Sexually dimorphic dynamics of the microtubule network in medaka (Oryzias latipes) germ cells.

Gametogenesis is the process through which germ cells differentiate into sexually dimorphic gametes, eggs and sperm. In the teleost fish medaka (Oryzias latipes), a germ cell-intrinsic sex determinant, foxl3, triggers germline feminization by activating two genetic pathways that regulate folliculogenesis and meiosis. Here, we identified a pathway involving a dome-shaped microtubule structure that may be the basis of oocyte polarity. This structure was first established in primordial germ cells in both sexes, but was maintained only during oogenesis and was destabilized in differentiating spermatogonia under the influence of Sertoli cells expressing dmrt1. Although foxl3 was dispensable for this pathway, dazl was involved in the persistence of the microtubule dome at the time of gonocyte development. In addition, disruption of the microtubule dome caused dispersal of bucky ball RNA, suggesting the structure may be prerequisite for the Balbiani body. Collectively, the present findings provide mechanistic insight into the establishment of sex-specific polarity through the formation of a microtubule structure in germ cells, as well as clarifying the genetic pathways implementing oocyte-specific characteristics.

A thrombospondin-dependent pathway for a protective ER stress response.

Thrombospondin (Thbs) proteins are induced in sites of tissue damage or active remodeling. The endoplasmic reticulum (ER) stress response is also prominently induced with disease where it regulates protein production and resolution of misfolded proteins. Here we describe a function for Thbs as ER-resident effectors of an adaptive ER stress response. Thbs4 cardiac-specific transgenic mice were protected from myocardial injury, whereas Thbs4(-/-) mice were sensitized to cardiac maladaptation. Thbs induction produced a unique profile of adaptive ER stress response factors and expansion of the ER and downstream vesicles. Thbs bind the ER lumenal domain of activating transcription factor 6α (Atf6α) to promote its nuclear shuttling. Thbs4(-/-) mice showed blunted activation of Atf6α and other ER stress-response factors with injury, and Thbs4-mediated protection was lost upon Atf6α deletion. Hence, Thbs can function inside the cell during disease remodeling to augment ER function and protect through a mechanism involving regulation of Atf6α.

Proteomic analysis of fatty liver induced by starvation of medaka fish larvae.

When medaka fish (Oryzias latipes) larvae are grown in the absence of exogenous nutrition, the liver becomes dark and positive to Oil Red O staining from 7 days post-hatch (dph). We determined the mechanism of this starvation-induced development of fatty liver by proteomic analysis using livers obtained from larvae grown in the presence or absence of 2% glucose at 5 dph. Results showed that changes in the expression levels of enzymes involved in glycolysis or the tricarboxylic acid cycle were modest, whereas the expression levels of enzymes involved in amino acid catabolism or ß-oxidation of fatty acids were significantly elevated, suggesting that they become major energy sources under starvation conditions. Expression levels of enzymes for the uptake and ß-oxidation of fatty acids as well as synthesis of triacylglycerol were elevated, whereas those for the synthesis of cholesterol as well as export of cholesterol and triacylglycerol were decreased under starvation conditions, which explains the accumulation of triacylglycerol in the liver. Our results provide the basis for future research to understand how gene malfunction(s) affects the development of fatty liver, which can lead to nonalcoholic steatohepatitis and then to liver cirrhosis.Key words: amino acid catabolism, ß-oxidation, triacylglycerol, cholesterol, export.

ATF6ß Deficiency Elicits Anxiety-like Behavior and Hyperactivity Under Stress Conditions.

Activating transcription factor 6 (ATF6) is an endoplasmic reticulum (ER) stress-regulated transcription factor that induces expression of major molecular chaperones in the ER. We recently reported that ATF6ß, a subtype of ATF6, promoted survival of hippocampal neurons exposed to ER stress and excitotoxicity, at least in part by inducing expression of calreticulin, an ER molecular chaperone with high Ca2+-binding capacity. In the present study, we demonstrate that ATF6ß deficiency in mice also decreases calreticulin expression and increases expression of glucose-regulated protein 78, another ER molecular chaperone, in emotional brain regions such as the prefrontal cortex (PFC), hypothalamus, hippocampus, and amygdala. Comprehensive behavioral analyses revealed that Atf6b-/- mice exhibit anxiety-like behavior in the light/dark transition test and hyperactivity in the forced swim test. Consistent with these results, PFC and hypothalamic corticotropin-releasing hormone (CRH) expression was increased in Atf6b-/- mice, as was circulating corticosterone. Moreover, CRH receptor 1 antagonism alleviated anxiety-like behavior in Atf6b-/- mice. These findings suggest that ATF6ß deficiency produces anxiety-like behavior and hyperactivity via a CRH receptor 1-dependent mechanism. ATF6ß could play a role in psychiatric conditions in the emotional centers of the brain.

Endoplasmic Reticulum-Associated Degradation Controls Virus Protein Homeostasis, Which Is Required for Flavivirus Propagation.

Many positive-stranded RNA viruses encode polyproteins from which viral proteins are generated by processing the polyproteins. This system produces an equal amount of each viral protein, though the required amounts for each protein are not the same. In this study, we found the extra membrane-anchored nonstructural (NS) proteins of Japanese encephalitis virus and dengue virus are rapidly and selectively degraded by the endoplasmic reticulum-associated degradation (ERAD) pathway. Our gene targeting study revealed that ERAD involving Derlin2 and SEL1L, but not Derlin1, is required for the viral genome replication. Derlin2 is predominantly localized in the convoluted membrane (CM) of the viral replication organelle, and viral NS proteins are degraded in the CM. Hence, these results suggest that viral protein homeostasis is regulated by Derlin2-mediated ERAD in the CM, and this process is critical for the propagation of these viruses. IMPORTANCE The results of this study reveal the cellular ERAD system controls the amount of each viral protein in virus-infected cells and that this "viral protein homeostasis" is critical for viral propagation. Furthermore, we clarified that the "convoluted membrane (CM)," which was previously considered a structure with unknown function, serves as a kind of waste dump where viral protein degradation occurs. We also found that the Derlin2/SEL1L/HRD1-specific pathway is involved in this process, whereas the Derlin1-mediated pathway is not. This novel ERAD-mediated fine-tuning system for the stoichiometries of polyprotein-derived viral proteins may represent a common feature among polyprotein-encoding viruses.

Effects of cordierite particulate filters on diesel engine exhaust emissions in terms of pollution prevention approaches for better environmental management.

In this study, a specific diesel fuel is experimentally tested in a 4-cylindered diesel engine with and without a cordierite-based diesel particulate filter (CPF) to show the prevention of emissions by using an after treatment system (ATS). In this context, engine exhaust emissions, total particle concentration (TPC) and soot concentration are investigated. The diesel engine is firstly evaluated with the data directly measured from the engine output (DEO) (without after treatment option), and then the changes in the exhaust emission are examined by using an ATS which is a cordierite-based diesel particulate filter to prevent pollution. In this regard, total particle concentration of DEO option is found to be 6134041.20 1/cm3 and total particle concentration by using CPF is obtained to be 707.84 1/cm3. 99.99% reduction in TPC is achieved thanks to the use of CPF. The soot concentration of DEO option is calculated to be 2.158 mg/m3. However, the soot concentration is found to be 0.014 mg/m3 by using the CPF. The particulate matters are burned at high temperatures after being filtered at the exhaust output thanks to the regeneration process within the CPF after treatment. CO emissions decreased from 0.7489 g/kWh to 0.7273 g/kWh with the CPF utilization, while HC emissions decreased from 0.0965 g/kWh to 0.0900 g/kWh via CPF. However, an increase in CO2 and NOx emissions are observed due to oxidation in the CPF.

Development of a Rapid in vivo Assay to Evaluate the Efficacy of IRE1-specific Inhibitors of the Unfolded Protein Response Using Medaka Fish.

Three types of transmembrane protein, IRE1α/IRE1ß, PERK, and ATF6α/ATF6ß, are expressed ubiquitously in vertebrates as transducers of the unfolded protein response (UPR), which maintains the homeostasis of the endoplasmic reticulum. IRE1 is highly conserved from yeast to mammals, and transmits a signal by a unique mechanism, namely splicing of mRNA encoding XBP1, the transcription factor downstream of IRE1 in metazoans. IRE1 contains a ribonuclease domain in its cytoplasmic region which initiates splicing reaction by direct cleavage of XBP1 mRNA at the two stem loop structures. As the UPR is considered to be involved in the development and progression of various diseases, as well as in the survival and growth of tumor cells, UPR inhibitors have been sought. To date, IRE1 inhibitors have been screened using cell-based reporter assays and fluorescent-based in vitro cleavage assays. Here, we used medaka fish to develop an in vivo assay for IRE1α inhibitors. IRE1α, IRE1ß, ATF6α and ATF6ß are ubiquitously expressed in medaka. We found that IRE1α/ATF6α-double knockout is lethal, similarly to IRE1α/IRE1ß- and ATF6α/ATF6ß-double knockout. Therefore, IRE1 inhibitors are expected to confer lethality to ATF6α-knockout medaka but not to wild-type medaka. One compound named K114 was obtained from 1,280 compounds using this phenotypic screening. K114 inhibited ER stress-induced splicing of XBP1 mRNA as well as reporter luciferase expression in HCT116 cells derived from human colorectal carcinoma, and inhibited ribonuclease activity of human IRE1α in vitro. Thus, this phenotypic assay can be used as a quick test for the efficacy of IRE1α inhibitors in vivo.Key words: endoplasmic reticulum, inhibitor screening, mRNA splicing, phenotypic assay, unfolded protein response.

Reinvestigation of Disulfide-bonded Oligomeric Forms of the Unfolded Protein Response Transducer ATF6.

ATF6α is an endoplasmic reticulum (ER)-embedded transcription factor which is rapidly activated by ER stress, and a major regulator of ER chaperone levels in vertebrates. We previously suggested that ATF6α occurs as a monomer, dimer and oligomer in the unstressed ER of Chinese hamster ovary cells due to the presence of two evolutionarily conserved cysteine residues in its luminal region (C467 and C618), and showed that ATF6α is reduced upon ER stress, such that only reduced monomer ATF6α is translocated to the Golgi apparatus for activation by proteolysis. However, mutagenesis analysis (C467A and C618A) revealed that the C618A mutant behaves in an unexpected manner (monomer and oligomer) during non-reducing SDS-PAGE, for reasons which remained unclear. Here, we used human colorectal carcinoma-derived HCT116 cells deficient in ATF6α and its relevant ATF6ß, and found that ATF6α dimer and oligomer are both dimers, which we designated C618-dimer and C467-dimer, respectively. We demonstrated that C467-dimer (previously considered an oligomer) behaved bigger than C618-dimer (previously considered a dimer) during non-reducing SDS-PAGE, based on their disulfide-bonded structures. Furthermore, ATF6α monomer physically associates with another ATF6α monomer in the absence of disulfide bonding, which renders two C467 residues in close proximity so that formation of C467-dimer is much easier than that of C618-dimer. In contrast, C618-dimer is more easily reduced upon ER stress. Thus, our analysis revealed that all forms of ATF6α, namely monomer, C618-dimer and C467-dimer, are activated by single reduction of a disulfide bond in response to ER stress, ensuring the rapidity of ATF6α activation.Key words: disulfide-bonded structure, endoplasmic reticulum, membrane-bound transcription factor, non-reducing SDS-PAGE, unfolded protein response.

The exercise-inducible bile acid receptor Tgr5 improves skeletal muscle function in mice.

TGR5 (also known as G protein-coupled bile acid receptor 1, GPBAR1) is a G protein-coupled bile acid receptor that is expressed in many diverse tissues. TGR5 is involved in various metabolic processes, including glucose metabolism and energy expenditure; however, TGR5's function in skeletal muscle is not fully understood. Using both gain- and loss-of-function mouse models, we demonstrate here that Tgr5 activation promotes muscle cell differentiation and muscle hypertrophy. Both young and old transgenic mice with muscle-specific Tgr5 expression exhibited increased muscle strength. Moreover, we found that Tgr5 expression is increased by the unfolded protein response (UPR), which is an adaptive response required for maintenance of endoplasmic reticulum (ER) homeostasis. Both ER stress response element (ERSE)- and unfolded protein response element (UPRE)-like sites are present in the 5' upstream region of the Tgr5 gene promoter and are essential for Tgr5 expression by Atf6α (activating transcription factor 6α), a well known UPR-activated transcriptional regulator. We observed that in the skeletal muscle of mice, exercise-induced UPR increases Tgr5 expression, an effect that was abrogated in Atf6α KO mice, indicating that Atf6α is essential for this response. These findings indicate that the bile acid receptor Tgr5 contributes to improved muscle function and provide an additional explanation for the beneficial effects of exercise on skeletal muscle activity.

ATF6α downregulation of PPARα promotes lipotoxicity-induced tubulointerstitial fibrosis.

Tubulointerstitial fibrosis is a strong predictor of progression in patients with chronic kidney disease, and is often accompanied by lipid accumulation in renal tubules. However, the molecular mechanisms modulating the relationship between lipotoxicity and tubulointerstitial fibrosis remain obscure. ATF6α, a transcription factor of the unfolded protein response, is reported to be an upstream regulator of fatty acid metabolism. Owing to their high energy demand, proximal tubular cells (PTCs) use fatty acids as their main energy source. We therefore hypothesized that ATF6α regulates PTC fatty acid metabolism, contributing to lipotoxicity-induced tubulointerstitial fibrosis. Overexpression of activated ATF6α transcriptionally downregulated peroxisome proliferator-activated receptor-α (PPARα), the master regulator of lipid metabolism, leading to reduced activity of fatty acid ß-oxidation and cytosolic accumulation of lipid droplets in a human PTC line (HK-2). ATF6α-induced lipid accumulation caused mitochondrial dysfunction, enhanced apoptosis, and increased expression of connective tissue growth factor (CTGF), as well as reduced cell viability. Atf6α-/- mice had sustained expression of PPARα and less tubular lipid accumulation following unilateral ischemia-reperfusion injury (uIRI), resulting in the amelioration of apoptosis; reduced expression of CTGF, α-smooth muscle actin, and collagen I; and less tubulointerstitial fibrosis. Administration of fenofibrate, a PPARα agonist, reduced lipid accumulation and tubulointerstitial fibrosis in the uIRI model. Taken together, these findings suggest that ATF6α deranges fatty acid metabolism in PTCs, which leads to lipotoxicity-mediated apoptosis and CTGF upregulation, both of which promote tubulointerstitial fibrosis.

Environmental pollution cost analyses of biodiesel and diesel fuels for a diesel engine.

In this study, Japanese Industrial Standard diesel no 2 and waste cooking oil biodiesel fuels are compared in terms of environmental pollution cost analysis. The experiments of the diesel and biodiesel fueled diesel engine are done at 100 Nm, 200 Nm and full load (294 Nm), while engine speed is constant at 1800 rpm. The method used in this study consists of a combination of economic and environmental parameters. According to the analyses, the total environmental pollution cost of the biodiesel is higher than the diesel fuel. On the other hand, the total cost of the CO2 emissions of the diesel fuel is generally found to be higher than biodiesel fuel in terms of the life cycle based environmental pollution cost. The specific environmental pollution cost is found as minimum at full load to be 2.217 US cent/kWh for the diesel fuel and 2.449 US cent/kWh for the biodiesel fuel at full load. On the other hand, the life cycle based specific environmental pollution cost is determined as minimum at full load to be 5.050 US cent/kWh for the diesel fuel and 5.309 US cent/kWh for the biodiesel fuel. The biodiesel fuel has higher values than diesel fuel in terms of the specific environmental pollution cost rates. The maximum total carbon dioxide emission rate is found as 0.2405 × 10-3 kg/kJ for the biodiesel fuel at 100 Nm engine torque and the minimum one is obtained as 0.1884 × 10-3 kg/kJ for the diesel fuel at full load. When the payback periods of the fuels are examined, biodiesel has longer period than diesel. The environmental payback period and life cycle based environmental payback period are also compared for fuels. In this context, the biodiesel has longer environmental payback periods rates than diesel. Consequently, the biodiesel fueled engine has higher environmental pollution cost rates than the diesel fueled engine, while the total carbon dioxide parameter of the diesel fuel is close to the biodiesel fuel's rate. Also, all of the other environmental parameters of diesel fuel is generally better than biodiesel. Consequently, the diesel fuel is generally better option than the biodiesel considering environmental aspects. For better environmental management, the diesel fuel utilization in the diesel engine is slightly better option than biodiesel fuel in terms of environmental pollution cost analysis.

SEL1L-dependent Substrates Require Derlin2/3 and Herp1/2 for Endoplasmic Reticulum-associated Degradation.

Accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). The ATF6 pathway is one of the three major pathways in vertebrates. Although ATF6, a transmembrane-type glycoprotein in the ER, functions as a UPR sensor/transducer, it is an unstable protein with a half-life of approximately 2 h and is constitutively subjected to the ER-associated degradation system with the location of the misfolded part in the ER lumen (ERAD-L). ERAD-L substrates are delivered to the cytosol through the retrotranslocon, which likely contains HRD1 (E3), gp78 (E3), SEL1L (a partner of HRD1), Derlin1/2/3 and Herp1/2. We previously showed that ATF6 represents a novel transmembrane-type ERAD-L substrate requiring both EDEM1/2/3-mediated mannose trimming and SEL1L. Here, by constructing and analyzing chicken DT40 cells deficient in various components of the retrotranslocon, we show that degradation of ATF6 requires Derlin2 or Derlin3 and that Derlin2 and Derlin3 are redundant for ERAD-L of ATF6. We further show that degradation of ATF6 requires Herp1 or Herp2 and that Herp1 and Herp2 are redundant for ERAD-L of ATF6. Furthermore, by investigating five more ERAD-L substrates, we show that SEL1L-dependent substrates require Derlin2/3 and Herp1/2 regardless of their soluble or transmembrane nature. Our results suggest that ERAD-L substrates take several routes to the cytosol. The HRD1-engaged route 1 requires SEL1L, Derlin2 or Derlin3, and Herp1 or Herp2, whereas the HRD1-engaged route 2 does not require them functionally. It remains to be determined whether the latter requires Derlin1 and whether these two routes are compositionally distinct.Key words: endoplasmic reticulum, proteasome, protein degradation, protein misfolding, ubiquitin.

Endoplasmic Reticulum (ER) Stress Induces Sirtuin 1 (SIRT1) Expression via the PI3K-Akt-GSK3ß Signaling Pathway and Promotes Hepatocellular Injury.

Sirtuin 1 (SIRT1), an NAD(+)-dependent histone deacetylase, plays crucial roles in various biological processes including longevity, stress response, and cell survival. Endoplasmic reticulum (ER) stress is caused by dysfunction of ER homeostasis and exacerbates various diseases including diabetes, fatty liver, and chronic obstructive pulmonary disease. Although several reports have shown that SIRT1 negatively regulates ER stress and ER stress-induced responses in vitro and in vivo, the effect of ER stress on SIRT1 is less explored. In this study, we showed that ER stress induced SIRT1 expression in vitro and in vivo. We further determined the molecular mechanisms of how ER stress induces SIRT1 expression. Surprisingly, the conventional ER stress-activated transcription factors XBP1, ATF4, and ATF6 seem to be dispensable for SIRT1 induction. Based on inhibitor screening experiments with SIRT1 promoter, we found that the PI3K-Akt-GSK3ß signaling pathway is required for SIRT1 induction by ER stress. Moreover, we showed that pharmacological inhibition of SIRT1 by EX527 inhibited the ER stress-induced cellular death in vitro and severe hepatocellular injury in vivo, indicating a detrimental role of SIRT1 in ER stress-induced damage responses. Collectively, these data suggest that SIRT1 expression is up-regulated by ER stress and contributes to ER stress-induced cellular damage.

Atf6α deficiency suppresses microglial activation and ameliorates pathology of experimental autoimmune encephalomyelitis.

Accumulating evidence suggests a critical role for the unfolded protein response in multiple sclerosis (MS) and in its animal model, experimental autoimmune encephalomyelitis (EAE). In this study, we investigated the relevance of activating transcription factor 6α (ATF6α), an upstream regulator of part of the unfolded protein response, in EAE. The expressions of ATF6α-target molecular chaperones such as glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94) were enhanced in the acute inflammatory phase after induction of EAE. Deletion of Atf6α suppressed the accumulation of T cells and microglia/macrophages in the spinal cord, and ameliorated the clinical course and demyelination after EAE induction. In contrast to the phenotypes in the spinal cord, activation status of T cells in the peripheral tissues or in the culture system was not different between two genotypes. Bone marrow transfer experiments and adoptive transfer of autoimmune CD4+ T cells to recipient mice (passive EAE) also revealed that CNS-resident cells are responsible for the phenotypes observed in Atf6α-/- mice. Further experiments with cultured cells indicated that inflammatory response was reduced in Atf6α-/- microglia, but not in Atf6α-/- astrocytes, and was associated with proteasome-dependent degradation of NF-κB p65. Thus, our results demonstrate a novel role for ATF6α in microglia-mediated CNS inflammation. We investigated the relevance of ATF6α, an upstream regulator of part of the UPR, in EAE. Deletion of Atf6α suppressed inflammation, and ameliorated demyelination after EAE. Bone marrow transfer experiments and adoptive transfer of autoimmune CD4+ T cells revealed that CNS-resident cells are responsible for the phenotypes in Atf6α-/- mice. Furthermore, inflammatory response was reduced in Atf6α-/- microglia, and was associated with degradation of NF-κB p65. Our results demonstrate a novel role for ATF6α in microglia-mediated inflammation. Cover image for this issue: doi: 10.1111/jnc.13346.

XBP 1-Deficiency Abrogates Neointimal Lesion of Injured Vessels Via Cross Talk With the PDGF Signaling.

OBJECTIVE: Smooth muscle cell (SMC) migration and proliferation play an essential role in neointimal formation after vascular injury. In this study, we intended to investigate whether the X-box-binding protein 1 (XBP1) was involved in these processes. APPROACH AND RESULTS: In vivo studies on femoral artery injury models revealed that vascular injury triggered an immediate upregulation of XBP1 expression and splicing in vascular SMCs and that XBP1 deficiency in SMCs significantly abrogated neointimal formation in the injured vessels. In vitro studies indicated that platelet-derived growth factor-BB triggered XBP1 splicing in SMCs via the interaction between platelet-derived growth factor receptor ß and the inositol-requiring enzyme 1α. The spliced XBP1 (XBP1s) increased SMC migration via PI3K/Akt activation and proliferation via downregulating calponin h1 (CNN1). XBP1s directed the transcription of mir-1274B that targeted CNN1 mRNA degradation. Proteomic analysis of culture media revealed that XBP1s decreased transforming growth factor (TGF)-ß family proteins secretion via transcriptional suppression. TGF-ß3 but not TGF-ß1 or TGF-ß2 attenuated XBP1s-induced CNN1 decrease and SMC proliferation. CONCLUSIONS: This study demonstrates for the first time that XBP1 is crucial for SMC proliferation via modulating the platelet-derived growth factor/TGF-ß pathways, leading to neointimal formation.

Deletion of Atf6α impairs astroglial activation and enhances neuronal death following brain ischemia in mice.

To dissect the role of endoplasmic reticulum (ER) stress and unfolded protein response in brain ischemia, we investigated the relevance of activating transcription factor 6α (ATF6α), a master transcriptional factor in the unfolded protein response, after permanent middle cerebral artery occlusion (MCAO) in mice. Enhanced expression of glucose-regulated protein78, a downstream molecular chaperone of ATF6α, was observed in both neurons and glia in the peri-infarct region of wild-type mice after MCAO. Analysis using wild-type and Atf6α(-/-) mice revealed a larger infarct volume and increased cell death in the peri-ischemic region of Atf6α(-/-) mice 5 days after MCAO. These phenotypes in Atf6α(-/-) mice were associated with reduced levels of astroglial activation/glial scar formation, and a spread of tissue damage into the non-infarct area. Further analysis in mice and cultured astrocytes revealed that signal transducer and activator of transcription 3 (STAT3)-glial fibrillary acidic protein signaling were diminished in Atf6α(-/-) astrocytes. A chemical chaperone, 4-phenylbutyrate, restored STAT3-glial fibrillary acidic protein signaling, while ER stressors, such as tunicamycin and thapsigargin, almost completely abolished signaling in cultured astrocytes. Furthermore, ER stress-induced deactivation of STAT3 was mediated, at least in part, by the ER stress-responsive tyrosine phosphatase, TC-PTP/PTPN2. These results suggest that ER stress plays critical roles in determining the level of astroglial activation and neuronal survival after brain ischemia.

An IFN-γ-stimulated ATF6-C/EBP-ß-signaling pathway critical for the expression of Death Associated Protein Kinase 1 and induction of autophagy.

The IFN family of cytokines operates a frontline defense against pathogens and neoplastic cells in vivo by controlling the expression of several genes. The death-associated protein kinase 1 (DAPK1), an IFN-γ-induced enzyme, controls cell cycle, apoptosis, autophagy, and tumor metastasis, and its expression is frequently down-regulated in a number of human tumors. Although the biochemical action of DAPK1 is well understood, mechanisms that regulate its expression are unclear. Previously, we have shown that transcription factor C/EBP-ß is required for the basal and IFN-γ-induced expression of DAPK1. Here, we show that ATF6, an ER stress-induced transcription factor, interacts with C/EBP-ß in an IFN-stimulated manner and is obligatory for Dapk1 expression. IFN-stimulated proteolytic processing of ATF6 and ERK1/2-mediated phosphorylation of C/EBP-ß are necessary for these interactions. More importantly, IFN-γ failed to activate autophagic response in cells lacking either ATF6 or C/EBP-ß. Consistent with these observations, the Atf6(-/-) mice were highly susceptible to lethal bacterial infections compared with the wild-type mice. These studies not only unravel an IFN signaling pathway that controls cell growth and antibacterial defense, but also expand the role of ATF6 beyond ER stress.

The unfolded protein response: the dawn of a new field.

Originating from cancer research in mammalian cultured cells, the entirely new field of the unfolded protein response (UPR) was born in 1988. The UPR is a transcriptional induction program coupled with intracellular signaling from the endoplasmic reticulum (ER) to the nucleus to maintain the homeostasis of the ER, an organelle which controls the quality of proteins destined for the secretory pathway. Extremely competitive analyses using the budding yeast Saccharomyces cerevisiae revealed that although signaling from both the ER and cell surface is initiated by activation of a transmembrane protein kinase, the mechanism downstream of ER-resident Ire1p, a sensor molecule of the UPR, is unique. Thus, unconventional spliceosome-independent mRNA splicing is utilized to produce the highly active transcription factor Hac1p. This is the autobiographical story of how a young and not yet independent scientist competed with a very famous full professor in the early days of UPR research, which ultimately lead to their sharing Lasker Basic Medical Research Award in 2014.

The unfolded protein response transducer ATF6 represents a novel transmembrane-type endoplasmic reticulum-associated degradation substrate requiring both mannose trimming and SEL1L protein.

Proteins misfolded in the endoplasmic reticulum (ER) are cleared by the ubiquitin-dependent proteasome system in the cytosol, a series of events collectively termed ER-associated degradation (ERAD). It was previously shown that SEL1L, a partner protein of the E3 ubiquitin ligase HRD1, is required for degradation of misfolded luminal proteins (ERAD-Ls substrates) but not misfolded transmembrane proteins (ERAD-Lm substrates) in both mammalian and chicken DT40 cells. Here, we analyzed ATF6, a type II transmembrane glycoprotein that serves as a sensor/transducer of the unfolded protein response, as a potential ERAD-Lm substrate in DT40 cells. Unexpectedly, degradation of endogenous ATF6 and exogenously expressed chicken and human ATF6 by the proteasome required SEL1L. Deletion analysis revealed that the luminal region of ATF6 is a determinant for SEL1L-dependent degradation. Chimeric analysis showed that the luminal region of ATF6 confers SEL1L dependence on type I transmembrane protein as well. In contrast, degradation of other known type I ERAD-Lm substrates (BACE457, T-cell receptor-α, CD3-δ, and CD147) did not require SEL1L. Thus, ATF6 represents a novel type of ERAD-Lm substrate requiring SEL1L for degradation despite its transmembrane nature. In addition, endogenous ATF6 was markedly stabilized in wild-type cells treated with kifunensine, an inhibitor of α1,2-mannosidase in the ER, indicating that degradation of ATF6 requires proper mannose trimming. Our further analyses revealed that the five ERAD-Lm substrates examined are classified into three subgroups based on their dependence on mannose trimming and SEL1L. Thus, ERAD-Lm substrates are degraded through much more diversified mechanisms in higher eukaryotes than previously thought.

Vascular endothelial cell growth-activated XBP1 splicing in endothelial cells is crucial for angiogenesis.

BACKGROUND: Vascular endothelial cell growth factor plays a pivotal role in angiogenesis via regulating endothelial cell proliferation. The X-box binding protein 1 (XBP1) is believed to be a signal transducer in the endoplasmic reticulum stress response. It is unknown whether there is crosstalk between vascular endothelial cell growth factor signaling and XBP1 pathway. METHODS AND RESULTS: We found that vascular endothelial cell growth factor induced the kinase insert domain receptor internalization and interaction through C-terminal domain with the unspliced XBP1 and the inositol requiring enzyme 1 α in the endoplasmic reticulum, leading to inositol requiring enzyme 1 α phosphorylation and XBP1 mRNA splicing, which was abolished by siRNA-mediated knockdown of kinase insert domain receptor. Spliced XBP1 regulated endothelial cell proliferation in a PI3K/Akt/GSK3ß/ß-catenin/E2F2-dependent manner and modulated the cell size increase in a PI3K/Akt/GSK3ß/ß-catenin/E2F2-independent manner. Knockdown of XBP1 or inositol requiring enzyme 1 α decreased endothelial cell proliferation via suppression of Akt/GSK3ß phosphorylation, ß-catenin nuclear translocation, and E2F2 expression. Endothelial cell-specific knockout of XBP1 (XBP1ecko) in mice retarded the retinal vasculogenesis in the first 2 postnatal weeks and impaired the angiogenesis triggered by ischemia. Reconstitution of XBP1 by Ad-XBP1s gene transfer significantly improved angiogenesis in ischemic tissue in XBP1ecko mice. Transplantation of bone marrow from wild-type o XBP1ecko mice could also slightly improve the foot blood reperfusion in ischemic XBP1ecko mice. CONCLUSIONS: These results suggest that XBP1 can function via growth factor signaling pathways to regulate endothelial proliferation and angiogenesis.