Abstract
BackgroundAdipose-derived stem cells (ADSCs) are increasingly used in regenerative medicine because of their potential to differentiate into multiple cell types, including osteogenic lineages. Sirtuin protein 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase that plays important roles in cell differentiation. NOTCH signaling has also been reported to involve in osteogenic differentiation. However, the function of SIRT6 in osteogenic differentiation of ADSCs and its relation to the NOTCH signaling pathways are yet to be explored.MethodsThe in vitro study with human ADSCs (hADSCs) and in vivo experiments with nude mice have been performed. Alkaline phosphatase (ALP) assays and ALP staining were used to detect osteogenic activity. Alizarin Red staining was performed to detect calcium deposition induced by osteogenic differentiation of ADSCs. Western blot, RT-qPCR, luciferase reporter assay, and co-immunoprecipitation assay were applied to explore the relationship between of SIRT6, DNA methyltransferases (DNMTs) and NOTCHs.ResultsSIRT6 promoted ALP activity, enhanced mineralization and upregulated expression of osteogenic-related genes of hADSCs in vitro and in vivo. Further mechanistic studies showed that SIRT6 deacetylated DNMT1, leading to its unstability at protein level. The decreased expression of DNMT1 prevented the abnormal DNA methylation of NOTCH1 and NOTCH2, resulting in the upregulation of their transcription. SIRT6 overexpression partially suppressed the abnormal DNA methylation of NOTCH1 and NOTCH2 by antagonizing DNMT1, leading to an increased capacity of ADSCs for their osteogenic differentiation.ConclusionThis study demonstrates that SIRT6 physical interacts with the DNMT1 protein, deacetylating and destabilizing DNMT1 protein, leading to the activation of NOTCH1 and NOTCH2, Which in turn promotes the osteogenic differentiation of ADSCs.
Highlights
Adipose-derived stem cells (ADSCs), a type of mesenchymal stem cells, are being increasingly accepted as an attractive cell type for regenerative medicine because of their potential to differentiate into multiple cell types, including adipogenic, osteogenic, and chondrogenic lineages (Caplan, 2007; Bianco et al, 2008; Worthley et al, 2015)
In order to elucidate the role of Sirtuin protein 6 (SIRT6) in osteogenic differentiation, human ADSCs (hADSCs) were cultured under osteogenic inductive conditions
Alizarin red S (ARS) staining, Alkaline phosphatase (ALP) staining and activity assays revealed that upregulated SIRT6 promoted mineralized bone matrix formation and ALP activity in hADSCs, SIRT6 knockdown inhibited osteogenic differentiation in hADSCs (Figures 1E–G)
Summary
Adipose-derived stem cells (ADSCs), a type of mesenchymal stem cells, are being increasingly accepted as an attractive cell type for regenerative medicine because of their potential to differentiate into multiple cell types, including adipogenic, osteogenic, and chondrogenic lineages (Caplan, 2007; Bianco et al, 2008; Worthley et al, 2015). Owing to their osteogenic capacity and ease of acquisition and culture, human ADSCs (hADSCs) are considered a suitable cellular source for the regeneration of bone loss and fractures (Schubert et al, 2013; Heilmeier et al, 2016). The function of SIRT6 in osteogenic differentiation of ADSCs and its relation to the NOTCH signaling pathways are yet to be explored
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