ER stress related factor ATF6 and caspase-12 trigger apoptosis in neonatal hypoxic-ischemic encephalopathy

The specific and available markers proteins of neonatal hypoxic-ischemic encephalopathy (HIE) injury are correlated with disease severity and the disability in childhood. Exploring the mechanism of HIE is very helpful to the targeted therapeutic approach in clinical. This study aims to explore the cell death-related proteins or biomarkers that plays roles in the HIE injury. In this study, 15 patients were included the 487 autopsies patients performed at the Department of Pathology. The lactate dehydrogenase (LDH) assay was used to detect the cell viability of NGF-differentiated PC12 cell. TUNEL assay was employed to examine the apoptotic cells in embedded slides samples. Three ER stress-related protein, including ATF6, p-Perk and IRE-1 were investigated using Western blot assay for the ER stress examination. The apoptosis associated caspase-12 and CHOP protein were detected by Western blot. The results indicated that LDH activity of living cells during hypoxia was significantly enhanced to 45% and 64% after 8 hours and 24 hours. The TUNEL results showed that plenty of the PC12 cells became the positive staining cells when treated with 0.1% O₂ hypoxia. ER stress UPR pathway protein, cleaved ATF6, was increased significantly when treated with 0.1% O₂ compared with the cells treated with 20% O₂. Furthermore, the caspase 12 activation was triggered when the cells treated with the 0.1% O₂. In conclusion, apoptosis is served as an important factor that triggers the HIE brain injury through cleaving the ATF6 and caspase-12 ER stress-related protein.     

周期性张应力作用下软骨细胞半胱氨酸蛋白酶12的表达规律

BACKGROUND: Excessive mechanical stimulation can lead to endoplasmic reticulum stress. Endoplasmic reticulum stress plays an important role in the occurrence and development of degenerative diseases. Caspase-12 is a specific molecule of endoplasmic reticulum stress, and the intensity of endoplasmic reticulum stress can be reflected by caspase-12 expression. OBJECTIVE: To observe the expression rule of Caspase-12 in chondrocytes under the cyclic tensile stress. METHODS: Human chondrocytes were used as test subjects. After group assignment, mechanical loading system was used. The loading group and corresponding Z-ATAD-FMK inhibitor group received 2, 12, 24, 36, and 48 hours of mechanical stimulation. The blank group (0 hour) and the paired Z-ATAD-FMK inhibitor group received the same process as the loading group except the loading. After loading, cell morphology and growth were observed under the microscope. RT-PCR and western blot assay were used to detect the expression changes of caspase-12 gene and protein in each group. RESULTS AND CONCLUSION: (1) Morphological observation results demonstrated that apoptosis appeared at 2 hours after loading, peaked at 24 hours. With prolonged time, cell growth showed the trend along stress, but apoptosis weakened. It is indicated that cyclic tensile stress could make chondrocyte apoptosis. Endoplasmic reticulum stress might be activated. The model of cells in vitro was established successfully. (2) Caspase-12 gene and protein expression showed consistent trend, and peaked at 24 hours, which showed significant differences as compared with the blank and inhibitor groups (P < 0.05). (3) Cyclic tensile stress can induce chondrocyte endoplasmic reticulum stress, and affect caspase-12 expression. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

美托洛尔对大鼠冠状动脉微栓塞后心肌细胞凋亡及caspase-12活化的影响及意义

目的： 探讨美托洛尔对大鼠冠状动脉微栓塞后心肌细胞凋亡及caspase-12活化的影响及意义。方法： 30只大鼠随机分为假手术组、微栓塞组、美托洛尔组（每组n＝10）,经左室注入42 μm微栓塞球,建立大鼠冠状动脉微栓塞模型,假手术组注射生理盐水代替微栓塞球,美托洛尔组为微栓塞术前30 min静脉注射美托洛尔。各组术后6 h分别心脏超声检测左室射血分数（LVEF）,TUNEL检测心肌细胞凋亡,Western blotting 检测凋亡蛋白caspase-12的活化。结果：① 与假手术组比较,微栓塞组LVEF显著下降（P＜0.05）;与微栓塞组比较,美托洛尔组LVEF没有显著差异。② 与假手术组比较,微栓塞组心肌细胞凋亡率、活化的caspase-12含量显著增加（均P＜0.05）;与微栓塞组比较,美托洛尔组心肌细胞凋亡率、活化的caspase-12含量显著减少（均P＜0.05）。结论： 美托洛尔抑制大鼠冠状动脉微栓塞后心肌细胞凋亡及caspase-12活化。     

骨桥蛋白通过内质网应激诱导C2C12肌细胞胰岛素抵抗

目的:探讨骨桥蛋白(osteopontin,OPN)对C2C12肌细胞胰岛素抵抗的影响及其可能的机制。方法:低血清培养辅以胰岛素处理诱导C2C12成肌细胞分化为C2C12肌细胞,蛋白免疫印迹检测蛋白质的表达,离心法分离细胞膜,葡萄糖摄取试剂盒定量葡萄糖摄取。结果:(1)骨桥蛋白以剂量依赖和时间依赖方式抑制胰岛素刺激的蛋白激酶B(Akt)的磷酸化,并抑制胰岛素所致的葡萄糖转运体4(Glut4)膜位移和葡萄糖的摄取;而特异性的抗OPN受体CD44抗体预处理可逆转上述变化。(2)OPN可诱导C2C12肌细胞的内质网应激,并促进c-Jun氨基端激酶(JNK)的磷酸化。(3)内质网应激抑制剂4-苯基丁酸(4-PBA)可降低OPN所增加的JNK磷酸化,恢复胰岛素刺激所致的Glut4的膜位移以及葡萄糖摄取。结论:骨桥蛋白通过诱导C2C12肌细胞内质网应激导致胰岛素抵抗。本研究为揭示骨桥蛋白在胰岛素抵抗和糖尿病中的作用提供了新的实验依据和理论解释。     

ER and aging—Protein folding and the ER stress response

The endoplasmic reticulum (ER) is a multifunctional organelle which co-ordinates protein folding, lipid biosynthesis, calcium storage and release. Perturbations that disrupt ER homeostasis lead to the misfolding of proteins, ER stress and up-regulation of a signaling pathway called the ER stress response or the unfolded protein response (UPR). The UPR is characterized by the induction of chaperones, degradation of misfolded proteins and attenuation of protein translation. Age-related declines and activity in key molecular chaperones and folding enzymes compromise proper protein folding and the adaptive response of the UPR. This review will highlight age-related changes in the protein folding machinery and in the UPR.     

Polyglutamine protein aggregation and toxicity are linked to the cellular stress response

Chronic exposure of cells to expanded polyglutamine proteins results in eventual cell demise. We constructed mouse cell lines expressing either the full-length androgen receptor (AR), or truncated forms of AR containing 25 or 65 glutamines to study the cellular consequences of chronic low-level exposure to these proteins. Expression of the polyglutamine-expanded truncated AR protein, but not the full-length expanded protein, resulted in the formation of cytoplasmic and nuclear aggregates and eventual cell death. Nuclear aggregates preferentially stained positive for heat shock protein (hsp)72, a sensitive indicator of a cellular stress response. Biochemical studies revealed that the presence of nuclear aggregates correlated with activation of the c-jun NH2-terminal kinase (JNK). Different metabolic insults, including heat shock treatment, and exposure to sodium arsenite or menadione, proved more toxic to those cells expressing the polyglutamine-expanded truncated protein than to cells expressing the non-expanded form. Cells containing cytoplasmic polyglutamine-protein aggregates exhibited a delayed expression of hsp72 after heat shock. Once expressed, hsp72 failed to localize normally and instead was sequestered within the protein aggregates. This was accompanied by an inability of the aggregate-containing cells to cease their stress response as evidenced by the continued presence of activated JNK. Finally, activation of the cellular stress response increased the overall extent of polyglutamine protein aggregation, especially within the nucleus. Inclusion of a JNK inhibitor reduced this stress-dependent increase in nuclear aggregates. Abnormal stress responses may contribute to enhanced cell vulnerability in cells expressing polyglutamine-expanded proteins and may increase the propensity of such cells to form cytoplasmic and nuclear inclusions.     

Molecular mechanism of satratoxin-induced apoptosis in HL-60 cells: activation of caspase-8 and caspase-9 is involved in activation of caspase-3

Satratoxins have been recognized as potential immunomodulatory agents in outbreaks of building-related illness. Here we report that satratoxin G-treated human leukemia HL-60 cells underwent apoptosis through the action of caspase-3 which was activated by both caspase-8 and caspase-9. Western blot analysis of caspase-3 in the satratoxin G-treated cells apparently indicated the appearance of a catalytically active fragment of 17 kDa. Increased caspase-3 activity was also detected by using a fluorogenic substrate, DEVD-AMC. Next, exposure to satratoxin G led to cleavage of PARP from its native 116 kDa form to a 85 kDa product. Moreover, DFF-45/ICAD were cleaved into a 12.5 kDa fragment via satratoxin G treatment. Enzymic assay on IETD-AMC revealed that caspase-8 is strongly activated by exposure to satratoxin G while T-2 toxin (T-2) could not activate caspase-8 at an early stage of apoptosis. Furthermore, satratoxin G caused a release of cytochrome c from mitochondria into the cytosol and increased the activity of caspase-9 against LEHD-AMC. These findings indicate that satratoxin G-induced apoptosis involves activation of caspase-3 and DFF-40/CAD through both activation of caspase-8 and cytosolic accumulation of cytochrome c along with activation of caspase-9.     

The equine arteritis virus induces apoptosis via caspase-8 and mitochondria-dependent caspase-9 activation

We have previously showed that equine arteritis virus (EAV), an arterivirus, induces apoptosis in vitro. To determine the caspase activation pathways involved in EAV-induced apoptosis, target cells were treated with peptide inhibitors of apoptosis Z-VAD-FMK (pan-caspase inhibitor), Z-IETD-FMK (caspase-8-specific inhibitor) or Z-LEHD-FMK (caspase-9-specific inhibitor) 4 h prior to infection with the EAV T1329 Canadian isolate. Significant inhibition of apoptosis was obtained with all peptide inhibitors used. Furthermore, apoptosis was inhibited in cells expressing the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase (HSV2-R1) or hsp70, two proteins which are known to inhibit apoptosis associated with caspase-8 activation and cytochrome c release-dependent caspase-9 activation, respectively. Given the activation of Bid and the translocation of cytochrome c within the cytoplasm, the overall results indicate that EAV induces apoptosis initiated by caspase-8 activation and subsequent mitochondria-dependent caspase-9 activation.     

Fibroblast apoptosis and caspase-8 activation in aseptic loosening

The presence of apoptosis has been investigated in the interface membranes collected during revision surgery of loosened total hip joint arthroplasty (THAs). Terminal deoxyrobonucleotidyl transferase (TdT) assay for apoptotic DNA fragmentation quantification revealed a statistically significant presence of apoptosis in aseptic samples, obtained from both cementless (2.37+/-0.6%) and cemented (12.01+/-1%) prosthesis compared to septic samples where apoptosis was almost absent. Activated caspase-8 immunostaining was almost undetectable in septic samples, while in the aseptic samples active caspase-8 was present weakly in the cementless samples (1.35+/-0.22%) and strongly in the cemented ones (9.0+/-0.40%). The caspase-8 cytoplasmatic staining allowed the morphological recognition of positive cells both as fibroblast-like and immunocompetent cells. In aseptic cemented samples fibroblast-like cells were the most represented subpopulation in the caspase-8 positive population scored (76.6%) compared to the immunocompetent cells (23.4%). Caspase-8 activation is an upstream event in the apoptotic pathway triggered by the activation of cytokines receptors such as TNF-alpha receptor 1 (TNFR-1), and the presence of caspase-8 activation in fibroblast-like cells in the aseptic interface membranes of THAs suggests a possible TNF-alpha dependent apoptosis.     

Activation of caspase-12 by endoplasmic reticulum stress induced by transient middle cerebral artery occlusion in mice

We sought to clarify the involvement of caspase-12, a representative molecule related to endoplasmic reticulum (ER) stress-induced cell-death signaling pathways, in neuronal death resulting from ischemia/reperfusion in mice. Transient focal cerebral ischemia (1 h) was produced by intraluminal occlusion of the middle cerebral artery (MCA). We assessed the expression patterns of caspase-12, Bip/GRP78, an ER-resident molecular chaperone whose expression serves as a good marker of ER stress, and caspase-7 by Western blotting and/or immunohistochemistry. Double-fluorescent staining of caspase-12 immunohistochemistry and the terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling (TUNEL) method was performed to clarify the involvement of caspase-12 in cell death. We confirmed that ER stress was induced during reperfusion in our model, as witnessed by up-regulated Bip/GRP78 expression in the MCA territory. Western blot analysis revealed that caspase-12 activation occurred at 5-23 h of reperfusion, and immunoreactivity for caspase-12 was enhanced mainly in striatal neurons on the ischemic side at the same time points. We found the co-localization of caspase-12 immunoreactivity and DNA fragmentation detectable by the TUNEL method. We did not detect the presence of caspase-7 in the ER fraction at the period of caspase-12 cleavage. Our results imply that cerebral ischemia/reperfusion induces ER stress and that caspase-12 activation concurred with ER stress. Caspase-12 seems to be involved in neuronal death induced by ischemia/reperfusion. Caspase-7 is not likely to contribute to the cleavage of caspase-12 in our experimental model.     

Reactive oxygen species-triggered trophoblast apoptosis is initiated by endoplasmic reticulum stress via activation of caspase-12, CHOP, and the JNK pathway in Toxoplasma gondii infection in mice

Toxoplasma gondii infection in pregnant women may result in abortion or in fetal teratogenesis; however, the underlying mechanisms are still unclear. In this paper, based on a murine model, we showed that maternal infection with RH strain T. gondii tachyzoites induced elevated production of reactive oxygen species (ROS), local oxidative stress, and subsequent apoptosis of placental trophoblasts. PCR array analysis of 84 oxidative stress-related genes demonstrated that 27 genes were upregulated at least 2-fold and that 9 genes were downregulated at least 2-fold in the T. gondii infection group compared with levels in the control group. The expression of NADPH oxidase 1 (Nox1) and glutathione peroxidase 6 (Gpx6) increased significantly, about 25-fold. The levels of malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) increased significantly with T. gondii infection, and levels of glutathione (GSH) decreased rapidly. T. gondii infection increased the early expression of endoplasmic reticulum stress (ERS) markers, followed by cleavage of caspase-12, activation of ASK1/JNK, and increased apoptosis of trophoblasts, both in vivo and in vitro. The apoptosis of trophoblasts, the activation of caspase-12 and the ASK1/JNK pathway, and the production of peroxides were dramatically inhibited by pretreatment with N-acetylcysteine (NAC). The upregulation of Nox1 was contact dependent and preceded the increase in levels of ERS markers and the activation of the proapoptosis cascade. Thus, we concluded that apoptosis in placental trophoblasts was initiated predominantly by ROS-mediated ERS via activation of caspase-12, CHOP, and the JNK pathway in acute T. gondii infection. Elevated ROS production is the central event in T. gondii-induced apoptosis of placental trophoblasts.     

Caspase-9和caspase-3在创伤后应激障碍大鼠海马神经元的表达及意义

目的:探讨caspase-9和caspase-3在创伤后应激障碍(PTSD)大鼠海马神经元中的表达及意义.方法:采用国际认定的无连续单一应激(SPS)方法刺激大鼠建立PTSD大鼠模型,取SPS刺激后1、4、7、14、28 d组和正常对照组.应用免疫组织化学、免疫荧光双标、激光共聚焦显微镜技术和免疫印迹法检测caspase-9和caspase-3蛋白的表达.结果:与正常对照组相比,caspase-9活性于SPS刺激后1 d升高,SPS刺激后7 d再次上调并达到高峰,之后逐渐下降.Caspase-3活性于SPS刺激后7 d达到高峰,之后逐渐下降.结论:caspase-9和caspase-3共同参与了PTSD大鼠海马神经元凋亡的调控.     

二硫苏糖醇通过激活calpain-2/caspase-12信号通路诱导BRL-3A细胞凋亡

目的:探讨二硫苏糖醇(DTT)诱导大鼠正常肝细胞株BRL-3A发生内质网应激时细胞内葡萄糖调节蛋白78(GRP78)、钙蛋白酶2 (calpain-2)、caspase-12及caspase-3的表达变化及对细胞凋亡的影响。方法:采用2. 5 mmol/L DTT分别处理BRL-3A细胞12 h和24 h,应用real-time PCR检测细胞内GRP78、calpain-2、caspase-12及caspase-3的mRNA水平;采用细胞免疫荧光检测细胞内GRP78、calpain-2、caspase-12及caspase-3的蛋白表达;应用Western blot检测cleaved caspase-12及cleaved caspase-3的表达变化;采用流式细胞术检测细胞凋亡情况。结果:BRL-3A细胞经DTT处理12 h及24 h后,GRP78、calpain-2及caspase-12的mRNA表达较正常对照组显著升高(P 0. 01),而caspase-3的mRNA水平与正常对照组比较无显著变化;细胞免疫荧光及Western blot检测发现,DTT处理细胞12 h及24 h后,BRL-3A细胞内GRP78、calpain-2、caspase-12及caspase-3的蛋白表达均较正常对照组显著增高,同时,cleaved caspase-12及cleaved caspase-3的表达也较正常对照组明显增多(P 0. 05);流式细胞术检测发现,经DTT处理后BRL-3A细胞的凋亡率较正常对照组显著增加(P 0. 05)。结论:二硫苏糖醇诱导BRL-3A细胞凋亡可能与calpain-2/caspase-12信号通路激活有关。     

Nonylphenol-induced thymocyte apoptosis involved caspase-3 activation and mitochondrial depolarization

Although the effect of 4-nonylphenol on cells of immune system have long been recognized, little is known about the effect of 4-nonylphenol on the induction of apoptosis and related signaling events in the lymphoid cells. In the present study, we used cultured thymocytes of mice to investigate the ability of 4-nonylphenol to induce the apoptosis of thymocytes and to explore the role of signal transduction pathway leading to apoptosis. The results showed that the cytotoxic effects of 4-nonyphenol involved DNA fragmentation (DNA ladder), characteristic of apoptosis. Staining of 4-nonyphenol-treated thymocytes with DNA-binding fluorochrome Hoechst 33258 showed the typical apoptotic nuclei condensation and fragmentation of chromatin. The rates of apoptosis of the 4-nonylphenol-treated thymocytes increased significantly at 4 and 6 h, which were determined by analysis of hypodiploid cells and FITC-Annexin V and PI double staining. Flow cytometer analysis also revealed that the loss of mitochondrial membrane potential and increased activity of caspase-3 occurred concomitantly with the onset of 4-nonyphenol-induced apoptosis. Furthermore, a caspase-3 inhibitor, z-DEVD-fmk protected thymocytes from apoptosis induced by 4-nonyphenol. These results suggest that 4-nonylphenol induces thymocyte apoptosis via caspase-3 activation and mitochondrial depolarization.     

ER stress abrogates the immunosuppressive effect of IL-10 on human macrophages through inhibition of STAT3 activation

Inflammation Research - To determine whether ER stress affects the inhibitory pathways of the human immune system, particularly the immunosuppressive effect of IL-10 on macrophages. In vitro...