Hereditary cataract of the Nakano mouse: Involvement of a hypomorphic mutation in the coproporphyrinogen oxidase gene

The Nakano cataract (NCT) is a recessive disorder in the mouse linked to the nct locus on chromosome 16. In this study, we positionally cloned the critical gene in the nct locus. Herein, we report that cataracts in the BALB/c-nct/nct mouse are caused by a hypomorphic mutation in the coproporphyrin oxidase gene (Cpox), encoding the enzyme responsible for catalyzing oxidative decarboxylation of the heme precursor, coproporphyrinogen III, in the heme biosynthetic pathway. BALB/c-nct/nct mice are homozygous for a G to T nucleotide substitution in the Cpox gene, which results in a p.R380L amino acid substitution in the CPDX protein. The CPDX isoform with the p.R380L substitution retained only 15% of the activity of the wild type isoform. BALB/c-nct/nct mice had excessive accumulation of coproporphyrin HI in the lens. The NCT phenotype was normalized by the introduction of a wild type Cpox transgene. The mechanisms by which impairment of CPDX leads to lens opacity in the NCT are elusive. However, our data illuminate a hitherto unanticipated involvement of the heme biosynthesis pathway in lens physiology.ArticleEXPERIMENTAL EYE RESEARCH. 112:45-50 (2013)journal articl

A-to-I RNA editing up-regulates human dihydrofolate reductase in breast cancer

Dihydrofolate reductase (DHFR) plays a key role in folate metabolism and is a target molecule of methotrexate. An increase in the cellular expression level of DHFR is one of the mechanisms of tumor resistance to methotrexate. The present study investigated the possibility that adenosine-to-inosine RNA editing, which causes nucleotide conversion by adenosine deaminase acting on RNA (ADAR) enzymes, might modulate DHFR expression. In human breast adenocarcinoma-derived MCF-7 cells, 26 RNA editing sites were identified in the 3′-UTR of DHFR. Knockdown of ADAR1 decreased the RNA editing levels of DHFR and resulted in a decrease in the DHFR mRNA and protein levels, indicating that ADAR1 up-regulates DHFR expression. Using a computational analysis, miR-25-3p and miR-125a-3p were predicted to bind to the non-edited 3′-UTR of DHFR but not to the edited sequence. The decrease in DHFR expression by the knockdown of ADAR1 was restored by transfection of antisense oligonucleotides for these miRNAs, suggesting that RNA editing mediated up-regulation of DHFR requires the function of these miRNAs. Interestingly, we observed that the knockdown of ADAR1 decreased cell viability and increased the sensitivity of MCF-7 cells to methotrexate. ADAR1 expression levels and the RNA editing levels in the 3′-UTR of DHFR in breast cancer tissues were higher than those in adjacent normal tissues. Collectively, the present study demonstrated that ADAR1 positively regulates the expression of DHFR by editing the miR-25-3p and miR-125a-3p binding sites in the 3′-UTR of DHFR, enhancing cellular proliferation and resistance to methotrexate. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.Embargo Period 12 month

Characterization of human UGT2A3 expression using a prepared specific antibody against UGT2A3

UDP-Glucuronosyltransferase (UGT) 2A3 belongs to a UGT superfamily of phase II drug-metabolizing enzymes that catalyzes the glucuronidation of many endobiotics and xenobiotics. Previous studies have demonstrated that UGT2A3 is expressed in the human liver, small intestine, and kidney at the mRNA level; however, its protein expression has not been determined. Evaluation of the protein expression of UGT2A3 would be useful to determine its role at the tissue level. In this study, we prepared a specific antibody against human UGT2A3 and evaluated the relative expression of UGT2A3 in the human liver, small intestine, and kidney. Western blot analysis indicated that this antibody is specific to UGT2A3 because it did not cross-react with other human UGT isoforms or rodent UGTs. UGT2A3 expression in the human small intestine was higher than that in the liver and kidney. Via treatment with endoglycosidase, it was clearly demonstrated that UGT2A3 was N-glycosylated. UGT2A3 protein levels were significantly correlated with UGT2A3 mRNA levels in a panel of 28 human liver samples (r = 0.64, p <0.001). In conclusion, we successfully prepared a specific antibody against UGT2A3. This antibody would be useful to evaluate the physiological, pharmacological, and toxicological roles of UGT2A3 in human tissues. (C) 2019 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.Peer reviewe

Serum-and Glucocorticoid-Regulated Kinase 2 Determines Drug-Activated Pregnane X Receptor to Induce Gluconeogenesis in Human Liver Cells

ABSTRACT Drug activation of the human nuclear pregnane X receptor (PXR) induced gluconeogenic genes and increased glucose production. In this study, we have determined that serum-and glucocorticoidregulated kinase 2 (SGK2) is an essential factor that mediates this PXR-regulated glucose 6-phosphatase (G6Pase) induction and glucose production. Both SGK2 and G6Pase mRNAs were increased in rifampicin-treated HepG2 cells stably expressing human PXR. Reporter and chromatin immunoprecipitation assays delineated PXR activation of the SGK2 gene to a distal and proximal DNA sequence within its promoter: distal PXR response element (22587/22209) and proximal PXR response element (2115/275), respectively. Small interfering RNA (siRNA) knockdown of SGK2 severely attenuated PXR-regulated induction of G6Pase as well as glucose production. SGK2 constitutes an insulin-independent signal pathway to regulate gluconeogenesis because siRNA knockdown of the insulin-responsive transcription factor forkhead box protein O1 did not affect rifampicin induction of G6Pase. Rifampicin treatment of two different samples of human primary hepatocytes revealed that PXR induces G6Pase in the presence of high levels of SGK2, whereas PXR represses G6Pase in its absence. Mediating PXR activation of the G6Pase gene is the first biological role found for hepatic SGK2 and might have therapeutic implications for side effects, such as diabetes, caused by drugs that activate PXR

薬剤誘発性糖尿病におけるPXR/SGK2 シグナル経路の機能的役割の解明

東京都医学総合研究所 / 金沢大学医薬保健研究域薬学系異物応答性核内受容体 PXR は、肝臓でのエネルギー代謝制御における機能的役割が示唆されているが、ヒトにおいてその分子機構は明らかではない。本研究では、ヒト肝癌細胞において薬剤処置により活性化した PXR が細胞内シグナル因子 SGK2 と共に、脂肪酸のβ酸化関連酵素遺伝子 CPT1A および ACSL1 の発現量を増加させることを見出した。さらに、CPT1A 遺伝子上流領域に薬剤処置依存的な PXR および SGK2 の結合サイトを同定した。これらの成果より、PXR は SGK2 と協調的に作用して脂肪酸のβ酸化を制御する新たな可能性を示した。Xenobiotic-sensing nuclear receptor, pregnane X receptor (PXR) may play a functional role in hepatic energy metabolism. However, the molecular mechanism remains unknown in humans. In this study, we demonstrated that drug-activated PXR increased mRNA expression of fatty acid β-oxidation-related genes, including CPT1A and ACSL1 in human hepatocellular carcinoma cells. PXR required serum/glucocorticoid regulated kinase 2 (SGK2) in the regulation. Furthermore, we identified PXR/SGK2 binding site within the 5’ upstream region of CPT1A gene in a drug-dependent manner. These results suggest the possibility that PXR utilizes SGK2 to regulate fatty acid β-oxidation.研究課題/領域番号:16K21055, 研究期間(年度):2016-04-01 – 2020-03-31出典：「薬剤誘発性糖尿病におけるPXR/SGK2 シグナル経路の機能的役割の解明」研究成果報告書　課題番号16K21055（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-16K21055/16K21055seika/)を加工して作

RNA編集によるヒト芳香族炭化水素受容体およびジヒドロ葉酸還元酵素の発現制御

博士論文要旨Abstract 以下に掲載：1.Journal of Biological Chemistry 291(2) pp.894-901 2017. American Society for Biochemistry and Molecular Biology 共著者：Masataka Nakano, Tatsuki Fukami, Saki Gotoh, Masataka Takamiya, Yasuhiro Aoki, Miki Nakajima 2.Journal of Biological Chemistry inpress. American Society for Biochemistry and Molecular Biology 共著者：Masataka Nakano, Tatsuki Fukami, Saki Gotoh, Miki Nakajim

The Role of Hypertriglyceridemia in the Development of Atherosclerosis and Endothelial Dysfunction

A hereditary postprandial hypertriglyceridemic rabbit (PHT rabbit) is a new dyslipidemic model showing remarkably high plasma triglycerides with only limited elevation of plasma total cholesterol. In PHT rabbits, plasma triglyceride was markedly elevated postprandially compared with healthy Japanese white (JW) rabbits. In physiological experiments, the ring preparation of the thoracic aorta was suspended in an organ bath filled with modified Krebs-Henseleit solution, and the developed tension was recorded. Endothelial function was evaluated by acetylcholine-induced vasorelaxation in each preparation with intact endothelium. The acetylcholine-induced endothelium-dependent relaxation was diminished in PHT compared with JW rabbits, suggesting endothelial dysfunction in PHT rabbits. Histological examination was carried out in adipose tissue, liver and aorta. They were fixed in formaldehyde and embedded in paraffin. The tissues were sliced (4 μm) and stained using hematoxylin-eosin solution. In the adipose tissue, the visceral fat accumulated, and the size of adipose cells was enlarged in PHT rabbits. The liver of the PHT rabbit was fatty and degenerated. In aorta, increased intimal thickness was observed, suggesting the progression of atherosclerosis in the PHT rabbit. This study suggests the important role of postprandial hypertriglyceridemia in atherosclerosis. By using PHT rabbits, the effects of hypertriglyceridemia on health and diseases could be evaluated precisely