The effect of delta-like 1 homologue on the proliferation and odontoblastic differentiation in human dental pulp stem cells

Abstract

Introduction: This study aimed to investigate the functions of delta-like homologue 1 (DLK1) in the proliferation and differentiation of human dental pulp stem cells (hDPSCs).

Methods: Immunohistochemical analysis was used to determine the expression of alkaline phosphatase (ALP), dentin sialophosphoprotein (DSPP), DLK1, NOTCH1 and p-ERK1/2 in the mouse first maxillary molar. Recombinant lentivirus was constructed to overexpress DLK1 stably in hDPSCs. The cell viability and proliferation of hDPSCs were examined by CCK8 and EdU incorporation assay respectively. The odontoblastic differentiation of hDPSCs was determined by detection of ALPase activity assay, ALP and alizarin red staining and the expression of mineralization-related genes including ALP, DSPP and dental matrix protein. The mRNA and protein levels of DLK1 and p-ERK1/2 protein expression were detected. ERK inhibitor was used to test the differentiation effect of DLK1 on hDPSCs.

Results: Delta-like homologue 1 was highly expressed on the odontoblasts and dental pulp cells on the first maxillary molar; the expression of p-ERK1/2 is similar with the DLK1 in the same process. The expression level of DLK1 increased significantly after the odontoblastic induction of hDPSCs. DLK1 overexpression increased the proliferation ability of hDPSCs and inhibited odontoblastic differentiation of hDPSCs. The protein level of p-ERK1/2 significantly increased in hDPSCs/dlk1-oe group. ERK signalling pathway inhibitor reversed the odontoblastic differentiation effects of DLK1 on hDPSCs.

Conclusions: The proliferation of hDPSCs was promoted after DLK1 overexpression. DLK1 inhibited the odontoblastic differentiation of hDPSCs, which maybe through ERK signalling pathway.

Figure 1

Alkaline phosphatase (ALP) and dentin sialophosphoprotein (DSPP) protein expression in the first maxillary molar in mice were detected by immunohistochemical analysis. A(a), B(b) and C(c) show the result of ALP. D(d), E(e) and F(f) show the result of DSPP. Both of them were increased during the development (IEE: inner enamel epithelium; OD: odontoblast; DP: dental pulp; E: enamel; D: dentin; A‐F: 200×; a‐f: 400×)

Figure 2

Delta‐like homologue 1 (DLK1) protein expression in the first maxillary molar in mice was detected by immunohistochemical analysis. A(a), At PN2,DLK1 was expressed in the ODs, and mildly expressed in a few dental pulp cells near ODs layer. B(b): At PN6, strong positive immunolabelling was observed in the whole ODs layer, and DLK1 was much higher in some DPCs near ODs layer. C(c): At PN6, DLK was not only highly expressed in ODs but also strongly located in a large number of DPCs in DP. Primarily, DLK1 was increasingly expressed in the ODs and DPCs. (IEE: inner enamel epithelium; OD: odontoblast; DP: dental pulp; CL: cervical loop; E: enamel; D: dentin; DPCs: dental pulp cells; white arrows mean DPCs; black arrows mean ODs; A‐C: 200×; a‐c: 400×)

Figure 3

The expression of delta‐like homologue 1 (DLK1) during odontoblastic differentiation of hDPSCs. A, B, C and E represent the up‐regulation of mRNA levels of DMP1, DSPP, ALP and DLK1 during the induction respectively. D shows the increased ALP activity in the process. I shows the Western blots results of DMP1, DSPP and DLK1 during the induction. F‐H show the quantification protein level of DMP1, DSPP and DLK1 relative to β‐actin according to the Western blots results, which showed the same trend with mRNA. The data represent the mean ± SD of three independent experiments

Figure 4

Stable overexpression of DLK1 in hDPSCs. A: hDPSCs transfected with lentiviral vector in vitro demonstrated GFP expression after being infected and selected for 1 wk (200×). B and D show the mRNA and protein levels of DLK1 after hDPSCs transfected with lentiviral vector in vitro for 7 and 14 d. C presents the quantification result of D

Figure 5

Delta‐like homologue 1 (DLK1) promotes the proliferation of hDPSCs. A: shows the EdU labelling in hDPSC/dlk1‐oe and hDPSC/control group (red: EdU labelling; blue: DAPI; 200×). B: shows the quantified results of EdU analyses (by measuring the positive cells); there were much more EdU‐positive cells in hDPSC/dlk1‐oe group than hDPSC/control groups (P<.0001). C: CCK‐8 assay indicated that the cell viability of hDPSC/dlk1‐oe groups was much higher than the hDPSC/control groups (P<.05) on days 1, 3, 5 and 7, and the data represent the mean ± SD of three independent experiments

Figure 6

ALP staining and mineralized nodule formation results. (A and B) A significantly lower ALP staining in the hDPSC/dlk1‐oe group on 7 d compared with in the hDPSC/wt and hDPSC/control groups was found. (C and D) The amount of mineralized nodules formed in the hDPSC/dlk1‐oe group was much less than control groups (A, C: scanner; B and D: phase‐contrast microscopy, 50×)

Figure 7

The inhibition effect of DLK1 on the odontoblastic differentiation of hDPSCs. A, B and C represent the mRNA level of DMP1, DSPP and ALP after DLK1 overexpression in hDPSCs during the odontoblastic differentiation. D shows the ALP activity after DLK1 overexpression. G shows the protein level of DMP1 and DSPP by Western blot analysis. E and F show the quantified protein level of DMP1 and DSPP relative to β‐actin during the induction according to Western blots. All the data represent the mean±SD of three independent experiments

Figure 8

p‐ERK1/2 and NOTCH1 protein expression in the first maxillary molar in mice were detected by immunohistochemical analysis. A(a), B(b) and C(c) show the result of p‐ERK1/2. D(d), E(e) and F(f) show the result of NOTCH1. p‐ERK1/2 expression pattern was much more similar with the DLK1 than NOTCH1 during the tooth development. (IEE: inner enamel epithelium; OD: odontoblast; DP: dental pulp; E: enamel; D: dentin; A, B, C, D, E and F: 200×; a, b, c, d, e and f:400×)

Figure 9

DLK1 regulates the differentiation through ERK signalling pathway. A shows activation of p‐ERK1/2 after DLK1 overexpression. B shows that the appropriate concentration of U0126 was 10 nmol/L, which shows the similar level with the control group. C and D show the quantification protein level of p‐ERK1/2 relative to total ERK1/2 of A and B respectively

Figure 10

A and B present the ALP staining results; C and D show mineralized nodule formation results, all of which suggested that the inhibitor effects of DLK1 on odontoblastic differentiation were reversed by U0126 (10 nmol/L)