Identification of Gli1-interacting proteins during simvastatin-stimulated osteogenic differentiation of bone marrow mesenchymal stem cells

Simvastatin has been shown to promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Our study aimed to illuminate the underlying mechanism, with a specific focus on the role of Hedgehog signaling in this process. BMSCs cultured with or without 10⁻⁷ mol/L simvastatin were subjected to evaluation of osteogenic differentiation capacity. Osteogenic markers such as type 1 collagen (COL1) and osteocalcin (OCN), as well as key molecules of Hedgehog signaling molecules, were examined by Western blot and real-time polymerase chain reaction (PCR). Co-immunoprecipitation and mass spectrometry assays were applied to screen for Gli1-interacting proteins. Cyclopamine (Cpn) was used as a Hedgehog signaling inhibitor. Our results indicated that simvastatin increased alkaline phosphatase (ALP) activity; mineralization of extracellular matrix; mRNA expression of ALP, COL1, and OCN; and expression and nuclear translocation of Gli1. Contrasting effects were observed in Cpn-exposed groups, but were partially rescued by the simvastatin treatment. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that Gli1-interacting proteins were primarily associated with mitogen-activated protein kinase (MAPK) (P = 7.04E⁻⁰⁴), hippo, insulin, and glucagon signaling. Further, hub genes identified by protein-protein interaction network analysis included Gli1-interacting proteins such as Ppp2r1a, Rac1, Etf1, and XPO1/CRM1. In summary, the current study showed that the mechanism by which simvastatin stimulates osteogenic differentiation of BMSCs involves activation of Hedgehog signaling, as indicated by interactions with Gli1 and, most notably, the MAPK signaling pathway.     

Bioinformatics analysis of the biological changes involved in the osteogenic differentiation of human mesenchymal stem cells

The mechanisms underlying the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) remain unclear. In the present study, we aimed to identify the key biological processes during osteogenic differentiation. To this end, we downloaded three microarray data sets from the Gene Expression Omnibus (GEO) database: GSE12266, GSE18043 and GSE37558. Differentially expressed genes (DEGs) were screened using the limma package, and enrichment analysis was performed. Protein‐protein interaction network (PPI) analysis and visualization analysis were performed with STRING and Cytoscape. A total of 240 DEGs were identified, including 147 up‐regulated genes and 93 down‐regulated genes. Functional enrichment and pathways of the present DEGs include extracellular matrix organization, ossification, cell division, spindle and microtubule. Functional enrichment analysis of 10 hub genes showed that these genes are mainly enriched in microtubule‐related biological changes, that is sister chromatid segregation, microtubule cytoskeleton organization involved in mitosis, and spindle microtubule. Moreover, immunofluorescence and Western blotting revealed dramatic quantitative and morphological changes in the microtubules during the osteogenic differentiation of human adipose‐derived stem cells. In summary, the present results provide novel insights into the microtubule‐ and cytoskeleton‐related biological process changes, identifying candidates for the further study of osteogenic differentiation of the mesenchymal stem cells.     

Induction of osteogenic differentiation of human mesenchymal stem cells by histone deacetylase inhibitors

Valproic acid (VPA) has been used as an anticonvulsant agent for the treatment of epilepsy, as well as a mood stabilizer for the treatment of bipolar disorder, for several decades. The mechanism of action for these effects remains to be elucidated and is most likely multifactorial. Recently, VPA has been reported to inhibit histone deacetylase (HDAC) and HDAC has been reported to play roles in differentiation of mammalian cells. In this study, the effects of HDAC inhibitors on differentiation and proliferation of human adipose tissue-derived stromal cells (hADSC) and bone marrow stromal cells (hBMSC) were determined. VPA increased osteogenic differentiation in a dose dependent manner. The pretreatment of VPA before induction of differentiation also showed stimulatory effects on osteogenic differentiation of hMSC. Trichostatin A (TSA), another HDAC inhibitor, also increased osteogenic differentiation, whereas valpromide (VPM), a structural analog of VPA which does not possess HDAC inhibitory effects, did not show any effect on osteogenic differentiation on hADSC. RT-PCR and Real-time PCR analysis revealed that VPA treatment increased osterix, osteopontin, BMP-2, and Runx2 expression. The addition of noggin inhibited VPA-induced potentiation of osteogenic differentiation. VPA inhibited proliferation of hADSC and hBMSC. Our results suggest that VPA enhance osteogenic differentiation, probably due to inhibition of HDAC, and could be useful for in vivo bone engineering using hMSC.     

Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation

Human MSCs have been studied to define the mechanisms involved in normal bone remodeling and the regulation of osteogenesis. During osteogenic differentiation, MSCs change from their characteristic fibroblast-like phenotype to near spherical shape. In this study, we analyzed the correlation between the organization of cytoskeleton of MSCs, changes in cell morphology, and the expression of specific markers (alkaline phosphatase activity and calcium deposition) of osteogenic differentiation. For osteoblastic differentiation, cells were cultured in a culture medium supplemented with 100 nM dexamethasone, 10 mM beta- glycerophosphate, and 50 microg/ml ascorbic acid. The organization of microfilaments and microtubules was examined by inmunofluorescence using Alexa fluor 594 phalloidin and anti alpha-tubulin monoclonal antibody. Cytochalasin D and nocodazole were used to alter reversibly the cytoskeleton dynamic. A remarkable change in cytoskeleton organization was observed in human MSCs during osteogenic differentiation. Actin cytoskeleton changed from a large number of thin, parallel microfilament bundles extending across the entire cytoplasm in undifferentiated MSCs to a few thick actin filament bundles located at the outermost periphery in differentiated cells. Under osteogenic culture conditions, a reversible reorganization of microfilaments induced by an initial treatment with cytochalasin D but not with nocodazole reduced the expression of differentiation markers, without affecting the final morphology of the cells. The results indicate that changes in the assembly and disassembly kinetics of microfilaments dynamic of actin network formation may be critical in supporting the osteogenic differentiation of human MSCs; also indicated that the organization of microtubules appears to have a regulatory role on the kinetic of this process.     

MiRNA-1271-5p regulates osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting forkhead box O1 (FOXO1)

Forkhead box O1 (FOXO1) is a key regulator of osteogenesis. The aim of this study was to identify the mechanisms of microRNAs (miRNAs) targeting FOXO1 in osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). Three miRNA target prediction programs were used to search for potential miRNAs that target FOXO1. Quantitative real-time polymerase chain reaction was conducted to detect the expression of miR-1271-5p and FOXO1 during osteogenic differentiation. Target gene prediction and screening, luciferase reporter assay was used to verify the downstream target gene of miR-1271-5p. The expression levels of FOXO1 and Runx2 were detected by RT-qPCR and Western blot analysis. Alkaline phosphatase (ALP) activity and matrix mineralization were detected by biochemical methods. The expression levels of Runx2, ALP, and osteocalcin were detected by RT-qPCR. Our results showed that miR-1271-5p was downregulated during osteogenic induction. And the expression levels of miR-1271-5p were higher in osteoporotic tissues than that in adjacent nonosteoporotic tissues. The expression levels of FOXO1 were lower in osteoporotic tissues than that in adjacent nonosteoporotic tissues. And a negative correlation was found between miR-1271-5p and FOXO1 in osteoporotic tissues. Overexpression of miR-1271-5p downregulated FOXO1 and inhibited osteogenic differentiation in hMSCs. Overexpression of miR-1271-5p downregulated the expression of osteogenic markers and reduced ALP activity. In addition, ectopic expression of FOXO1 reversed the effect of miR-1271-5p on osteogenic differentiation. In conclusion, miR-1271-5p functioned as a therapeutic target of osteogenic differentiation in hMSCs by inhibiting FOXO1, which provides valuable insights into the use of miR-1271-5p as a target in the treatment of osteoporosis and other bone metabolic diseases.     

NT-3 promotes osteogenic differentiation of mouse bone marrow mesenchymal stem cells by regulating the Akt pathway

Objectives:To investigate the effect of neurotrophin-3 (NT-3) on osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).Methods:Osteogenic differentiation was detected by alkaline phosphatase (ALP) staining and alizarin red staining (ARS). Adipogenic differentiation was detected by oil red O (ORO) staining. The expression of bone-related genes (Runx2, Osterix, OCN, ALP) and lipogenic genes (FABP4, PPAR, CEBP, LPL) was detected by real-time quantitative polymerase chain reaction (real-time qPCR). The expression of p-Akt and Akt protein was detected by Western blot assay.Results:ALP staining and ARS staining showed that the overexpression of NT-3 could promote the differentiation into osteoblasts, while knockdown of NT-3 could inhibit that. Real-time qPCR showed that the overexpression of NT-3 could increase the expression of osteoblast genes, while knockdown of NT-3 could inhibit that. ORO staining showed that the overexpression of NT-3 could inhibit the differentiation into adipogenesis, while knockdown of NT-3 can promote that. Real-time qPCR showed that the overexpression of NT-3 could reduce the expression of lipogenic genes. while knockdown NT-3 could increase that. In addition, the overexpression of NT-3 increased p-Akt/Akt levels significantly, while knockdown NT-3 reduced that significantly.Conclusion:NT-3 could promote the differentiation of mouse BMSCs into osteoblasts and inhibit their differentiation into adipogenesis.     

Hypermethylation of microRNA-149 activates SDF-1/CXCR4 to promote osteogenic differentiation of mesenchymal stem cells

MicroRNAs (miRs) involve in osteogenic differentiation and osteogenic potential of mesenchymal stem cells (MSCs). Accordingly, the present study aimed to further uncover role miR-149 plays in osteogenic differentiation of MSCs with the involvement of the stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor-4 (CXCR4) pathway. Initially, the osteogenic differentiation model was induced. Next, the positive expression of STRO-1 in periosteum, alkaline phosphatase (ALP) activity, osteocalcin (OCN) protein content, and the calcium deposition in MSCs were determined. MSCs were treated with DNA methyltransferase inhibitor 5-aza-CdR, SDF-1 neutralizing antibody, or CXCR4 antagonist AMD3100 to investigate their roles in osteogenic differentiation; with the expression of CD44, CD90, CD14, and CD45 detected. Furthermore, the levels of SDF-1 and CXCR4, and the genes related to stemness (Nanog, Oct-4, and Sox-2) were measured to explore the effects of miR-149. The obtained data revealed the upregulation of STRO-1 in the periosteum. miR-149 could specifically bind to SDF-1. Besides, increased miR-149 methylation, higher ALP activity and OCN content, decreased positive rates of CD44 and CD90, and increased positive rates of CD14 and CD45 were found in osteogenic differentiation of MSCs. Subsequently, 5-Aza-CdR treatment reversed the above-mentioned effects. MSCs were finally treated with SDF-1 neutralizing antibody or AMD3100 to decrease Nanog, Oct-4, and Sox-2 expression. Taken together these results, miR-149 hypermethylation has the potential to activate the SDF-1/CXCR4 pathway and further promote osteogenic differentiation of MSCs.     

Secreted microvesicular miR‐31 inhibits osteogenic differentiation of mesenchymal stem cells

Damage to cells and tissues is one of the driving forces of aging and age‐related diseases. Various repair systems are in place to counteract this functional decline. In particular, the property of adult stem cells to self‐renew and differentiate is essential for tissue homeostasis and regeneration. However, their functionality declines with age (Rando, 2006). One organ that is notably affected by the reduced differentiation capacity of stem cells with age is the skeleton. Here, we found that circulating microvesicles impact on the osteogenic differentiation capacity of mesenchymal stem cells in a donor‐age‐dependent way. While searching for factors mediating the inhibitory effect of elderly derived microvesicles on osteogenesis, we identified miR‐31 as a crucial component. We demonstrated that miR‐31 is present at elevated levels in the plasma of elderly and of osteoporosis patients. As a potential source of its secretion, we identified senescent endothelial cells, which are known to increase during aging in vivo (Erusalimsky, 2009). Endothelial miR‐31 is secreted within senescent cell‐derived microvesicles and taken up by mesenchymal stem cells where it inhibits osteogenic differentiation by knocking down its target Frizzled‐3. Therefore, we suggest that microvesicular miR‐31 in the plasma of elderly might play a role in the pathogenesis of age‐related impaired bone formation and that miR‐31 might be a valuable plasma‐based biomarker for aging and for a systemic environment that does not favor cell‐based therapies whenever osteogenesis is a limiting factor.     

GNAS knockdown suppresses osteogenic differentiation of mesenchymal stem cells via activation of Hippo signaling pathway

Bone marrow-derived mesenchymal stem cells (BMSCs) are a suitable option for cell-based tissue engineering therapies due to their ability to renew and differentiate into multiple different tissue types, such as bone. Over the last decade, the effect of GNAS on the regulation of osteoblast differentiation has attracted great attention. Herein, this study aimed to explore the role of GNAS in osteogenic differentiation of MSCs. A total of 85 GNAS^f/f male mice were selected for animal experiments and 10 GNAS^f/f male mice for BMSC isolation to conduct cell experiments. The mice and BMSCs were treated with Verteporfin (a Hippo signaling pathway inhibitor) to inhibit the Hippo signaling pathway or recombinant adenovirus-expressing Cre to knockout the GNAS expression. Next, computed tomography scan, Von Kossa staining, and alizarin red staining were performed to detect osteogenic differentiation ability. Moreover, immunohistochemistry and alkaline phosphatase (ALP) staining were used to assess the expression of Oc and Osx in femur tissues and ALP activity. At last, the expression of GNAS, osteogenic markers, and factors related to the Hippo signaling pathway was evaluated. Initially, the results displayed successful knockout of the GNAS gene from mice and BMSCs. Moreover, the data indicated that GNAS knockout inhibits expression of Oc, Osx, ALP, BMP-2, and Runx2, and ALP activity. Additionally, GNAS knockout promotes activation of the Hippo signaling pathway, so as to repress osteogenic differentiation. Collectively, depleted GNAS exerts an inhibitory role in osteogenic differentiation of MSCs by activating Hippo signaling pathway, providing a candidate mediator for osteoporosis.     

Sonic hedgehog enhances the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells

MSCs (mesenchymal stem cells) may be promising seed cells for tissue regeneration because of their self-renewal and multi-differentiation potential. Shh (sonic hedgehog) is involved in the skeletal formation during embryo development and skeletal regeneration. However, how Shh regulates the biological characteristics of BM-MSCs (bone marrow-derived MSCs) is poorly understood. We have investigated the effect of rShh-N (recombinant N-terminal Shh) on the proliferation and osteogenic differentiation of rBM-MSCs (rat BM-MSCs) in vitro. rBM-MSCs were treated with rShh-N at concentrations up to 200 ng/ml. Proliferation and colony-forming ability of rBM-MSCs were increased in a dose-dependent manner. rShh-N increased the ratio of cells in S and G2/M phase, as well as the number of Ki-67+ cells. In addition, ALP (alkaline phosphatase) activity and matrix mineralization were enhanced by 200 ng/ml rShh-N. Real-time PCR showed that rShh-N (200 ng/ml) up-regulated the expression of genes encoding Cbfa-1 (core-binding factor α1), osteocalcin, ALP and collagen type I in rBM-MSCs. This information reveals some potential of rShh-N in the therapeutics of bone-related diseases.