Biomedical subjects

Satoshi Inoue

Publications and source records attributed to Satoshi Inoue.

At least 19 recordsLinked to original sources

PubMed2025

Membrane topology inversion of GGCX mediates cytoplasmic carboxylation for antiviral defense.

Mitochondrial antiviral signaling protein (MAVS) is an adaptor involved in antiviral immunity, but its regulation is not fully understood. We identified carboxylation of MAVS by vitamin K (VK)-dependent γ-glutamyl carboxylase (GGCX), which was unexpected owing to the reported membrane topology of GGCX. We found that GGCX could undergo topology inversion to carboxylate MAVS within the cytoplasm. This carboxylation enhanced the ability of MAVS to induce type I interferons while suppressing the induction of apoptosis. Genetic knockout of GGCX, a VK-free diet, or depletion of VK by inhibiting VK epoxide reductase 1 with warfarin increased viral susceptibility in mice. Thus, we identified a MAVS regulatory mechanism-the existence of cytoplasmic protein carboxylation and topological inversion of GGCX-and demonstrated how modulating VK levels may influence antiviral defense.

Animals↗

PubMed2007

Q89R polymorphism in the LDL receptor-related protein 5 gene is associated with spinal osteoarthritis in postmenopausal Japanese women.

STUDY DESIGN: An association study investigating the genetic etiology for spinal osteoarthritis. OBJECTIVE: To determine the association of single-nucleotide polymorphism (SNP) causing an amino-acid change (Q89R) in the low-density lipoprotein receptor-related protein 5 (LRP5) coding region with spinal osteoarthritis. SUMMARY OF BACKGROUND DATA: Wnt/beta-catenin signaling pathway regulates bone density through a Wnt coreceptor LRP5. This pathway is also involved in cartilage development and homeostasis, suggesting that genetic variation in LRP5 gene may affect the pathogenesis of cartilage-related diseases, such as osteoarthritis. METHODS: We evaluated the presence of osteophytes, endplate sclerosis, and narrowing of disc spaces in 357 Japanese postmenopausal women. Missense coding SNP for Q89R of LRP5 gene was determined using TaqMan polymerase chain reaction (PCR) method. RESULTS: We found that subjects without the R allele (QQ; n = 321) had a significantly lower osteophyte formation score than did subjects bearing at least one R allele (QR + RR; n = 36) (7.80 vs. 10.89, P = 0.0019 by analysis of covariance). CONCLUSIONS: We suggest that a genetic variation at the LRP5 gene locus is associated with spinal osteoarthritis, in line with the involvement of the LRP5 gene in the bone and cartilage metabolism.

Aged↗

PubMed2006

alpha-Synuclein stimulates differentiation of osteosarcoma cells: relevance to down-regulation of proteasome activity.

Because a limited study previously showed that alpha-synuclein (alpha-syn), the major pathogenic protein for Parkinson disease, was expressed in differentiating brain tumors as well as various peripheral cancers, the main objective of the present study was to determine whether alpha-syn might be involved in the regulation of tumor differentiation. For this purpose, alpha-syn and its non-amyloidogenic homologue beta-syn were stably transfected to human osteosarcoma MG63 cell line. Compared with beta-syn-overexpressing and vector-transfected cells, alpha-syn-overexpressing cells exhibited distinct features of differentiated osteoblastic phenotype, as shown by up-regulation of alkaline phosphatase and osteocalcin as well as inductive matrix mineralization. Further studies revealed that proteasome activity was significantly decreased in alpha-syn-overexpressing cells compared with other cell types, consistent with the fact that proteasome inhibitors stimulate differentiation of various osteoblastic cells. In alpha-syn-overexpressing cells, protein kinase C (PKC) activity was significantly decreased, and reactivation of PKC by phorbol ester significantly restored the proteasome activity and abrogated cellular differentiation. Moreover, activity of lysosome was up-regulated in alpha-syn-overexpressing cells, and treatment of these cells with autophagy-lysosomal inhibitors resulted in a decrease of proteasome activity associated with up-regulation of alpha-syn expression, leading to enhance cellular differentiation. Taken together, these results suggest that the stimulatory effect of alpha-syn on tumor differentiation may be attributed to down-regulation of proteasome, which is further modulated by alterations of various factors, such as protein kinase C signaling pathway and a autophagy-lysosomal degradation system. Thus, the mechanism of alpha-syn regulation of tumor differentiation and neuropathological effects of alpha-syn may considerably overlap with each other.

Autophagy↗

PubMed2006

Serological survey of Ehrlichia and Anaplasma infection of feral raccoons (Procyon lotor) in Kanagawa Prefecture, Japan.

Numbers of feral raccoon; the possible reservoir animal of Ehrlichia and Anaplasma, are increasing in Japan. Thus serological methods were utilized to examine Ehrlichia and Anaplasma infection in raccoons from Kanagawa Prefecture, Japan. By using an indirect immunofluorescence assay, among 187 feral raccoons examined, 1 (0.5%) serologically reacted with Ehrlichia canis, 3 (1.6%) with Ehrlichia chaffeensis and 1 (0.5%) with Anaplasma phagocytophilum with the titers of 1:40 or more. Although screening PCR for Ehrlichia and Anaplasma species failed to detect the presence of ehrlichial DNA in serum samples, results of the serological tests suggested that the feral raccoons might be infected with some species of Ehrlichia and Anaplasma.

Anaplasma↗

PubMed2006

Role of GPI-anchored NCAM-120 in rabies virus infection.

Although the neural cell adhesion molecule (NCAM) -140 and -180 have been shown to serve as a receptor for rabies virus (RV), it was not known whether the other major isoform of NCAM, GPI-anchored NCAM-120 functions as RV receptor. In this study, we have established HEp-2 cells stably expressing NCAM-120 or NCAM-140, and their susceptibilities to RV infection were compared. The results demonstrated that NCAM-120 served as virus attachment protein; however, the cells expressing NCAM-120 did not support efficient RV replication. Furthermore, the level of IFN-ss mRNA was apparently elevated in NCAM-120 expressing cells but not in NCAM-140 expressing cells, suggesting that GPI-anchored NCAM-120 suppressed RV replication via induction of IFN-ss even though NCAM-120 was able to promote virus penetration into the cells.

Cell Line↗

PubMed2006

A ubiquitin E3 ligase Efp is up-regulated by interferons and conjugated with ISG15.

Interferon (IFN) regulates various target genes that mediate important roles in immune responses such as antiviral state. Protein ISG15-conjugation (ISGylation) is implicated in the IFN-induced antiviral response. Here we demonstrate that Efp mRNA as well as protein expression could be up-regulated by Type I IFN in HeLa cells and HepG2 cells. Luciferase assay reveals that the first intron of Efp gene contains a functional interferon-stimulated response element (ISRE) and electrophoretic mobility shift assay shows that the ISRE binds to signal transducer and activator of transcription 1 (STAT1). Chromatin immunoprecipitation assays have shown that the ISRE recruits STAT1 in vivo IFN-dependently. Moreover, we demonstrate that Efp protein could be conjugated with not only ubiquitin but also ISG15. These data suggest that Efp is an IFN-responsive gene that potentially mediates IFN actions, involved in ISGylation and ubiquitination of proteins including Efp itself.

Cell Line, Tumor↗

PubMed2006

A functional single nucleotide polymorphism in the vitamin-K-dependent gamma-glutamyl carboxylase gene (Arg325Gln) is associated with bone mineral density in elderly Japanese women.

The vitamin-K-dependent gamma-glutamyl carboxylase (GGCX) carboxylates vitamin-K-dependent proteins including bone Gla protein (osteocalcin) and matrix Gla protein, which play important roles in bone metabolism. Therefore, GGCX polymorphism might explain in part individual susceptibility to osteoporosis. In the present study, polymorphisms in the exons of this gene were screened in Japanese elderly women and a non-synonymous single nucleotide polymorphisms (SNP) were found; c.8762 G>A; (Arg325Gln). When the kinetic parameters of GGCX325-Gln and GGCX325-Arg were compared in vitro, Vmax/Km was significantly higher for GGCX325-Gln (944.4+/-9.21 pmol/30 min/mg/mM FLEEL) than for GGCX325-Arg (671.9+10.79 pmol/30 min/mg/mM FLEEL) (p=0.018). Then, association study of this polymorphism with forearm bone mineral density (BMD) of Japanese postmenopausal women (n=500, age 73.6+/-5.74) was conducted. As a result, the body mass index (BMI)-adjusted Z score in the subpopulation older than 75 years (n=207) was higher in those with 325-Gln (0.650+/-0.883, mean+/-SD) than those with 325-Arg/Gln or 325-Arg (0.133+/-0.650) (p=0.0383). This is the first report to demonstrate the different activities of GGCX between the common genotypes and their association with BMD.

Aged↗

PubMed2006

Inhibitory effects of B cells on antitumor immunity.

B-cell functions in antitumor immunity are not well understood. In this study, we evaluated the role of B cells in the development of antitumor immunity using Friend murine leukemia virus gag-expressing mouse EL-4 (EL-4 gag), D5 mouse melanoma, or MCA304 mouse sarcoma cells. To screen tumors for susceptibility to B-cell-deficient immune environments, spleen cells from naive C57BL/6 [wild-type (WT)] and B-cell knockout (BKO) mice were cultured with irradiated tumor cells in vitro. When cells were stimulated with EL-4 gag or D5 (but not MCA304 tumors), IFN-gamma production from CD8 T cells and natural killer cells was markedly decreased in WT compared with BKO cultures. IFN-gamma production was correlated with CD40 ligand expression on the tumor and inversely with interleukin-10 (IL-10) production by B cells. Sorted WT B cells produced more IL-10 than CD40 knockout (CD40KO) B cells when cocultured with EL-4 gag or D5 (but not MCA304). IFN-gamma production by BKO cells was reduced by the addition of sorted naive WT B cells (partially by CD40KO B cells) or recombinant mouse IL-10. In vivo tumor progression mirrored in vitro studies in that WT mice were unable to control tumor growth whereas EL-4 gag and D5 tumors (but not MCA304) were eliminated in BKO mice. Robust in vivo antitumor CTLs developed only in BKO tumor-challenged mice. Our studies provide the first mechanistic basis for the concept that B-cell depletion could therapeutically enhance antitumor immune responses to certain tumors by decreasing IL-10 production from B cells.

Animals↗

PubMed2006

Identification and Herc5-mediated ISGylation of novel target proteins.

ISG15, a protein containing two ubiquitin-like domains, is an interferon-stimulated gene product that functions in antiviral response and is conjugated to various cellular proteins (ISGylation) upon interferon stimulation. ISGylation occurs via a pathway similar to the pathway for ubiquitination that requires the sequential action of E1/E2/E3: the E1 (UBE1L), E2 (UbcH8), and E3 (Efp/Herc5) enzymes for ISGylation have been hitherto identified. In this study, we identified six novel candidate target proteins for ISGylation by a proteomic approach. Four candidate target proteins were demonstrated to be ISGylated in UBE1L- and UbcH8-dependent manners, and ISGylation of the respective target proteins was stimulated by Herc5. In addition, Herc5 was capable of binding with the respective target proteins. Thus, these results suggest that Herc5 functions as a general E3 ligase for protein ISGylation.

Cytokines↗

PubMed2006

NaOCl degradation of a HEMA-free all-in-one adhesive bonded to enamel and dentin following two air-blowing techniques.

OBJECTIVE: Phase-separation within HEMA-free all-in-one dental adhesives may result in the entrapment of droplets within the adhesive resin. Strongly air-blowing prior to polymerization, can remove most of these droplets. The objective of this study was to evaluate the effect these droplets may have on the resistance of the adhesive-tooth interface to NaOCl degradation. METHODS: The micro-tensile bond strength (microTBS) to enamel and dentin was determined when a HEMA-free all-in-one adhesive was applied either following a mild or strong air-blowing technique. The bonds were also exposed to an aqueous sodium hypochlorite (NaOCl) solution for 1h, following a recently introduced methodology to mimic in vivo bond degradation. RESULTS: This study revealed that strong air-blowing of the adhesive only resulted in a significantly higher micro-tensile bond strength (microTBS) to dentin, but not to enamel. Likewise, NaOCl only reduced the microTBS to dentin for both the mild and strong air-blowing technique, but again not the microTBS to enamel. Failure analysis by SEM clearly revealed that strong air-blowing is less effective in droplet removal when the adhesive was applied in small and narrow class-I cavities, as compared to when it was applied to flat surfaces. CONCLUSIONS: NaOCl did preferentially dissolve the hybrid layer at dentin, and more for the mild than for the strong air-blowing technique. A strong air-blowing procedure resulted in a more NaOCl-resistant hybrid layer, so that it can be concluded that a HEMA-free one-step adhesive definitely benefits from a strong air-blowing technique.

Air↗

PubMed2006

Inhibition of histone deacetylase class I but not class II is critical for the sensitization of leukemic cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis.

From work done largely on derived cell lines, it has been suggested that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) might be a therapeutic target for many forms of malignancy. However, use of primary tumor cells, including chronic lymphocytic leukemic (CLL) cells, has shown inherent resistance to TRAIL. Although the molecular basis for this resistance remains unknown, treatment with histone deacetylase inhibitors (HDACi) often sensitizes resistant cells to TRAIL-induced apoptosis. We used structurally diverse HDACi to ascertain which HDAC needs to be inhibited for the sensitization. Inhibition of HDAC class I but not class II is required for sensitization to TRAIL-induced apoptosis of CLL cells and various cell lines. Using different HDACi together with small interfering RNA for HDAC1, HDAC2, HDAC3, and HDAC6, we report that inhibition of HDAC1 and HDAC2 but not HDAC3, HDAC6, and HDAC8 are primarily responsible for sensitization to TRAIL-induced apoptosis. Based on these data and our previous studies, we propose that a clinical trial in CLL is warranted using a combination of a selective HDACi that inhibits HDAC1 and/or HDAC2 together with a form of TRAIL that signals through TRAIL receptor 1.

Apoptosis↗

PubMed2006

Chelation-assisted transformation: synthesis of 1,4-dicarboxylate esters by the Rh-catalyzed carbonylation of internal alkynes with pyridin-2-ylmethanol.

The reaction of internal alkynes 1 with CO and pyridin-2-ylmethanol (2) in the presence of Rh(4)(CO)(12) results in a double-hydroesterification leading to 1,4-dicarboxylate esters 3. The reaction does not proceed via two consecutive hydroesterifications of alkynes, but the intermediacy of ketene intermediates is proposed. The coordination of the pyridine nitrogen in 2 to rhodium is essential for the reaction to proceed. [reaction: see text]

Journal Article↗

PubMed2006

Steroid and xenobiotic receptor SXR mediates vitamin K2-activated transcription of extracellular matrix-related genes and collagen accumulation in osteoblastic cells.

Vitamin K2 is a critical nutrient required for blood coagulation. It also plays a key role in bone homeostasis and is a clinically effective therapeutic agent for osteoporosis. We previously demonstrated that vitamin K2 is a transcriptional regulator of bone marker genes in osteoblastic cells and that it may potentiate bone formation by activating the steroid and xenobiotic receptor, SXR. To explore the SXR-mediated vitamin K2 signaling network in bone homeostasis, we identified genes up-regulated by both vitamin K2 and the prototypical SXR ligand, rifampicin, in osteoblastic cells using oligonucleotide microarray analysis and quantitative reverse transcription-PCR. Fourteen genes were up-regulated by both ligands. Among these, tsukushi, matrilin-2, and CD14 antigen were shown to be primary SXR target genes. Moreover, collagen accumulation in osteoblastic MG63 cells was enhanced by vitamin K2 treatment. Gain- and loss-of-function analyses showed that the small leucine-rich proteoglycan, tsukushi, contributes to vitamin K2-mediated enhancement of collagen accumulation. Our results suggest a new function for vitamin K2 in bone formation as a transcriptional regulator of extracellular matrix-related genes, that are involved in the collagen assembly.

Animals↗

PubMed2006

Epigenetic and proteolytic inactivation of 14-3-3sigma in breast and prostate cancers.

14-3-3sigma is an epithelial marker whose expression is induced by DNA damage through a p53-dependent pathway. 14-3-3sigma functions sequesters cyclin B1-CDC2 complexes outside the nucleus and thereby contributes to a G2 arrest. Down-regulation or lack of 14-3-3sigma is a frequent event in breast and prostate cancers. Epigenetic silencing by CpG methylation, p53 inactivation, and proteasome-dependent proteolysis leads to loss of 14-3-3sigma. Hypermethylation of the 14-3-3sigma gene is often observed in precancerous lesions and likely to be causally linked to the onset of cancer. Proteolytic inactivation of 14-3-3sigma has been recently found in breast and prostate cancers. In breast cancer, the estrogen-responsive E3 ubiquitin ligase Efp specifically targets 14-3-3sigma for degradation. The E2 ubiquitin conjugating enzyme UBC8 and Efp also mediates ISG15 modification of 14-3-3sigma. Detection of 14-3-3sigma inactivation on the protein or DNA methylation level may be used for cancer prognosis. Furthermore, 14-3-3sigma may be a potential therapeutic target in breast and prostate cancer.

14-3-3 Proteins↗

PubMed2006

17Beta-estradiol protects against oxidative stress-induced cell death through the glutathione/glutaredoxin-dependent redox regulation of Akt in myocardiac H9c2 cells.

The GSH/glutaredoxin (GRX) system is involved in the redox regulation of certain enzyme activities, and this system protects cells from H2O2-induced apoptosis by regulating the redox state of Akt (Murata, H., Ihara, Y., Nakamura, H., Yodoi, J., Sumikawa, K., and Kondo, T. (2003) J. Biol. Chem. 278, 50226-50233). Estrogens, such as 17beta-estradiol (E2), play an important role in development, growth, and differentiation and appear to have protective effects on oxidative stress mediated by estrogen receptor alpha (ERalpha). However, the role of the ERbeta-mediated pathway in this cytoprotection and the involvement of E2 in the redox regulation are not well understood. In the present study, we demonstrated that E2 protected cardiac H9c2 cells, expressing ERbeta from H2O2-induced apoptosis concomitant with an increase in the activity of Akt. E2 induced the expression of glutaredoxin (GRX) as well as gamma-glutamylcysteine synthetase, a rate-limiting enzyme for the synthesis of GSH. Inhibitors for both gamma-glutamylcysteine synthetase and GRX and ICI182,780, a specific inhibitor of ERs, abolished the protective effect of E2 on cell survival as well as the activity of Akt, suggesting that ERbeta is involved in the cytoprotection and redox regulation by E2. Transcription of the GRX gene was enhanced by E2. The promoter activity of GRX was up-regulated by an ERbeta-dependent element. These results suggest that the GRX/GSH system is involved in the cytoprotective and genomic effects of E2 on the redox state of Akt, a pathway that is mediated, at least in part, by ERbeta. This mechanism may also play an antiapoptotic role in cancer cells during carcinogenesis or chemotherapy.

Animals↗

PubMed2006

Antitumor activity and downregulation of pro-angiogenic molecules in human prostate cancer cells by a novel thiazolidione compound.

BACKGROUND: Current treatments for prostate cancer are effective in many patients with locally advanced disease, but many of these patients eventually have recurrence. It is therefore important to develop alternative therapeutic agents with improved efficacy and tolerability. We recently identified a synthetic thiazolidin compound, 5-(2,4-dihydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidione (DBPT), that induces apoptosis in human colon cancer cells, independent of p53 and P-glycoprotein status. Here, we investigated the antitumor properties and mechanisms of action of this compound in human prostate cancer cell lines. METHODS: The effect of DBPT on cell-cycle progression and apoptosis in LNCaP and DU145 cells was examined by flow cytometry and Western blotting. The effect of DBPT on pro-angiogenic molecules was analyzed by Western blotting and by an enzyme-linked immunosorbent assay. RESULTS: DBPT inhibited the growth of LNCaP and DU145 cells with 50% inhibitory concentrations ranging from 1.6 to 5.9 microM. Treating LNCaP and DU145 cells with DBPT led to a time-dependent cell-cycle arrest in the G(2)/M phase and increased levels of G(2)/M checkpoint proteins, such as cyclin B1, cdc25C, phosphorylated histone H(3), and MPM-2. DBPT induced the phosphorylation of Bcl-xL and Bim, and induced apoptosis, as evidenced by cleavage of caspase and poly (ADP-ribose) polymerase. DBPT also effectively induced apoptosis in Bcl-2-overexpressing DU145 cells. Furthermore, DBPT decreased hypoxia-inducible factor 1 alpha and vascular endothelial growth factor expression in LNCaP cells under both normoxia and hypoxia. CONCLUSIONS: DBPT can suppress proliferation, induce apoptosis, and down regulate pro-angiogenic molecules in prostate cancer cells, and might be useful in treating prostate cancer.

Apoptosis↗

PubMed2006

A highly attenuated rabies virus HEP-Flury strain reverts to virulent by single amino acid substitution to arginine at position 333 in glycoprotein.

An amino acid at position 333 in the glycoprotein of several fixed rabies virus strains is responsible for the pathogenicity in adult mice. Substitution of arginine at this position largely reduces the viral pathogenicity in adult mice. Attenuation by this single amino acid substitution has been established by using escape mutants selected by monoclonal antibodies and point-mutated virus generated by reverse-genetics. A highly attenuated HEP-Flury strain, which was selected by serial passages in cell cultures, has glutamine at this position. In this study, a point-mutated rHEP333R virus, having arginine at position 333, was generated and examined for the responsibility of this substitution in rabies pathogenicity. The rHEP333R acquired an ability to spread and propagate in mouse brain but the parental rHEP did not. The pathogenicity of rHEP333R to adult mice by intracerebral inoculation largely increased. We confirmed that an arginine at position 333 contributed to reversion of the pathogenicity in a highly attenuated HEP-Flury strain.

Amino Acid Substitution↗

PubMed2006

Diethylstilbestrol increases the density of prolactin cells in male mouse pituitary by inducing proliferation of prolactin cells and transdifferentiation of gonadotropic cells.

Diethylstilbestrol (DES) has been implicated in mammalian abnormalities. We examined the effects of DES on follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) cells in the pituitaries of male mice treated with various doses of DES for 20 days. DES reduced the density of FSH and LH cells in a dose-dependent manner, but increased that of PRL cells. When the expression of estrogen receptor (ER) alpha and beta was assessed, an induction of ERbeta by DES was found predominantly in PRL cells. However, since these effects were abolished in ERalpha knockout mice, DES appears to act primarily through ERalpha. When the expression of Ki-67 and Pit-1 in PRL cells was examined at various time-points after DES treatment, some PRL cells became Ki-67 positive at 10-15 days, and Pit-1-positive cells were increased at 5-15 days. Furthermore, some FSH and LH cells became Pit-1 positive, and co-localized with PRL at 5-10 days. Our results indicate that DES increases PRL cells by inducing proliferation of PRL cells and transdifferentiation of FSH/LH cells to PRL cells.

Animals↗