Nobuya Sakai

Himeji Dokkyo University, Himezi, Hyōgo, Japan

Are you Nobuya Sakai?

Claim your profile

Publications (8)35.18 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The candidate tumor suppressor gene RASSF1A (Ras-association domain family 1, isoform A) is inactivated in many types of adult and childhood cancers. However, the mechanisms by which RASSF1A exerts tumor suppressive functions have yet to be elucidated. In this report, we sought to identify candidate proteins that interact with RASSF1A using proteomic screening. Using peptide mass fingerprinting, we identified protein arginine N-methyltransferase 5 (PRMT5), a type II protein arginine N-methyltransferase that monomethylates and symmetrically dimethylates arginine residues, as a novel protein that interacts with RASSF1A. The association between the two proteins was confirmed by co-immunoprecipitation and immunofluorescence staining. Co-expressing RASSF1A and PRMT5 led to a redistribution of PRMT5 from the cytosol to stabilized microtubules, where RASSF1A and PRMT5 became co-localized. Our results demonstrate that PRMT5 translocates to bundled microtubules on stabilization by RASSF1A expression. Our results show that the tumor suppressor RASSF1A interacts with PRMT5 in vivo and in vitro. Notably, this is the first demonstration of RASSF1A-dependent microtubule recruitment of PRMT5, suggesting a novel role for RASSF1A in the anchoring of cytosolic PRMT5 to microtubules.
    Biochemical and Biophysical Research Communications 10/2015; DOI:10.1016/j.bbrc.2015.10.065 · 2.30 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Only limited information is available on agonist-dependent changes in the subcellular localization of vasopressin V1B receptors. Our radioligand binding study of membrane preparations and intact cells revealed that a large fraction of the V1B receptor is located in the cytoplasm in unstimulated CHO cells, which is in contrast to the plasma membrane localization of the V1A and V2 receptors. Moreover, when the affinity of radiolabeled arginine-vasopressin ([(3)H]AVP) was compared between membrane preparations and intact cells, the affinity of [(3)H]AVP to the cell surface V1B receptors, but not the V1A receptors, was significantly reduced. Although the number and affinity of cell surface V1B receptors decreased, they became extensively internalized upon binding with [(3)H]AVP. Approximately 87% of cell surface-bound [(3)H]AVP was internalized and became resistant to acid wash during incubation with 1nM [(3)H]AVP. By contrast, less ligand (35%) was internalized in the cells expressing the V1A receptor. Extensive internalization of the V1B receptors was partially attenuated by inhibitors of cytoskeletal proteins, siRNA against β-arrestin 2, or the removal of sodium chloride from the extracellular buffer, indicating that this internalization involves clathrin-coated pits. Together, these results indicate that the mechanism that regulates the number and affinity of V1B receptors in the plasma membrane is markedly distinct from the corresponding mechanisms for the V1A and V2 receptors and plays a critical role under stress conditions, when vasopressin release is augmented. Copyright © 2015. Published by Elsevier B.V.
    European journal of pharmacology 08/2015; 765. DOI:10.1016/j.ejphar.2015.08.043 · 2.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A group of synthetic substance P (SP) antagonists, such as [Arg(6),D-Trp(7,9),N(Me)Phe(8)]-substance P(6-11) and [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]-substance P, bind to a range of distinct G-protein-coupled receptor (GPCR) family members, including V1a vasopressin receptors, and they competitively inhibit agonist binding. This extended accessibility enabled us to identify a GPCR subset with a partially conserved binding site structure. By combining pharmacological data and amino acid sequence homology matrices, a pharmacological lineage of GPCRs that are sensitive to these two SP antagonists was constructed. We found that sensitivity to the SP antagonists was not limited to the Gq-protein-coupled V1a and V1b receptors; Gs-coupled V2 receptors and oxytocin receptors, which couple with both Gq and Gi, also demonstrated sensitivity. Unexpectedly, a dendrogram based on the amino acid sequences of 222 known GPCRs showed that a group of receptors sensitive to the SP antagonists are located in close proximity to vasopressin/oxytocin receptors. Gonadotropin-releasing peptide receptors, located near the vasopressin receptors in the dendrogram, were also sensitive to the SP analogs, whereas α1B adrenergic receptors, located more distantly from the vasopressin receptors, were not sensitive. Our finding suggests that pharmacological lineage analysis is useful in selecting subsets of candidate receptors that contain a conserved binding site for a ligand with broad-spectrum binding abilities. The knowledge that the binding site of the two broad-spectrum SP analogs partially overlaps with that of distinct peptide agonists is valuable for understanding the specificity/broadness of peptide ligands. Copyright © 2015. Published by Elsevier B.V.
    European Journal of Pharmacology 01/2015; 749. DOI:10.1016/j.ejphar.2015.01.001 · 2.53 Impact Factor

  • Cancer Research 08/2013; 73(8 Supplement):2818-2818. DOI:10.1158/1538-7445.AM2013-2818 · 9.33 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Effective detection of potential nephrotoxicity is crucial for pre-clinical drug development. We evaluated a sensitive animal model for drug-induced kidney injury, which includes hemi-nephrectomy of mice. Although bucillamine and d-penicillamine are used for the treatment of rheumatoid arthritis in Japan, drug-related adverse effects on the kidney can limit their therapeutic utilities. When bucillamine (1000 or 2000 mg/kg/day) or d-penicillamine (2000 mg/kg/day) were orally administered to hemi-nephrectomised BALB/c mice, the urinary protein levels of bucillamine-treated mice, but not of those treated with d-penicillamine, the vehicle, or in bucillamine treated unnephrectomized mice, were significantly increased and remained high during the 4-week drug-loading period. Membranous glomerulonephropathy occasionally seen in bucillamine/d-penicillamine-treated arthritis patients was not reproduced in mice. Instead, our mouse model showed proximal tubular injury and podocyte foot process effacement in the bucillamine-treated kidneys. These two cell types are also the primary targets of the experimental Heymann membranous glomerulonephropathy. Gene expression profiling of the bucillamine-treated mice identified lipocalin 2 as a significantly up-regulated transcript together with cytochrome P450 CYP4a14, a group-specific component, and proprotein convertase subtilisin/kexin type 9. Moreover, large amounts of lipocalin 2 were detected in the urine of the bucillamine-treated mice, but not in the hemi-nephrectomised control mice. These results indicate that hemi-nephrectomy effectively promotes acute kidney injury by bucillamine, which is accompanied by up-regulation of the urinary biomarker lipocalin 2. Our mouse model with initial stage of kidney injury should be useful to analyse the pathogenesis of drug-induced glomerular and tubular injuries.
    European journal of pharmacology 09/2011; 670(1):208-15. DOI:10.1016/j.ejphar.2011.08.051 · 2.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Patients with clinical stage I/II (T1-2N0M0) oral tongue squamous cell carcinoma (TSCC) usually undergo partial glossectomy alone. However, 14-48% of them develop delayed neck metastasis (DNM), which may lead to an unfavorable course. Recently epithelial-to-mesenchymal transition (EMT) has been thought to play a crucial role in cancer metastasis. The present study aimed to examine the associations of EMT-involved molecular factors and clinicopathological factors with DNM in stage I/II TSCC. mRNA expression levels of E-cadherin and its transcriptional repressors (snail, SIP1, and twist) in 7 head and neck squamous cell carcinoma (HNSCC) cell lines were evaluated by quantitative real-time PCR. Clinicopathological parameters and immunohistochemical expressions of E-cadherin and its repressors were examined in surgical specimens of 37 stage I/II TSCC patients who underwent partial glossectomy alone. In HNSCC cells, E-cadherin expression was inversely correlated with SIP1 expression (P = 0.023). Univariate analysis of immunohistochemistry showed that overexpression of SIP1 and loss of E-cadherin were significantly correlated with DNM, although no inverse correlation was found between E-cadherin and its repressors. Multiple logistic regression analysis including clinicopathological and molecular factors revealed that overexpression of SIP1 (P = 0.005), loss of E-cadherin (P = 0.046), and vascular invasion (P = 0.024) were independently correlated with DNM. These results suggest that development of DNM in stage I/II TSCC is closely related to induction of EMT in primary tumor cells. Especially, SIP1 and E-cadherin are considered to be the possible markers for selecting patients at high risk of DNM.
    Annals of Surgical Oncology 09/2011; 19(2):612-9. DOI:10.1245/s10434-011-2052-1 · 3.93 Impact Factor

  • Cancer Research 07/2011; 71(8 Supplement):5213-5213. DOI:10.1158/1538-7445.AM2011-5213 · 9.33 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A noncoding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), is upregulated in several malignant tumors. Its expression in neuroblastoma, however, is not known, and the regulatory mechanisms of MALAT1 gene expression have not been elucidated. The aim of this study is to clarify how MALAT1 gene expression is altered by extracellular signals in the SK-N-SH neuroblastoma cell line and to define its proximal promoter in order to study the mechanism of MALAT1 gene expression. Transcript amounts were analyzed by real-time semiquantitative polymerase chain reaction (qPCR). Genes coregulated with MALAT1 were identified by DNA microarray analysis. The structure of the MALAT1 transcript was delineated using a tiling microarray, and the 5'-end was determined using the rapid amplification of cDNA ends (RACE) method. We investigated binding of the cyclic AMP-responsive element binding (CREB) transcription factor to the MALAT1 promoter by using chromatin immunoprecipitation (ChIP) followed by tiling array analysis, and the results were confirmed using ChIP-qPCR. The posterior pituitary hormone oxytocin increased the levels of MALAT1 and immediate early gene transcripts as early as 15 min after stimulation. Although the expression of immediate early genes returned to basal levels after 3h, MALAT1 transcript levels peaked 6-24h after stimulation. We identified a shorter transcriptional initiation site and found that CREB binds to the defined proximal promoter of the MALAT1 gene. The expression of the tumor marker MALAT1 ncRNA is sensitive to cell surface receptor activation by oxytocin in a neuroblastoma cell line.
    Life sciences 02/2010; 86(11-12):455-60. DOI:10.1016/j.lfs.2010.02.001 · 2.70 Impact Factor