Int J Stem Cells.  2022 Nov;15(4):384-394. 10.15283/ijsc21240.

NBCe1 Regulates Odontogenic Differentiation of Human Dental Pulp Stem Cells via NF-κB

• Li Q
• Ju Y
• Jin C
• Liu L
• Zhao S

• KMID: 2536208
• DOI: http://doi.org/10.15283/ijsc21240

Abstract

Background and Objectives
Dental pulp stem cells (DPSCs) play an important role in the repair of tooth injuries. Electrogenic sodium bicarbonate cotransporter 1 (NBCe1) is a Na^＋ -coupled HCO₃⁻ transporter encoded by the solute carrier 4A4 (SLC4A4) gene and plays a crucial role in maintaining the pH of DPSCs. Our previous research confirmed that NBCe1 is highly expressed in odontoblasts during the development of the tooth germ. Therefore, in this study, we aimed to investigate the effect of NBCe1 on odontogenic differentiation of DPSCs and further clarify the underlying mechanisms.
Methods and Results
DPSCs were isolated and identified, and the selective NBCe1 inhibitor S0859 was used to treat DPSCs. We used a cell counting Kit-8 assay to detect cell proliferative ability, and intracellular pH was assessed using confocal microscopy. Odontogenic differentiation of DPSCs was analyzed using real-time PCR and Alizarin Red S staining, and the NF-κB pathway was assessed using western blotting. Our results indicated that 10 μM S0859 was the optimal concentration for DPSC induction. Intracellular pH was decreased upon treatment with S0859. The mRNA expressions of DSPP, DMP1, RUNX2, OCN, and OPN were upregulated in the NBCe1 inhibited group compared to the controls. Moreover, NBCe1 inhibition significantly activated the NF-κB pathway, and a NF-κB inhibitor reduced the effect of NBCe1 on DPSC differentiation.
Conclusions
NBCe1 inhibition significantly promotes odontogenic differentiation of DPSCs, and this process may be regulated by activating the NF-κB signaling pathway.

Keyword

Dental pulp stem cells; NBCe1; Intracellular pH; Odontogenic differentiation; NF-κB

Figure

• 

  Fig. 1 Characterization of human dental pulp stem cells (DPSCs). (A) Primary DPSCs showed typical fibroblast or spindle morphology. (B) DPSCs were fused and grown in a swirl on the 13th day of primary culture (Scale bar=50 μm). (C) Control group for alizarin red S staining. (D) Alizarin red S staining of mineralized nodules after osteogenic-induced culture (Scale bar=200 μm). (E) Control group for oil red O staining. (F) Oil red O positive staining after adipogenic differentiation (Scale bar=200 μm). (G) Flow cytometry assay showing DPSCs with positive expression for CD44, CD73, CD90 and CD105, and negative expression CD34, CD11b, CD19, CD45 and HLA-DR.

• 

  Fig. 2 Effect of S0859 on prolifera-tion and intracellular pH of DPSCs. (A) A CCK-8 assay revealed that low S0859 concentration had little effect on DPSCs viability, and a high dose inhibited proliferation (*p<0.05). (B) Standard curve for pH and its effect on DPSCs obtained using BCECF-AM. The pH/fluorescence ratio (530/640) conforms to the linear regression equation y=0.1897x＋5.9766 (R2= 0.9903). (C) Effect of S0859 on intracellular pH of DPSCs observed using BCECF-AM. The intracellular pH of DPSCs treated with S0859 decreased compared to the control group (*p< 0.05).

• 

  Fig. 3 NBCe1 inhibition promoted the odontogenic differentiation of DPSCs. (A) Real-time PCR indicated higher expressions of odontogenic genes (DMP1, DSPP, Runx2, OCN and OPN) in the MM＋S0859 group compared to the NC or MM groups (**p<0.01; *p<0.05). (B) Alizarin red staining showed obvious mineralized nodules in the MM＋S0859 group compared to the control group (Scale bar=50 μm). (C) A CPC assay quantified the mineralized nodules and revealed that the concentration of mineralized nodules was higher in the MM＋S0859 group compared to the MM group (***p<0.001, *p<0.05). (D) Protein expression levels of p65 and p-p65. (E) The ratio of p-p65/p65 in the S0859-treated group was higher compared to the NC and MM group (*p<0.05, **p<0.01). NC: control medium, MM: mineralized-inducing medium, CPC: cetylpyridinium chloride.

• 

  Fig. 4 Effect of NF-κB inhibitor on the odontogenic differentiation of DPSCs treated with S0859. (A) A Real-time PCR assay demonstrated that the mRNA expression levels of DSPP, DMP1, Runx2, OCN, and OPN in the S0859＋PDTC group were decreased compared to that in the S0859 group (*p<0.05, **p<0.01). (B) Alizarin red staining of mineralized nodules was more obvious in the S0859 group than in the S0859＋PDTC group (Scale bar=50 μm). (C) CPC quantification showed that the concentration of mineralized nodules in the S0859 group was higher than in the S0859＋PDTC group (*p<0.05, **p<0.01). (D) Protein levels of p65 and p-p65 in the MM group, MM＋S0859 group, MM＋PDTC group, and MM＋S0859＋PDTC group. (E) Quantification of the p-p65/p65 ratios after inhibition of the NF-κB pathway (*p<0.05, **p<0.01). NC: control medium, MM: mineralized-inducing medium, CPC: cetylpyridinium chloride.

• 

  Fig. 5 Schematic diagram of the pro-posed mechanism for NBCe1 regulation of the odontogenic differentia-tion of DPSCs. P: phosphorylation.

Reference

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