Hidetoshi Fujita

Cytoplasmic destruction of p53 by the endoplasmic reticulum-resident ubiquitin ligase ‘Synoviolin'

Synoviolin, also called HRD1, is an E3 ubiquitin ligase and is implicated in endoplasmic reticulum ‐associated degradation. In mammals, Synoviolin plays crucial roles in various physiological and pathological processes, including embryogenesis and the pathogenesis of arthropathy. However, little is known about the molecular mechanisms of Synoviolin in these actions. To clarify these issues, we analyzed the profile of protein expression in synoviolin‐null cells. Here, we report that Synoviolin targets tumor suppressor gene p53 for ubiquitination. Synoviolin sequestrated and metabolized p53 in the cytoplasm and negatively regulated its cellular level and biological functions, including transcription, cell cycle regulation and apoptosis. Furthermore, these p53 regulatory functions of Synoviolin were irrelevant to other E3 ubiquitin ligases for p53, such as MDM2, Pirh2 and Cop1, which form autoregulatory feedback loops. Our results provide novel insights into p53 signaling mediated by Synoviolin.

Abnormally High Levels of Virus-Infected IFN-γ+CCR4+CD4+CD25+ T Cells in a Retrovirus-Associated Neuroinflammatory Disorder

Background Human T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus associated with both HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic neuroinflammatory disease, and adult T-cell leukemia (ATL). The pathogenesis of HAM/TSP is known to be as follows: HTLV-1-infected T cells trigger a hyperimmune response leading to neuroinflammation. However, the HTLV-1-infected T cell subset that plays a major role in the accelerated immune response has not yet been identified. Principal Findings Here, we demonstrate that CD4+CD25+CCR4+ T cells are the predominant viral reservoir, and their levels are increased in HAM/TSP patients. While CCR4 is known to be selectively expressed on T helper type 2 (Th2), Th17, and regulatory T (Treg) cells in healthy individuals, we demonstrate that IFN-γ production is extraordinarily increased and IL-4, IL-10, IL-17, and Foxp3 expression is decreased in the CD4+CD25+CCR4+ T cells of HAM/TSP patients as compared to those in healthy individuals, and the alteration in function is specific to this cell subtype. Notably, the frequency of IFN-γ-producing CD4+CD25+CCR4+Foxp3− T cells is dramatically increased in HAM/TSP patients, and this was found to be correlated with disease activity and severity. Conclusions We have defined a unique T cell subset—IFN-γ+CCR4+CD4+CD25+ T cells—that is abnormally increased and functionally altered in this retrovirus-associated inflammatory disorder of the central nervous system.

Severe loss of invariant NKT cells exhibiting anti-HTLV-1 activity in patients with HTLV-1-associated disorders.

Invariant natural killer T (iNKT) cells are unique T cells that regulate the immune response to microbes, cancers, and autoimmunity. We assessed the characteristics of iNKT cells from persons infected with human T-lymphotropic virus type 1 (HTLV-1). Whereas most infected persons remain asymptomatic carriers (ACs) throughout their lives, a small proportion, usually with high equilibrium proviral loads,develop 2 diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia (ATL). We demonstrated that the frequency of iNKT, NK, and dendritic cells in the peripheral blood of HAM/TSP and ATL patients is decreased. We also observed an inverse correlation between the iNKT cell frequency and the HTLV-1 proviral load in the peripheral blood of infected persons. Notably, in vitro stimulation of peripheral blood cells with alpha-galactosylceramide led to an increase in the iNKT cell number and a subsequent decrease in the HTLV-1-infected T-cell number in samples from ACs but not HAM/TSP or ATL patients. Our results suggest that iNKT cells contribute to the immune defense against HTLV-1, and iNKT-cell depletion plays an important role in the pathogenesis of HAM/TSP and ATL. Therefore, iNKT cell-based immunotherapy may be an effective strategy for preventing these HTLV-1-associated disorders.

E3 ubiquitin ligase synoviolin is involved in liver fibrogenesis.

Background and Aim Chronic hepatic damage leads to liver fibrosis, which is characterized by the accumulation of collagen-rich extracellular matrix. However, the mechanism by which E3 ubiquitin ligase is involved in collagen synthesis in liver fibrosis is incompletely understood. This study aimed to explore the involvement of the E3 ubiquitin ligase synoviolin (Syno) in liver fibrosis. Methods The expression and localization of synoviolin in the liver were analyzed in CCl4-induced hepatic injury models and human cirrhosis tissues. The degree of liver fibrosis and the number of activated hepatic stellate cells (HSCs) was compared between wild type (wt) and Syno+/− mice in the chronic hepatic injury model. We compared the ratio of apoptosis in activated HSCs between wt and Syno+/− mice. We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished. Furthermore, we compared collagen synthesis between wt and Syno−/− mice embryonic fibroblasts (MEF) using quantitative RT-PCR, western blotting, and collagen assay; then, we immunohistochemically analyzed the localization of collagen in Syno−/− MEF cells. Results In the hepatic injury model as well as in cirrhosis, synoviolin was upregulated in the activated HSCs, while Syno+/− mice developed significantly less liver fibrosis than in wt mice. The number of activated HSCs was decreased in Syno+/− mice, and some of these cells showed apoptosis. Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression. Moreover, in Syno−/− MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum. Conclusion Our findings demonstrate the importance of the E3 ubiquitin ligase synoviolin in liver fibrosis.

Temporal lobe epilepsy in a genius of natural history MRI volumetric study of postmortem brain

Kumagusu Minakata (1867-1941), a Japanese genius devoted to natural history and folklore, is famous for his immense range of works, including his discovery of many new varieties of mycetozoa, or slime molds. His diary reveals that he was affected by epilepsy. In this study of his brain, we adopted a method of measuring the volume of the hippocampi by MRI of postmortem brain and found evidence of right hippocampal atrophy. This finding, together with the striking parallels between his behavior and the known behavioral syndrome in temporal lobe epilepsy (TLE), suggests that he was affected by TLE. The postmortem imaging analysis of brain, as performed in this study, offers a bridge between neuroscience and classic psychopathologic approaches to the creativity of geniuses.

Activation of synoviolin promoter in rheumatoid synovial cells by a novel transcription complex of interleukin enhancer binding factor 3 and GA binding protein α

OBJECTIVE Synoviolin is an E3 ubiquitin ligase, and its overexpression is implicated in the pathogenesis of rheumatoid arthritis (RA). We reported previously that Ets binding site 1 (EBS-1) within the synoviolin promoter is crucial for the expression of synoviolin, and GA binding protein (GABP) binds to this site. This study was undertaken to elucidate the precise mechanisms of transcriptional regulation via EBS-1. METHODS We performed purification and identification of complex components that bind to EBS-1 and inspected their contributions to the transcriptional regulation of synoviolin in rheumatoid synovial cells. We biochemically purified proteins that had EBS-1 binding activity and identified the proteins using liquid chromatography tandem mass spectrometry analysis. The identified proteins were verified to recruit and form the complex on EBS-1 using electrophoretic mobility shift assay and coimmunoprecipitation assay. Furthermore, their transcription activities were tested by reporter assays and RNA interference experiments. RESULTS We identified interleukin enhancer binding factor 3 (ILF-3) as a novel factor in the complex. ILF-3 was demonstrated to activate the synoviolin promoter via association with GABPalpha in rheumatoid synovial cells. In addition, further activation was observed with ILF-2 and GABPbeta, previously reported interactants of ILF-3 and GABPalpha, respectively. Moreover, ILF-3-knockdown experiments showed reduced expression of the synoviolin gene. CONCLUSION Our findings indicate that ILF-3, which has been known to regulate IL-2 expression in T cells, up-regulates synoviolin expression with GABPalpha in rheumatoid synovial cells. ILF-3 might be a target for RA treatment through its effect on IL-2 in T cells and synoviolin in rheumatoid synovial cells.

Strain-dependent Damage in Mouse Lung After Carbon Ion Irradiation

PURPOSE To examine whether inherent factors produce differences in lung morbidity in response to carbon ion (C-ion) irradiation, and to identify the molecules that have a key role in strain-dependent adverse effects in the lung. METHODS AND MATERIALS Three strains of female mice (C3H/He Slc, C57BL/6J Jms Slc, and A/J Jms Slc) were locally irradiated in the thorax with either C-ion beams (290 MeV/n, in 6 cm spread-out Bragg peak) or with ¹³⁷Cs γ-rays as a reference beam. We performed survival assays and histologic examination of the lung with hematoxylin-eosin and Massons trichrome staining. In addition, we performed immunohistochemical staining for hyaluronic acid (HA), CD44, and Mac3 and assayed for gene expression. RESULTS The survival data in mice showed a between-strain variance after C-ion irradiation with 10 Gy. The median survival time of C3H/He was significantly shortened after C-ion irradiation at the higher dose of 12.5 Gy. Histologic examination revealed early-phase hemorrhagic pneumonitis in C3H/He and late-phase focal fibrotic lesions in C57BL/6J after C-ion irradiation with 10 Gy. Pleural effusion was apparent in C57BL/6J and A/J mice, 168 days after C-ion irradiation with 10 Gy. Microarray analysis of irradiated lung tissue in the three mouse strains identified differential expression changes in growth differentiation factor 15 (Gdf15), which regulates macrophage function, and hyaluronan synthase 1 (Has1), which plays a role in HA metabolism. Immunohistochemistry showed that the number of CD44-positive cells, a surrogate marker for HA accumulation, and Mac3-positive cells, a marker for macrophage infiltration in irradiated lung, varied significantly among the three mouse strains during the early phase. CONCLUSIONS This study demonstrated a strain-dependent differential response in mice to C-ion thoracic irradiation. Our findings identified candidate molecules that could be implicated in the between-strain variance to early hemorrhagic pneumonitis after C-ion irradiation.

Intravenous dendritic cell administration enhances suppression of lung metastasis induced by carbon-ion irradiation

Abstract Carbon-ion radiotherapy (CIRT) is an advanced radiotherapy and has achieved good local control, even in tumors that are resistant to conventional photon beam radiotherapy (PBRT). However, distant metastasis control is an important issue. Recently, the combination of radiotherapy and immunotherapy has attracted the attention. In immunotherapy, dendritic cells (DCs) play a pivotal role in the anti-tumor immune system. However, the mechanisms underlying the combination therapy of DCs and radiotherapy have been unclear. In the present study, we evaluated anti-metastatic effects of this combination therapy, focused on the irradiation type and the route of DC administration, using a mouse model. C3H/He mice bearing NR-S1 cells were treated with CIRT or PBRT, using biologically equivalent doses. Subsequently, DCs were administered intratumorally (IT) or intravenously (IV). IV and IT DC administrations combined with CIRT to the local tumor, but not alone, significantly suppressed pulmonary metastasis, whereas the combination of DCs with PBRT suppressed metastasis at a relatively higher dose. Additionally, the anti-metastatic effect was greater in IV DC administration compared with in IT DC administration in both CIRT and PBRT. The expression levels of CD40 and IL-12 in DCs were significantly increased after co-culturing with CIRT-treated NR-S1 cells. In addition, IV administration of those co-cultured DCs significantly suppressed pulmonary metastasis. Furthermore, ecto-calreticulin levels from CIRT-treated NR-S1 cells significantly increased compared with those of a PBRT-treated tumor. Taken together, these results suggest that local CIRT combined with IV DCs augments an immunogenicity of the tumor cells by ecto-calreticulin expression and the maturation of DCs to stimulate anti-tumor immunity to decrease lung metastases.