Abstract
Simvastatin (SVS) promotes the osteogenic differentiation of mesenchymal stem cells (MSCs) and has been studied for MSC-based bone regeneration. However, the mechanism underlying SVS-induced osteogenesis is not well understood. We hypothesize that α5 integrin mediates SVS-induced osteogenic differentiation. Bone marrow MSCs (BMSCs) derived from BALB/C mice, referred to as D1 cells, were used. Alizarin red S (calcium deposition) and alkaline phosphatase (ALP) staining were used to evaluate SVS-induced osteogenesis of D1 cells. The mRNA expression levels of α5 integrin and osteogenic marker genes (bone morphogenetic protein-2 (BMP-2), runt-related transcription factor 2 (Runx2), collagen type I, ALP and osteocalcin (OC)) were detected using quantitative real-time PCR. Surface-expressed α5 integrin was detected using flow cytometry analysis. Protein expression levels of α5 integrin and phosphorylated focal adhesion kinase (p-FAK), which is downstream of α5 integrin, were detected using Western blotting. siRNA was used to deplete the expression of α5 integrin in D1 cells. The results showed that SVS dose-dependently enhanced the gene expression levels of osteogenic marker genes as well as subsequent ALP activity and calcium deposition in D1 cells. Upregulated p-FAK was accompanied by an increased protein expression level of α5 integrin after SVS treatment. Surface-expressed α5 integrin was also upregulated after SVS treatment. Depletion of α5 integrin expression significantly suppressed SVS-induced osteogenic gene expression levels, ALP activity, and calcium deposition in D1 cells. These results identify a critical role of α5 integrin in SVS-induced osteogenic differentiation of BMSCs, which may suggest a therapeutic strategy to modulate α5 integrin/FAK signaling to promote MSC-based bone regeneration.
Highlights
Bone injuries are the most common traumatic injuries in humans, and mesenchymal stem cells (MSCs) are considered a valuable source for bone regeneration due to their osteogenic differentiation property [1,2,3,4]
To test the effect of SVS treatment on the osteogenesis of D1 cells, we examined the mRNA expression levels of osteogenic marker genes in D1 cells after SVS treatment
The mRNA expression level of bone morphogenic protein-2 (BMP-2) was dose-dependently increased by SVS treatment in D1 cells (Figure 1)
Summary
Bone injuries are the most common traumatic injuries in humans, and mesenchymal stem cells (MSCs) are considered a valuable source for bone regeneration due to their osteogenic differentiation property [1,2,3,4]. The important issue for MSC-based bone regeneration is finding a new effective strategy to promote the osteogenic differentiation of MSCs. Statins are lipid-lowering drugs, known as a hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, that are used to inhibit cholesterol biosynthesis [9]. Statins have been reported to enhance the expression of bone morphogenic protein-2 (BMP-2), which is an important growth factor for osteogenic differentiation of stem cells [3,9,10,11,12]. Simvastatin (SVS) is a statin that has been shown to enhance BMP-2 expression and has been clinically studied to stimulate bone formation [9,12,13,14,15,16]. Cell death has been shown when using SVS to induce osteogenic differentiation of MSCs [17]. These findings raise questions regarding the strategy of using SVS to induce osteogenic differentiation of MSCs for bone regeneration
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