Identification of GRP78 as a molecular target of medicarpin in osteoblast cells by proteomics

Osteogenic activity was identified in medicarpin (Med), a natural pterocarpan. Further, it was decided to study the differentially regulated protein expression during osteoblast differentiation in the presence of Med. Using 2D proteomic approach, we found that Med treatment to osteoblasts significantly downregulated GRP78, an ER chaperone with anti-apoptotic properties which also controls the activation of unfolded protein response signaling, a pro-survival strategy for normal ER functioning. However, severe stress leads to triggering of apoptotic responses and signaling switches to pro-apoptotic. In order to elucidate the effect of Med downregulation of GRP78, osteoblasts were transfected with SiGRP78 or SiGRP78+ Med or Med alone. It was seen that mRNA and protein levels of ER stress markers like GRP78, ATF-4, and CHOP were decreased in all the three groups with maximum reduction in SiGRP78+ Med group. Med targets GRP78 by inhibiting mitochondrial-mediated apoptosis which is evident by reduced levels of cytochrome c, caspase-3, Bax/BCL2 ratio, and enhanced expression of survivin. Finally, Annexin-PI staining of apoptotic cells revealed that MED inhibition of GRP78 leads to reduced osteoblast apoptosis and increased osteoblast survival. Altogether, our data show that Med inhibits ER stress-induced apoptosis and promotes osteoblast cell survival by targeting GRP78.

     

Mitochondrial TRAP1 regulates the unfolded protein response in the endoplasmic reticulum

Stress in mitochondria or the endoplasmic reticulum (ER) independently causes cell death. Recently, it was reported that ER stress causes mitochondrial dysfunction via p53-upregulated modulator of apoptosis (PUMA). However, little is known regarding the mitochondria molecules that mediate ER dysfunction. The present study revealed that tumor necrosis factor receptor-associated protein 1 (TRAP1), which localizes in the mitochondria, is associated with the unfolded protein response (UPR) in the ER. TRAP1 knockdown activated the ER-resident caspase-4, which is activated by ER stress, to induce cell death in humans. However, TRAP1 knockdown cells did not show a significant increase in the level of cell death at least within 24 h after early phase of ER stress in comparison with that of the control cells. This finding could be attributed to a number of reasons. TRAP1 knockdown failed to activate caspase-9, which is activated by activated caspase-4. In addition, TRAP1 knockdown increased the basal level of GRP78/BiP expression, which protects cells, and decreased the basal level of C/EBP homologous protein (CHOP) expression, which induces cell death, even under ER stress. Thus, the present study revealed that mitochondria could be a potential regulator of the UPR in the ER through mitochondrial TRAP1.     

Translocation of Bim to the endoplasmic reticulum (ER) mediates ER stress signaling for activation of caspase-12 during ER stress-induced apoptosis

Endoplasmic reticulum (ER) stress activates caspase-12 in murine cells, triggering the ER stress-specific cascade for implementation of apoptosis. In C2C12 murine myoblast cells, activation of the cascade occurs without release of cytochrome c from mitochondria, suggesting that the cascade is independent of mitochondrial damage. Stable overexpression of Bcl-xL in C2C12 cells suppressed activation of caspase-12 and apoptosis. In ER-stressed cells, but not in normal cells, Bcl-xL was co-immunoprecipitated with Bim, a pro-apoptotic member of the Bcl-2 family, suggesting that Bcl-xL sequesters Bim, thereby inhibiting the apoptotic signaling. Fractionation of C2C12 cells revealed that ER stress led to translocation of Bim from a dynein-rich compartment to the ER, while stable overexpression of Bcl-xL suppressed accumulation of Bim on the ER. Although the toxic effect of Bim had been previously observed only at the mitochondrial outer membrane, overexpression of a Bim derivative, Bim(ER), targeted at the surface of the ER led to apoptosis. A C2C12 transfectant overexpressing the caspase-12 suppressor protein was resistant to Bim(ER), suggesting that the toxic effect of Bim on the ER is dependent on activation of caspase-12. Knockdown of Bim by RNA interference provided cells resistant to ER stress. These results suggest that translocation of Bim to the ER in response to ER stress is an important step toward activation of caspase-12 and initiation of the ER stress-specific caspase cascade.     

Induction of apoptosis by cigarette smoke via ROS-dependent endoplasmic reticulum stress and CCAAT/enhancer-binding protein-homologous protein (CHOP)

In this report, we investigated a role of endoplasmic reticulum (ER) stress in cigarette smoke (CS)-induced apoptosis of human bronchial epithelial cells (hBEC). Exposure of hBEC to CS or CS extract (CSE) caused expression of endogenous ER stress markers GRP78 and CHOP and induction of apoptosis evidenced by nuclear condensation, membrane blebbing, and activation of caspase-3 and caspase-4. In vivo exposure of mice to CS also caused induction of GRP78 and CHOP in the lung. Attenuation of ER stress by overexpression of ER chaperone GRP78 or ORP150 significantly attenuated CSE-triggered apoptosis. Exposure of hBEC to CSE caused generation of reactive oxygen species, and treatment with antioxidants inhibited CSE-induced apoptosis. Interestingly, antioxidants including a scavenger of O(2)(*-) blunted induction of CHOP by CSE without affecting the level of GRP78, and dominant-negative inhibition of CHOP abolished CSE-induced apoptosis. Furthermore, a generator of O(2)(*-) selectively induced CHOP and apoptosis in hBEC. Our results revealed that: (1) CS induces ER stress in vitro and in vivo, (2) ER stress mediates CS-triggered apoptosis downstream of oxidative stress, (3) CS-initiated apoptosis is caused through oxidative stress-dependent induction of CHOP, (4) O(2)(*-) may play a dominant role in this process, and (5) oxidative stress-independent induction of GRP78 counterbalances the proapoptotic action of CHOP.     

人胃癌BGC-823细胞中去甲斑蝥素抑癌作用机理的蛋白质组学研究

去甲斑蝥素是我国自行研制的抗肿瘤药物,在临床上主要用于消化道肿瘤的治疗．实验表明,去甲斑蝥素可引起人胃癌BGC-823细胞发生 M期阻滞及细胞凋亡．进一步利用双向电泳和质谱技术,筛选出了去甲斑蝥素抑癌作用相关蛋白．研究显示,线粒体热休克蛋白CH60、线粒体ATP合酶d亚单位、内质网葡萄糖调节蛋白GRP78、线粒体Hsp70的辅助因子GRPE1、SH3L3以及染色质组装因子1小亚基RBBP4参与了去甲斑蝥素的抑癌作用．研究提示,去甲斑蝥素可能通过促进线粒体热休克蛋白及p53的表达进而激活caspase-3依赖的凋亡通路,并且去甲斑蝥素在引发内质网协迫之后,可通过抑制胞外信号调节激酶(extracellular signal regulated kinase, ERK)的活性促进肿瘤细胞的凋亡．进一步分析了去甲斑蝥素与线粒体ATP合酶抑制剂寡霉素A的联合用药对人胃癌细胞生长的影响,结果表明,联合用药的抑瘤效果比单独用药的抑瘤效果显著,提示去甲斑蝥素可能通过抑制线粒体ATP合酶功能抑制BGC-823生长．上述结果为优化去甲斑蝥素的联合用药方案提供了新线索．     

cFLIP downregulation is an early event required for endoplasmic reticulum stress-induced apoptosis in tumor cells

Protein misfolding or unfolding and the resulting endoplasmic reticulum (ER) stress frequently occur in highly proliferative tumors. How tumor cells escape cell death by apoptosis after chronic ER stress remains poorly understood. We have investigated in both two-dimensional (2D) cultures and multicellular tumor spheroids (MCTSs) the role of caspase-8 inhibitor cFLIP as a regulator of the balance between apoptosis and survival in colon cancer cells undergoing ER stress. We report that downregulation of cFLIP proteins levels is an early event upon treatment of 2D cultures of colon cancer cells with ER stress inducers, preceding TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) upregulation, caspase-8 activation, and apoptosis. Maintaining high cFLIP levels during ER stress by ectopic expression of cFLIP markedly inhibits ER stress-induced caspase-8 activation and apoptosis. Conversely, cFLIP knockdown by RNA interference significantly accelerates caspase-8 activation and apoptosis upon ER stress. Despite activation of the proapoptotic PERK branch of the unfolded protein response (UPR) and upregulation of TRAIL-R2, MCTSs are markedly more resistant to ER stress than 2D cultures of tumor cells. Resistance of MCTSs to ER stress-induced apoptosis correlates with sustained cFLIP_L expression. Interestingly, resistance to ER stress-induced apoptosis is abolished in MCTSs generated from cFLIP_L knockdown tumor cells. Overall, our results suggest that controlling cFLIP levels in tumors is an adaptive strategy to prevent tumor cell’s demise in the unfavorable conditions of the tumor microenvironment.Subject terms: Cancer microenvironment, Apoptosis     

Involvement of caspase-2 and caspase-9 in endoplasmic reticulum stress-induced apoptosis: a role for the IAPs

Dysregulation of apoptosis is involved in a wide spectrum of disease ranging from proliferative to degenerative disorders. An emerging area of study in apoptosis is the critical contribution of the endoplasmic reticulum (ER) in both mitochondrial and ER specific apoptosis pathways. Here we show that brefeldin A and tunicamycin-mediated ER stress lead to caspase-dependent apoptosis involving caspase-2. Confocal microscopy and subcellular fractionation indicate that caspase-2 is localized to the ER, and following ER stress, the processing of caspase-2 and -9 is an early event preceding the activation of caspase-3 and -7 and the cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP). Inhibition and silencing of either caspase-2 or caspase-9 suppress ER stress-induced apoptosis, as demonstrated by annexin V binding. Similarly, transduction with an adenovirus encoding either Inhibitors of Apoptosis (IAP) protein HIAP1/c-IAP2 or HIAP2/c-IAP1 also suppresses ER stress-induced apoptosis. However, among HIAP1, HIAP2 and XIAP, only HIAP2 binds and inhibits caspase-2. Our results thus indicate a novel mechanism by which HIAP2 can regulate ER-initiated apoptosis by modulating the activity of caspase-2.     

Down-regulation of glucose-regulated protein (GRP) 78 potentiates cytotoxic effect of celecoxib in human urothelial carcinoma cells

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor that has been reported to elicit anti-proliferative response in various tumors. In this study, we aim to investigate the antitumor effect of celecoxib on urothelial carcinoma (UC) cells and the role endoplasmic reticulum (ER) stress plays in celecoxib-induced cytotoxicity. The cytotoxic effects were measured by MTT assay and flow cytometry. The cell cycle progression and ER stress-associated molecules were examined by Western blot and flow cytometry. Moreover, the cytotoxic effects of celecoxib combined with glucose-regulated protein (GRP) 78 knockdown (siRNA), (-)-epigallocatechin gallate (EGCG) or MG132 were assessed. We demonstrated that celecoxib markedly reduces the cell viability and causes apoptosis in human UC cells through cell cycle G1 arrest. Celecoxib possessed the ability to activate ER stress-related chaperones (IRE-1α and GRP78), caspase-4, and CCAAT/enhancer binding protein homologous protein (CHOP), which were involved in UC cell apoptosis. Down-regulation of GRP78 by siRNA, co-treatment with EGCG (a GRP78 inhibitor) or with MG132 (a proteasome inhibitor) could enhance celecoxib-induced apoptosis. We concluded that celecoxib induces cell cycle G1 arrest, ER stress, and eventually apoptosis in human UC cells. The down-regulation of ER chaperone GRP78 by siRNA, EGCG, or proteosome inhibitor potentiated the cytotoxicity of celecoxib in UC cells. These findings provide a new treatment strategy against UC.     

Mdivi-1 Protects Epileptic Hippocampal Neurons from Apoptosis via Inhibiting Oxidative Stress and Endoplasmic Reticulum Stress in Vitro

Mitochondrial division inhibitor 1 (mdivi-1), a selective inhibitor of the mitochondrial fission protein dynamin-related protein 1, has been proposed to have a neuroprotective effect on hippocampal neurons in animal models of epilepsy. However, the effect of mdivi-1 on epileptic neuronal death in vitro remains unknown. Therefore, we investigated the effect of mdivi-1 and the underlying mechanisms in the hippocampal neuronal culture (HNC) model of acquired epilepsy (AE) in vitro. We found that mitochondrial fission was increased in the HNC model of AE and inhibition of mitochondrial fission by mdivi-1 significantly decreased neuronal apoptosis induced by AE. In addition, mdivi-1 pretreatment significantly attenuated oxidative stress induced by AE characterized by decrease of reactive oxygen species (ROS) production and malondialdehyde level and by increase of superoxide dismutase activity. Moreover, mdivi-1 pretreatment significantly decreased endoplasmic reticulum (ER) stress markers glucose-regulated protein 78, C/EBP homologous protein expression and caspase-3 activation. Altogether, our findings suggest that mdivi-1 protected against AE-induced hippocampal neuronal apoptosis in vitro via decreasing ROS-mediated oxidative stress and ER stress.     

Involvement of dopamine receptors in binge methamphetamine-induced activation of endoplasmic reticulum and mitochondrial stress pathways

Single large doses of methamphetamine (METH) cause endoplasmic reticulum (ER) stress and mitochondrial dysfunctions in rodent striata. The dopamine D(1) receptor appears to be involved in these METH-mediated stresses. The purpose of this study was to investigate if dopamine D(1) and D(2) receptors are involved in ER and mitochondrial stresses caused by single-day METH binges in the rat striatum. Male Sprague-Dawley rats received 4 injections of 10 mg/kg of METH alone or in combination with a putative D(1) or D(2) receptor antagonist, SCH23390 or raclopride, respectively, given 30 min prior to each METH injection. Rats were euthanized at various timepoints afterwards. Striatal tissues were used in quantitative RT-PCR and western blot analyses. We found that binge METH injections caused increased expression of the pro-survival genes, BiP/GRP-78 and P58(IPK), in a SCH23390-sensitive manner. METH also caused up-regulation of ER-stress genes, Atf2, Atf3, Atf4, CHOP/Gadd153 and Gadd34. The expression of heat shock proteins (HSPs) was increased after METH injections. SCH23390 completely blocked induction in all analyzed ER stress-related proteins that included ATF3, ATF4, CHOP/Gadd153, HSPs and caspase-12. The dopamine D(2)-like antagonist, raclopride, exerted small to moderate inhibitory influence on some METH-induced changes in ER stress proteins. Importantly, METH caused decreases in the mitochondrial anti-apoptotic protein, Bcl-2, but increases in the pro-apoptotic proteins, Bax, Bad and cytochrome c, in a SCH23390-sensitive fashion. In contrast, raclopride provided only small inhibition of METH-induced changes in mitochondrial proteins. These findings indicate that METH-induced activation of striatal ER and mitochondrial stress pathways might be more related to activation of SCH23390-sensitive receptors.     

Control of ER stress by a chemical chaperone counteracts apoptotic signals in IFN-γ-treated murine hepatocytes

Apoptosis of hepatocytes plays a key role in the pathogenesis of immune-mediated hepatitis. However, the detailed mechanisms of apoptotic signaling remain unclear. In this study, we investigated the involvement of ER stress in a model of IFN-γ-induced apoptosis of hepatocytes in vitro, using a chemical chaperone reagent, glycerol. IFN-γ-induced apoptotic events (mitochondrial release of cytochrome c, enzymatic activation of caspase-3 and -9) were markedly inhibited by glycerol. Glycerol induced partial inhibition of cytotoxicity indicated by lactate dehydrogenase release from the cytosol but had no inhibitory effect on the induction of IRF-1 gene expression and reactive oxygen species, required for hepatocyte apoptosis by IFN-γ. Induction of caspase-4 and -12 gene expression, positively correlated with ER stress, was attenuated by glycerol. Gene analysis revealed that induction of ER stress-related genes, C/EBP homologue protein (CHOP/GADD153) and TRB3, was suppressed completely by glycerol treatment. These results suggest that ER stress plays a crucial role in mediating apoptosis of hepatocytes induced by IFN-γ, and a chemical chaperone is an effective inhibitor of the ER stress.     

HSP105 interacts with GRP78 and GSK3 and promotes ER stress-induced caspase-3 activation

Stress of the endoplasmic reticulum (ER stress) is caused by the accumulation of misfolded proteins, which occurs in many neurodegenerative diseases. ER stress can lead to adaptive responses or apoptosis, both of which follow activation of the unfolded protein response (UPR). Heat shock proteins (HSP) support the folding and function of many proteins, and are important components of the ER stress response, but little is known about the role of one of the major large HSPs, HSP105. We identified several new partners of HSP105, including glycogen synthase kinase-3 (GSK3), a promoter of ER stress-induced apoptosis, and GRP78, a key component of the UPR. Knockdown of HSP105 did not alter UPR signaling after ER stress, but blocked caspase-3 activation after ER stress. In contrast, caspase-3 activation induced by genotoxic stress was unaffected by knockdown of HSP105, suggesting ER stress-specificity in the apoptotic action of HSP105. However, knockdown of HSP105 did not alter cell survival after ER stress, but instead diverted signaling to a caspase-3-independent cell death pathway, indicating that HSP105 is necessary for apoptotic signaling after UPR activation by ER stress. Thus, HSP105 appears to chaperone the responses to ER stress through its interactions with GRP78 and GSK3, and without HSP105 cell death following ER stress proceeds by a non-caspase-3-dependent process.     

TRAIL-induced apoptosis of human melanoma cells involves activation of caspase-4

Although it is conventionally regarded as an inflammatory caspase, recent studies have shown that caspase-4 plays a role in induction of apoptosis by endoplasmic reticulum (ER) stress. We report here that activation of caspase-4 is also involved in induction of apoptosis by TNF-related apoptosis-inducing ligand (TRAIL) in human melanoma cells. Treatment with TRAIL resulted in activation of caspase-4. This appeared to be mediated by caspase-3, in that caspase-4 was activated later than caspase-8, -9, and -3, and that inhibition of caspase-3 blocked TRAIL-induced caspase-4 activation. Notably, TRAIL triggered ER stress in melanoma cells as shown by up-regulation of the GRP78 protein and the spliced form of XBP-1 mRNA. This seemed to be necessary for activation of caspase-4, as activation of caspase-3 by agents that did not trigger ER stress did not cause activation of caspase-4. Importantly, inhibition of caspase-4 also partially blocked caspase-3 activation, suggesting that activation of caspase-4 may be positive feed-back mechanism to further enhance caspase-3 activation. Collectively, these results show that activation of caspase-4 contributes to TRAIL-induced apoptosis and is associated with induction of ER stress by TRAIL in melanoma cells, and may have important implications for improving therapeutic efficacies of TRAIL in melanoma.     

Caspase-2 cleavage of BID is a critical apoptotic signal downstream of endoplasmic reticulum stress

The accumulation of misfolded proteins stresses the endoplasmic reticulum (ER) and triggers cell death through activation of the multidomain proapoptotic BCL-2 proteins BAX and BAK at the outer mitochondrial membrane. The signaling events that connect ER stress with the mitochondrial apoptotic machinery remain unclear, despite evidence that deregulation of this pathway contributes to cell loss in many human degenerative diseases. In order to "trap" and identify the apoptotic signals upstream of mitochondrial permeabilization, we challenged Bax-/- Bak-/- mouse embryonic fibroblasts with pharmacological inducers of ER stress. We found that ER stress induces proteolytic activation of the BH3-only protein BID as a critical apoptotic switch. Moreover, we identified caspase-2 as the premitochondrial protease that cleaves BID in response to ER stress and showed that resistance to ER stress-induced apoptosis can be conferred by inhibiting caspase-2 activity. Our work defines a novel signaling pathway that couples the ER and mitochondria and establishes a principal apoptotic effector downstream of ER stress.     

GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis

The chaperone glucose-regulated protein, 78/immunoglobulin binding protein (GRP78/Bip), protects cells from cytotoxicity induced by DNA damage or endoplasmic reticulum (ER) stress. In this study, we showed that GRP78 is a major inducible protein in human non-small cell lung cancer H460 cells treated with ER stress inducers, including A23187 and thapsigargin. AEBSF, an inhibitor of serine protease, diminished GRP78 induction, enhanced mitochondrial permeability, and augmented apoptosis in H460 cells during ER stress. Simultaneously, AEBSF promoted Raf-1 degradation and suppressed phosphorylation of Raf-1 at Ser338 and/or Tyr340 during ER stress. Coimmunoprecipitation assays and subcellular fractionations showed that GRP78 associated and colocalized with Raf-1 on the outer membrane of mitochondria, respectively. While treatment of cells with ER stress inducers inactivated BAD by phosphorylation at Ser75, a Raf-1 phosphorylation site; AEBSF attenuated phosphorylation of BAD, leading to cytochrome c release from mitochondria. Additionally, overexpression of GRP78 and/or Raf-1 protected cells from ER stress-induced apoptosis. Taken together, our results indicate that GRP78 may stabilize Raf-1 to maintain mitochondrial permeability and thus protect cells from ER stress-induced apoptosis.