Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response, involving the induction of molecular chaperones, translational attenuation, and ER-associated degradation, to prevent cell death. Here, we found that the autophagy system is activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes, which were recognized at the ultrastructural level. The formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3 "dots"), representing autophagosomes, was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, the autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation after ER stress. In contrast, PERK-deficient cells and ATF6 knockdown cells showed that autophagy was induced after ER stress in a manner similar to the wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, suggesting that autophagy plays important roles in cell survival after ER stress.

Eukaryotic cells have signalling pathways from the endoplasmic reticulum (ER) to cytosol and nuclei, to avoid excess accumulation of unfolded proteins in the ER. We previously identified a new type of ER stress transducer, OASIS, a bZIP (basic leucine zipper) transcription factor, which is a member of the CREB/ATF family and has a transmembrane domain. OASIS is processed by regulated intramembrane proteolysis (RIP) in response to ER stress, and is highly expressed in osteoblasts. OASIS(-/-) mice exhibited severe osteopenia, involving a decrease in type I collagen in the bone matrix and a decline in the activity of osteoblasts, which showed abnormally expanded rough ER, containing of a large amount of bone matrix proteins. Here we identify the gene for type 1 collagen, Col1a1, as a target of OASIS, and demonstrate that OASIS activates the transcription of Col1a1 through an unfolded protein response element (UPRE)-like sequence in the osteoblast-specific Col1a1 promoter region. Moreover, expression of OASIS in osteoblasts is induced by BMP2 (bone morphogenetic protein 2), the signalling of which is required for bone formation. Additionally, RIP of OASIS is accelerated by BMP2 signalling, which causes mild ER stress. Our studies show that OASIS is critical for bone formation through the transcription of Col1a1 and the secretion of bone matrix proteins, and they reveal a new mechanism by which ER stress-induced signalling mediates bone formation.

We have previously described a panel of serially transplantable glioblastoma multiforme xenograft lines established by direct subcutaneous injection of patient tumor tissue in the flanks of nude mice. Here we report the characterization of four of these lines with respect to their histopathologic, genetic, and growth properties following heterotopic-to-orthotopic (flank-to-intracranial) transfer. Cells from short-term cultures, established from excised flank xenografts, were harvested and injected into the brains of nude mice (10(6) cells per injection). The intracranial tumors generated from these injections were all highly mitotic as well as highly invasive, but they lacked necrotic features in most instances and failed to show endothelial cell proliferation in all instances. For mice receiving injections from a common explant culture, tumor intracranial growth rate was consistent, as indicated by relatively narrow ranges in survival time. In contrast to the loss of epidermal growth factor receptor gene (EGFR) amplification in cell culture, high-level amplification and overexpression of EGFR were retained in intracranial tumors established from two EGFR-amplified flank tumors. A third intracranial tumor retained patient tumor amplification and high-level expression of platelet-derived growth factor receptor alpha gene. Because the heterotopic-to-orthotopic transfer and propagation of glioblastoma multiforme preserves the receptor tyrosine kinase (RTK) gene amplification of patient tumors, this approach should facilitate investigations for determining the extent to which RTK amplification status influences tumor response to RTK-directed therapies. The fact that such studies were carried out by using an invasive tumor model in an anatomically appropriate context should ensure a rigorous preclinical assessment of agent efficacy.

Adipose tissue plays a central role in maintaining metabolic homeostasis under normal conditions. Metabolic diseases such as obesity and type 2 diabetes are often accompanied by chronic inflammation and adipose tissue dysfunction. In this study, we observed that endoplasmic reticulum (ER) stress and the inflammatory response occurred in adipose tissue of mice fed a high-fat diet for a period of 16 weeks. After 16 weeks of feeding, ER stress markers increased and chronic inflammation occurred in adipose tissue. We found that ER stress is induced by free fatty acid (FFA)-mediated reactive oxygen species (ROS) generation and up-regulated gene expression of inflammatory cytokines in 3T3-L1 adipocytes. Oral administration to obese mice of chemical chaperons, which alleviate ER stress, improved chronic inflammation in adipose tissue, followed by the suppression of increased body weight and improved insulin signaling. These results indicate that ER stress plays important pathophysiological roles in obesity-induced adipose tissue dysfunction.

Many tissues have a specific signal transduction system for endoplasmic reticulum (ER) dysfunction; however, the mechanisms underlying the ER stress response in cartilage remain unclear. BBF2H7 (BBF2 human homologue on chromosome 7), an ER-resident basic leucine zipper transcription factor, is activated in response to ER stress and is highly expressed in chondrocytes. In this study, we generated Bbf2h7(-/-) mice to assess the in vivo function of BBF2H7. The mice showed severe chondrodysplasia and died by suffocation shortly after birth because of an immature chest cavity. The cartilage showed a lack of typical columnar structure in the proliferating zone and a decrease in the size of the hypertrophic zone, resulting in a significant reduction of extracellular matrix proteins. Interestingly, proliferating chondrocytes showed abnormally expanded ER, containing aggregated type II collagen (Col2) and cartilage oligomeric matrix protein (COMP). We identified Sec23a, which encodes a coat protein complex II component responsible for protein transport from the ER to the Golgi, as a target of BBF2H7, which directly bound to a CRE-like sequence in the promoter region of Sec23a to activate its transcription. When Sec23a was introduced to Bbf2h7(-/-) chondrocytes, the impaired transport and secretion of cartilage matrix proteins was totally restored, indicating that by activating protein secretion the BBF2H7-Sec23a pathway has a crucial role in chondrogenesis. Our findings provide a new link by which ER stress is converted to signalling for the activation of ER-to-Golgi trafficking.

To avoid excess accumulation of unfolded proteins in the endoplasmic reticulum (ER), eukaryotic cells have signaling pathways from the ER to the cytosol or nucleus. These processes are collectively termed the ER stress response. Double stranded RNA activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) is a major transducer of the ER stress response and directly phosphorylates eIF2α, resulting in translational attenuation. Phosphorylated eIF2α specifically promotes the translation of the transcription factor ATF4. ATF4 plays important roles in osteoblast differentiation and bone formation. Perk−/− mice are reported to exhibit severe osteopenia, and the phenotypes observed in bone tissues are very similar to those of Atf4−/− mice. However, the involvement of the PERK-eIF2α-ATF4 signaling pathway in osteogenesis is unclear. Phosphorylated eIF2α and ATF4 protein levels were attenuated in Perk−/− calvariae, and the gene expression levels of osteocalcin (Ocn) and bone sialoprotein (Bsp), which are targets for ATF4, were also down-regulated. Treatment of wild-type primary osteoblasts with BMP2, which is required for osteoblast differentiation, induced ER stress, leading to an increase in ATF4 protein expression levels. In contrast, the level of ATF4 in Perk−/− osteoblasts was severely diminished. The results indicate that PERK signaling is required for ATF4 activation during osteoblast differentiation. Perk−/− osteoblasts exhibited decreased alkaline phosphatase activities and delayed mineralized nodule formation relative to wild-type cultures. These abnormalities were almost completely restored by the introduction of ATF4 into Perk−/− osteoblasts. Taken together, ER stress occurs during osteoblast differentiation and activates the PERK-eIF2α-ATF4 signaling pathway followed by the promotion of gene expression essential for osteogenesis, such as Ocn and Bsp.

Endoplasmic reticulum (ER) stress transducers IRE1 (inositol requiring 1), PERK (PKR-like endoplasmic reticulum kinase), and ATF6 (activating transcription factor 6) are well known to transduce signals from the ER to the cytoplasm and nucleus when unfolded proteins accumulate in the ER. Recently, we identified OASIS (old astrocyte specifically induced substance) as a novel ER stress transducer expressed in astrocytes. We report here that BBF2H7 (BBF2 human homolog on chromosome 7), an ER-resident transmembrane protein with the bZIP domain in the cytoplasmic portion and structurally homologous to OASIS, is cleaved at the membrane in response to ER stress. The cleaved fragments of BBF2H7 translocate into the nucleus and can bind directly to cyclic AMP-responsive element sites to activate transcription of target genes. Interestingly, although BBF2H7 protein is not expressed under normal conditions, it is markedly induced at the translational level during ER stress, suggesting that BBF2H7 might contribute to only the late phase of unfolded protein response signaling. In a mouse model of focal brain ischemia, BBF2H7 protein is prominently induced in neurons in the peri-infarction region. Furthermore, in a neuroblastoma cell line, BBF2H7 overexpression suppresses ER stress-induced cell death, while small interfering RNA knockdown of BBF2H7 promotes ER stress-induced cell death. Taken together, our results suggest that BBF2H7 is a novel ER stress transducer and could play important roles in preventing accumulation of unfolded proteins in damaged neurons.

Eukaryotic cells can adapt to endoplasmic reticulum (ER) dysfunction by producing diverse signals from the ER to the cytosol or nucleus. These signalling pathways are collectively known as the unfolded protein response (UPR). The canonical branches of the UPR are mediated by three ER membrane-bound proteins: PERK, IRE1 and ATF6. These ER stress transducers basically play important roles in cell survival after ER stress. Recently, novel types of ER stress transducers that share a region of high sequence similarity with ATF6 have been identified. They have a transmembrane domain, which allows them to associate with the ER, and possess a transcription-activation domain and a bZIP domain. These membrane-bound bZIP transcription factors include Luman, OASIS, BBF2H7, CREBH and CREB4. Despite their structural similarities with ATF6, differences in activating stimuli, tissue distribution and response element binding indicate specialized functions of each member on regulating the UPR in specific organs and tissues. Here, we summarize our current understanding of the biochemical characteristics and physiological functions of the ER-resident bZIP transcription factors.