O2 for h Then, the cells were further incubated in fresh medium at 37 °C for h Cell viability was determined by a neutral red uptake assay Values are shown as the means ± SD of four independent experiments The significance of differences was assessed by an unpaired Student’s t-test: *P < 0.05 for cell viability in +DOX versus )DOX (B) HeLa-tet ⁄ Hsp105 cells maintained in the medium with lgỈmL)1 DOX were exposed to 0.4 mM H2O2 for h, and further incubated in fresh medium at 37 °C for h The cells were then stained with annexin V-fluorescein isothiocyanate and Hoechst 33342 Phosphatidylserine externalization and nuclear morphology were observed using a confocal laser scanning microscope (C) HeLa-tet ⁄ Hsp105 cells were exposed to 0.4 mM H2O2 for h, and further incubated in fresh medium at 37 °C for h The cells were then stained with Hoechst 33342, and nuclear morphology of cells was observed using a fluorescence microscope (left panels) Rates of apoptosis were calculated using at least 200 cells in each experiment Values are the means ± SD of four independent experiments (right panel) The significance of differences was assessed by an unpaired Student’s t-test: *P < 0.05 for apoptosis in +DOX versus )DOX 4560 FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS Suppression of H2O2-induced apoptosis by Hsp105 N Yamagishi et al A B C Fig Hsp105 does not suppress apoptosis induced by heat shock in HeLa cells HeLa-tet ⁄ Hsp105 cells were heated at 45 °C for 15–60 min, and then the cells were further incubated at 37 °C for h Cell viability (A), phosphatidylserine externalization and nuclear morphology (B), and rates of apoptosis (C) were determined as described in Fig Values are the means ± SD of four independent experiments The significance of differences was assessed by an unpaired Student’s t-test There were no significant differences between groups cytosol, in which cytochrome c forms a complex with Apaf-1 and procaspase-9, and activates procaspase-9 and procaspase-3 successively [25–27] Therefore, we next examined whether caspase-9 is activated in HeLa cells treated with H2O2 When HeLa cells were treated with H2O2, procaspase-9 was processed to active forms (37 and 35 kDa fragments), and the activation of caspase-9 was suppressed by overexpression of Hsp105a or Hsp105b (Fig 4A) In contrast, caspase-9 was not activated by heat shock (Fig 4B) Furthermore, when the cellular distribution of cytochrome c in HeLa-tet ⁄ Hsp105a cells was examined by immunofluorescence microscopy, cytochrome c was found exclusively in the mitochondria of control cells, but was observed throughout the cytoplasm after treatment with H2O2 in most cells without overexpression FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS 4561 Suppression of H2O2-induced apoptosis by Hsp105 N Yamagishi et al A B Fig Hsp105 suppresses the activation of caspase-3 induced by H2O2 but not by heat shock in HeLa cells HeLa-tet ⁄ Hsp105 cells were treated with 0.5 mM H2O2 for 0.5 or h (A) or heat shock at 45 °C for h (B) Aliquots (20 lg of protein) of the cell extracts were separated by SDS–PAGE and immunostained using anti-Hsp105, anti-caspase-3 or anti-PARP The intensity of bands was quantified using public-domain NIH IMAGE software (http://rsb.info.nih.gov/nih-image), and normalized to the total protein content detected by Coomassie Brilliant Blue staining Values are the means of two independent experiments of Hsp105a However, release of cytochrome c was suppressed in cells overexpressing Hsp105a (Fig 5A) In addition, when the cells were fractionated into a soluble fraction and a particulate fraction containing mitochondria by treatment with digitonin, a large amount of cytochrome c was detected in the mitochondrial fraction of untreated cells with or without over4562 expression of Hsp105a or Hsp105b (Fig 5B) However, in the cells treated with H2O2, the amount of cytochrome c increased in the soluble fraction concomitant with its decrease in the particulate fraction The release of cytochrome c from mitochondria was suppressed by the overexpression of Hsp105a or Hsp105b Several studies have demonstrated that Bax, FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS Suppression of H2O2-induced apoptosis by Hsp105 N Yamagishi et al A Fig Effect of Hsp105 overexpression on the activation of caspase-9 induced by treatment with H2O2 HeLa-tet ⁄ Hsp105 cells were treated with 0.5 mM H2O2 for 0.5 or h (A) or heat shock at 45 °C for h (B) Aliquots (20 lg of protein) of the cell extracts were separated by SDS–PAGE and immunostained using anti-Hsp105 or anticaspase-9 The intensity of bands was quantified by public-domain NIH IMAGE software, and normalized to the total protein content detected by Coomassie Brilliant Blue staining Values are the means of two independent experiments B a pro-apoptotic Bcl-2 family member, is translocated from the cytoplasm to the mitochondria, and leads to the release of cytochrome c and other proteins from the mitochondrial intermembrane space [28,29] However, we did not detect Bax translocation to the mitochondria in HeLa cells treated with H2O2 (Fig 5B) Under these conditions, a mitochondrial matrix protein, Hsp60, was recovered exclusively in the particulate fraction, whereas a cytosolic protein, glyceraldehyde-3-phosphate dehydrogenase (G3DPH), was recovered exclusively in the soluble fraction Thus, Hsp105a and Hsp105b suppress H2O2-induced apoptosis at or upstream of cytochrome c release from mitochondria Hsp105a and Hsp105b suppress H2O2-induced apoptosis by inhibition of the p38 MAPK pathway but not the JNK pathway Both JNK and p38 MAPK have been implicated as key regulators in some forms of stress-induced apoptosis [30,31] Phosphorylation of JNK at Thr183 and Typ185 is required for JNK activation [32] Activation of p38 MAPK occurs through phosphorylation of Thr180 and Tyr182 Therefore, we examined whether JNK and p38 MAPK are phosphorylated at Thr183 ⁄ Tyr185 and Thr180 ⁄ Tyr182, respectively, in HeLa cells treated with H2O2 (Fig 6) Phosphorylated JNK and p38 MAPK were observed within 15 after treatment with H2O2, and the phosphorylation of these kinases was maintained for more than h However, the phosphorylation of JNK and p38 MAPK was markedly reduced in the cells overexpressing Hsp105a or Hsp105b Next, we examined whether the activation of JNK or p38 MAPK is required for H2O2-induced apoptosis, using a p38 MAPK inhibitor, SB203580, and a JNK inhibitor, SP600125 As shown in Fig 7A, H2O2induced apoptosis was suppressed by treatment with 10 lm SB203580, but not by treatment with 10 lm SP600125 Under these conditions, SB203580 markedly suppressed phosphorylation of MAPK-activating protein kinase-2 (a substrate of p38 MAPK), but did not affect the phosphorylation of c-Jun (a substrate of JNK) Conversely, SP600125 markedly suppressed the phosphorylation of JNK with no effect on p38 MAPK activity (Fig 7B) These results suggest that Hsp105a and Hsp105b suppress H2O2-induced apoptosis by inhibiting the p38 MAPK pathway but not the JNK pathway FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS 4563 Suppression of H2O2-induced apoptosis by Hsp105 N Yamagishi et al A B Fig Hsp105 suppresses the release of cytochrome c from mitochondria induced by treatment with H2O2 in HeLa cells (A) HeLa-tet ⁄ Hsp105a cells were exposed to 0.5 mM H2O2 for h, and then the cells were further incubated in fresh medium at 37 °C for h The cellular distribution of cytochrome c was then detected by indirect immunofluorescence microscopy using anti-cytochrome c antibody (green) Mitochondria were detected by staining with Mitotracker (red) Arrows indicate cells in which cytochrome c has been released into the cytoplasm (B) HeLa-tet ⁄ Hsp105 cells were exposed to 0.5 mM H2O2 for h, and then the cells were further incubated in fresh medium at 37 °C for h The cells were then harvested, and were fractionated into cytosolic and mitochondrial fractions Both fractions were subjected to 15% SDS–PAGE, followed by immunostaining using anti-cytochrome c, anti-BAX, anti-Hsp60 or anti-G3PDH Hsp60 and G3PDH are marker proteins for the mitochondrial and cytoplasmic fractions, respectively The intensity of bands was quantified by public-domain NIH IMAGE software, and normalized to the total protein content detected by Coomassie Brilliant Blue staining The amount of cytochrome c released from mitochondria was estimated from the amounts of cytochrome c in the immunoblots of mitochondrial fraction Values are the means ± SD of three independent experiments Statistical significance was determined by an unpaired Student’s t-test: *P < 0.05 for cytochrome c release in +DOX versus )DOX Discussion We showed previously that Hsp105a has opposing effects on H2O2-induced apoptosis depending on the cell type; an anti-apoptotic effect in neuronal PC12 cells and a pro-apoptotic effect in embryonic F9 cells [17,20] However, it is not fully understood how Hsp105a regulates H2O2-induced apoptosis In addition, the role of Hsp105b in stress-induced apoptosis remains to be clarified In the present study, we showed that, although Hsp105a and Hsp105b suppress the activation of JNK and p38 MAPK by treatment with H2O2 in HeLa cells, H2O2-induced apoptosis was suppressed by the inhibition of p38 MAPK but not 4564 JNK Thus, Hsp105a and Hsp105b were suggested to suppress oxidative stress-induced apoptosis at or upstream of activation of p38 MAPK Several studies have demonstrated that Bax, a proapoptotic Bcl-2 family member, is one of the targets for JNK ⁄ p38 MAPK signaling [33,34] In response to stress, Bax undergoes a conformational change and is redistributed to the mitochondria [35] It is likely that Bax (and related members of this subfamily) mediates the release of cytochrome c from mitochondria [34] Here, we showed that Hsp105a or Hsp105b suppress H2O2induced apoptosis by inhibiting the release of cytochrome c from mitochondria in HeLa cells However, we did not detect Bax translocation to the mitochondria FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS Suppression of H2O2-induced apoptosis by Hsp105 N Yamagishi et al Fig Effects of Hsp105 overexpression on the activation of JNK and p38 induced by treatment with H2O2 HeLa-tet ⁄ Hsp105 cells were exposed to 0.5 mM H2O2 for 30 min, then further incubated in fresh medium at 37 °C for 15–120 Aliquots (20 lg of protein) of the cell extracts were separated by SDS–PAGE and immunostained using anti-phospho-SAPK ⁄ JNK (Thr183 ⁄ Tyr185), anti-SAPK ⁄ JNK, anti-phosphop38 MAPK (Thr180 ⁄ Tyr182) or anti-p38 MAPK Similar results were obtained from three independent experiments, and typical blots are shown in HeLa cells treated with H2O2 On the other hand, we observed previously that Hsp105a and Hsp105b suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to the mitochondria in HeLa cells [19] Therefore, H2O2 may induce apoptosis by the release of cytochrome c from mitochondria without Bax translocation to the mitochondria in HeLa cells It has been established that JNK phosphorylates the antiapoptotic proteins Bcl-2 and Bcl-XL and inhibits their prosurvival function [36–39] In addition to Bcl-2 and Bcl-XL, previous studies have also implicated the proapoptotic BH3-only proteins Bid and Bim in JNK ⁄ p38 MAPK-stimulated apoptosis [32,38] Further studies will be necessary to clarify the targets of p38 MAPK signaling in HeLa cells treated with H2O2 Apoptosis signal-regulating kinases (ASKs) are serine ⁄ threonine kinases that activate both the p38 MAPK and JNK signaling pathways as MAPK kinase kinase ASKs are activated in response to various cytotoxic stresses, including oxidative stress and UV irradiation, and play an essential role in stress-induced apoptosis [40,41] Therefore, it is likely that Hsp105a and Hsp105b suppress oxidative stress-induced apoptosis through the prevention of ASK activity ASK1 activity is stimulated by phosphorylation of ASK1 at Thr838, and suppressed by phosphorylation at Ser83 [42,43] However, overexpression of Hsp105a and Hsp105b did not suppress the phosphorylation of ASK1 at Thr838, and did not affect to the phosphorylation of ASK1 at Ser83 in HeLa cells treated with H2O2 (data not shown) Although Hsp105a and Hsp105b may suppress oxidative stress-induced apoptosis at steps between the activation of ASK1 and p38 MAPK, further studies are necessary to clarify the targets of Hsp105 in the suppression of oxidative stress-induced apoptosis Two pathways are known to be important for transducing a death signal to the apoptotic machinery The ‘extrinsic’ pathway involves activation of death receptors such as tumor necrosis factor and Fas by binding of their respective ligands and the subsequent recruitment of caspase-8 [44] Caspase-8 can activate effector caspases, such as caspase-3 and caspase-7, directly [45] or indirectly by cleaving Bid and inducing the release of cytochrome c from the mitochondria [46–49] In contrast, the ‘intrinsic’ pathway is activated directly by death stimuli, and induces the release of cytochrome c from the mitochondria into the cytosol [25,26,50–53] Cytosolic cytochrome c binds to Apaf-1 and induces caspase-9-dependent activation of caspase-3 [54–57] Here, we have shown that Hsp105a and Hsp105b suppress cytochrome c release from mitochondria and activate of caspase-9 and caspase-3 in H2O2-treated cells In contrast, Hsp105a and Hsp105b failed to prevent heat-induced apoptosis Interestingly, heat shock treatment activated procaspase-3 but not procaspase-9 Therefore, Hsp105a and Hsp105b may not be able to FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS 4565 Suppression of H2O2-induced apoptosis by Hsp105 N Yamagishi et al Fig H2O2-induced apoptosis was suppressed by treatment with a p38 MAPK inhibitor but not a JNK inhibitor in HeLa cells (A) HeLa-tet ⁄ Hsp105 cells that did not overexpress Hsp105a or Hsp105b (+DOX) were exposed to 0.5 mM H2O2 for 45 min, then further incubated in fresh medium with or without 10 lM SB203580 or 10 lM SP600125 at 37 °C for h Then the cells were stained with Hoechst 33342 and the nuclear morphology of the cells was observed using a fluorescence microscope Rates of apoptosis were calculated using at least 200 cells in each experiment Values are the mean ± SD of three independent experiments Statistical significance was determined by an unpaired Student’s t-test A probability level (P) < 0.05 was considered statistically significant and is indicated by asterisks (B) HeLatet ⁄ Hsp105 cells (+DOX) were exposed to 0.5 mM H2O2 for 45 min, then further incubated in fresh medium with SB203580 or SP600125 at 37 °C for h Aliquots (20 lg of protein) of the cell extracts were separated by SDS–PAGE and immunostained using anti-phospho-SAPK ⁄ JNK (Thr183 ⁄ Tyr185), anti-SAPK ⁄ JNK, antiphospho-p38 MAPK (Thr180 ⁄ Tyr182), anti-p38 MAPK, anti-phospho-MAPKAP-2 (Thr222) or anti-phospho-c-Jun (Ser63) A against heat-induced apoptosis [58] Recently, mammalian Hsp105 ⁄ 110 and its yeast homologues Sse1p ⁄ 2p have been shown to act as efficient nucleotide exchange factors for Hsp70 and for its orthologues in Saccharomyces cerevisiae, Ssa1p and Ssb1p, respectively, and enhance Hsp70-mediated chaperone activity [59–61] However, although HSP105 family proteins are important components of the Hsp70 chaperone machinery, excess Hsp110 seems to have a negative effect on Hsp70-mediated chaperone activity because it accelerates substrate cycling to such an extent that the reaction becomes unproductive for folding [60] Therefore, Hsp105a and Hsp105b seem not to simply suppress oxidative stress-induced apoptosis by regulation of Hsp70-mediated chaperone activity In summary, we have shown here that Hsp105a and Hsp105b suppress H2O2-induced apoptosis by suppression of p38 MAPK Although further studies are necessary to understand the precise mechanism by which Hsp105 regulates stress-induced apoptosis, understanding the action of Hsp105a and Hsp105b in apoptosis may offer novel ways of treating apoptosis-related diseases, such as cancer, injury after ischemia, and neurodegenerative disorders B suppress apoptosis induced by the mitochondria-independent pathway Several Hsps have been shown to modulate the pathway of apoptosis positively In particular, Hsp70 has been shown to protect against apoptosis by a variety of stressors through suppression of JNK activation and apoptosome formation [7–12] Interestingly, the chaperone activity of Hsp70 is required for protection 4566 Experimental procedures Antibodies The following antibodies were used for immunoblotting and immunofluorescence experiments: Hsp105, rabbit polyclonal anti-mouse Hsp105, which only reacts with FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS N Yamagishi et al mouse Hsp105 [62], or rabbit anti-human Hsp105, which reacts with human, mouse, rat, and monkey Hsp105 [15]; PARP, rabbit polyclonal anti-PARP (#sc-7150; Santa Cruz Biotechnology, Santa Cruz, CA, USA); cleaved caspase-3, rabbit polyclonal anti-cleaved caspase-3 (Asp175) (#9661; Cell Signaling Technology, Danvers, MA, USA); caspase-9, rabbit polyclonal anti-caspase-9 (#9502; Cell Signaling Technology); cytochrome c, rabbit polyclonal anti-cytochrome c (#sc-7159; Santa Cruz); Bax, rabbit polyclonal anti-Bax (#06-499; Upstate Biotechnology, Lake Placid, NY, USA); Hsp60, mouse monoclonal antiHsp60 clone LK-1 (#SR-B806; Medical & Biological Laboratories; Nagoya, Japan); G3DPH, rabbit polyclonal anti-G3PDH (#2275-PC-1; Trevigen, Gaithersburg, MD, USA); JNK phosphorylated at Thr183 and Tyr185, rabbit polyclonal anti-phospho-SAPK ⁄ JNK (Thr183 ⁄ Tyr185) (#9251; Cell Signaling Technology); JNK, rabbit polyclonal anti-SAPK ⁄ JNK (#9252; Cell Signaling Technology); p38 MAPK phosphorylated at Thr180 and Tyr182, rabbit polyclonal anti-phospho-p38 MAPK (Thr180 ⁄ Tyr182) (#9211; Cell Signaling Technology); p38 MAPK, rabbit polyclonal anti-p38 MAPK (#9212; Cell Signaling Technology); MAPKAP-2 phosphorylated at Thr222, rabbit polyclonal anti-phospho-MAPKAP-2 (Thr222) (#9211; Cell Signaling Technology); phosphorylated c-Jun at Ser63, rabbit polyclonal anti-phospho-c-Jun (Ser63) (#9261; Cell Signaling Technology) Cells HeLa-tet ⁄ Hsp105a and HeLa-tet ⁄ Hsp105b cells, which overexpress either mouse Hsp105a or Hsp105b by removing DOX from the medium, have been described previously [19] These cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and lgỈmL)1 DOX at 37 °C with 95% air and 5% CO2 The induction of mouse Hsp105a and Hsp105b expression was performed as follows HeLa-tet ⁄ Hsp105a and HeLa-tet ⁄ Hsp105b cells were trypsinized, washed twice with 10 mL of fresh medium, and then grown in fresh medium without DOX for 24 h The following day, the cells were washed once with NaCl ⁄ Pi and incubated in fresh medium without DOX at 37 °C for an additional 24 h For treatment with H2O2, cells were treated with 0.2– 0.6 mm H2O2 in NaCl ⁄ Pi containing 0.9 mm CaCl2 and 0.5 mm MgCl2 at 37 °C for h, washed with NaCl ⁄ Pi, and further incubated in fresh medium at 37 °C For heat shock treatment, cells were treated in a water bath set at 45 °C for 15–120 Cell viability assay Cells (1 · 104 cells per well) in 96-well plates were incubated at 37 °C for h with 50 lgỈmL)1 neutral red, and Suppression of H2O2-induced apoptosis by Hsp105 fixed with The with 50% absorbance 1% formaldehyde containing 1% CaCl2 for dye incorporated into viable cells was extracted ethanol containing 1% acetic acid, and at 540 nm was measured Detection of phosphatidylserine externalization Cells grown on collagenized cover slips (2 · 104 cells per cm2 in 24-well plates) were washed with buffer A (10 mm Hepes pH 7.5, 140 mm NaCl and 2.5 mm CaCl2), and stained with 0.5 lgỈmL)1 annexin V–fluorescein isothiocyanate solution (Sigma, St Louis, MO, USA) in buffer A containing 10 lm Hoechst 33342 for 10 in the dark Cells displaying phosphatidylserine externalization were observed using a confocal laser scanning microscope (LSM410; Zeiss, Jena, Germany) Morphological examination of apoptotic cells Cells grown on collagenized cover slips (2 · 104 cells per cm2 in 24-well plates) were fixed with 3.7% formaldehyde for 30 at room temperature After washing twice with NaCl ⁄ Pi, the cells were stained with 10 lm Hoechst 33342 for 10 in the dark and observed using a fluorescence microscope (Nikon, Tokyo, Japan) The cells detached from cover slips were harvested in a microtube, and stained with 10 lm Hoechst 33342 Cells were scored as apoptotic if they displayed nuclear fragmentation and ⁄ or chromatin condensation For assessment of proportion of apoptotic cells, the number of cells on cover slips and the number of detached cells were determined Immunoblot analysis Cells (2 · 105 cells per 35 mm diameter dish) were lysed with 0.1% SDS and boiled for Aliquots (20 lg of protein) of cell extracts in SDS sample buffer (62.5 mm Tris ⁄ HCl pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol and 0.00125% bromophenol blue) were subjected to SDS–PAGE, and then transferred onto nitrocellulose membranes by electrotransfer The membranes were blocked with 5% skim milk in Tris–buffered saline (20 mm Tris ⁄ HCl pH 7.6, 137 mm NaCl) containing 0.1% Tween20 (Wako Pure Chemical, Osaka, Japan), and incubated with the indicated primary antibodies Then, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG, and the antibody–antigen complexes were detected using an ECL ⁄ western blot detection system (GE Healthcare, Little Chalfont, UK) Intracellular distribution of cytochrome c Cells grown on collagenized cover slips (2 · 104 cells per cm2 in 24-well plates) were stained with 0.2 lgỈmL)1 FEBS Journal 275 (2008) 4558–4570 ª 2008 The Authors Journal compilation ª 2008 FEBS 4567 Suppression of H2O2-induced apoptosis by Hsp105 N Yamagishi et al Mitotracker Orange (Invitrogen, Carlsbad, CA, USA) for 15 Then, cells were fixed with 3.7% formaldehyde for 30 at room temperature, and permeabilized with 0.4% Triton X-100 (Wako Pure Chemical) in NaCl ⁄ Pi After blocking with 5% BSA in NaCl ⁄ Pi, anti-cytochrome c at a : 100 dilution was added to the cover slips, which were incubated in a moist chamber for h at 37 °C After washing with NaCl ⁄ Pi, fluorescein isothiocyanate-conjugated anti-rabbit IgG (1 ⁄ 50, Vector, Burlingame, CA, USA) was added to the cover slips, which were incubated further at 37 °C for h After another wash with NaCl ⁄ Pi, the cells were observed using a confocal laser scanning microscope For fractionation into a soluble fraction and a particulate fraction containing mitochondria, cells (1 · 106 cells per 100 mm diameter dish) were lysed with 100 lgỈmL)1 digitonin in NaCl ⁄ Pi, and then incubated at 25 °C for After centrifugation at 3000 g for min, the supernatants were recovered and further centrifuged at 15 000 g for The supernatants were recovered as cytosolic fractions Pellets were dissolved in 0.1% SDS, and centrifuged at 15 000 g for 15 The supernatants were recovered as mitochondrial fractions Both fractions were subjected to 15% SDS–PAGE, and analyzed by immunoblotting using anti-cytochrome c, anti-Hsp60 and anti-G3PDH 10 11 12 13 14 Acknowledgements This study was supported in part by a grant from the Ministry of Education, Science, Sports and Culture of Japan (to T H and N.Y.) 15 References Samali A & Orrenius S (1998) Heat shock proteins: regulators of stress 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compilation ª 2008 FEBS ... JNK pathway Results Hsp10 5a and Hsp105b suppress apoptosis induced by H2O2 but not by heat shock in HeLa cells Previously, we have established human HeLa cell lines (HeLa- tet ⁄ Hsp10 5a and HeLa- tet... necessary to clarify the targets of p38 MAPK signaling in HeLa cells treated with H2O2 Apoptosis signal-regulating kinases (ASKs) are serine ⁄ threonine kinases that activate both the p38 MAPK and... caspase-9 and caspase-3 in H2O2-treated cells In contrast, Hsp10 5a and Hsp105b failed to prevent heat- induced apoptosis Interestingly, heat shock treatment activated procaspase-3 but not procaspase-9