Non-canonical Wnt signaling participates in Jagged1-induced osteo/odontogenic differentiation in human dental pulp stem cells

Abstract

Osteoblast differentiation requires the interaction of various cell signaling pathways to modulate cell responses. Notch and Wnt signaling are among the crucial pathways that control numerous biological processes, including osteo/odontogenic differentiation. The aim of the present study was to examine the involvement of Wnt signaling in the Jagged1-induced osteo/odontogenic differentiation in human dental pulp stem cells (hDPSCs). The Wnt-related gene expression was analyzed from publicly available data of Jagged1-treated human dental pulp cells. The mRNA expression of Wnt ligands (WNT2B, WNT5A, WNT5B, and WNT16) and Wnt inhibitors (DKK1, DKK2, and SOST) were confirmed using real-time polymerase chain reaction. Among the Wnt ligands, WNT2B and WNT5A mRNA levels were upregulated after Jagged1 treatment. In contrast, the Wnt inhibitors DKK1, DKK2, and SOST mRNA levels were downregulated. Recombinant WNT5A, but not WNT2B, significantly promoted in vitro mineral deposition by hDPSCs. Wnt signaling inhibition using IWP-2, but not DKK1, inhibited Jagged1-induced alkaline phosphatase (ALP) activity, mineralization, and osteo/odontogenic marker gene expression in hDPSCs. In conclusion, Jagged1 promoted hDPSC osteo/odontogenic differentiation by modulating the non-canonical Wnt pathway.

Figure 1

Indirect immobilized Jagged1 promotes hDPSC osteo/odontogenic differentiation. Mesenchymal stem cell surface markers were examined using (a) flow cytometry. Multilineage differentiation towards the osteo/odontogenic and adipogenic lineages was examined using (b) alizarin red s staining and (c) oil red o staining, respectively. Cells were seeded on Jagged1 immobilized surface and maintained in osteo/odontogenic medium. Cells on hFc-immobilized surfaces were used as the control. The cells were cultured in osteo/odontogenic medium for 3, 7, and 14 days. Osteo/odontogenic differentiation was evaluated by (d) ALP staining and (e) in vitro mineral deposition using alizarin red s staining. The alizarin red s staining was solubilized, and (f) the absorbance was measured at 570 nm. Bars indicate a significant difference between groups (p < 0.05).

Figure 2

Notch regulates Wnt-related gene expression. (a) Heatmap demonstrating the significantly differential expression of Wnt-related genes in the Jagged1-treated condition. (b) The differential gene expression of selected genes in Jagged1-treated hDPSCs was confirmed using real-time polymerase chain reaction. Bars indicate a significant difference between groups (p < 0.05).

Figure 3

Dose and time course experiment of Jagged1 promotes Wnt related gene expression in hDPSCs. Correlation of Notch and Wnt pathways. (a) The mRNA levels of Notch-related genes (HES1 and HEY1) and Wnt-related genes (WNT2B, WNT5A, WNT5B, WNT16, DKK1, DKK2, and SOST) were measured using real-time polymerase chain reaction after exposed to different Jagged1 concentrations for 24 h. (b) The mRNA expression of hDPSCs treated with 10 nM Jagged1 was evaluated at day 1, 3, and 7. Bars indicate a significant difference between groups (p < 0.05).

Figure 4

WNT5A enhances the osteo/odontogenic differentiation in hDPSCs. The cells were maintained in osteo/odontogenic medium with either (a–c) recombinant WNT5A or (d–f) WNT2B supplementation. Cells cultured in osteo/odontogenic medium were used as the control. (a and d) Mineral deposition was evaluated at day 14. (b and e) The solubilized staining was measured and quantitated. (c and f) The mRNA levels of osteo/odontogenic marker genes were measured using real-time polymerase chain reaction on day 7. Bars indicate a significant difference between groups (p < 0.05).

Figure 5

IWP-2 attenuates the osteo/odontogenic differentiation induced by Jagged1. Cells were pretreated with DKK1 or IWP-2 30 min prior to seeding on Jagged1 surfaces and further maintain in osteo/odontogenic induction medium. (a and d) ALP enzymatic activity and mineral deposition was examined at day 7 and 14, respectively. (b and e) The solubilized alizarin red s staining was measured and quantitated. (c and f) The mRNA levels of osteo/odontogenic marker genes were measured using real-time polymerase chain reaction. Bars indicate a significant difference between groups (p < 0.05).

Figure 6

Reciprocal control between Notch and Wnt pathway. The cells were cultured in osteo/odontogenic medium supplemented with 200 ng/ml WNT5A for 7 days. The effects of WNT5A on Notch signaling were determined by the mRNA levels of Notch-related genes (HES1 and HEY1). Bars indicate a significant difference between groups (p < 0.05).