[Show abstract][Hide abstract] 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.
PLoS ONE 01/2012; 7(3):e34298. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: N-myc downstream-regulated gene 2 (Ndrg2) is a differentiation- and stress-associated molecule predominantly expressed in astrocytes in the central nervous system (CNS). To study the expression and possible role of Ndrg2 in quiescent and activated astrocytes, mice were administrated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP), a Parkinson disease (PD)-related neurotoxin which causes both neurodegeneration and glial activation. Immunohistological analysis revealed that Ndrg2 was highly expressed in both types of astrocytes, but less so in astrocytes during the early process of activation. Ndrg2 was also expressed in astrocyte-like cells, but not in neurons, in human brains from PD and Cortico-basal degeneration (CBD) patients. In cultured astrocytes, gene silencing of Ndrg2 significantly enhanced the numbers of 5-bromo-2'-deoxy-uridine (BrdU)-incorporated and proliferating cell nuclear antigen (PCNA)-positive cells, and reduced the length of cell processes and the amount of F-actin. In contrast, adenovirus-mediated overexpression of Ndrg2 significantly reduced the numbers of BrdU-incorporated and PCNA-positive cells, and enhanced the amount of F-actin. Fractionation and immunocytochemical analysis further revealed that Ndrg2 was located in different cellular fractions including the cytosol and cell surface membranes. These results suggest that Ndrg2 may regulate astroglial activation through the suppression of cell proliferation and stabilization of cell morphology.
Neurochemistry International 06/2011; 59(1):21-7. · 2.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The N-myc downstream-regulated gene (NDRG) family consists of four related proteins, NDRG1-NDRG4, in mammals. We previously generated NDRG1-deficient mice that were unable to maintain myelin sheaths in peripheral nerves. This condition was consistent with human hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease type 4D, caused by a nonsense mutation of NDRG1. In contrast, the effects of genetic defects of the other NDRG members remain unknown. In this study, we focused on NDRG4, which is specifically expressed in the brain and heart. In situ mRNA hybridization on the brain revealed that NDRG4 was expressed in neurons of various areas. We generated NDRG4-deficient mice that were born normally with the expected Mendelian frequency. Immunochemical analysis demonstrated that the cortex of the NDRG4-deficient mice contained decreased levels of brain-derived neurotrophic factor (BDNF) and normal levels of glial cell line-derived neurotrophic factor, NGF, neurotrophin-3, and TGF-β1. Consistent with BDNF reduction, NDRG4-deficient mice had impaired spatial learning and memory but normal motor function in the Morris water maze test. When temporary focal ischemia of the brain was induced, the sizes of the infarct lesions were larger, and the neurological deficits were more severe in NDRG4-deficient mice compared with the control mice. These findings indicate that NDRG4 contributes to the maintenance of intracerebral BDNF levels within the normal range, which is necessary for the preservation of spatial learning and the resistance to neuronal cell death caused by ischemic stress.
Journal of Biological Chemistry 06/2011; 286(29):26158-65. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CMT4D disease is a severe autosomal recessive demyelinating neuropathy with extensive axonal loss leading to early disability, caused by mutations in the N-myc downstream regulated gene 1 (NDRG1). NDRG1 is expressed at particularly high levels in the Schwann cell (SC), but its physiological function(s) are unknown. To help with their understanding, we characterise the phenotype of a new mouse model, stretcher (str), with total Ndrg1 deficiency, in comparison with the hypomorphic Ndrg1 knock-out (KO) mouse. While both models display normal initial myelination and a transition to overt pathology between weeks 3 and 5, the markedly more severe str phenotype suggests that even low Ndrg1 expression results in significant phenotype rescue. Neither model replicates fully the features of CMT4D: although axon damage is present, regenerative capacity is unimpaired and the mice do not display the early severe axonal loss typical of the human disease. The widespread large fibre demyelination coincides precisely with the period of rapid growth of the animals and the dramatic (160-500-fold) increase in myelin volume and length in large fibres. This is followed by stabilisation after week 10, while small fibres remain unaffected. Gene expression profiling of str peripheral nerve reveals non-specific secondary changes at weeks 5 and 10 and preliminary data point to normal proteasomal function. Our findings do not support the proposed roles of NDRG1 in growth arrest, terminal differentiation, gene expression regulation and proteasomal degradation. Impaired SC trafficking failing to meet the considerable demands of nerve growth, emerges as the likely pathogenetic mechanism in NDRG1 deficiency.
Neurobiology of Disease 02/2011; 42(3):368-80. · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The N-myc downstream-regulated gene (NDRG) family consists of four proteins: NDRG1, NDRG2, NDRG3, and NDRG4 in mammals. NDRG1 has been thoroughly studied as an intracellular protein associated with stress response, cell growth, and differentiation. A nonsense mutation in the NDRG1 gene causes hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease type 4D. We previously generated Ndrg1-deficient mice and found that they exhibited peripheral nerve degeneration caused by severe demyelination, but that the complicated motor abilities were retained. These results implied that other NDRG family proteins may compensate for the NDRG1 deficiency in the central nervous system. In this study we raised specific antibodies against each member of the NDRG protein family and examined their cellular expression patterns in the mouse brain. In the cerebrum, NDRG1 and NDRG2 were localized in oligodendrocytes and astrocytes, respectively, whereas NDRG3 and NDRG4 were ubiquitous. In the cerebellum, NDRG1 and NDRG4 were localized in Purkinje cells and NDRG2 in Bergmann glial cells. NDRG3 was detected in the nuclei in most cells. These expression patterns demonstrated the cell type-specific and ubiquitous localization of the NDRG family proteins. Each NDRG may play a partially redundant role in specific cells in the brain.
Journal of Histochemistry and Cytochemistry 03/2008; 56(2):175-82. · 2.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously reported that N-myc downstream regulated gene-1 (NDRG1) is an early inducible protein during the maturation of mouse bone marrow-derived mast cells (BMMCs) toward a connective tissue mast cell-like phenotype. To clarify the function of NDRG1 in mast cells and allergic responses, we herein analyzed mast cell-associated phenotypes of mice lacking the Ndrg1 gene. Allergic responses including IgE-mediated passive systemic and cutaneous anaphylactic reactions were markedly attenuated in Ndrg1-deficient mice as compared with those in wild-type mice. In Ndrg1-deficient mice, dermal and peritoneal mast cells were decreased in number and morphologically abnormal with impaired degranulating ability. Ex vivo, Ndrg1-deficient BMMCs cocultured with Swiss 3T3 fibroblasts in the presence of stem cell factor, a condition that facilitates the maturation of BMMCs toward a CTMC-like phenotype, displayed less exocytosis than replicate wild-type cells after the cross-linking of FcepsilonRI or stimulation with compound 48/80, even though the exocytotic response of IL-3-maintained, immature BMMCs from both genotypes was comparable. Unlike degranulation, the production of leukotriene and cytokines by cocultured BMMCs was unaffected by NDRG1 deficiency. Taken together, the altered phenotypes of Ndrg1-deficient mast cells both in vivo and ex vivo suggest that NDRG1 has roles in the terminal maturation and effector function (degranulation) of mast cells.
The Journal of Immunology 07/2007; 178(11):7042-53. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Catechol-O-methyltransferase (COMT) is an enzyme that inactivates catecholamines. Several studies have suggested that this enzyme may play a role in blood pressure regulation. We previously reported that the expression levels of Comt mRNA in Dahl salt-sensitive (DS) rats were lower than those in Lewis (LEW) rats. However, the physiological significance of this phenomenon has not been investigated. The purpose of the present study was to evaluate the significance of lower expression of Comt in Dahl salt-sensitive hypertension. The Comt gene in DS rats has a palindromic insertion in 3'-untranslated region, which appears to be responsible for reduced mRNA stability. A genome-wide quantitative trait loci (QTL) analysis of blood pressure using 107 F2 rats indicated that a statistically significant QTL for pulse pressure was located at the Comt locus in chromosome 11. Microarray analysis confirmed that Comt was the only gene differentially expressed between DS and LEW rats in this chromosomal region. However, COMT inhibitors had no significant effects on blood pressure in either DS or LEW rats. Thus, Comt was excluded from the candidate genes contributing to salt-sensitive hypertension in DS rats. A true gene responsible for pulse pressure in this chromosome 11 region remains to be determined.
Hypertension Research 06/2007; 30(5):459-67. · 2.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genetically hypertensive rats provide an excellent model to investigate the genetic mechanisms of hypertension. We previously identified three differentially expressed genes, Acadsb (short/branched chain acyl-CoA dehydrogenase), Comt (catecholamine-O-methyltransferase), and Pnpo (pyridoxine 5'-phosphate oxidase), in hypertensive and normotensive rat kidneys as potential susceptibility genes for rat hypertension. We examined the association of human homologues of these genes with human hypertension.
We sequenced three genes using samples from 48 or 96 hypertensive patients, identified single nucleotide polymorphisms, and genotyped them in a population-based sample of 1818 Japanese individuals (771 hypertensive individuals and 1047 controls).
After adjustments for age, body mass index, present illness (hyperlipidaemia, diabetes mellitus), and lifestyle (smoking, alcohol consumption), multivariate logistic regression analysis revealed that -512A>G in ACADSB was associated with hypertension in women (AA vs AG + GG: odds ratio = 0.70, 95% confidence interval = 0.53-0.94). This single nucleotide polymorphism was in tight linkage disequilibrium with -254G>A. Furthermore, -1187G>C in COMT was associated with hypertension in men (GG vs CG + CC: odds ratio = 0.69, 95% confidence interval = 0.52-0.93) and was in tight linkage disequilibrium with 186C>T. After adjustments described above, -512 A>G and -254G>A in ACADSB were associated with variations in systolic blood pressure. ACADSB was in tight linkage disequilibrium with MGC35392 across a distance of 18.3 kb. COMT was not in linkage disequilibrium with any adjacent genes. Analysis indicated that two haplotypes of COMT were significantly associated with hypertension in men.
Our study suggests the possible involvement of genetic polymorphisms in ACADSB and COMT in essential hypertension in the Japanese population.
Journal of Hypertension 02/2007; 25(1):103-10. · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We previously selected a group of hypertension candidate genes by a key word search using the OMIM database of NCBI and validated 525 coding single nucleotide polymorphisms (SNPs) in 179 hypertension candidate genes by DNA sequencing in a Japanese population. In the present study, we examined the association between 61 non-synonymous SNPs and blood pressure variations and hypertension. We used DNA samples taken from 1,880 subjects in the Suita study, a population-based study using randomly selected subjects. Analyses of covariance adjusting for age, body mass index, hyperlipidemia, diabetes, smoking, drinking, and antihypertensive medication revealed that 17 polymorphisms in 16 genes (APOB, CAST, CLCNKB, CTNS, GHR, GYS1, HF1, IKBKAP, KCNJ11, LIPC, LPL, P2RY2, PON2, SLC4A1, TRH, VWF) were significantly associated with blood pressure variations. Multivariate logistic regression analysis with adjustment for the same factors revealed that 11 polymorphisms in 11 genes (CAST, CTLA4, F5, GC, GHR, LIPC, PLA2G7, SLC4A1, SLCI8A1, TRH, VWF) showed significant associations with hypertension. Five polymorphisms in five genes, CAST(calpastatin), LIPC (hepatic lipase), SLC4A1 (band 3 anion transporter), TRH (thyrotropin-releasing hormone), and VWF (von Willebrand factor), were significantly associated with both blood pressure variation and hypertension. Thus, our study suggests that these five genes were susceptibility genes for essential hypertension in this Japanese population.
Hypertension Research 09/2006; 29(8):611-9. · 2.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ADAMTS13 is a plasma metalloproteinase that regulates platelet adhesion and aggregation through cleavage of von Willebrand factor (VWF) multimers. In humans, genetic or acquired deficiency in ADAMTS13 causes thrombotic thrombocytopenic purpura (TTP), a condition characterized by thrombocytopenia and hemolytic anemia with microvascular platelet thrombi. In this study, we report characterization of mice bearing a targeted disruption of the Adamts13 gene. ADAMTS13-deficient mice were born in the expected mendelian distribution; homozygous mice were viable and fertile. Hematologic and histologic analyses failed to detect any evidence of thrombocytopenia, hemolytic anemia, or microvascular thrombosis. However, unusually large VWF multimers were observed in plasma of homozygotes. Thrombus formation on immobilized collagen under flow was significantly elevated in homozygotes in comparison with wild-type mice. Thrombocytopenia was more severely induced in homozygotes than in wild-type mice after intravenous injection of a mixture of collagen and epinephrine. Thus, a complete lack of ADAMTS13 in mice was a prothrombotic state, but it alone was not sufficient to cause TTP-like symptoms. The phenotypic differences of ADAMTS13 deficiencies between humans and mice may reflect differences in hemostatic system functioning in these species. Alternatively, factors in addition to ADAMTS13 deficiency may be necessary for development of TTP.
[Show abstract][Hide abstract] ABSTRACT: NDRG1 is an intracellular protein that is induced under a number of stress and pathological conditions, and it is thought to be associated with cell growth and differentiation. Recently, human NDRG1 was identified as a gene responsible for hereditary motor and sensory neuropathy-Lom (classified as Charcot-Marie-Tooth disease type 4D), which is characterized by early-onset peripheral neuropathy, leading to severe disability in adulthood. In this study, we generated mice lacking Ndrg1 to analyze its function and elucidate the pathogenesis of Charcot-Marie-Tooth disease type 4D. Histological analysis showed that the sciatic nerve of Ndrg1-deficient mice degenerated with demyelination at about 5 weeks of age. However, myelination of Schwann cells in the sciatic nerve was normal for 2 weeks after birth. Ndrg1-deficient mice showed muscle weakness, especially in the hind limbs, but complicated motor skills were retained. In wild-type mice, NDRG1 was abundantly expressed in the cytoplasm of Schwann cells rather than the myelin sheath. These results indicate that NDRG1 deficiency leads to Schwann cell dysfunction, suggesting that NDRG1 is essential for maintenance of the myelin sheaths in peripheral nerves. These mice will be used for future analyses of the mechanisms of myelin maintenance.
Molecular and Cellular Biology 06/2004; 24(9):3949-56. · 5.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The protein kinase Akt mediates several metabolic and mitogenic effects of insulin, whereas activation of protein kinase C (PKC) isoforms has been implicated in the inhibition of insulin action. We have previously shown that both PKC and PKCepsilon are activated in skeletal muscle of insulin-resistant high fat-fed rats, and to identify potential substrates for these kinases, we incubated recombinant PKC isoforms with rat muscle fractions in vitro. PKC specifically phosphorylated a 48-kDa protein that was subsequently identified by mass spectrometry as Ndrg2. Ndrg2 is highly related to N-Myc downstream-regulated protein 1, which has been linked to stress responses, cell proliferation, and differentiation, although Ndrg2 itself is not repressed by N-Myc. Ndrg2 contains several potential phosphorylation sites, including three Akt consensus sequences. Ndrg2 phosphorylation was enhanced in [32P]orthophosphate-labeled C2C12 muscle cells co-overexpressing either PKC or Akt. Phosphorylation of Ndrg2 was examined further using a phospho (Ser/Thr) Akt substrate antibody. Insulin increased Ndrg2 phosphorylation in C2C12 cells in a wortmannin- and palmitate-inhibitable manner, whereas rapamycin, PD98059, and bisindoylmaleimide I had no effect, supporting a direct role for Akt. Mutation of Ndrg2 indicated that Thr-348 is the major phosphorylation site detected by the antibody and that Akt stimulates phosphorylation of this site, whereas PKC phosphorylates Ser-332. PKC overexpression, however, diminished the effect of insulin on Thr-348 phosphorylation without reducing Akt activation, suggesting that this is mediated through phosphorylation of Ndrg2 at Ser-332. Our data identify Ndrg2 as a novel insulin-dependent phosphoprotein and suggest that PKC may inhibit insulin action in part by reducing its phosphorylation by Akt.
Journal of Biological Chemistry 05/2004; 279(18):18623-32. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To identify candidate genes responsible for hypertension in Dahl salt-sensitive rats (Dahl-S), an oligonucleotide microarray analysis was performed to find differentially expressed genes in kidneys of Dahl-S and Lewis rats. We obtained 101 F2 male rats from Dahl-S and Lewis rats and performed precise measurements of blood pressure (BP) and heart rate by telemetric monitoring at 14 weeks of age after 9 weeks of salt-loading. The correlation analysis between genotypes of differentially expressed genes and BP in F2 rats indicated that pyridoxine 5'-phosphate oxidase (Pnpo) and catecholamine-O-methyltransferease (Comt) showed a highly significant association with BP. However, in the case of Comt, the Dahl-S genotype correlated with low BP. Short/branched chain acyl-CoA dehydrogenase and Sah also showed a significant association with systolic blood pressure. The present study provided evidence that Pnpo is a candidate gene responsible for hypertension in Dahl-S rats.
Biochemical and Biophysical Research Communications 02/2004; 313(3):647-53. · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Spontaneously hypertensive rats (SHR) are a well-known animal model for hypertension. We have previously identified eleven differentially expressed genes in the kidneys between SHR/Hos and Wistar-Kyoto rats (WKY/Hos) using an oligonucleotide microarray and analyzed the correlation between these genes and hypertension. In the present study, we analyzed the differentially expressed genes in the kidneys between SHR/NCrj and WKY/NCrj obtained from an other source to clarify the common and/or specific gene expression between the different sources. Furthermore, expression changes in the representative genes were characterized by Northern blot analysis using samples prepared from a third source, the Izm strain. The comparison revealed quite different changes in the differentially expressed genes among them. Sequence analysis of one of the differentially expressed genes, cytosolic epoxide hydrolase, revealed that two haplotypes could in part explain the expression level. Our study showed the complex nature of the genetic heterogeneity between SHR and WKY from different sources.
Biochemical and Biophysical Research Communications 09/2002; 296(3):537-43. · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We identified genes that were differentially expressed between spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) using cDNA microarray analysis, and analyzed the correlation between these genes and hypertension. Twenty four genes were found to be up-regulated and 20 were down-regulated in SHR. We selected 11 genes (6 up-regulated genes: SAH, Hsp70, MCT1, RBP, IDI1, Prion; and 5 down-regulated genes: Thrombin, Dyn, SOD3, Ela1, Gst Y(b)) and subjected them to an F2 cosegregation analysis. One hundred five F2 rats were obtained from the same strains used for microarray analysis, and blood pressure was measured directly with a catheter implanted in the femoral artery. The genotypes of monocarboxylate transporter 1 and glutathione S-transferase Y(b) subunit significantly affected diastolic blood pressure in F2 rats, and these two genes are located near each other on chromosome 2. However, quantitative trait loci (QTL) analysis in this region revealed that the QTL for diastolic blood pressure were from these two genes. Antihypertensive treatment with either enalapril or hydralazine only affected the expression level of Hsp70, which was up-regulated by hydralazine, probably through compensatory sympathetic activation. We were unable to associate the other 10 genes with hypertension in SHR. Based on these results, the identification of differentially expressed genes may not be an efficient method for selecting candidate genes for hypertension in the SHR-WKY system.
Hypertension Research 04/2002; 25(2):249-55. · 2.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Single-nucleotide polymorphisms (SNPs) located in coding regions (coding SNPs; cSNPs) with amino acid substitution can potentially alter protein function. Therefore, identification of the nonsynonymous cSNPs of the genes of common diseases is valuable in tests of association with phenotypes. In this study, we validated 525 candidate cSNPs from 179 hypertension candidate genes deposited in the publicly available database dbSNP by DNA sequencing of samples from 32 Japanese individuals. We identified a total of 143 SNPs (27%) in 93 hypertension candidate genes. We also identified 16 new SNPs, for a total of 159 SNPs. Of the 159 SNPs thus identified, 104 were nonsynonymous. We estimate that approximately 20% of the SNPs deposited in dbSNP database showed a minor allele frequency of over 5%. The candidate SNPs for hypertension identified in this study would be valuable for association studies with hypertension to accelerate the identification of hypertension genes.
Journal of Human Genetics 02/2002; 47(8):387-94. · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The rostral ventrolateral medulla (RVLM) plays a critical role in the tonic and reflexive regulation of arterial blood pressure. Recent studies have demonstrated that injection of kynurenic acid (KYN) into the RVLM of spontaneously hypertensive rats (SHR) decreases arterial blood pressure. We hypothesized that a relative increase in the excitatory amino acid-mediated drive of RVLM vasomotor neurons in SHR may be due to derangement of one of the enzymes that affect the KYN level in the brain. We selected kynureninase, kynureninase hydroxylase, kynurenine aminotransferase type I, and kynurenine aminotransferase type II as candidates that may affect the KYN level in the brainstem. We conducted association studies between polymorphisms of these genes and blood pressure in an F2 population derived from SHR and Wistar-Kyoto rats (WKY). The cosegregation analysis indicated that only the kynureninase gene (KYNU) polymorphism influenced systolic blood pressure (SBP) and residuals of systolic blood pressure after adjusting for heart rate and body weight (RSBP). KYNU was found to be located on rat chromosome 3, and quantitative trait loci analysis at this locus indicated that the logarithms of the odds scores for KYNU in terms of SBP and RSBP were 2.0 and 3.3, respectively. This association with blood pressure decreased in proportion to the distance from KYNU. The expression level of KYNU mRNA in the brainstem was about 3.1 and 2.9 times higher in 10-week-old and 16-week-old SHR than in age-matched WKY, respectively. The increased expression of KYNU in SHR is thought to decrease the KYN level. KYNU seems to be one of the genes that contributes to hypertension in SHR.
Hypertension Research 02/2002; 25(1):135-40. · 2.79 Impact Factor