Korean J Physiol Pharmacol.  2022 Jan;26(1):37-45. 10.4196/kjpp.2022.26.1.37.

Nicotinamide phosphoribosyltransferase regulates the cell differentiation and mineralization in cultured odontoblasts

• Kang KR
• Kim JS
• Seo JY
• Lim H
• Kim TH
• Yu SK
• Kim HJ
• Kim CS
• Chun HS
• Park JC
• Kim DK

• KMID: 2524529
• DOI: http://doi.org/10.4196/kjpp.2022.26.1.37

Abstract

The aim of the present study was to investigate the physiological role of nicotinamide phosphoribosyltransferase (NAMPT) associated with odontogenic differentiation during tooth development in mice. Mouse dental papilla cell-23 (MDPC-23) cells cultured in differentiation media were stimulated with the specific NAMPT inhibitor, FK866, and Visfatin (NAMPT) for up to 10 days. The cells were evaluated after 0, 4, 7, and 10 days. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The mineralization assay was performed by staining MDPC-23 cells with Alizarin Red S solution. After cultivation, MDPC-23 cells were harvested for quantitative PCR or Western blotting. Analysis of variance was performed using StatView 5.0 software (SAS Institute Inc., Cary, NC, USA). Statistical significance was set at p < 0.05. The expression of NAMPT increased during the differentiation of murine odontoblast-like MDPC-23 cells. Furthermore, the up-regulation of NAMPT promoted odontogenic differentiation and accelerated mineralization through an increase in representative odontoblastic biomarkers, such as dentin sialophosphoprotein, dentin matrix protein-1, and alkaline phosphatase in MDPC-23 cells. However, treatment of the cells with the NAMPT inhibitor, FK866, attenuated odontogenic differentiation, as evidenced by the suppression of odontoblastic biomarkers. These data indicate that NAMPT regulated odontoblastic differentiation through the regulation of odontoblastic biomarkers. The increase in NAMPT expression in odontoblasts was closely related to the formation of the extracellular matrix and dentin via the Runx signaling pathway. Therefore, these data suggest that NAMPT is a critical regulator of odontoblast differentiation during tooth development.

Keyword

Differentiation; Nicotinamide phosphoribosyltransferase; Odontoblasts; Tooth development

Figure

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  Fig. 1 The expression of NAMPT during odontoblastic differentiation of MDPC-23 cells. (A) Mineralized nodule formation in MDPC-23 cells. MDPC-23 cells were cultured with odontoblastic induction medium for 10 days, and mineralization was evaluated by alizarin Red S staining (upper panel). The mineralization was quantified by colorimetric spectrophotometry (lower panel). (B) mRNAs were prepared on day 0, 4, 7, and 10 after induction. qPCR was performed to detect the expression levels of ALP, Col-1, DMP-1, DSPP, BSP, and NAMPT. Quantitative data for mRNA expressions (B, upper panel) were analyzed by using ImageJ software after GAPDH normalization (B, lower panel). Each data point represents the mean ± SEM of three experiments. NAMPT, nicotinamide phosphoribosyltransferase; MDPC-23, mouse dental papilla cell-23; BSP, bone sialoprotein; DMP-1, dentin matrix protein-1; DSPP, dentin sialophosphoprotein; ALP, alkaline phosphatase; Col-1, collagen type-1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 2 The effects of Visfatin on mineralization in MDPC-23 cells. (A) MDPC-23 cell viability after Visfatin treatment as determined by MTT assays. (B) Alizarin Red S stain (upper panel) showing mineralized nodule formation in MDPC-23 cells, cultured in differentiation media and treated with Visfatin for up to 10 days. The mineralization was quantified using colorimetric spectrophotometry (lower panel). (C) Treatment with Visfatin at 50 ng/ml according to the defined conditions. The expression level of odontoblast biomarkers were assayed by Western blotting according to standard methods. Quantitative data for protein expressions (C, upper panel) were analyzed by using ImageJ software after β-actin normalization (C, lower panel). Each data point represents the mean ± SEM of three experiments. MDPC-23, mouse dental papilla cell-23; DSPP, dentin sialophosphoprotein; DMP-1, dentin matrix protein-1; NAMPT, nicotinamide phosphoribosyltransferase; BSP, bone sialoprotein. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 3 The effects of the specific NAMPT inhibitor, FK866, on mineralization in MDPC-23 cells. (A) MDPC-23 cell viability after FK866 treatment as determined by MTT assays. (B) Alizarin Red S stain (left panel) showing mineralized nodule formation in MDPC-23 cells, cultured in differentiation media and treated with FK866 for up to 10 days. The mineralization was quantified using colorimetric spectrophotometry (right panel). Each data point represents the mean ± SEM of three experiments. NAMPT, nicotinamide phosphoribosyltransferase; MDPC-23, mouse dental papilla cell-23. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 4 The role of the specific NAMPT inhibitor, FK866, on differentiation in MDPC-23. qRT-PCR was performed to detect the expression levels of BSP (A), ALP (B), Col-1 (C), DMP-1 (D), DSPP (E), and NAMPT (F). GAPDH served as the experimental control. All data are based on three independent experiments. (G) Treatment with FK866 at 1 nM according to the defined conditions. Western blotting was used to assess the expression level of odontoblast biomarkers. Quantitative data for protein expressions (G, left panel) were analyzed by using ImageJ software after β-actin normalization (G, right panel). Each data point represents the mean ± SEM of three experiments. MDPC-23, mouse dental papilla cell-23; BSP, bone sialoprotein; ALP, alkaline phosphatase; Col-1, collagen type-1; DMP-1, dentin matrix protein-1; DSPP, dentin sialophosphoprotein; NAMPT, nicotinamide phosphoribosyltransferase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 5 Analysis of the NAMPT synthesis pathway in MDPC-23 odontoblast differentiation. Western blotting was performed to detect the protein expression levels of Runx-1, Runx-2, and Runx-3 in MDPC-23 cells treated with FK866 (A) or Visfatin (B). β-Actin served as the experimental control. The expression level of Runx family proteins were assayed using Western blotting. NAMPT, nicotinamide phosphoribosyltransferase; MDPC-23, mouse dental papilla cell-23.

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