Background
Mutations in isocitrate dehydrogenase (IDH) have been observed in many human malignancies [
1]. The initial mutations identified were IDH1 and IDH2 in ~ 80% of intermediate grade gliomas [
2] and in ~ 20% of de novo acute myeloid leukemia [
3]. Further investigation revealed that frequent IDH mutations were present in both benign and malignant types of cartilaginous tumors, including 71% of conventional chondrosarcomas, 57% of dedifferentiated chondrosarcomas, periosteal chondromas, sporadic central cartilaginous tumors and enchondromas [
4‐
6]. IDH1 mutations cause substitutions at codon R132, while IDH2 mutations affect codon R172 or R140. Recently, IDH2-R172S mutation has been proved to exist in giant cell tumors of bone and osteosarcomas patients [
7].
Cytosolic IDH1 and mitochondrial IDH2 are NADP
+-dependent enzymes that assists in metabolizing isocitrate to α-ketoglutarate (α-KG) in the tricarboxylic acid cycle [
1]. Generally, the cancer-associated IDH mutations involves loss of normal catalytic activity of IDH in producing α-KG and gain a neomorphic function of producing R-enantiomer of 2-hydroxylglutarate (R-2HG) [
8,
9]. Thus, the IDH1/2 mutant cells are expected to have increased levels of R-2HG, which, are at extremely low concentrations under physiological conditions [
10]. Also, the cartilaginous tumors and other neoplasms with IDH1/2 mutations have increased levels of R-2HG [
4]. Compelling evidence indicated that IDH1/2 mutation is sufficient to initiate enchondromas and sarcomas in vivo [
11,
12]. However, the underlying mechanisms require to investigate further. It is believed that the IDH mutations promote tumorigenesis through putative “oncometabolite” R-2HG accumulation [
13]. R-2-HG was also found elevated in colon [
14] and breast cancer cells [
15] harboring IDH1/2 wild type, despite the R-2-HG levels are lower than IDH mutant. However, the role of R-2-HG in IDH wild type cells in oncogenesis still controversial.
The oncometabolite R-2HG had structural similarities with α-KG, and due to this R-2HG competitively inhibited α-KG dependent enzymes, thereby inducing epigenetic changes including histone modification and DNA hypermethylation [
12]. DNA methylation is regarded as an important epigenetic modification that regulates various cellular processes such as differentiation or proliferation. However, dysregulation of it could result in disordered stem cell function or cellular transformation [
16]. The epigenetic changes caused mutant IDH protein impaired the differentiation of hematopoietic stem cells and neurogenic precursor cells [
13,
17,
18].
Most of the cartilaginous tumors develop from the intramedullary region, and tumor cells were found to be chondrocyte-like in morphology [
19]. The clinical findings suggested that bone marrow cells with the capability to differentiate into chondrogenic cells are considered as precursors of this tumor type. Mesenchymal stromal cells (MSCs) could differentiate into chondrogenic, osteogenic and adipocytic lineages, and reside in bone marrow, and are therefore regarded as reasonable precursor cells of cartilaginous tumors [
20‐
22]. Notably, increasing studies have reported that the phenotypic, molecular and gene expressions that run parallelly between the development of chondrosarcoma and the chondrogenic differentiation of MSCs are similar [
23,
24]
. Hence, in this study, the effect of oncometabolite R-2HG on differentiation, proliferation and DNA methylation status of bone marrow MSCs was investigated.
Methods
Isolation, culture, and expansion of MSCs
This study was approved by the Ethics Committee of First Affiliated Hospital, School of Medicine, Zhejiang University. Bone marrow (BM) samples were obtained from the healthy adult donors after taking their consent. The mononuclear cells were collected by density gradient centrifugation (Ficoll 1.077 g/mL; Haoyang Biological Manufacture, Co., Ltd., Tianjin, China). The cells were then seeded at a density of 4 × 105 cells/cm2 in low-glucose Dulbecco’s modified Eagle’s medium (LG-DMEM; Gibco, Carlsbad, CA, USA) addied with 10% fetal bovine serum (FBS; Gibco) and 100 IU/mL penicillin/streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. After 48 h, the non-adherent cells were removed and the medium was replaced every 3 days. The cells after reaching 70–80% confluence were trypsinized and reseeded at a density of 8 × 103 cells/cm2. MSCs at passages 3–4 were used in this study.
Compounds
R-2HG (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in phosphate buffered saline (PBS). The MSCs were treated with 0 to 1.5 mM concentrations of R-2HG.
Cell proliferation analysis
Proliferation of MSCs was determined using Cell Counting Kit-8 assay (CCK-8; Dojin, Tokyo, Japan). Briefly, the cells were plated at the density of 3000 cells/well in 96-well plates, and then were exposed to R-2HG at a concentration of 0 to 1.5 mM. After culturing at 37 °C in a humidified incubator with 5% CO2 for 0, 2, 4, 6, or 8 days, the cells were incubated at 37 °C with 20 μl CCK-8 solution for 2 h, and the absorbance was measured by a multiwell spectrophotometer (Bio-Rad Laboratories, Tokyo, Japan) at 490 nm.
Flow cytometry assay
MSCs exposed to 0, 1.0, 1.5 mM R-2HG were determined by flow cytometry assay. A total of 5 × 105 cells from single-cell suspensions were incubated for 30 min at room temperature with fluorochrome-conjugated monocolonal antibodies against CD34-PE, CD73-APC, CD90-FITC, CD105-PE (eBioscience, San Diego, CA, USA), CD45-FITC, and HLA-DR-PE-Cy5 (Biolegend, San Diego, CA, USA). After washing with PBS, immunofluorescence analysis was performed by flow cytometry using a FACS Calibur system (Beckman Coulter, Miami, FL, USA) and data were calculated using the FlowJo Software. Appropriate isotype-matched antibodies were used as controls.
Osteogenic differentiation
MSCs were seeded into 0.1% gelatin coated 6-well plates at a density of 10,000 cells/cm2 in LG-DMEM supplemented with 10% FBS. After 2 days, cells were transferred to osteogenic induction medium for 14 days. The medium consists LG-DMEM containing 10% FBS, 10 mM β-glycerophosphate, 0.1 μM dexamethasone and 50 μM ascorbic acid (Sigma-Aldrich). R-2HG at a concentration of 0–1.5 mM was added to the osteogenic induction medium. The medium should be changed for every 3 days. The mineralized areas were revealed using alizarin red staining.
Chondrogenic differentiation
The cells after reaching 80% confluence were trypsinized, washed, and resuspended in high-glucose DMEM with 1 mM sodium pyruvate (Invitrogen), 0.1 μM dexamethasone (Sigma-Aldrich), 200 μM ascorbic acid (Sigma-Aldrich), 1 × insulin-transferrin-selenium (Invitrogen) and 10 ng/ml transforming growth factor-1 (Peprotech, London, UK). The viable cells were seeded in 15-ml conical tubes at a density of 5 × 105 cells per pellet. Next, the cells were gently allowed to centrifuge to the bottom of the tubes to form compact cell pellets, and then incubated in a humidified atmosphere in 5% CO2 at 37 °C. The medium should be changed every 3 days. R-2HG at a concentration of 0–1.5 mM was used for treatment from day 1.
Sections of paraffin-embedded MSCs pellets were processed for immunohistochemistry using rabbit anti-human collagen type II (Abcam, Cambridge, MA, USA). EnVision detection kit (Dako, Carpinteria, CA) was applied to analyze the immunoreactivity of the sections. Non-immune rabbit- IgG antibody was used as the negative control.
Adipogenic differentiaion
The MSCs were seeded into 6-well plates in a density of 20,000 cells/cm2. While cells were grown to confluence, they were transferred to adipogenic induction medium containing LG-DMED and adipogenic stimulatory supplement (Stem Cell Technologies, Hangzhou, China) and the system was cultured for 21 days. The medium was changed every 3 days. R-2HG at a concentration of 0–1.5 mM was added to the adipogenic induction medium. The adipogenic differentiation was mesured by cellular accumulation of large lipid vacuoles that are stained with oil red O (Sigma-Aldrich).
Real-time quantitative polymerase chain reaction analysis
Messenger RNA (mRNA) expressions of osteogenic (BGLAP, IBSP, LPL, SP7), adipogenic (CEBPA, PPARG, ADIPOQ and FABP4) and chondrogenic differentiation related markers (SOX9, RUNX2, COL2A1 and COL10A1) were quantified using real-time quantitative polymerase chain reaction analysis (RT-PCR). The cultured cell layers or pellets of each group were collected on Day 6 of induction medium incubation. The total RNA was extracted from MSCs using Trizol reagent (Invitrogen) and then was reversely transcribed into complementary DNA (cDNA) by PrimeScript RT reagent Kit (Takara, Tokyo, Japan). Equal amounts of cDNA were used and amplified with SYBR Premix Ex Taq using SYBR Premix Ex Taq (Takara). Every sample was performed in in three independent experiments and all the results were normalized to the levels of glyceraldehydes 3-phosphate dehydrogenase (GAPDH).
The expressions of the components of Sonic Hedgehog signaling pathway including Sonic Hedgehog ligand (SHH), Patched 1 (PTCH1), Smoothened (SMO), and Gli transcription factors (GLI-1, 2 and 3) were quantified by RT-PCR. RNA was prepared from MSCs treated in the absence or presence of 1.0 mM and 1.5 mM R-2HG during osteogenic induction for 6 days.
Illumina Infinium methylation assay
The changes in DNA methylation of MSCs exposed to R-2HG, and the genome-scale methylation profiles were explored as described previously [
25]. MSCs were cultured in proliferation medium in the absence or presence of 1.0 mM R-2HG for 6 days and collected. Bisulfite conversion of genomic DNA was prepared using EZ DNA methylation Kit (Zymo Research, D5002, USA). A total amount of 500 ng of DNA was bisulfite converted and subsequently processed for hybridization onto an Infinium Human Methylation 450 Bead Array (Illumina, San Diego, CA, USA) under the manufacturer’s instructions. This array can interrogate 27,578 CpG dinucleotides encompassing 14,495 genes. In brief, the DNA was mixed with bisulfite, and the nonmethylated C nucleotides were converted to U (T), whereas the methylated C nucleotides remained to be unaffected. Subsequently the bisulfite-treated DNA was amplified, fragmented, and hybridized to locus-specific oligonucleotides on the BeadArray. C or T nucleotides were detected by fluorescence signaling in order to obtain the single-nucleotide extension of the DNA fragments. The results were interpreted as a ratio (β value) of methylated signal (C) when compared with the sum of methylated and unmethylated signal (C-T) for each locus, where 0 was regarded as fully unmethylated DNA and 1 as fully methylated DNA.
To investigate the methylation state of MSCs during osteogenic and chondrogenic differentiation, the cultured cell layers or pellets of MSCs treated either in absence or presence of 1.0 mM R-2HG were collected on Day 6 of induction medium incubation. Bisulfite conversion of genomic DNA was prepared using EZ DNA methylation Kit (Zymo Research, D5002, USA). A total amount of 500 ng of DNA was bisulfite converted and subsequently processed for hybridization onto an Infinium Human Methylation 850 Bead Array (Illumina, San Diego, CA, USA) under the manufacturer’s instructions. Methylation analysis was performed using the R/Bioconductor package Minfi. Methylated CpG sites in promotor region of related genes (BGLAP, IBSP, LPL, SP7, SOX9, RUNX2, COL2A1, COL10A1, SHH, PTCH1, SMO, GLI-1, 2 and 3) were analyzed from the array-based data.
Heat maps
The heat maps were designed by Mev software. The Euclidean distance within the two groups of samples was calculated using the average linkage measure [the mean of all pair-wise distances (linkages) between the members of the two concerned groups]. Gene annotation and enrichment analyses were performed by KEGG databases using the DAVID Bioinformatics Resources (
http://david.abcc.ncifcrf.gov/) interfaces and WebGestalt (
http://bioinfo.vanderbilt.edu/webgestalt/), respectively.
Gene pathway analysis
To determine the biological processes enriched within genes of differential methylation in the comparisons, we uploaded the gene lists into the Ingenuity Pathway Analysis (IPA; Ingenuity Systems, Redwood City, CA, USA). Each gene symbol was linked to its corresponding gene object in the Ingenuity Pathways Knowledge Base. Then the IPA integrates the genes and molecules that share part of the same biological functions or regulatory networks interacting together. The over-represented cellular and molecular functions were ranked according to the calculated P-value.
Statistical analysis
The results are expressed as mean ± standard error (SE), each performed in duplicates. Statistical analysis was performed by analysis of variance (ANOVA). All analyses used SPSS software (Paris, France). A p-value of < 0.05 was considered significant.
Discussion
Some metabolites play a critical role as regulators of some important enzymes in various biological pathways. According to recent studies, metabolic alterations promote the initiation and development of malignant cells. R-2HG that is produced by mutant IDH proteins is regarded as a prototype of these oncometabolites, and a serious of studies have proved the role of R-2HG in malignant transformation [
13,
27]. Elevated levels of R-2HG that are caused due to mutations in IDH1 and IDH2 are frequently shown (up to 87%) in enchondromas [
4]. Impaired differentiation by R-2HG has been reported in central nervous system and during hematopoietic differentiation processes [
13,
27]. We therefore examined the effects of R-2HG on the characteristics, especially on the differentiation properties of human MSCs, which presumably act as precursors of cartilaginous tumors.
The results of the present study showed that R-2HG impaired the calcification of MSCs and reduced the expression of both early and late osteoblast differentiation-related genes in a dose-dependent manner, indicating the inhibition of osteogenic differentiation of MSCs by R-2HG. In consistent with our data, Suijker et al. [
28] recently reported impaired development of vertebrate rings in zebrafish with the presence of R-2HG, suggesting that R-2HG blocks osteoblast differentiation in vivo. Interestingly, the results of our study along with the previous study [
28] demonstrated that R-2HG inhibited osteogenic differentiation, while mutations are virtually rare in osteosarcoma and very frequently found in cartilaginous tumors [
4].
We next investigated the effect of R-2HG on chondrogenic differentiation of MSCs. The results indicated that R-2HG suppressed chondrogenic differentiation of human MSCs, but might promote the onset of chondrocyte hypertrophy at lower concentration (1.0 mM). Lu et al. [
11] demonstrated that the expression of mutant IDH2 in 3 T3-L1 cells results in a profound impairment in chondrocyte differentiation and was consistent with our study results. Recently, Hirata et al. [
29] reported dysregulation of chondrogenic differentiation with persistence of hypertrophic chondrocytes from mice with the mutant IDH1 or control chondrocytes treated with R-2HG, preventing the bone from normal replacement of cartilage. However, Suijker et al. [
28] indicated that half of the MSCs showed an increase in differentiation towards chondrogenic lineage in the presence of R-2HG. Variations in these results might occur due to differences in concentrations of R-2HG used in the experiments. In this study, hypertrophic markers including Runx2 and Col10a were up-regulated under chondrogenic differentiation conditions in the presence of 1.0 mM R-2HG. Runx2 is an important transcription factor in chondrocyte hypertrophy that promotes the expressions of Col 10, thus disturbing chondrocyte homeostasis [
30].
Compelling evidences indicated that IDH1/2 mutation is sufficient to initiate enchondromas and sarcomas in vivo [
11,
29]. MSCs give rise to variations in differentiated cells, including adipocytes, osteocytes, neural cells, stromal cells, chondrocytes, muscle cells and fibroblasts,, which are thought to be the progenitor cells of many different types of sarcomas [
23]. Our data indicated that increased levels of R-2HG blocked osteogenic differentiation and disturbed the normal chondrogenic differentiation of MSCs, partly explaining the mechanism of cartilage tumor formation induced by IDH mutation. The mechanisms of tumorigenesis is therefore comparable to other tumors caused by IDH mutations, as the differentiation was impaired in hematopoietic precursor cells [
17], neurogenic precursor cells [
18] and liver [
31] progenitor cells.
Besides osteogenic and chondroblastic differentiation, MSCs is also able to differentiate into adipocytes. We herein showed that R-2HG promoted adipogenic differentiation of MSCs as measured by increased lipid vacuoles and enhanced gene marker expression. This was opposite to the reduced adipogenic differentiation caused by R-2HG or by introduction of an IDH2 mutation in 3 T3-L1 cells [
11]. These murine 3 T3-L1 cells involve spontaneous adipogenic differentiation. The effect on human MSCs was studied to explain the differences in the results. MSCs are delicately balanced during their adipo-osteogenic differentiation. Many in vitro investigations have proved that fat-induction factors inhibited osteogenesis, and in contrast, the bone-induction factors hindered adipogenesis [
32]. Our results also confirmed that R-2HG inhibited osteogenesis, while promoted the adipogenesis of MSCs.
A key issue in IDH1/2 mutation-induced tumorigenesis is the blockage of cellular differentiation [
18]. Though the precise oncogenic consequences of IDH mutations remained unclear, high levels of R-2HG is widely believed to be essential in the process. R-2HG competitively inhibited multiple a-KG-dependent dioxygenases, including key epigenetic regulators, histone demethylases and DNA-demethylating agents for example [
10]. According to a previous study, CpG island methylation was found increased significantly in IDH mutant chondrosarcoma samples [
11]. In addition, Jin et al. recently showed that IDH1 R132C mutation increased histone methylation in both cartilage- and bone-related genes and global histone methylation [
33]. However, the effects of R-2HG on DNA methylation status of MSCs are still unknown.
We herein showed that R-2HG induced a pronounced DNA hypermethylation state of MSCs both in proliferation and osteogenic differentiation conditions. R-2HG treated MSCs revealed a high ratio of hypermethylation among CpG islands during chondrogenic differentiation, however, there were more hypomethylation sites in some gene regions. The varies of methylation status might be result from the different induction condition, such as a higher concentration of ascorbic acid, which induces TET-dependent DNA demethylation [
34]. Our data further confirmed that active DNA methylation does occur on lineage-specific gene promotors during R-2HG induced osteogenic and chondrogenic differentiation. DNA hypermethylation is regarded as a barrier in the differentiation of MSCs [
35]. Thus, hypermethylation of related genes induced by R-2HG might contribute to the impairment in the differentiation of MSCs.
In functional analysis, the top canonical pathway is the Shh signaling. Previous studies indicated that increased Shh signaling promoted osteogenesis in various bone-forming cells, and in contrast, Shh signaling repressed adipogenic differentiation in preadipocytes [
36,
37]. In addition, Shh promoted chondrogenesis in MSCs by inducing the expression of Sox9 [
38]. A recent study indicated that mechanical stimulation promoted osteogenic differentiation of MSCs through epigenetic regulation of Shh [
39]. Moreover, Shh and Gli genes play an essential role during cartilage development [
40]. Our results showed that R-2HG impaired the osteogenic differentiation of MSCs, which in turn was accompanied by down-regulation of Shh signaling, implying that Shh signaling might play a role in this process.
Due to lack of effective treatment strategies for advanced diseases, the clinical management of chondrosarcomas remains exceptionally challenging [
41]. Somatic mutations of IDH genes exist in more than 50% of primary conventional chondrosarcomas. More recently, the first mutant IDH2 inhibitor, enasidenib (AG-221), in patients with relapsed or refractory IDH2-mutated AML has been approved by FDA [
42]. The development of IDH inhibitors is an emerging treatment option for patients with chondrosarcoma.
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