Identification of caspase-independent PKCepsilon-JNK/p38 MAPK signaling module in response to metabolic inhibition in H9c2 cells

To understand the molecular mechanism of ischemia-induced cardiac myocyte cell death, H9c2 cells were studied by chemical hypoxia (CH), using metabolic inhibition buffer. CH suppressed the activities of caspase-3, -8, and -9. c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) were activated, whereas extracellular regulated kinase (ERK) was inactivated. Only protein kinase Cepsilon (PKCepsilon) among PKC isotypes was translocated to the membrane fraction implying its activation. Moreover, the administration of PKCepsilon inhibitor suppressed the phosphorylations of JNK/p38 MAPK and reduced CH-induced cell death. An administration of JNK/p38 MAPK inhibitors also decreased CH-induced cell deaths, implying JNK/p38 MAPK's causative roles in the deaths. Collectively, this study identified a novel caspase-independent PKCepsilon-JNK/p38 MAPK signaling module induced by CH in cardiac myocytes. Our data show that the PKCepsilon-JNK/p38 MAPK signaling module contributes to CH-induced H9c2 cell death. This contrasts with previous notions, i.e., PKCepsilon's protective effect against ischemic death. Thus our data suggest that PKCepsilon can mediate alternative signals, i.e., beneficiary or deleterious signals, depending on the cell type, intensity, and/or type of injury.     

Simultaneous induction of apoptotic and survival signaling pathways in macrophage-like THP-1 cells by Shiga toxin 1

Shiga toxins have been shown to induce apoptosis in many cell types. However, Shiga toxin 1 (Stx1) induced only limited apoptosis of macrophage-like THP-1 cells in vitro. The mechanisms regulating macrophage death or survival following toxin challenge are unknown. Differentiated THP-1 cells expressed tumor necrosis factor receptors and membrane-associated tumor necrosis factor alpha (TNF-alpha) and produced soluble TNF-alpha after exposure to Stx1. However, the cells were refractory to apoptosis induced by TNF-alpha, although the cytokine modestly increased apoptosis in the presence of Stx1. Despite the partial resistance of macrophage-like THP-1 cells to Stx1-mediated killing, treatment of these cells with Stx1 activated a broad array of caspases, disrupted the mitochondrial membrane potential (DeltaPsi(m)), and released cytochrome c into the cytoplasm. The DeltaPsi(m) values were greatest in cells that had detached from plastic surfaces. Specific caspase inhibitors revealed that caspase-3, caspase-6, caspase-8, and caspase-9 were primarily involved in apoptosis induction. The antiapoptotic factors involved in macrophage survival following toxin challenge include inhibitors of apoptosis proteins and X-linked inhibitor of apoptosis protein. NF-kappaB and JNK mitogen-activated protein kinases (MAPKs) appeared to activate survival pathways, while p38 MAPK was involved in proapoptotic signaling. The JNK and p38 MAPKs were shown to be upstream signaling pathways which may regulate caspase activation. Finally, the protein synthesis inhibitors Stx1 and anisomycin triggered limited apoptosis and prolonged JNK and p38 MAPK activation, while macrophage-like cells treated with cycloheximide remained viable and showed transient activation of MAPKs. Collectively, these data suggest that Stx1 activates both apoptotic and cell survival signaling pathways in macrophage-like THP-1 cells.     

Role of PKC-delta during hypoxia in heart-derived H9c2 cells

In the present study, we investigated the role of protein kinase C (PKC) isoforms during hypoxia in heart-derived H9c2 cells. Hypoxia caused a rapid translocation of PKC-delta from soluble to particulate fraction and a downregulation of PKC-epsilon and PKC-zeta, whereas PKC-alpha and PKC-beta I remained unaltered. When H9c2 cells were pretreated with PKC-delta inhibitor rottlerin (3 microM), hypoxia-induced apoptotic and necrotic cell death were significantly increased. Hypoxic insult also caused an activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK with no change in c-Jun NH(2)-terminal protein kinase (JNK) phosphorylation. Hypoxia-induced cell death was increased by treatment with ERK1/2 inhibitor U0126 (10 microM), but attenuated by p38 MAPK inhibitor SB202190 (10 microM). Treatment with rottlerin completely blocked the hypoxia-induced ERK phosphorylation, whereas it significantly increased p38 MAPK phosphorylation. The hypoxia-induced translocation of PKC-delta was not altered by U0126 and/or SB202190. From these results, it is suggested that hypoxia causes a rapid translocation of PKC-delta and subsequently ERK activation and p38 inactivation, rendering H9c2 cells resistant to hypoxia-induced cell death.     

Cyclic adenosine monophosphate inhibits nitric oxide-induced apoptosis of cardiac muscle cells in a c-Jun N-terminal kinase-dependent manner

Cyclic adenosine monophosphate (cAMP) modulates various agent-induced apoptosis. In this study, we observed that cAMP had a significantly protective effect on nitric oxide (NO)-induced cytotoxicity in H9c2 cardiac muscle cells. Pretreatment with DBcAMP (cAMP analogue) or forskolin (adenylyl cyclase activator) also significantly prevented the SNP-induced apoptosis in H9c2 cells. In contrast, H-89 or KT5720 (PKA inhibitor) reversed the protective effects of DBcAMP. In this study, DBcAMP or forskolin reduced SNP-induced JNK/SAPK activation to the basal level, but KT5720 reversed the inhibitory effects of these two agents. In contrast to JNK/SAPK activation, DBcAMP and forskolin significantly enhanced SNP-activated p38 MAPK phosphorylation and did not affect SNP-mediated ERK activation. KT5720 reversed the effects of DBcAMP and forskolin on p38 MAPK phosphorylation. The inhibition of the JNK pathway by transfection of a dominant negative mutant of JNK/SAPK markedly reduced the extent of SNP-induced cell death. Taken together, we suggest that JNK/SAPK is related to cAMP-protective effect in SNP-induced apoptosis. In addition, c-AMP relating agents protected SNP-induced cell death in neonatal rat ventricular cardiomyocytes. The cAMP-relating agent-induced protective effect is not resricted in H9c2 cardiac muscle cells.     

Cyclic Adenosine Monophosphate Inhibits Nitric Oxide‐Induced Apoptosis of Cardiac Muscle Cells in a c‐Jun N‐Terminal Kinase‐Dependent Manner

Cyclic adenosine monophosphate (cAMP) modulates various agent‐induced apoptosis. In this study, we observed that cAMP had a significantly protective effect on nitric oxide (NO)‐induced cytotoxicity in H9c2 cardiac muscle cells. Pretreatment with DBcAMP (cAMP analogue) or forskolin (adenylyl cyclase activator) also significantly prevented the SNP‐induced apoptosis in H9c2 cells. In contrast, H‐89 or KT5720 (PKA inhibitor) reversed the protective effects of DBcAMP. In this study, DBcAMP or forskolin reduced SNP‐induced JNK/SAPK activation to the basal level, but KT5720 reversed the inhibitory effects of these two agents. In contrast to JNK/SAPK activation, DBcAMP and forskolin significantly enhanced SNP‐activated p38 MAPK phosphorylation and did not affect SNP‐mediated ERK activation. KT5720 reversed the effects of DBcAMP and forskolin on p38 MAPK phosphorylation. The inhibition of the JNK pathway by transfection of a dominant negative mutant of JNK/SAPK markedly reduced the extent of SNP‐induced cell death. Taken together, we suggest that JNK/SAPK is related to cAMP‐protective effect in SNP‐induced apoptosis. In addition, c‐AMP relating agents protected SNP‐induced cell death in neonatal rat ventricular cardiomyocytes. The cAMP‐relating agent‐induced protective effect is not resricted in H9c2 cardiac muscle cells.     

c-Jun N-terminal kinase and p38 mitogen-activated protein kinase mediate double-strand RNA-induced inducible nitric oxide synthase expression in microglial cells

Double-stranded RNA (dsRNA) has been implicated as a potential immune stimulant in activating microglia, which can cause chronic neurodegeneration. In this study, we examined the involvement of different types of mitogen-activated protein kinases (MAPKs) in the induction of inducible nitric oxide synthase (iNOS) by dsRNA in microglial cells. Nitric oxide production was increased after exposure of microglia to 50 μg/mL dsRNA. Levels of dsRNA-induced nitrite production in a line of immortalized murine microglia (BV2) and in primary cultures of murine microglia were decreased by inhibition of JNK or p38 MAPK, but were increased by inhibition of extracellular signal-regulated kinase. Similar results were shown in the levels of dsRNA-induced iNOS gene expression in BV2 cells. Phosphorylation levels of p38 MAPK were increased, depending on p38 MAPK inhibitor concentrations, while activation levels of MAPKAPK2, a known p38 substrate, were inhibited. Thus, it is likely that SB203580 inhibited the kinase activity of p38 MAPK, resulting in the loss of a feedback inhibition regulatory loop of p38 MAPK in BV2 cells. These findings suggest that dsRNA stimulated iNOS expression via MAPK signaling pathways, including JNK and p38 MAPK.     

Close association of p38 and JNK with plasminogen-dependent upregulation of PAI-1 in rat astrocytes in vitro

As reported previously, stimulation of astrocytes with plasminogen (PLGn) remarkably enhances their production/release of plasminogen activator inhibitor-1 (PAI-1). In addition, both p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) are activated in these astrocytes. However, it remains to be determined whether the MAPK activation is associated with the PAI-1 induction in PLGn-stimulated astrocytes. In the present study, we investigated the relationship between MAPK activity and PAI-1 induction in PLGn-stimulated astrocytes. PLGn stimulation led to definitive phosphorylation of three MAPKs: external signal regulated kinase (ERK), JNK and p38. These results suggest that all of these MAPKs, either alone or in combination, are involved in PAI-1 induction. To verify this association, an inhibition experiment was carried out by using inhibitors specific for each MAPK. The results of the immunoblotting analysis indicated that 20 μM SB203580 (the p38 inhibitor) or SP600125 (the JNK inhibitor) suppressed approximately 85% or 40% of PLGn-inducible PAI-1, respectively. Only 20% inhibition was achieved by pretreatment of astrocytes with 20 μM PD98059 (the inhibitor of MEK1/2, an upstream kinase of ERK). In conclusion, p38 and JNK were shown to be the major MAPKs involved in the signaling cascade leading to PAI-1 induction in astrocytes stimulated with PLGn.     

三七皂苷Rg1抑制脂多糖诱导小胶质细胞激活的机制研究

目的:探讨中药有效成分三七皂苷Rg1(Ginsenoside Rg1,Rg1)对抑制脂多糖(lipopolysaccharide,LPS)诱导的小胶质细胞株BV-2细胞激活的机制。方法:用LPS刺激BV-2细胞构建激活模型,采用四甲基偶氮唑蓝比色法(MTT)检测Rg1对BV-2细胞的活力影响,蛋白质免疫印迹(Western Blot)方法检测不同浓度Rg1(10、20和40μmol/L)对磷酸化的核因子-κB抑制蛋白-α(inhibitorκB-α,IκB-α)和反应结合蛋白(cAMP-responseelement binding protein,CREB)以及促分裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)家族的细胞外信号调节激酶(extracellular signal-regulated kinase 1/2,ERK1/2)、c-Jun氨基端激酶(c-Jun N-terminal kinase,JNK)和p38促分裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)等细胞信号通路蛋白的表达及其变化规律。结果:不同浓度的Rg1明显抑制了LPS诱导的磷酸化IκB-α和CREB蛋白表达以及MAPKs通路(ERK1/2,JNK,p38 MAPK)磷酸化蛋白表达,并且对p38 MAPK表达的影响呈剂量依赖性。结论:Rg1可能通过抑制MAPKs的磷酸化来调控LPS诱导的小胶质细胞株BV-2细胞激活,发挥其神经抗炎的作用。     

Ethanol-induced oxidative stress is mediated by p38 MAPK pathway in mouse hippocampal cells

It has been known that ethanol causes neuronal cell death through oxidative stress. Ethanol itself and reactive oxygen species (ROS) produced by ethanol modulate intracellular signaling pathways including mitogen-activated protein kinase (MAPK) cascades. This study was conducted to examine the impact of ethanol on MAPK signaling in HT22 cells. Ethanol (100 and 400 mM) caused activation of ERK, p38 MAPK, and JNK. ERK activation occurred in early time and p38 MAPK activation was evident when ERK activation was diminished. Specific inhibitor of p38 MAPK (SB203580) protected HT22 cells against ethanol, which was accompanied by an inhibition of ROS accumulation. However, inhibitors of ERK (U0126) and JNK (SP600125) had no effects on ethanol-induced neuronal cell death when they are treated with ethanol for 24 h. These results suggest that p38 MAPK may have important roles in ROS accumulation during ethanol-induced oxidative stress in HT22 cells.     

Differential modulation of MAP kinases by zinc deficiency in IMR-32 cells: role of H(2)O(2)

The influence of zinc deficiency on the modulation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK) was studied. Using human IMR-32 cells as a model of neuronal cells, the role of oxidants on MAPKs and activator protein-1 (AP-1) activation in zinc deficiency was investigated, characterizing the participation of these events in the triggering of apoptosis. Relative to controls, cells incubated in media with low zinc concentrations showed increased cell oxidants and hydrogen peroxide (H(2)O(2)) release, increased JNK and p38 activation, high nuclear AP-1-DNA binding activity, and AP-1-dependent gene expression. Catalase addition to the media prevented the increase of cellular oxidants and inhibited JNK, p38, and AP-1 activation. Low levels of ERK1/2 phosphorylation were observed in the zinc-deficient cells in association with a reduction in cell proliferation. Catalase treatment did not prevent the above events nor the increased rate of apoptosis in the zinc-deficient cells. It is first demonstrated that a decrease in cellular zinc triggers H(2)O(2)-independent, as well as H(2)O(2)-dependent effects on MAPKs. Zinc deficiency-induced increases in cellular H(2)O(2) can trigger the activation of JNK and p38, leading to AP-1 activation, events that are not involved in zinc deficiency-induced apoptosis.     

Calpain-mediated regulation of the distinct signaling pathways and cell migration in human neutrophils

We studied the mechanisms underlying calpain inhibition-mediated human neutrophil migration. MAPKs, including ERK, p38, and JNK, MEK1/2, MAPK kinase 3/6 (MKK3/6), PI-3K/Akt, c-Raf, and p21-activated kinase (PAK; an effector molecule of Rac) were rapidly (within 30 s) activated in neutrophils upon exposure to calpain inhibitors (PD150606 and N-acetyl-Leu-Leu-Nle-CHO) but not PD145305 (inactive analog of PD150606). Following activation of these pathways, neutrophils displayed active migration (chemotaxis), which was sustained for more than 45 min. The studies with pharmacological inhibitors suggest that calpain inhibition-mediated neutrophil migration is mediated by activation of MEK/ERK, p38, JNK, PI-3K/Akt, and Rac. NSC23766 (Rac inhibitor) and pertussis toxin (PTX) suppressed calpain inhibitor-induced phosphorylation of distinct signaling molecules (PAK, c-Raf, MEK1/2, ERK, MKK3/6, p38, JNK, and Akt) as well as cell migration, suggesting that the PTX-sensitive G protein and Rac axis may be a possible key target of calpain inhibitors. Differentiated neutrophil-like HL-60 cells but not undifferentiated cells displayed cell migration and activation of MAPKs and PI-3K/Akt on calpain inhibition. These findings suggest that constitutively active calpain negatively regulates activation of the distinct signaling pathways and cell migration in resting neutrophils, and this regulatory system develops during differentiation into mature neutrophils.     

Minocycline protects melanocytes against H2O2-induced cell death via JNK and p38 MAPK pathways

Vitiligo is an acquired and progressive disorder manifested by the selective destruction of melanocytes in the skin. An extremely high level of hydrogen peroxide (H2O2) in plasma as well as in lesional skin has been reported in vitiligo patients. High H2O2 level has been suggested to be responsible for the disappearance of melanocytes in vitiligo. JNK and p38 MAPK are strongly induced by oxidative stress and related to neuron loss in neurodegenerative disorders. Minocycline, an antibiotic possessing antioxidant activity, is capable of attenuating oxidative stress-induced neurotoxicity. To investigate whether minocycline rescues melanocytes from H2O2-induced apoptosis, cultured mouse melanocytes (B10BR) were treated with H2O2 in the presence or absence of minocycline. Our data showed that H2O2 decreases cell viability in a concentration-dependent manner which is attenuated by minocycline. Also, H2O2 treatment activates JNK and p38 MAPK, and executive caspase 3 in B10BR cells. Minocycline significantly inhibits H2O2-induced activation of JNK, p38 MAPK and caspase 3. Collectively, we concluded that minocycline protects melanocytes against H2O2-induced apoptosis in vitro. Its protective effect is associated with the inhibition of JNK and p38 MAPK. Our findings suggest that minocycline, a clinically well-tolerated, safe antibiotic, may be used to prevent melanocyte loss in the early stage of vitiligo.     

磷酸化的丝裂原活化蛋白激酶在大鼠睾丸的表达

目的研究丝裂原活化蛋白激酶(MAPKs)的3个亚家族成员细胞外信号调节激酶(ERK)、c-Jun N-端激酶(JNK)和p38 MAPK的活化形式在睾丸中的定位,了解MAPKs在生精过程中所起的作用。方法免疫组织化学方法检测正常大鼠睾丸中磷酸化的p-ERKJ、NK、p38 MAPK的表达情况。结果正常大鼠睾丸中p-ERK主要分布于精原细胞、细线前期到粗线期的初级精母细胞以及9~12期长形精子细胞的细胞核,p-JNK则主要位于支持细胞与支持细胞、支持细胞与生精细胞(尤其是19期精子细胞)之间,而p-p38 MAPK除了在生精小管的部分细胞胞质中有分布外,其表达最明显的部位是在间质细胞的细胞质。结论ERKJ、NK和p-38 MAPK分别定位于正常大鼠睾丸内的不同部位,提示MAPKs不同的亚家族成员分别在精子发生的不同环节中发挥主要作用。ERK可能参与生精细胞增殖、分化的信号转导,JNK则可能通过调节细胞的黏附而最终影响生精细胞的迁移与精子释放过程,而p38 MAPK除了可能与JNK一起参与精子释放的调节外,最主要的作用可能是睾酮合成分泌的调节。     

Paclitaxel causes mouse splenic lymphocytes to a state hyporesponsive to lipopolysaccharide stimulation

Multiple immune system actions have been ascribed to paclitaxel (taxol), a novel anticancer drug, including the capacity to induce macrophage antitumor cytotoxic molecule production. In the present studies, we demonstrated that paclitaxel produced a selective inhibition of lipopolysaccharide (LPS)-induced B cell proliferation. Similarly, in vitro polyclonal antibody-forming cell responses also were found to be inhibited by paclitaxel. Conversely, paclitaxel exhibited no inhibitory effects on concanavalin A (Con A)-induced T cell proliferation. To study the pathway leading to paclitaxel-induced immunosuppression, we analyzed Raf-1/ERK and JNK/p38 MAPK pathways, both of which have been reported to be involved in LPS signaling. Our results indicate that taxol treatment inhibits Raf-1 kinase activation while having no effect on ERK activation suggesting that ERK activation is distinct from upstream Raf-1 kinase in taxol-induced immunomodulation. Furthermore, paclitaxel pretreatment caused down-regulation of stress-activated MAPKs, JNK and p38 MAPK in lipopolysaccharide (LPS)-stimulated mouse splenic lymphocytes, demonstrating that spleen cells are induced to a state hyporesponsive to LPS stimulation by pre-exposing them to paclitaxel. Taken together, these results suggest that down-regulation of JNK/p38 MAP kinase may contribute to paclitaxel-induced immunosuppression in mouse splenic lymphocytes.     

活性氧与丝裂原激活蛋白激酶通路的相互作用介导高糖引起的心肌细胞损伤

目的探讨活性氧(ROS)与丝裂原激活蛋白激酶(MAPK)通路的相互作用在高糖损伤H9c2心肌细胞中的作用。方法应用细胞计数盒(CCK-8)检测细胞存活率;Hoechst 33258核染色检测凋亡细胞形态及数量的改变;双氯荧光素(DCFH-DA)染色荧光显微镜照相检测细胞内ROS水平;Western blot测定蛋白质表达水平。结果高糖(35 mmol/L葡萄糖)处理H9c2心肌细胞24 h可引起明显的损伤,表现为细胞存活率下降,凋亡细胞数量及ROS水平明显升高。另方面,高糖可明显地上调磷酸化(p)p38MAPK、细胞外信号调节蛋白激酶1/2(ERK1/2)及c-Jun N端激酶(JNK)(为MAPK家族的3个成员)的表达水平。N-乙酰半胱氨酸(NAC,为ROS清除剂)能抑制高糖引起的心肌细胞毒性和细胞凋亡,也能阻断高糖对p-p38MAPK、p-ERK1/2及p-JNK表达的上调作用。此外,p38MAPK、ERK1/2和JNK的选择性抑制剂均能抑制高糖引起的心肌损伤,并能抑制ROS生成增多。结论在高糖损伤H9c2心肌细胞中,存在ROS与MAPK通路的正相互作用,这种相互作用可能在高糖引起的心肌细胞损伤中起着重要的作用。     

MNNG对哺乳类细胞JNK/SAPK及p38MAPK作用及其信号源研究

目的:研究低浓度烷化剂N-甲基-N' -硝基-N-亚硝基胍(MNNG)对JNK/SAPK及p38 MAPK通路的作用及其信号源。方法: 分别测定完整Vero细胞和脱核Vero细胞的JNK/SAPK及p38 MAPK酶活性,并比较其结果。 结果:低浓度MNNG在完整Vero细胞和脱核Vero细胞中均抑制JNK/SAPK酶活性;在p38 MAPK通路中,完整Vero细胞表现酶活性升高,而脱核Vero细胞该激活作用消失。 结论: 低浓度MNNG抑制JNK/SAPK的作用不依赖于核内信号,而对p38 MAPK的激活作用依赖与于核内信号。     

p38 MAPK as a negative regulator of VEGF/VEGFR2 signaling pathway in serum deprived human SK-N-SH neuroblastoma cells

Evidence suggests that vascular endothelial growth factor (VEGF) mediates neuroprotection to prevent an apoptotic cell death. The p38 mitogen-activated protein kinase (MAPK) pathway is implicated as an important mediator of neuronal apoptosis but its role in VEGF-mediated neuroprotection is unclear. Herein, we show that treatments with the p38 MAPK inhibitor, SB202190, enhanced VEGF-mediated survival in serum deprived SK-N-SH neuroblastoma cells by decreasing caspase-3/7 activation while increasing the phosphorylation of the extracellular signal-regulated kinase (ERK1/2) and Akt signaled through the VEGF receptor, VEGFR2. A blockade of VEGFR2 signaling with a selective inhibitor, SU1498 or gene silencing with VEGFR2 siRNA in SB202190 treated cells abrogated this prosurvival response and induced high activation levels of caspase-3/7. These findings suggested that the protection elicited by p38 MAPK inhibition in serum starved cells was dependent on a functional VEGF/VEGFR2 pathway. However, p38 MAPK inhibition attenuated caspase-3 cleavage in SU1498/SB202190 treated cells, indicating that p38 MAPK and caspase-3 only contributed in part to the total levels of caspase-3/7 induced by VEGFR2 inhibition. Pretreatments with the pan caspase inhibitor, z-VAD-fmk, prevented the apoptosis induced by VEGFR2 inhibition and promoted survival in serum starved cells irrespective of p38 MAPK inhibition. Collectively, our findings suggest that p38 MAPK exerts a negative effect on VEGF-mediated signaling through VEGFR2 in serum starved neuroblastoma cells. Furthermore, VEGF signals protection against a caspase-mediated cell death that is regulated by p38 MAPK-dependent and -independent mechanisms.     

Low concentrations of nitric oxide (NO) induced cell death in PC12 cells through activation of p38 mitogen-activated protein kinase (p38 MAPK) but not via extracellular signal-regulated kinases (ERK1/2) or c-Jun N-terminal protein kinase (JNK)

Nitric oxide (NO), a highly reactive gaseous molecule, has been previously reported to induce apoptosis-like cell death even at a low concentration in PC12 cells. In this study, we examined NO-induced activation of members of the mitogen-activated protein kinase (MAPK) family, i.e., p38 MAPK, extracellular signal-regulated kinases (ERK1/2), and c-Jun N-terminal protein kinase (JNK). Following the exposure of PC12 cells to an NO donor, (+)-(E)-4-ethyl-2-[hydroxyimino]-5-nitro-3-hexenamide (NOR3; 100 muM), the phosphorylation level of p38 MAPK increased time dependently from 2 to 6 h, but that of both ERK1/2 and JNK did not. Treatment with a p38 MAPK inhibitor SB203580 partially blocked the NOR3-induced cell death. Neither PD98059, U0126 (inhibitors of ERK1/2) nor SP600125 (a specific inhibitor of JNK) treatments had any significant effect on the NOR3-induced cell death. These findings suggest that the activation of a p38 MAPK pathway, but not that of ERK1/2 or JNK, plays an essential role in the apoptosis-like cell death induced by low concentrations of NO.     

p38β MAPK affords cytoprotection against oxidative stress-induced astrocyte apoptosis via induction of αB-crystallin and its anti-apoptotic function

The activation of p38 mitogen-activated protein kinases (MAPKs) has been implicated in many cellular processes, such as, inflammation, cell death, and survival. In mammals, four distinct genes encode the four known members of p38 MAPKs, p38α, p38β, p38γ, and p38δ. Despite the fact that p38α and p38β MAPKs share over 75% homology sequences, they have distinct, perhaps even opposite roles under stress conditions. In our previous report, we showed that p38β MAPK is induced in activated astrocytes in the penumbra of the postischemic brain, wherein it was co-localized with αB-crystallin and MAPKAPK-2. To investigate the functional significance of p38β MAPK in astrocytes, a C6 astroglioma cell line stably over-expressing p38β MAPK was generated. In these cells, hydrogen peroxide-induced apoptosis was reduced to 44.3% of that obtained from normal C6 cells. Interestingly, we found that expression of a small heat shock protein, αB-crystallin, was significantly increased in these cells, but that the expressions of HSP27 and HSP70 were not. Repression of αB-crystallin expression by αB-crystallin siRNA transfection suppressed the protective effect and recovered caspase 3 activity, indicating that αB-crystallin induction plays a crucial role in the protection against H₂O₂-induced apoptosis observed in p38β-overexpressing C6 astroglioma cells. We found that the binding between αB-crystallin and partially processed caspase-3 (a p24 intermediate) was significantly increased in p38β-overexpressing cells, which might result in suppression of caspase 3 activity in these cells. These results indicate that p38β confers protection against H₂O₂-induced astrocytes apoptosis by inducing a small heat shock protein, αB-crystallin, which inhibits caspase-3 activation.