The unfolded protein response is shaped by the NMD pathway

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), an essential adaptive intracellular pathway that relieves the stress. Although the UPR is an evolutionarily conserved and beneficial pathway, its chronic activation contributes to the pathogenesis of a wide variety of human disorders. The fidelity of UPR activation must thus be tightly regulated to prevent inappropriate signaling. The nonsense-mediated RNA decay (NMD) pathway has long been known to function in RNA quality control, rapidly degrading aberrant mRNAs, and has been suggested to regulate subsets of normal mRNAs. Here, we report that the NMD pathway regulates the UPR. NMD increases the threshold for triggering the UPR in vitro and in vivo, thereby preventing UPR activation in response to normally innocuous levels of ER stress. NMD also promotes the timely termination of the UPR. We demonstrate that NMD directly targets the mRNAs encoding several UPR components, including the highly conserved UPR sensor, IRE1α, whose NMD-dependent degradation partly underpins this process. Our work not only sheds light on UPR regulation, but demonstrates the physiological relevance of NMD''s ability to regulate normal mRNAs.     

Degradation of the endoplasmic reticulum by autophagy in plants

Eukaryotic cells have developed sophisticated strategies to contend with environmental stresses faced in their lifetime. Endoplasmic reticulum (ER) stress occurs when the accumulation of unfolded proteins within the ER exceeds the folding capacity of ER chaperones. ER stress responses have been well characterized in animals and yeast, and autophagy has been suggested to play an important role in recovery from ER stress. In plants, the unfolded protein response signaling pathways have been studied, but changes in ER morphology and ER homeostasis during ER stress have not been analyzed previously. Autophagy has been reported to function in tolerance of several stress conditions in plants, including nutrient deprivation, salt and drought stresses, oxidative stress, and pathogen infection. However, whether autophagy also functions during ER stress has not been investigated. The goal of our study was to elucidate the role and regulation of autophagy during ER stress in Arabidopsis thaliana.     

The unfolded protein response in melanocytes: activation in response to chemical stressors of the endoplasmic reticulum and tyrosinase misfolding

Accumulation of proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), comprising three signaling pathways initiated by Ire1, Perk and Atf6 respectively. Unfolded protein response activation was compared in chemically stressed murine wildtype melanocytes and mutant melanocytes that retain tyrosinase in the ER. Thapsigargin, an ER stressor, activated all pathways in wildtype melanocytes, triggering Caspase 12-mediated apoptosis at toxic doses. Albino melanocytes expressing mutant tyrosinase showed evidence of ER stress with increased Ire1 expression, but the downstream effector, Xbp1, was not activated even following thapsigargin treatment. Attenuation of Ire1 signaling was recapitulated in wildtype melanocytes treated with thapsigargin for 8 days, with diminished Xbp1 activation observed after 4 days. Atf6 was also activated in albino melanocytes, with no response to thapsigargin, while the Perk pathway was not activated and thapsigargin treatment elicited robust expression of the downstream effector CCAAT-enhancer-binding protein homologous protein. Thus, melanocytes adapt to ER stress by attenuating two UPR pathways.     

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.     

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.     

Activation of the unfolded protein response in Parkinson's disease

Parkinson's disease (PD) is, at the neuropathological level, characterized by the accumulation of misfolded proteins. The presence of misfolded proteins can trigger a cellular stress response in the endoplasmic reticulum (ER) called the Unfolded Protein Response (UPR). The UPR has been shown to be involved in cellular models for PD. In this study, we investigated UPR activation in the substantia nigra of control and PD patients. Immunoreactivity for the UPR activation markers phosphorylated pancreatic ER kinase (pPERK) and phosphorylated eukaryotic initiation factor 2alpha (peIF2alpha) is detected in neuromelanin containing dopaminergic neurons in the substantia nigra of PD cases but not in control cases. In addition, pPERK immunoreactivity is colocalized with increased alpha-synuclein immunoreactivity in dopaminergic neurons. These data show that the UPR is activated in PD and that UPR activation is closely associated with the accumulation and aggregation of alpha-synuclein.     

Brain trauma induces X-box protein 1 processing indicative of activation of the endoplasmic reticulum unfolded protein response

Brain trauma was induced in mice using a closed head injury (CHI) model. At 1, 6 or 24 h after trauma, brains were dissected into the cortex, striatum and hippocampus. Changes in levels of processed X-box protein 1 (xbp1), glucose-regulated protein 78 (grp78), growth arrest and DNA damage-inducible gene 153 (gadd153) and heat-shock protein 70 (hsp70) mRNA, indicating impaired endoplasmic reticulum (ER) and cytoplasmic functioning, were evaluated by quantitative PCR. In the cortex, processed xbp1 mRNA levels rose to 2000% of control 1 h after CHI, and stayed high throughout the experiments. In the hippocampus and striatum, processed xbp1 mRNA levels rose in a delayed fashion, peaking at 6 h (1000% of control) and 24 h after CHI (1500% of control) respectively. Levels of grp78 mRNA were only slightly increased in the cortex 24 h after CHI (150% of control), and were unchanged or transiently decreased in the hippocampus and striatum. Levels of gadd153 mRNA did not change significantly after trauma. A transient rise in hsp70 mRNA levels was observed only in the cortex, peaking at 1 h after CHI (600% of control). Processing of xbp1 mRNA is a sign of activation of the unfolded protein response indicative of ER dysfunction. The results suggest that brain trauma induces ER dysfunction, which spreads from the ipsilateral cortex to the hippocampus and striatum. These observations may have clinical implications and should therefore be considered for future investigations on therapeutic intervention of brain injury caused by contusion-induced neurotrauma.     

Oscillation of mitogen-activated protein kinases in response to endoplasmic reticulum stress

Periodic patterns of biochemical systems determine the function, behavior, and fate of eukaryotic cells. The cascades of mitogen-activated protein (MAP) kinases play crucial roles in a diverse range of cell function, and recent reports indicate that oscillation of extracellular signal-regulated kinase (ERK) activity is observed following stimulation by some growth factors. In the current report, we provide evidence that ERK and c-Jun N-terminal kinase, but not p38 MAP kinase, are activated periodically in response to endoplasmic reticulum stress. When activity of MAP kinases is evaluated, their oscillatory property should be considered carefully, especially under stress conditions.     

Blocking of N-acetylglucosaminyltransferase V induces cellular endoplasmic reticulum stress in human hepatocarcinoma 7,721 cells

N-acetylglucosaminyltransferase V （GnT-V） is an important tumorigenesis and metastasis-associated enzyme. To study its biofunction, the GnT-V stably suppressed cell line （GnT-V-AS/7721） was constructed from 7721 hepatocarcinoma cells in previous study. In this study, cDNA array gene expression profiles were compared between GnT-V-AS/7721 and parental 7721 cells. The data indicated that GnT-V-AS/7721 showed a characteristic expression pattern consistent with the ER stress. The molecular mechanism of the ER stress was explored in GnT-V-AS/7721 by the analysis on key molecules in both two unfolded protein response （UPR） pathways. For ATF6 and Irel/XBP-1 pathway, it was evidenced by the up-regulation of BIP at mRNA and protein level, and the appearance of the spliced form ofXBP-1. As for PERK/eIF2α pathway, the activation of ER eIF2α kinase PERK was observed. To confirm the results from GriT-V-AS/7721 cells, the key molecules in the UPR were examined again in 7721 cells interfered with the GnT-V by the specific RNAi treatment. The results were similar with those from GnT-V-AS/7721, indicating that blocking of GnT-V can specifically activate ER stress in 7721 cells. Rate of 3H-Man incorporation corrected with rate of 3H-Leu incorporation in GnT-V-AS/7721 was down-regulated greatly compared with the control, which demonstrated the deficient function of the enzyme synthesizing N-glycans after GnT-V blocking. Moreover, the faster migrating form of chaperone GRP94 associated with the underglycosylation, and the extensively changed N-glycans structures of intracellular glycoproteins were also detected in GnT-V-AS/7721. These results supported the mechanism that blocking of GnT-V expression impaired functions of chaperones and N-glycan-synthesizing enzymes, which caused UPR in vivo.     

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.