Expression of Concern: Sox9 Potentiates BMP2-Induced Chondrogenic Differentiation and Inhibits BMP2-Induced Osteogenic Differentiation.
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ABSTRACT:
SUBMITTER: PLOS ONE Editors
PROVIDER: S-EPMC8016310 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundBone morphogenetic protein 2 (BMP2) is a promising chondrogenic growth factor for cartilage tissue-engineering, but it also induces robust endochondral ossification. Human synovial-derived mesenchymal stromal cells (hSMSCs) have attracted great interest due to their poor potential for differentiation into osteogenic lineages. Smad7 plays a significant in the endochondral ossification. In this study, we explored a new method to amplify the BMP2-induced chondrogenic differentiation of hSMSCs by downregulating Smad7 and applying a cellular scaffold.MethodshSMSCs were isolated from human knee joint synovium from 3 donors through adhesion growth. In vitro and in vivo models of the chondrogenic differentiation of hSMSCs were established. Transgenic expression of BMP2 and silencing of Smad7 and Smad7 was achieved by adenoviral vectors. The osteogenic differentiation was detected by alkaline phosphatase staining, alizarin red staining, and RT-PCR analysis of the osteogenic genes RUNX2, Osterix, and Osteocalcin. The chondrogenic differentiation was detected by Alcian blue staining and RT-PCR analysis of the chondrogenic genes SOX9, COL2, and aggrecan. Hypertrophic differentiation was detected by the markers COL10 and MMP13. A subcutaneous stem cell implantation model was established with polyethylene glycol citrate-co-N-isopropylacrylamide (PPCN) scaffolds and athymic nude mice (3/group, 4-6 week-old female) and evaluated by micro-CT, H&E staining, and Alcian blue staining. An immunohistochemistry assay was used to detected COL1 and COL2, and an immunofluorescence assay was used to detect COL10 and MMP13.ResultsThese hSMSCs identified by flow cytometry. These hSMSCs exhibited lower osteo-differentiation potential than iMads and C3H10T1/2-cells. When Smad7 was silenced in BMP2-induced hSMSCs, the chondrogenic differentiation genes SOX9, COL2, and aggrecan were enhanced in vitro. Additionally, it silencing Smad7 led to a decrease in the hypertrophic differentiation genes COL10 and MMP13. In subcutaneous stem cell implantation assays, immunofluorescence and immunohistochemical staining demonstrated that silencing Smad7 increased the number of COL2-positive cells and decreased the expression of COL1, COL10, and MMP13.ConclusionThis study suggests that the application of hSMSCs, cell scaffolds, and silencing Smad7 can potentiate BMP2-induced chondrogenic differentiation and inhibit endochondral ossification. Thus, inhibiting the expression of Smad7 in BMP2-induced hSMSC differentiation may be a new strategy for cartilage tissue-engineering.
| S-EPMC7885459 | biostudies-literature
Project description:The aim of this study was to identify new microRNAs (miRNAs) that are modulated during the differentiation of mesenchymal stem cells (MSCs) toward chondrocytes. Using large scale miRNA arrays, we compared the expression of miRNAs in MSCs (day 0) and at early time points (day 0.5 and 3) after chondrogenesis induction. Transfection of premiRNA or antagomiRNA was performed on MSCs before chondrogenesis induction and expression of miRNAs and chondrocyte markers was evaluated at different time points during differentiation by RT-qPCR. Among miRNAs that were modulated during chondrogenesis, we identified miR-574-3p as an early up-regulated miRNA. We found that miR-574-3p up-regulation is mediated via direct binding of Sox9 to its promoter region and demonstrated by reporter assay that retinoid X receptor (RXR)? is one gene specifically targeted by the miRNA. In vitro transfection of MSCs with premiR-574-3p resulted in the inhibition of chondrogenesis demonstrating its role during the commitment of MSCs towards chondrocytes. In vivo, however, both up- and down-regulation of miR-574-3p expression inhibited differentiation toward cartilage and bone in a model of heterotopic ossification. In conclusion, we demonstrated that Sox9-dependent up-regulation of miR-574-3p results in RXR? down-regulation. Manipulating miR-574-3p levels both in vitro and in vivo inhibited chondrogenesis suggesting that miR-574-3p might be required for chondrocyte lineage maintenance but also that of MSC multipotency.
| S-EPMC3633883 | biostudies-literature
Project description:BACKGROUND: MicroRNAs (miRNAs) are a family of small, non-coding single-stranded RNA molecules involved in post-transcriptional regulation of gene expression. As such, they are believed to play a role in regulating the step-wise changes in gene expression patterns that occur during cell fate specification of multipotent stem cells. Here, we have studied whether terminal differentiation of C2C12 myoblasts is indeed controlled by lineage-specific changes in miRNA expression. RESULTS: Using a previously generated RNA polymerase II (Pol-II) ChIP-on-chip dataset, we show differential Pol-II occupancy at the promoter regions of six miRNAs during C2C12 myogenic versus BMP2-induced osteogenic differentiation. Overexpression of one of these miRNAs, miR-378, enhances Alp activity, calcium deposition and mRNA expression of osteogenic marker genes in the presence of BMP2. CONCLUSIONS: Our results demonstrate a previously unknown role for miR-378 in promoting BMP2-induced osteogenic differentiation.
| S-EPMC3905160 | biostudies-literature
Project description:While human mesenchymal stem cells (hMSCs), either in the bone marrow or in tumour microenvironment could be targeted by radiotherapy, their response is poorly understood. The oxic effects on radiosensitivity, cell cycle progression are largely unknown, and the radiation effects on hMSCs differentiation capacities remained unexplored. Here we analysed hMSCs viability and cell cycle progression in 21% O2 and 3% O2 conditions after medical X-rays irradiation. Differentiation towards osteogenesis and chondrogenesis after irradiation was evaluated through an analysis of differentiation specific genes. Finally, a 3D culture model in hypoxia was used to evaluate chondrogenesis in conditions mimicking the natural hMSCs microenvironment. The hMSCs radiosensitivity was not affected by O2 tension. A decreased number of cells in S phase and an increase in G2/M were observed in both O2 tensions after 16 hours but hMSCs released from the G2/M arrest and proliferated at day 7. Osteogenesis was increased after irradiation with an enhancement of mRNA expression of specific osteogenic genes (alkaline phosphatase, osteopontin). Osteoblastic differentiation was altered since matrix deposition was impaired with a decreased expression of collagen I, probably through an increase of its degradation by MMP-3. After induction in monolayers, chondrogenesis was altered after irradiation with an increase in COL1A1 and a decrease in both SOX9 and ACAN mRNA expression. After induction in a 3D culture in hypoxia, chondrogenesis was altered after irradiation with a decrease in COL2A1, ACAN and SOX9 mRNA amounts associated with a RUNX2 increase. Together with collagens I and II proteins decrease, associated to a MMP-13 expression increase, these data show a radiation-induced impairment of chondrogenesis. Finally, a radiation-induced impairment of both osteogenesis and chondrogenesis was characterised by a matrix composition alteration, through inhibition of synthesis and/or increased degradation. Alteration of osteogenesis and chondrogenesis in hMSCs could potentially explain bone/joints defects observed after radiotherapy.
| S-EPMC4383487 | biostudies-literature
Project description:BackgroundNicotine has negative effects on tissue repair, little research concerns its effect on the cartilage repair of tissue engineering stem cells. The present study aimed to investigate the effects of nicotine on the bone marrow-derived mesenchymal stem cells' (BMSCs) chondrogenic repair function of cartilage defects and explored the molecular mechanism.MethodsA cartilage defect model of rat was repaired by BMSC transplantation, and treated with nicotine or saline at 2.0 mg/kg/d in 12 weeks. Nicotine's effect on chondrogenic differentiation was studied by exposing BMSCs to nicotine at 0.1, 1, 10, and 100 μM, and methyllycaconitine (MLA), which is a selective α7-nicotinic acetylcholine receptor (nAChR) inhibitor and si-RNA of nuclear factor of activated T cells 2 (NFATc2), were used to verify the molecular mechanism of nicotine's effect.ResultsData showed that nicotine inhibited cartilage repair function by suppressing SRY-type high-mobility group box 9 (Sox9) in regenerated tissues. Further in vitro study demonstrated that nicotine enhanced intracellular Ca2+ and activity of calcineurin (CaN) through α7-nAChR, increased the nucleic expressions of NFATc2 and the bindings to SOX9 promoter, and thus reduced the acetylation of H3K9 and H3K14 in SOX9 promoter.ConclusionsFindings from this study demonstrated that nicotine suppressed the chondrogenic differentiation of BMSCs in vivo and in vitro, which offers insight into the risk assessment of cartilage defect repair in a nicotine exposure population and its therapeutic target.
| S-EPMC5891899 | biostudies-literature
Project description:To investigate the effects of Genistein on the osteogenic related gene expression profiles during osteoblastic differentiation of human bone marrow mesenchymal stem cell (hBMSC) cultures, the hBMSCs were cultured under osteogenic differentiation medium with the addition of Genistein (10(-8)?10(-5) M) for 12 days. The cell proliferation was measured by BrdU incorporation, while the osteoblastic differentiation in hBMSC cultures was assessed by cellular alkaline phosphatase (ALP) activity. The cell apoptosis was determined by caspase 3/7 activation. GEArray Q series human osteogenesis gene array was used to analyze large-scale gene expression in Genistein-treated hBMSC cultures compared to the control group. Quantitative real-time RT-PCR, small interfering RNA (siRNA), and western blot analysis were used to confirm the microarray data in five representative transcripts. Genistein (10(-8)?10(-6) M) dose- and time-dependently increased cell proliferation and cellular ALP activity, but had no significant effect on cell apoptosis in hBMSC cultures. The 96-gene array analysis indicated that 22 genes were upregulated more than 2-fold and 7 genes were downregulated at least 1.5-fold. The expressions of bone morphogenetic proteins (BMPs), small mothers against decapentaplegic homologs (SMADs), and Runt-related transcription factor 2 (RUNX2) were concomitantly increased under Genistein treatment while insulin-like growth factor 2 and inhibitory SMADs 6 and 7 expressions were significantly decreased. The results of the real-time RT-PCR had a correlation with the results of microarray analysis and were estrogen-receptor dependent. Specific gene siRNAs knock-down further confirmed the osteogenic effects of Genistein on BMP2, SMAD5 and RUNX2 protein expression. Genistein enhanced osteogenic differentiation in cultured hBMSCs mainly through the BMP-dependent SMADs and RUNX2 signaling.
| S-EPMC3858582 | biostudies-literature
Project description:The osteogenic differentiation of mesenchymal stem cells (MSCs) is governed by multiple mechanisms. Growing evidence indicates that ubiquitin-dependent protein degradation is critical for the differentiation of MSCs and bone formation; however, the function of ubiquitin-specific proteases, the largest subfamily of deubiquitylases, remains unclear. Here we identify USP34 as a previously unknown regulator of osteogenesis. The expression of USP34 in human MSCs increases after osteogenic induction while depletion of USP34 inhibits osteogenic differentiation. Conditional knockout of Usp34 from MSCs or pre-osteoblasts leads to low bone mass in mice. Deletion of Usp34 also blunts BMP2-induced responses and impairs bone regeneration. Mechanically, we demonstrate that USP34 stabilizes both Smad1 and RUNX2 and that depletion of Smurf1 restores the osteogenic potential of Usp34-deficient MSCs in vitro. Taken together, our data indicate that USP34 is required for osteogenic differentiation and bone formation.
| S-SCDT-EMBOJ-2018-99398 | biostudies-other
Project description:Chondrogenic differentiation of mesenchymal stem cells (MSCs) is accurately regulated by essential transcription factors and signaling cascades. However, the precise mechanisms involved in this process still remain to be defined. MicroRNAs (miRNAs) regulate various biological processes by binding target mRNA to attenuate protein synthesis. To investigate the mechanisms for miRNAs-mediated regulation of chondrogenic differentiation, we identified that miR-145 was decreased during transforming growth factor beta 3 (TGF-?3)-induced chondrogenic differentiation of murine MSCs. Subsequently, dual-luciferase reporter gene assay data demonstrated that miR-145 targets a putative binding site in the 3'-UTR of SRY-related high mobility group-Box gene 9 (Sox9) gene, the key transcription factor for chondrogenesis. In addition, over-expression of miR-145 decreased expression of Sox9 only at protein levels and miR-145 inhibition significantly elevated Sox9 protein levels. Furthermore, over-expression of miR-145 decreased mRNA levels for three chondrogenic marker genes, type II collagen (Col2a1), aggrecan (Agc1), cartilage oligomeric matrix protein (COMP), type IX collagen (Col9a2) and type XI collagen (Col11a1) in C3H10T1/2 cells induced by TGF-?3, whereas anti-miR-145 inhibitor increased the expression of these chondrogenic marker genes. Thus, our studies demonstrated that miR-145 is a key negative regulator of chondrogenic differentiation by directly targeting Sox9 at early stage of chondrogenic differentiation.
| S-EPMC3140487 | biostudies-literature
Project description:BRE is a multifunctional adapter protein involved in DNA repair, cell survival and stress response. To date, most studies of this protein have been focused in the tumor model. The role of BRE in stem cell biology has never been investigated. Therefore, we have used HUCPV progenitor cells to elucidate the function of BRE. HUCPV cells are multipotent fetal progenitor cells which possess the ability to differentiate into a multitude of mesenchymal cell lineages when chemically induced and can be more easily amplified in culture. In this study, we have established that BRE expression was normally expressed in HUCPV cells but become down-regulated when the cells were induced to differentiate. In addition, silencing BRE expression, using BRE-siRNAs, in HUCPV cells could accelerate induced chondrogenic and osteogenic differentiation. Hence, we postulated that BRE played an important role in maintaining the stemness of HUCPV cells. We used microarray analysis to examine the transcriptome of BRE-silenced cells. BRE-silencing negatively regulated OCT4, FGF5 and FOXO1A. BRE-silencing also altered the expression of epigenetic genes and components of the TGF-?/BMP and FGF signaling pathways which are crucially involved in maintaining stem cell self-renewal. Comparative proteomic profiling also revealed that BRE-silencing resulted in decreased expressions of actin-binding proteins. In sum, we propose that BRE acts like an adaptor protein that promotes stemness and at the same time inhibits the differentiation of HUCPV cells.
| S-EPMC3720665 | biostudies-literature
Project description:Muscle satellite cells make up a stem cell population that is capable of differentiating into myocytes and contributing to muscle regeneration upon injury. In this work we investigate the mechanism by which these muscle progenitor cells adopt an alternative cell fate, the cartilage fate. We show that chick muscle satellite cells that normally would undergo myogenesis can be converted to express cartilage matrix proteins in vitro when cultured in chondrogenic medium containing TGFß3 or BMP2. In the meantime, the myogenic program is repressed, suggesting that muscle satellite cells have undergone chondrogenic differentiation. Furthermore, ectopic expression of the myogenic factor Pax3 prevents chondrogenesis in these cells, while chondrogenic factors Nkx3.2 and Sox9 act downstream of TGFß or BMP2 to promote this cell fate transition. We found that Nkx3.2 and Sox9 repress the activity of the Pax3 promoter and that Nkx3.2 acts as a transcriptional repressor in this process. Importantly, a reverse function mutant of Nkx3.2 blocks the ability of Sox9 to both inhibit myogenesis and induce chondrogenesis, suggesting that Nkx3.2 is required for Sox9 to promote chondrogenic differentiation in satellite cells. Finally, we found that in an in vivo mouse model of fracture healing where muscle progenitor cells were lineage-traced, Nkx3.2 and Sox9 are significantly upregulated while Pax3 is significantly downregulated in the muscle progenitor cells that give rise to chondrocytes during fracture repair. Thus our in vitro and in vivo analyses suggest that the balance of Pax3, Nkx3.2 and Sox9 may act as a molecular switch during the chondrogenic differentiation of muscle progenitor cells, which may be important for fracture healing.
| S-EPMC3388093 | biostudies-literature