Mesenchymal stem cells (MSCs) are able to differentiate into adipocytes, chondroblasts or cartilage under different stimulation conditions. Identifying a mechanism that triggers the differentiation of MSCs into cartilage may help the development of novel therapeutic approaches for heterotopic ossification, the pathological formation of lamellar bone in soft tissue outside the skeleton that may lead to debilitating immobility. Bone morphogenetic proteins (BMPs), including BMP-7, are the most potent growth factors for enhancing bone formation. The current study aimed to understand the potential involvement of the Wnt/β-catenin signaling pathway in the BMP-7-induced growth of rabbit MSCs (rMSCs). Different concentrations of BMP-7 were applied to cultured rMSCs, and proliferation was evaluated by MTT assay. Changes in the phosphorylation state of glycogen synthase kinase (GSK)-3β, in addition to the expression levels of alkaline phosphatase, β-catenin and runt-related transcription factor 2 were observed by western blot analysis. Following treatment with BMP-7, the phosphorylation of GSK-3β was stimulated and the expression of β-catenin, ALP and Runx2 was increased. Furthermore, inhibiting β-catenin signaling with XAV-939 suppressed the BMP-7-mediated changes. The results indicated that the BMP-7-induced differentiation of rMSCs into cartilage was promoted primarily by the Wnt/β-catenin pathway.
ABSTRACT. Osteoporosis poses a major public health threat in aging societies. Adipose-derived stem cells (ADSCs) are multipotent adult stem cells that have the ability to yield mesenchymal stem cells, and have the potential to undergo osteogenesis and bone regeneration. Bone morphogenetic proteins (BMPs) have been demonstrated to upregulate bone gene expression after mechanical injury and to improve bone injury repair. This study aimed to produce BMP-2 expression in ADSCs by using lentiviral vectors. Subcutaneous adipose tissue from 4-week-old male Sprague-Dawley rats was used. Oil red O staining was used to detect adipocyte formation from ADSCs. Induction of ADSC osteogenesis was confirmed with Alizarin red S staining. The recombinant lenti-hBMP-2/ BMP-2 promotes chondrogenesis of rat adipose neo was constructed to infect ADSCs, BMP-2 expression was measured by immunoblotting analysis, and cellular alkaline phosphatase levels were examined. We found that >70% of ADSC cells could be induced to differentiate into osteocytes or adipocytes. Under osteogenic induction, ADSCs showed increased intracellular calcium deposition, the formation of calcium tubercles, and the disappearance of cellular structures in calcium tubercles. After infection of ADSCs by lenti-hBMP-2/neo, BMP-2 was expressed after doxycycline induction. We, thus, conclude that ADSCs maintain vigorous growth ex vivo and possess stem celllike properties. When infected with lenti-hBMP-2/neo, ADSCs can be induced to promote BMP-2 expression.
FOXD3 has been found previously to positively regulate miR-26b, a tumor inhibitor of nasopharyngeal carcinoma (NPC). However, its precise function and associated mechanism of action in NPC has yet been investigated. In this study, FOXD3 mRNA and protein expression was evaluated using RT-qPCR, western blotting and immunohistochemistry (IHC). Protein levels involved in the PI3K/Akt pathway were assessed by western blot and cell proliferation was determined by MTT and colony forming assays. Additionally, cell apoptosis was assessed by flow cytometric assay. Finally, the migration and invasion capabilities of the NPC cells were determined using wound healing and Transwell assays. We found that FOXD3 levels were relatively low in NPC tissue and cells, whereas its increase caused the inhibition of the PI3K/Akt pathway. Functional experiments found that overexpression of FOXD3 suppressed cell proliferation, migration and invasionand enhanced cell apoptosis in C6661 cells. IGF-1, an activator of the PI3K/Akt pathway, reversed the inhibitory effect of FOXD3. Furthermore, we found an upregulation of the PI3K/Akt pathway and inhibitory effects of FOXD3 upregulation on C6661 cellular activities. In conclusion, FOXD3 negatively mediated the PI3K/Akt pathway to restrain the processes involved in C6661 cell pathology. These findings further exposed the function and downstream axis of FOXD3 in NPC and displayed a promising new target for NPC therapy.
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