小胞体ストレスセンサーBBF2H7は成長軟骨においてATF5-MCL1経路を活性化させアポトーシスを抑制する

広島大学(Hiroshima University)博士(医学)Doctor of Philosophy in Medical Sciencedoctora

Unfolded protein response, activated by OASIS family transcription factors, promotes astrocyte differentiation

OASIS is a member of the CREB/ATF family of transcription factors and modulates cell- or tissue-specific unfolded protein response signalling. Here we show that this modulation has a critical role in the differentiation of neural precursor cells into astrocytes. Cerebral cortices of mice specifically deficient in OASIS (Oasis−/−) contain fewer astrocytes and more neural precursor cells than those of wild-type mice during embryonic development. Furthermore, astrocyte differentiation is delayed in primary cultured Oasis−/− neural precursor cells. The transcription factor Gcm1, which is necessary for astrocyte differentiation in Drosophila, is revealed to be a target of OASIS. Introduction of Gcm1 into Oasis−/− neural precursor cells improves the delayed differentiation of neural precursor cells into astrocytes by accelerating demethylation of the Gfap promoter. Gcm1 expression is temporally controlled by the unfolded protein response through interactions between OASIS family members during astrocyte differentiation. Taken together, our findings demonstrate a novel mechanism by which OASIS and its associated family members are modulated by the unfolded protein response to finely control astrocyte differentiation.This work was partly supported by grants from the Japan Society for the Promotion of Science KAKENHI (#22020030, #22800049), Sumitomo Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Astellas Foundation for Research on Metabolic Disorders, Takeda Science Foundation, The Pharmacological Research Foundation Tokyo, Daiichi-Sankyo Foundation of Life Science, The Naito Foundation, Senri Life Science Foundation, Hokuto Foundation for Bioscience, and The Japan Prize Foundation

大腸杯細胞の最終分化における小胞体ストレスセンサーOASISの役割

広島大学(Hiroshima University)博士(医学)Doctor of Philosophy in Medical Sciencedoctora

Control of glucose metabolism is important in tenogenic differentiation of progenitors derived from human injured tendons.

Glucose metabolism is altered in injured and healing tendons. However, the mechanism by which the glucose metabolism is involved in the pathogenesis of tendon healing process remains unclear. Injured tendons do not completely heal, and often induce fibrous scar and chondroid lesion. Because previous studies have shown that tendon progenitors play roles in tendon repair, we asked whether connective tissue progenitors appearing in injured tendons alter glucose metabolism during tendon healing process. We isolated connective tissue progenitors from the human injured tendons, obtained at the time of primary surgical repair of rupture or laceration. We first characterized the change in glucose metabolism by metabolomics analysis using [1,2-13C]-glucose using the cells isolated from the lacerated flexor tendon. The flux of glucose to the glycolysis pathway was increased in the connective tissue progenitors when they proceeded toward tenogenic and chondrogenic differentiation. The influx of glucose to the tricarboxylic acid (TCA) cycle and biosynthesis of amino acids from the intermediates of the TCA cycle were strongly stimulated toward chondrogenic differentiation. When we treated the cultures with 2-deoxy-D-glucose (2DG), an inhibitor of glycolysis, 2DG inhibited chondrogenesis as characterized by accumulation of mucopolysaccharides and expression of AGGRECAN. Interestingly, 2DG strongly stimulated expression of tenogenic transcription factor genes, SCLERAXIS and MOHAWK under both chondrogenic and tenogenic differentiation conditions. The findings suggest that control of glucose metabolism is beneficial for tenogenic differentiation of connective tissue progenitors

Transcriptional Regulation of VEGFA by the Endoplasmic Reticulum Stress Transducer OASIS in ARPE-19 Cells

<div><h3>Background</h3><p>Vascular endothelial growth factor-A (VEGFA) is the main mediator of angiogenesis. Angiogenesis plays important roles not only in many physiological processes, but also in the pathophysiology of many diseases. VEGFA is one of the therapeutic targets of treatment for ocular diseases with neovascularization. Therefore, elucidation of the regulatory mechanisms for VEGFA expression is important for the development of pharmaceutical drugs. Recent studies have demonstrated that the unfolded protein response is involved in the transcriptional regulation of VEGFA. However, the precise regulation of VEGFA in the human retina is not fully understood.</p> <h3>Principal Findings</h3><p>When human retinal pigment epithelial cells, ARPE-19, were exposed to endoplasmic reticulum stressors, VEGFA mRNA was significantly upregulated. The unfolded protein response-related transcription factors XBP1, ATF4, ATF6, and OASIS were expressed in ARPE-19 cells. To determine which transcription factors preferentially contribute to the induction of VEGFA expression after endoplasmic reticulum stress, we carried out reporter assays using an approximately 6-kbp 5′-upstream region of the human VEGFA gene. Among these transcription factors, OASIS acted most effectively on the VEGFA promoter in ARPE-19 cells. Based on data obtained for certain deleted and mutated reporter constructs, we determined that OASIS promoted VEGFA expression by acting on a cyclic AMP-responsive element-like site located at around –500 bp relative to the VEGFA transcription start site. Furthermore, we confirmed that OASIS directly bound to the promoter region containing this site by chromatin immunoprecipitation assays.</p> <h3>Conclusions and Significance</h3><p>We have demonstrated a novel regulatory mechanism for VEGFA transcription by OASIS in human retinal pigment epithelial cells. Chemical compounds that regulate the binding of OASIS to the promoter region of the VEGFA gene may have potential as therapeutic agents for ocular diseases with neovascularization.</p> </div

OASIS directly binds to the promoter region in the human VEGFA gene.

<p>(A) Schematic representation of the VEGFA promoter and the annealing sites of the primer set used in the ChIP assays. (B) PCR amplification of the VEGFA promoter region including the CRE-like site 4. ARPE-19 cells were transfected with a vector expressing the FLAG-tagged OASIS N-terminus. A GFP expression vector was used as a control. Immunoprecipitation was performed with anti-histone H3, anti-mouse IgG, or anti-FLAG antibodies, followed by the PCR using the specific primer sets.</p

VEGFA mRNA is upregulated by ER stressors.

<p>(A) RT-PCR analysis of VEGFA and β-actin in ARPE-19 cells treated with ER stressors (1 µM thapsigargin or 3 µg/ml tunicamycin) for 3, 6, 12, and 24 h. The bottom panels show the results of real-time RT-PCR. Data are means ± SD (n = 3). *p<0.05, **p<0.01, ***p<0.001, by Student’s <i>t</i>-test. (B) RT-PCR analyses of UPR-related transcription factors in ARPE-19 cells under normal condition and ER stress with 1 µM thapsigargin for 6 h. Unspliced; unspliced forms of XBP1 mRNA, spliced; spliced forms of XBP1 mRNA. (C) Western blot analyses of XBP1, ATF4, ATF6, and OASIS in ARPE-19 cells under ER stress with 1 µM thapsigargin for 12 h. Activated forms of these four molecules were upregulated under ER stress conditions.</p

OASIS modulates VEGFA promoter activities via the region between –709 and –437 bp.

<p>(A) Schematic diagrams of the deleted reporter constructs from the 6-kbp 5′-upstream promoter of the human VEGFA gene. Five putative CRE-like sites (containing an ACGT core) exist in the 6-kbp VEGFA promoter region. (B) Reporter assays using ARPE-19 cells. Each deletion reporter vector and the OASIS N-terminus expression vector were co-transfected. Reporter assays were performed at 48 h after the transfection. Note that reporter activities significantly decreased in cells transfected with the 200-bp construct, suggesting that OASIS acts on a site in the region between –709 and –437 bp of the VEGFA promoter. Data are means ± SD (n = 6). *p<0.05, **p<0.01, by Student’s <i>t</i>-test. (C) Western blot analysis shows the FLAG-tagged OASIS N-terminus was expressed at equal levels in each sample.</p