ヒト網膜色素上皮はヒドロキノンと最終糖化産物の共刺激により、VEGF遺伝子の発現上昇を伴って増殖する

Although recent research showed that advanced glycation endproduct (AGE) and hydroquinone (HQ) are related to the pathogenesis of age-related macular degeneration (AMD), the mechanism how AGE and HQ induce or accelerate AMD remains elusive. In the present study, we examined the effects of AGE and HQ on changes of human retinal pigment epithelial (RPE) cell numbers and found that the viable cell numbers were markedly reduced by HQ by apoptosis and that AGE prevented the decreases of HQ-treated cell numbers by increased replicative DNA synthesis of RPE cells without changing apoptosis. Real-time RT-PCR revealed that vascular endothelial growth factor (VEGF)-A mRNA was increased by HQ treatment and the addition of HQ+AGE resulted in a further increment. The increase of VEGF secretion was confirmed by ELISA, and inhibition of VEGF signaling by chemical inhibitors and small interfering RNA decreased the HQ+AGE-induced increases in RPE cell numbers. The deletion analysis demonstrated that −102 to −43 region was essential for the VEGF-A promoter activation. Site-directed mutaions of specificity protein 1 (SP1) binding sequences in the VEGF-A promoter and RNA interference of SP1 revealed that SP1 is an essential transcription factor for VEGF-A expression. These results indicate that HQ induces RPE cell apoptosis, leading to dry AMD, and suggest that AGE stimulation in addition to HQ enhances VEGF-A transcription via the AGE-receptor for AGE pathway in HQ-damaged cells. As a result, the secreted VEGF acts as an autocrine/paracrine growth factor for RPE and/or adjacent vascular cells, causing wet AMD.博士（医学）・甲第640号・平成27年11月27日Copyright © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

シェーグレン症候群の新規自己抗原であるREG 蛋白の唾液導管細胞における発現誘導にはIL-6/STAT 経路が重要である

The regenerating gene, Reg, was originally isolated from a rat regenerating islet cDNA library, and its human homolog was named REG Iα. Recently, we reported that REG Iα mRNA as well as its product were overexpressed in ductal epithelial cells in the minor salivary glands of Sjögren׳s syndrome (SS) patients. This study was undertaken to elucidate the role of cytokines and the subsequent intracellular mechanism for induction of REG Iα in the salivary glands of SS patients. We prepared a reporter plasmid containing REG Iα promoter (−1190/+26) upstream of a luciferase reporter gene. The promoter plasmid was introduced by lipofection into human NS-SV-DC and rat A5 salivary ductal cells. The cells were treated with interleukin (IL)-6, IL-8, and a combination of the two. Thereafter transcriptional activity of REG Iα was measured by luciferase assay. We found that IL-6 stimulation, but not IL-8, significantly enhanced the REG Iα promoter activity in salivary ductal cells. Deletion analysis revealed that the region of −141 to −117 of the REG Iα gene was responsible for the promoter activation by IL-6, which contains a consensus sequence for signal transduction and activation of transcription (STAT). The introduction of siRNA for human STAT3 abolished IL-6-induced REG Iα transcription. These results showed that IL-6 stimulation induced REG Iα transcription through STAT3 activation and binding to the consensus sequence of REG Iα promoter in salivary ductal cells. This IL-6/STAT dependent REG Iα induction might play a role in the pathogenesis of SS.博士（医学）・甲第641号・平成27年11月27日Copyright © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CCBY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Interleukin-6/STAT pathway is responsible for the induction of gene expression of REG Iα, a new auto-antigen in Sjögren׳s syndrome patients, in salivary duct epithelial cells

The regenerating gene, Reg, was originally isolated from a rat regenerating islet cDNA library, and its human homolog was named REG Iα. Recently, we reported that REG Iα mRNA as well as its product were overexpressed in ductal epithelial cells in the minor salivary glands of Sjögren׳s syndrome (SS) patients. This study was undertaken to elucidate the role of cytokines and the subsequent intracellular mechanism for induction of REG Iα in the salivary glands of SS patients. We prepared a reporter plasmid containing REG Iα promoter (−1190/+26) upstream of a luciferase reporter gene. The promoter plasmid was introduced by lipofection into human NS-SV-DC and rat A5 salivary ductal cells. The cells were treated with interleukin (IL)-6, IL-8, and a combination of the two. Thereafter transcriptional activity of REG Iα was measured by luciferase assay. We found that IL-6 stimulation, but not IL-8, significantly enhanced the REG Iα promoter activity in salivary ductal cells. Deletion analysis revealed that the region of −141 to −117 of the REG Iα gene was responsible for the promoter activation by IL-6, which contains a consensus sequence for signal transduction and activation of transcription (STAT). The introduction of siRNA for human STAT3 abolished IL-6-induced REG Iα transcription. These results showed that IL-6 stimulation induced REG Iα transcription through STAT3 activation and binding to the consensus sequence of REG Iα promoter in salivary ductal cells. This IL-6/STAT dependent REG Iα induction might play a role in the pathogenesis of SS

VGLL3 is a mechanosensitive protein that promotes cardiac fibrosis through liquid–liquid phase separation

Myofibroblasts cause tissue fibrosis by producing extracellular matrix proteins, such as collagens. Humoral factors like TGF-β, and matrix stiffness are important for collagen production by myofibroblasts. However, the molecular mechanisms regulating their ability to produce collagen remain poorly characterised. Here, we show that vestigial-like family member 3 (VGLL3) is specifically expressed in myofibroblasts from mouse and human fibrotic hearts and promotes collagen production. Further, substrate stiffness triggers VGLL3 translocation into the nucleus through the integrin β1-Rho-actin pathway. In the nucleus, VGLL3 undergoes liquid-liquid phase separation via its low-complexity domain and is incorporated into non-paraspeckle NONO condensates containing EWS RNA-binding protein 1 (EWSR1). VGLL3 binds EWSR1 and suppresses miR-29b, which targets collagen mRNA. Consistently, cardiac fibrosis after myocardial infarction is significantly attenuated in Vgll3-deficient mice, with increased miR-29b expression. Overall, our results reveal an unrecognised VGLL3-mediated pathway that controls myofibroblasts’ collagen production, representing a novel therapeutic target for tissue fibrosis

Prospective study of daily low-dose nedaplatin and continuous 5-fluorouracil infusion combined with radiation for the treatment of esophageal squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Protracted low-dose concurrent chemotherapy combined with radiation has been proposed for enhanced treatment results for esophageal cancer. We evaluated the efficacy and the toxicity of a novel regimen of daily low-dose nedaplatin (cis-diammine-glycolatoplatinum) and continuous infusion of 5-fluorouracil (5-FU) with radiation in patients with esophageal squamous cell carcinoma.</p> <p>Methods</p> <p>Between January 2003 and June 2008, 33 patients with clinical stage I to IVB esophageal squamous cell carcinoma were enrolled. Nedaplatin (10 mg/body/day) was administered daily and 5-FU (500 mg/body/day) was administered continuously for 20 days. Fractionated radiotherapy for a total dose of 50.4-66 Gy was administered together with chemotherapy. Additional chemotherapy with nedaplatin and 5-FU was optionally performed for a maximum of 5 courses after chemoradiotherapy. The primary end-point of this study was to evaluate the tumor response, and the secondary end-points were to evaluate the toxicity and the overall survival.</p> <p>Results</p> <p>Twenty-two patients (72.7%) completed the regimen of chemoradiotherapy. Twenty patients (60.6%) achieved a complete response, 10 patients (30.3%) a partial response. One patient (3.0%) had a stable disease, and 2 (6.1%) a progressive disease. The overall response rate was 90.9% (95% confidence interval: 75.7%-98.1%). For grade 3-4 toxicity, leukopenia was observed in 75.8% of the cases, thrombocytopenia in 24.2%, anemia in 9.1%, and esophagitis in 36.4%, while late grade 3-4 cardiac toxicity occurred in 6.1%. Additional chemotherapy was performed for 26 patients (78.8%) and the median number of courses was 3 (range, 1-5). The 1-, 2- and 3-year survival rates were 83.9%, 76.0% and 58.8%, respectively. The 1- and 2-year survival rates were 94.7% and 88.4% in patients with T1-3 M0 disease, and 66.2% and 55.2% in patients with T4/M1 disease.</p> <p>Conclusion</p> <p>The treatment used in our study may yield a high complete response rate and better survival for each stage of esophageal squamous cell carcinoma.</p> <p>Trial registration</p> <p>ClinicalTrials.gov Identifier: NCT00197444</p

Regenerating Gene Protein as a Novel Autoantigen in the Pathogenesis of Sjögren’s Syndrome

Sjögren’s syndrome, an autoimmune disease characterized by exocrine gland dysfunction leading to dry mouth and dry eye diseases, is typified by lymphoplasmacytic infiltrations and a progressive destruction of the salivary and lacrimal glands. Despite an ever-increasing focus on identifying the underlying etiology of Sjögren’s syndrome, the factors that initiate this autoimmune disease and the mechanisms that cause the subsequent exocrine gland dysfunction remain a mystery. The original explanatory concept for the pathogenesis of Sjögren’s syndrome proposed a specific, self-perpetuating, immune-mediated loss of acinar and ductal cells as the principal cause of salivary gland dysfunction. We highlight the possible involvement of regenerating gene (Reg) in the regeneration and destruction of salivary gland acinar and ductal cells in Sjögren’s syndrome. The Reg gene was originally isolated as a gene specifically overexpressed in regenerating pancreatic islets and constitutes a growth factor family (Reg family). We describe how salivary gland dysfunction is initiated and maintained and how it can be regenerated or progressed, mediated by the Reg gene, Reg protein, and anti-REG autoantibodies in Sjögren’s syndrome

Human retinal pigment epithelial cell proliferation by the combined stimulation of hydroquinone and advanced glycation end-products via up-regulation of VEGF gene.

Although recent research showed that advanced glycation endproduct (AGE) and hydroquinone (HQ) are related to the pathogenesis of age-related macular degeneration (AMD), the mechanism how AGE and HQ induce or accelerate AMD remains elusive. In the present study, we examined the effects of AGE and HQ on changes of human retinal pigment epithelial (RPE) cell numbers and found that the viable cell numbers were markedly reduced by HQ by apoptosis and that AGE prevented the decreases of HQ-treated cell numbers by increased replicative DNA synthesis of RPE cells without changing apoptosis. Real-time RT-PCR revealed that vascular endothelial growth factor (VEGF)-A mRNA was increased by HQ treatment and the addition of HQ+AGE resulted in a further increment. The increase of VEGF secretion was confirmed by ELISA, and inhibition of VEGF signaling by chemical inhibitors and small interfering RNA decreased the HQ+AGE-induced increases in RPE cell numbers. The deletion analysis demonstrated that −102 to −43 region was essential for the VEGF-A promoter activation. Site-directed mutaions of specificity protein 1 (SP1) binding sequences in the VEGF-A promoter and RNA interference of SP1 revealed that SP1 is an essential transcription factor for VEGF-A expression. These results indicate that HQ induces RPE cell apoptosis, leading to dry AMD, and suggest that AGE stimulation in addition to HQ enhances VEGF-A transcription via the AGE-receptor for AGE pathway in HQ-damaged cells. As a result, the secreted VEGF acts as an autocrine/paracrine growth factor for RPE and/or adjacent vascular cells, causing wet AMD.博士（医学）・甲第640号・平成27年11月27日Copyright © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).identifier:Biochemistry and biophysics reports Vol.2 p.123-131(2015 Jul)identifier:24055808identifier:http://ginmu.naramed-u.ac.jp/dspace/handle/10564/3101identifier:Biochemistry and biophysics reports, 2: 123-13