Overexpression of Dlx2 enhances osteogenic differentiation of BMSCs and MC3T3-E1 cells via direct upregulation of Osteocalcin and Alp

Abstract

Genetic studies have revealed a critical role of Distal-homeobox (Dlx) genes in bone formation, and our previous study showed that Dlx2 overexpressing in neural crest cells leads to profound abnormalities of the craniofacial tissues. The aim of this study was to investigate the role and the underlying molecular mechanisms of Dlx2 in osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) and pre-osteoblast MC3T3-E1 cells. Initially, we observed upregulation of Dlx2 during the early osteogenesis in BMSCs and MC3T3-E1 cells. Moreover, Dlx2 overexpression enhanced alkaline phosphatase (ALP) activity and extracellular matrix mineralization in BMSCs and MC3T3-E1 cell line. In addition, micro-CT of implanted tissues in nude mice confirmed that Dlx2 overexpression in BMSCs promoted bone formation in vivo. Unexpectedly, Dlx2 overexpression had little impact on the expression level of the pivotal osteogenic transcription factors Runx2, Dlx5, Msx2, and Osterix, but led to upregulation of Alp and Osteocalcin (OCN), both of which play critical roles in promoting osteoblast maturation. Importantly, luciferase analysis showed that Dlx2 overexpression stimulated both OCN and Alp promoter activity. Through chromatin-immunoprecipitation assay and site-directed mutagenesis analysis, we provide molecular evidence that Dlx2 transactivates OCN and Alp expression by directly binding to the Dlx2-response cis-acting elements in the promoter of the two genes. Based on these findings, we demonstrate that Dlx2 overexpression enhances osteogenic differentiation in vitro and accelerates bone formation in vivo via direct upregulation of the OCN and Alp gene, suggesting that Dlx2 plays a crucial role in osteogenic differentiation and bone formation.

Immunocytochemical characterization of primary teeth pulp stem cells from three stages of resorption in serum‐free medium

“…The expression of Dlx2 homeobox proteins involved in early craniofacial development has been used to distinguish odontoblast precursor cells from other mesenchymal progenitors 55 . Cultured primary pulp cells in this study variably expressed Dlx2 protein in a clustered pattern, possibly associated with its role in the formation of cell condensations which form cartilage and bone in vitro 56 . These findings not only suggest that these cells are from an odontoblastic lineage but also support the view that in serum‐free culture nestin and Dlx2‐positive primary pulp cells have retained the overall features of active odontoblasts in vitro.…”

Berberine mediates root remodeling in an immature tooth with apical periodontitis by regulating stem cells from apical papilla differentiation

“…Alkaline phosphatase and alizarin red staining hSCAPs were cultured in osteogenic media for 4 or 14 days and fixed with 4% paraformaldehyde. 45 The ALP staining kit (Byotime Biotech, Shanghai, China) and alizarin red staining kit (Cyagen, CA, USA) were used in line with the manufacturers' instructions.…”

Morusin induces osteogenic differentiation of bone marrow mesenchymal stem cells by canonical Wnt/β-catenin pathway and prevents bone loss in an ovariectomized rat model

“…*P < 0.05 and **P < 0.01 in comparison to the control group. "ns" refers to no statistical significance compared with the control group previous studies, promoting BMSC osteogenic differentiation could effectively prevent or slow down the occurrence and development of osteoporosis [30][31][32].…”