SRO9基因参与调控酿酒酵母内质网应激反应

【目的】研究酵母SRO9基因在内质网应激(Endoplasmic reticulum stress,ERS)中的作用。【方法】利用PCR介导的同源重组方法构建SRO9基因缺失菌株,检测其在内质网应激诱导剂衣霉素处理条件下的克隆形成能力;通过比色法检测细胞内的H2O2含量,超氧化物歧化酶SOD活性和细胞增殖能力;通过实时荧光定量PCR检测内质网应激靶基因和超氧化物歧化酶编码基因SOD1及SOD2的转录水平。【结果】相对于野生型酵母菌株,SRO9基因缺失酵母菌株对内质网应激诱导剂衣霉素的抗性增强,参与内质网应激反应的靶基因转录上调;细胞内H2O2含量下降,SOD1、SOD2转录水平降低,总SOD活性降低;对氧化剂CHP和VK3的抵抗性减弱,复制寿命明显缩短。【结论】SRO9基因缺失酵母细胞对内质网应激诱导剂衣霉素的抗性增强,原因可能是由于SRO9基因缺失激活了细胞的内质网应激反应。     

Methoxyflavones protect cells against endoplasmic reticulum stress and neurotoxin

Enhanced endoplasmic reticulum (ER) stress leads to cell death in various pathophysiological situations. During a search for compounds that regulate ER stress, we identified methoxyflavones, a group of flavonoids, as strong protective agents against ER stress. Analysis in mouse insulinoma MIN6 cells revealed that methoxyflavones mildly activated the eukaryotic initiation factor 2 and nuclear factor erythroid 2-related factor pathways, but not the XBP1 pathway, and induced downstream genes, including glucose-regulated protein (GRP) 78, a molecular chaperone in the ER. The protective effect of methoxyflavones was enhanced by agents that increase intracellular cAMP levels such as forskolin, dibutyryl-cAMP and IBMX, but suppressed by the protein kinase A (PKA) inhibitor H-89, suggesting involvement of the PKA pathway in the regulation of ER stress by methoxyflavones. Consistent with the results in cultured cells, pretreatment of mice with tangeretin, a methoxyflavone, enhanced expression of GRP78 and HO-1 without causing ER stress in renal tubular epithelium and prevented tunicamycin-induced cell death. Furthermore, preadministration of tangeretin in mice enhanced expression of GRP78 in the substantia nigra pars compacta and protected dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin that induces both oxidative and ER stress. These results suggest that methoxyflavones play an important role in the regulation of ER stress and could be a therapeutic target for the ER stress-related diseases. neuronal degeneration; flavonoids     

Calnexin is involved in apoptosis induced by endoplasmic reticulum stress in the fission yeast

Stress conditions affecting the functions of the endoplasmic reticulum (ER) cause the accumulation of unfolded proteins. ER stress is counteracted by the unfolded-protein response (UPR). However, under prolonged stress the UPR initiates a proapoptotic response. Mounting evidence indicate that the ER chaperone calnexin is involved in apoptosis caused by ER stress. Here, we report that overexpression of calnexin in Schizosaccharomyces pombe induces cell death with apoptosis markers. Cell death was partially dependent on the Ire1p ER-stress transducer. Apoptotic death caused by calnexin overexpression required its transmembrane domain (TM), and involved sequences on either side of the ER membrane. Apoptotic death caused by tunicamycin was dramatically reduced in a strain expressing endogenous levels of calnexin lacking its TM and cytosolic tail. This demonstrates the involvement of calnexin in apoptosis triggered by ER stress. A genetic screen identified the S. pombe homologue of the human antiapoptotic protein HMGB1 as a suppressor of apoptotic death due to calnexin overexpression. Remarkably, overexpression of human calnexin in S. pombe also provoked apoptotic death. Our results argue for the conservation of the role of calnexin in apoptosis triggered by ER stress, and validate S. pombe as a model to elucidate the mechanisms of calnexin-mediated cell death.     

CHOP is involved in endoplasmic reticulum stress-induced apoptosis by enhancing DR5 expression in human carcinoma cells

It has been shown that excess stress to the endoplasmic reticulum (ER) triggers apoptosis, but the mechanisms underlying these processes remain unclear. We and others have reported previously that DR5 expression is up-regulated in thapsigargin (THG)-treated human cancer cells. Here, we provide evidence that CHOP is involved in THG up-regulation of DR5, which is a critical step for ER stress-induced apoptosis in human cancer cells. In human colon cancer HCT116 cells, knockdown of DR5 by siRNA blocked THG-induced Bax conformational change along with caspase-3 activation and cell death. Moreover, inhibition of CHOP expression attenuated DR5 up-regulation and apoptosis induced by THG, whereas ectopic expression of DR5 restored the sensitivity of CHOP siRNA-transfected cells to THG-induced apoptosis. In addition to HCT116 cells, inhibition of CHOP or DR5 induction also attenuated THG-induced cell death in other cancer cell lines including LNCaP, A2780S, and DU145, indicating that CHOP and DR5 are critical for ER stress-mediated apoptosis in human carcinoma cells. Furthermore, we identified a potential CHOP-binding site in the 5'-flanking region of the DR5 gene. Mutation of this site abrogated the enhanced reporter activity in response to THG treatment. Together, our findings suggest that CHOP regulates ER stress-induced apoptosis, at least in part, through enhancing DR5 expression in some types of human cancer cells.     

Taurine protection of PC12 cells against endoplasmic reticulum stress induced by oxidative stress

Background

Taurine is a free amino acid present in high concentrations in a variety of organs of mammalians. As an antioxidant, taurine has been found to protect cells against oxidative stress, but the underlying mechanism is still unclear.

Methods

In this report, we present evidence to support the conclusion that taurine exerts a protective function against endoplasmic reticulum (ER) stress induced by H₂O₂ in PC 12 cells. Oxidative stress was introduced by exposure of PC 12 cells to 250 uM H₂O₂ for 4 hours.

Results

It was found that the cell viability of PC 12 cells decreased with an increase of H₂O₂ concentration ranging from approximately 76% cell viability at 100 uM H₂O₂ down to 18% at 500 uM H₂O₂. At 250 uM H₂O₂, cell viability was restored to 80% by taurine at 25 mM. Furthermore, H₂O₂ treatment also caused a marked reduction in the expression of Bcl-2 while no significant change of Bax was observed. Treatment with taurine restored the reduced expression of Bcl-2 close to the control level without any obvious effect on Bax. Furthermore, taurine was also found to suppress up-regulation of GRP78, GADD153/CHOP and Bim induced by H₂O₂, suggesting that taurine may also exert a protective function against oxidative stress by reducing the ER stress.

Conclusion

In summary, taurine was shown to protect PC12 cells against oxidative stress induced by H₂O₂. ER stress was induced by oxidative stress and can be suppressed by taurine.

     

Bufotalin-induced apoptosis in osteoblastoma cells is associated with endoplasmic reticulum stress activation

The search for novel and more efficient chemo-agents against malignant osteoblastoma is important. In this study, we examined the potential anti-osteoblastoma function of bufotalin, and studied the underlying mechanisms. Our results showed that bufotalin induced osteoblastoma cell death and apoptosis in dose- and time-dependent manners. Further, bufotalin induced endoplasmic reticulum (ER) stress activation in osteoblastoma cells, the latter was detected by the induction of C/EBP homologous protein (CHOP), phosphorylation of inositol-requiring enzyme 1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK), as well as caspase-12 activation. Conversely, the ER stress inhibitor salubrinal, the caspase-12 inhibitor z-ATAD-fmk as well as CHOP depletion by shRNA significantly inhibited bufotalin-induced osteoblastoma cell death and apoptosis. Finally, by using a mice xenograft model, we demonstrated that bufotalin inhibited U2OS osteoblastoma cell growth in vivo. In summary, our results suggest that ER stress contributes to bufotalin-induced apoptosis in osteoblastoma cells. Bufotalin might be investigated as a novel anti-osteoblastoma agent.     

Activation of autophagy by unfolded proteins during endoplasmic reticulum stress

Endoplasmic reticulum stress is defined as the accumulation of unfolded proteins in the endoplasmic reticulum, and is caused by conditions such as heat or agents that cause endoplasmic reticulum stress, including tunicamycin and dithiothreitol. Autophagy, a major pathway for degradation of macromolecules in the vacuole, is activated by these stress agents in a manner dependent on inositol‐requiring enzyme 1b (IRE1b), and delivers endoplasmic reticulum fragments to the vacuole for degradation. In this study, we examined the mechanism for activation of autophagy during endoplasmic reticulum stress in Arabidopsis thaliana. The chemical chaperones sodium 4–phenylbutyrate and tauroursodeoxycholic acid were found to reduce tunicamycin‐ or dithiothreitol‐induced autophagy, but not autophagy caused by unrelated stresses. Similarly, over‐expression of BINDING IMMUNOGLOBULIN PROTEIN (BIP), encoding a heat shock protein 70 (HSP70) molecular chaperone, reduced autophagy. Autophagy activated by heat stress was also found to be partially dependent on IRE1b and to be inhibited by sodium 4–phenylbutyrate, suggesting that heat‐induced autophagy is due to accumulation of unfolded proteins in the endoplasmic reticulum. Expression in Arabidopsis of the misfolded protein mimics zeolin or a mutated form of carboxypeptidase Y (CPY*) also induced autophagy in an IRE1b‐dependent manner. Moreover, zeolin and CPY* partially co‐localized with the autophagic body marker GFP–ATG8e, indicating delivery to the vacuole by autophagy. We conclude that accumulation of unfolded proteins in the endoplasmic reticulum is a trigger for autophagy under conditions that cause endoplasmic reticulum stress.     

Rtn1p is involved in structuring the cortical endoplasmic reticulum

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Delta in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Delta cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.     

FCN3 functions as a tumor suppressor of lung adenocarcinoma through induction of endoplasmic reticulum stress

In this study, we report a novel function of FCN3 (Ficolin 3), a secreted lectin capable of activating the complement pathway, as a tumor suppressor of lung adenocarcinoma (LUAD). First, the expression of FCN3 was strongly down-regulated in cancer tissues compared to matched normal lung tissues, and down-regulation of FCN3 was shown to be significantly correlated with increased mortality among LUAD patients. Interestingly, while ectopic expression of FCN3 led to cell cycle arrest and apoptosis in A549 and H23 cells derived from LUAD, the secreted form of the protein had no effect on the cells. Rather, we found evidence indicating that activation of the unfolded protein response from endoplasmic reticulum (ER) stress is induced by ectopic expression of FCN3. Consistently, inhibition of ER stress response led to enhanced survival of the LUAD cells. Of note, the fibrinogen domain, which is not secreted, turned out to be both necessary and sufficient for induction of apoptosis when localized to ER, consistent with our proposed mechanism. Collectively, our data indicate that FCN3 is a tumor suppressor gene functioning through induction of ER stress.Subject terms: Non-small-cell lung cancer, Apoptosis, Endoplasmic reticulum     

Curcumin induces pro-apoptotic endoplasmic reticulum stress in human leukemia HL-60 cells

Curcumin has been shown to induce apoptosis in many cancer cells. However, the molecular mechanism(s) responsible for curcumin-induced apoptosis is not well understood and most probably involves several pathways. In HL-60 cells, curcumin induced apoptosis and endoplasmic reticulum (ER) stress as evidenced by the survival molecules such as phosphorylated protein kinase-like ER-resident kinase, phosphorylated eukaryotic initiation factor-2alpha, glucose-regulated protein-78, and the apoptotic molecules such as caspase-4 and CAAT/enhancer binding protein homologous protein (CHOP). Inhibition of caspase-4 activity by z-LEVD-FMK, blockage of CHOP expression by small interfering RNA, and treatment with salubrinal, an ER inhibitor, significantly reduced curcumin-induced apoptosis. Removing two double bonds in curcumin, which was speculated to form Michael adducts with thiols in secretory proteins, resulted in a loss of the ability of curcumin to induce apoptosis as well as ER stress. Thus, the present study shows that curcumin-induced apoptosis is associated with its ability to cause ER stress.     

Apoptosis induced by endoplasmic reticulum stress involved in diabetic kidney disease

Endoplasmic reticulum stress has been suggested to play a crucial role in the pathogenesis of diabetic complications. However, whether it is involved in the renal injury of diabetic nephropathy is still not known. We investigated the involvement of ER-associated apoptosis in kidney disease of streptozocin (STZ)-induced diabetic rats. We used albuminuria examination, hematoxylin & eosin (H&E) staining and TUNEL analysis to identify the existence of diabetic nephropathy and enhanced apoptosis. We performed immunohistochemistry, Western blot, and real-time PCR to analyze indicators of ER molecule chaperone and ER-associated apoptosis. GRP78, the ER chaperone, was up-regulated significantly in diabetic kidney compared to control. Furthermore, three hallmarks of ER-associated apoptosis, C/EBP homologous protein (CHOP), c-JUN NH2-terminal kinase (JNK) and caspase-12, were found to have activated in the diabetic kidney. Taken together, those results suggested that apoptosis induced by ER stress occurred in diabetic kidney, which may contribute to the development of diabetic nephropathy.     

Hydrogen sulfide protects SH-SY5Y cells against 6-hydroxydopamine-induced endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress has been implicated in several neurodegenerative diseases, including Parkinson's disease. The present study attempted to investigate the effect of hydrogen sulfide (H(2)S) on 6-hydroxydopamine (6-OHDA)-induced ER stress in SH-SY5Y cells. We found in the present study that exogenous application of sodium hydrosulfide (NaHS; an H(2)S donor, 100 μM) significantly attenuated 6-OHDA (50 μM)-induced cell death. NaHS also reversed the upregulation of cleaved poly(ADP-ribose) polymerase and caspase 9 in 6-OHDA-treated cells. Consistent with its cytoprotective effects, NaHS markedly reduced 6-OHDA induced-ER stress responses, including the upregulated levels of eukaryotic initiation factor-2α phosphorylation, glucose-regulated protein 78, and C/EBP homologous protein expression. The protective effect of H(2)S on ER stress was attenuated by blockade of Akt activity with an Akt inhibitor or inhibition of heat shock protein (Hsp)90 with geldanamycin but not by suppression of ERK1/2 with PD-98059. Blockade of Akt also significantly decreased the protein abundance of Hsp90 in SH-SY5Y cells. Moreover, overexpression of cystathionine β-synthase (a main H(2)S-synthesizing enzyme in the brain) elevated the Hsp90 protein level and suppressed 6-OHDA-induced ER stress. In conclusion, the protective effect of H(2)S against 6-OHDA-induced ER stress injury in SH-SY5Y cells involves the Akt-Hsp90 pathway.     

Deferoxamine induces endoplasmic reticulum stress in PC12 cells

Deferoxamine (DFA, N'-[5-(acetyl-hydroxy-amino)-pentyl]-N-[5-[3-(5-aminopentyl-hydroxy-carbamoyl) propanoylamino]pentyl]-N-hydroxy-butane diamide) is a chelating agent used to remove excess iron from the body and to reduce organ and tissue damage. DFA enhances both iron regulatory protein 1 (IRP1) expression and its endoplasmic reticulum (ER) membrane-binding activity, as occurs in hypoxia, an ER stress, in cultured cells. Here, we show that DFA promotes ER stress via an ER signal pathway.     

Lipolysis response to endoplasmic reticulum stress in adipose cells

In obesity and diabetes, adipocytes show significant endoplasmic reticulum (ER) stress, which triggers a series of responses. This study aimed to investigate the lipolysis response to ER stress in rat adipocytes. Thapsigargin, tunicamycin, and brefeldin A, which induce ER stress through different pathways, efficiently activated a time-dependent lipolytic reaction. The lipolytic effect of ER stress occurred with elevated cAMP production and protein kinase A (PKA) activity. Inhibition of PKA reduced PKA phosphosubstrates and attenuated the lipolysis. Although both ERK1/2 and JNK are activated during ER stress, lipolysis is partially suppressed by inhibiting ERK1/2 but not JNK and p38 MAPK and PKC. Thus, ER stress induces lipolysis by activating cAMP/PKA and ERK1/2. In the downstream lipolytic cascade, phosphorylation of lipid droplet-associated protein perilipin was significantly promoted during ER stress but attenuated on PKA inhibition. Furthermore, ER stress stimuli did not alter the levels of hormone-sensitive lipase and adipose triglyceride lipase but caused Ser-563 and Ser-660 phosphorylation of hormone-sensitive lipase and moderately elevated its translocation from the cytosol to lipid droplets. Accompanying these changes, total activity of cellular lipases was promoted to confer the lipolysis. These findings suggest a novel pathway of the lipolysis response to ER stress in adipocytes. This lipolytic activation may be an adaptive response that regulates energy homeostasis but with sustained ER stress challenge could contribute to lipotoxicity, dyslipidemia, and insulin resistance because of persistently accelerated free fatty acid efflux from adipocytes to the bloodstream and other tissues.