Derlin-1 Deficiency Is Embryonic Lethal, Derlin-3 Deficiency Appears Normal, and Herp Deficiency Is Intolerant to Glucose Load and Ischemia in Mice

Abstract

Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) causes a cellular condition called ER stress. To overcome ER stress, unfolded proteins are eliminated by an ER-associated degradation (ERAD) system. To explore the physiological requirements for ERAD-related membrane proteins in mammals, we generated Derlin-1–, Derlin-3–, and Herp-deficient mice by gene targeting. Complete loss of Derlin-1 caused embryonic lethality at around E7–E8 (early somite stages). In contrast, Derlin-3– and Herp-deficient mice were born alive with the expected Mendelian frequency, and were superficially indistinguishable from wild-type mice. However, in the Derlin-3– and Herp-deficient mouse organs, the expression levels of ERAD-related proteins were affected under both normal and ER stress conditions; specific effects differed among the organs. Degradation of ERAD substrates was reduced in the Herp-deficient liver, and Herp-deficient mice exhibited impaired glucose tolerance and vulnerability to brain ischemic injury, both of which are known to be implicated in ER stress. Our findings indicate that ERAD or uncharacterized functions involving Derlin-1 are essential in early embryonic development. Derlin-3– and Herp-deficient mice may become useful model animals for investigations of the physiological contribution of ERAD under stressful or pathological conditions.

About the Authors:

Yuka Eura

Affiliation: Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Suita, Osaka, Japan

Hiroji Yanamoto

Affiliation: Laboratory of Neurology and Neurosurgery, National Cerebral and Cardiovascular Center, Suita, Osaka, Japan

Yuji Arai

Affiliation: Department of Bioscience and Genetics, National Cerebral and Cardiovascular Center, Suita, Osaka, Japan

Tomohiko Okuda

Affiliation: Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Suita, Osaka, Japan

Toshiyuki Miyata

Affiliation: Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Suita, Osaka, Japan

Koichi Kokame

* E-mail: [email protected]

Affiliation: Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Suita, Osaka, Japan

Introduction

Unfolded or misfolded proteins accumulated in the endoplasmic reticulum (ER) can induce ER stress, which leads to defects in protein homeostasis. To overcome ER stress, the cells operate several survival systems, one of which is a disposal mechanism called ER-associated degradation (ERAD). In ERAD, misfolded proteins are recognized in the ER and retrotranslocated to the cytosol to be degraded by the ubiquitin-proteasome system [1], [2], [3], [4]. Multiple proteins participate in the process; in particular, ER-membrane proteins including HRD1, Derlin, and Herp work together in functional complexes termed ERAD complexes.

HRD1 is a transmembrane E3 ubiquitin ligase that mediates ubiquitination of ERAD substrates. Yeast Hrd1p/Der3p is essential for degradation of HMG-CoA reductase and mutated carboxypeptidase Y (CPY*) [5], [6]. A protein complex consisting of Hrd1p, Hrd3p, Der1p, and Usa1p deals with ER-luminal misfolded proteins; Hrd1p is the central membrane component of this complex [7]. Mammalian HRD1 also functions in ERAD, forming a complex with other proteins [8], [9], [10].

Mammalian Derlin family proteins, Derlin-1, Derlin-2, and Derlin-3, are orthologs of yeast Der1p, which was identified as a gene that complemented a yeast mutant that cannot degrade CPY* [11]. All Derlin family members contain transmembrane domains and are embedded in the ER membrane [12], [13]. In human, Derlin-1 and Derlin-2 mRNAs were expressed ubiquitously, and expression of Derlin-3 mRNA was relatively restricted to several tissues including placenta, pancreas, spleen, and small intestine [13]. Previous reports have delineated their functional roles in ERAD. Derlin-1 is the prime candidate for the ERAD retrotranslocation [12], [14]: anti-Derlin-1 antibody inhibited retrotranslocation of ERAD substrates in an in vitro retrotranslocation assay [15], and overexpression of a dominant-negative Derlin-1 mutant inhibits ERAD [12]. Overexpression of Derlin-2 or Derlin-3 accelerated ERAD of misfolded glycoproteins, and knockdown of either gene inhibited the degradation [13].

Herp was first reported as an ER stress–responsive protein expressed in cultured human vascular endothelial cells [16]. Herp is a representative target gene of the unfolded protein response (UPR); the induction of Herp mRNA expression following ER stress is mediated via two cis-acting ER stress–response elements, ERSE and ERSE-II [17]. Although Herp may play a broad range of biological roles [18], [19], [20], [21], [22], recent reports focus on its function in ERAD. Degradation of several ERAD substrates, including connexin 43 [20], CD3δ [9], [23], κLC [24], polycystin-2 [25], and α1-antitrypsin null Hong Kong (NHK) [10] is dependent on Herp. Usa1p, the yeast ortholog of Herp, may be the scaffold protein of the functional complex containing Hrd1p [26]; it is also involved in the degradation of Hrd1p itself [27].

Although growing evidence suggests that ERAD-complex components play important roles in cellular functions, little data exist regarding their systemic significance in mammals. HRD1-deficient mice die in utero around embryonic day 13.5, largely due to impairment of definitive erythropoiesis [28]. Derlin-2–deficient mice exhibit perinatal lethality and represented only 4% of mice at weaning [29]. To understand the physiological roles of the Derlin family members and Herp, we generated three mouse knockouts.

Results and Discussion

Offspring from the Cross of Heterozygous Mice

To generate Derlin-1–, Derlin-3–, and Herp-deficient mice, we constructed targeting vectors for disruption of the mouse Derl1, Derl3, and Herpud1 genes (Figure 1). All the F1 heterozygous mice, Derl1+/−, Derl3+/−, and Herpud1+/−, were viable and grew with no apparent phenotypic abnormalities under normal breeding conditions. Next, the heterozygous mice were crossed to generate homozygous mutants, and the genotypes of newborn offspring were determined by genomic PCR. From these matings, Derl3−/− and Herpud1−/− newborn mice were born in the expected Mendelian ratios, whereas Derl1−/− mice were not detected, indicating that the complete loss of Derlin-1 severely affected normal embryogenesis (Table 1).

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Figure 1. Targeted disruption of Derlin-1, Derlin-3, and Herp.

Structures of the mouse Derl1, Derl3, and Herpud1 loci, and the targeting vectors for disrupting each gene. Neo; neomycin resistance gene, IRES; internal ribosome entry site, DTA; diphtheria toxin A chain.

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Table 1. Genotype of offspring from the cross of heterozygous mice.

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Early Embryonic Death of Derl1−/−

To identify the embryonic stages at which Derl1−/− mice die, we analyzed the embryos in uteri of pregnant Derl1+/− female mice that had been crossed with Derl1+/− male mice (Table 2). We collected embryos and determined their genotypes by PCR. In the Derl1+/− crosses, only three (2.6%) Derl1−/− embryos were detected at E10.5, whereas 23 Derl1+/+ and 60 Derl1+/− embryos were present. At E8.5, however, the numbers of embryos, 13 (Derl1+/+), 28 (Derl1+/−), and 11 (Derl1−/−), were not significantly different from the expected Mendelian frequency. This suggested that most Derl1−/− embryos are resorbed between E8.5 and E10.5. Indeed, by E10.5, the Derl1+/− crosses resulted in a high frequency (26.5%) of the embryonic resorption compared with 4.5% and 1.9% resorption in wild-type C57BL/6 and ICR, respectively (Table 2). Genotypes of the resorbed embryos were not determined, to avoid miscounting caused by commingling of maternal tissues with embryos. If resorbed embryos were assumed to be Derl1−/−, the numbers of embryos [23 (Derl1+/+), 60 (Derl1+/−), and 34 (Derl1−/−)] conformed to the expected Mendelian frequency. The appearances of embryos collected at E8.5 are shown in Figure 2. In contrast to the normal development of Derl1+/+ and Derl1+/− embryos, all 11 Derl1−/− embryos showed obvious delay in their development; they appeared as wild-type up to E7.5. Western blotting analysis of the embryos at E7.5 demonstrated the presence of Derlin-1 in Derl1+/+ and Derl1+/− and the absence in Derl1−/− embryos (Figure S1). In conclusion, the Derl1−/− mice were embryonic lethal at E7–E8.

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Figure 2. Stereomicroscopic appearance of Derlin-1–deficient mouse embryos.

Derl1−/− embryos at E8.5 exhibited developmental delay and resorption, respectively, whereas Derl1+/+ and Derl1+/− embryos were normal. Bars, 50 µm.

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Table 2. Genotype of embryos from the cross of Derl1+/− heterozygous mice.

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In contrast, Derl3−/− and Herpud1−/− mice were born and grew normally. These findings indicated that the requirement for each Derlin in embryogenesis was different: Derlin-1 was essential in the early developmental stages (the beginning of the organogenesis), deletion of Derlin-2 results in perinatal death [29], and Derlin-3 was not essential in development. The loss of Derlin-3 may be compensated by Derlin-1 or Derlin-2. Our findings also indicated that Derlin-1, Derlin-3, and Herp are not essential for cell survival, because even embryonically lethal Derl1−/− survived until at least E7.5. This is consistent with the fact that Der1p, Dfm1p (Der1p homolog), and Usa1p are not essential for yeast survival.

Effects of Derlin-3 and Herp Deficiency in Adult Mouse Organs

Although both Derl3−/− and Herpud1−/− mice were born and grew normally, deficiency of Derlin-3 or Herp may affect functionally related proteins. To investigate the effect of the knockouts on the expression levels of ERAD-related proteins, we prepared tissue homogenates and total RNA from the liver, pancreas, and kidneys of control wild type (WT), Derl3−/−, and Herpud1−/− male mice that had been injected intraperitoneally 12 h before dissection with either PBS (control) or the protein N-glycosylation inhibitor tunicamycin (Tm), in order to induce pharmacological ER stress. The samples were subjected to Western blotting (Figures 3 and S2) and RT-PCR analyses (Figure 4).

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Figure 3. Effects of Derlin-3 and Herp deficiency on the levels of ERAD-related proteins.

WT, Derl3−/−, and Herpud1−/− mice were intraperitoneally injected with PBS as control (C) or Tm (2 µg/g body weight) 12 h before sacrifice. Liver, pancreas, and kidney homogenates were subjected to Western blotting using the indicated antibodies. Quantitative data are shown in Figure S2. Total RNAs prepared from the organs were subjected to RT-PCR in order to analyze the splicing of XBP1 (lowest panels). Quantitative data on the ratios (spliced/unspliced+spliced) are shown in Figure 4. *unspliced and **spliced forms of XBP1 mRNA.

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Figure 4. RT-PCR analysis of liver, pancreas, and kidneys from wild-type (WT), Derl3−/−, and Herpud1−/− mice.

Mice were intraperitoneally injected with PBS as control (C) or Tm (2 µg/g body weight) 12 h before sacrifice. Total RNAs were prepared from the liver, pancreas, and kidneys and subjected to RT-PCR. The products were analyzed by gel electrophoresis, followed by staining with SYBR Green I. Each band intensity was normalized to the mean intensity for PBS-injected WT mice. Data are expressed as means with range (n = 2). XBP1 (s); spliced form of XBP1 mRNA.

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In the WT liver, protein levels of Derlin-3 and Herp were quite low under normal conditions (Figure 3, left panels, C), but dramatically elevated 12 h after Tm treatment (left panels, Tm). HRD1, GRP78 (an ER-resident molecular chaperone), and VIMP (VCP-interacting membrane protein) were also increased by Tm treatment in WT mice. Similarly, in the Derl3−/− and Herpud1−/− liver, the levels of HRD1, GRP78, and VIMP were increased by Tm treatment. Although the Herp level was also increased by Tm treatment in the Derl3−/− liver, its levels were reduced by approximately 30% as compared with those in the Tm-treated WT (Figure S2), suggesting some physical or functional interactions between Derlin-3 and Herp. In the Herpud1−/− liver, regardless of Tm treatment, HRD1 level was higher than in WT. This may be partly explained by the fact that degradation of yeast Hrd1p is dependent on Usa1p (Herp ortholog) [27]. The levels of Derlin-1, Derlin-2, and AAA-ATPase p97/VCP were little affected by Derlin-3 or Herp deficiencies in the liver.

In the pancreas (Figure 3, middle panels), the levels of Derlin-1 and Derlin-2 were remarkably decreased by deficiency of Derlin-3 but not Herp. In the Herpud1−/− pancreas, the level of VIMP was lower than in WT and Derl3−/− pancreas, whereas the levels of Derlin-3, HRD1, and GRP78 were higher. In all three mouse strains, Tm had very little effect on expression levels of ERAD-related proteins in the pancreas, in contrast to the situation in the liver and kidneys. Although Derlin-3 was almost absent in the liver (left panels) and kidneys (right panels) under normal conditions, Derlin-3 could be clearly detected in the pancreas even without Tm treatment (middle panels). In the kidneys (right panels), responses to Tm treatment were broadly similar to those in the liver (left panels), but Tm-dependent upregulation of Derlin-3, GRP78, and VIMP in the kidneys was moderate compared to the liver. Thus, these findings indicated that the responses to pharmacologically induced ER stress vary among different organs.

The differences among organs in the effects of Tm and genetic deficiencies were observed not only at the protein level but also the mRNA level: the splicing of XBP1 mRNA, an indicator of the activation of ER stress sensor IRE1 [30], was induced by Tm treatment in the liver and kidneys, but not in the pancreas, of all the three mouse strains (Figures 3 and 4). The mRNA levels of other ERAD-related proteins were also investigated by RT-PCR (Figure 4): some of them (e.g., Derlin-3 and Herp mRNAs) exhibited dynamic changes similar to those observed in their protein levels, in all three organs; others (e.g., Derlin-1, Derlin-2, and HRD1 mRNAs) exhibited changes distinct from those observed in their protein levels. The decrease in the Derlin-1 and Derlin-2 protein levels in the Derl3−/− pancreas was likely post-translational because their mRNA levels were not decreased by Derlin-3 deficiency (compare Figures S2 and 4). The increased protein levels of HRD1 in the Herpud1−/− pancreas and kidney may indicate its resistance to Herp-dependent degradation as is the case in the liver. The inconsistency in the protein and mRNA levels of ERAD-related factors may be reflective of the complex, systemic, and organ-specific regulation of each protein at the level of transcription, translation, co-/post-translational modification, and degradation.

Effect of Herp Deficiency on ERAD Substrate Degradation

Partial loss and quantitative imbalance in ERAD-related factors were expected to affect the function of ERAD. Then, we utilized a hydrodynamics-based in vivo gene transfection technique, to observe the degradation of ERAD substrates in mice. This technique provides introduction and expression of exogenous genes in whole animals, especially in the liver [31]. We injected a plasmid DNA encoding NHK with a C-terminal green fluorescent protein (GFP) tag to Herpud1+/+ and Herpud1−/− mice. NHK, a truncated mutant of human α1-antitrypsin mainly produced by liver cells, and NHK-GFP are well-characterized as model substrates for ERAD [32], [33], [34], [35], [36]. Herp participates in the degradation of NHK in HeLa cells [10]. Western blotting of the Herpud1+/+ liver expressing NHK-GFP detected the expected band of NHK-GFP, and its levels were elevated by proteasome inhibition (Figure 5A). The exogenous expression of NHK-GFP in the Herpud1+/+ liver produced not only the intact band but also its fragment bands (Figure 5B), suggesting that NHK-GFP is degraded by ERAD in the mouse liver. In contrast, in the Herpud1−/− liver, the levels of the fragment bands were decreased with the accumulation of the intact band. These results indicated that Herp deficiency led to a partial defect in ERAD at least in the liver. Similarly, the other organs of Herpud1−/− mice and of Derl3−/− mice may also have some defect in ERAD. In that case, Herpud1−/− and Derl3−/− mice were expected to exhibit abnormality in challenge tests.

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Figure 5. Effect of Herp deficiency on NHK-GFP degradation.

(A) NHK-GFP was expressed in the livers of Herpud1+/+ mice in the absence or presence of epoxomicin (n = 2), and detected by Western blotting using anti-GFP antibodies. (B) NHK-GFP was expressed in the livers of Herpud1+/+ and Herpud1−/− mice (n = 2), and detected by Western blotting using anti-GFP antibodies.

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Glucose Tolerance and Insulin Response

The changes in ERAD-related proteins and defect in ERAD may induce some systemic phenotypes in Derlin-3– and Herp-deficient mice, especially under stressful conditions. Then, we compared tolerance to conditions associated with ER stress between Derl3+/+ and Derl3−/− and between Herpud1+/+ and Herpud1−/− mice. We performed a glucose tolerance test, because blood glucose levels are a good index of vulnerability to ER stress [37], [38]. After glucose was intraperitoneally injected in fasted mice, blood glucose levels in the tails were measured over time (Figure 6). At all time points examined, blood glucose levels in Derl3−/− mice were quite similar to those of Derl3+/+ mice (Figure 6A). In contrast, although blood glucose levels in Herpud1−/− mice were normal before glucose administration, Herpud1−/− mice showed significantly higher levels of blood glucose than Herpud1+/+ mice after glucose administration (Figure 6B). Because blood glucose levels were normal in Herpud1−/− mice after insulin injections (Figure 6C), Herp deficiency does not appear to cause aberrant responses to insulin. These results indicated that glucose tolerance was impaired not in Derl3−/− mice but in Herpud1−/− mice. Hyperglycemia has been reported in PERK- [39] and IRE1α-deficient mice [40], both of which are UPR trigger proteins that sense ER stress. Our finding in Herpud1−/− mice is the first case of abnormal glucose homeostasis caused by genetic disruption of one UPR target protein contained in ERAD complexes.

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Figure 6. Glucose tolerance and insulin response tests.

Blood glucose levels were examined at the indicated time points after intraperitoneal injection of glucose (A and B) or insulin (C) into Derl3−/− (A, open symbols), Derl3+/+ (A, filled symbols), Herpud1−/− (B and C, open symbols), and Herpud1+/+ mice (B and C, filled symbols). Data are expressed as the means with error bars of standard deviation (A, n = 12; B, n = 18; C, n = 16). Asterisks indicate p<0.01 (t-test) for Herpud1−/− vs. Herpud1+/+ mice.

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Cerebral Infarction Model

ER stress is induced in ischemic areas of the cerebrum, in which an infarction area forms as a result of lethal stress to neuronal cells [41], [42]. Herp contributes to protecting neural cells against ER stress-induced apoptosis in vitro [21], [22]. In this study, to examine the effects of Herp deficiency on resistance to cerebral ischemia in vivo, we measured the size of infarct lesions after three-vessel occlusion to cause temporary focal ischemia limited in the neocortex (Figure 7). Twenty-four hours after a 30-min ischemic period, infarction volumes were significantly larger in the neocortices of Herpud1−/− mice compared with Herpud1+/+ mice, indicating that Herp mediates resistance to the cerebral ischemia and reperfusion, probably via increasing resistance of the ER to ischemia-derived ER stress.

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Figure 7. Cerebral infarction models.

Neocortical infarction was induced using the temporary three-vessel occlusion method. Representative images of cerebral infarctions from Herpud1+/+ and Herpud1−/− mice stained with 2,3,5-triphenyl-tetrazolium chloride 24 h after ischemia are shown (A). White areas indicate infarct regions. Infarction volumes (n = 6) are expressed as the means with error bars of standard deviation (B).

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In summary, our study demonstrated that Derlin-1–deficient mice were embryonically lethal at much earlier stages (E7–E8), in contrast to HRD1- and Derlin-2–deficient mice, which die at E13.5 [28] and perinatally [29], respectively. At present, it is not clear whether the embryonic death of Derl1−/− mice is caused by a defect in ERAD function or by a loss of some as-yet uncharacterized function of the gene. We also showed that Derlin-3– and Herp-deficient mice were born and grew normally, but exhibited some changes in ERAD-related protein components in the liver, pancreas, and kidneys. In addition, the in vivo challenge tests indicated that Herp plays a protective role under certain stressful conditions to the ER. These mice may be useful for investigations of the physiological contribution of ERAD under stressful or pathological conditions such as diabetes and ischemia.

Materials and Methods

Ethics Statement

All live mouse experiments were approved by the Animal Care and Use Committee of the National Cerebral and Cardiovascular Center (approval number: 11019), and were performed in accordance with institutional and national guidelines and regulations.

Generation of Knockout Mice

F1 heterozygous mice, Derl1+/− and Derl3+/−, were generated from C57BL/6J-derived Bruce-4 embryonic stem cells (Millipore), and crossed to wild-type C57BL/6JJcl mice (CLEA Japan) for breeding. Herpud1+/− mice were generated from 129/SvJ-derived Go Germline embryonic stem cells (Incyte Genomics), and backcrossed to wild-type C57BL/6CrSlc mice (Japan SLC) for 10 generations. Targeting strategies for each gene are shown in Figure 1. The genotypes of offspring were examined by PCR analysis of DNA isolated from ear biopsy. The primer sequences and product sizes are listed in Table S1.

Analysis of Embryos

In crosses of Derl1+/− mice, embryos were microscopically isolated from uteri 7, 8, or 10 days after successful copulation, and the developmental stages were confirmed by appearance. The genotypes of embryos were determined by PCR as described above. To increase gestation efficiency, Derl1+/− mice with mixed genetic backgrounds, C57BL/6JJcl and Jcl:ICR (CLEA Japan), were used for the embryonic studies.

Tissue Preparation

To examine the effects of pharmacologically induced ER stress in vivo, 18-h fasted male mice were intraperitoneally injected with PBS or tunicamycin (WAKO, 200 µg/ml dissolved in PBS) at a dose of 2 µg/g body weight. After 12 h, the anesthetized mice were perfused with PBS, and their liver, pancreas, and kidneys were excised. The organ tissues were homogenized by Polytron PT1200 (Kinematica) in SDS-PAGE sample buffer. Tissue homogenates were subjected to Western blotting. Equal loading of the samples was confirmed by quantitating protein levels in gels stained with GelCode Blue Stain Reagent (Thermo Scientific). Alternatively, for RT-PCR analysis, total RNA was prepared from the excised tissues using the RNeasy Mini Kit (Qiagen).

Western Blotting

Anti-Derlin-3 antibody was raised in rabbits against a keyhole limpet hemocyanin–conjugated peptide, D215PDYLPLPEEQPEL228, derived from mouse Derlin-3. Anti-Herp was raised against S122DGLRQREVLRNLS135, derived from mouse Herp. These antibodies were subjected to affinity purification before use. Anti-Derlin-1 (MBL), anti-Derlin-2 (MBL), anti-HRD1 (Abgent), anti-VIMP (Sigma), anti-p97 (Santa Cruz Biotechnology), anti-KDEL (Stressgen Biotechnologies), anti-ß-actin (Sigma), and anti-GFP (Clontech) antibodies were purchased for use. Tissue homogenates were subjected to SDS-PAGE and transferred to PVDF membranes (Bio-Rad). The membranes were blocked with 5% skim milk, incubated with primary antibodies, and then incubated with HRP-labeled anti-rabbit or mouse IgG (Kirkegaard & Perry Laboratories). Signals were developed using Immobilon Western Chemiluminescent HRP Substrate (Millipore), and detected using an Image Analyzer LAS-3000 (Fujifilm).

RT-PCR

Total RNA prepared from mouse tissues was subjected to RT-PCR using the OneStep RT-PCR Kit (Qiagen). The primer sequences and product sizes are listed in Table S1. RT-PCR for XBP1 was designed to amplify both 140-bp (unspliced form) and 114-bp (spliced form) products. The PCR products were separated by polyacrylamide gel electrophoresis and stained with SYBR Green I Nucleic Acid Gel Stain (Lonza). The bands were detected with the LAS-3000 and quantified using the Image Gauge software (Fujifilm). Band intensities were normalized against GAPDH for target genes other than XBP1. For XBP1, the ratios of the spliced form to the unspliced plus spliced forms were evaluated.

Hydrodynamics-Based In Vivo Gene Transfection

The open reading frame of NHK was inserted into pAcGFP1-N1 plasmid vector (Clontech) for expression of NHK-GFP. The plasmid DNA was injected into male mice using a hydrodynamics-based transfection technique [31]. Briefly, 5 µg of the plasmid DNA was diluted in 1.5–2.2 ml (a volume equivalent to 8% of the body weight) of TransIT-EE Delivery Solution (Mirus Bio) and injected via the tail vein within 5–10 s. After 12 h, the anesthetized mice were perfused with PBS, and their liver was excised. Alternatively, the proteasome inhibitor epoxomicin (Peptide Institute, 0.2 mM dissolved in PBS) were intraperitoneally injected into mice at a dose of 15 µl/g body weight 12 h after hydrodynamics-based transfection, and after 12 h, the liver was excised. The liver tissues were homogenized by Polytron PT1200 in an ice-cold buffer (10 mM HEPES, 220 mM mannitol, 70 mM sucrose; pH 7.4) containing Complete Protease Inhibitor Cocktail (Roche), and subjected to subcellular fractionation using centrifugation. The microsomes were solubilized by a buffer containing 1% digitonin on ice, and subjected to Western blotting after removal of debris by centrifugation.

Glucose Tolerance and Insulin Response Tests

Glucose (200 mg/ml) was intraperitoneally injected into 13-week-old fasted male mice (2 mg/g body weight). Alternatively, human insulin (100 mU/ml, Lilly) was intraperitoneally injected into 14-week-old fasted male mice (1 mU/g body weight). Before the injection and 15, 30, 60, and 120 min postinjection, a drop of blood was collected from the tail end, and the glucose level was measured using GlucoCard GT-1810 (Arkray). All mice examined were confirmed to carry the normal Nnt (nicotinamide nucleotide transhydrogenase) gene by PCR-based genotyping using primer pairs, CAGGTAAGAAAGCTCCTGTTT/GTGCATTGAACCCTCAAAGG for the normal allele product (235 bp) and GTCTGATACGTCCTTCATGGT/CTAGCCTTTCAGTTTTCAGGA for the deleted allele product (344 bp). Nnt is occasionally disrupted by deletion of exons 7–11 in the C57BL/6J mouse substrains, which may lead to glucose intolerance and reduced insulin secretion [43], [44].

Ischemia Stroke Model

To assess tolerance to cerebral ischemia, 9-week-old male mice were subjected to the three-vessel occlusion technique [45], [46]. Briefly, the bilateral common carotid arteries were exposed and clipped under anesthesia to cause occlusion for the desired period of time. The left middle cerebral artery (MCA) was exposed by drilling a burr hole in the skull, and cauterized at the lateral edge of the olfactory tract to induce focal ischemia for cortical neurons in the MCA region. After 30 min of the three-vessel occlusion, the clips on the common carotid arteries were removed, which effectively terminate the induction of focal ischemia by increasing the collateral cerebral blood flow toward the targeted MCA territory up to the level that normal neurons never die. After 24 h, the mice were administered an overdose of pentobarbital and intracardially perfused with ice-cold PBS. The brain was removed, cut into six 1-mm-thick slices beginning at the frontal tip, and immersed in 2% 2,3,5-triphenyl-tetrazolium chloride to stain the viable tissues. The slices were then immersed in 4% paraformaldehyde/PBS, and the infarct lesion and hemispheric areas of each slice were measured by computer-assisted image analysis system. Finally, the infarct lesion size was adjusted for edema (infarction index) and calculated for each animal.

Supporting Information

[Figure omitted. See PDF.]

Figure S1.

Expression of Derlin-1 in Derl1+/+, Derl1+/−, and Derl1−/− mouse embryos. The embryos resulting from Derl1+/− matings were isolated from uteri at E7.5. Approximately half of each embryo was used for DNA preparation followed by PCR-genotyping, and the other half was subjected to Western blotting using anti-Derlin-1 and anti-ß-actin antibodies.

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(PDF)

Figure S2.

Western blotting analysis of liver, pancreas, and kidneys from wild-type (WT), Derl3−/−, and Herpud1−/− mice. Mice were intraperitoneally injected with PBS as control (C) or tunicamycin (Tm) 12 h before sacrifice. Liver, pancreas, and kidney homogenates were subjected to Western blotting using the indicated antibodies. Band intensities were normalized to the mean intensity of PBS-injected WT mice (for samples other than Derlin-3 and Herp) or Tm-injected WT mice (for Derlin-3 and Herp). Data are expressed as means with range (n = 2). The original blots are shown in Figure 3.

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(PDF)

Table S1.

Primer sequences for genotyping PCR and RT-PCR.

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(PDF)

Author Contributions

Conceived and designed the experiments: YE TM KK. Performed the experiments: YE HY YA TO KK. Analyzed the data: YE HY TM KK. Wrote the paper: YE TM KK.

Citation: Eura Y, Yanamoto H, Arai Y, Okuda T, Miyata T, Kokame K (2012) Derlin-1 Deficiency Is Embryonic Lethal, Derlin-3 Deficiency Appears Normal, and Herp Deficiency Is Intolerant to Glucose Load and Ischemia in Mice. PLoS ONE7(3): e34298. https://doi.org/10.1371/journal.pone.0034298

References

1. Tsai B, Ye Y, Rapoport TA (2002) Retro-translocation of proteins from the endoplasmic reticulum into the cytosol. Nat Rev Mol Cell Biol 3: 246–255.B. TsaiY. YeTA Rapoport2002Retro-translocation of proteins from the endoplasmic reticulum into the cytosol.Nat Rev Mol Cell Biol3246255

2. Meusser B, Hirsch C, Jarosch E, Sommer T (2005) ERAD: the long road to destruction. Nat Cell Biol 7: 766–772.B. MeusserC. HirschE. JaroschT. Sommer2005ERAD: the long road to destruction.Nat Cell Biol7766772

3. Vembar SS, Brodsky JL (2008) One step at a time: endoplasmic reticulum-associated degradation. Nat Rev Mol Cell Biol 9: 944–957.SS VembarJL Brodsky2008One step at a time: endoplasmic reticulum-associated degradation.Nat Rev Mol Cell Biol9944957

4. Hirsch C, Gauss R, Horn SC, Neuber O, Sommer T (2009) The ubiquitylation machinery of the endoplasmic reticulum. Nature 458: 453–460.C. HirschR. GaussSC HornO. NeuberT. Sommer2009The ubiquitylation machinery of the endoplasmic reticulum.Nature458453460

5. Hampton RY, Gardner RG, Rine J (1996) Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein. Mol Biol Cell 7: 2029–2044.RY HamptonRG GardnerJ. Rine1996Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein.Mol Biol Cell720292044

6. Bordallo J, Plemper RK, Finger A, Wolf DH (1998) Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins. Mol Biol Cell 9: 209–222.J. BordalloRK PlemperA. FingerDH Wolf1998Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins.Mol Biol Cell9209222

7. Carvalho P, Stanley AM, Rapoport TA (2010) Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p. Cell 143: 579–591.P. CarvalhoAM StanleyTA Rapoport2010Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p.Cell143579591

8. Lilley BN, Ploegh HL (2005) Multiprotein complexes that link dislocation, ubiquitination, and extraction of misfolded proteins from the endoplasmic reticulum membrane. Proc Natl Acad Sci USA 102: 14296–14301.BN LilleyHL Ploegh2005Multiprotein complexes that link dislocation, ubiquitination, and extraction of misfolded proteins from the endoplasmic reticulum membrane.Proc Natl Acad Sci USA1021429614301

9. Schulze A, Standera S, Buerger E, Kikkert M, van Voorden S, et al. (2005) The ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway. J Mol Biol 354: 1021–1027.A. SchulzeS. StanderaE. BuergerM. KikkertS. van Voorden2005The ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway.J Mol Biol35410211027

10. Kny M, Standera S, Hartmann-Petersen R, Kloetzel PM, Seeger M (2011) Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like domain-dependent manner. J Biol Chem 286: 5151–5156.M. KnyS. StanderaR. Hartmann-PetersenPM KloetzelM. Seeger2011Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like domain-dependent manner.J Biol Chem28651515156

11. Knop M, Finger A, Braun T, Hellmuth K, Wolf DH (1996) Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast. EMBO J 15: 753–763.M. KnopA. FingerT. BraunK. HellmuthDH Wolf1996Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast.EMBO J15753763

12. Lilley BN, Ploegh HL (2004) A membrane protein required for dislocation of misfolded proteins from the ER. Nature 429: 834–840.BN LilleyHL Ploegh2004A membrane protein required for dislocation of misfolded proteins from the ER.Nature429834840

13. Oda Y, Okada T, Yoshida H, Kaufman RJ, Nagata K, et al. (2006) Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation. J Cell Biol 172: 383–393.Y. OdaT. OkadaH. YoshidaRJ KaufmanK. Nagata2006Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation.J Cell Biol172383393

14. Ye Y, Shibata Y, Yun C, Ron D, Rapoport TA (2004) A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol. Nature 429: 841–847.Y. YeY. ShibataC. YunD. RonTA Rapoport2004A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol.Nature429841847

15. Wahlman J, DeMartino GN, Skach WR, Bulleid NJ, Brodsky JL, et al. (2007) Real-time fluorescence detection of ERAD substrate retrotranslocation in a mammalian in vitro system. Cell 129: 943–955.J. WahlmanGN DeMartinoWR SkachNJ BulleidJL Brodsky2007Real-time fluorescence detection of ERAD substrate retrotranslocation in a mammalian in vitro system.Cell129943955

16. Kokame K, Agarwala KL, Kato H, Miyata T (2000) Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress. J Biol Chem 275: 32846–32853.K. KokameKL AgarwalaH. KatoT. Miyata2000Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress.J Biol Chem2753284632853

17. Kokame K, Kato H, Miyata T (2001) Identification of ERSE-II, a new cis-acting element responsible for the ATF6-dependent mammalian unfolded protein response. J Biol Chem 276: 9199–9205.K. KokameH. KatoT. Miyata2001Identification of ERSE-II, a new cis-acting element responsible for the ATF6-dependent mammalian unfolded protein response.J Biol Chem27691999205

18. Sai X, Kawamura Y, Kokame K, Yamaguchi H, Shiraishi H, et al. (2002) Endoplasmic reticulum stress-inducible protein, Herp, enhances presenilin-mediated generation of amyloid ß-protein. J Biol Chem 277: 12915–12920.X. SaiY. KawamuraK. KokameH. YamaguchiH. Shiraishi2002Endoplasmic reticulum stress-inducible protein, Herp, enhances presenilin-mediated generation of amyloid ß-protein.J Biol Chem2771291512920

19. Sai X, Kokame K, Shiraishi H, Kawamura Y, Miyata T, et al. (2003) The ubiquitin-like domain of Herp is involved in Herp degradation, but not necessary for its enhancement of amyloid ß-protein generation. FEBS Lett 553: 151–156.X. SaiK. KokameH. ShiraishiY. KawamuraT. Miyata2003The ubiquitin-like domain of Herp is involved in Herp degradation, but not necessary for its enhancement of amyloid ß-protein generation.FEBS Lett553151156

20. Hori O, Ichinoda F, Yamaguchi A, Tamatani T, Taniguchi M, et al. (2004) Role of Herp in the endoplasmic reticulum stress response. Genes Cells 9: 457–469.O. HoriF. IchinodaA. YamaguchiT. TamataniM. Taniguchi2004Role of Herp in the endoplasmic reticulum stress response.Genes Cells9457469

21. Chan SL, Fu W, Zhang P, Cheng A, Lee J, et al. (2004) Herp stabilizes neuronal Ca2+ homeostasis and mitochondrial function during endoplasmic reticulum stress. J Biol Chem 279: 28733–28743.SL ChanW. FuP. ZhangA. ChengJ. Lee2004Herp stabilizes neuronal Ca2+ homeostasis and mitochondrial function during endoplasmic reticulum stress.J Biol Chem2792873328743

22. Chigurupati S, Wei Z, Belal C, Vandermey M, Kyriazis GA, et al. (2009) The homocysteine-inducible endoplasmic reticulum stress protein counteracts calcium store depletion and induction of CCAAT enhancer-binding protein homologous protein in a neurotoxin model of Parkinson disease. J Biol Chem 284: 18323–18333.S. ChigurupatiZ. WeiC. BelalM. VandermeyGA Kyriazis2009The homocysteine-inducible endoplasmic reticulum stress protein counteracts calcium store depletion and induction of CCAAT enhancer-binding protein homologous protein in a neurotoxin model of Parkinson disease.J Biol Chem2841832318333

23. Kim TY, Kim E, Yoon SK, Yoon JB (2008) Herp enhances ER-associated protein degradation by recruiting ubiquilins. Biochem Biophys Res Commun 369: 741–746.TY KimE. KimSK YoonJB Yoon2008Herp enhances ER-associated protein degradation by recruiting ubiquilins.Biochem Biophys Res Commun369741746

24. Okuda-Shimizu Y, Hendershot LM (2007) Characterization of an ERAD pathway for nonglycosylated BiP substrates, which require Herp. Mol Cell 28: 544–554.Y. Okuda-ShimizuLM Hendershot2007Characterization of an ERAD pathway for nonglycosylated BiP substrates, which require Herp.Mol Cell28544554

25. Liang G, Li Q, Tang Y, Kokame K, Kikuchi T, et al. (2008) Polycystin-2 is regulated by endoplasmic reticulum-associated degradation. Hum Mol Genet 17: 1109–1119.G. LiangQ. LiY. TangK. KokameT. Kikuchi2008Polycystin-2 is regulated by endoplasmic reticulum-associated degradation.Hum Mol Genet1711091119

26. Horn SC, Hanna J, Hirsch C, Volkwein C, Schutz A, et al. (2009) Usa1 functions as a scaffold of the HRD-ubiquitin ligase. Mol Cell 36: 782–793.SC HornJ. HannaC. HirschC. VolkweinA. Schutz2009Usa1 functions as a scaffold of the HRD-ubiquitin ligase.Mol Cell36782793

27. Carroll SM, Hampton RY (2010) Usa1p is required for optimal function and regulation of the Hrd1p endoplasmic reticulum-associated degradation ubiquitin ligase. J Biol Chem 285: 5146–5156.SM CarrollRY Hampton2010Usa1p is required for optimal function and regulation of the Hrd1p endoplasmic reticulum-associated degradation ubiquitin ligase.J Biol Chem28551465156

28. Yagishita N, Ohneda K, Amano T, Yamasaki S, Sugiura A, et al. (2005) Essential role of synoviolin in embryogenesis. J Biol Chem 280: 7909–7916.N. YagishitaK. OhnedaT. AmanoS. YamasakiA. Sugiura2005Essential role of synoviolin in embryogenesis.J Biol Chem28079097916

29. Dougan SK, Hu CC, Paquet ME, Greenblatt MB, Kim J, et al. (2011) Derlin-2-deficient mice reveal an essential role for protein dislocation in chondrocytes. Mol Cell Biol 31: 1145–1159.SK DouganCC HuME PaquetMB GreenblattJ. Kim2011Derlin-2-deficient mice reveal an essential role for protein dislocation in chondrocytes.Mol Cell Biol3111451159

30. Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K (2001) XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107: 881–891.H. YoshidaT. MatsuiA. YamamotoT. OkadaK. Mori2001XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor.Cell107881891

31. Liu F, Song Y, Liu D (1999) Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther 6: 1258–1266.F. LiuY. SongD. Liu1999Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA.Gene Ther612581266

32. Hosokawa N, Tremblay LO, You Z, Herscovics A, Wada I, et al. (2003) Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong α1-antitrypsin by human ER mannosidase I. J Biol Chem 278: 26287–26294.N. HosokawaLO TremblayZ. YouA. HerscovicsI. Wada2003Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong α1-antitrypsin by human ER mannosidase I.J Biol Chem2782628726294

33. Greenblatt EJ, Olzmann JA, Kopito RR (2011) Derlin-1 is a rhomboid pseudoprotease required for the dislocation of mutant α-1 antitrypsin from the endoplasmic reticulum. Nat Struct Mol Biol 18: 1147–1152.EJ GreenblattJA OlzmannRR Kopito2011Derlin-1 is a rhomboid pseudoprotease required for the dislocation of mutant α-1 antitrypsin from the endoplasmic reticulum.Nat Struct Mol Biol1811471152

34. Hosokawa N, Wada I, Nagasawa K, Moriyama T, Okawa K, et al. (2008) Human XTP3-B forms an endoplasmic reticulum quality control scaffold with the HRD1-SEL1L ubiquitin ligase complex and BiP. J Biol Chem 283: 20914–20924.N. HosokawaI. WadaK. NagasawaT. MoriyamaK. Okawa2008Human XTP3-B forms an endoplasmic reticulum quality control scaffold with the HRD1-SEL1L ubiquitin ligase complex and BiP.J Biol Chem2832091420924

35. Francisco AB, Singh R, Li S, Vani AK, Yang L, et al. (2010) Deficiency of suppressor enhancer Lin12 1 like (SEL1L) in mice leads to systemic endoplasmic reticulum stress and embryonic lethality. J Biol Chem 285: 13694–13703.AB FranciscoR. SinghS. LiAK VaniL. Yang2010Deficiency of suppressor enhancer Lin12 1 like (SEL1L) in mice leads to systemic endoplasmic reticulum stress and embryonic lethality.J Biol Chem2851369413703

36. Christianson JC, Olzmann JA, Shaler TA, Sowa ME, Bennett EJ, et al. (2012) Defining human ERAD networks through an integrative mapping strategy. Nat Cell Biol 14: 93–105.JC ChristiansonJA OlzmannTA ShalerME SowaEJ Bennett2012Defining human ERAD networks through an integrative mapping strategy.Nat Cell Biol1493105

37. Marciniak SJ, Ron D (2006) Endoplasmic reticulum stress signaling in disease. Physiol Rev 86: 1133–1149.SJ MarciniakD. Ron2006Endoplasmic reticulum stress signaling in disease.Physiol Rev8611331149

38. Scheuner D, Kaufman RJ (2008) The unfolded protein response: a pathway that links insulin demand with ß-cell failure and diabetes. Endocr Rev 29: 317–333.D. ScheunerRJ Kaufman2008The unfolded protein response: a pathway that links insulin demand with ß-cell failure and diabetes.Endocr Rev29317333

39. Harding HP, Zeng H, Zhang Y, Jungries R, Chung P, et al. (2001) Diabetes mellitus and exocrine pancreatic dysfunction in perk−/− mice reveals a role for translational control in secretory cell survival. Mol Cell 7: 1153–1163.HP HardingH. ZengY. ZhangR. JungriesP. Chung2001Diabetes mellitus and exocrine pancreatic dysfunction in perk−/− mice reveals a role for translational control in secretory cell survival.Mol Cell711531163

40. Iwawaki T, Akai R, Kohno K (2010) IRE1α disruption causes histological abnormality of exocrine tissues, increase of blood glucose level, and decrease of serum immunoglobulin level. PLoS One 5: e13052.T. IwawakiR. AkaiK. Kohno2010IRE1α disruption causes histological abnormality of exocrine tissues, increase of blood glucose level, and decrease of serum immunoglobulin level.PLoS One5e13052

41. DeGracia DJ, Montie HL (2004) Cerebral ischemia and the unfolded protein response. J Neurochem 91: 1–8.DJ DeGraciaHL Montie2004Cerebral ischemia and the unfolded protein response.J Neurochem9118

42. Ogawa S, Kitao Y, Hori O (2007) Ischemia-induced neuronal cell death and stress response. Antioxid Redox Signal 9: 573–587.S. OgawaY. KitaoO. Hori2007Ischemia-induced neuronal cell death and stress response.Antioxid Redox Signal9573587

43. Toye AA, Lippiat JD, Proks P, Shimomura K, Bentley L, et al. (2005) A genetic and physiological study of impaired glucose homeostasis control in C57BL/6J mice. Diabetologia 48: 675–686.AA ToyeJD LippiatP. ProksK. ShimomuraL. Bentley2005A genetic and physiological study of impaired glucose homeostasis control in C57BL/6J mice.Diabetologia48675686

44. Freeman H, Shimomura K, Horner E, Cox RD, Ashcroft FM (2006) Nicotinamide nucleotide transhydrogenase: a key role in insulin secretion. Cell Metab 3: 35–45.H. FreemanK. ShimomuraE. HornerRD CoxFM Ashcroft2006Nicotinamide nucleotide transhydrogenase: a key role in insulin secretion.Cell Metab33545

45. Yanamoto H, Xue JH, Miyamoto S, Nagata I, Nakano Y, et al. (2004) Spreading depression induces long-lasting brain protection against infarcted lesion development via BDNF gene-dependent mechanism. Brain Res 1019: 178–188.H. YanamotoJH XueS. MiyamotoI. NagataY. Nakano2004Spreading depression induces long-lasting brain protection against infarcted lesion development via BDNF gene-dependent mechanism.Brain Res1019178188

46. Yamamoto H, Kokame K, Okuda T, Nakajo Y, Yanamoto H, et al. (2011) NDRG4 protein-deficient mice exhibit spatial learning deficits and vulnerabilities to cerebral ischemia. J Biol Chem 286: 26158–26165.H. YamamotoK. KokameT. OkudaY. NakajoH. Yanamoto2011NDRG4 protein-deficient mice exhibit spatial learning deficits and vulnerabilities to cerebral ischemia.J Biol Chem2862615826165