MiR-26a functions oppositely in osteogenic differentiation of BMSCs and ADSCs depending on distinct activation and roles of Wnt and BMP signaling pathway.
ABSTRACT: MicroRNAs (miRNAs) emerge as important regulators of stem cell lineage commitment and bone development. MiRNA-26a (miR-26a) is one of the important miRNAs regulating osteogenic differentiation of both bone marrow-derived mesenchymal stem cells (BMSCs) and adipose tissue-derived mesenchymal stem cells (ADSCs). However, miR-26a functions oppositely in osteogenic differentiation of BMSCs and ADSCs, suggesting distinct post-transcriptional regulation of tissue-specific MSC differentiation. However, the molecular basis is largely unknown. Here, we report that the function of miR-26a is largely depended on the intrinsic signaling regulation network of MSCs. Using bioinformatics and functional assay, we confirmed that miR-26a potentially targeted on GSK3? and Smad1 to regulate Wnt and BMP signaling pathway. Overall comparative analysis revealed that Wnt signaling was enhanced more potently and played a more important role than BMP signaling in osteogenic differentiation of BMSCs, whereas BMP pathway was more essential for promoting osteogenic differentiation of ADSCs. The distinct activation pattern and role of signaling pathways determined that miR-26a majorly targeted on GSK3? to activate Wnt signaling for promoting osteogenic differentiation of BMSCs, whereas it inhibited Smad1 to suppress BMP signaling for interfering with the osteogenic differentiation of ADSCs. Taken together, our study demonstrated that BMSCs and ADSCs applied different signaling pathway to facilitate their osteogenic differentiation, which determined the inverse function of miR-26a. The distinct transcriptional regulation and post-transcriptional regulation network suggested the intrinsic molecular differences between tissue-specific MSCs and the complexity in MSC research and MSC-based cell therapy.
Project description:BACKGROUND:Many studies have shown that long noncoding RNAs (lncRNAs) are closely related to the stimulation of osteogenic differentiation of adipose-derived stem cells (ADSCs) and the prevention of osteoporosis. Current research aimed to investigate the novel lncRNA and explored the function and molecular mechanism of the LINC00314/miR-129-5p/GRM5 axis in regulating osteogenic differentiation of ADSCs. METHODS:LncRNA and miRNA sequencing was performed in normal and osteogenic differentiation-induced ADSCs (osteogenic group). Abnormally expressed lncRNAs and miRNAs were obtained by the R software and the relative expression of LINC00314, miR-129-5p, and GRM5 during osteogenic induction was measured by RT-PCR. ADSCs were then transfected with pcDNA3.1-sh-LINC00314 and agomiR-129-5p. Alizarin red staining (ARS) and alkaline phosphatase (ALP) staining were performed to identify the mechanism of the LINC00314/miR-129-5p/GRM5 axis in regulating osteogenic differentiation of ADSCs. RESULTS:LINC00314 was significantly upregulated in the group of osteogenic-induced ADSCs. LINC00314 and GRM5 mimics increased the early and late markers of osteogenic differentiation, which manifest in not only the markedly increased ALP activity but also higher calcium deposition, while miR-129-5p mimic had the opposite effects. LINC00314 directly targeted miR-129-5p through luciferase reporter assay, and miR-129-5p suppressed GRM5 expression. Moreover, the LINC00314/miR-129-5p/GRM5 regulatory axis activated the Wnt/?-catenin signaling pathway. CONCLUSIONS:LINC00314 confers contributory function in the osteogenic differentiation of ADSCs and thus the LINC00314/miR-129-5p/GRM5 axis may be a novel mechanism for osteogenic-related disease.
Project description:OBJECTIVES:It is of profound significance for clinical bone regeneration to clarify the specific molecular mechanism from which we found that osteogenic differentiation of adipose-derived stem cells (ADSCs) will be probably promoted by exosomes. MATERIALS AND METHODS:By means of lentiviral transfection, miR-130a-3p overexpression and knockdown ADSCs were constructed. Alizarin Red S was used to detect the calcium deposits, and qPCR was used to detect osteogenesis-related genes, to verify the effect of miR-130a-3p on the osteogenic differentiation of ADSCs. CCK-8 was used to detect the effect of miR-130a-3p on the proliferation of ADSCs. The target binding between miR-130a-3p and SIRT7 was verified by dual-luciferase reporter gene assay. Furthermore, the role of Wnt signalling pathway in the regulation of ADSCs osteogenesis and differentiation by miR-130a-3p was further verified by detecting osteogenic-related genes and proteins and alkaline phosphatase activity. RESULTS:(a) Overexpression of miR-130a-3p can enhance the osteogenic differentiation of ADSCs while reducing protein and mRNA levels of SIRT7, a target of miR-130a-3p. (b) Our study further found that overexpression of miR-130a-3p leads to down-regulation of SIRT7 expression with up-regulation of Wnt signalling pathway-associated protein. (c) Overexpression of miR-130a-3p inhibited proliferation of ADSCs, while knockdown promoted it. CONCLUSIONS:The obtained findings indicate that exosomal miR-130a-3p can promote osteogenic differentiation of ADSCs partly by mediating SIRT7/Wnt/?-catenin axis, which will hence promote the application of exosomal microRNA in the field of bone regeneration.
Project description:Disrupted Wnt signaling in osteoblastic-lineage cells leads to bone formation defect in osteoporosis. However, the factors repressing Wnt signaling are unclear. In our study, we found that Wnt signaling was suppressed persistently in bone marrow-derived mesenchymal stem cells (BMSCs) during osteoporosis. Accordingly, histone acetylation levels on Wnt genes (Wnt1, Wnt6, Wnt10a, and Wnt10b) were declined in BMSCs from OVX mice. By screening the family of histone acetyltransferase, we identified that GCN5 expression increased during osteogenic differentiation of BMSCs, whereas decreased after osteoporosis. Further analysis revealed that GCN5 promoted osteogenic differentiation of BMSCs by increasing acetylation on histone 3 lysine 9 loci on the promoters of Wnt genes. Reduced GCN5 expression suppressed Wnt signaling, resulting in osteogenic defect of BMSCs from OVX mice. Moreover, restoring GCN5 levels recovered BMSC osteogenic differentiation, and attenuated bone loss in OVX mice. Taken together, our study demonstrated that disrupted histone acetylation modification in BMSCs lead to bone formation defect during osteoporosis. The findings also introduced a novel therapeutic target for osteoporosis.
Project description:Transplantation of mesenchymal stem cells (MSCs) is a promising therapy for ischemic injury; however, inadequate survival of implanted cells in host tissue is a substantial impediment in the progress of cellular therapy. Secreted Frizzled-related protein 2 (sFRP2) has recently been highlighted as a key mediator of MSC-driven myocardial and wound repair. Notably, sFRP2 mediates significant enhancement of MSC engraftment in vivo. We hypothesized that sFRP2 improves MSC engraftment by modulating self-renewal through increasing stem cell survival and by inhibiting differentiation. In previous studies we demonstrated that sFRP2-expressing MSCs exhibited an increased proliferation rate. In the current study, we show that sFRP2 also decreased MSC apoptosis and inhibited both osteogenic and chondrogenic lineage commitment. sFRP2 activity occurred through the inhibition of both Wnt and bone morphogenic protein (BMP) signaling pathways. sFRP2-mediated inhibition of BMP signaling, as assessed by levels of pSMAD 1/5/8, was independent of its effects on the Wnt pathway. We further hypothesized that sFRP2 inhibition of MSC lineage commitment may reduce heterotopic osteogenic differentiation within the injured myocardium, a reported adverse side effect. Indeed, we found that sFRP2-MSC-treated hearts and wound tissue had less ectopic calcification. This work provides important new insight into the mechanisms by which sFRP2 increases MSC self-renewal leading to superior tissue engraftment and enhanced wound healing.
Project description:Elucidating the molecular mechanisms responsible for osteogenesis of human adipose-derived mesenchymal stem cells (hADSCs) will provide deeper insights into the regulatory mechanisms of this process and help develop more efficient methods for cell-based therapies. In this study, we analysed the role of miR-26a in the regulation of hADSC osteogenesis. The endogenous expression of miR-26a increased during the osteogenic differentiation. The overexpression of miR-26a promoted hADSC osteogenesis, whereas osteogenesis was repressed by miR-26a knockdown. Additionally, miR-26a directly targeted the 3'UTR of the GSK3?, suppressing the expression of GSK3? protein. Similar to the effect of overexpressing miR-26a, the knockdown of GSK3? promoted osteogenic differentiation, whereas GSK3? overexpression inhibited this process, suggesting that GSK3? acted as a negative regulator of hADSC osteogenesis. Furthermore, GSK3? influences Wnt signalling pathway by regulating ?-catenin, and subsequently altered the expression of its downstream target C/EBP?. In turn, C/EBP? transcriptionally regulated the expression of miR-26a by physically binding to the CTDSPL promoter region. Taken together, our data identified a novel feedback regulatory circuitry composed of miR-26a, GSK3? and C/EBP?, the function of which might contribute to the regulation of hADSC osteogenesis. Our findings provided new insights into the function of miR-26a and the mechanisms underlying osteogenesis of hADSCs.
Project description:BACKGROUND: Transforming growth factor-? (TGF-?) is a key cytokine during differentiation of mesenchymal stem cells (MSC) into vascular smooth muscle cells (VSMC). High phosphate induces a phenotypic transformation of vascular smooth muscle cells (VSMC) into osteogenic-like cells. This study was aimed to evaluate signaling pathways involved during VSMC differentiation of MSC in presence or not of high phosphate. RESULTS: Our results showed that TGF-? induced nuclear translocation of Smad3 as well as the expression of vascular smooth muscle markers, such as smooth muscle alpha actin, SM22?, myocardin, and smooth muscle-myosin heavy chain. The addition of high phosphate to MSC promoted nuclear translocation of Smad1/5/8 and the activation of canonical Wnt/?-catenin in addition to an increase in BMP-2 expression, calcium deposition and alkaline phosphatase activity. The administration of TGF-? to MSC treated with high phosphate abolished all these effects by inhibiting canonical Wnt, BMP and TGF-? pathways. A similar outcome was observed in high phosphate-treated cells after the inhibition of canonical Wnt signaling with Dkk-1. Conversely, addition of both Wnt/?-catenin activators CHIR98014 and lithium chloride enhanced the effect of high phosphate on BMP-2, calcium deposition and alkaline phosphatase activity. CONCLUSIONS: Full VSMC differentiation induced by TGF-? may not be achieved when extracellular phosphate levels are high. Moreover, TGF-? prevents high phosphate-induced osteogenesis by decreasing the nuclear translocation of Smad 1/5/8 and avoiding the activation of Wnt/?-catenin pathway.
Project description:The success rate of oral implants is lower in type 2 diabetes mellitus (T2DM) patients than in nondiabetic subjects; functional impairment of bone marrow-derived mesenchymal stem cells (BMSCs) is an important underlying cause. Many factors in the blood can act on BMSCs to regulate their biological functions and influence implant osseointegration, but which factors play important negative roles in T2DM patients is still unclear. This study is aimed at screening differentially expressed genes in the blood from T2DM and nondiabetic patients, identifying which genes impact the osteogenic differentiation potential of alveolar BMSCs in T2DM patients, exploring drug intervention regimens, and providing a basis for improving implant osseointegration. Thus, a whole-blood gene expression microarray dataset (GSE26168) of T2DM patients and nondiabetic controls was analyzed. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) results, differentially expressed genes and signaling pathways related to BMSC osteogenic differentiation were screened, and major risk genes were extracted based on the mean decrease Gini coefficient calculated using the random forest method. Bone morphogenetic protein-4 (BMP-4), with significantly low expression in T2DM blood, was identified as the most significant factor affecting BMSC osteogenic differentiation potential. Subsequently, metformin, a first-line clinical drug for T2DM treatment, was found to improve the osteogenic differentiation potential of BMSCs from T2DM patients via the BMP-4/Smad/Runx2 signaling pathway. These results demonstrate that low BMP-4 expression in the blood of T2DM patients significantly hinders the osteogenic function of BMSCs and that metformin is effective in counteracting the negative impact of BMP-4 deficiency.
Project description:Extracorporeal shockwave (ESW) has been shown of great potential in promoting the osteogenesis of bone marrow mesenchymal stem cells (BMSCs), but it is unknown whether this osteogenic promotion effect can also be achieved in other MSCs (i.e., tendon-derived stem cells (TDSCs) and adipose-derived stem cells (ADSCs)). In the current study, we aimed not only to compare the osteogenic effects of BMSCs induced by ESW to those of TDSCs and ADSCs; but also to investigate the underlying mechanisms. We show here that ESW (0.16?mj/mm(2)) significantly promoted the osteogenic differentiation in all the tested types of MSCs, accompanied with the downregulation of miR-138, but the activation of FAK, ERK1/2, and RUNX2. The enhancement of osteogenesis in these MSCs was consistently abolished when the cells were pretreated with one of the following conditions: overexpression of miR-138, FAK knockdown using specific siRNA, and U0126, implying that all of these elements are indispensable for mediating the effect of ESW. Moreover, our study provides converging genetic and molecular evidence that the miR-138-FAK-ERK1/2-RUNX2 machinery can be generally activated in ESW-preconditioned MSCs, suggesting that ESW may be a promising therapeutic strategy for the enhancement of osteogenesis of MSCs, regardless of their origins.
Project description:Oleanolic acid (OA), a pentacyclic triterpenoid, has been shown to modulate multiple signaling pathways in a variety of cell linages. But the mechanisms underlying OA-mediated mesenchymal stromal cell (MSC) osteogenic differentiation are not known. In this study, we examined effects of OA on cell viability, osteogenic differentiation in MSCs, and the involvement of Notch and BMP signaling. OA induced bone marrow derived MSC differentiation towards osteoprogenitor cells and inhibited Notch signaling in a dose dependent manner. Constitutive activation of Notch signaling fully blocked OA induced MSC osteogenic differentiation. The expression level of early osteogenic marker genes, ALP, Runx2, and type I collagen, which play a critical role in MSC to osteoblast transition and servers as a downstream target of BMP signaling, was significantly induced by OA. Furthermore, BMP2 mediated MSC osteogenic differentiation was significantly enhance by OA treatment, indicating a synergistic effect between BMP2 and OA. Our results suggest that OA is a promising bioactive agent for bone tissue regeneration, and inhibition of Notch signaling is required for its osteogenic effects on MSCs.