Abstract
Our previous study found that the level of CCN1 increases as osteogenic differentiation progresses in tonsil-derived mesenchymal stem cells (TMSCs). This study investigated how CCN1 is regulated through HDAC inhibition in TMSCs and their relationship with osteogenesis. Valproic acid (VPA) (1–5 mM), a well-known histone deacetylase (HDAC) inhibitor, strongly inhibited TMSC proliferation without altering MSC-specific surface markers, CD14, 34, 45, 73, 90 and 105. However, CD146 expression increased at 5 mM VPA. VPA increased osteogenic differentiation of TMSCs but decreased adipogenesis and chondrogenesis, as evidenced by the cell-specific staining of differentiation. The former was validated by the increased osteocalcin (OCN). The changes in CCN1 by VPA was biphasic; it increased until 48 h and decreased thereafter. Knockdown of CCN1 by using siRNA inhibited the osteogenic effect of VPA. VPA had no effect on CCN1 mRNA expression, but inhibition of protein synthesis by cycloheximide showed that VPA slowed down the CCN1 protein degradation. Moreover, overexpression of HDAC1 completely inhibited VPA-induced CCN1. Our results indicate that VPA inhibits the HDAC1, inducing CCN1 protein stability rather than gene expression, thereby promoting osteogenic differentiation of TMSCs. These findings present the noble implication of VPA as an inhibitor of HDAC1 to facilitate CCN1-induced osteogenic differentiation of MSCs.
Highlights
Tonsil-derived mesenchymal stem cells (TMSCs) are multipotent MSCs that have increasingly been studied due to their relatively high proliferation rate and low allogenicity compared to other tissue-derived MSCs [1]
MTT assay showed that valproic acid (VPA) exposure for 72 h decreased the proliferation of the tonsil-derived mesenchymal stem cells (TMSCs) in a concentration-dependent manner, where its effect was significant at 1 mM (p < 0.05), 2.5 mM (p < 0.01) and 5 mM (p < 0.001) (Figure 1A)
Our results showed that VPA increased the osteogenic differentiation of the TMSCs as visualized by the calcium mineralization stained by Alizarin Red S (Figure 3A), even though it decreased the adipogenic and chondrogenic differentiation of the TMSCs, and this was assessed by the decreased formation of lipid droplets stained with Oil Red O and sulfated proteoglycan deposits stained with Alcian blue, respectively (Figure 3A)
Summary
Tonsil-derived mesenchymal stem cells (TMSCs) are multipotent MSCs that have increasingly been studied due to their relatively high proliferation rate and low allogenicity compared to other tissue-derived MSCs [1]. CCN1 was shown to promote the differentiation of endothelial progenitor cells [7], and a more recent study reported that CCN1 stimulated the proliferation and differentiation of osteoblasts, altering bone remodeling of myeloma bone disease [8]. Mechanical strain, for example, induces acetylation of histone 3 and 4 at the CCN1 gene-coding region by activating CREB-binding protein (CBP) histone acetyltransferase (HAT) through signaling cascade of p38 stress-activated protein kinase (SAPK), thereby increasing promoter activity and expression of CCN1 [11]. The results from our study indicated that the expression of CCN1 is required for the proper induction for the osteogenic differentiation of TMSCs [7], which made us to further investigate the detailed molecular mechanism associated with CCN1 and osteogenesis
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