Shin-ichiro Hino

Autophagy Is Activated for Cell Survival after Endoplasmic Reticulum Stress

ABSTRACT 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.

Phosphorylation of β-Catenin by Cyclic AMP-Dependent Protein Kinase Stabilizes β-Catenin through Inhibition of Its Ubiquitination

ABSTRACT The mechanism of cross talk between the Wnt signaling and cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]) pathways was studied. Prostaglandin E1 (PGE1), isoproterenol, and dibutyryl cAMP (Bt2cAMP), all of which activate PKA, increased the cytoplasmic and nuclear β-catenin protein level, and these actions were suppressed by a PKA inhibitor and RNA interference for PKA. PGE1 and Bt2cAMP also increased T-cell factor (Tcf)-dependent transcription through β-catenin. Bt2cAMP suppressed degradation of β-catenin at the protein level. Although PKA did not affect the formation of a complex between glycogen synthase kinase 3β (GSK-3β), β-catenin, and Axin, phosphorylation of β-catenin by PKA inhibited ubiquitination of β-catenin in intact cells and in vitro. Ser675 was found to be a site for phosphorylation by PKA, and substitution of this serine residue with alanine in β-catenin attenuated inhibition of the ubiquitination of β-catenin by PKA, PKA-induced stabilization of β-catenin, and PKA-dependent activation of Tcf. These results indicate that PKA inhibits the ubiquitination of β-catenin by phosphorylating β-catenin, thereby causing β-catenin to accumulate and the Wnt signaling pathway to be activated.

Signalling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation

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.

Regulation of endoplasmic reticulum stress response by a BBF2H7-mediated Sec23a pathway is essential for chondrogenesis

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.

BBF2H7, a Novel Transmembrane bZIP Transcription Factor, Is a New Type of Endoplasmic Reticulum Stress Transducer

ABSTRACT 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.

Inhibition of the Wnt Signaling Pathway by Idax, a Novel Dvl-Binding Protein

ABSTRACT In attempting to clarify the roles of Dvl in the Wnt signaling pathway, we identified a novel protein which binds to the PDZ domain of Dvl and named it Idax (for inhibition of the Dvl and Axin complex). Idax and Axin competed with each other for the binding to Dvl. Immunocytochemical analyses showed that Idax was localized to the same place as Dvl in cells and that expression of Axin inhibited the colocalization of Dvl and Idax. Further, Wnt-induced accumulation of β-catenin and activation of T-cell factor in mammalian cells were suppressed by expression of Idax. Expression of Idax inXenopus embryos induced ventralization with a reduction in the expression of siamois, a Wnt-inducible gene. Idax inhibited Wnt- and Dvl- but not β-catenin-induced axis duplication. It is known that Dvl is a positive regulator in the Wnt signaling pathway and that the PDZ domain is important for this activity. Therefore, these results suggest that Idax functions as a negative regulator of the Wnt signaling pathway by directly binding to the PDZ domain of Dvl.

Glycogen Synthase Kinase 3 and h-prune Regulate Cell Migration by Modulating Focal Adhesions

ABSTRACT h-prune, which has been suggested to be involved in cell migration, was identified as a glycogen synthase kinase 3 (GSK-3)-binding protein. Treatment of cultured cells with GSK-3 inhibitors or small interfering RNA (siRNA) for GSK-3 and h-prune inhibited their motility. The kinase activity of GSK-3 was required for the interaction of GSK-3 with h-prune. h-prune was localized to focal adhesions, and the siRNA for GSK-3 or h-prune delayed the disassembly of paxillin. The tyrosine phosphorylation of focal adhesion kinase (FAK) and the activation of Rac were suppressed in GSK-3 or h-prune knocked-down cells. GSK-3 inhibitors suppressed the disassembly of paxillin and the activation of FAK and Rac. Furthermore, h-prune was highly expressed in colorectal and pancreatic cancers, and the positivity of the h-prune expression was correlated with tumor invasion. These results suggest that GSK-3 and h-prune cooperatively regulate the disassembly of focal adhesions to promote cell migration and that h-prune is useful as a marker for tumor aggressiveness.

A Novel β-Catenin-binding Protein Inhibits β-Catenin-dependent Tcf Activation and Axis Formation

β-Catenin is efficiently phosphorylated by glycogen synthase kinase-3β in the Axin complex in the cytoplasm, resulting in the down-regulation. In response to Wnt, β-catenin is stabilized and translocated into the nucleus where it stimulates gene expression through Tcf/Lef. Here we report a novel protein, designated Duplin (for axis duplication inhibitor), which negatively regulates the function of β-catenin in the nucleus. Duplin was located in the nucleus. Duplin bound directly to the Armadillo repeats of β-catenin, thereby inhibiting the binding of Tcf to β-catenin. It did not affect the stability of β-catenin but inhibited Wnt- or β-catenin-dependent Tcf activation. Furthermore, expression of Duplin in Xenopus embryos inhibited the axis formation and β-catenin-dependent axis duplication, and prevented the β-catenins ability to rescue ventralizing phenotypes induced by ultraviolet light irradiation. Thus, Duplin is a nuclear protein that inhibits β-catenin signaling.

Activation of OASIS family, ER stress transducers, is dependent on its stabilization

Endoplasmic reticulum (ER) stress transducers transduce signals from the ER to the cytoplasm and nucleus when unfolded proteins accumulate in the ER. BBF2 human homolog on chromosome 7 (BBF2H7) and old astrocyte specifically induced substance (OASIS), ER-resident transmembrane proteins, have recently been identified as novel ER stress transducers that have roles in chondrogenesis and osteogenesis, respectively. However, the molecular mechanisms that regulate the activation of BBF2H7 and OASIS under ER stress conditions remain unresolved. Here, we showed that BBF2H7 and OASIS are notably unstable proteins that are easily degraded via the ubiquitin-proteasome pathway under normal conditions. ER stress conditions enhanced the stability of BBF2H7 and OASIS, and promoted transcription of their target genes. HMG-CoA reductase degradation 1 (HRD1), an ER-resident E3 ubiquitin ligase, ubiquitinated BBF2H7 and OASIS under normal conditions, whereas ER stress conditions dissociated the interaction between HRD1 and BBF2H7 or OASIS. The stabilization of OASIS in Hrd1−/− cells enhanced the expression of collagen fibers during osteoblast differentiation, whereas a knockdown of OASIS in Hrd1−/− cells suppressed the production of collagen fibers. These findings suggest that ER stress stabilizes OASIS family members and this is a novel molecular mechanism for the activation of ER stress transducers.

Regulation of ER molecular chaperone prevents bone loss in a murine model for osteoporosis

Endoplasmic reticulum (ER) stress response is important for protein maturation in the ER. Some murine models for bone diseases have provided significant insight into the possibility that pathogenesis of osteoporosis is related to ER stress response of osteoblasts. We examined a possible correlation between osteoporosis and ER stress response. Bone specimens from 8 osteoporosis patients and 8 disease-controls were used for immunohistochemical analysis. We found that ER molecular chaperones, such as BiP (immunoglobulin heavy-chain binding protein) and PDI (protein-disulfide isomerase) are down-regulated in osteoblasts from osteoporosis patients. Based on this result, we hypothesized that up-regulation of ER molecular chaperones in osteoblasts could restore decreased bone formation in osteoporosis. Therefore, we investigated whether treatment of murine model for osteoporosis with BIX (BiP inducer X), selective inducer BiP, could prevent bone loss. We found that oral administration of BIX effectively improves decline in bone formation through the activation of folding and secretion of bone matrix proteins. Considering these results together, BIX may be a potential therapeutic agent for the prevention of bone loss in osteoporosis patients.