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.     

Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics

Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined. Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation. Finally, we highlight how dysregulation of ER-mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders.     

Chemical chaperones reduce ionizing radiation-induced endoplasmic reticulum stress and cell death in IEC-6 cells

Radiotherapy, which is one of the most effective approaches to the treatment of various cancers, plays an important role in malignant cell eradication in the pelvic area and abdomen. However, it also generates some degree of intestinal injury. Apoptosis in the intestinal epithelium is the primary pathological factor that initiates radiation-induced intestinal injury, but the mechanism by which ionizing radiation (IR) induces apoptosis in the intestinal epithelium is not clearly understood. Recently, IR has been shown to induce endoplasmic reticulum (ER) stress, thereby activating the unfolded protein response (UPR) signaling pathway in intestinal epithelial cells. However, the consequences of the IR-induced activation of the UPR signaling pathway on radiosensitivity in intestinal epithelial cells remain to be determined. In this study, we investigated the role of ER stress responses in IR-induced intestinal epithelial cell death. We show that chemical ER stress inducers, such as tunicamycin or thapsigargin, enhanced IR-induced caspase 3 activation and DNA fragmentation in intestinal epithelial cells. Knockdown of Xbp1 or Atf6 with small interfering RNA inhibited IR-induced caspase 3 activation. Treatment with chemical chaperones prevented ER stress and subsequent apoptosis in IR-exposed intestinal epithelial cells. Our results suggest a pro-apoptotic role of ER stress in IR-exposed intestinal epithelial cells. Furthermore, inhibiting ER stress may be an effective strategy to prevent IR-induced intestinal injury.     

Endoplasmic reticulum stress associated responses in cancer

The endoplasmic reticulum (ER) is responsible for many housekeeping functions within the cell and is an important site for pathways that regulates its state of homeostasis. When cellular states perturb ER functions, a phenomenon termed “ER stress” activates a number of pathways to counteract the associated damages; these pathways are together called the unfolded protein response (UPR). The UPR has a dualistic function; it exists to alleviate damage associated with ER stress, however, if this is not possible, then it signals for cell death through apoptosis. Cancer cells are shown to be very resilient under extreme environmental stress and an increasing number of studies have indicated that this may be largely due to an altered state of the UPR. The role of ER stress and the UPR in cancer is still not clear, however many components are involved and may prove to be promising targets in future anti-cancer therapy. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Effect of Liraglutide on endoplasmic reticulum stress in diabetes

Endoplasmic reticulum (ER) stress is associated with the development of diabetes. The present study sought to investigate the effect of Liraglutide, a glucagon like peptide 1 analogue, on ER stress in β-cells. We found that Liraglutide protected the pancreatic INS-1 cells from thapsigargin-induced ER stress and the ER stress associated cell apoptosis, mainly by suppressing the PERK and IRE1 pathways. We further tested the effects of Liraglutide in the Akita mouse, an ER-stress induced type 1 diabetes model. After administration of Liraglutide for 8 weeks, p-eIF2α and p-JNK were significantly decreased in the pancreas of the Akita mouse, while the treatment showed no significant impact on the levels of insulin of INS-cells. Taken together, our findings suggest that Liraglutide may protect pancreatic cells from ER stress and its related cell death.     

Advanced glycation end products suppress osteoblastic differentiation of stromal cells by activating endoplasmic reticulum stress

Advanced glycation end products (AGEs) are involved in bone quality deterioration in diabetes mellitus. We previously showed that AGE2 or AGE3 inhibited osteoblastic differentiation and mineralization of mouse stromal ST2 cells, and also induced apoptosis and decreased cell growth. Although quality management for synthesized proteins in endoplasmic reticulum (ER) is crucial for the maturation of osteoblasts, the effects of AGEs on ER stress in osteoblast lineage are unknown. We thus examined roles of ER stress in AGE2- or AGE3-induced suppression of osteoblastogenesis of ST2 cells. An ER stress inducer, thapsigargin (TG), induced osteoblastic differentiation of ST2 cells by increasing the levels of Osterix, type 1 collagen (Col1), alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA. AGE2 or AGE3 suppressed the levels of ER stress sensors such as IRE1α, ATF6 and OASIS, while they increased the levels of PERK and its downstream molecules, ATF4. A reduction in PERK level by siRNA did not affect the AGEs-induced suppression of the levels of Osterix, Col1 and OCN mRNA. In conclusion, AGEs inhibited the osteoblastic differentiation of stromal cells by suppressing ER stress sensors and accumulating abnormal proteins in the cells. This process might accelerate AGEs-induced suppression of bone formation found in diabetes mellitus.     

Prednisolone-induced beta cell dysfunction is associated with impaired endoplasmic reticulum homeostasis in INS-1E cells

Glucocorticoids (GCs), such as prednisolone (PRED), are widely prescribed anti-inflammatory drugs, but their use may induce glucose intolerance and diabetes. GC-induced beta cell dysfunction contributes to these diabetogenic effects through mechanisms that remain to be elucidated. In this study, we hypothesized that activation of the unfolded protein response (UPR) following endoplasmic reticulum (ER) stress could be one of the underlying mechanisms involved in GC-induced beta cell dysfunction. We report here that PRED did not affect basal insulin release but time-dependently inhibited glucose-stimulated insulin secretion in INS-1E cells. PRED treatment also decreased both PDX1 and insulin expression, leading to a marked reduction in cellular insulin content. These PRED-induced detrimental effects were found to be prevented by prior treatment with the glucocorticoid receptor (GR) antagonist RU486 and associated with activation of two of the three branches of the UPR. Indeed, PRED induced a GR-mediated activation of both ATF6 and IRE1/XBP1 pathways but was found to reduce the phosphorylation of PERK and its downstream substrate eIF2α. These modulations of ER stress pathways were accompanied by upregulation of calpain 10 and increased cleaved caspase 3, indicating that long term exposure to PRED ultimately promotes apoptosis. Taken together, our data suggest that the inhibition of insulin biosynthesis by PRED in the insulin-secreting INS-1E cells results, at least in part, from a GR-mediated impairment in ER homeostasis which may lead to apoptotic cell death.     

Apelin alleviates diabetes-associated endoplasmic reticulum stress in the pancreas of Akita mice

Apelin, a newly identified bioactive adipokine, has been found to play important roles in multiple diseases, including diabetes, hypertension and cardiovascular diseases with unclear molecular mechanisms. The present study aimed to investigate the effects of apelin on endoplasmic reticulum (ER) stress in the pancreas of Akita mice, a well-established type 1 diabetic model. Apelin-13 (400 pmol/kg) was injected from tail vein for 10 weeks. The physiological characters of experimental animals were evaluated, pancreatic islet morphology and insulin content were assessed by immunohistochemistry, and ER stress markers in the pancreas were examined by Western blots. Our results indicate apelin treatment significantly ameliorates diabetes-induced reduction in pancreatic islet mass and insulin content. Further studies suggested apelin treatment alleviates ER stress by inhibiting the diabetes induced up-regulation of PERK and IRE1α and chaperones (GRP78, calnexin and Hsp70) levels in Akita mice. We also demonstrated that apelin treatment normalizes the diabetes induced alteration of AKT and ERK activations in the pancreas of Akita mice. Taken together, these results suggest a novel physiological role of apelin in alleviating ER stress in the pancreas of type 1 diabetes.     

Direct proteasome binding and subsequent degradation of unspliced XBP-1 prevent its intracellular aggregation

The non-canonical splicing of XBP-1 mRNA is a hallmark of the mammalian unfolded protein response (UPR). The proteasomal degradation of unspliced XBP-1 (XBP-1u) facilitates the termination of the UPR. Thus, understanding the mechanism of XBP-1u degradation may allow control over UPR duration and intensity.We show that XBP-1u interacts with purified 20S proteasomes through its unstructured C-terminus, which leads to its degradation in a manner that autonomously opens the proteasome gate. In living cells, the C-terminus of XBP-1u accumulates in aggresome structures in the presence of proteasome inhibitors. We propose that direct proteasomal degradation of XBP-1u prevents its intracellular aggregation.

Structured summary

MINT-7302217: XBP1-u (uniprotkb:P17861-1) binds (MI:0407) to Proteasome subunit alpha 7.2 (uniprotkb:O14818) by pull down (MI:0096)MINT-7302148: Vimentin (uniprotkb:P08670) and XBP1-u (uniprotkb:P17861-1) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7302163: XBP1-u (uniprotkb:P17861-1) binds (MI:0407) to Proteasome subunit alpha 5 (uniprotkb:P28066) by pull down (MI:0096)MINT-7302186: XBP1-u (uniprotkb:P17861-1) binds (MI:0407) to Proteasome subunit alpha 6 (uniprotkb:P60900) by pull down (MI:0096)     

Endoplasmic reticulum stress induces inverse regulations of major functions in portal myofibroblasts during liver fibrosis progression

Portal myofibroblasts (PMF) form a sub-population of highly proliferative and proangiogenic liver myofibroblasts that derive from portal mesenchymal progenitors. Endoplasmic reticulum (ER) stress was previously shown to modulate fibrogenesis, notably in the liver. Our aim was to determine if ER stress occurred in PMF and affected their functions. PMF were obtained after their expansion in vivo from bile duct-ligated (BDL) rats and referred to as BDL PMF. Compared to standard PMF obtained from normal rats, BDL PMF were more myofibroblastic, as assessed by higher alpha-smooth muscle actin expression and collagen 1 production. Their proangiogenic properties were also higher, whereas their proliferative and migratory capacities were lower. CHOP expression was detected in the liver of BDL rats, at the leading edge of portal fibrosis where PMF accumulate. BDL PMF displayed ER dilatation and an overexpression of the PERK pathway downstream targets, Chop, Gadd34 and Trb3, in comparison with standard PMF. In vitro, the induction of ER stress by tunicamycin in standard PMF, caused a decrease in their proliferative and migratory activity, and an increase in their proangiogenic activity, without affecting their myofibroblastic differentiation. Conversely, the treatment of BDL PMF with the PERK inhibitor GSK2656157 reduced ER stress, which caused a decrease in their angiogenic properties, and restored their proliferative and migratory capacity. In conclusion, PMF develop ER stress as they expand with the progression of fibrosis, which further increases their proangiogenic activity, but also inhibits their proliferation and migration. This phenotypic switch may restrict PMF expansion while they support angiogenesis.     

Neuropeptide Y mitigates ER stress–induced neuronal cell death by activating the PI3K–XBP1 pathway

The unfolded protein response (UPR) is an evolutionarily conserved adaptive reaction that increases cell survival under endoplasmic reticulum (ER) stress conditions. ER stress–associated neuronal cell death pathways play roles in the pathogenesis of neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and Huntington’s disease. Neuropeptide Y (NPY) has an important role in neuroprotection against neurodegenerative diseases. In this study, we investigated whether NPY has a protective role in ER stress–induced neuronal cell death in SK-N-SH human neuroblastoma cells. An ER stress–inducing chemical, tunicamycin, increased the activities of caspase-3 and -4, whereas pretreatment with NPY decreased caspase-3 and -4 activities during the ER stress response. In addition, NPY suppressed the activation of three major ER stress sensors during the tunicamycin-induced ER stress response. NPY-mediated activation of PI3K increased nuclear translocation of XBP1s, which in turn induced expression of Grp78/BiP. Taken together, our data indicated that NPY plays a protective role in ER stress–induced neuronal cell death through activation of the PI3K–XBP1 pathway, and that NPY signaling can serve as therapeutic target for ER stress–mediated neurodegenerative diseases.     

Endoplasmic reticulum stress response in yeast and humans

Stress pathways monitor intracellular systems and deploy a range of regulatory mechanisms in response to stress. One of the best-characterized pathways, the UPR (unfolded protein response), is an intracellular signal transduction pathway that monitors ER (endoplasmic reticulum) homoeostasis. Its activation is required to alleviate the effects of ER stress and is highly conserved from yeast to human. Although metazoans have three UPR outputs, yeast cells rely exclusively on the Ire1 (inositol-requiring enzyme-1) pathway, which is conserved in all Eukaryotes. In general, the UPR program activates hundreds of genes to alleviate ER stress but it can lead to apoptosis if the system fails to restore homoeostasis. In this review, we summarize the major advances in understanding the response to ER stress in Sc (Saccharomyces cerevisiae), Sp (Schizosaccharomyces pombe) and humans. The contribution of solved protein structures to a better understanding of the UPR pathway is discussed. Finally, we cover the interplay of ER stress in the development of diseases.     

The unfolded protein response to endoplasmic reticulum stress in cultured astrocytes and rat brain during experimental diabetes

Oxidative-nitrosative stress and inflammatory responses are associated with endoplasmic reticulum (ER) stress in diabetic retinopathy, raising the possibility that disturbances in ER protein processing may contribute to CNS dysfunction in diabetics. Upregulation of the unfolded protein response (UPR) is a homeostatic response to accumulation of abnormal proteins in the ER, and the present study tested the hypothesis that the UPR is upregulated in two models for diabetes, cultured astrocytes grown in 25 mmol/L glucose for up to 4 weeks and brain of streptozotocin (STZ)-treated rats with diabetes for 1–7 months. Markers associated with translational blockade (phospho-eIF2α and apoptosis (CHOP), inflammatory response (inducible nitric oxide synthase, iNOS), and nitrosative stress (nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase, GAPDH) were not detected in either model. Nrf2 was present in nuclei of low- and high-glucose cultures, consistent with oxidative stress. Astrocytic ATF4 expression was not altered by culture glucose concentration, whereas phospho-IRE and ATF6 levels were higher in low- compared with high-glucose cultures. The glucose-regulated chaperones, GRP78 and GRP94, were also expressed at higher levels in low- than high-glucose cultures, probably due to recurrent glucose depletion between feeding cycles. In STZ-rat cerebral cortex, ATF4 level was transiently reduced at 4 months, and p-IRE levels were transiently elevated at 3 months. However, GRP78 and GRP94 expression was not upregulated, and iNOS, amyloid-β, and nuclear accumulation of GAPDH were not evident in STZ-diabetic brain. High-glucose cultured astrocytes and STZ-diabetic brain are relatively resistant to diabetes-induced ER stress, in sharp contrast with cultured retinal Müller cells and diabetic rodent retina.     

Modulation of endoplasmic reticulum chaperone GRP78 by high glucose in hippocampus of streptozotocin-induced diabetic mice and C6 astrocytic cells

Diabetes mellitus is known to increase the risk of neurodegeneration, and both diseases are reported to be linked to dysfunction of endoplasmic reticulum (ER). Astrocytes are important in the defense mechanism of central nervous system (CNS), with great ability of tolerating accumulation of toxic substances and sensitivity in Ca²⁺ homeostasis which are two key functions of ER. Here, we investigated the modulation of the glucose-regulated protein 78 (GRP78) in streptozotocin (STZ)-induced diabetic mice and C6 cells cultured in high glucose condition. Our results showed that more reactive astrocytes were presented in the hippocampus of STZ-induced diabetic mice. Simultaneously, decrease of GRP78 expression was found in the astrocytes of diabetic mice hippocampus.