Effects of neurotrophin receptor-mediated MAGE homology on proliferation and odontoblastic differentiation of mouse dental pulp cells

Abstract

Objectives: This study aimed to investigate effects of neurotrophin receptor-mediated melanoma antigen-encoding gene homology (NRAGE) on proliferation and odontoblastic differentiation of mouse dental pulp cells (mDPCs).

Materials and methods: Mouse dental pulp cells were infected with recombinant lentivirus to stably knockdown expression of NRAGE, and biological effects of NRAGE on the cells were detected. Proliferation and odontoblastic differentiation of mDPCs were observed. Simultaneously, mRNA and protein levels of NRAGE and nuclear factor-κB (NF-κB) protein expression were detected. Immunofluorescence assay was used to detect expression and location of NRAGE and NF-κB.

Results: NRAGE mRNA and protein levels reduced significantly after mDPC odontoblastic induction. Knockdown of NRAGE inhibited the proliferation of mDPCs. However, knockdown of NRAGE enhanced their odontoblastic differentiation with up-regulated ALPase activity. It also promoted mineral nodule formation as well as mRNA and protein expressions of ALP, DSPP and DMP1. Protein levels of NF-κB/p50 significantly increased, whereas NF-κB/p105 protein expression decreased in the mDPC/shNRG group. Immunofluorescence revealed that relocation of NF-κB was similar to that of NRAGE during odontoblastic induction, in which NF-κB translocated from the cytoplasm to the nucleus.

Conclusion: NRAGE is a potent regulator of proliferation and odontoblastic differentiation of mDPCs, which might be via the NF-κB signalling pathway.

Figure 1

Expression of NRAGE during odontoblastic differentiation of mDPCs. mRNA expression of mineralization‐related markers and NRAGE on days 0, 7 and 14 after induction: (a) DMP1, (b) DSPP, (c) ALP, (d) ALP activity and (e) NRAGE. Glyceraldehyde‐3‐phosphate (GAPDH) was used as normalization control. (f) DSP, DMP1 and NRAGE protein levels on days 0, 7 and 14 after induction. *Significant difference (P < 0.05) versus day 0, **Significant difference (P < 0.01) versus day 0.

Figure 2

Stable knockdown of NRAGE in mDPCs. mRNA level (a) and protein level (b) on days 0 and 14 after mDPC infection. **Significant difference (P < 0.01) versus control.

Figure 3

Effect of NRAGE knockdown on mDPC proliferation. *Significant differences (P < 0.05) versus control and **Significant difference (P < 0.01) versus control. OD, optical density.

Figure 4

Mineralization and ALP staining of mDPC/wt, mDPC/shCon and mDPC/shNRG after odontoblastic induction for 14 days. (a) Result of ALP staining was higher in the mDPC/shNRG group than in mDPC/wt and mDPC/shCon groups (original magnification 50×). (b) Formation of mineralization nodules was much more in mDPC/shNRG group than in mDPC/wt and mDPC/shCon groups (original magnification 50×).

Figure 5

Effects of NRAGE knockdown on odontoblastic differentiation of mDPCs. Effect of NRAGE knockdown on mRNA level of odontoblastic marker genes in mDPCs on days 0, 7 and 14: (a) DMP1, (b) DSPP and (c) ALP. GAPDH was used as normalization control. (d) ALP activity in mDPC/shNRG group after 0, 7 and 14 days induction. Protein levels of DMP1 and DSP by western blot analysis on days 0, 7 and 14. *Significant difference (P < 0.05) versus control, **Significant difference (P < 0.01) versus control.

Figure 6

Protein levels of NRAGE and NF‐κB after odontoblastic induction for 0, 7 and 14 days in mDPC/wt, mDPC/shCon and mDPC/shNRG groups. NRAGE was stably knocked down during induction in the mDPC/shNRG group. Protein levels of NF‐κB/p50 and NF‐κB/p105 by western blot analysis.

Figure 7

Immunofluorescence staining of NRAGE and NF‐κB relocation in mDPCs during odontoblastic induction for days 0, 3, 7, 11 and 14 (400× magnification). Immunofluorescence staining with anti‐NRAGE antibody (green), anti‐NF‐κB antibody (red) and Hoechst (blue) was performed. Negative control staining was performed with immunoglobulin G control antibody.