Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1080/25785826.2019.1657377.

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.ebiom.2018.09.048.

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DOI： 10.1186/s13099-018-0251-z.

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Language：Japanese   Publisher：日本膵臓学会  

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Language：English  

Early-staged cholangiocarcinoma (CCA) is difficult to diagnose due to its high potential for invasion and metastasis. Epithelial-mesenchymal transition (EMT) is induced by transforming growth factor-β (TGF-β) in a process thought to be important for invasion and metastasis in several cancers, including CCA. Although microRNAs (miRNAs) have been implicated in the pathogenesis of several malignancies, their roles to CCA are not clearly understood. Some miRNAs were reported to be included in extracellular vesicles (EVs) and transferred from their donor cells to other cells, modulating recipient cell behaviors. In this study, the involvement and functional roles of EV-contained miRNAs during EMT in human CCA were determined. Expression profiling identified a subset of miRNAs that were reduced by TGF-β in CCA cells. Among these, miR-30e was highly downregulated by TGF-β and predicted to target Snail, which is an EMT-inducible transcription factor. MiR-30e overexpression suppressed cell invasion and migration via inhibiting EMT, whereas miR-30e inhibition promoted EMT, cell invasion and migration. Moreover, miR-30e was enriched in EVs derived from CCA cells after miR-30e overexpression, and miR-30e intercellular transfer through EVs suppressed EMT, cell invasion and migration in recipient CCA cells. Together, our results suggest that EV-mediated miR-30e transfer could inhibit EMT via directly targeting Snail, which subsequently suppresses CCA cell invasion and migration. These findings provide several new insights into regulatory mechanisms of tumor invasion and metastasis in human CCA.

DOI： 10.18632/oncotarget.24711

PubMed

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1530/EDM-17-0088

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Objective: Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) possess multiple bioactive isoforms that are rendered non-insulinotropic by the enzyme dipeptidyl peptidase-4 (DPP-4). Recently, some ELISA kits have been developed to specifically measure " active" GIP and GLP-1, but it is unclear if these kits can accurately quantify all bioactive forms. Therefore, it remains uncertain to what extent treatment with a DPP-4 inhibitor boosts levels of biologically active GIP and GLP-1. Thus, we evaluated our novel receptor-mediated incretin bioassays in comparison to commercially available ELISA kits using plasma samples from healthy subjects before and after DPP-4 inhibitor administration.
Methods: We utilized cell lines stably co-transfected with human GIP or GLP-1 receptors and a cAMP-inducible luciferase expression construct for the bioassays and commercially available ELISA kits. Assays were tested with synthetic GIP and GLP-1 receptor agonists and plasma samples collected from subjects during a 75 g oral glucose tolerance test (OGTT) performed before or following 3-day administration of a DPP-4 inhibitor.
Results: A GIP isoform GIP(1e30) NH2 increased luciferase activity similarly to GIP(1e42) in the GIP bioassay but was not detectable by either a total or active GIP ELISA kit. During an OGTT, total GIP levels measured by ELISA rapidly increased from 0 min to 15 min, subsequently reaching a peak of 59.2 +/- 8.3 pmol/l at 120 min. In contrast, active GIP levels measured by the bioassay peaked at 15 min (43.4 +/- 6.4 pmol/l) and then progressively diminished at all subsequent time points. Strikingly, at 15 min, active GIP levels as determined by the bioassay reached levels approximately 20-fold higher after the DPP-4 inhibitor treatment, while total and active GIP levels determined by ELISA were increased just 1.5 and 2.1-fold, respectively. In the absence of DPP-4 inhibition, total GLP-1 levels measured by ELISA gradually increased up to 90 min, reaching 23.5 +/- 2.4 pmol/l, and active GLP-1 levels determined by the bioassay did not show any apparent peak. Following administration of a DPP-4 inhibitor there was an observable peak of active GLP-1 levels as determined by the bioassay at 15 min after oral glucose load, reaching 11.0 +/- 0.62 pmol/l, 1.4-fold greater than levels obtained without DPP-4 inhibitor treatment. In contrast, total GLP-1 levels determined by ELISA were decreased after DPP-4 inhibitor treatment.
Conclusion: Our results using bioassays indicate that there is a greater increase in plasma levels of bioactive GIP than GLP-1 in subjects treated with DPP-4 inhibitors, which may be unappreciated using conventional ELISAs. (C) 2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

DOI： 10.1016/j.molmet.2016.12.009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

A low carbohydrate diet (LCHD) as well as sodium glucose cotransporter 2 inhibitors (SGLT2i) may reduce glucose utilization and improve metabolic disorders. However, it is not clear how different or similar the effects of LCHD and SGLT2i are on metabolic parameters such as insulin sensitivity, fat accumulation, and especially gluconeogenesis in the kidney and the liver. We conducted an 8-week study using non-diabetic mice, which were fed ad-libitum with LCHD or a normal carbohydrate diet (NCHD) and treated with/without the SGLT-2 inhibitor, ipragliflozin. We compared metabolic parameters, gene expression for transcripts related to glucose and fat metabolism, and glycogen content in the kidney and the liver among the groups. SGLT2i but not LCHD improved glucose excursion after an oral glucose load compared to NCHD, although all groups presented comparable non-fasted glycemia. Both the LCHD and SGLT2i treatments increased calorie-intake, whereas only the LCHD increased body weight compared to the NCHD, epididimal fat mass and developed insulin resistance. Gene expression of certain gluconeogenic enzymes was simultaneously upregulated in the kidney of SGLT2i treated group, as well as in the liver of the LCHD treated group. The SGLT2i treated groups showed markedly lower glycogen content in the liver, but induced glycogen accumulation in the kidney. We conclude that LCHD induces deleterious metabolic changes in the non-diabetic mice. Our results suggest that SGLT2i induced gluconeogenesis mainly in the kidney, whereas for LCHD it was predominantly in the liver.

DOI： 10.1371/journal.pone.0157672

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Glucose-dependent insulinotropic polypepide (GIP) was first extracted from porcine gut mucosa and identified as incretin decades ago. Though early studies have shown the possible GIP isoforms by gel filtration profiles from porcine or human intestinal extracts analyzed by radioimmunoassay (RIA), GIP is currently believed to consist of 42 amino acids (GIP1-42), which are released from gut K-cells and promote postprandial insulin release. In fact, GIP1-42 is usually processed from proGIP by the action of prohormone convertase (PC) 1/3 in the gut. GIP expression is occasionally found in the intestinal glucagon-like peptide-1-secreting cells, suggesting gene expression of both GIP and proglucagon can co-exist in identical cells. However, GIP1-42 immunoreactivity is rarely found in -cells or other pancreatic endocrine cells of wild-type mammals. Interestingly, we found that short-form GIP1-30 is expressed in and released from pancreatic -cells and a subset of enteroendocrine cells through proGIP processing by PC2. GIP1-30 is also insulinotropic and modulates glucose-stimulated insulin secretion in a paracrine manner. It is also suggested that short-form GIP1-30 possibly plays a crucial role for the islet development. It has not been well elucidated whether expression of GIP1-30 is modulated in the diabetic status, and whether GIP1-30 might have therapeutic potentials. Our preliminary data suggest that short-form GIP1-30 might play important roles in glucose metabolism.

DOI： 10.1111/jdi.12445

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Tokyo  

DOI： 10.1007/s13340-015-0246-7

Scopus

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Tubular injury is one of the important determinants of progressive renal failure in diabetic nephropathy (DN), and TGF-beta 1 has been implicated in the pathogenesis of tubulointerstitial disease that characterizes proteinuric renal disease. The aim of this study was to identify novel therapeutic target molecules that play a role in the tubule damage of DN. We used an LC-MS/MS-based proteomic technique and human renal proximal epithelial cells (HRPTECs). Urine samples from Japanese patients with type 2 diabetes (n = 46) were used to quantify the candidate protein. Several proteins in HRPTECs in cultured media were observed to be driven by TGF-beta 1, one of which was 33-kDa IGFBP7, which is a member of IGFBP family. TGF-beta 1 up-regulated the expressions of IGFBP7 mRNA and protein in a dose-and time-dependent fashion via Smad2 and 4, but not MAPK pathways in HRPTECs. In addition, the knockdown of IGFBP7 restored the TGF-beta 1-induced epithelial to mesenchymal transition (EMT). In the immunohistochemical analysis, IGFBP7 was localized to the cytoplasm of tubular cells but not that of glomerular cells in diabetic kidney. Urinary IGFBP7 levels were significantly higher in the patients with macroalbuminuria and were correlated with age (r = 0.308, p = 0.037), eGFR (r = -0.376, p = 0.01), urinary beta(2)-microglobulin (r = 0.385, p = 0.008), and urinary N-acetyl-beta-D-glucosaminidase (NAG) (r = 0.502, p = 0.000). A multivariate regression analysis identified urinary NAG and age as determinants associated with urinary IGFBP7 levels. In conclusion, our data suggest that TGF-beta 1 enhances IGFBP7 via Smad2/4 pathways, and that IGFBP7 might be involved in the TGF-beta 1-induced tubular injury in DN.

DOI： 10.1371/journal.pone.0150897

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Persistent high concentration of glucose causes cellular stress and damage in diabetes via derangement of gene expressions. We previously reported high glucose activates hypoxia-inducible factor-1αand downstream gene expression in mesangial cells, leading to an extracellular matrix expansion in the glomeruli. A glucose-responsive transcription factor carbohydrate response element-binding protein (ChREBP) is a key mediator for such perturbation of gene regulation. To provide insight into glucose-mediated gene regulation in mesangial cells, we performed chromatin immunoprecipitation followed byDNAmicroarray analysis and identified platelet-derived growth factor-C (PDGF-C) as a novel target gene of ChREBP In streptozotocin-induced diabetic mice, glomerular cells showed a significant increase inPDGF-C expression; the ratio ofPDGF-C-positive cells to the total number glomerular cells demonstrated more than threefold increase when compared with control animals. In cultured human mesangial cells, high glucose enhanced expression ofPDGF-C protein by 1.9-fold. Knock-down of ChREBPabrogated this induction response. UpregulatedPDGF-C contributed to the production of typeIVand typeVIcollagen, possibly via an autocrine mechanism. Interestingly, urinaryPDGF-C levels in diabetic model mice were significantly elevated in a fashion similar to urinary albumin. Taken together, we hypothesize that a high glucose-mediated induction ofPDGF-C via ChREBPin mesangial cells contributes to the development of glomerular mesangial expansion in diabetes, which may provide a platform for novel predictive and therapeutic strategies for diabetic nephropathy.

DOI： 10.14814/phy2.12730

PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Aims/hypothesis Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone released from gut K cells. While the predominant form is GIP(1-42), a shorter form, GIP(1-30), is produced by pancreatic alpha cells and promotes insulin secretion in a paracrine manner. Here, we elucidated whether GIP(1-30) expression is modulated in mouse models of diabetes. We then investigated whether PEGylated GIP(1-30) can improve islet function and morphology as well as suppress the progression to hyperglycaemia in mice treated with low-dose streptozotocin (LD-STZ).
Methods We examined pancreatic GIP immunoreactivity in rodent diabetic models. We synthesised [D-Ala(2)]GIP(1-30) and modified the C-terminus with polyethylene glycol (PEG) to produce a dipeptidyl peptidase-4 (DPP-4)-resistant long-acting GIP analogue, [D-Ala(2)]GIP(1-30)-PEG. We performed i.p.GTT and immunohistochemical analysis in nondiabetic and LD-STZ diabetic mice, with or without administration of [D-Ala(2)]GIP(1-30)-PEG.
Results Pancreatic GIP expression was concomitantly enhanced with alpha cell expansion in rodent models of diabetes. Treatment with DPP-4 inhibitor decreased both the GIP-and glucagon-positive areas and preserved the insulin-positive area in LD-STZ diabetic mice. Body weight was not affected by [D-Ala(2)]GIP(1-30)-PEG in LD-STZ or non-diabetic mice. Treatment with GIP significantly ameliorated chronic hyperglycaemia and improved glucose excursions in LD-STZ mice. Treatment with GIP also reduced alpha cell expansion in the islets and suppressed plasma glucagon levels compared with non-treated LD-STZ mice. Additionally, [D-Ala2] GIP(1-30)-PEG preserved beta cell area via inhibition of apoptosis in LD-STZ mice.
Conclusions/interpretation Our data suggest that GIP(1-30) expression is upregulated in diabetes, and PEGylated GIP(1-30) can suppress the progression to STZ-induced hyperglycaemia by inhibiting beta cell apoptosis and alpha cell expansion.

DOI： 10.1007/s00125-015-3842-y

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Inhibitory PAS domain protein (IPAS), a dominant negative regulator of hypoxia-inducible gene expression, is generated from hypoxia inducible transcription factor-3 alpha (HIF-3 alpha) pre-mRNA by an alternative splicing mechanism. Inactivation of the IPAS transcript in mice leads to the neo-vascularization of the cornea, suggesting that IPAS is an important regulator of anti-angiogenesis in this tissue. For the first time we demonstrate that serine-arginine (SR) proteins are involved in oxygen tension-dependent changes in pre-mRNA splicing. SR proteins isolated from hypoxic cells differentially interact with RNA (compared with proteins isolated from cells cultured under normoxic conditions). They possess the differential ability to activate hypoxia-dependent splice sites, and they are more phosphorylated than those isolated from normoxic HeLa cells. We also show that expression of SR protein kinases (CLK1, SRPK1, SRPK2) in hypoxic cells is elevated at mRNA and protein levels. Increased expression of CLK1 kinase is regulated by HIFs. Reduction of CLK1 cellular expression levels reduces hypoxia-dependent full-length carbonic anhydrase IX (CAIX) mRNA and CAIX protein formation and changes hypoxia-dependent cysteine-rich angiogenic inducer 61 (Cyr61) mRNA isoform formation profiles.

DOI： 10.1074/jbc.M115.639690

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.2169/internalmedicine.54.4735

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

Defects in glucose uptake by the skeletal muscle cause diseases linked to metabolic disturbance such as type 2 diabetes. The molecular mechanism determining glucose disposal in the skeletal muscle in response to cellular stimuli including insulin, however, remains largely unknown. The hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a transcription factor operating in the cellular adaptive response to hypoxic conditions. Recent studies have uncovered pleiotropic actions of HIF-1 alpha in the homeostatic response to various cellular stimuli, including insulin under normoxic conditions. Thus we hypothesized HIF-1 alpha is involved in the regulation of glucose metabolism stimulated by insulin in the skeletal muscle. To this end, we generated C2C12 myocytes in which HIF-1 alpha is knocked down by short-hairpin RNA and examined the intracellular signaling cascade and glucose uptake subsequent to insulin stimulation. Knockdown of HIF-1 alpha expression in the skeletal muscle cells resulted in abrogation of insulin-stimulated glucose uptake associated with impaired mobilization of glucose transporter 4 (GLUT4) to the plasma membrane. Such defect seemed to be caused by reduced phosphorylation of the protein kinase B substrate of 160 kDa (AS160). AS160 phosphorylation and GLUT4 translocation by AMP-activated protein kinase activation were abrogated as well. In addition, expression of the constitutively active mutant of HIF-1 alpha (CA-HIF-1 alpha) or upregulation of endogenous HIF-1 alpha in C2C12 cells shows AS160 phosphorylation comparable to the insulin-stimulated level even in the absence of insulin. Accordingly GLUT4 translocation was increased in the cells expressing CA-HIF1 alpha. Taken together, HIF-1 alpha is a determinant for GLUT4-mediated glucose uptake in the skeletal muscle cells thus as a possible target to alleviate impaired glucose metabolism in, e.g., type 2 diabetes.

DOI： 10.1152/ajpendo.00597.2012

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Language：English   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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DOI： 10.1007/s00535-012-0651-7

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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DOI： 10.1007/s00125-011-2365-4

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER DIABETES ASSOC  

OBJECTIVE-Chronic hypoxia has been recognized as a key regulator in renal tubulointerstitial fibrosis, as seen in diabetic nephropathy, which is associated with the activation of hypoxia-inducible factor (HIF)-1 alpha. We assess here the effects of the biguanide, metformin, on the expression of HIF-1 alpha in diabetic nephropathy using renal proximal tubular cells and type 2 diabetic rats.
RESEARCH DESIGN AND METHODS-We explored the effects of metformin on the expression of HIF-1 alpha using human renal proximal tubular epithelial cells (HRPTECs). Male Zucker diabetic fatty (ZDF; Gmi-fa/fa) rats were treated from 9 to 39 weeks with metformin (250 mg kg(-1) . day(-1)) or insulin.
RESULTS- Metformin inhibited hypoxia-induced HIF-1 alpha accumulation and the expression of HIF-1-targeted genes in HRPTECs. Although metformin activated the downstream pathways of AMP-activated protein kinase (AMPK), neither the AMPK activator, AICAR, nor the mTOR inhibitor, rapamycin, suppressed hypoxia-induced HIF-1 alpha expression. In addition, knockdown of AMPK-a did not abolish the inhibitory effects of metformin on HIF-1 alpha expression. The proteasome inhibitor, MG-132, completely eradicated the suppression of hypoxia-induced HIF-1 alpha accumulation by metformin. The inhibitors of mitochondrial respiration similarly suppressed hypoxia-induced HIF-1 alpha expression. Metformin significantly decreased ATP production and oxygen consumption rates, which subsequently led to increased cellular oxygen tension. Finally, metformin, but not insulin, attenuated tubular HIF-1 alpha expression and pimonidazole staining and ameliorated tubular injury in ZDF rats.
CONCLUSIONS-Our data suggest that hypoxia-induced HIF-1 alpha accumulation in diabetic nephropathy could be suppressed by the antidiabetes drug, metformin, through the repression of oxygen consumption. Diabetes 60:981-992, 2011

DOI： 10.2337/db10-0655

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

High glucose evokes a variety of signals in mesangial cells that alter cellular functions responsible for the development of diabetic glomerulopathy. The hypoxia-inducible factor-1 alpha (HIF-1 alpha) regulates cellular homeostasis under hypoxic conditions, but it also has pleiotropic effects in response to cellular stresses at normoxia. Here we determined whether HIF-1 alpha has a role in the regulation of mesangial cells in hyperglycemia. In the streptozotocin-induced diabetic mouse model, glomerular mesangial cells had a significant increase in HIF-1 alpha expression in the nucleus. In cultured mesangial cells, high glucose enhanced the expression of HIF-1 alpha and its target genes known to be involved in the development of diabetic glomerulopathy. A glucose-responsive carbohydrate response element binding protein (ChREBP) was found to have a critical role in the transcriptional upregulation of HIF-1 alpha and downstream gene expression in mesangial cells exposed to high glucose. Knockdown of HIF-1 alpha or ChREBP in mesangial cells abrogated the high glucose-mediated perturbation of gene expression. Our results show that ChREBP and HIF-1 alpha mediate gene regulation in mesangial cells. Further studies will be needed to find out whether these findings are relevant to the development of the diabetic nephropathy. Kidney International (2010) 78, 48-59; doi: 10.1038/ki.2010.99; published online 7 April 2010

DOI： 10.1038/ki.2010.99

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC IMMUNOLOGISTS  

Peripheral T cells encounter rapid decrease in oxygen tension because they are activated by Ag recognition and migrate into inflammatory sites or tumors. Activated T cells, therefore, are thought to have such machineries that enable them to adapt to hypoxic conditions and execute immune regulation in situ. We have recently shown that survival of CD3-engaged human peripheral blood T cells is prolonged under hypoxic conditions and hypoxia-inducible factor-1 (HIF-1) and its target gene product adrenomedullin play a critical role for the process. It is also shown that hypoxia alone is not sufficient, but TCR-mediated signal is required for accumulation of HIF-1 alpha in human peripheral T cells. In the present study, we showed that TCR engagement does not influence hypoxia-dependent stabilization but stimulates protein synthesis of HIF-1a, most possibly via PI3K/mammalian target of rapamycin system, and that expression of HIF-1a and its target genes is blocked by treatment with rapamycin. Since some of those gene products, e.g., glucose transporters and phosphoglycerokinase, are considered to be essential for glycolysis and energy production under hypoxic conditions and adequate immune reaction in T cells, this TCR-mediated synthesis of HIF-1 alpha may play a pivotal role in peripheral immune response. Taken together, our results may highlight a novel aspect of downstream signal from Ag recognition by TCR and a unique pharmacological role of rapamycin as well.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Glucocorticoids are secreted from the adrenal glands and act as a peripheral effector of the hypothalamic-pituitary-adrenal axis, playing an essential role in stress response and homeostatic regulation. In target cells, however, it remains unknown how glucocorticoids fine-tune the cellular pathways mediating tissue and systemic adaptation. Recently, considerable evidence indicates that adaptation to hypoxic environments is influenced by glucocorticoids and there is cross-talk between hypoxia-dependent signals and glucocorticoid-mediated regulation of gene expression. We therefore investigated the interaction between these important stress-responsive pathways, focusing on the glucocorticoid receptor (GR) and hypoxia-inducible transcription factor HIF-1. Here we show that, under hypoxic conditions, HIF-1-dependent gene expression is further up-regulated by glucocorticoids via the GR. This up-regulation cannot be substituted by the other steroid receptors and is suggested to result from the interaction between the GR and the transactivation domain of HIF-1alpha. Moreover, our results also indicate that the ligand binding domain of the GR is essential for this interaction, and the critical requirement for GR agonists suggests the importance of the ligand-mediated conformational change of the GR. Because these proteins are shown to colocalize in the distinct compartments of the nucleus, we suggest that these stress-responsive transcription factors have intimate communication in close proximity to each other, thereby enabling the fine-tuning of cellular responses for adaptation.

DOI： 10.1074/jbc.M302581200

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Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1046/j.1365-2443.2003.00618.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Ligand-receptor coupling is one of the important constituents of signal transduction and is essential for physiological transmission of actions of endogenous substances including steroid hormones. However, molecular mechanisms of the redundancy between glucocorticoid and mineralocorticoid actions remain unknown because of complicated cross-talk among, for example, these adrenal steroids, their cognate receptors, and target genes. Receptor-specific ligand that can distinctly modulate target gene expression should be developed to overcome this issue. In this report, we showed that a pyrazolosteroid cortivazol (CVZ) does not induce either nuclear translocation or transactivation function of the mineralocorticoid receptor (MR) but does both for the glucocorticoid receptor (GR). Moreover, deletion analysis of the C-terminal end of the GR has revealed that CVZ interacts with the distinct portion of the ligand binding-domain (LBD) and differentially modulates the ligand-dependent interaction between transcription intermediary factor 2 and the LBD when compared with cortisol, dexamethasone, and aldosterone. Thus, it is indicated that CVZ may not be only a molecular probe for the analysis of the redundancy between the GR and MR in vivo but also a useful reagent to clarify structure-function relationship of the GR LBD.

DOI： 10.1074/jbc.M107946200

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DOI： 10.1385/1-59259-274-0:171

PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Ursodeoxycholic acid (UDCA) is the current mainstay of treatment for various liver diseases including primary biliary cirrhosis. UDCA acts as a bile secretagogue, cytoprotective agent, immunomodulator, and inhibitor of cellular apoptosis. Despite this cumulative evidence of the cytoprotective and immunosuppressive effects of UDCA, both the target molecule and pathway of UDCA action remain unknown. We previously described that, in the absence of glucocorticoid ligand, UDCA activates the glucocorticoid receptor (GR) into DNA binding species but does not elicit its transactivational function in a transient transfection assay. Here we further studied the molecular mechanism of UDCA action and revealed that the ligand binding domain of the GR is responsible for UDCA-dependent nuclear translocation of the GR. Indeed, we demonstrated that UDCA acts on the distinct region of the ligand binding domain when compared with the classical GR agonist dexamethasone, resulting in loss of coactivator recruitment and differential regulation of gene expression by the GR. Our data clearly indicated that UDCA, at least in part via activation of the GR, suppresses NF-kappaB-dependent transcription through the intervention of GR-p65 interaction. Together with the established clinical safety of UDCA, we may propose that UDCA could be a prototypical compound for development of a novel and selective GR modifier.

DOI： 10.1074/jbc.M107098200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We examined the effect of the novel antioxidant EPC-K1 (L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,1 2-trimethyltridecyl)-2H-1-benzopyran-6-yl hydrogen phosphate] potassium salt) on glu cocorticoid receptor function. We used cloned CHOpMTGR cells in which human glucocorticoid receptor cDNA was stably transfected and the glucocorticoid receptor was expressed at high levels. We recently suggested that glucocorticoid-mediated gene expression is modulated via the cellular redox state [Makino et at., J Clin Invest 98: 2469-2477, 1996]. In the present study, this issue was clearly evidenced by the finding that cellular treatment with H2O2 decreased the ligand binding and transcriptional activity of the glucocorticoid receptor, and we showed that these inhibitory effects of H2O2 were effectively titrated by the addition of EPC-K1. Moreover, DNA-binding activity of the bacterially expressed DNA-binding domain of the glucocorticoid receptor was repressed by the thiol-oxidizing reagent diamide; EPC-K1 also counteracted this repressive effect of diamide. Thus, the redox state was indicated to influence glucocorticoid receptor function at various steps, and EPC-K1 may be useful in restoring the cellular glucocorticoid-responsiveness in oxidative conditions. BIOCHEM PHARMACOL 56;1:79-86, 1998. (C) 1998 Elsevier Science Inc.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Redox regulation of transcription factors has recently been demonstrated for AP-1, NF-kappa B, Sp-1 and glucocorticoid receptor in vitro and in vivo. The redox state in estrogen-dependent cells possibly influences the function of estrogen receptor (ER), and the regulation of the function of ER is essential for understanding of growth and differentiation of these cells, as well as promotion and progression of estrogen-associated cancer, In this paper, we first analyzed the effects of redox state on transcriptional activity of ER in terms of pS2 mRNA expression and transfection of ERE-CAT plasmid in human breast cancer cells. Addition of H2O2 at low concentrations lowered levels of pS2 mRNA and also down-regulated ERE-CAT activity, which was recovered by transfection of thioredoxin (TRX) expression vector, Next, the transfection of antisense TRX plasmid diminished ERE-CAT activity, and the activity was recovered by co-transfected sense TRX. Furthermore, specific DNA binding activity of recombinant ER was inhibited by sulfhydryl-modifying reagents and restored by the addition of recombinant TRX protein in electrophoretic mobility shift assay. These results in vitro and in vivo revealed that the transcription activity of ER is strongly influenced by its redox state, which is reversibly modulated by endogenous redox effector protein, TRX.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILLIAMS & WILKINS  

The water-soluble gold preparation aurothiomalate, which contains gold as Au(I), is frequently prescribed for patients with rheumatoid arthritis as a disease-modifying agent. We report that aurothiomalate negatively modulates glucocorticoid hormone action; it represses the ligand- and DNA-binding activities and the transactivation function of the glucocorticoid receptor. We suggested the existence of endogenous titrating activities of Au(I) because otherwise administration of aurothiomalate to a patient with rheumatoid arthritis would be expected to result in peripheral insensitivity to glucocorticoids and worsen the patient's status. Focusing on metal ions that are present in vivo, we found that Zn(II) counteracts the inhibitory effect of Au(I) on glucocorticoid receptor function. This complementary effect of Zn(II) was observed at physiological concentrations. We suggest that Zn(II) preserves glucocorticoid receptor function in target tissues and maintains hormone responsiveness, even with chrysotherapy.

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/0922-4106(95)90133-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILLIAMS & WILKINS  

The neutral metalloproteinase collagenase is known to be, among others, one of the key enzymes promoting joint destruction in patients with rheumatoid arthritis. Because inflammatory cytokines, e.g., interleukin-l and tumor necrosis factor-alpha, are considered to activate collagenase gene expression through activation of the transcription factor activator protein-1, we examined whether the water-soluble gold compound aurothiomalate (AuTM) influenced collagenase gene expression, using phorbol ester-treated human fibroblasts. However, AuTM did not prevent phorbol ester-mediated activation of activator protein-1 DNA-binding activity and subsequent induction of collagenase gene expression. In contrast, AuTM counteracted the repressive effects of glucocorticoids on collagenase gene expression and restored collagenase mRNA levels. The molecular target of this paradoxical AuTM action was suggested to be the glucocorticoid receptor.

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Language：Japanese   Book type：Scholarly book

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Language：Japanese   Book type：Scholarly book

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Language：Japanese   Book type：Scholarly book

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Language：Japanese   Book type：Dictionary, encyclopedia

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER DIABETES ASSOC  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (bulletin of university, research institution)   Publisher：日本糖尿病合併症学会  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：メディカルレビュー社  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：ライフサイエンス出版  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER DIABETES ASSOC  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：医学出版  

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Language：English   Publisher：WILEY-BLACKWELL  

DOI： 10.1111/j.2040-1124.2011.00118.x

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：医事出版社  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER DIABETES ASSOC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER DIABETES ASSOC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER DIABETES ASSOC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPAN ENDOCRINE SOC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER DIABETES ASSOC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER DIABETES ASSOC  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：医学出版  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：日本臨床社  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER DIABETES ASSOC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER DIABETES ASSOC  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：先端医学社  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：医歯薬出版株式会社  

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Language：English   Publisher：AMER SOC MICROBIOLOGY  

Hypoxia-inducible factors (HIFs) are crucial for oxygen homeostasis during both embryonic development and postnatal life. Here we show that a novel HIF family basic helix-loop-helix (bHLH) PAS (Per-Arnt-Sim) protein, which is expressed predominantly during embryonic and neonatal stages and thereby designated NEPAS (neonatal and embryonic PAS), acts as a negative regulator of HIF-mediated gene expression. NEPAS mRNA is derived from the HIF-3 alpha gene by alternative splicing, replacing the first exon of HIF-3 alpha with that of inhibitory PAS. NEPAS can dimerize with Arnt and exhibits only low levels of transcriptional activity, similar to that of HIF-3a. NEPAS suppressed reporter gene expression driven by HIF-1 alpha and HIF-2 alpha. By generating mice with a targeted disruption of the NEPAS/HIF-3 alpha locus, we found that homozygous mutant mice (NEPAS/ HIF-3 alpha(-/-)) were viable but displayed enlargement of the right ventricle and impaired lung remodeling. The expression of endothelin I and platelet-derived growth factor beta was increased in the lung endothelial cells of NEPAS/HIF-3 alpha-null mice. These results demonstrate a novel regulatory mechanism in which the activities of HIF-1 alpha and HIF-2 alpha are negatively regulated by NEPAS in endothelial cells, which is pertinent to lung and heart development during the embryonic and neonatal stages.

DOI： 10.1128/MCB.01332-07

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER DIABETES ASSOC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER DIABETES ASSOC  

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS), a dominant negative regulator of hypoxia-inducible transcription factors (HIFs), is potentially implicated in negative regulation of angiogenesis in such tissues as the avascular cornea of the eye. We have previously shown IPAS mRNA expression is up-regulated in hypoxic tissues, which at least in part involves hypoxia-dependent alternative splicing of the transcripts from the IPAS/HIF-3 alpha locus. In the present study, we demonstrate that a hypoxia-driven transcriptional mechanism also plays a role in augmentation of IPAS gene expression. Isolation and analyses of the promoter region flanking to the first exon of IPAS gene revealed a functional hypoxia response element at position -834 to -799, whereas the sequence upstream of the HIF-3 alpha first exon scarcely responded to hypoxic stimuli. A transient transfection experiment demonstrated that HIF-1 alpha mediates IPAS promoter activation via the functional hypoxia response element under hypoxic conditions and that a constitutively active form of HIF-1 alpha is sufficient for induction of the promoter in normoxic cells. Moreover, chromatin immunoprecipitation and electrophoretic mobility shift assays showed binding of the HIF-1 complex to the element in a hypoxia-dependent manner. Taken together, HIF-1 directly up-regulates IPAS gene expression through a mechanism distinct from RNA splicing, providing a further level of negative feedback gene regulation in adaptive responses to hypoxic/ischemic conditions.

DOI： 10.1074/jbc.M700732200

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Language：Japanese   Publisher：(一社)日本リウマチ学会  

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Language：Japanese   Publisher：(一社)日本リウマチ学会  

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Language：Japanese   Publisher：(有)科学評論社  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：科学評論社  

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Language：Japanese   Publisher：(一社)日本リウマチ学会  

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Language：English   Publisher：AMER ASSOC IMMUNOLOGISTS  

Peripheral T lymphocytes undergo activation by antigenic stimulation and function in hypoxic areas of inflammation. We demonstrated in CD3-positive human T cells accumulating in inflammatory tissue expression of the hypoxia-inducible factor-1alpha (HIF-1alpha), indicating a role of hypoxia-mediated signals in regulation of T cell function. Surprisingly, accumulation of HIF-1alpha in human T cells required not only hypoxia but also TCR/CD3-mediated activation. Moreover, hypoxia repressed activation-induced cell death (AICD) by TCR/CD3 stimulation, resulting in an increased survival of the cells. Microarray analysis suggested the involvement of HIF-1 target gene product adrenomedullin (AM) in this process. Indeed, AM receptor antagonist abrogated hypoxia-mediated repression of AICD. Moreover, synthetic AM peptides repressed AICD even in normoxia. Taken together, we propose that hypoxia is a critical determinant of survival of the activated T cells via the HIF-1alpha-AM cascade, defining a previously unknown mode of regulation of peripheral immunity.

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Language：Japanese   Publisher：(一社)日本内分泌学会  

DOI： 10.1507/endocrine1927.79.1_175

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本腎臓学会  

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Language：Japanese   Publisher：(一社)日本リウマチ学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：BIOMED CENTRAL LTD  

DOI： 10.1186/ar868

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, functions as a dominant negative regulator of hypoxia-inducible transcription factors (HIFs) by forming complexes with those proteins that fail to bind to hypoxia response elements of target genes. We have previously observed that IPAS is predominantly expressed in mice in Purkinje cells of the cerebellum and in corneal epithelium of the eye where it appears to play a role in negative regulation of angiogenesis and maintenance of an avascular phenotype. Sequencing of the mouse IPAS genomic structure revealed that IPAS is a splicing variant of the HIF-3alpha locus. Thus, in addition to three unique exons (1a, 4a, and 16) IPAS shares three exons (2, 4, and 5) with HIF-3alpha as well as alternatively spliced variants of exons 3 and 6. In experiments using normal mice and mice exposed to hypoxia (6% O-2) for 6 h we observed alternative splicing of the HIF-3alpha transcript in the heart and lung. The alternatively spliced transcript was only observed under hypoxic conditions, thus defining a novel mechanism of hypoxia-dependent regulation of gene expression. Importantly, this mechanism may establish negative feedback loop regulation of adaptive responses to hypoxia/ischemia in these tissues.

DOI： 10.1074/jbc.C200328200

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, functions as a dominant negative regulator of hypoxia-inducible transcription factors (HIFs) by forming complexes with those proteins that fail to bind to hypoxia response elements of target genes. We have previously observed that IPAS is predominantly expressed in mice in Purkinje cells of the cerebellum and in corneal epithelium of the eye where it appears to play a role in negative regulation of angiogenesis and maintenance of an avascular phenotype. Sequencing of the mouse IPAS genomic structure revealed that IPAS is a splicing variant of the HIF-3alpha locus. Thus, in addition to three unique exons (1a, 4a, and 16) IPAS shares three exons (2, 4, and 5) with HIF-3alpha as well as alternatively spliced variants of exons 3 and 6. In experiments using normal mice and mice exposed to hypoxia (6% O-2) for 6 h we observed alternative splicing of the HIF-3alpha transcript in the heart and lung. The alternatively spliced transcript was only observed under hypoxic conditions, thus defining a novel mechanism of hypoxia-dependent regulation of gene expression. Importantly, this mechanism may establish negative feedback loop regulation of adaptive responses to hypoxia/ischemia in these tissues.

DOI： 10.1074/jbc.C200328200

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Language：Japanese   Publisher：医歯薬出版(株)  

個体において,肺胞気,動脈血,毛細血管,組織・細胞に至る酸素分圧のグラジエントが存在する.とくに細胞レベルでは酸素分圧は10mmHg前後であり,酸素代謝は極めて厳格にコントロールされている.ここで,転写因子HIF-1は低酸素で活性化され,VEGFなど多くの遺伝子の発現を転写レベルで正に制御し,細胞・臓器・個体レベルでの適応をコントロールしている.近年,虚血性心疾患などを対象に,HIF-1遺伝子の治療的応用も試みられている.一方,固形腫瘍などではHIF-1がVHLの変異等によって活性化され,腫瘍血管新生等に関与している.IPASはHIF-1に拮抗する分子であり,血管新生のフィードバック機構の解明のみならず,血管新生病への治療的応用も期待される

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Language：English   Publisher：NATL ACAD SCIENCES  

Insufficient oxygen and nutrient supply often restrain solid tumor growth, and the hypoxia-inducible factors (HIF) 1alpha and HIF-2alpha are key transcription regulators of phenotypic adaptation to low oxygen levels. Moreover, mouse gene disruption studies have implicated HIF-2alpha in embryonic regulation of tyrosine hydroxylase, a hallmark gene of the sympathetic nervous system. Neuroblastoma tumors originate from immature sympathetic cells, and therefore we investigated the effect of hypoxia on the differentiation status of human neuroblastoma cells. Hypoxia stabilized HIF-1alpha and HIF-2alpha proteins and activated the expression of known hypoxia-induced genes, such as vascular endothelial growth factor and tyrosine hydroxylase. These changes in gene expression also occurred in hypoxic regions of experimental neuroblastoma xenografts grown in mice. In contrast, hypoxia decreased the expression of several neuronal/neuroendocrine marker genes but induced genes expressed in neural crest sympathetic progenitors, for instance c-kit and Notch-1. Thus, hypoxia apparently causes dedifferentiation both in vitro and in vivo. These findings suggest a novel mechanism for selection of highly malignant tumor cells with stem-cell characteristics.

DOI： 10.1073/pnas.102660199

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本リウマチ学会  

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Language：Japanese   Publisher：(一社)日本リウマチ学会  

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Language：Japanese   Publisher：(一社)日本リウマチ学会  

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The cytokine receptor common 0 subunit (P.) transmits intracellular signals upon binding ligand such as granulocyte-macrophage colony-stimulating factor or interleukin-3 (IL-3); however, transcriptional regulation under the control of signaling events downstream of the P,, is not fully understood. Using murine Ba/F3 cells, here we demonstrate that the beta(c)-mediated signals stimulate NF-kappaB-driven gene expression of not only the reporter construct but also endogenous target genes such as IL-6. Analyzing the effects of several inhibitors or mutant receptors revealed that this NF-kappaB activation is mediated neither by MEK/ERK/MAPK nor by the phosphatidylinositol 3-kinase pathway but by STAT5. Overexpression experiments of the wild-type or constitutive active form of STAT5 further confirmed this notion. In addition, STAT5-dependent NF-kappaB activation is mediated not through an inducible nuclear translocation but via up-regulation of both DNA binding activity and transactivation potential of NF-kappaB. Furthermore, we also show that as yet undefined humoral factor(s) may be involved in this NF-kappaB activation process. Taken together, we may propose that cytokine receptor-mediated STAT5 activation and expression of its target genes culminates in a unique mode of NF-kappaB activation and gene expression.

DOI： 10.1074/jbc.M109878200

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Language：English   Publisher：MACMILLAN PUBLISHERS LTD  

Alteration of gene expression is a crucial component of adaptive responses to hypoxia. These responses are mediated by hypoxia-inducible transcription factors (HIFs)(1,2). Here we describe an inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, which is a basic helix-loop-helix (bHLH)/PAS protein structurally related to HIFs. IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. In mice, IPAS was predominantly expressed in Purkinje cells of the cerebellum and in corneal epithelium of the eye. Expression of IPAS in the cornea correlates with low levels of expression of the VEGF gene under hypoxic conditions. Application of an IPAS antisense oligonucleotide to the mouse cornea induced angiogenesis under normal oxygen conditions, and demonstrated hypoxia-dependent induction of VEGF gene expression in hypoxic corneal cells. These results indicate a previously unknown mechanism for negative regulation of angiogenesis and maintenance of an avascular phenotype.

DOI： 10.1038/35107085

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Language：English   Publisher：MACMILLAN PUBLISHERS LTD  

Alteration of gene expression is a crucial component of adaptive responses to hypoxia. These responses are mediated by hypoxia-inducible transcription factors (HIFs)(1,2). Here we describe an inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, which is a basic helix-loop-helix (bHLH)/PAS protein structurally related to HIFs. IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. In mice, IPAS was predominantly expressed in Purkinje cells of the cerebellum and in corneal epithelium of the eye. Expression of IPAS in the cornea correlates with low levels of expression of the VEGF gene under hypoxic conditions. Application of an IPAS antisense oligonucleotide to the mouse cornea induced angiogenesis under normal oxygen conditions, and demonstrated hypoxia-dependent induction of VEGF gene expression in hypoxic corneal cells. These results indicate a previously unknown mechanism for negative regulation of angiogenesis and maintenance of an avascular phenotype.

DOI： 10.1038/35107085

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Language：Japanese   Publisher：日本癌学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本リウマチ学会  

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Language：Japanese   Publisher：(一社)日本リウマチ学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(有)医学の世界社  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：English   Publisher：OXFORD UNIV PRESS  

In normoxic cells the hypoxia-inducible factor-1 alpha (HIF-1 alpha) is rapidly degraded by the ubiquitin-proteasome pathway, and activation of HIF-1 alpha to a functional form requires protein stabilization. Here we show that the product of the von Hippel-Lindau (VHL) tumor suppressor gene mediated ubiquitylation and proteasomal degradation of HIF-1 alpha under normoxic conditions via interaction with the core of the oxygen-dependent degradation domain of HIF-1 alpha. The region of VHL mediating interaction with HIF-1 alpha overlapped with a putative macromolecular binding site observed within the crystal structure of VHL, This motif of VHL also represents a mutational hotspot in tumors, and one of these mutations impaired interaction with HIF-1 alpha and subsequent degradation. Interestingly, the VHL binding site within HIF-1 alpha overlapped with one of the minimal transactivation domains. Protection of HIF-1 alpha against degradation by VHL was a multistep mechanism, including hypoxia-induced nuclear translocation of HIF-1 alpha and an intranuclear hypoxia-dependent signal. VHL was not released from HIF-1 alpha during this process. Finally, stabilization of HIF-1 alpha protein levels per se did not totally bypass the need of the hypoxic signal for generating the transactivation response.

DOI： 10.1093/emboj/19.16.4298

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：The Japanese Society of Inflammation and Regeneration  

Glucocorticoids are not only essential hormones to maintain homeostasis but also effective drugs for controlling inflammatory disorders. Glucocorticoids inhibit expression of proinflammatory genes such as cytokines, chemokines, and adhesion molecules. The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR) . The GR is localized in the cytoplasm in the absence of hormonal ligand. Upon binding ligand, the GR dissociates from molecular chaperones such as heat shock proteins and translocates into the nucleus. The GR interacts with other transcription factors such as NF-κB and AP-1 which activate proinflammatory genes and inhibits inflammation through negative regulation of these transcription factors and/or competition with these transcription factors for association with coactivators. Therefore, nuclear translocation of the GR is an important step for glucocorticoid action and is controlled by nuclear localization signals within the GR molecule and various cellular factors. It is suggested that cellular oxidation-reduction (redox) status also regulates nuclear translocation of GR by targeting the cysteine within the NL1. We demonstrated that various functions of GR are regulated by redox status.<BR>Taken together, it is important to understand the mechanisms of redox dependent regulation of GR function, to aid development of novel drugs and therapies for various disorders including inflammatory diseases.

DOI： 10.2492/jsir1981.20.657

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Language：Japanese   Publisher：(有)医学の世界社  

単球系細胞株U937細胞とヒトマクロファージのAhRがリガンドに応答して機能することを示した.単球系細胞株U937細胞,ヒトマクロファージ及びヒトリンパ球に,リガンドである3MCによって時間と濃度依存性に活性化され,標的DNA配列であるXREへの結合能を有するAhRの存在が明らかとなった

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Language：Japanese   Publisher：(有)医学の世界社  

ナトリウムの恒常性維持に必須のMRのリガンド依存性転写活性が酸化的条件下で負に調節されること,cDDPがおそらくはROI産生を介してMRのリガンド依存性転写活性を抑制することを示した.MR-GFPの核移行の検討及びVP16-MRDBDを用いたレポーターアッセイから,cDDPはMRのリガンド結合から核移行の過程には殆ど影響を与えず,MRとDNAとの相互作用を抑制する可能性が考えられた

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

A number of transcription factors including the glucocorticoid receptor (GR) are regulated in a redox-dependent fashion. We have previously reported that the functional activity of the GR is suppressed under oxidative conditions and restored in the presence of reducing reagents. In the present study, we have used a chimeric human GR fused to the Aequorea green fluorescent protein and demonstrated that both ligand-dependent and -independent nuclear translocation of the GR is impaired under oxidative conditions in living cells. Substitution of Cys-481 for Ser within NL1 of the human GR resulted in reduction of sensitivity to oxidative treatment, strongly indicating that Cys-481 is one of the target amino acids for redox regulation of the receptor. Taken together, we may conclude that redox-dependent regulation of nuclear translocation of the GR constitutes an important mechanism for modulation of glucocorticoid-dependent signal transduction.

DOI： 10.1074/jbc.274.15.10363

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本リウマチ学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

HIF-1 alpha (hypoxia-inducible factor 1 alpha) is a basic-helix-loop-helix PAS (Per/Arnt/Sim) transcription factor that, under hypoxic conditions, dimerizes with a partner factor, the basic-helix-loop-helix/PAS protein Arnt, to recognize hypoxia-responsive elements of target genes. It has recently been demonstrated that HLF-1 alpha protein but not mRNA levels are dramatically up-regulated in response to hypoxia, Here we show that inhibitors of 26 S proteasome activity produced a dramatic accumulation of endogenous as well as transfected HIF-1 alpha protein under normoxic conditions, whereas the levels of Amt protein were not affected. HIF-1 alpha was polyubiquitinated in vivo under normoxic conditions, indicating rapid degradation via the ubiquitin-proteasome pathway. This degradation process appeared to target a region within the C terminus of HIF-1 alpha. Importantly, HIF-1 alpha ubiquitination was drastically decreased under hypoxic conditions. Up-regulation of HIF-1 alpha protein by proteasome inhibitors did not result in transcriptional activation of reporter genes, indicating either the requirement of additional regulatory steps to induce functional activity of HIF-1 alpha: or the inability of polyubiquitinated forms of HIF-1 alpha to mediate hypoxic signal transduction, In support of both these notions, we demonstrate that HIF-1 alpha showed hypoxia-dependent translocation from the cytoplasm to the nucleus and that this regulatory mechanism was severely impaired in the presence of proteasome inhibitors. Taken together, these data demonstrate that the mechanism of hypoda-dependent activation of HIF-1 alpha is a complex multistep process and that stabilization of HIF-1 alpha protein levels is not sufficient to generate a functional form.

DOI： 10.1074/jbc.274.10.6519

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The glucocorticoid receptor (GR) is considered to belong to a class of transcription factors, the functions of which are exposed to redox regulation. We have recently demonstrated that thioredoxin (TRX), a cellular reducing catalyst, plays an important role in restoration of GR function in vivo under oxidative conditions. Although both the ligand binding domain and other domains of the GR have been suggested to be modulated by TRX, the molecular mechanism of the interaction is largely unknown. In the present study, we hypothesized that the DNA binding domain (DBD) of the GR, which is highly conserved among the nuclear receptors, is also responsible for communication with TRX in vivo, Mammalian two-hybrid assay and glutathione S-transferase pull-down assay revealed the direct association between TRX and the GR DBD. Moreover, analysis of subcellular localization of TRX and the chimeric protein harboring herpes simplex viral protein 16 transactivation domain and the GR DBD indicated that the interaction might take place in the nucleus under oxidative conditions. Together these observations indicate that TRX, via a direct association with the conserved DBD motif, may represent a key mediator operating in interplay between cellular redox signaling and nuclear receptor-mediated signal transduction.

DOI： 10.1074/jbc.274.5.3182

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：ACADEMIC PRESS INC  

DOI： 10.1016/S0083-6729(08)60643-3

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Language：English   Publisher：OXFORD UNIV PRESS  

In response to decreased cellular oxygen concentrations the basic helix-loop-helix (bHLH)/PAS (Per, Amt, Sim) hypoxia-inducible transcription factor, HIF-1 alpha, mediates activation of networks of target genes involved in angiogenesis, erythropoiesis and glycolysis. Here we demonstrate that the mechanism of activation of HIF-1 alpha is a multi-step process which includes hypoxia-dependent nuclear import and activation (derepression) of the transactivation domain, resulting in recruitment of the CREB-binding protein (CBP)/p300 coactivator. Inducible nuclear accumulation was shown to be dependent on a nuclear localization signal (NLS) within the C-terminal end of HIF-1 alpha which also harbors the hypoxia-inducible transactivation domain. Nuclear import of HIF-1 alpha was inhibited by either deletion or a single amino acid substitution within the NLS sequence motif and, within the context of the full-length protein, these mutations also resulted in inhibition of the transactivation activity of HIF-1 alpha and recruitment of CBP. However, nuclear localization per se was not sufficient for transcriptional activation, since fusion of HIF-1 alpha to the heterologous GAL4 DNA-binding domain generated a protein which showed constitutive nuclear localization but required hypoxic stimuli for-function as a CBP-dependent transcription factor, Thus, hypoxia-inducible nuclear import and transactivation by recruitment of CBP can be functionally separated from one another and play critical roles in signal transduction by HIF-1 alpha.

DOI： 10.1093/emboj/17.22.6573

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Language：Japanese   Publisher：(有)医学の世界社  

GRの核移行が細胞内のレドックス状態によっても制御されうることを明らかにした.酸化条件下ではGRのリガンド結合能,リガンド依存性のhsp90の解離が低下し,更にGRの核移行も抑制される.しかし,酸化ストレスによるリガンド依存性のhsp90の解離の抑制はGRの核移行が完全に抑制される濃度のH2O2存在下においても完全ではなく,酸化ストレスはGRの核移行自体にも作用する可能性が示唆された

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Language：Japanese   Publisher：北海道医学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：FEDERATION AMER SOC EXP BIOL  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：FEDERATION AMER SOC EXP BIOL  

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Language：Japanese   Publisher：(有)科学評論社  

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Language：Japanese   Publisher：ライフサイエンス出版(株)  

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Language：Japanese   Publisher：(株)アークメディア  

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Language：English   Publisher：ROCKEFELLER UNIV PRESS  

Adaptation to stress evokes a variety of biological responses, including activation of the hypothalamic-pituitary-adrenal (HPA) axis and synthesis of a panel of stress-response proteins at cellular levels: for example, expression of thioredoxin (TRX) is significantly induced under oxidative conditions. Glucocorticoids, as a peripheral effector of the HPA axis, exert their actions via interaction with a ligand-inducible transcription factor glucocorticoid receptor (GR). However, how these stress responses coordinately regulate cellular metabolism is still unknown. In this study, we demonstrated that either antisense TRX expression or cellular treatment with H2O2 negatively modulates GR function and decreases glucocorticoid-inducible gene expression. Impaired cellular response to glucocorticoids is rescued by overexpression of TRX, most possibly through the functional replenishment of the GR. Moreover, not only the ligand binding domain but the DNA binding domain of the GR is also suggested to be a direct target of TRX.
Together, we here present evidence showing that cellular glucocorticoid responsiveness is coordinately modulated by redox state and TRX level and propose that cross talk between neuroendocrine control of stress responses and cellular antioxidant systems may be essential for mammalian adaptation processes.

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Language：English   Publisher：WILLIAMS & WILKINS  

We examined the effects of various metal ions on the DNA-binding activity of the glucocorticoid receptor. Electrophoretic mobility shift assays demonstrated that the sequence-specific DNA binding activity of the receptor was decreased by metal ions in a dose-dependent fashion. The most potent inhibitor was Au(I). Cu(II), Cd(II), and Zn(II) were, in that order, less potent as inhibitors, whereas Fe(III), AI(III), and Mg(II) had no apparent effect, The inhibitory actions of metal ions were efficiently counteracted by the sulfhydryl reducing reagents 2-mercaptoethanol and N-acetyl-L-cysteine, indicating that metal ions interfere with the DNA binding activity of the glucocorticoid receptor through modification of sulfhydryl groups in the receptor molecule. Modification of sulfhydryls by metals seems to involve neither disulfide bond formation nor permanent destruction of the GR protein and is reversible, We also show that metal ions inhibit glucocorticoid-inducible gene transcription in vivo, presumably by interfering with the interaction between the glucocorticoid receptor and cognate DNA target sequences. In summary, these data demonstrate that metal ions are capable of modulating glucocorticoid receptor mediated intracellular signalling pathways.

Web of Science

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Language：English   Publisher：OXFORD UNIV PRESS UNITED KINGDOM  

We studied the effects of bile acids on inducibility of the transcription factor AP-1 in human colon carcinoma LoVo cells, Firstly, cells were treated with chenodeoxycholic acid and the nuclear extracts from those cells were processed by electrophoretic mobility shift assays to analyze nuclear AP-1 DNA-binding activity, We demonstrated that chenodeoxycholic acid induced AP-1 DNA-binding activity in a dose- and time-dependent fashion, Antibody supershift experiments clearly revealed that the majority of protein components in induced AP-1 DNA-binding activity were the products of oncogenes c-fos and c-jun, On the other hand, DNA-binding activity in the nuclear extracts for either NF kappa B, Sp1, or ATF/CREB was not affected by bile acids, suggesting that the effect of bile acids was rather specific for AP-1, Transient transfection experiments supported this notion: expression of the AP-1-luciferase reporter construct was induced by bile acids in a dose-dependent manner, and expression of either reporter construct for NF kappa B, Sp1, or ATF/CREB was not influenced by treatment of the cells with bile acids, We also demonstrated that those bile acids efficiently activated AP-1-dependent promoter in DLD-1 cells, which (as well as LoVo cells), are derived from colon adenocarcinoma, but not in COLO320DM cells which are from colon carcinoid tumor, Thus, we may indicate that bile acids exclusively induce nuclear AP-1 activity in colon adenocarcinoma cells.

DOI： 10.1093/carcin/17.3.427

Web of Science

PubMed

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Language：English   Publisher：AMER ASSOC IMMUNOLOGISTS  

The therapeutic effectiveness of ursodeoxycholic acid (UDCA) for various autoimmune liver diseases strongly indicates that UDCA possesses immunomodulatory activities. Experimental evidence also supports this notion, since, for example, UDCA has been shown to suppress secretion of IL-2, IL-4, and IFN-gamma from activated T lymphocytes, and Ig production from B lymphocytes. To investigate the mechanical background of UDCA-mediated immunomodulation, we asked whether UDCA interacts with the intracellular signal transduction pathway, especially whether it is involved in immunosuppressive glucocorticoid hormone action. For this purpose, we used a cloned Chinese hamster ovary cell line, CHOpMTGR, in which glucocorticoid receptor cDNA was stably integrated. In immunocytochemical analysis, we found that treatment with UDCA promoted the nuclear translocation of the glucocorticoid receptor in a ligand-independent fashion, which was further confirmed by immunoprecipitation assays. Moreover, the translocated glucocorticoid receptor demonstrated sequence-specific DNA binding activity. Transient transfection experiments revealed that treatment of the cells with UDCA marginally enhanced glucocorticoid-responsive gene expression. We also showed that UDCA suppressed IFN-gamma-mediated induction of MHC class II gene expression via the glucocorticoid receptor-mediated pathway. Together, UDCA-dependent promotion of translocation of the glucocorticoid receptor may be associated with, at least in part, its immunomodulatory action through glucocorticoid receptor-mediated gene regulation.

Web of Science

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Language：Japanese   Publisher：(有)医学の世界社  

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DOI： 10.1507/endocrj.41.623

PubMed

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Event date： 2023.5 - 2023.6

Language：English   Presentation type：Poster presentation  

Venue：Milan, Italy  

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Event date： 2023.5 - 2023.6

Language：English   Presentation type：Poster presentation  

Venue：Milan, Italy  

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Event date： 2023.5 - 2023.6

Language：English   Presentation type：Poster presentation  

Venue：Milan, Italy  

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Event date： 2023.4

Language：English   Presentation type：Poster presentation  

Venue：福岡市  

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Event date： 2023.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌市  

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Event date： 2023.2

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：旭川市  

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Event date： 2023.1

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：旭川市  

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Event date： 2022.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌市  

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Event date： 2022.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌市  

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Event date： 2022.6

Language：English   Presentation type：Poster presentation  

Venue：Copenhagen, Denmark  

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Event date： 2022.4

Language：Japanese   Presentation type：Poster presentation  

Venue：横浜市  

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Event date： 2021.4 - 2021.5

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2021.2

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2020.8 - 2020.9

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2020.8 - 2020.9

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2020.8 - 2020.9

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2020.8 - 2020.9

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2020.6

Language：English   Presentation type：Poster presentation  

Venue：Frankfurt, Germany  

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Event date： 2019.11

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

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Event date： 2019.4

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：京都国際会館  

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Event date： 2018.6

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2017.4

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2016.11 - 2016.12

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：横浜市  

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Event date： 2015.11

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：盛岡市  

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Event date： 2015.11

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：岩手県盛岡市  

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Event date： 2015.11

Language：English   Presentation type：Poster presentation  

Venue：San Francisco, USA  

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Event date： 2015.10 - 2015.11

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Seoul, Korea  

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Event date： 2015.9

Language：English   Presentation type：Poster presentation  

Venue：Stockholm, Sweden  

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Event date： 2015.5

Language：English   Presentation type：Poster presentation  

Venue：Dublin, Ireland  

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Event date： 2014.11

Language：Japanese   Presentation type：Oral presentation (invited, special)  

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Event date： 2014.11

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2014.10

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：札幌市  

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Event date： 2014.10

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：東京都  

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Event date： 2014.9

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2014.9

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Kanazawa  

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Event date： 2014.9

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：kanazawa, Japan  

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Event date： 2014.6

Language：English   Presentation type：Poster presentation  

Venue：San Francisco  

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Event date： 2014.4

Language：English   Presentation type：Poster presentation  

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Event date： 2014.3

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：札幌市  

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Event date： 2013.10

Language：English   Presentation type：Poster presentation  

Venue：Kyoto, Japan  

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Event date： 2013.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：仙台市  

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Event date： 2013.2

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：四日市市  

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Event date： 2013.2

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：四日市市  

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Event date： 2012.11

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：福岡市  

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Event date： 2012.8

Language：English   Presentation type：Oral presentation (invited, special)  

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Event date： 2012.7

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：札幌市  

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Event date： 2012.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：札幌市  

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Event date： 2011.10

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：札幌市  

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Event date： 2011.5

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：札幌市  

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Event date： 2010.10

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：大津市  

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Event date： 2010.7

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：札幌市  

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Event date： 2010.3

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：大阪市  

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Event date： 2010.2

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：鎌倉市  

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Event date： 2009.4

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：東京都  

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Event date： 2009.4

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：札幌市  

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Event date： 2009.3

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：東京都  

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Event date： 2008.4

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Sapporo, Japan  

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Event date： 2006.4

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：東京都  

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Event date： 2005.4

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：東京都  

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Event date： 2004.4

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：川崎市  

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Event date： 2004.4

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Vilinius, Lithuania  

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Event date： 2004.4

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Kouchi, Japan  

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Event date： 2004.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌市  

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北海道難病連医療講演会で「膠原病〜基礎知識と最近の話題」を講演した。

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高校生を対象に地域医療体験のための病院実習指導を函館中央病院で行った

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地域医療の実情、本学の役割などについて函館中部高校で講演

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講演：高尿酸血症・痛風に対する薬物療法の重要性

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全国膠原病友の会北海道支部医療講演会で「膠原病の治療〜最近の話題と展望〜」を講演した

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講演「関節リウマチ 診療の進化ー診断・治療・マネージメントー」を行った

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講演「関節リウマチ〜どんな病気？」を行った

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講演「リウマチ性疾患の病態と治療 ー最近の話題ー」を行った

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"北海道メディカルミュージアム
橋本病について " 平成25年5月30日（木）旭川医科大学遠隔医療センター

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講演：関節リウマチについて H24.12.12

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講演：関節リウマチ治療における生物学的製剤の適正使用に向けて

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第30回北海道メディカルミュージアム「痛風・高尿酸血症」（旭川医科大学遠隔医療センター）H24.5.28

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講演

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【教員評価対象外項目：地方学会】

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