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The transcription factor DLX3 regulates the osteogenic differentiation of human dental follicle precursor cells.

ABSTRACT: The transcription factor DLX3 plays a decisive role in bone development of vertebrates. In neural-crest derived stem cells from the dental follicle (DFCs), DLX3 is differentially expressed during osteogenic differentiation, while other osteogenic transcription factors such as DLX5 or RUNX2 are not highly induced. DLX3 has therefore a decisive role in the differentiation of DFCs, but its actual biological effects and regulation are unknown. This study investigated the DLX3-regulated processes in DFCs. After DLX3 overexpression, DFCs acquired a spindle-like cell shape with reorganized actin filaments. Here, marker genes for cell morphology, proliferation, apoptosis, and osteogenic differentiation were significantly regulated as shown in a microarray analysis. Further experiments showed that DFCs viability is directly influenced by the expression of DLX3, for example, the amount of apoptotic cells was increased after DLX3 silencing. This transcription factor stimulates the osteogenic differentiation of DFCs and regulates the BMP/SMAD1-pathway. Interestingly, BMP2 did highly induce DLX3 and reverse the inhibitory effect of DLX3 silencing in osteogenic differentiation. However, after DLX3 overexpression in DFCs, a BMP2 supplementation did not improve the expression of DLX3 and the osteogenic differentiation. In conclusion, DLX3 influences cell viability and regulates osteogenic differentiation of DFCs via a BMP2-dependent pathway and a feedback control.

SUBMITTER: Viale-Bouroncle S

PROVIDER: S-EPMC3396153 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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The transcription factor DLX3 regulates the osteogenic differentiation of human dental follicle precursor cells.

Viale-Bouroncle Sandra S Felthaus Oliver O Schmalz Gottfried G Brockhoff Gero G Reichert Torsten E TE Morsczeck Christian C

Stem cells and development 20120207 11

The transcription factor DLX3 plays a decisive role in bone development of vertebrates. In neural-crest derived stem cells from the dental follicle (DFCs), DLX3 is differentially expressed during osteogenic differentiation, while other osteogenic transcription factors such as DLX5 or RUNX2 are not highly induced. DLX3 has therefore a decisive role in the differentiation of DFCs, but its actual biological effects and regulation are unknown. This study investigated the DLX3-regulated processes in ...[more]

PMID: 22107079

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Project description:Several studies have indicated that dietary silicon (Si) is beneficial for bone homeostasis and skeletal health. Furthermore, Si-containing bioactive glass biomaterials have positive effects on bone regeneration when used for repair of bone defects. Si has been demonstrated to stimulate osteoblast differentiation and bone mineralisation in vitro. However, the mechanisms underlying these effects of Si are not well understood. The aim of the present study was to investigate the effects of soluble Si on osteogenic differentiation and connexin 43 (CX43) gap junction communication in cultured pluripotent cells from human dental follicles (hDFC). Neutral Red uptake assay demonstrated that 25 μg/ml of Si significantly stimulated hDFC cell proliferation. Dosages of Si above 100 μg/ml decreased cell proliferation. Alizarin Red staining showed that osteogenic induction medium (OIM) by itself and in combination with Si (25 μg/ml) significantly increased mineralisation in hDFC cultures, although Si alone had no such effect. The expression of osteoblast-related markers in hDFC was analysed with RT-qPCR. OSX, RUNX2, BMP2, ALP, OCN, BSP and CX43 genes were expressed in hDFC cultured for 1, 7, 14 and 21 days. Expression levels of BMP-2 and BSP were significantly upregulated by OIM and Si (25 μg/ml) and were also induced by Si alone. Notably, the expression levels of OCN and CX43 on Day 21 were significantly increased only in the Si group. Flow cytometric measurements revealed that Si (50 μg/ml) significantly increased CX43 protein expression and gap junction communication in hDFC. Next-generation sequencing (NGS) and bioinformatics processing were used for the identification of differentially regulated genes and pathways. The influence of OIM over the cell differentiation profile was more prominent than the influence of Si alone. However, Si in combination with OIM increased the magnitude of expression (up or down) of the differentially regulated genes. The gene for cartilage oligomeric matrix protein (COMP) was the most significantly upregulated. Genes for the regulator of G protein signalling 4 (RGS4), regulator of G protein signalling 2 (RGS2), and matrix metalloproteinases (MMPs) 1, 8, and 10 were also strongly upregulated. Our findings reveal that soluble Si stimulates Cx43 gap junction communication in hDFC and induces gene expression patterns associated with osteogenic differentiation. Taken together, the results support the conclusion that Si is beneficial for bone health.

Project description:Dental follicle cells (DFCs) are stem/progenitor cells of the periodontium and give rise to alveolar osteoblasts. However, understanding of the molecular mechanisms of osteogenic differentiation, which is required for cell-based therapies, is delimited. This study is aimed at analyzing the energy metabolism during the osteogenic differentiation of DFCs. Human DFCs were cultured, and osteogenic differentiation was induced by either dexamethasone or bone morphogenetic protein 2 (BMP2). Previous microarray data were reanalyzed to examine pathways that are regulated after osteogenic induction. Expression and activity of metabolic markers were evaluated by western blot analysis and specific assays, relative amount of mitochondrial DNA was measured by real-time quantitative polymerase chain reaction, the oxidative state of cells was determined by a glutathione assay, and the lipidome of cells was analyzed via mass spectrometry (MS). Moreover, osteogenic markers were analyzed after the inhibition of fatty acid synthesis by 5-(tetradecyloxy)-2-furoic acid or C75. Pathway enrichment analysis of microarray data revealed that carbon metabolism was amongst the top regulated pathways after osteogenic induction in DFCs. Further analysis showed that enzymes involved in glycolysis, citric acid cycle, mitochondrial activity, and lipid metabolism are differentially expressed during differentiation, with most markers upregulated and more markedly after induction with dexamethasone compared to BMP2. Moreover, the cellular state was more oxidized, and mitochondrial DNA was distinctly upregulated during the second half of differentiation. Besides, MS of the lipidome revealed higher lipid concentrations after osteogenic induction, with a preference for species with lower numbers of C-atoms and double bonds, which indicates a de novo synthesis of lipids. Concordantly, inhibition of fatty acid synthesis impeded the osteogenic differentiation of DFCs. This study demonstrates that energy metabolism is highly regulated during osteogenic differentiation of DFCs including changes in the lipidome suggesting enhanced de novo synthesis of lipids, which are required for the differentiation process.

Project description:BackgroundHuman dental pulp stromal cells (hDPSCs) are promising sources of mesenchymal stem cells (MSCs) for bone tissue regeneration. Circular RNAs (circRNAs) have been demonstrated to play critical roles in stem cell osteogenic differentiation. Herein, we aimed to investigate the role of circAKT3 during osteogenesis of hDPSCs and the underlying mechanisms of its function.MethodsWe performed circRNA sequencing to investigate the expression profiles of circular RNAs during osteogenesis of hDPSCs. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect the expression pattern of circAKT3 and miR-206 in hDPSCs during osteogenesis. We knocked down circAKT3 and interfered the expression of miR-206 to verify their regulatory role in hDPSC osteogenesis. We detected hDPSCs mineralization by alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining and used dual-luciferase reporter assay to validate the direct binding between circAKT3 and miR-206. To investigate in vivo mineralization, we performed subcutaneous transplantation in nude mice and used hematoxylin and eosin, Masson's trichrome, and immunohistochemistry staining.ResultsTotally, 86 circRNAs were differentially expressed during hDPSC osteogenesis, in which 29 were downregulated while 57 were upregulated. circAKT3 was upregulated while miR-206 was downregulated during hDPSC osteogenesis. Knockdown of circAKT3 inhibited ALP/ARS staining and expression levels of osteogenic genes. circAKT3 directly interacted with miR-206, and the latter one suppressed osteogenesis of hDPSCs. Silencing miR-206 partially reversed the inhibitory effect of circAKT3 knockdown on osteogenesis. Connexin 43 (CX43), which positively regulates osteogenesis of stem cells, was predicted as a target of miR-206, and overexpression or knockdown of miR-206 could correspondingly decrease and increase the expression of CX43. In vivo study showed knockdown of circAKT3 suppressed the formation of mineralized nodules and expression of osteogenic proteins.ConclusionDuring osteogenesis of hDPSCs, circAKT3 could function as a positive regulator by directly sponging miR-206 and arresting the inhibitive effect of miR-206 on CX43 expression.

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The transcription factor DLX3 regulates the osteogenic differentiation of human dental follicle precursor cells.

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The transcription factor DLX3 regulates the osteogenic differentiation of human dental follicle precursor cells.

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