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 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 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

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 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

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

Secreted BBF2H7 C-terminus is involved in the proliferation of cancer cells.

<p>U251MG cells were knocked down by siRNAs, followed by treatment with conditioned medium collected from HEK293T cells transfected with an empty vector (Mock) or BBF2H7 C-terminus (C-Sup.), or by treatment with C-Sup. depleted of BBF2H7 C-terminus by immunoprecipitation using an anti-BBF2H7 C-terminus antibody (C-Sup. + Ab.), and then cell proliferation was monitored by cell counting and WST-8 assays. Scramble and siBBF2H7-1 indicate non-targeting siRNA and <i>Bbf2h7</i>-targeting siRNA, respectively. (A, B) U251MG cells were cultured for 5 days after conditioned medium treatment, and then analyzed by cell counting at day 5 (A) and WST-8 assays at the indicated days (B). Error bars represent the mean ± SD of five independent experiments. <b>*</b><i>P</i> < 0.05, <b>**</b><i>P</i> < 0.01, <b>***</b><i>P</i> < 0.001. (C) RT-PCR analysis of Hh target genes in U251MG cells at 5 days after conditioned medium treatment. (D) Quantitative real-time PCR analyses of Hh target genes expression in C. Error bars represent the mean ± SD of five independent experiments. <b>*</b><i>P</i> < 0.05.</p

The structure of BBF2H7 and its expression in tumors or cancer cell lines.

<p>(A) Increased expression of <i>Bbf2h7</i> in human cancers. Microarray datasets of tumors were accessed in the ONCOMINE Cancer Profiling Database (version 4.4.4.4, <a href="http://www.oncomine.org/" target="_blank">www.oncomine.org</a>). Box plots showing increased expression of <i>Bbf2h7</i> during tumorigenesis of various cancers were constructed from ONCOMINE. The y-axis represents the log₂ median-centered intensity (normalized expression). The line within the box represents the median expression value for each group, and the upper and lower edges of the box indicate the 75^th and 25^th percentiles of the distribution, respectively. The lines (whiskers) from each box extend to the 90^th and 10^th percentiles of the distribution. The black dots outside the ends of the whiskers represent the largest and smallest data points. Box plots depicting the distribution of <i>Bbf2h7</i> expression within each sample and a Student’s <i>t</i>-test giving a <i>P</i> value for the comparison of <i>Bbf2h7</i> expression between normal and malignant tissue samples were obtained directly through ONCOMINE. Normal colon tissue samples included ascending colon, descending colon and rectum. (B) Predicted peptide features of human BBF2H7. Transcription activation, basic leucine zipper (bZIP) and transmembrane domains, as well as a site-1 protease (S1P) site are indicated. (C) Under ER stress conditions, BBF2H7 is transported from the ER to the Golgi apparatus and cleaved at the S1P site [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125982#pone.0125982.ref009" target="_blank">9</a>]. The cleaved BBF2H7 N-terminus acts as a transcription factor via binding to the cyclic AMP response element (CRE) of target genes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125982#pone.0125982.ref015" target="_blank">15</a>]. In contrast, the cleaved BBF2H7 C-terminus is extracellularly secreted [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125982#pone.0125982.ref016" target="_blank">16</a>]. (D) Western blotting of the endogenous full-length BBF2H7 and the N- or C-terminus of BBF2H7 using cell lysates (left panel) or culture media (right panel) of several human cancer cell lines. BBF2H7 N-terminus, BBF2H7 C-terminus or IL-6 in the culture media was immunoprecipitated using anti-BBF2H7 N-terminus (top panel), anti-BBF2H7 C-terminus (middle panel), or anti-IL-6 antibodies (bottom panel), respectively. β-actin or IL-6 was used as loading controls. (E) RT-PCR analysis of <i>Xbp1</i> mRNA expression in several human cancer cell lines. <i>Xbp1-s</i> and <i>Xbp1-u</i> indicate spliced and unspliced forms of <i>Xbp1</i>, respectively. Note that <i>Xbp1-s</i> was detected in all cancer cell lines, indicating that these cells are undergoing ER stress. (F) RT-PCR analysis of <i>Xbp1</i> mRNA expression in U251MG cells treated with 2 mM 4-PBA, a chemical chaperon, for 3 h. (G) Quantification of <i>Xbp1-s</i> expression in F. Error bars represent the mean ± SD of five independent experiments. <b>**</b><i>P</i> < 0.01. (H) Western blotting of BBF2H7 in U251MG cells treated with 1 μM thapsigargin (TG), an inhibitor of ER Ca²⁺-ATPase, for 6 h. (I) Immunohistochemistry of brain sections from glioblastoma patients using an anti-BBF2H7 C-terminus-specific antibody. Strong signals were detected both in the cytosol of the cancer cells (indicated with an arrowhead) and in their surrounding extracellular space (indicated with arrows). Left panel shows a low magnification, and right panel shows a higher magnification of the left panel. Scale bars indicate 50 μm (left panel) and 10 μm (right panel).</p

Cancer cell proliferation in an Shh-dependent manner.

<p>(A) RT-PCR analysis of Hh target genes in several cancer cell lines treated with 500 ng/ml recombinant Shh for 48 h. (B) Quantitative real-time PCR analyses of Hh target genes expression in A. Error bars represent the mean ± SD of five independent experiments. <b>*</b><i>P</i> < 0.05, <b>**</b><i>P</i> < 0.01. (C) The cancer cell lines were cultured for 5 days after treatment with 500 ng/ml recombinant Shh and analyzed by WST-8 assays at day 5. Error bars represent the mean ± SD of five independent experiments. <b>*</b><i>P</i> < 0.05.</p

Suppressed proliferation of cancer cells by knockdown of <i>Bbf2h7</i>.

<p>(A) Western blotting of BBF2H7 in U251MG cells knocked down by siRNAs. Scramble and siBBF2H7 indicate non-targeting siRNA and <i>Bbf2h7</i>-targeting siRNAs, respectively. The sequences of siBBF2H7-1 and siBBF2H7-2 have different nucleotide positions. (B) Quantification of full-length BBF2H7 expression in A. Error bars represent the mean ± SD of five independent experiments. <b>***</b><i>P</i> < 0.001. (C) Western blotting of full-length BBF2H7 or the C-terminus of BBF2H7 using cell lysates (left panel) or culture media (right panel) of U251MG cells knocked down by siRNAs. BBF2H7 C-terminus or IL-6 in the culture media was immunoprecipitated using anti-BBF2H7 C-terminus (upper panel) or anti-IL-6 antibodies (lower panel), respectively. β-actin or IL-6 was used as loading controls. (D–G) U251MG cells were knocked down by siRNAs, and then cell growth was monitored by cell counting and WST-8 assays. (D, E) U251MG cells were cultured for 5 days after transfection with each siRNA in the absence or presence of 2 μM PPN, and then analyzed by cell counting at day 5 (D) and WST-8 assays at the indicated days (E). Error bars represent the mean ± SD of five independent experiments. <b>*</b><i>P</i> < 0.05, <b>**</b><i>P</i> < 0.01, <b>***</b><i>P</i> < 0.001. (F) RT-PCR analysis of Hh target genes in U251MG cells at 5 days after transfection with each siRNA in the absence or presence of 2 μM PPN. (G) Quantitative real-time PCR analyses of Hh target genes expression in F. Error bars represent the mean ± SD of five independent experiments. <b>*</b><i>P</i> < 0.05.</p