1,25D3 Promotes Mineralization of Osteoblasts by Activating C/EBP beta-DKK1 Axis

Abstract

Background/Purpose: 1,25-Dihydroxyvitamin D3 (1,25D3) has a positive therapeutic effect on osteoporosis by activating osteoblasts. Bone formation is a dynamic and sequential process that comprises osteoblasts proliferation, increased ALP activity, collagen synthesis, and mineralization. In addition, the tight regulation between Wnt/β-catenin signaling and Dickkopf-1 (Dkk-1), Wnt antagonist, is critical for these series of steps to induce osteoblast differentiation and bone formation. Therefore, it is possible that 1,25D3 promote osteoblastic differentiation through Wnt activation; however, the potential role of Dkk1 in 1,25D3-induced osteoblastic differentiation is not fully elucidated.

Methods: Bone tissues were obtained from knee replacement surgery and primary preosteoblasts were isolated using outgrowth method. Preosteoblasts cultures underwent microarray analysis on day 14 either with stimuli for differentiation or without. The microarray data was verified using qPCR and ELISA. Also, the effect of 1,25D3 during preosteoblasts differentiation to osteoblasts was assessed by intracellular alkaline phosphatase (ALP) activity analysis, alizarin red (ARS) staining for calcium deposit, and hydroxyapatite staining. To investigate the indirect regulation of genes by 1,25D3 in unmineralized osteoblasts, preosteoblasts were incubated with ascorbic acid (AA) for 3 days and then exposed to 1,25D3 for 1 day. Subsequently, what happened at the molecular level by 1,25D3 was investigated using qPCR, Dkk1 promoter assay, immunostaining, immunoblotting, and Chromatin Immunoprecipitation (ChIP) assay.

Results: Gene expression microarrays revealed upregulation of DKK1 expression in differentiated osteoblasts. Increase in DKK1 expression and secretion in differentiated osteoblasts were confirmed by qPCR and ELISA, respectively. 1,25D3 promoted osteoblast maturation of preosteoblasts and obviously induced the expression of C/EBPβ and DKK1 at day 7 during differentiation. Interestingly, mRNA and protein levels of C/EBPβ and DKK1 were markedly increased by 1,25D3 treatment following AA stimuli. To demonstrate the relationship between the two genes, we performed knockdown of C/EBPβ with siRNA, which reduced the up-regulation of DKK1 induced by 1,25D3. In contrast, overexpression of C/EBPβ boosted enhancement of DKK1 by 1,25D3. Furthermore, we also found that C/EBPβ bind to human DKK1 promoter in response to 1,25D3. Intriguingly, blocking DKK1 attenuated calcified nodule of mineralized osteoblasts, but not ALP activity and collagen synthesis.

Conclusion: Taken together, these observations suggest that 1,25D3 promotes the mineralization of osteoblasts through activation of DKK1 followed by an increase in C/EBPβ.