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Taha Toros Arşivi, Dosya Adı: Galatasarayİstanbul Kalkınma Ajansı (TR10/14/YEN/0033) İstanbul Development Agency (TR10/14/YEN/0033

Mutant IDH1 Dysregulates the Differentiation of Mesenchymal Stem Cells in Association with Gene-Specific Histone Modifications to Cartilage- and Bone-Related Genes

<div><p>Somatic mutations in the <i>isocitrate dehydrogenase</i> (<i>IDH)1/2</i> genes endow encoding proteins with neomorphic activity to produce the potential oncometabolite, 2-hydroxyglutarate (2-HG), which induces the hypermethylation of histones and DNA. The incidence of IDH1/2 mutations in cartilaginous tumors was previously shown to be the highest among various types of tumors, except for those in the central nervous system. Mutations have been detected in both benign (enchondromas) and malignant (chondrosarcomas) types of cartilaginous tumors, whereas they have rarely been found in other mesenchymal tumors such as osteosarcomas. To address this unique tumor specificity, we herein examined the effects of IDH1 R132C, which is the most prevalent mutant in cartilaginous tumors, on the differentiation properties of human mesenchymal stem cells (hMSCs). The induction of the <i>IDH1</i> R132C gene into MSCs markedly increased the amount of 2-HG and up-regulated global histone methylation. The induction of IDH1 R132C promoted the chondrogenic differentiation of hMSCs by enhancing the expression of <i>SOX9</i> and <i>COL2A1</i> genes in association with an increase in the active mark (H3K4me3), but disrupted cartilage matrix formation. On the other hand, IDH1 R132C inhibited expression of the <i>ALPL</i> gene in association with an increase in the repressive mark (H3K9me3), and subsequently inhibited the osteogenic properties of hMSCs and human osteosarcoma cells. Since osteogenic properties are an indispensable feature for the diagnosis of osteosarcoma, the inhibitory effects of IDH1 R132C on osteogenic properties may contribute to the lack of osteosarcomas with the <i>IDH1</i> R132C mutation. These results suggested that IDH1 R132C contributed to the formation of cartilaginous tumors by dysregulating the chondrogenic and osteogenic differentiation of hMSCs via gene-specific histone modulation.</p></div

Direct regulation of the <i>FZD10</i> gene by the SS18-SSX2 fusion protein.

<p>A) Promoter activity in the regulatory region of the <i>FZD10</i> gene. The upper panel indicated the 5’ region of the <i>FZD 10</i> gene with amplified regions in the ChIP-qPCR analysis. The number indicates relative to the transcription start site, and the positions of the amplified region are: -1206 to -955 bp; -825 to -569 bp; -93 to +47 bp; +621 to +869 bp. The lower panel showed the promoter activities of fragments derived from this 5’ region in SYO-1. Error bars reflect SD in 3 experiments. B) Induction of SS18-SSX2 in U2OS. U2OS was transfected with an empty vector (EV) or 3xHA-tagged <i>SS18-SSX2</i>, and 48 h after the transfection, the expression of SS18-SSX2 was analyzed by Western blotting. The SS18-SSX2 and SS18 proteins were detected by an anti-SS18 antibody (top panel), and the 3xHA-SS18-SSX2 protein was detected by an anti-HA antibody (middle panel). C) Induction of <i>FZD10</i> expression by SS18-SSX2 in U2OS. The expression of <i>FZD10</i> was analyzed by RT-qPCR. Expression levels were normalized to those of human <i>ACTB</i> and calculated as fold changes relative to SYO-1. Error bars reflect SD in 3 experiments. **, p<0.01 by the <i>t</i>-test. D) Binding of SS18-SSX2 to the <i>FZD10</i> locus. A ChIP assay with an anti-HA antibody and RT-qPCR were performed. The values indicate relative to rabbit IgG. Error bars reflect SD in 3 experiments.</p

Induction of SS18-SSX2 in hESCs, hNCCs, and hNCC-derived MSCs.

<p>A-C) DOX dose-dependently induced the SS18-SSX2 protein in KhES1-HA (A), KhES1-NCC-FL (B), and KhES1-MSC-FL (C) cells. Cells with Stuffer (-Control) and SS18-SSX2 were treated with the indicated concentrations of DOX for 24 h, and the expression of SS18-SSX2 was analyzed by Western blotting. The SS18-SSX2 and SS18 proteins were detected using an anti-SS18 antibody (top panel), and the 3xHA-SS18-SSX2 or FLAG-SS18-SSX2 protein was detected by an anti-HA or anti-FLAG antibody (middle panel). D and E) Morphology (left panels) and expression of mCherry (right panels) in KhES1-HA cells treated with 0 (D) or 0.3 (E) μg/ml of DOX for 24 and 96 h. Scale bar, 200 μm. F) Effects of SS18-SSX2 on the cell viability of KhES1-NCC-FL and KhES1-MSC-FL cells. Cells with Stuffer (-Control) or SS18-SSX2 were treated with the indicated concentrations of DOX for 48 h, and cell viability was measured using the AlamarBlue assay. n.s. means not significant. Error bars reflect SD in 4 experiments. G) Induction of <i>FZD10</i> expression by SS18-SSX2 in KhES1-HA, KhES1-NCC-FL, and KhES1-MSC-FL cells. Cells with Stuffer (-Control) or SS18-SSX2 were treated with the indicated concentrations of DOX for 24 h, and the expression of <i>FZD10</i> was analyzed by RT-qPCR. Expression levels were normalized to those of human <i>ACTB</i> and calculated as fold changes relative to SYO-1. Error bars reflect SD in 3 experiments. **, p<0.01 by the <i>t</i>-test.</p

Cell type-dependent effects of SS18-SSX2 on histone modifications at the <i>FZD10</i> locus.

<p>A-C) Changes in histone modifications at the <i>FZD10</i> locus by SS18-SSX2 in KhES1-HA, KhES1-NCC-FL, and KhES1-MSC-FL cells. Cells with Stuffer (-Control) and SS18-SSX2 were treated for 24 h with DOX (0.1, 0.3, and 1.0 μg/ml for KhES1-HA, KhES1-NCC-FL, and KhES1-MSC-FL cells, respectively). The levels of H3Ac (A), H3K4me3 (B), and H3K27me3 (C) were analyzed by ChIP-qPCR. The values indicate relative to the input. Error bars reflect SD in 3 experiments. **, p<0.01 by the <i>t</i>-test.</p

Cell type-dependent differences in BAF complexes.

<p>A and B) The mRNA (A) and protein (B) expression of BAF47 in KhES1-HA, KhES1-NCC-FL, and KhES1-MSC-FL cells. Cells with Stuffer (-Control) and SS18-SSX2 cells were treated with the indicated concentrations of DOX for 24 h, and the expression of BAF47 was analyzed. A) mRNA expression analysis using RT-qPCR. Expression levels were normalized to those of human <i>ACTB</i>. Error bars reflect SD in 3 experiments. B) Protein expression analysis by Western blotting. The BAF47 protein was detected using an anti-BAF47 antibody. 293T cells were used as a positive control. C) Recruitment of SS18-SSX2 to the <i>FZD10</i> core promoter region (from -93 to +47 bp) in KhES1-HA, KhES1-NCC-HA, and KhES1-MSC-HA cells. Cells were treated with DOX (0.1, 1.0, and 3.0 μg/ml for KhES1-HA, KhES1-NCC-HA, and KhES1-MSC-HA cells, respectively) for 24 h. A ChIP assay with an anti-HA antibody and RT-qPCR were performed. The values indicate relative to rabbit IgG. Error bars reflect SD in 3 experiments.</p

IDH1 R132C inhibited the osteogenic differentiation of human osteosarcoma cells.

<p><b>A.</b> mRNA expression of the <i>ALPL</i> gene. RNAs were extracted from ANOS cells transduced with doxycycline (Dox) inducible expression vectors containing the wild-type <i>IDH1</i> (WT) or <i>IDH1</i> R132C (R132) gene before and after osteogenic induction (OI) and analyzed by qRT-PCR. Data are shown as a value relative to that of WT-ANOS cells before OI after normalizing to expression of the <i>ACTB</i> gene expression. **, <i>p</i><0.01 by Dunnett`s multiple comparisons test compared to the WT cells or R132C cells without Dox treatment. <b>B.</b> Calcium (Ca) deposition after osteogenic induction in WT- and R132C-ANOS cells. Data were normalized to the culture area. **, <i>p</i><0.01 by the Student`s <i>t</i>-test. <b>C.</b> Calcified nodule formation after osteogenic induction. WT- or R132C-ANOS cells were cultured under osteogenic induction conditions with or without doxycycline and then stained with Alizarin Red S. Error bars indicate the average ± SD from three biological replicates.</p

IDH1 R132C differentially regulated the expression of chondrocyte- and osteocyte-related genes by gene-specific epigenetic modulation.

<p>Active and repressive histone marks associated with the promoter regions of the <i>SOX9</i> (<b>A</b>), <i>COL2A1</i> (<b>B</b>), and <i>ALPL</i> (<b>C</b>) genes. The status of histone modification in each hMSC (Par, EV, MT, or R132C, as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131998#pone.0131998.g001" target="_blank">Fig 1</a>) was analyzed by chromatin immunoprecipitation (ChIP) using antibodies against H3K4me3, H3K9me3, and H3K27me3. The target region of each locus was shown in the scheme at the top of each graph. Data were presented by qPCR, and the values were indicated relative to the input. Error bars reflect SD in 3 experiments. TSS, transcription start site. **, <i>p</i><0.01 by Dunnett`s multiple comparisons test compared to the parental cells.</p

The effect of IDH1 R132C on osteogenic properties of hMSCs.

<p><b>A.</b> The mRNA expression of <i>RUNX2</i>, <i>OSX</i>, <i>ALPL</i> and <i>COL1A1</i> genes in hMSCs. RNAs were extracted from each hMSC and analyzed by qRT-PCR. Data are shown as a value relative to that of parental cells after normalizing to expression of the <i>ACTB</i> gene. <b>B.</b> Calcium (Ca) deposition after osteogenic induction in each hMSC. Data were normalized to the culture area. Results (<b>A</b> and <b>B</b>) are the mean ± SE from the results of three different donors. <b>C.</b> Calcified nodule formation after osteogenic induction. Each type of BM01 cell was cultured under osteogenic induction conditions and then stained with Alizarin Red S. **, <i>p</i><0.01 by Dunnett`s multiple comparisons test compared to the parental cells. Par, EV, MT, and R132C were as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131998#pone.0131998.g001" target="_blank">Fig 1</a>.</p

IDH1 R132C produced 2-HG and increased global histone methylation in hMSCs.

<p><b>A.</b> Detection of 2-HG by GC-MS. Intracellular 2-HG was detected in the extracts of BM01 cells expressing the <i>IDH1</i> R132C gene. Peaks with <i>m/z</i> 246 and 247 were selected as quantification ions for glutamate and 2-HG, respectively. <b>B.</b> Relative amount of 2-HG in hMSCs. The amount 2-HG and glutamate was measured by GC-MS in cell extracts from each type of cell and the ratio was demonstrated. The mean ± SE from the results of three donors was shown. <b>C.</b> Expression of active (H3K4me3) and repressive (H3K9me3 and H3K27me3) histone marks. Protein lysates were prepared from each type of BM01 cell and used for western blotting to detect exogenous (Exo) and endogenous (Endo) IDH1 and indicated histone H3. <b>D.</b> Quantitative analyses of active and repressive histone marks. The expression level of each mark in infected hMSCs (EV, WT, or R132C) was demonstrated as a value relative to those in parental hMSCs (Par). The mean ± SE from the results of three donors was shown. Par, parental hMSC; EV, hMSCs infected with the empty vector; WT and R132C, hMSCs infected with the vector containing the wild-type <i>IDH1</i> or <i>IDH1</i> R132C gene, respectively. *, <i>p</i><0.05 and **, <i>p</i><0.01 by Dunnett`s multiple comparisons test compared to the parental cells.</p