ATF6-DGAT pathway is involved in TLR7-induced innate immune response in Ctenopharyngodon idellus kidney cells.

DGAT1 and DGAT2 are two acyl-CoA:diacylglycerol O-acyltransferase (DGAT) enzymes that catalyze the final step in triglyceride (TG) synthesis. TGs are the primary constituents of lipid droplets (LDs). Although it has been demonstrated that LDs modulate immune and inflammatory responses in CIK cells, little is known about whether DGAT1 and DGAT2 involve in this process. Firstly, grass carp DGAT2 was isolated and characterized, encoding 361 amino acids, and all DGAT2 proteins in genomic structures are conserved in vertebrates. Then, using TLR7 agonist, we induced LDs accumulation in CIK cells. Only DGAT1b and DGAT2 were upregulated in forming TLR7 agonist induced-LDs. Next, we utilized small-molecule inhibitors of DGAT1 and DGAT2. The results indicated that DGAT1 inactivation attenuated TG content and the relative expressions of IFNα3, NF-κB, IL-1ß, and TNFα genes, whereas DGAT2 inhibition decreased TG content and the relative expressions of MyD88, IRF7, IFNα3, NF-κB, IL-1ß, and TNFα genes, implying that DGAT1-generated LDs and DGAT2-generated LDs contribute to TLR7-induced immune response via different signaling pathways. Finally, inhibiting ATF6 effectively decreased DGAT-generated LDs accumulation and the expression of TLR7 signaling-related genes induced by TLR7 agonist, implying that ATF6 UPR pathway may mediate the role of DGAT-generated LDs in TLR7 signaling. Overall, we demonstrate that DGAT1 and DGAT2-catalyzed TAG synthesis may generate different LDs to provide distinct signaling platforms for innate TLR7 signaling.

ALA protects against ERS-mediated apoptosis in a cochlear cell model with low citrate synthase expression.

A/J mouse is a model of age-related hearing loss (AHL). Mutation in the citrate synthase (Cs) gene of the mouse plays an important role in the hearing loss and degeneration of cochlear cells. To investigate the pathogenesis of cochlear cell damage in A/J mice resulted from Cs mutation, we downregulated the expression level of CS in HEI-OC1, a cell line of mouse cochlea, by shRNA. The results showed that low CS expression led to low ability of cell proliferation. Further study revealed an increase level of reactive oxygen species (ROS), activation of ATF6 mediated endoplasmic reticulum stress (ERS) and high expression levels of caspase12 and Bax in the cells. Moreover, the AEBSF, an ATF6 inhibitor, could reduce the expression levels of caspase-12 and Bax by inhibiting the hydrolysis of ATF6 in the cells. Finally, antioxidant alpha-lipoic acid (ALA) reduced the ROS levels and the apoptotic signals in the cell model with low CS expression. We therefore conclude that the ERS mediated apoptosis, which is triggered by ROS, may be involved in the cell degeneration in the cochleae of A/J mice.

Ceapins block the unfolded protein response sensor ATF6α by inducing a neomorphic inter-organelle tether.

The unfolded protein response (UPR) detects and restores deficits in the endoplasmic reticulum (ER) protein folding capacity. Ceapins specifically inhibit the UPR sensor ATF6α, an ER-tethered transcription factor, by retaining it at the ER through an unknown mechanism. Our genome-wide CRISPR interference (CRISPRi) screen reveals that Ceapins function is completely dependent on the ABCD3 peroxisomal transporter. Proteomics studies establish that ABCD3 physically associates with ER-resident ATF6α in cells and in vitro in a Ceapin-dependent manner. Ceapins induce the neomorphic association of ER and peroxisomes by directly tethering the cytosolic domain of ATF6α to ABCD3's transmembrane regions without inhibiting or depending on ABCD3 transporter activity. Thus, our studies reveal that Ceapins function by chemical-induced misdirection which explains their remarkable specificity and opens up new mechanistic routes for drug development and synthetic biology.

Targeting ER stress/ER stress response in myopathies.

There is more skeletal muscle tissue in the body than any other tissue and, as it is the organ of the majority of metabolic activity, muscle defect can affect the health of the entire body. Endoplasmic reticulum (ER) stress due to defects in protein folding/degradation balance, altered calcium and lipid levels and alterations in ER-mitochondria contacts has recently been recognised as the pathogenic cause of many different myopathies. In addition, a maladaptive ER stress response triggered by ER stress and mediated by three ER stress sensors (PERK, IRE1 and ATF6) is involved in a failure to relieve muscle tissue from this stress. Targeting ER stress and the ER stress response pathway offers a broad range of opportunities for treating myopathies but, as the inhibition of the three ER stress sensors may not be safe because it could lead to unexpected effects; it therefore calls for careful analysis of the changes in downstream signal transduction in the different myopathies so these sub-pathways can be pharmacologically targeted. This review summarises the known inhibitors of the ER stress response and the successful results obtained using some of them in mouse models of muscle diseases caused by ER stress/ER stress response.

The metastasis suppressor, NDRG1, differentially modulates the endoplasmic reticulum stress response.

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), is a stress response protein that is involved in the inhibition of multiple oncogenic signaling pathways. Initial studies have linked NDRG1 and the endoplasmic reticulum (ER) stress response. Considering this, we extensively examined the mechanism by which NDRG1 regulates the ER stress response in pancreatic and colon cancer cells. We also examined the anti-cancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), which induces NDRG1 expression and causes ER stress. The expression of NDRG1 was demonstrated to regulate the three main arms of the ER stress response by: (1) increasing the expression of three major ER chaperones, binding immunoglobulin protein (BiP), calreticulin, and calnexin; (2) suppressing the protein kinase, RNA-activated (PKR)-like ER kinase (PERK); (3) inhibiting the inositol-requiring kinase 1α (IRE1α) arm; and (4) increasing the cleavage of activating transcription factor 6 (ATF6). An important finding was that NDRG1 enhances the anti-proliferative and anti-migratory activity of Dp44mT. This increased efficacy could be related to the following effects in the presence of Dp44mT and NDRG1, namely: markedly increased activation of the PERK target, eukaryotic translation initiation factor 2α (eIF2α); the maintenance of activating transcription factor 4 (ATF4) expression; high cytosolic Ca+2 that increases the sensitivity of cells to apoptosis via activation of the calmodulin-dependent kinase II (CaMKII) signaling cascade; and increased pro-apoptotic C/EBP-homologous protein (CHOP) expression. Collectively, this investigation dissects the molecular mechanisms through which NDRG1 manipulates the ER stress response and its ability to potentiate the activity of the potent anti-cancer agent, Dp44mT.

ATF6α Activation Enhances Survival against Chemotherapy and Serves as a Prognostic Indicator in Osteosarcoma.

Patients with metastatic or relapsed/refractory osteosarcoma (OS) have a 5-year survival rate of <30%. This has remained unchanged over several decades. One of the factors contributing to lack of improvement in survival is the development of chemoresistance. Hence, elucidating and targeting the mechanisms that promote survival against chemotherapy and lead to chemoresistance is pivotal to improving outcomes for these patients. We identified that endoplasmic reticulum (ER) stress-activated transcription factor, ATF6α, is essential for the survival of OS cells against chemotherapy induced cell death. ATF6α cleavage and activity were enhanced in OS cells compared to normal osteoblasts and knockdown of ATF6α expression enhanced sensitivity of OS cells against chemotherapy induced cell death. This was in part due to increased Bax activation. Pharmacologic inhibition or knock-down of downstream targets of ATF6α, protein disulfide isomerases (PDI) and ERO1ß, a thiol oxidase that is involved in the re-oxidation of PDIs also independently induced pronounced killing of OS cells following chemotherapy. Analysis of primary tumors from OS patients reveals that patients with high levels of nuclear ATF6α: (1) also had increased expression of its downstream targets the chaperone BiP and enzyme PDI, (2) had a significant likelihood of developing metastasis at diagnosis, (3) had significantly poorer overall and progression free survival, and (4) had poorer response to chemotherapy. These findings suggest that targeting survival signaling by the ATF6α pathway in OS cells may favor eradication of refractory OS tumor cells and ATF6α could be a useful predictor for chemo-responsiveness and prognosis.

Pharmacologic inhibition of S1P attenuates ATF6 expression, causes ER stress and contributes to apoptotic cell death.

Mammalian cells express unique transcription factors embedded in the endoplasmic reticulum (ER) membrane, such as the sterol regulatory element-binding proteins (SREBPs), that promote de novo lipogenesis. Upon their release from the ER, the SREBPs require proteolytic activation in the Golgi by site-1-protease (S1P). As such, inhibition of S1P, using compounds such as PF-429242 (PF), reduces cholesterol synthesis and may represent a new strategy for the management of dyslipidemia. In addition to the SREBPs, the unfolded protein response (UPR) transducer, known as the activating transcription factor 6 (ATF6), is another ER membrane-bound transcription factor that requires S1P-mediated activation. ATF6 regulates ER protein folding capacity by promoting the expression of ER chaperones such as the 78-kDa glucose-regulated protein (GRP78). ER-resident chaperones like GRP78 prevent and/or resolve ER polypeptide accumulation and subsequent ER stress-induced UPR activation by folding nascent polypeptides. Here we report that pharmacological inhibition of S1P reduced the expression of ATF6 and GRP78 and induced the activation of UPR transducers inositol-requiring enzyme-1α (IRE1α) and protein kinase RNA-like ER kinase (PERK). As a consequence, S1P inhibition also increased the susceptibility of cells to ER stress-induced cell death. Our findings suggest that S1P plays a crucial role in the regulation of ER folding capacity and also identifies a compensatory cross-talk between UPR transducers in order to maintain adequate ER chaperone expression and activity.

miR-103/107 promote ER stress-mediated apoptosis via targeting the Wnt3a/ß-catenin/ATF6 pathway in preadipocytes.

Both miR-103 and miR-107 have been demonstrated to restrain cell proliferation and regulate lipid metabolism and inflammation. However, the effects of miR-103/107 on preadipocyte apoptosis remain unknown. In the present research, we have investigated how miR-103/107 regulated preadipocyte apoptosis. We found that miR-103/107 aggravated endoplasmic reticulum (ER) stress-mediated apoptosis in preadipocytes. We confirmed that miR-103/107 targeted WNT family member 3a (Wnt3a) in preadipocytes. It was found that overexpressing Wnt3a resulted in suppression of ER stress-mediated apoptosis, while restoration of miR-103/107 counteracted the effects of Wnt3a in preadipocytes. Moreover, bioinformatics and luciferase assays indicated that activating transcription factor (ATF)6 is a key player linking miR-103/107-induced ER stress to apoptosis. ATF6 is regulated by lymphoid enhancer-binding factor 1, a transcription factor downstream of the Wnt3a/ß-catenin signaling pathway, and ATF6 binds to the B-cell lymphoma 2 (Bcl2) promoter to regulate apoptosis further. In conclusion, miR-103/107 promoted ER stress-mediated apoptosis by targeting the Wnt3a/ß-catenin/ATF6 signaling pathway in preadipocytes. This study revealed that the miR-103/107-Wnt3a/ß-catenin-ATF6 pathway is critical to the progression of apoptosis in preadipocytes, which suggested that approaches to activate miR-103/107 could potentially be useful as new therapies for treating obesity and metabolic syndrome-related disorders.

Melatonin, a novel selective ATF-6 inhibitor, induces human hepatoma cell apoptosis through COX-2 downregulation.

AIM: To clarify the mechanisms involved in the critical endoplasmic reticulum (ER) stress initiating unfolded protein response pathway modified by melatonin. METHODS: Hepatoma cells, HepG2, were cultured in vitro. Flow cytometry and TUNEL assay were used to measure HepG2 cell apoptosis. Western blotting and quantitative reverse transcription-polymerase chain reaction methods were used to determine the protein and messenger RNA levels of ER stress and apoptosis related genes' expression, respectively. Tissue microarray construction from patients was verified by immunohistochemical analysis. RESULTS: In the present study, we first identified that melatonin selectively blocked activating transcription factor 6 (ATF-6) and then inhibited cyclooxygenase-2 (COX-2) expression, leading to enhanced liver cancer cell apoptosis under ER stress condition. Dramatically increased CCAAT-enhancer-binding protein homologous protein level, suppressed COX-2 and decreased Bcl-2/Bax ratio by melatonin or ATF-6 siRNA contributed the enhanced HepG2 cell apoptosis under tunicamycin (an ER stress inducer) stimulation. In clinical hepatocellular carcinoma patients, the close relationship between ATF-6 and COX-2 was further confirmed. CONCLUSION: These findings indicate that melatonin as a novel selective ATF-6 inhibitor can sensitize human hepatoma cells to ER stress inducing apoptosis.

Sublethal endoplasmic reticulum stress caused by the mutation of immunoglobulin heavy chain-binding protein induces the synthesis of a mitochondrial protein, pyrroline-5-carboxylate reductase 1.

Most human neurodegenerative diseases are sporadic and appear later in life. Aging and neurodegeneration are closely associated, and recent investigations reveal that endoplasmic reticulum (ER) stress is involved in the progression of these features. Immunoglobulin heavy chain-binding protein (BiP) is an ER chaperone that is central to ER functions. We produced knock-in mice expressing a mutant BiP that lacked the retrieval sequence to elucidate the effect of a functional defect in an ER chaperone in multicellular organisms. The homozygous mutant BiP mice died within several hours after birth because of respiratory failure with an impaired biosynthesis of pulmonary surfactant by alveolar type II cells. The heterozygous mutant BiP mice grew up to be apparently normal adults, although some of them revealed motor disabilities as they aged. Here, we report that the synthesis of a mitochondrial protein, pyrroline-5-carboxylate reductase 1 (PYCR1), is enhanced in the brains of homozygous mutant BiP mice. We performed a two-dimensional gel analysis followed by liquid chromatography-tandem mass spectrometry. PYCR1 was identified as one of the enhanced proteins. We also found that sublethal ER stress caused by tunicamycin treatment induced the synthesis of PYCR1 in murine fibroblasts. PYCR1 has been shown to be related to the aging process. Mutations in the PYCR1 gene cause cutis laxa with progeroid features and mental retardation. These findings suggest a pathophysiological interaction between ER stress and a mitochondrial function in aging.

Ceapins are a new class of unfolded protein response inhibitors, selectively targeting the ATF6α branch.

The membrane-bound transcription factor ATF6α plays a cytoprotective role in the unfolded protein response (UPR), required for cells to survive ER stress. Activation of ATF6α promotes cell survival in cancer models. We used cell-based screens to discover and develop Ceapins, a class of pyrazole amides, that block ATF6α signaling in response to ER stress. Ceapins sensitize cells to ER stress without impacting viability of unstressed cells. Ceapins are highly specific inhibitors of ATF6α signaling, not affecting signaling through the other branches of the UPR, or proteolytic processing of its close homolog ATF6ß or SREBP (a cholesterol-regulated transcription factor), both activated by the same proteases. Ceapins are first-in-class inhibitors that can be used to explore both the mechanism of activation of ATF6α and its role in pathological settings. The discovery of Ceapins now enables pharmacological modulation all three UPR branches either singly or in combination.

Ceapins inhibit ATF6α signaling by selectively preventing transport of ATF6α to the Golgi apparatus during ER stress.

The membrane-bound transcription factor ATF6α is activated by proteolysis during endoplasmic reticulum (ER) stress. ATF6α target genes encode foldases, chaperones, and lipid biosynthesis enzymes that increase protein-folding capacity in response to demand. The off-state of ATF6α is maintained by its spatial separation in the ER from Golgi-resident proteases that activate it. ER stress induces trafficking of ATF6α. We discovered Ceapins, a class of pyrazole amides, as selective inhibitors of ATF6α signaling that do not inhibit the Golgi proteases or other UPR branches. We show that Ceapins block ATF6α signaling by trapping it in ER-resident foci that are excluded from ER exit sites. Removing the requirement for trafficking by pharmacological elimination of the spatial separation of the ER and Golgi apparatus restored cleavage of ATF6α in the presence of Ceapins. Washout of Ceapins resensitized ATF6α to ER stress. These results suggest that trafficking of ATF6α is regulated by its oligomeric state.

Induction of Endoplasmic Reticulum Stress via Reactive Oxygen Species Mediated by Luteolin in Melanoma Cells.

BACKGROUND: This study aimed to investigate whether luteolin, a flavonoid, induces apoptosis in human melanoma cells via endoplasmic reticulum (ER) stress. MATERIALS AND METHODS: To investigate the effects of luteolin in human melanoma cells, the anti-proliferation, apoptosis, ER stress induction and reactive oxygen species (ROS) generation were evaluated using MTT, Hoechst 33342, ER-tracker Blue White DPX and DCF-DA staining assays, respectively. RESULTS: Luteolin inhibited cell proliferation and increased apoptotic body formation. Luteolin induced ER stress, as shown by ER staining and mitochondrial Ca(2+) overloading. Luteolin increased expression of the ER stress-related proteins; protein kinase RNA-like ER kinase, phospho eukaryotic translation initiation factor 2α, activating transcription factor (ATF) 6, CCAAT/enhancer-binding protein-homologous protein (CHOP), and cleaved caspase 12. Furthermore, luteolin increased the level of intracellular ROS, leading to ROS-mediated apoptosis and ER stress. However, N-acetyl cysteine, a ROS scavenger, decreased ROS levels, apoptosis, and ER stress induced by luteolin treatment. In addition, knockdown of CHOP and ATF6 by small-interfering RNA inhibited luteolin-induced cell death. CONCLUSION: Luteolin induces apoptosis by ER stress via increasing ROS levels.

High-content screening identifies inhibitors of the nuclear translocation of ATF6.

Activating transcription factor 6 (ATF6) is a transmembrane protein that consists of a cytoplasmic domain and an endoplasmic reticulum (ER) luminal domain. As unfolded protein levels arise in the ER, the ER cytoplasmic domain of ATF6 moves to the nucleus, where it activates the transcription of a range of genes, including those involved in apoptosis. As ATF6 only becomes functional once it has moved to the nucleus, compounds that inhibit its re-localization are of therapeutic interest. The aim of the present study was to rapidly and accurately identify such compounds using a novel image­based, high­content screening (HCS) technique. The results from the HCS analysis were then confirmed by luciferase reporter assays, western blot analysis and the measurement of cell viability. We found that HCS identified compounds which inhibited ATF6 nuclear translocation with high specificity, as confirmed by the luciferase reporter assay and western blot analysis. Moreover, we demonstrated that 3 of the 80 identified compounds impaired ATF6-mediated induced cell death. The data from this study support the theory that HCS is a novel, high throughput method which can be used for accurate and rapid compound screening.

Insulin demand regulates ß cell number via the unfolded protein response.

Although stem cell populations mediate regeneration of rapid turnover tissues, such as skin, blood, and gut, a stem cell reservoir has not been identified for some slower turnover tissues, such as the pancreatic islet. Despite lacking identifiable stem cells, murine pancreatic ß cell number expands in response to an increase in insulin demand. Lineage tracing shows that new ß cells are generated from proliferation of mature, differentiated ß cells; however, the mechanism by which these mature cells sense systemic insulin demand and initiate a proliferative response remains unknown. Here, we identified the ß cell unfolded protein response (UPR), which senses insulin production, as a regulator of ß cell proliferation. Using genetic and physiologic models, we determined that among the population of ß cells, those with an active UPR are more likely to proliferate. Moreover, subthreshold endoplasmic reticulum stress (ER stress) drove insulin demand-induced ß cell proliferation, through activation of ATF6. We also confirmed that the UPR regulates proliferation of human ß cells, suggesting that therapeutic UPR modulation has potential to expand ß cell mass in people at risk for diabetes. Together, this work defines a stem cell-independent model of tissue homeostasis, in which differentiated secretory cells use the UPR sensor to adapt organ size to meet demand.

Endoplasmic reticulum stress impairment in the spinal dorsal horn of a neuropathic pain model.

Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, but its role in neuropathic pain remains unclear. In this study, we examined the ER stress and the unfolded protein response (UPR) activation in a L5 spinal nerve ligation (SNL)-induced rat neuropathic pain model. SNL-induced neuropathic pain was assessed behaviorally using the CatWalk system, and histologically with microglial activation in the dorsal spinal horn. L5 SNL induced BIP upregulation in the neuron of superficial laminae of dorsal spinal horn. It also increased the level of ATF6 and intracellular localization into the nuclei in the neurons. Moreover, spliced XBP1 was also markedly elevated in the ipsilateral spinal dorsal horn. The PERK-elF2 pathway was activated in astrocytes of the spinal dorsal horn in the SNL model. In addition, electron microscopy revealed the presence of swollen cisternae in the dorsal spinal cord after SNL. Additionally, inhibition of the ATF6 pathway by intrathecal treatment with ATF6 siRNA reduced pain behaviors and BIP expression in the dorsal horn. The results suggest that ER stress might be involved in the induction and maintenance of neuropathic pain. Furthermore, a disturbance in UPR signaling may render the spinal neurons vulnerable to peripheral nerve injury or neuropathic pain stimuli.

ATF6 mediates a pro-inflammatory synergy between ER stress and TLR activation in the pathogenesis of liver ischemia-reperfusion injury.

Although the roles of the metabolic stress in organ ischemia-reperfusion injury (IRI) have been well recognized, the question of whether and how these stress responses regulate innate immune activation against IR remains unclear. In a murine liver partial warm ischemia mode, we showed that prolonged ischemia triggered endoplasmic reticulum (ER) stress response, particularly, the activating transcription factor 6 (ATF6) branch, in liver Kupffer cells (KCs) and altered their responsiveness against Toll-like receptor (TLR) stimulation. Ischemia-primed cells increased pro-, but decreased anti-, inflammatory cytokine productions. Alleviation of ER stress in vivo by small chemical chaperon 4-phenylbutyrate or ATF6 small interfering RNA (siRNA) diminished the pro-inflammatory priming effect of ischemia in KCs, leading to the inhibition of liver immune response against IR and protection of livers from IRI. In vitro, ATF6 siRNA abrogated the ER stress-mediated pro-inflammatory enhancement of macrophage TLR4 response, by restricting NF-κB and restoring Akt activations. Thus, ischemia primes liver innate immune cells by ATF6-mediated ER stress response. The IR-induced metabolic stress and TLR activation function in synergy to activate tissue inflammatory immune response.

ATF6 upregulates XBP1S and inhibits ER stress-mediated apoptosis in osteoarthritis cartilage.

As we previously reported, transcription factor XBP1S enhances BMP2-induced chondrocyte differentiation and acts as a positive mediator of chondrocyte hypertrophy. The purpose of this study was to determine (1) whether XBP1S influences ER stress-mediated apoptosis in osteoarthritis (OA); (2) whether ATF6 regulates IRE1/XBP1 signal pathway in OA cartilage; (3) what are the associated molecules affecting apoptosis in osteoarthritis and the molecular events underlying this process. Herein, we examined and found that ER stress-associated molecules were activated in OA patients, specifically XBP1S splice and expression were increased markedly by TNF-α and IL-1ß treatments. Transcription factor ATF6 can specifically bind to the promoter of XBP1 gene and enhance the expression of XBP1S spliced by IRE1α in osteoarthritis cartilage. Furthermore, siXBP1S can enhance ER stress-mediated apoptosis and main matrix degradation in osteoarthritis. Whereas AdXBP1S can inhibit ER stress-mediated apoptosis and TNFα induced nitrite production in OA cartilage. In a word, our observations demonstrate the importance of XBP1S in osteoarthritis. ATF6 and IRE1α can regulate endogenous XBP1S gene expression synergistically in OA cartilage. More significantly, XBP1S was a negative regulator of apoptosis in osteoarthritis by affecting caspase 3, caspase 9, caspase 12, p-JNK1, and CHOP.

Sustained IRE1 and ATF6 signaling is important for survival of melanoma cells undergoing ER stress.

Apoptosis triggered by endoplasmic reticulum (ER) stress is associated with rapid attenuation of the IRE1α and ATF6 pathways but persistent activation of the PERK branch of the unfolded protein response (UPR) in cells. However, melanoma cells are largely resistant to ER stress-induced apoptosis, suggesting that the kinetics and durations of activation of the UPR pathways are deregulated in melanoma cells undergoing ER stress. We show here that the IRE1α and ATF6 pathways are sustained along with the PERK signaling in melanoma cells subjected to pharmacological ER stress, and that this is, at least in part, due to increased activation of the MEK/ERK pathway. In contrast to an initial increase followed by rapid reduction in activation of IRE1α and ATF6 signaling in control cells that were relatively sensitive to ER stress-induced apoptosis, activation of IRE1α and ATF6 by the pharmacological ER stress inducer tunicamycin (TM) or thapsigargin (TG) persisted in melanoma cells. On the other hand, the increase in PERK signaling lasted similarly in both types of cells. Sustained activation of IRE1α and ATF6 signaling played an important role in protecting melanoma cells from ER stress-induced apoptosis, as interruption of IRE1α or ATF6 rendered melanoma cells sensitive to apoptosis induced by TM or TG. Inhibition of MEK partially blocked IRE1α and ATF6 activation, suggesting that MEK/ERK signaling contributed to sustained activation of IRE1α and ATF6. Taken together, these results identify sustained activation of the IRE1α and ATF6 pathways of the UPR driven by the MEK/ERK pathway as an important protective mechanism against ER stress-induced apoptosis in melanoma cells.

Mmu-miR-702 functions as an anti-apoptotic mirtron by mediating ATF6 inhibition in mice.

MicroRNAs (miRNAs) are a group of endogenous, small, noncoding RNAs that function as key post-transcriptional regulators. miRNAs are involved in many biological processes including apoptosis. In this study, mouse miR-702 (mmu-miR-702), a mirtron derived from the 13th intron of the Plod3 gene, was identified as a regulator of anti-apoptosis. mmu-miR-702 was down-regulated after treatment with the apoptosis-inducer isoproterenol both in vivo and in vitro. According to over-expression experiments, mmu-miR-702 inhibited apoptosis as well as the expression levels of a subset of apoptosis-related genes including activating transcription factor 6 (ATF6). An interaction between mmu-miR-702 and the ATF6 3'-UTR binding site was confirmed using luciferase reporter and western blot assays. This is the first report of ATF6 interaction with miRNA. Although the possible existence of miR-702 in the human genome is low, our results indicate that mirtrons also participate in the process of apoptosis and may provide a novel study strategy for apoptosis.