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CREB/ATF transcription factor OASIS/CREB3L1 is upregulated in long-term-cultured astrocytes undergoing cell-cycle arrest due to loss of DNA integrity by repeated replication. However, the roles of OASIS in the cell cycle remain unexplored. We find that OASIS arrests the cell cycle at G 2 /M phase after DNA damage via direct induction of p21. Cell-cycle arrest by OASIS is dominant in astrocytes and osteoblasts, but not in fibroblasts, which are dependent on p53. In a brain injury model, Oasis -/- reactive astrocytes surrounding the lesion core show sustained growth and inhibition of cell-cycle arrest, resulting in prolonged gliosis. We find that some glioma patients exhibit low expression of OASIS due to high methylation of its promoter. Specific removal of this hypermethylation in glioblastomas transplanted into nude mice by epigenomic engineering suppresses the tumorigenesis. These findings suggest OASIS as a critical cell-cycle inhibitor with potential to act as a tumor suppressor., Competing Interests: Declaration of interests All authors declare no competing financial interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mucopolysaccharidosis type II (MPS II) results from the dysfunction of a lysosomal enzyme, iduronate-2-sulfatase (IDS). Dysfunction of IDS triggers the lysosomal accumulation of its substrates, glycosaminoglycans, leading to mental retardation and systemic symptoms including skeletal deformities and valvular heart disease. Most patients with severe types of MPS II die before the age of 20. The administration of recombinant IDS and transplantation of hematopoietic stem cells are performed as therapies for MPS II. However, these therapies either cannot improve functions of the central nervous system or cause severe side effects, respectively. To date, 729 pathogenetic variants in the IDS gene have been reported. Most of these potentially cause misfolding of the encoded IDS protein. The misfolded IDS mutants accumulate in the endoplasmic reticulum (ER), followed by degradation via ER-associated degradation (ERAD). Inhibition of the ERAD pathway or refolding of IDS mutants by a molecular chaperone enables recovery of the lysosomal localization and enzyme activity of IDS mutants. In this review, we explain the IDS structure and mechanism of activation, and current findings about the mechanism of degradation-dependent loss of function caused by pathogenetic IDS mutation. We also provide a potential therapeutic approach for MPS II based on this loss-of-function mechanism.

The nuclear envelope (NE) safeguards the genome and is pivotal for regulating genome activity as the structural scaffold of higher-order chromatin organization. NE had been thought as the stable during the interphase of cell cycle. However, recent studies have revealed that the NE can be damaged by various stresses such as mechanical stress and cellular senescence. These types of stresses are called NE stress. It has been proposed that NE stress is closely related to cellular dysfunctions such as genome instability and cell death. Here, we found that an endoplasmic reticulum (ER)-resident transmembrane transcription factor, OASIS, accumulates at damaged NE. Notably, the major components of nuclear lamina, Lamin proteins were depleted at the NE where OASIS accumulates. We previously demonstrated that OASIS is cleaved at the membrane domain in response to ER stress. In contrast, OASIS accumulates as the full-length form to damaged NE in response to NE stress. The accumulation to damaged NE is specific for OASIS among OASIS family members. Intriguingly, OASIS colocalizes with the components of linker of nucleoskeleton and cytoskeleton complexes, SUN2 and Nesprin-2 at the damaged NE. OASIS partially colocalizes with BAF, LEM domain proteins, and a component of ESCRT III, which are involved in the repair of ruptured NE. Furthermore, OASIS suppresses DNA damage induced by NE stress and restores nuclear deformation under NE stress conditions. Our findings reveal a novel NE stress response pathway mediated by OASIS.

Aggregation, fibril formation, and deposition of amyloid β (Aβ) protein are believed to be the central pathogeneses of Alzheimer's disease (AD). Numerous studies have shown that fibril formation is promoted by preformed seeds at the beginning of the aggregation process. Therefore, aggregated molecules that promote fibrillization of Aβ protein as seeds could affect the pathology. We recently found that approximately 40 amino acid hydrophobic peptides, BBF2H7-derived small peptide (BSP) fragments, are generated via intramembranous cleavage under endoplasmic reticulum (ER) stress conditions. Interestingly, similar to Aβ protein, the fragments exhibit a high aggregation propensity and form fibril structures. It has been noted that ER stress is involved in the pathogenesis of AD. In this study, we examined the effect of BSP fragments on aggregation and cytotoxicity of Aβ 1-40 protein, which is generated as a major species of Aβ protein, but has a lower aggregative property than Aβ 1-42 protein. We demonstrated that BSP fragments promote aggregation of Aβ 1-40 protein. Aggregates of Aβ 1-40 protein mediated by BSP fragments also exhibited potent neurotoxicity. Our findings suggest the possibility that BSP fragments affect accumulation of Aβ proteins and are involved in the pathogenesis of AD., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

We previously reported that RNF183, a member of the RING finger (RNF) protein family, is specifically expressed in the renal collecting duct and that RNF183 mRNA is induced by the activity of nuclear factor of activated T cells 5 (NFAT5), which regulates the transcription of essential proteins for adaptation to hypertonic conditions. The renal medulla is the only tissue that is continuously hypertonic; therefore, RNF183 possibly plays an important role in adaptation to continuous hypertonic conditions. However, the mechanism of how cells adapt to long-term hypertonicity via RNF183 remains unclear. In this study, the Na, K-ATPase α1 subunit was identified as a candidate substrate of RNF183 by the BirA proximity-biotinylation technique. The Na, K-ATPase α1 subunit acts as an ion transporter along with the Na, K-ATPase β1 subunit at the plasma membrane. We confirmed that RNF183 interacted with both α1 and β1 subunits; however, we found that RNF183 ubiquitinated only the β1 subunit, not the α1 subunit. Furthermore, RNF183 translocated both α1 and β1 subunits from the plasma membrane to lysosomes. In addition, the expression levels of α1 and β1 subunits in HEK293 cells stably expressing RNF183 were significantly decreased compared with mock control cells, and were restored by siRNA-mediated knockdown of RNF183. Moreover, in RNF183-expressing cells, chloroquine treatment increased the protein levels of the α1 and β1 subunits. Therefore, our results suggest that Na, K-ATPase α1 and β1 subunits are degraded in lysosomes by RNF183-mediated ubiquitination of β1 subunit., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Intramembrane cleavage of transmembrane proteins is a fundamental cellular process to produce important signals that elicit biological responses. These proteolytic events are known as regulated intramembrane proteolysis (RIP). ATF6 and BBF2H7 are transmembrane basic leucine zipper transcription factors and are subjected to RIP by site-1 protease (S1P) and site-2 protease (S2P) sequentially in response to endoplasmic reticulum (ER) stress. However, the detailed mechanisms responsible for RIP of the transcription factors, including the precise cutting sites, are still unknown. In this study, we demonstrated that S1P cleaves BBF2H7 just before the RXXL S1P recognition motif. Conversely, S2P cut at least three different sites in the membrane (next to Leu380, Met381, and Leu385), indicating that S2P cleaves the substrates at variable sites or via a multistep process. Interestingly, we found BBF2H7-derived small peptide (BSP) fragments located between the S1P and S2P cleavage sites in cells exposed to ER stress. Major type of BSP fragments was composed of 45 amino acid including partial transmembrane and luminal regions and easily aggregates like amyloid β (Aβ) protein. These results advance the understanding of poorly characterized ER stress-dependent RIP. Furthermore, the aggregable peptides produced by ER stress could link to the pathophysiology of neurodegenerative disorders., (© 2019 The Authors. The FASEB Journal published by Wiley Periodicals, Inc. on behalf of Federation of American Societies for Experimental Biology.)

RNF183 is a ubiquitin ligase containing RING-finger and transmembrane domains, and its expression levels are increased in patients with inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, and in 2,4,6-trinitrobenzene sulfonic acid-induced colitis mice. Here, we further demonstrate that RNF183 was induced to a greater degree in the dextran sulfate sodium (DSS)-treated IBD model at a very early stage than were inflammatory cytokines. In addition, fluorescence-activated cell sorting and polymerase chain reaction analysis revealed that RNF183 was specifically expressed in epithelial cells of DSS-treated mice, which suggested that increased levels of RNF183 do not result from the accumulation of immune cells. Furthermore, we identified death receptor 5 (DR5), a member of tumour necrosis factor (TNF)-receptor superfamily, as a substrate of RNF183. RNF183 mediated K63-linked ubiquitination and lysosomal degradation of DR5. DR5 promotes TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis signal through interaction with caspase-8. Inhibition of RNF183 expression was found to suppress TRAIL-induced activation of caspase-8 and caspase-3. Thus, RNF183 promoted not only DR5 transport to lysosomes but also TRAIL-induced caspase activation and apoptosis. Together, our results provide new insights into potential roles of RNF183 in DR5-mediated caspase activation in IBD pathogenesis.

Nuclear factor of activated T-cells 5 (NFAT5) directly binds to the promoter of the RING finger protein 183 (RNF183) gene and induces its transcription under hypertonic conditions in mouse inner-medullary collecting duct (mIMCD-3) cells. However, there is no specific anti-RNF183 antibody for immunostaining; therefore, it is unclear whether NFAT5 regulates RNF183 expression in vivo and where RNF183 is localized in the kidney. This study investigated NFAT5-regulated in vivo RNF183 expression and localization using CRISPR/Cas9-mediated RNF183-green fluorescent protein (RNF183-GFP) knock-in mice. GFP with linker sequences was introduced upstream of an RNF183 open reading frame in exon 3 by homologous recombination through a donor plasmid. Immunofluorescence staining using GFP antibody revealed that GFP signals gradually increase from the outer medulla down to the inner medulla and colocalize with aquaporin-2. Furosemide treatment dramatically decreased RNF183 expression in the renal medulla, consistent with the decrease in NFAT5 protein and target gene mRNA expression. Furosemide treatment of mIMCD-3 cells did not affect mRNA expression and RNF183 promoter activities. These results indicated that RNF183 is predominantly expressed in the renal medullary collecting ducts, and that decreased renal medullary tonicity by furosemide treatment decreases RNF183 expression by NFAT5 downregulation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mucopolysaccharidosis type II (MPS II) is one of the most common mucopolysaccharidoses, which is caused by mutation of the gene encoding iduronate 2-sulfatase (IDS). The loss of function of IDS leads to the accumulation of heparan sulfate and dermatan sulfate of glycosaminoglycans throughout the body, resulting in skeletal deformities, mental retardation, rigid joints, and thick skin. Recently, enzyme replacement therapy has become a common strategy for treating this condition. However, its effectiveness on the central nervous system (CNS) is limited because intravenously administered recombinant IDS (rIDS) cannot pass through the blood brain barrier. Therefore, several methods for delivering rIDS to the CNS, using anti-human transferrin receptor antibody and adeno-associated virus 9, have been explored. To investigate additional approaches for treatment, more cognition about the intracellular dynamics of mutant IDS is essential. We have already found that mutant IDS accumulated in the endoplasmic reticulum (ER) and was degraded by ER-associated degradation (ERAD). Although the dynamics of degradation of mutant IDS was revealed, the molecular mechanism related to the folding of mutant IDS in the ER remained unclear. In this research, we confirmed that mutant IDS retained in the ER would be folded by binding with calnexin (CNX). Thus, knockdown of CNX reduced the translocation of mutant IDS from ER to lysosome and its enzyme activity, indicating that the correct folding of this protein via interaction with CNX ensures its functional activity. These findings reveal the possibility that modifying the interaction of mutant IDS and CNX could contribute to alternative therapeutic strategies for MPS II., (Copyright © 2019. Published by Elsevier Inc.)

We previously reported that among the 37 RING finger protein (RNF) family members, RNF183 mRNA is specifically expressed in the kidney under normal conditions. However, the mechanism supporting its kidney-specific expression pattern remains unclear. In this study, we elucidated the mechanism of the transcriptional activation of murine Rnf183 in inner-medullary collecting duct cells. Experiments with anti-RNF183 antibody revealed that RNF183 is predominantly expressed in the renal medulla. Among the 37 RNF family members, Rnf183 mRNA expression was specifically increased in hypertonic conditions, a hallmark of the renal medulla. RNF183 up-regulation was consistent with the activation of nuclear factor of activated T cells 5 (NFAT5), a transcription factor essential for adaptation to hypertonic conditions. Accordingly, siRNA-mediated knockdown of NFAT5 down-regulated RNF183 expression. Furthermore, the -3,466 to -3,136-bp region upstream of the mouse Rnf183 promoter containing the NFAT5-binding motif is conserved among mammals. A luciferase-based reporter vector containing the NFAT5-binding site was activated in response to hypertonic stress, but was inhibited by a mutation at the NFAT5-binding site. ChIP assays revealed that the binding of NFAT5 to this DNA site is enhanced by hypertonic stress. Of note, siRNA-mediated RNF183 knockdown increased hypertonicity-induced caspase-3 activation and decreased viability of mIMCD-3 cells. These results indicate that (i) RNF183 is predominantly expressed in the normal renal medulla, (ii) NFAT5 stimulates transcriptional activation of Rnf183 by binding to its cognate binding motif in the Rnf183 promoter, and (iii) RNF183 protects renal medullary cells from hypertonicity-induced apoptosis., (© 2019 Maeoka et al.)

Mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome, is a devastating progressive disease caused by mutations in the iduronate 2-sulfatase (IDS) gene. IDS is one of the sulfatase enzymes required for lysosomal degradation of glycosaminoglycans. Mutant proteins linked to diseases are often prone to misfolding. These misfolded proteins accumulate in the endoplasmic reticulum (ER) and are degraded by the ubiquitin-proteasome pathway (ER-associated degradation (ERAD)). The decreased enzyme activities of IDS mutants may be due to accelerated degradation by ERAD. However, intracellular dynamics including degradation of IDS mutants is unexplored. In this report, we examined biochemical and biological characteristics of wild-type (WT) IDS and IDS mutants expressed in HeLa cells. IDS was shown to be glycosylated in the ER and Golgi apparatus and proteolytically cleaved to generate the mature forms in the Golgi apparatus. The mature WT IDS was translocated to the lysosome. In contrast, all IDS mutants we examined were found to accumulate in the ER and could not efficiently translocate to the lysosome. Accumulated IDS mutants in the ER were ubiquitinated by ERAD-related ubiquitin E3 ligase HRD1 followed by degradation via ERAD. Suppressed degradation of 'attenuated' mutant A85T IDS (the late-onset form of MPS II) by inhibiting ERAD components improved translocation to the lysosome and its activities. Our novel findings provide alternative targets to current principal therapies for MPS II. These perspectives provide a potenti al framework to develop fundamental therapeutic strategies and agents.

Adult mammalian peripheral neurons have an intrinsic regrowth capacity in response to axonal injury. The induction of calcium ion (Ca 2+ ) oscillations at an injured site is critical for the regulation of regenerative responses. In polarized neurons, distal axonal segments contain a well-developed endoplasmic reticulum (ER) network that is responsible for Ca 2+ homeostasis. Although these characteristics implicate the relevance among injury-induced Ca 2+ dynamics, axonal ER-derived signaling, and regenerative responses propagated along the axons, the details are not fully understood. In the present study, we found that Ca 2+ release from the axonal ER was accelerated in response to injury. Additionally, axonal injury-dependent Ca 2+ release from the ER activated unfolded protein response (UPR) signaling at injured sites. Inhibition of axonal UPR signaling led to fragmentation of the axonal ER and disrupted growth cone formation, suggesting that activation of axonal UPR branches following axonal injury promotes regeneration via regulation of ER reconstruction and formation of growth cones. Our studies revealed that local activation of axonal UPR signaling by injury-induced Ca 2+ release from the ER is critical for regeneration. These findings provide a new concept for the link between injury-induced signaling at a distant location and regulation of organelle and cytoskeletal formation in the orchestration of axonal regeneration., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lentinula edodes mycelia solid culture extract (MSCE) is used as a medical food ingredient and provides beneficial effects to patients with cancer and chronic type C hepatitis. Low molecular weight lignin (LM-lignin), which is an active component of MSCE, exhibits hepatoprotective, antitumor, antiviral, and immunomodulatory effects. In this study, we investigated the effect of LM-lignin/lignosulfonic acid on intestinal barrier function. Lignosulfonic acid enhanced transepithelial membrane electrical resistance in human intestinal Caco-2 cell monolayers. In Caco-2 cells treated with lignosulfonic acid, expression of claudin-2, which forms high conductive cation pores in tight junctions (TJs), was decreased. Lignosulfonic acid also attenuated the barrier dysfunction that is caused by tumor necrosis factor (TNF)-α and interferon (IFN)-γ in Caco-2 cells. TNF-α- and IFN-γ-induced activation of NF-κB, such as translocation of NF-κB p65 into the nucleus and induction of gene expression, was inhibited by lignosulfonic acid treatment. Furthermore, lignosulfonic acid decreased the TNF-α- and IFN-γ-induced increase in interleukin (IL)-1β and IL-6 expression in Caco-2 cells. These results suggest that lignosulfonic acid not only enhances TJ barrier function but also restores TJ barrier integrity impaired by inflammatory cytokines. Therefore, lignosulfonic acid may be beneficial for the treatment of inflammation-induced intestinal barrier dysfunction observed in inflammatory bowel disease.

We identified 37 ubiquitin ligases containing RING-finger and transmembrane domains. Of these, we found that RNF183 is abundantly expressed in the kidney. RNF183 predominantly localizes to the endoplasmic reticulum (ER), Golgi, and lysosome. We identified Sec16A, which is involved in coat protein complex II vesicle formation, as an RNF183-interacting protein. RNF183 colocalized with Sec16A and interacted through the central conserved domain (CCD) of Sec16A. Although Sec16A is not a substrate for RNF183, RNF183 was more rapidly degraded by the ER-associated degradation (ERAD) in the absence of Sec16A. Sec16A also stabilized the interacting ubiquitin ligase RNF152, which localizes to the lysosome and has structural similarity with RNF183. These results suggest that Sec16A appears to regulate the protein stability and localization of lysosomal ubiquitin ligases.

Unfolded protein response (UPR) has roles not only in resolving the accumulation of unfolded proteins owing to endoplasmic reticulum (ER) stress, but also in regulation of cellular physiological functions. ER stress transducers providing the branches of UPR signaling are known to localize in distal dendritic ER of neurons. These reports suggest that local activation of UPR branches may produce integrated outputs for distant communication, and allow regulation of local events in highly polarized neurons. Here, we demonstrated that synaptic activity- and brain-derived neurotrophic factor (BDNF)-dependent local activation of UPR signaling could be associated with dendritic functions through retrograde signal propagation by using murine neuroblastoma cell line, Neuro-2A and primary cultured hippocampal neurons derived from postnatal day 0 litter C57BL/6 mice. ER stress transducer, inositol-requiring kinase 1 (IRE1), was activated at postsynapses in response to excitatory synaptic activation. Activated dendritic IRE1 accelerated accumulation of the downstream transcription factor, x-box-binding protein 1 (XBP1), in the nucleus. Interestingly, excitatory synaptic activation-dependent up-regulation of XBP1 directly facilitated transcriptional activation of BDNF. BDNF in turn drove its own expression via IRE1-XBP1 pathway in a protein kinase A-dependent manner. Exogenous treatment with BDNF promoted extension and branching of dendrites through the protein kinase A-IRE1-XBP1 cascade. Taken together, our findings indicate novel mechanisms for communication between soma and distal sites of polarized neurons that are coordinated by local activation of IRE1-XBP1 signaling. Synaptic activity- and BDNF-dependent distinct activation of dendritic IRE1-XBP1 cascade drives BDNF expression in cell soma and may be involved in dendritic extension. Cover Image for this issue: doi. 10.1111/jnc.14159., (© 2017 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Claudins are key functional and structural components of tight junctions (TJs) in epithelial cell sheets. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds to claudin-4 and reversibly modulates intestinal TJ seals, thereby enhancing paracellular transport of solutes. However, the use of C-CPE as an absorption enhancer is limited by the molecule's immunogenicity and manufacturing cost. Here, we developed a high-throughput screening system based on the Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) method to identify claudin-4 binders in a library collection of 32,560 compounds. Thiostrepton, identified from the screen, decreased transepithelial electrical resistance and increased flux of 4-kDa fluorescein isothiocyanate-labelled dextran (FD-4) in Caco-2 cell monolayers, a model of intestinal epithelium. Thiostrepton changed the expression, but not the localisation, of TJ components. Treatment of rat jejunum with thiostrepton increased the absorption of FD-4 without tissue toxicity, indicating that thiostrepton is a novel claudin-4 binder that enhances intestinal permeability. The screening system may therefore be a useful tool for identifying claudin-4 binders to enhance drug absorption in mucosa.

Secretory and membrane proteins are synthesized in ribosomes, then mature in the endoplasmic reticulum (ER), but if ER function is impaired, immature defective proteins accumulate in the ER. This situation is called ER stress: in response, a defensive mechanism called the unfolded protein response (UPR) is activated in cells to reduce the defective proteins. During the UPR, the ER transmembrane sensor molecules inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and RNA-dependent protein kinase (PKR)-like ER kinase (PERK) are activated, stress signals are transduced to the outside of the ER, and various cell responses, including gene induction, occur. In ER-associated degradation (ERAD), one type of UPR, defective proteins are eventually expelled from the ER and degraded in the cytoplasm through the ubiquitin proteasome system. Since ER stress has been reported to have relationships with neurodegenerative diseases, diabetes, metabolic syndromes, and cancer, it is the focus of increased attention from the perspectives of elucidating pathogenic mechanisms, and in the development of therapeutics.

Androgen-Induced bZIP (AIbZIP) is structurally a bZIP transmembrane transcription factor belonging to the CREB/ATF family. This molecule is highly expressed in androgen-sensitive prostate cancer cells and is transcriptionally upregulated by androgen treatment. Here, we investigated molecular mechanism of androgen-dependent expression of AIbZIP and its physiological function in prostate cancer cells. Our data showed that SAM pointed domain-containing ETS transcription factor (SPDEF), which is upregulated by androgen treatment, directly activates transcription of AIbZIP. Knockdown of AIbZIP caused a significant reduction in the proliferation of androgen-sensitive prostate cancer cells with robust expression of p21. Mechanistically, we demonstrated that AIbZIP interacts with old astrocyte specifically induced substance (OASIS), which is a CREB/ATF family transcription factor, and prevents OASIS from promoting transcription of its target gene p21. These findings showed that AIbZIP induced by the androgen receptor (AR) axis plays a crucial role in the proliferation of androgen-sensitive prostate cancer cells, and could be a novel target of therapy for prostate cancer.

The endoplasmic reticulum (ER) plays a pivotal role in maintaining cellular homeostasis. However, numerous environmental and genetic factors give rise to ER stress by inducing an accumulation of unfolded proteins. Under ER stress conditions, cells initiate the unfolded protein response (UPR). Here, we demonstrate a novel aspect of the UPR by electron microscopy and immunostaining analyses, whereby multivesicular body (MVB) formation was enhanced after ER stress. This MVB formation was influenced by inhibition of ER stress transducers inositol required enzyme 1 (IRE1) and PKR-like ER kinase (PERK). Furthermore, exosome release was also increased during ER stress. However, in IRE1 or PERK deficient cells, exosome release was not upregulated, indicating that IRE1- and PERK-mediated pathways are involved in ER stress-dependent exosome release., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a mechanism by which unfolded proteins that accumulate in the ER are transported to the cytosol for ubiquitin-proteasome-mediated degradation. Ubiquitin ligases (E3s) are a group of enzymes responsible for substrate selectivity and ubiquitin chain formation. The purpose of this study was to identify novel E3s involved in ERAD. Thirty-seven candidate genes were selected by searches for proteins with RING-finger motifs and transmembrane regions, which are the major features of ERAD E3s. We performed gene expression profiling for the identified E3s in human and mouse tissues. Several genes were specifically or selectively expressed in both tissues; the expression of four genes (RNFT1, RNF185, CGRRF1 and RNF19B) was significantly upregulated by ER stress. To determine the involvement of the ER stress-responsive genes in ERAD, we investigated their ER localisation, in vitro autoubiquitination activity and ER stress resistance. All were partially localised to the ER, whereas CGRRF1 did not possess E3 activity. RNFT1 and RNF185, but not CGRRF1 and RNF19B, exhibited significant resistance to ER stressor in an E3 activity-dependent manner. Thus, these genes are possible candidates for ERAD E3s.

Luman (also known as CREB3) is a type-II transmembrane transcription factor belonging to the OASIS family that localizes to the endoplasmic reticulum (ER) membrane under normal conditions. In response to ER stress, OASIS-family members are subjected to regulated intramembrane proteolysis (RIP), following which the cleaved N-terminal fragments translocate to the nucleus. In this study, we show that treatment of bone marrow macrophages (BMMs) with cytokines - macrophage colony-stimulating factor (M-CSF) and RANKL (also known as TNFSF11) - causes a time-dependent increase in Luman expression, and that Luman undergoes RIP and becomes activated during osteoclast differentiation. Small hairpin (sh)RNA-mediated knockdown of Luman in BMMs prevented the formation of multinucleated osteoclasts, concomitant with the suppression of DC-STAMP, a protein that is essential for cell-cell fusion in osteoclastogenesis. The N-terminus of Luman facilitates promoter activity of DC-STAMP, resulting in upregulation of DC-STAMP expression. Furthermore, Luman interacts with DC-STAMP, and controls its stability and localization. These results suggest that Luman regulates the multinucleation of osteoclasts by promoting cell fusion of mononuclear osteoclasts through DC-STAMP induction and intracellular distribution during osteoclastogenesis., (© 2015. Published by The Company of Biologists Ltd.)

The unfolded protein response (UPR) not only resolves endoplasmic reticulum (ER) stress, but also regulates cellular physiological functions. In this study, we first linked the UPR to the physiological roles of brown adipose tissue (BAT). BAT is one of the tissues that control energy homeostasis in the body. Brown adipocytes are able to dissipate energy in the form of heat owing to their mitochondrial protein, uncoupling protein 1 (UCP1). We found that one of the UPR branches, the IRE1α-XBP1 pathway, was activated during the transcriptional induction of Ucp1. Inhibiting the IRE1α-XBP1 pathway reduced the induction of Ucp1 expression. However, the activation of the IRE1α-XBP1 pathway by ER stress never upregulated Ucp1. On the other hand, the activation of protein kinase A (PKA) induced Ucp1 transcription through the activation of IRE1α-XBP1. The inhibition of PKA abrogated the activation of IRE1α-XBP1 pathway, while the inhibition of a p38 mitogen activated protein kinase (p38 MAPK), which is one of the downstream molecules of PKA, never suppressed the activation of IRE1α-XBP1 pathway. These data indicate that PKA-dependent IRE1α-XBP1 activation is crucial for the transcriptional induction of Ucp1 in brown adipocytes, and they demonstrate a novel, ER stress -independent role of the UPR during thermogenesis.

OASIS/CREB3L1, an endoplasmic reticulum (ER)-resident transcription factor, plays important roles in osteoblast differentiation. In this study, we identified new crosstalk between OASIS and the hypoxia signaling pathway, which regulates vascularization during bone development. RT-PCR and real-time PCR analyses revealed significant decreases in the expression levels of hypoxia-inducible factor-1α (HIF-1α) target genes such as vascular endothelial growth factor A (VEGFA) in OASIS-deficient (Oasis(-/-)) mouse embryonic fibroblasts. In coimmunoprecipitation experiments, the N-terminal fragment of OASIS (OASIS-N; activated form of OASIS) bound to HIF-1α through the bZIP domain. Luciferase assays showed that OASIS-N promoted the transcription activities of a reporter gene via a hypoxia-response element (HRE). Furthermore, the expression levels of an angiogenic factor Vegfa was decreased in Oasis(-/-) osteoblasts. Immunostaining and metatarsal angiogenesis assay showed retarded vascularization in bone tissue of Oasis(-/-) mice. These results suggest that OASIS affects the expression of HIF-1α target genes through the protein interaction with HIF-1α, and that OASIS-HIF-1α complexes may play essential roles in angiogenesis during bone development.

Lentinula edodes mycelia solid culture extract (MSCE) contains several bioactive molecules, including some polyphenolic compounds, which exert immunomodulatory, antitumor, and hepatoprotective effects. In this study, we examined the anti-hepatitis C virus (HCV) activity of MSCE and low-molecular-weight lignin (LM-lignin), which is the active component responsible for the hepatoprotective effect of MSCE. Both MSCE and LM-lignin inhibited the entry of two HCV pseudovirus (HCVpv) types into Huh7.5.1 cells. LM-lignin inhibited HCVpv entry at a lower concentration than MSCE and inhibited the entry of HCV particles in cell culture (HCVcc). MSCE also inhibited HCV subgenome replication. LM-lignin had no effect on HCV replication, suggesting that MSCE contains additional active substances. We demonstrate here for the first time the anti-HCV effects of plant-derived LM-lignin and MSCE. The hepatoprotective effect of LM-lignin suggests that lignin derivatives, which can be produced in abundance from existing plant resources, may be effective in the treatment of HCV-related diseases., (Copyright © 2015 Elsevier Inc. All rights reserved.)

BBF2H7 is an endoplasmic reticulum (ER)-resident transmembrane basic leucine zipper (bZIP) transcription factor that is cleaved at the transmembrane domain by regulated intramembrane proteolysis in response to ER stress. The cleaved cytoplasmic N-terminus containing transcription activation and bZIP domains translocates into the nucleus to promote the expression of target genes. In chondrocytes, the cleaved luminal C-terminus is extracellularly secreted and facilitates proliferation of neighboring cells through activation of Hedgehog signaling. In the present study, we found that Bbf2h7 expression levels significantly increased by 1.070-2.567-fold in several tumor types including glioblastoma compared with those in respective normal tissues, using the ONCOMINE Cancer Profiling Database. In some Hedgehog ligand-dependent cancer cell lines including glioblastoma U251MG cells, the BBF2H7 C-terminus was secreted from cells into the culture media and promoted cancer cell proliferation through activation of Hedgehog signaling. Knockdown of Bbf2h7 expression suppressed the proliferation of U251MG cells by downregulating Hedgehog signaling. The impaired cell proliferation and Hedgehog signaling were recovered by addition of BBF2H7 C-terminus to the culture medium of Bbf2h7-knockdown U251MG cells. These data suggest that the secreted luminal BBF2H7 C-terminus is involved in Hedgehog ligand-dependent cancer cell proliferation through activation of Hedgehog signaling. Thus, the BBF2H7 C-terminus may be a novel target for the development of anticancer drugs.

Intercellular spaces between adjacent mucosal epithelial cells are sealed by tight junctions (TJs) that prevent the free movement of solutes across the epithelium. Claudins (CLs), a family of 27 integral membrane proteins, are essential components for TJ seals. We previously used a CL-3/-4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), to show that CL modulation is a promising method to enhance mucosal absorption. Recently, by using a C-CPE mutant library, we developed a CL binder (m19) with broad specificity to CL-1, -2, -4, and -5. Here, we compared the mucosal absorption-enhancing activity of C-CPE and m19. Both CL binders enhanced jejunal absorption of dextran with a molecular mass of 4000 and 150,000 Da and nasal absorption of dextran with a mass of 4000 Da but not 150,000 Da in rats. Although both binders showed similar nasal absorption-enhancing activity of dextran (4000 Da), m19 exhibited a more potent jejunal absorption-enhancing effect than that of C-CPE. These findings suggest that mucosal absorption-enhancing activity may be modified by modulating CL specificity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Claudins (CL) are a family of tetra-transmembrane proteins that are the structural and functional components of tight junctions (TJ). CLs are promising targets for drug development because of their role in mucosal drug absorption and cancer. However, CL-targeted drug development has been delayed because CLs have low antigenicity and preparing CL proteins is difficult. We developed a CL binder by using the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) and a baculoviral display system. After screening CL binders from a C-CPE mutant-displaying library by using CL-displaying budded baculovirus (BV) we isolated a C-CPE mutant called m19, which bound to CL1, CL2, CL4 and CL5. A 3-dimensional analysis showed that m19 has a structural backbone similar to C-CPE. The charge density of the CL-binding domains of m19 and C-CPE differed, suggesting that electrostatic interactions may occur between m19 and CLs. Treatment of epithelial cells with m19 decreased the paracellular but not transcellular integrity, and m19 enhanced jejunal absorption. Thus, we successfully created a CL binder with broad specificity. These findings will contribute to future preparation of CL binders for CL-targeted drug development., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Tight junctions (TJs) maintain cellular polarity between the apical and basolateral region of epithelial cells. Claudin, a tetra-transmembrane protein, plays a pivotal role in the barrier function of TJs. We previously found that a claudin modulator, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), may be a promising candidate for improving the mucosal absorption of drugs. C-CPE is a fragment of enterotoxin, and putative CPE claudin receptors are highly expressed in liver and kidney. The safety and antigenicity of C-CPE must be evaluated for future clinical application. Therefore, we evaluated whether C-CPE administration in mice leads to tissue injury or production of antibodies. Intravenous administration of C-CPE at 5 mg/kg, which is a more than 25-fold higher dose than that used in a murine mucosal absorption model, did not increase biochemical markers of liver and kidney injury even after 11 injections once a week. Nasal C-CPE administration (2 mg/kg) once a week for 11 administrations also did not increase these biochemical markers, but 6 administrations of C-CPE resulted in elevation of C-CPE-specific serum IgG. These results indicate that development of a less antigenic claudin modulator will be essential for future clinical application of a C-CPE-based mucosal absorption enhancer.

Tumor metastasis of epithelium-derived tumors is the major cause of death from malignant tumors. Overexpression of claudin is observed frequently in malignant tumors. However, claudin-targeting antimetastasis therapy has never been investigated. We previously prepared a claudin-4-targeting antitumor molecule that consisted of the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) fused to protein synthesis inhibitory factor (PSIF) derived from Pseudomonas exotoxin. In the present study, we investigated whether claudin CPE receptors can be a target for tumor metastasis by using the C-CPE-fused PSIF as a claudin-targeting agent. One of the most popular murine metastasis models is the lung metastasis of intravenously injected B16 cells. Therefore, we first investigated the effects of the C-CPE-fused PSIF on lung metastasis of claudin-4-expressing B16 (CL4-B16) cells. Intravenous administration of the C-CPE-fused PSIF suppressed lung metastasis of CL4-B16 cells but not B16 cells. Injection of C-CPE-fused PSIF also inhibited tumor growth and spontaneous lung metastasis of murine breast cancer 4T1 cells inoculated into the subcutis. Treatment with C-CPE-fused PSIF did not show apparent side effects in mice. These findings indicate that claudin targeting may be a novel strategy for inhibiting some tumor metastases.

Purpose: This study was undertaken to clarify to what extentintervention by dental hygienist visits and leaflet mailing, once or three times a year, increased the dental health scores of Tobishima Village residents in a 3 year period., Methods: The oral health status and oral health score (the Oral Salutogenic Score) of 786 residents were examined in May, 2004. Of the total, 446 people had oral health scores equal to or less than 15. They were divided into three groups at random, receiving: dental hygienist visits (visit group); leaflet mailing three times a year (three times mail group); and leaflet mailing only once a year (single mail group). Oral health status and scores were examined a second time by dentists in May, 2007 and average increases of scores over 3 years were calculated for each of the three groups. The Mann-Whitney test and cost-effectiveness analysis were employed for comparisons., Results: The average increase in oral health score was 46.2 +/- 8.1% for the visit group, 31.2 +/- 4.4% for the three times mail group and 25.0+/-5.2% single mail group over 3 years. The visit group value was significantly higher than the single mail group (P<0.05). Of the resi-dents who had an oral health score equal to or less than 15 points, increase of their oral health score those who received a dental hygienist visit had a higher than the groups who were sent leaflets. With the latter, the greater frequency of leaflet distribution was more effective for increasing the oral health score., Conclusion: It was concluded that visits by dental hygienists are more effective than mailing of leaflets. In addition, mailing 2 or 3 times a year is more effective than once a year.

Recent progress in pharmaceutical technology based on genomic and proteomic research has provided many drug candidates, including not only chemicals but peptides, antibodies and nucleic acids. These candidates do not show pharmaceutical activity without their absorption into systemic flow and movement from the systemic flow into the target tissue. Epithelial and endothelial cell sheets play a pivotal role in the barrier between internal and external body and tissues. Tight junctions (TJs) between adjacent epithelial cells limit the movement of molecules through the intercellular space in epithelial and endothelial cell sheets. Thus, a promising strategy for drug delivery is the modulation of TJ components to allow molecules to pass through the TJ-based cellular barriers. In this review, we discuss recent progress in the development of TJ modulators and the possibility of absorption enhancers and drug-delivery systems based on TJ components.

Purpose: To investigate the possibility of using polylactic acid (PLA) as a surgical adjuvant for visualizing the vitreous body during vitrectomy., Methods: After a core vitrectomy, 1 mL of PLA suspension was injected into the rabbit vitreous in two groups: group A, 2.5% PLA (n = 5), and group B, 1% PLA (n = 9). Vehicle injection instead of PLA was used as a control (group C, n = 5). The clinical signs and electroretinogram (ERG) were evaluated for 28 days, and histologic findings were evaluated on day 28. Next, intraocular pressure (IOP) after intracameral injection of a PLA suspension was evaluated in the rabbits (n = 6). Last, the visualization of the vitreous body by PLA suspension was evaluated during vitrectomy in monkey eyes (n = 4)., Results: The white granules of PLA disappeared from the vitreous cavity in 10 eyes within 3 weeks; however, a small amount of PLA remained in four eyes for 4 weeks. Mild inflammation of the anterior chamber was observed in one eye in group B and 1 eye in group C. No cataract or retinal hemorrhage was found in any eyes. The amplitude of ERG on each time point did not differ between the groups. IOP remained within normal range except for the initial spike. Retinal structure was well preserved histologically. During vitrectomy in monkey eyes, the vitreous body was well visualized, and the posterior vitreous separation was performed easily and safely., Conclusions: PLA can be a new surgical adjuvant to visualize the vitreous body during vitrectomy.

Uniformity and precision of single dose are required for ophthalmic suspensions including water-insoluble ingredients. Solid sediments formed after standing still must be immediately re-dispersible and distributed homogeneously before use. However, selection of an appropriate water-soluble polymer as suspending agent is a challenging problem. In this report, the relationship between the surface tension and the re-dispersibility of suspensions was investigated. The surface tension of 0.1 w/v% fluorometholone suspensions began to decline from 74 mN/m at 0.0001 w/v% of hydroxypropylmethylcellulose (HPMC) and became almost constant at 52 mN/m at 0.01 w/v% of HPMC. Re-dispersion time was less than 4 s when HPMC was present at concentrations between 0.0001 w/v% and 0.01 w/v%. At these concentrations, aggregation of suspended particles was not observed. When indomethacin suspensions at 1.0 w/v% concentration were used, the surface tension began to decline from 73 mN/m at 0.0005 w/v% HPMC and became constant at 50 mN/m at 0.005 w/v% HPMC. The suspension also showed good re-dispersibility, and a uniform suspension was obtained between 0.0005 w/v% and 0.005 w/v% of HPMC. The time required for re-dispersion was less than 17 s. The change of surface tension showed a good correlation with the concentration of HPMC in ophthalmic suspensions having good re-dispersibility. Measurement of the surface tension of suspensions provided the optimal concentration of the water-soluble polymers for the suspensions of well re-dispersible characteristics. Evaluation of ophthalmic suspension using surface tension is a good strategy for formulation of suspending pharmaceutical products in the ophthalmic area.