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

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

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

OASIS promotes the transcription of VEGFA.

<p>(A) Schematic diagram of the human VEGFA promoter region. The first intron of the human VEGFA gene contains an ATF4-binding site (○), and that the 6-kbp 5′-upstream promoter has two potential binding sites for XBP1 (•) and CRE-like sites (▪). TSS: transcription start site. (B) Reporter assays using ARPE-19 cells. A reporter vector derived from the 6-kbp 5′-upstream region of the human VEGFA gene and expression vectors for XBP1, ATF4, ATF6, or OASIS were co-transfected. At 48 h after the transfection, luciferase activities were measured. Data are means ± SD (n = 4). *p<0.05, ***p<0.001, by Student’s <i>t</i>-test. (C) RT-PCR analysis of VEGFA in ARPE-19 cells infected with an adenovirus vector carrying OASIS. The right panel shows the results of real-time RT-PCR. The VEGFA mRNA expression level is increased by 5.5-fold after the transfection of OASIS. Data are means ± SD (n = 3). **p<0.01, by Student’s <i>t</i>-test.</p

OASIS acts on the CRE-like site at –509 to –506 bp in the VEGFA promoter.

<p>(A) The top panel shows schematic diagrams of the mutated reporter constructs. The bottom panel shows schematic representations of the wild-type CRE-like site (containing an ACGT core) and the mutated CRE-like sites (containing an AaGg core). (B) Reporter assays using ARPE-19 cells. Each mutated 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 mutated CRE-like site 4 construct. Data are means ± SD (n = 4). ***p<0.001, 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

DS_10.1177_0363546518778789 – Supplemental material for Modulating Glucose Metabolism and Lactate Synthesis in Injured Mouse Tendons: Treatment With Dichloroacetate, a Lactate Synthesis Inhibitor, Improves Tendon Healing

<p>Supplemental material, DS_10.1177_0363546518778789 for Modulating Glucose Metabolism and Lactate Synthesis in Injured Mouse Tendons: Treatment With Dichloroacetate, a Lactate Synthesis Inhibitor, Improves Tendon Healing by Kairui Zhang, Michael W. Hast, Soutarou Izumi, Yu Usami, Snehal Shetye, Ngozi Akabudike, Nancy J. Philp, Masahiro Iwamoto, Itzhak Nissim, Louis J. Soslowsky and Motomi Enomoto-Iwamoto in The American Journal of Sports Medicine</p