Takanori Fujimura

Pancreatic β cell proliferation by intermittent hypoxia via up-regulation of Reg family genes and HGF gene

AIMS Although accumulating evidence suggests the associations between sleep apnea syndrome (SAS) and type 2 diabetes, the direct effect of intermittent hypoxia (IH) on pancreatic β cell proliferation remains a missing piece of the puzzle. MAIN METHODS Rat RINm5F β cells, hamster HIT-T15 β cells, and human 1.1B4 β cells were exposed to normoxia (21% O2, 5% CO2, and balance N2), to sustained hypoxia (SH: 1% O2, 5% CO2, and balance N2), or to intermittent hypoxia (IH: 64 cycles of 5 min SH and 10 min normoxia) for 24 h. After the treatment, cellular proliferation and apoptosis were measured by WST-8 assay and TUNEL method, respectively. The expression of regenerating gene (Reg) family, interleukin (IL)-6, and hepatocyte growth factor (HGF) was determined by real-time RT-PCR. KEY FINDINGS The cellular proliferation of HIT-T15, RINm5F and 1.1B4 cells by IH was significantly increased, whereas apoptosis of these cells was unchanged. Real-time RT-PCR revealed that the mRNA levels of Reg family genes, IL-6, a typical Reg family gene inducer, and HGF, an inhibitor of high-concentration of Reg protein-induced apoptosis, were increased in IH-treated cells. In addition, siRNAs against rat Reg family genes except for PAP I/Reg 2 attenuated IH-induced β cell proliferation. SIGNIFICANCE IH stress stimulates pancreatic β cell to induce IL-6 gene expression. By the IL-6 stimulation, β cells over-express Reg family genes as well as HGF gene. Reg family proteins stimulate β cell proliferation and HGF inhibits apoptosis of β cells. As a result, β cell numbers are increased by IH.

Synergistic activations of REG I α and REG I β promoters by IL-6 and Glucocorticoids through JAK/STAT pathway in human pancreatic β cells.

Reg (Regenerating gene) gene was originally isolated from rat regenerating islets and its encoding protein was revealed as an autocrine/paracrine growth factor for β cells. Rat Reg gene is activated in inflammatory conditions for β cell regeneration. In human, although five functional REG family genes (REG Iα, REG Iβ, REG III, HIP/PAP, and REG IV) were isolated, their expressions in β cells under inflammatory conditions remained unclear. In this study, we found that combined addition of IL-6 and dexamethasone (Dx) induced REG Iα and REG Iβ expression in human 1.1B4 β cells. Promoter assay revealed that a signal transducer and activator of transcription- (STAT-) binding site in each promoter of REG Iα (TGCCGGGAA) and REG Iβ (TGCCAGGAA) was essential for the IL-6+Dx-induced promoter activation. A Janus kinase 2 (JAK2) inhibitor significantly inhibited the IL-6+Dx-induced REG Iα and REG Iβ transcription. Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed that IL-6+Dx stimulation increased STAT3 binding to the REG Iα promoter. Furthermore, small interfering RNA-mediated targeting of STAT3 blocked the IL-6+Dx-induced expression of REG Iα and REG Iβ. These results indicate that the expression of REG Iα and REG Iβ should be upregulated in human β cells under inflammatory conditions through the JAK/STAT pathway.

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.

Significance of Interleukin-6/STAT Pathway for the Gene Expression of REG Iα, a New Autoantigen in Sjögren’s Syndrome Patients, in Salivary Duct Epithelial Cells

The regenerating gene, Reg, was originally isolated from a rat regenerating islet complementary DNA (cDNA) library, and its human homologue was named REG Iα. Recently, we reported that REG Iα messenger RNA (mRNA), as well as its product, was overexpressed in ductal epithelial cells in the salivary glands of Sjögren’s syndrome patients. Furthermore, autoantibodies against REG Iα were found in the sera of Sjögren’s syndrome patients, and the patients who were positive for the anti-REG Iα antibody showed significantly lower saliva secretion than antibody-negative patients. We found the mechanism of REG Iα induction in salivary ductal epithelial cells. Reporter plasmid containing REG Iα promoter (−1190/+26) upstream of a luciferase gene was introduced into human NS-SV-DC and rat A5 salivary ductal cells. The cells were treated with several cytokines (interleukin (IL)-6, IL-8, etc.), upregulated in Sjögren’s syndrome salivary ducts, and the transcriptional activity was measured. IL-6 stimulation significantly enhanced the REG Iα promoter activity in both cells. Deletion analysis revealed that the −141∼−117 region of the REG Iα gene was responsible for the promoter activation by IL-6, which contains a consensus sequence for signal transducer and activator of transcription (STAT) binding. The introduction of small interfering RNA for human STAT3 abolished IL-6-induced REG Iα transcription. These results indicated that IL-6 stimulation induced REG Iα transcription through STAT3 activation and binding to the REG Iα promoter in salivary ductal cells. This dependence of REG Iα induction upon IL-6/STAT in salivary duct epithelial cells may play an important role in the pathogenesis/progression of 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.

Drug retention and discontinuation reasons between seven biologics in patients with rheumatoid arthritis -The ANSWER cohort study-

The purpose of this study was to evaluate the retention and discontinuation reasons of seven biological disease-modifying antirheumatic drugs (bDMARDs) in a real-world setting of patients with rheumatoid arthritis (RA). 1,037 treatment courses with bDMARDs from 2009 to 2016 [female, 81.8%; baseline age, 59.6 y; disease duration 7.8 y; rheumatoid factor positivity 81.5%; Disease Activity Score in 28 joints using erythrocyte sedimentation rate (DAS28-ESR), 4.4; concomitant prednisolone 43.5% and methotrexate 68.6%; Bio-naïve, 57.1%; abatacept (ABT), 21.3%; tocilizumab (TCZ), 20.7%; golimumab (GLM), 16.9%; etanercept (ETN), 13.6%; adalimumab (ADA), 11.1%; infliximab (IFX), 8.5%; certolizumab pegol (CZP), 7.9%] were included in this multi-center, retrospective study. Drug retention and discontinuation reasons at 36 months were estimated using the Kaplan-Meier method and adjusted by potent confounders using Cox proportional hazards modeling. As a result, 455 treatment courses (43.9%) were stopped, with 217 (20.9%) stopping due to inefficacy, 113 (10.9%) due to non-toxic reasons, 86 (8.3%) due to toxic adverse events, and 39 (3.8%) due to remission. Drug retention rates in the adjusted model were as follows: total retention (ABT, 60.7%; ADA, 32.7%; CZP, 43.3%; ETN, 51.9%; GLM, 45.4%; IFX, 31.1%; and TCZ, 59.2%; P < 0.001); inefficacy (ABT, 81.4%; ADA, 65.7%; CZP, 60.7%; ETN, 71.3%; GLM, 68.5%; IFX, 65.0%; and TCZ, 81.4%; P = 0.015), toxic adverse events (ABT, 89.8%; ADA, 80.5%; CZP, 83.9%; ETN, 89.2%; GLM, 85.5%; IFX, 75.6%; and TCZ, 77.2%; P = 0.50), and remission (ABT, 95.5%; ADA, 88.1%; CZP, 91.1%; ETN, 97.5%; GLM, 94.7%; IFX, 86.4%; and TCZ, 98.4%; P < 0.001). In the treatment of RA, ABT and TCZ showed higher overall retention, and TCZ showed lower inefficacy compared to IFX, while IFX showed higher discontinuation due to remission compared to ABT, ETN, GLM, and TCZ in adjusted modeling.

Subclinical articular involvement in primary Sjögren's syndrome assessed by ultrasonography and its negative association with anti-centromere antibody

Objectives. To evaluate the subclinical articular involvement in patients with primary Sjögrens syndrome (pSS) using musculoskeletal ultrasound (MSUS), and to correlate the findings with laboratory results and clinical manifestations. Methods. Forty-eight consecutive patients with pSS were enrolled. The bilateral metacarpophalangeal, proximal interphalangeal, and interphalangeal joints were examined using MSUS, and the synovial hypertrophy and power Doppler signal were recorded for each joint using semi-quantitative scores (0 = normal, 1 = mild change compared with undamaged joint, 2 = moderate change, and 3 = severe change). Results. Mild or moderate synovial hypertrophy was found in 151 (15.7%) and 2 (0.2%) out of 960 hand joints, respectively, and power Doppler signals were present in 19 (2.0%) of the 960 joints. While anti-centromere antibody (ACA) was found in 10 patients (20.8%), none of the patients with MSUS-confirmed synovitis was positive for ACA. No other autoantibodies, laboratory tests, or clinical manifestations correlated with MSUS-confirmed synovitis. Conclusion. MSUS is useful for detecting subclinical synovitis in pSS patients. MSUS showed that ACA-positive pSS patients had a low prevalence of synovitis.

AB0189 Interleukin-6/Stat Pathway is Responsible for the Induction of REG Iα, A New Auto-Antigen in SjÖGren's Syndrome Patients, in Salivary Duct Epithelial Cells

Background The regenerating gene, Reg, was originally isolated from a rat regenerating islet cDNA library, and its human homologue was named REG Iα. Reg gene product acts as a growth factor and is important for various inflammatory diseases. Sjögrens syndrome (SS) is chronic autoimmune disease characterized by inflammation of salivary and lacrimal glands, and local or systemic overexpression of pro-inflammatory cytokines is involved with the pathogenesis. Recently, we reported that REG Iα mRNA as well as its product (REG Iα protein) were overexpressed in ductal epithelial cells in the minor salivary glands (MSG) of SS patients (1). Furthermore, auto-antibodies against REG Iα were found in SS patients and the anti-REG Iα auto-antibody positive group showed significant lower saliva secretion (1). Although some correlations between expressions of REG family genes and cytokines were reported (1), which cytokine is responsible for REG Iα expression in MSG has been elusive. Objectives This study was undertaken to elucidate the role of cytokines for induction of REG Iα in salivary glands of Sjögrens syndrome patients. We examined which cytokines are responsible for REG Iα transcription in salivary ductal epithelial cells. Methods REG Iα promoter (-1190/+26) was inserted upstream of a luciferase reporter gene in pGL3-Basic vector. The promoter plasmid was introduced by lipofection into human NS-SV-DC cells and A5 rat salivary ductal cells. After 6 hours, the cells were treated with interleukin (IL)-6 (20 ng/ml), IL-8 (100 nM), IL-22 (10 ng/ml), interferon (IFN)-β (1,500 units/ml), and combination of them. After further 24 hour, the cells were harvested and transcriptional activity of REG Iα was measured by luciferase assay. Results We found that IL-6 stimulation significantly enhanced the REG Iα promoter activity in human NS-SV-DC cells and A5 rat salivary ductal cells. Treatment with neither IL-8, IL-22, nor IFN-β changed the transcriptional activity of REG Iα. To identify the regions necessary for activation of REG Iα promoter by IL-6, progressive deletions of the REG Iα promoter were performed. Deletion analysis revealed that the region of -141 to -117 of the REG Iα gene was responsible for the promoter activation by IL-6. This region contains a consensus sequence for signal transduction and activation of transcription (STAT). Site-directed mutations of STAT-binding site in the region of REG Iα significantly attenuated promoter activation by IL-6. Conclusions The present study showed that REG Iα transcription in salivary ductal cells was stimulated by IL-6. Our study also suggested STAT3 bound the consensus sequence of REG Iα promoter and regulated transcription in ductal epithelial cells in response to IL-6 stimulation. It was suggested that overexpression of REG Iα protein in salivary ductal cells is dependent on IL-6/STAT pathway and IL-6/STAT dependent REG Iα induction may play a role in the pathogenesis of SS. References Yoshimoto K et al. Clin Exp Immunol 2013; 174: 1-9. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2813

Regulators of Beta-Cell Death and Regeneration

Pancreatic β-cell deficiency underlies both type 1 and type 2 diabetes, and restoration or replacement of β-cell mass/function is therefore the logical long-term solution to therapy. While it has long been held that type 1 diabetes results from an irreversible loss of β-cells, and that type 2 diabetes is primarily caused by impaired insulin action, there is now increasing evidence linking both types of diabetes to defects in β-cell mass and insulin secretion. Pancreatic β-cells have traditionally been viewed as a quiescent cell population. However, several recent lines of evidence indicated that like most tissues the β-cell mass is dramatically regulated with ongoing β-cell regeneration throughout life to replenish lost or damaged β-cells. Based on our recent data concerning β-cell death, dysfunction, and regeneration, we would like to describe regulation of pancreatic β-cell death, functioning, and regeneration.

AB0310 Prognostic Factor for Forefoot Deformity in Early Rheumatoid Arthritis

Background Disease activity score (DAS) 28 and simplified disease activity index (SDAI) and clinical disease activity index (CDAI) are often used for clinical assessment, and these criteria dose not include assessment of joints in the feet. Residual synovitis of the MTP joints and various factors such as inflammatory marker, drugs and weght-bearing possibly cause various deformities of the forefoot and lead to functional, cosmetic problems. Objectives To determine prognostic factors of forefoot deformity in early rheumatoid arthritis (RA). Methods Twenty-four outpatients with RA before tight control were enrolled. At baseline, the mean age of patients was 57.8 years and the mean disease duration was 11.2 months. The mean body mass index (BMI) was 22.0 kg/m2. The mean DAS28, SDAI and modified health assessment questionnaire (mHAQ) at baseline was 4.84, 21.9 and 0.29. The mean CRP and MMP-3 was 1.7 mg/dl and 153 ng/ml. Eleven patients (46%) were treated with conventional synthetic disease modified anti-rheumatic drugs (csDMARDs) and 8 patients (33%) were treated with glucocorticoids (GCs, mean dose of prednisolone 5.6mg). Nine patients (38%) were treated biological agents at final follow up. As ultrasonographic assessment, bilateral first IP joint, the second to fifth PIP joint, the first to fifth MCP and MTP joints were assessed by semi-quantitative assessment (0 - 3) using power Doppler ultrasonography (PDUS). The sum score of all joints and MTP joints was used as total PDUS (TPD) and foot PDUS (FPD). Radiographic damages of feet were evaluated by using van der Heijde modified total sharp score (footTSS, 0 - 168). Radiographic forefoot deformities were evaluated by using hallux valgus angle (HV), 1st to 2nd metatarsal angle (M1M2) and 1st to 5th metatarsal angle (M1M5). Both radiographic assessment were performed at baseline and final follow up (mean 2.7 years), and the sum of these angle (HV + M1M2 + M1M5) was used as total deformity score (TDS). Results Twenty-one (87.5%) and 18 (75%) patients achieved DAS28 and SDAI remission after tight control. At final follow up, all deformity angle were significantly progressed and mean ΔHV, ΔM1M2, ΔM1M5 and ΔTDS was 3.58, 2.29, 3.88 and 9.75 respectively. ΔTDS were not correlated with ΔfootTSS (ρ=0.09, p=0.68). DAS28, SDAI, TPD, FPD, CRP, MMP-3 and BMI were not correlated with ΔTDS, whereas univariate linear analysis identified significant correlation with GCs and ΔTDS (p=0.009), and multiple regression analysis revealed GCs was a independent risk for forefoot deformity (p=0.01). Conclusions Forefoot deformity in RA progressed even if clinical remission was achieved. Baseline prognostic factor for forefoot deformity in early RA was oral low dose GCs in the early stage of treatment. Disclosure of Interest None declared