Dual Signaling of the Fas Receptor: Initiation of Both Apoptotic and Necrotic Cell Death Pathways

Murine L929 fibrosarcoma cells were transfected with the human Fas (APO-1/CD95) receptor, and the role of various caspases in Fas-mediated cell death was assessed. Proteolytic activation of procaspase-3 and -7 was shown by Western analysis. Acetyl-Tyr-Val-Ala-Asp-chloromethylketone and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone, tetrapeptide inhibitors of caspase-1– and caspase-3–like proteases, respectively, failed to block Fas-induced apoptosis. Unexpectedly, the broad-spectrum caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone and benzyloxycarbonyl-Asp(OMe)-fluoromethylketone rendered the cells even more sensitive to Fas-mediated cell death, as measured after 18 h incubation. However, when the process was followed microscopically, it became clear that anti-Fas–induced apoptosis of Fas-transfected L929 cells was blocked during the first 3 h, and subsequently the cells died by necrosis. As in tumor necrosis factor (TNF)-induced necrosis, Fas treatment led to accumulation of reactive oxygen radicals, and Fas-mediated necrosis was inhibited by the oxygen radical scavenger butylated hydroxyanisole. However, in contrast to TNF, anti-Fas did not activate the nuclear factor κB under these necrotic conditions. These results demonstrate the existence of two different pathways originating from the Fas receptor, one rapidly leading to apoptosis, and, if this apoptotic pathway is blocked by caspase inhibitors, a second directing the cells to necrosis and involving oxygen radical production.     

游离胆固醇经P38-MAPK信号途径激活未折叠蛋白反应诱导巨噬细胞凋亡

目的:探讨p38-MAPK信号通路对未折叠蛋白反应的影响及在游离胆固醇(FC)诱导巨噬细胞凋亡中的作用。方法:收集小鼠腹腔巨噬细胞进行体外培养,使用100μg/mL乙酰低密度脂蛋白及10μg/mL胆固醇乙酰转移酶抑制剂-58035促进FC聚集,以p38特异性抑制剂SB203580进行干预,Annexin-V和PI双染后流式细胞仪检测细胞凋亡,Western-bolt检测磷酸化p38和CHOP表达。结果:普通培养基孵育的巨噬细胞无磷酸化p38和CHOP表达,只有少量细胞凋亡;而在促进FC聚集条件下孵育的巨噬细胞磷酸化p38和CHOP表达明显,8 h后凋亡细胞为(21.8±0.6)%;使用SB203580干预后无磷酸化p38表达,CHOP表达减少,凋亡细胞为(6.9±0.3)%。结论:FC聚集是诱导巨噬细胞凋亡的重要原因,p38通过激活未折叠反应参与这一过程,而SB203580通过抑制p38活性对FC诱导的巨噬细胞凋亡具有保护作用。     

N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine induces apoptosis through the activation of caspases-3 and -8 in human platelets. A role for endoplasmic reticulum stress

Summary. Background: Apoptosis or programmed cell death involves a number of biochemical events, including the activation of caspases, which lead to specific cell morphology changes and ultimately cell death. Traditionally, two apoptotic pathways have been described: the cell‐surface death receptor‐dependent extrinsic pathway and the mitochondria‐dependent intrinsic pathway. Alternatively, apoptosis has been reported to be induced by endoplasmic reticulum (ER) stress, which is mainly induced by a reduction in intraluminal free Ca²⁺ concentration ([Ca²⁺]_ER). Objectives: The present study aimed to investigate the development of apoptotic events after ER stress induced by N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN), an ER Ca²⁺ chelator, in human platelets. Methods: Changes in cytosolic free Ca²⁺ concentration, caspase activity and phosphatidylserine externalization were determined by fluorimetric techniques. Results: Our results indicate that TPEN reduces the amount of free Ca²⁺ releasable by the Ca²⁺‐mobilizing agonist thrombin. TPEN induced activation of caspase‐3, ‐8 and ‐9 and subsequent phosphatidylserine externalization. The ability of TPEN to induce phosphatidylserine externalization was smaller than that of thrombin. In addition, TPEN was able to induce phosphorylation of the eukaryotic initiation factor 2α (eIF2α). TPEN‐mediated caspase‐3 activation requires functional caspase‐8, but is independent of H₂O₂ generation. Activation of caspase‐3 and ‐8 by TPEN was prevented by salubrinal, an agent that prevents ER stress‐induced apoptosis. Conclusion: These findings provide experimental evidence for the existence of ER stress‐mediated apoptosis in human platelets, a process that might limit platelet life span upon prolonged stimulation with agonists.     

特发性血小板减少性紫癜患者外周血T细胞Fas-FasL和caspase-3凋亡相关蛋白的表达及其意义

目的 探讨Fas、FasL及caspase-3凋亡相关蛋白在特发性血小板减少性紫癜(ITP)患者T淋巴细胞的表达及其意义.方法 应用流式细胞术测定T细胞亚群表面Fas、FasL的表达率及细胞质中活化caspase-3的表达率,用Western blot法检测T细胞亚群caspase-3蛋白的表达.结果 与健康对照组[(29.4±8.2)%]相比,ITP患者组CD4+ T细胞表面Fas的表达率显著增加[(42.1±9.5)%](P＜0.05),CD8+ T细胞表面Fas的表达率略有增加但差异无统计学意义[(9.3±6.0)%与(13.4±5.8)%](P＞0.05).ITP患者组T细胞亚群表面FasL的表达率均较健康对照组显著增加(P＜0.05).ITP患者组T细胞亚群胞质中活化caspase-3的表达率,明显高于健康对照组(P＜0.05).Western blot检测结果显示,与治疗前相比,ITP患者治疗后CD4+ T细胞表达pro-caspase-3和cleaved-caspase-3均明显减少(P＜0.05).结论ITP患者外周血T细胞Fas、FasL及caspase-3表达明显增加,激素治疗可干预Fas-FasL、caspase-3的表达水平,提示Fas、FasL及caspase-3信号通路在ITP发生机制中起一定作用.     

P38MAPK regulates caspase-3 by binding to caspase-3 in nucleus of human hepatoma Bel-7402 cells during anti-Fas antibody- and actinomycin D-induced apoptosis

Anti-Fas antibody- and actinomycin D (FA/AD) has been shown to have anti-tumor activity in some tumor cells. However, many of the molecular mechanism of FA/AD-induced apoptosis of human hepatoma Bel-7402 cells have not been fully clarified. In the present study, therefore, the effect of FA/AD in presence or absence of p38MAPK inhibitor SB203580 on the proliferation, apoptosis, p38MAPK, caspase-3, location of p38MAPK and caspase-3, and interaction between p38MAPK and caspase-3 in Bel-7402 cell was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), annexin V-FITC/propidium iodide (PI) double staining, electron microscopy, immunoblot, immunofluorescence and immunoprecipitation/immunoblot assay, respectively. We found that FA/AD significantly resulted in the inhibition of proliferation, induction of apoptosis, activation and up-regulation of p38MAPK, activation and up-regulation of caspase-3, translocation of p38MAPK and caspase-3 from cytosol to nucleus, and formation of p38MAPK/caspase-3 complex in Bel-7402 cells. In contrast, SB203580, a p38MAPK-specific inhibitor, apparently blocked induction of apoptosis, activation and up-regulation of p38MAPK, activation and up-regulation of caspase-3, and translocation of p38MAPK and caspase-3 from cytosol to nucleus in FA/AD-treated Bel-7402 cells. Taken together, we conclude that p38MAPK regulates caspase-3 by binding to caspase-3 in nucleus of Bel-7402 cells during FA/AD-induced apoptosis.     

Hypertonicity regulates the function of human neutrophils by modulating chemoattractant receptor signaling and activating mitogen-activated protein kinase p38.

Excessive neutrophil activation causes posttraumatic complications, which may be reduced with hypertonic saline (HS) resuscitation. We tested if this is because of modulated neutrophil function by HS. Clinically relevant hypertonicity (10-25 mM) suppressed degranulation and superoxide formation in response to fMLP and blocked the activation of the mitogen activated protein kinases (MAPK) ERK1/2 and p38, but did not affect Ca2+ mobilization. HS did not suppress oxidative burst in response to phorbol myristate acetate (PMA). This indicates that HS suppresses neutrophil function by intercepting signal pathways upstream of or apart from PKC. HS activated p38 by itself and enhanced degranulation in response to PKC activation. This enhancement was reduced by inhibition of p38 with SB203580, suggesting that p38 up-regulation participates in HS-induced enhancements of degranulation. HS had similar effects on the degranulation of cells that were previously stimulated with fMLP, but had no effect on its own, suggesting that HS enhancement of degranulation requires another signal. We conclude that depending on other stimuli, HS can suppress neutrophil activation by intercepting multiple receptor signals or augment degranulation by enhancing p38 signaling. In patients HS resuscitation may reduce posttraumatic complications by preventing neutrophil activation via chemotactic factors released during reperfusion.     

应激诱导的Caspase-3和Livin在早孕胎盘组织表达的研究

【目的】研究在应激诱导下早孕胎盘组织中caspase3和Livin的表达,探讨Livin的凋亡调节作用。【方法】选取健康早孕而自愿终止妊娠妇女35例,随机分组,相应的应激原作用后行人工流产取绒毛组织,用半定量RTPCR分析绒毛组织中Caspase3mRNA和LivinmRNA的表达。【结果】不同强度的应激原诱导后早孕胎盘组织Caspase3mRNA的表达为(0.814±0.023)(10min组)、(0.842±0.045)(15min组),相比较于(0.190±0.021)(正常组)差异有显著性(P<0.05);LivinαmRNA的表达为(0.4155±0.012)(5min组)、(0.8903±0.079)(10min组)、(0.9113±0.070)(15min组),相比较于(0.2527±0.015)(正常组)差异有显著性(P<0.05);LivinβmRNA的表达与正常组比较差异无显著性(P>0.05)。【结论】在正常早孕胎盘组织中低表达Caspase3的同时相对高表达Livinβ和低表达Livinα,在外界应激原的诱导下Livinα的表达随Caspase3表达的增加而呈平行上升趋势,上调“凋亡阈值”,从而保证了生理和病理状况下早孕胎盘组织滋养层细胞的适度凋亡;因此Livin可能在早孕胎盘组织的凋亡调节中起着内源性安全阈值的作用。     

Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils

Activation of leukocytes by proinflammatory stimuli selectively initiates intracellular signal transduction via sequential phosphorylation of kinases. Lipopolysaccharide (LPS) stimulation of human neutrophils is known to result in activation of p38 mitogen-activated protein kinase (MAPk); however, the upstream activator(s) of p38 MAPk is unknown, and consequences of p38 MAPk activation remain largely undefined. We investigated the MAPk kinase (MKK) that activates p38 MAPk in response to LPS, the p38 MAPk isoforms that are activated as part of this pathway, and the functional responses affected by p38 MAPk activation. Although MKK3, MKK4, and MKK6 all activated p38 MAPk in experimental models, only MKK3 was found to activate recombinant p38 MAPk in LPS-treated neutrophils. Of p38 MAPk isoforms studied, only p38alpha and p38delta were detected in neutrophils. LPS stimulation selectively activated p38alpha. Specific inhibitors of p38alpha MAPk blocked LPS-induced adhesion, nuclear factor-kappa B (NF-kappaB) activation, and synthesis of tumor necrosis factor-alpha (TNF-alpha). Inhibition of p38alpha MAPk resulted in a transient decrease in TNF-alpha mRNA accumulation but persistent loss of TNF-alpha synthesis. These findings support a pathway by which LPS stimulation of neutrophils results in activation of MKK3, which in turn activates p38alpha MAPk, ultimately regulating adhesion, NF-kappaB activation, enhanced gene expression of TNF-alpha, and regulation of TNF-alpha synthesis.     

Differential Regulation and ATP Requirement for Caspase-8 and Caspase-3 Activation during CD95- and Anticancer Drug–induced Apoptosis

Apoptosis is induced by different stimuli, among them triggering of the death receptor CD95, staurosporine, and chemotherapeutic drugs. In all cases, apoptosis is mediated by caspases, although it is unclear how these diverse apoptotic stimuli cause protease activation. Two regulatory pathways have been recently identified, but it remains unknown whether they are functionally independent or linked to each other. One is mediated by recruitment of the proximal regulator caspase-8 to the death receptor complex. The other pathway is controlled by the release of cytochrome c from mitochondria and the subsequent ATP-dependent activation of the death regulator apoptotic protease-activating factor 1 (Apaf-1). Here, we report that both pathways can be dissected by depletion of intracellular ATP. Prevention of ATP production completely inhibited caspase activation and apoptosis in response to chemotherapeutic drugs and staurosporine. Interestingly, caspase-8, whose function appeared to be restricted to death receptors, was also activated by these drugs under normal conditions, but not after ATP depletion. In contrast, inhibition of ATP production did not affect caspase activation after triggering of CD95. These results suggest that chemotherapeutic drug–induced caspase activation is entirely controlled by a receptor-independent mitochondrial pathway, whereas CD95-induced apoptosis can be regulated by a separate pathway not requiring Apaf-1 function.     

Co-localization of iron binding on silica with p62/sequestosome1 (SQSTM1) in lung granulomas of mice with acute silicosis

The cellular mechanisms involved in the development of silicosis have not been fully elucidated. This study aimed to examine influence of silica-induced lung injury on autophagy. Suspensions of crystalline silica particles were administered transnasally to C57BL/6j mice. Immunohistochemical examination for Fas and p62 protein expression was performed using lung tissue specimens. Two-dimensional and quantitative analysis of silica deposits in the lungs were performed in situ using lung tissue sections by an in-air microparticle induced X-ray emission (in-air micro-PIXE) analysis system, which was based on irrradiation of specimens with a proton ion microbeam. Quantitative analysis showed a significant increase of iron levels on silica particles (assessed as the ratio of Fe relative to Si) on day 56 compared with day 7 (p<0.05). Fas and p62 were expressed by histiocytes in granulomas on day 7, and the expressions persisted for day 56. Fas- and p62-expressing histiocytes were co-localized in granulomas with silica particles that showed an increase of iron levels on silica particles in mouse lungs. Iron complexed with silica induces apoptosis, and may lead to dysregulations of autophagy in histiocytes of granulomas, and these mechanisms may contribute to granuloma development and progression in silicosis.     

急性肝损伤大鼠肝脏Fas和FasL的表达及其意义

目的 研究急性肝损伤大鼠肝脏Fas和FasL的表达情况，探讨细胞凋亡在中毒性肝损伤发病中的地位及其意义。方法 健康雄性Wistar大鼠35只，随机分为正常对照组和实验组，实验组再分为3、9、16、24、36和48h6个亚组，每组5只。制备四氯化碳中毒性肝损伤动物模型，采用苏木素-伊红（HE）染色，光镜下观察肝组织损伤情况，采用免疫组化方法测定不同时间点肝组织Fas和FasL的表达，采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）观察肝细胞凋亡情况。同时测定各时间点血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）和肝组织超氧化物歧化酶（SOD）活性、肝组织丙二醛（MDA）含量变化。结果 Fas和FasL在正常大鼠肝细胞中未见表达，实验组3h后即开始有明显表达，并随时间延长表达相应增强；病理学和TUNEL检测结果均显示肝脏有严重损伤，大量肝细胞发生凋亡。大鼠染毒后血清ALT、AST活性和肝组织MDA含量明显升高，肝组织SOD活性显著降低，与正常对照组比较差异均十分显著（P〈O．05或P〈O．01）。结论 大鼠急性肝损伤时Fas和FasL表达显著增加，和肝细胞凋亡变化相一致，提示Fas／Fasl。系统及介导的细胞凋亡反应在中毒性肝损伤的发病机制中可能占有重要地位。     

Caspase-3在戊四氮急性致痫大鼠脑组织中的表达

目的 研究caspase-3在戊四氮(pentylenetetrazole，PTZ)急性致痫大鼠脑组织中的表达情况。方法 免疫组织化学SP法显示海马及皮层caspase-3阳性细胞，CMIAS图像分析系统半定量分析caspase-3蛋白的含量。结果 癫痫发作后海马及皮层caspase-3蛋白的表达水平均增加，且海马组织较皮层组织增加更为明显。结论 Caspas-3参与了癫痫后脑组织神经元的凋亡过程；在癫痫发作所致的神经元损伤中，海马较皮层可能更为敏感。     

Stimulation of Toll-like receptor 3 and 4 induces interleukin-1beta maturation by caspase-8

The cytokine interleukin (IL)-1β is a key mediator of the inflammatory response and has been implicated in the pathophysiology of acute and chronic inflammation. IL-1β is synthesized in response to many stimuli as an inactive pro–IL-1β precursor protein that is further processed by caspase-1 into mature IL-1β, which is the secreted biologically active form of the cytokine. Although stimulation of membrane-bound Toll-like receptors (TLRs) up-regulates pro–IL-1β expression, activation of caspase-1 is believed to be mainly initiated by cytosolic Nod-like receptors. In this study, we show that polyinosinic:polycytidylic acid (poly[I:C]) and lipopolysaccharide stimulation of macrophages induces pro–IL-1β processing via a Toll/IL-1R domain–containing adaptor-inducing interferon-β–dependent signaling pathway that is initiated by TLR3 and TLR4, respectively. Ribonucleic acid interference (RNAi)–mediated knockdown of the intracellular receptors NALP3 or MDA5 did not affect poly(I:C)-induced pro–IL-1β processing. Surprisingly, poly(I:C)- and LPS-induced pro–IL-1β processing still occurred in caspase-1–deficient cells. In contrast, pro–IL-1β processing was inhibited by caspase-8 peptide inhibitors, CrmA or vFLIP expression, and caspase-8 knockdown via RNAi, indicating an essential role for caspase-8. Moreover, recombinant caspase-8 was able to cleave pro–IL-1β in vitro at exactly the same site as caspase-1. These results implicate a novel role for caspase-8 in the production of biologically active IL-1β in response to TLR3 and TLR4 stimulation.     

西洋参茎叶皂苷对大鼠局灶性脑缺血后半胱氨酸天冬氨酸蛋白酶8表达的影响

目的 研究西洋参茎叶皂苷(PQS)对大鼠局灶性脑缺血后半胱氨酸天冬氨酸蛋白酶8(caspase-8)表达的影响,并进一步探讨其机制.方法 采用线栓法阻断大鼠一侧大脑中动脉(MCAO),制备局灶性脑缺血模型,逆转录-聚合酶链反应(RT-PCR)技术检测大脑皮质凋亡相关基因caspase-8mRNA表达.结果 模型组caspase-8mRNA表达较PQS高剂量组、PQS低剂量组、尼莫地平组、假手术组均明显增加(0.713±0.117 vs 0.467±0.089、0.552±0.099、0.468±0.097、0.422±0.077,P＜0.01);而其他组间caspase-8mRNA表达差异均无统计学意义.结论 PQS对大鼠局灶性脑缺血具有保护作用,其机制可能与西洋参茎叶皂苷抑制神经元细胞凋亡有关.     

缺血预处理星形胶质细胞GDNF抑制p38MAPK信号通路减轻神经元凋亡

目的研究缺血预处理星形胶质细胞分泌GDNF抑制p38MAPK信号通路发挥脑保护作用。方法原代培养神经元及星形胶质细胞,给予缺血预处理,将星形胶质细胞培养基制备成条件培养基,沉默GDNF基因的培养基作为另一种条件培养基,将神经元分为对照组、预处理组、预处理+缺血组、缺血组,应用不同条件培养基孵育神经元,流式细胞术检测神经元凋亡,Western Blot法检测神经元p38MAPK及p-p38MAPK的表达。结果预处理+缺血组和缺血组神经元凋亡率明显增高(P<0.05),加入ACM2后凋亡率较ACM1和ACM3两组均减低(P<0.05)。每组神经元的p38MAPK蛋白表达均无明显变化(P>0.05);预处理+缺血组及缺血组p-p38MAPK的蛋白表达均明显高于对照组和预处理组(P<0.05),预处理+缺血组p-p38MAPK的蛋白表达均较缺血组低(P<0.05),加入ACM2组的p-p38MAPK蛋白表达低于ACM1和ACM3两组(P<0.05)。结论缺血预处理后星形胶质细胞分泌GDNF抑制p38MAPK信号通路的激活从而减少神经元凋亡,起到脑保护作用。     

Suppression of phosphorylation of extracellular-signal-regulated kinase and p38 mitogen-activated protein kinase in polymorphonuclear leukocytes by the proton pump inhibitor lansoprazole

Lansoprazole (LPZ) is a proton pump inhibitor that suppresses gastric secretion and exerts anti-inflammatory effects on immune cells. Recently, LPZ has been used for the treatment of peptic ulcer and gastritis, which can be caused by Helicobacter pylori, due to its potent acid-suppressive effects. We focused the aim to the anti-inflammatory effects on the over-activation of neutrophils, and investigated the effects of LPZ on the signal transduction of the mitogen-activated protein kinase (MAPK) family. LPZ slightly phosphorylated p38 MAPK of neutrophils at a concentration of 10 μg/ml, but did not phosphorylate extracellular-signal regulated kinase (ERK) 1/2. Pretreatment of neutrophils with (1–5 μg/ml) LPZ strongly attenuated the phorbol-12-myristate-13-acetate-stimulated phosphorylation of ERK1/2, and LPZ slightly suppressed the lipopolysaccharide (LPS)- and N-formylmethionylleucylphenylalanine-stimulated phosphorylation of p38. ERK1/2 produces the mitochondrial anti-apoptotic proteins, and the signaling pathway from LPS and N-formylmethionylleucylphenylalanine to p38 is the main pathway for reactive oxygen species production. The mechanism of anti-inflammatory effect of LPZ on hyper-activated neutrophils is suggested to be the suppression of signal transduction of ERK1/2 and p38 MAPK.     

Selective inhibitor of p38 mitogen-activated protein kinase inhibits lipopolysaccharide-induced interleukin-8 expression in human pulmonary vascular endothelial cells

Adult respiratory distress syndrome (ARDS) characterized by permeability edema is observed in severe insults such as bacteremia sepsis. Interleukin (IL)-8, which chemoattracts and activates neutrophils, has been suggested to play an important role in the production of ARDS. Therefore, the inhibition of IL-8 production is an important strategy for the treatment of ARDS. Recent studies have revealed the role of p38 mitogen-activated protein (MAP) kinase in cytokine expression and the inhibition by a selective inhibitor of p38 MAP kinase activity of cytokine expression in a variety of cell types. However, little is known about the role of p38 MAP kinase in lipopolysaccharide (LPS)-induced IL-8 expression in pulmonary vascular endothelial cells and the effect of a selective p38 MAP kinase inhibitor on it. In the present study, we therefore attempted to clarify these issues. The results showed that LPS induced p38 MAP kinase phosphorylation and activity, and SB 203580 as a selective inhibitor of p38 MAP kinase activity inhibited p38 MAP kinase activity and IL-8 expression in LPS-stimulated pulmonary vascular endothelial cells. These results indicate that p38 MAP kinase regulates LPS-induced IL-8 expression in pulmonary vascular endothelial cells. Although it is currently not known whether SB 203580 is capable of producing beneficial effects on ARDS, a strategy of inhibiting p38 MAP kinase activity by a selective p38 MAP kinase inhibitor may apply to the therapy for ARDS.