Koji Matsuhisa

IRE1α-XBP1 is a novel branch in the transcriptional regulation of Ucp1 in brown adipocytes

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.

Multivesicular body formation enhancement and exosome release during endoplasmic reticulum stress

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.

OASIS modulates hypoxia pathway activity to regulate bone angiogenesis

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.

Genome-wide identification and gene expression profiling of ubiquitin ligases for endoplasmic reticulum protein degradation

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 is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization

ABSTRACT 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. Highlighted Article: Luman, an ER-resident transcription factor, regulates the multinucleation of osteoclasts by promoting cell fusion of mononuclear osteoclasts through DC-STAMP induction and transportation during osteoclastogenesis.

Neuronal activity-dependent local activation of dendritic unfolded protein response promotes expression of brain-derived neurotrophic factor in cell soma

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.

Lignosulfonic acid attenuates NF-κB activation and intestinal epithelial barrier dysfunction induced by TNF-α/IFN-γ in Caco-2 cells

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.

ER Stress and Disease: Toward Prevention and Treatment

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.

Sec16A, a key protein in COPII vesicle formation, regulates the stability and localization of the novel ubiquitin ligase RNF183

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.

Axonal Activation of the Unfolded Protein Response Promotes Axonal Regeneration Following Peripheral Nerve Injury

Adult mammalian peripheral neurons have an intrinsic regrowth capacity in response to axonal injury. The induction of calcium ion (Ca2+) 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 Ca2+ homeostasis. Although these characteristics implicate the relevance among injury-induced Ca2+ 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 Ca2+ release from the axonal ER was accelerated in response to injury. Additionally, axonal injury-dependent Ca2+ 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 Ca2+ 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.