The Glucocorticoid Receptor Regulates the <i>ANGPTL4</i> Gene in a CTCF-Mediated Chromatin Context in Human Hepatic Cells

<div><p>Glucocorticoid signaling through the glucocorticoid receptor (GR) plays essential roles in the response to stress and in energy metabolism. This hormonal action is integrated to the transcriptional control of GR-target genes in a cell type-specific and condition-dependent manner. In the present study, we found that the GR regulates the <i>angiopoietin-like 4</i> gene (<i>ANGPTL4</i>) in a CCCTC-binding factor (CTCF)-mediated chromatin context in the human hepatic HepG2 cells. There are at least four CTCF-enriched sites and two GR-binding sites within the <i>ANGPTL4</i> locus. Among them, the major CTCF-enriched site is positioned near the <i>ANGPTL4</i> enhancer that binds GR. We showed that CTCF is required for induction and subsequent silencing of <i>ANGPTL4</i> expression in response to dexamethasone (Dex) and that transcription is diminished after long-term treatment with Dex. Although the <i>ANGPTL4</i> locus maintains a stable higher-order chromatin conformation in the presence and absence of Dex, the Dex-bound GR activated transcription of <i>ANGPTL4</i> but not that of the neighboring three genes through interactions among the <i>ANGPTL4</i> enhancer, promoter, and CTCF sites. These results reveal that liganded GR spatiotemporally controls <i>ANGPTL4</i> transcription in a chromosomal context.</p></div

Enrichment of GR, CTCF, acetyl-H3K27, and RNA polymerase II at the <i>ANGPTL4</i> gene locus in cells treated with Dex.

<p>(A) Enrichment of glucocorticoid receptor (GR) in cells treated with Dex. As shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169225#pone.0169225.g001" target="_blank">Fig 1B</a></b>, HepG2 cells were treated with Dex for 24 h. ChIP-qPCR analysis was performed using an anti-GR antibody and an anti-rabbit IgG (control), followed by quantitative PCR using specific primers for each AG site and the control (NC). (B–D) Enrichment of CTCF, acetyl-H3K27 (H3K27ac), and active RNA polymerase II (Pol2 ser5-P) in cells treated with Dex. ChIP-qPCR analyses were performed using an anti-CTCF antibody and an anti-rabbit IgG (control) (B), anti-H3K27ac (C), and anti-Pol2 ser5-P (D), followed by quantitative PCR using specific primers for each indicated site. Relative enrichment of the control (NC) site was normalized to 1 (D). Asterisks indicate statistically significance between control (Dex 0 h) and Dex-treated cells at each time point. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.005.</p

Long-term dexamethasone treatment inhibits the induction of <i>ANGPTL4</i> transcription, together with down-regulation of the GR.

<p><b>(A)</b> Protocol for <u>L</u>ong-<u>T</u>erm <u>D</u>examethasone <u>T</u>reatment (LTDT). For LTDT, HepG2 cells were initially cultured in DMEM medium supplemented with 10% DCC-treated FBS and 100 nM dexamethasone (Dex) for 14 days. Black arrows show sampling times. (B) Decrease in <i>ANGPTL4</i> induction in LTDT cells. <b>(C)</b> Decreased enrichment of GR after Dex treatment of LTDT cells. ChIP-qPCR analysis was performed using an anti-GR antibody and an anti-rabbit IgG (control), followed by quantitative PCR using specific primers for each AG site and the control (NC). (D) CTCF enrichment in control and LTDT cells. ChIP-qPCR analysis was performed using an anti-CTCF antibody and anti-rabbit IgG (control), followed by quantitative PCR using specific primers for each AC site. (E) Expression of CTCF and GR after Dex treatment of control and LTDT cells. The amount of GR decreased in most LTDT cells. The relative level of GR normalized to that of β-tubulin is shown below. Uncropped image of western blot analysis is shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169225#pone.0169225.s006" target="_blank">S6 Fig</a></b>. Asterisks indicate statistically significance between control and LTDT cells at each time point. **<i>P</i> < 0.01, ***<i>P</i> < 0.005.</p

Specific changes in higher-order chromatin conformation of the <i>ANGPTL4</i> locus in cells treated with dexamethasone.

<p>(A) Chromosome conformation capture (3C) assays were performed using of DpnII-digested fragments containing each AC/AG site and the <i>ANGPTL4</i> promoter. AC3/AG2 sites reside in the same fragment, and AG2 is the <i>ANGPTL4</i> enhancer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169225#pone.0169225.ref015" target="_blank">15</a>]. The relative interaction frequencies of the reference AC3/AG2 fragment (indicated with red) with other DpnII fragments were determined using qPCR analysis of at least three distinct samples from HepG2 cells treated with Dex. Gray arrowheads indicate the orientation of CTCF-binding sites. (B) The relative interaction frequencies of the reference <i>ANGPTL4</i> promoter (indicated with red) with other DpnII fragments in Dex-treated cells. PCR amplification using internal primers derived from the <i>ANGPTL4</i> locus was used as a loading control to normalize the amount of DNA fragments. The efficiencies of DpnII digestions and subsequent ligations were determined at each restriction site. The relative frequencies of interactions between the reference and its closest site in the control state (Dex 0 h) were normalized to 1. Asterisks indicate statistically significance between control (Dex 0 h) and Dex-treated cells (Dex 3h). *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Distribution of glucocorticoid receptor and CTCF in human <i>ANGPTL4</i> gene locus.

<p><b>(A)</b> Enrichment of the glucocorticoid receptor (GR), CTCF, and modified histone H3 in the <i>ANGPTL4</i> locus of HepG2 cells. <i>KANK3</i>, <i>ANGPTL4</i>, <i>RAB11B-AS</i>, and <i>RAB11B</i> are located across an approximately 80-kb region. The arrow at the transcription start site of each gene indicates the direction of transcription. According to publically available data and our ChIP-Seq results, two GR-binding sites (designated AG1 and AG2) and four CTCF-enriched sites (designated AC1–AC4) are indicated in orange and green, respectively. NC, negative control. Modifications of histone H3, such as acetylation and methylation, are shown. AG2/AC3 sites are close to each other, and AG2 is an enhancer that has been demonstrated in rat cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169225#pone.0169225.ref015" target="_blank">15</a>]. <b>(B)</b> Induction of <i>ANGPTL4</i> transcription by dexamethasone (Dex). HepG2 cells were grown in DMEM medium supplemented with 10% dextran-coated charcoal (DCC)-treated FBS and were treated with Dex (100 nM). Black arrows show the sampling times of the assays. <b>(C)</b> <i>ANGPTL4</i> as a direct GR target in HepG2 cells. The GR antagonist mifepristone was added to the medium (100 μM for 1 h) before Dex treatment. The relative expression level is indicated as a value normalized to the level of <i>36B4</i> mRNA. Asterisks indicate statistically significance between control (Dex 0 h) and Dex-treated cells at each time point. ***<i>P</i> < 0.005.</p

The role of CTCF in regulating <i>ANGPLT4</i> transcription.

<p>(A) qRT-PCR analysis of HepG2 cells transfected with the siRNAs (siCont, siCTCF, and si<i>RAB11B AS</i>) for 48 h and then treated with Dex (100 nM). Expression levels were normalized to those of <i>36B4</i> transcripts. <b>(B)</b> Western blot analysis of CTCF and GR expression in siRNA-transfected cells. Asterisks indicate statistically significance among siRNA-transfected cells at each time point. <b>(C)</b> qRT-PCR analysis of <i>ANGPTL4</i> mRNA expression in siRNA-transfected HepG2 cells treated with Dex (see <b>Fig 4A</b>). <b>(D)</b> qRT-PCR analysis of <i>ANGPTL4</i> mRNA expression in siRNA-transfected LTDT cells treated with Dex. Expression levels were normalized to those of <i>36B4</i> transcripts. <b>(E)</b> Enrichment of CTCF at AC3 and GR at AG sites in siRNA-transfected cells. ChIP-qPCR analysis was performed using anti-CTCF, anti-GR, and anti-rabbit IgG (control) antibodies, followed by quantitative PCR using primers specific for each site. Asterisks indicate statistically significance between control and CTCF-knockdown cells at each time point. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.005.</p

Additional file 2: Figure S2. of Pre-treatment neutrophil to lymphocyte ratio predicts the chemoradiotherapy outcome and survival in patients with oral squamous cell carcinoma: a retrospective study

The relationship between the NLR status and cancer-specific survival in patients with OSCC. In the Kaplan-Meier survival analysis of patients with oral squamous cell carcinoma (OSCC), the patients were divided into three groups based on their NLR status (Tertiles 1, 2 and 3). (A) The overall survival (OS) of the 124 OSCC patients stratified by their NLR status. (B) The disease-free survival (DFS) of the 124 OSCC patients stratified by their NLR status. (JPG 974 kb

Additional file 1: Figure S1. of Pre-treatment neutrophil to lymphocyte ratio predicts the chemoradiotherapy outcome and survival in patients with oral squamous cell carcinoma: a retrospective study

The relationships between the NLR status and cancer-specific survival in patients with OSCC. In the Kaplan-Meier survival analysis of patients with oral squamous cell carcinoma (OSCC), the patients were divided into two groups (low and high groups) based on the average NLR value (=2.7). (A) Overall survival (OS) of the 124 OSCC patients based on their NLR status. (B) Disease-free survival (DFS) of the 124 OSCC patients based on their NLR status. (JPG 867 kb

Generation of Large Numbers of Antigen-Expressing Human Dendritic Cells Using CD14-ML Technology

<div><p>We previously reported a method to expand human monocytes through lentivirus-mediated introduction of cMYC and BMI1, and we named the monocyte-derived proliferating cells, CD14-ML. CD14-ML differentiated into functional DC (CD14-ML-DC) upon addition of IL-4, resulting in the generation of a large number of DC. One drawback of this method was the extensive donor-dependent variation in proliferation efficiency. In the current study, we found that introduction of BCL2 or LYL1 along with cMYC and BMI1 was beneficial. Using the improved method, we obtained CD14-ML from all samples, regardless of whether the donors were healthy individuals or cancer patients. <i>In vitro</i> stimulation of peripheral blood T cells with CD14-ML-DC that were loaded with cancer antigen-derived peptides led to the establishment of CD4⁺ and CD8⁺ T cell lines that recognized the peptides. Since CD14-ML was propagated for more than 1 month, we could readily conduct genetic modification experiments. To generate CD14-ML-DC that expressed antigenic proteins, we introduced lentiviral antigen-expression vectors and subjected the cells to 2 weeks of culture for drug-selection and expansion. The resulting antigen-expressing CD14-ML-DC successfully induced CD8⁺ T cell lines that were reactive to CMVpp65 or MART1/MelanA, suggesting an application in vaccination therapy. Thus, this improved method enables the generation of a sufficient number of DC for vaccination therapy from a small amount of peripheral blood from cancer patients. Information on T cell epitopes is not necessary in vaccination with cancer antigen-expressing CD14-ML-DC; therefore, all patients, irrespective of HLA type, will benefit from anti-cancer therapy based on this technology.</p></div

Induction of CD8⁺ T cell lines that are reactive to cancer antigens by CD14-ML-DC.

<p>(A) Protocol for the induction of cancer antigen-specific CD8⁺ T cells by CD14-ML-DC. In order to generate CD14-ML-DC, we added IL-4 to CD14-ML. After 3 days, we added OK432. CD14-ML-DC were pulsed with peptides for 3 h, X-ray-irradiated (45 Gy), and subsequently mixed with autologous CD8⁺ T cells. Cells were cultured with rIL-7 (10 ng/ml) in AIM-V with 5% human decomplemented plasma. On days 7 and 14, the T cells were restimulated with the autologous peptide-pulsed CD14-ML-DC and on days 9 and 16, and were supplemented with rIL-2 (20 IU/ml). CD14-ML-DC were prepared each time, and we only added IL-4 (did not add OK432). IFN-γ ELISPOT assay and flow cytometry were performed after 6 or 7 days from the third round of peptide stimulation. (B, C) Peripheral blood CD8⁺ T cells were obtained from a HLA-A*24:02-positive healthy donor (healthy donor 1) and were co-cultured with 4 peptides (CDCA1_56-64, KIF20A_66-75, LY6K_177-186 and IMP-3_508–516)-loaded autologous CD14-ML-DC. (B) On day 21, the number of IFN-γ producing CD8⁺ T cells were analyzed by ELISPOT assay (Day 21). The results of the T cells before stimulation culture are also shown (Day 0). The HIV-peptide was used as a control peptide. (C) On day 21, the T cells were recovered and stained with anti-CD8 mAb and the HLA-A*24:02/CDCA1_56-64 or HLA-A*24:02/LY6K_177-186 tetramer. The numbers in the figure indicate the percentage of the CD8⁺ T cells that were positively stained with the tetramer of the HLA-peptide complex (Day 21). The results of the T cells before stimulation culture are also shown (day 0). (D, E) A similar experiment as in (B, C) was done with the cells obtained from a HLA-A*02:01-positive donor (healthy donor 2). We used 4 peptides (CDCA1_351-359, KIF20A_809-817, MART1_26-35 and IMP3_515-523) for the stimulation of the T cells. (D) The number of IFN-γ producing CD8⁺ T cells was analyzed by ELISPOT assay. (E) The T cells were recovered and stained with an anti-CD8 mAb and a HLA-A*02:01/MART1_26-35 dextramer, HLA-A*02:01/CDCA1_351-359 tetramer or HLA-A*02:01/IMP3_515-523 tetramer. The numbers in the figure indicate the percentage of the CD8⁺ T cells that were positively stained with the dextramer or tetramer of HLA-peptide complex.</p