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Promoter methylation and expression pattern of DLX3, ATF4, and FRA1 genes during osteoblastic differentiation of adipose-derived mesenchymal stem cells.

ABSTRACT: Introduction: Nowadays, mesenchymal stem cells are touted as suitable cell supply for the restoration of injured bone tissue. The existence of osteogenic differentiation makes these cells capable of replenishing damaged cells in the least possible time. It has been shown that epigenetic modifications, especially DNA methylation, contribute to the regulation of various transcription factors during phenotype acquisition. Hence, we concentrated on the correlation between the promoter methylation and the expression of genes DLX3, ATF4 , and FRA1 during osteoblastic differentiation of adipose-derived mesenchymal stem cells in vitro after 21 days. Methods: Adipose-derived mesenchymal stem cells were cultured in osteogenesis differentiation medium supplemented with 0.1 µM dexamethasone, 10 mM ?-glycerol phosphate, and 50 µM ascorbate-2-phosphate for 21 days. RNA and DNA extraction was done on days 0, 7, 14, and 21. Promoter methylation and expression levels of genes DLX3 , ATF4 , and FRA1 were analyzed by methylation-specific quantitative PCR and real-time PCR assays, respectively. Results: We found an upward expression trend with the increasing time for genes DLX3, ATF4, and FRA1 in stem cells committed to osteoblast-like lineage compared to the control group (P <0.05). On the contrary, methylation-specific quantitative PCR displayed decreased methylation rates of DLX3 and ATF4 genes, but not FRA1 , over time compared to the non-treated control cells (P <0.05). Bright-field images exhibited red-colored calcified deposits around Alizarin Red S-stained cells after 21 days compared to the control group. Statistical analysis showed a strong correlation between the transcription of genes DLX3 and ATF4 and methylation rate (P <0.05). Conclusion: In particular, osteoblastic differentiation of adipose-derived mesenchymal stem cells enhances DLX3 and ATF4 transcriptions by reducing methylation rate for 21 days.

SUBMITTER: Rahimzadeh S

PROVIDER: S-EPMC7502906 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Promoter methylation and expression pattern of DLX3, ATF4, and FRA1 genes during osteoblastic differentiation of adipose-derived mesenchymal stem cells.

Rahimzadeh Sevda S Rahbarghazi Reza R Aslani Somayeh S Rajabi Hadi H Latifi Zeinab Z Farshdousti Hagh Majid M Nourazarian Alireza A Nozad Charoudeh Hojjatollah H Nouri Mohammad M Abhari Alireza A

BioImpacts : BI 20191125 4

Introduction: Nowadays, mesenchymal stem cells are touted as suitable cell supply for the restoration of injured bone tissue. The existence of osteogenic differentiation makes these cells capable of replenishing damaged cells in the least possible time. It has been shown that epigenetic modifications, especially DNA methylation, contribute to the regulation of various transcription factors during phenotype acquisition. Hence, we concentrated on the correlation between the promoter ...[more]

PMID: 32983940

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Project description:BackgroundOsteogenesis Imperfecta (OI) (OMIM %259450) is a heterogeneous group of inherited disorders characterized by increased bone fragility, with clinical severity ranging from mild to lethal. The majority of OI cases are caused by mutations in COL1A1 or COL1A2. Bruck Syndrome (BS) is a further recessively-inherited OI-like phenotype in which bone fragility is associated with the unusual finding of pterygia and contractures of the large joints. Notably, several studies have failed to show any abnormalities in the biosynthesis of collagen 1 in BS patientes. Evidence was obtained for a specific defect of the procollagen telopeptide lysine hydroxylation in BS, whereas mutations in the gene PLOD2 have been identified. Recently, several studies described FKBP10 mutations in OI-like and BS patients, suggesting that FKBP10 is a bonafide BS locus.MethodsWe analyzed the coding region and intron/exon boundaries of COL1A1, COL1A2, PLOD2 and FKBP10 genes by sequence analysis using an ABI PRISM 3130 automated sequencer and Big Dye Terminator Sequencing protocol. Mononuclear cells obtained from the bone marrow of BS, OI patients and healthy donors were cultured and osteogenic differentiation was induced. The gene expression of osteoblast specific markers were also evaluated during the osteoblastic differentiation of mesenchymal stem cell (MSC) by qRT-PCR using an ABI7500 Sequence Detection System.ResultsNo mutations in COL1A1, COL1A2 or PLOD2 were found in BS patient. We found a homozygous 1-base-pair duplication (c.831dupC) that is predicted to produce a translational frameshift mutation and a premature protein truncation 17 aminoacids downstream (p.Gly278ArgfsX95). The gene expression of osteoblast specific markers BGLAP, COL1A1, MSX2, SPARC and VDR was evaluated by Real Time RT-PCR during differentiation into osteoblasts and results showed similar patterns of osteoblast markers expression in BS and healthy controls. On the other hand, when compared with OI patients, the expression pattern of these genes was found to be different.ConclusionsOur work suggests that the gene expression profiles observed during mesenchymal stromal cell differentiation into osteoblast are distinct in BS patients as compared to OI patients. The present study shows for the first time that genes involved in osteogenesis are differentially expressed in BS and OI patients.

Project description:BackgroundDifferentiation of mesenchymal stem cells to osteoblasts is widely performed in research laboratories. Classical tests to prove this differentiation employ procedures such as cell fixation, cell lysis or cell scraping. Very few studies report gentle dissociation of mesenchymal stem cells undergoing an osteodifferentiation process. Here we used this technique to reveal the presence of several cell layers during osteogenesis and to study their different properties.MethodsThrough the sequential enzymatic detachment of the cells, we confirm the presence of several layers of differentiated cells and we compare them in terms of enzymatic sensitivity for dissociation, expression of cluster of differentiation, cytosolic calcium oscillations and osteogenic potential. Adipogenic and neurogenic differentiations were also performed in order to compare the cell layers.ResultsThe cells undergoing differentiation formed one layer in the neurogenic differentiation, two layers in the adipogenic differentiation and at least four layers in the osteogenic differentiation. In the latter, the upper layers, maintained by a collagen I extracellular matrix, can be dissociated using collagenase I, while the remaining lowest layer, attached to the bottom of the dish, is sensitive only to trypsin-versene. The action of collagenase I is more efficient before the mineralization of the extracellular matrix. The collagenase-sensitive and trypsin-sensitive layers differ in their cluster of differentiation expression. The dissociation of the cells on day 15 reveals that cells could resume their growth (increase in cell number) and rapidly differentiate again in osteoblasts, in 2 weeks (instead of 4 weeks). Cells from the upper layers displayed a higher mineralization.ConclusionsMSCs undergoing osteogenic differentiation form several layers with distinct osteogenic properties. This could allow the investigators to use upper layers to rapidly produce differentiated osteoblasts and the lowest layer to continue growth and differentiation until an ulterior dissociation.

Dataset Information

Promoter methylation and expression pattern of DLX3, ATF4, and FRA1 genes during osteoblastic differentiation of adipose-derived mesenchymal stem cells.

Publications

Promoter methylation and expression pattern of <i>DLX3</i>, <i>ATF4</i>, and <i>FRA1</i> genes during osteoblastic differentiation of adipose-derived mesenchymal stem cells.

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