Amphiregulin regulates odontogenic differentiation of dental pulp stem cells by activation of mitogen-activated protein kinase and the phosphatidylinositol 3-kinase signaling pathways

Abstract

Background: Human dental pulp stem cells (hDPSCs) have received widespread attention in the fields of tissue engineering and regenerative medicine. Although amphiregulin (AREG) has been shown to play a vital function in the biological processes of various cell types, its effects on DPSCs remain largely unknown. The aim of this study was to explore the specific role of AREG as a biologically active factor in the regeneration of dental pulp tissue.

Methods: The growth of hDPSCs, together with their proliferation and apoptosis, in response to AREG was examined by CCK-8 assay and flow cytometry. We explored the effects of AREG on osteo/odontogenic differentiation in vitro and investigated the regeneration and mineralization of hDPSCs in response to AREG in vivo. The effects of AREG gain- and loss-of-function on DPSC differentiation were investigated following transfection using overexpression plasmids and shRNA, respectively. The involvement of the mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K)/Akt pathways in the mineralization process and the expression of odontoblastic marker proteins after AREG induction were investigated by using Alizarin Red S staining and Western blotting, respectively.

Results: AREG (0.01-0.1 µg/mL) treatment of hDPSCs from 1 to 7 days increased hDPSCs growth and affected apoptosis minimally compared with negative controls. AREG exposure significantly promoted hDPSC differentiation, shown by increased mineralized nodule formation and the expression of odontoblastic marker protein expression. In vivo micro-CT imaging and quantitative analysis showed significantly greater formation of highly mineralized tissue in the 0.1 μg/mL AREG exposure group in DPSC/NF-gelatin-scaffold composites. AREG also promoted extracellular matrix production, with collagen fiber, mineralized matrix, and calcium salt deposition on the composites, as shown by H&E, Masson, and Von Kossa staining. Furthermore, AREG overexpression boosted hDPSC differentiation while AREG silencing inhibited it. During the differentiation of hDPSCs, AREG treatment led to phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and PI3K/Akt. Notably, a specific inhibitor of ERK, JNK, and PI3K/Akt signaling markedly reduced AREG-induced differentiation, as well as levels of phosphorylated ERK and JNK in hDPSCs.

Conclusions: The data indicated that AREG promoted odontoblastic differentiation and facilitated regeneration and mineralization processes in hDPSCs.

Keywords: Cell differentiation; Signal transduction; Tissue regeneration.

Fig. 1

Culture and characterization of DPSCs. A (a) DPSCs isolated and cultured from pulp tissue samples, (b) clonal DPSCs, (c) were cultured for 2 weeks. B (a) Mineralized nodule formation in osteo/odontogenic induction medium demonstrated by Alizarin Red S staining, (b) lipid droplet formation in adipogenic induction medium using Oil Red O staining. C Flow cytometry of molecular surface antigen markers in DPSCs. (a) Negative control for DPSCs. Representative profiles are shown for the expression of the immunophenotypic markers CD34 (b), CD45 (c), CD90 (d), CD105 (e), CD29 (f), CD146 (g) and STRO-1 (h) in DPSCs

Fig. 2

Effects of AREG on the growth of DPSCs. A The CCK-8 assay was used to detect cell proliferation in different AREG-treated groups (0.01–1 mg/mL) and untreated DPSCs at 1, 3, 5 and 7 days (n = 5, *P < 0.05). B The cell cycle proliferation index (PI = G2/M + S) in AREG-treated and control groups analyzed by flow cytometry. C Flow cytometry analysis of AREG-treated and control groups

Fig. 3

AREG enhances the oriented differentiation potential of DPSCs. A Alizarin Red S staining of DPSCs after 14 days’ culture with a range of AREG concentrations in mineralizing medium. B Quantitative analysis of Alizarin Red S staining (n = 5, **P < 0.05). C Western blot analysis showing expression of the odontoblastic markers DSPP, RUNX2, BSP, and OCN. The full-length gels and blots are included in Additional file 1: Fig. S1A. D ImageJ software analysis of the gray level of the panel. Data are expressed as means ± SDs, n = 3, *P < 0.05, **P < 0.01

Fig. 4

The effect of AREG on the regeneration and mineralization of hDPSCs in vivo. A Micro-CT images of the DPSC/S-scaffold construct in nude mice after subcutaneous implantation for 4 weeks. A (a) and (b) show the DPSC/NF-gelatin-scaffold composites of the control and AREG group, respectively. B Quantitative analysis of micro-CT images (a) BV/TV (Bone Volume to Tissue Volume); (b) Tb.Th. (Trabecular Thickness); (c) Tb.N. (Trabecular Number); (d) Tb.Sp. (Trabecular Separation). C Histological staining of the DPSC/S-scaffold construct after subcutaneous implantation in nude mice for 4 weeks. (a, d, g) H&E staining; (b, e, h) Masson staining; (c, f, i) Von Kossa staining. *P < 0.05 represents a significant change compared with the control

Fig. 5

The effects of AREG overexpression/knockdown on the differentiation of DPSCs. A Alizarin Red S staining showing mineralized nodule formation in the AREG ( +) and AREG (–) groups. B Quantitative analysis of A (n = 5, **P < 0.05). C Protein expression of odontoblastic markers DSPP, RUNX2, BSP, and OCN shown by Western blotting in the AREG ( +) group. The full-length gels and blots are included in Additional file 1: Fig. S1B. D ImageJ analysis of the gray level of the panel. E Protein expression of odontoblastic markers DSPP, RUNX2, BSP, and OCN shown by Western blotting in the AREG ( −) group. The full-length gels and blots are included in Additional file 1: Fig. S1C. F ImageJ was used to analyze the gray level of the panel. Data represent means ± SDs, n = 3, *P < 0.05, **P < 0.01

Fig. 6

AREG promotes differentiation of DPSCs by activation of the ERK, JNK, and AKT pathways. A Protein levels of ERK and p-ERK, JNK and p-JNK, p38 and p-p38, and ATK and p-AKT treated with AREG at different time points (0, 30, 60, and 90 min) shown by Western blotting in DPSCs. The full-length gels and blots are included in Additional file 1: Fig. S2A. B Quantitative analysis of the gray intensity of A. C Treatment of DPSCs with specific ERK, JNK, or PI3K inhibitors (U0126, SP600125, and LY294002, respectively). Protein levels of ERK and p-ERK, JNK and p-JNK, p-AKT/AKT are shown by Western blotting at the indicated times. The full-length gels and blots are included in Additional file 1: Fig. S2B. D Quantitative analysis of the ratio of p-ERK/ERK, p-JNK/JNK and p-AKT/AKT from C. E Alizarin Red S staining showing mineralized nodule formation in DPSCs treated with the specific inhibitors. F Quantitative analysis of Alizarin Red S staining (n = 5, *P < 0.05). G Western blots showing protein expression of odontoblastic markers (DSPP, RUNX2, BSP, and OCN) in different groups at day 14. The full-length gels and blots are included in Additional file 1: Fig. S2C. H Quantitative analysis of data presented in G. Data represent means ± SDs, n = 3, *P < 0.05, **P < 0.01