Biomedical subjects

Kunikazu Tanji

Publications and source records attributed to Kunikazu Tanji.

At least 19 recordsLinked to original sources

PubMed2006

Oncogenic protein UnpEL/Usp4 deubiquitinates Ro52 by its isopeptidase activity.

UnpEL (also known as Usp4 or Unph) is an oncogenic protein, because its expression with a strong promoter results in the tumorigenic transformation of NIH3T3 cells injected into nude mice. Although the structure of UnpEL is that of a deubiquitinating enzyme, neither its precise function in mammalian cells nor the mechanism of UnpEL-mediated tumorigenesis is known. Here, we show that UnpEL functions as a deubiquitinating enzyme in human HEK293T cells and its isopeptidase activity deconjugates ubiquitin specifically from a UnpEL-interacting protein Ro52. We further show that UnpEL translocates to the cytoplasmic rod-like structures and colocalizes with Ro52 when Ro52 is overexpressed in HEK293 cells. These results suggest that UnpEL colocalizes with the unubiquitinated form of Ro52 to the cytoplasmic rod-like structures, where it keeps Ro52 unubiquitinated. The continuous deubiquitination of Ro52 might be involved in tumorigenesis.

Cell Line↗

PubMed2006

Interleukin-1beta enhances the angiotensin-induced expression of plasminogen activator inhibitor-1 through angiotensin receptor upregulation in human astrocytes.

Plasminogen activator inhibitor-1 (PAI-1) regulates not only fibrinolysis but extracellular matrix remodeling, and angiotensin II is known to play an important role in controlling the expression of PAI-1 in astrocytes. We have studied the effect of interleukin-1beta (IL-1beta), one of major cytokines also active in the nervous system, on the angiotensin II-induced expression of PAI-1 in human astrocytes. Cultures of normal human astrocytes were stimulated with IL-1beta and angiotensin II, and the expression of mRNAs for angiotensin II type 1 receptor (AT1) and PAI-1 was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) or real-time quantitative PCR. PAI-1 protein in astrocyte-conditioned medium was measured by enzyme-linked immunosorbent assay (ELISA). IL-1beta enhanced the expression of AT1 in astrocytes in time- and concentration-dependent manners. After 24-h stimulation, 10 ng/ml IL-1beta and 10 nM angiotensin II increased the levels of PAI-1 protein in astrocyte-conditioned medium by 1.9-fold and 1.8-fold of the basal value, respectively. There was no synergistic effect when the cells were stimulated simultaneously with IL-1beta and angiotensin II. When the cells were stimulated, with angiotensin II, 16 h after the stimulation with IL-1beta, the production of PAI-1 was enhanced by 1.4-fold as compared to the cells stimulated only with IL-1beta. CV-11794, an AT1 antagonist, inhibited the enhanced PAI-1 production in response to angiotensin II. We conclude that IL-1beta increases angiotensin II-induced PAI-1 secretion by astrocytes through the induction of AT1, and the enhanced secretion of PAI-1 may modulate functions of plasminogen activators in the nervous system.

Angiotensin II↗

PubMed2006

NUB1 suppresses the formation of Lewy body-like inclusions by proteasomal degradation of synphilin-1.

NUB1 is a potent down-regulator of the ubiquitin-like protein NEDD8, because it targets NEDD8 to the proteasome for proteolytic degradation. From results in this study, we found that NUB1 physically interacts with synphilin-1 through its NEDD8-binding site, implying that NUB1 also targets synphilin-1 to the proteasome for degradation. Synphilin-1 is a major component of inclusion bodies found in the brains of patients with neurodegenerative alpha-synucleinopathies, including Parkinson's disease. In this study, we immunostained sections of brains from patients with Parkinson's disease and other alpha-synucleinopathies and demonstrated that NUB1, as well as synphilin-1, accumulates in the inclusion bodies. To define the role of NUB1 in the formation of these inclusion bodies, we performed a co-transfection assay using cultured HEK293 cells. This assay showed that NUB1 suppresses the formation of synphilin-1-positive inclusions. Further, biochemical assays revealed that NUB1 overexpression leads to the proteasomal degradation of synphilin-1. These results and our previous observations suggest that NUB1 indeed targets synphilin-1 to the proteasome for its efficient degradation, which, because of the resultant reduction in synphilin-1, suppresses the formation of synphilin-1-positive inclusions.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Function and subcellular location of Ro52beta.

Autoantigen Ro52alpha was recently identified as an E3 ubiquitin ligase. Its splicing variant Ro52beta, which lacks a leucine zipper, has not been characterized yet. We therefore characterized Ro52beta in contrast to Ro52alpha. Our biochemical assays revealed that both Ro52alpha and Ro52beta function as E3 ubiquitin ligases and self-ubiquitinate in cooperation with UbcH5B in vitro. In addition, both Ro52alpha and Ro52beta are ubiquitinated when overexpressed with ubiquitin in HEK293T cells, suggesting that both function as E3 ligases and self-ubiquitinate in vivo. However, cytological studies revealed that Ro52alpha mainly localizes to the cytoplasmic rod-like structures, whereas Ro52beta diffusely localizes to both the cytoplasm and the nucleus. Since the leucine zipper plays a role in the homodimerization and heterodimerization of Ro52alpha, the dimerization might be required for the localization of Ro52alpha to the rod-like structures. On the basis of these results, Ro52alpha and Ro52beta appear to ubiquitinate their particular substrates at different locations.

Alternative Splicing↗

PubMed2005

Interaction of NUB1 with the proteasome subunit S5a.

NUB1 interacts with a ubiquitin-like protein NEDD8 to target the NEDD8 monomer and neddylated proteins to the proteasome for degradation. Therefore, NUB1 is thought to be a potent downregulator of NEDD8 conjugation system. Since NUB1 possesses a UBL domain, which was previously shown to be an S5a-interacting motif in RAD23/HHR23, we initially hypothesized that NUB1 interacts with the S5a subunit of the proteasome through its UBL domain. To examine this, we performed an in vitro GST pull-down assay and a yeast two-hybrid assay. Unexpectedly, our studies revealed that NUB1 directly interacts with the S5a subunit through its C-terminal region between amino acid residues 536 and 584, not through its UBL domain. Although the UBL domain was not an S5a-interacting motif in NUB1, our further studies revealed that the UBL domain is required for the function of NUB1.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Platelet-activating factor enhances the expression of nerve growth factor in normal human astrocytes under hypoxia.

Nerve growth factor (NGF) is required for the survival of neurons. We have addressed the effect of platelet-activating factor (PAF), one of the mediators of ischemic injury of the brain, on NGF expression in astrocytes. Normal human astrocytes in culture were stimulated with PAF, and levels of NGF mRNA and protein were analyzed by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA). PAF increased the expressions of NGF mRNA and protein in astrocytes in time- and concentration-dependent manners. After 48-h stimulation, 10 nmol/L PAF increased the levels of NGF protein in astrocyte-conditioned medium by 1.4-fold. The PAF-induced stimulation of NGF expression was further enhanced (2.1-fold of the control) in the cells under hypoxic culture condition. BN52021 (Ginkgolide B), an antagonist for PAF binding sites, suppressed the effect of PAF. We conclude that PAF enhances NGF gene expression in human astrocytes, and the PAF-induced increase in the expression of NGF under hypoxia may benefit the protection of the nervous tissue by promoting neuronal survival.

Astrocytes↗

PubMed2003

Expression of metallothionein-III induced by hypoxia attenuates hypoxia-induced cell death in vitro.

Metallothioneins (MTs) are metal-binding proteins that are expressed in many tissues including brain. MTs protect cells and organs against metal toxicity and oxidants. Among MTs, a brain-predominant subtype MT-III has prominent neuroprotective activity against various types of damage. Here we show that the expression of MT-III is induced in cultured normal human astrocytes by hypoxia, and that overexpressed MT-III protects human embryonic kidney cells from hypoxia, suggesting that MT-III can protect the brain from hypoxic damage.

Astrocytes↗

PubMed2003

Interleukin-1 induces tau phosphorylation and morphological changes in cultured human astrocytes.

Tau phosphorylation is associated with neurite outgrowth and morphogenesis in neurons. Since inflammatory stimuli induce marked morphological changes in astrocytes, we examined the effect of interleukin 1alpha (IL-1alpha), an inflammatory cytokine, on the expression and phosphorylation of tau protein in cultured human astrocytes. Western blot analysis showed a rapid and transient increase (3-30 min) of tau phosphorylation at the AT8 epitope. The cells began to extend processes from 24 h after IL-1alpha stimulation. It is known that tau phosphorylation at the AT8 epitope reduces its ability to bind to, and stabilize, microtubules. The early tau phosphorylation induced by IL-1alpha might create a plastic environment for morphological changes in astrocytes.

Astrocytes↗

PubMed2003

15-Deoxy-delta 12,14-prostaglandin J2 inhibits the expression of granulocyte-macrophage colony-stimulating factor in endothelial cells stimulated with lipopolysaccharide.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), one of major hematopoietic growth factors, activates mature leukocytes. GM-CSF is produced by endothelial cells stimulated with lipopolysaccharide (LPS), and the LPS-induced GM-CSF production may play an important role in the activation of neutrophils on the endothelial surface. 15-Deoxy-delta 12,14-prostaglandin J2 (15d-PGJ2) is a ligand for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and modulates inflammatory reactions by regulating the expression of various genes. We studied the effect of 15d-PGJ2 on the LPS-induced GM-CSF expression in endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured and the expressions of GM-CSF mRNA and protein were analyzed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. 15d-PGJ2 inhibited the LPS-induced GM-CSF expression in a concentration-dependent manner; but ciglitazone, another agonist for PPAR-gamma, had no effect. This suggests that 15d-PGJ2 inhibits GM-CSF expression through a mechanism unrelated to PPAR-gamma. 15d-PGJ2 induced, by itself, the expression of interleukin-8, a potent proinflammatory chemokine, in HUVEC. 15d-PGJ2 may regulate inflammatory reactions by controlling the balance of various cytokines.

Cells, Cultured↗

PubMed2003

Glycogen synthase kinase-3beta phosphorylates synphilin-1 in vitro.

alpha-Synuclein is known to be a major component of Lewy bodies and glial cytoplasmic inclusions in the brains of patients with alpha-synucleinopathies. Synphilin-1, an alpha-synuclein-associated protein, is also present in these inclusions. However, little is known about the post-translational modifications of synphilin-1. In the present study, it is reported that synphilin-1 is phosphorylated by glycogen synthase kinase-3beta in vitro. It is well known that protein phosphorylation is involved in various physiological phenomena, including signal transduction and protein degradation. Therefore, phosphorylation of synphilin-1 may play an important role in the function of this protein in the brain.

Blotting, Western↗

PubMed2002

Alpha-synuclein pathology affecting Bergmann glia of the cerebellum in patients with alpha-synucleinopathies.

We carried out immunohistochemical examinations of the brains (cerebella) of patients who had suffered from Parkinson's disease (PD), diffuse Lewy body disease (DLBD) or multiple system atrophy (MSA), using antibodies specific for alpha-synuclein. Alpha-synuclein-positive doughnut-shaped structures were found occasionally in the cerebellar molecular layer in some of these patients. Double-labeling immunofluorescence and immunoelectron microscopy studies revealed that these alpha-synuclein-positive doughnut-shaped structures were located in the glial fibrillary acidic protein-positive radial processes of Bergmann glia, corresponding to the outer area of Lewy body-like inclusions, and consisted of granulo-filamentous structures. These findings indicate that, although not frequently, Bergmann glia of the cerebellum are also the targets of alpha-synuclein pathology in alpha-synucleinopathies such as PD, DLBD and MSA.

Aged↗

PubMed2002

Platelet-activating factor enhances the expression of vascular endothelial growth factor in normal human astrocytes.

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for vascular endothelial cells. To examine whether platelet-activating factor (PAF) induces the expression of VEGF in human astrocytes, we stimulated cultured normal astrocytes with PAF and performed semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) or real-time quantitative PCR for VEGF mRNA and enzyme-linked immunosorbent assay for VEGF protein. PAF increased the expression of VEGF in astrocytes in time- and dose-dependent manners. After 24-h stimulation, 10 nM PAF increased the levels of VEGF protein in astrocyte-conditioned medium by 1.3-fold. When the cells were subjected to hypoxia, the PAF-induced production of VEGF was enhanced by 6.7-fold as compared to the unstimulated cells incubated under normoxia. Dexamethasone was found to inhibit the enhanced VEGF production in response to the stimulation with PAF under hypoxia. We conclude that PAF induces VEGF gene expression in human astrocytes, and the PAF-induced increase in the expression of VEGF may modulate nervous tissue injury due to hypoxia.

Anti-Inflammatory Agents↗

PubMed2002

Immunohistochemical comparison of alpha- and beta-synuclein in adult rat central nervous system.

We investigated the immunohistochemical localization of alpha- and beta-synuclein (alphaS and betaS) in the adult rat CNS and compared the distribution of these proteins with that of synaptophysin (SYN), a well-established presynaptic marker. Anti-SYN, alphaS and betaS antibodies immunolabeled the neuropil in a punctate pattern. No SYN immunoreactivity was found in the neuronal cytoplasm. In contrast, both anti-alphaS and betaS immunostained the neuronal cytoplasm and nucleus to various extents depending on the brain region. alphaS-positive neurons were found in the olfactory bulb, deeper layers of the cerebral neocortex, substantia nigra and pontine nucleus. Immunoreactivity was most intense in neurons of the substantia nigra. betaS-positive neurons were more widely distributed throughout the CNS: more than 50% of neurons were positive for betaS in the granular cell layer of the olfactory bulb, layer IV of the cerebral cortex, CA3 of the hippocampus, basal ganglia and thalamic reticular nuclei. Moreover, there were differences among these proteins in the distribution pattern of immunoreactivity in the neuropil in some CNS regions. These findings suggest that although alphaS and betaS are concentrated in presynaptic terminals, significant amounts of these proteins are also present in the neuronal perikarya. Furthermore, the distribution of alphaS-positive neurons is similar to that of intraneuronal inclusions in alphaS transgenic animals as well as in human alpha-synucleinopathies.

Animals↗

PubMed2002

Expression of alpha-synuclein, the precursor of non-amyloid beta component of Alzheimer's disease amyloid, in human cerebral blood vessels.

The non-amyloid beta component of Alzheimer's disease amyloid (NAC) is detected in cerebral amyloid angiopathy; and the precursor of NAC is now known to be identical to alpha-synuclein (alpha-S), a major component of Lewy bodies in Parkinson's disease. We studied if cerebral vascular cells express alpha-S. Immunohistochemical studies of human cerebral tissues from control and cerebral amyloid angiopathy patients revealed the expression of alpha-S in vascular endothelial and smooth muscle cells. Then we studied the expression of alpha-S in vitro using cultures of vascular cells. Cultures of human umbilical vein endothelial cells and umbilical artery smooth muscle cells were found to constitutively express alpha-S messenger RNA and protein. alpha-S is normally expressed in vascular cells and may play some physiological role in the vascular wall.

Aged↗

PubMed2002

Retinoic acid-inducible gene-I is induced in endothelial cells by LPS and regulates expression of COX-2.

Bacterial lipopolysaccharides (LPS) induce expression of multiple genes in endothelial cells, which are critical cellular effectors in various pathologic syndromes. Using subtractive hybridization to identify genes that are differentially induced in human endothelial cells treated with LPS, we found that retinoic acid-inducible gene I (RIG-I) is induced in endothelial cells stimulated with LPS. RIG-I encodes a protein belonging to the DExH-box family which has diverse roles in regulation of gene expression and cellular functions. Cyclooxygenase-2 (COX-2) is also induced in endothelial cells by LPS. Overexpression of RIG-I selectively upregulated expression of COX-2 and also induced COX-2 promoter activity. RIG-I is an inducible gene in stimulated endothelial cells that may have important roles in vascular pathology by virtue of its ability to regulate expression of the COX-2 gene product.

Cell Line↗

PubMed2002

Demonstration of alpha-synuclein immunoreactivity in neuronal and glial cytoplasm in normal human brain tissue using proteinase K and formic acid pretreatment.

alpha-Synuclein (alphaS), a presynaptic nerve terminal protein, is now known to be a major component of neuronal and glial cytoplasmic inclusions in alpha-synucleinopathies (Lewy body disease and multiple system atrophy). However, alphaS has not been identified in either neuronal or glial cytoplasm in formalin-fixed, paraffin-embedded tissue sections from the normal human brain. Previous studies have shown that pretreatment with either proteinase K or formic acid enhances alphaS immunoreactivity. The aim of the present study was, therefore, to study the effects of pretreatment with proteinase K and formic acid on alphaS immunoreactivity in vibratome sections of brain tissue taken from normal human subjects. In addition to presynaptic staining, alphaS immunostaining was recognized in neuronal perikarya in the pretreated sections; this immunoreactivity was more intense in sections taken from the deeper layers of the cerebral neocortex, the CA2/3 region of the hippocampus, and the substantia nigra. This pattern of alphaS expression coincides with the distribution of intraneuronal inclusions in alphaS transgenic animals as well as in human autopsy tissue taken from patients with Lewy body disease. Furthermore, intense immunoreactivity was also found in the cytoplasm of astrocytes and oligodendrocytes throughout the brain. These findings suggest that a significant amount of alphaS is also present in the neuronal and glial cytoplasm in the normal human brain.

Aged↗

PubMed2002

15-Deoxy-D12,14-prostaglandin J2 inhibits CX3CL1/fractalkine expression in human endothelial cells.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma)is a member of nuclear hormone receptor superfamily, and is knownto play a role in various biological processes including inflammatoryresponses and adipocyte differentiation. CX3CL1/fractalkineis a potent agonist for chemotaxis and adhesion of monocytes and lymphocytes. Endothelial cells produce fractalkine when stimulated with cytokinessuch as interleukin-1 (IL-1), tumour necrosis factor-alpha andinterferon-gamma (IFN-gamma). We herein report that 15-deoxy-n12,14 -prostaglandinJ2 (15d-PGJ2), a PPAR-gamma agonist,inhibits the expression of fractalkine induced by IFN-gamma orIL-1beta in human endothelial cells. Agonist for PPAR-alpha (WY14643)or PPAR-gamma (ciglitazone) did not inhibit the cytokine-inducedfractalkine expression, and the effect of 15d-PGJ2 maybe independent of PPAR. 15-Deoxy-D12,14 prostaglandinJ2 also inhibited the adhesion of blood mononuclear cellsto endothelial monolayers treated with IFN-gamma or IL-1beta. The data suggest that 15d-PGJ2 regulates inflammatoryreactions, at least in part, through the inhibition of fractalkineexpression and leucocyte traffic through the endothelium.

Cell Adhesion↗

PubMed2002

Thalamic retrograde degeneration in the congenitally hydrocephalic rat is attributable to apoptotic cell death.

Congenitally hydrocephalic HTX rats develop ventricular dilatation with extensive damage of the cerebral white matter. Recently, we have reported that neuronal cell death also occurs in the thalamus of HTX rats. To investigate the mechanism underlying this thalamic degeneration in these animals, we carried out a histopathological study of the brain at different phases of postnatal development. Eosinophilic neurons with condensed chromatin or fragmented nuclei were observed in the thalamus from postnatal day 17 onward. The incidence of cell death in the thalamus increased with the progression of hydrocephalus. Ultrastructurally, thalamic neurons occasionally had apoptotic features including nuclear chromatin condensation and marginalization. Immunohistochemically, single-stranded DNA-positive neuronal nuclei were found in the thalamus. They were also positively stained with the TUNEL method. Marked loss of myelin and axons with many TUNEL-positive oligodendrocytes were found in the cerebral white matter. These findings suggest that the neuronal cell death observed in the thalamus in hydrocephalic HTX rats is retrograde degeneration due to extensive damage of axons in the cerebral white matter and that the thalamic retrograde degeneration is attributable to apoptotic cell death.

Animals↗