Indirect immobilized Jagged1 suppresses cell cycle progression and induces odonto/osteogenic differentiation in human dental pulp cells

Abstract

Notch signaling regulates diverse biological processes in dental pulp tissue. The present study investigated the response of human dental pulp cells (hDPs) to the indirect immobilized Notch ligand Jagged1 in vitro. The indirect immobilized Jagged1 effectively activated Notch signaling in hDPs as confirmed by the upregulation of HES1 and HEY1 expression. Differential gene expression profiling using an RNA sequencing technique revealed that the indirect immobilized Jagged1 upregulated genes were mainly involved in extracellular matrix organization, disease, and signal transduction. Downregulated genes predominantly participated in the cell cycle, DNA replication, and DNA repair. Indirect immobilized Jagged1 significantly reduced cell proliferation, colony forming unit ability, and the number of cells in S phase. Jagged1 treated hDPs exhibited significantly higher ALP enzymatic activity, osteogenic marker gene expression, and mineralization compared with control. Pretreatment with a γ-secretase inhibitor attenuated the Jagged1-induced ALP activity and mineral deposition. NOTCH2 shRNA reduced the Jagged1-induced osteogenic marker gene expression, ALP enzymatic activity, and mineral deposition. In conclusion, indirect immobilized Jagged1 suppresses cell cycle progression and induces the odonto/osteogenic differentiation of hDPs via the canonical Notch signaling pathway.

Figure 1

Isolated cell characterization. Cell morphology was evaluated using a light microscope (A). The mRNA expression of mesenchymal markers was examined using semi-quantitative polymerase chain reaction (B). Surface marker expression was analyzed using flow cytometry (C–G).

Figure 2

Indirect immobilized Jagged1 effectively activated Notch signaling in hDPs. Cells were seeded on direct or indirect immobilized Jagged1 tissue culture plates for 24 h (A,B). hDPs were seeded on indirect immobilized Jagged1 or treated with soluble Jagged1 for 24 h (C,D). HES1 and HEY1 mRNA expression was evaluated using real-time polymerase chain reaction. Bars indicate a significant difference between groups (p < 0.05). Black dots (•) indicate outlier data points.

Figure 3

Differentially expressed pathways in Jagged1 treated hDPs determined by Reactome pathway database analysis. The differentially expressed genes were analyzed using an online bioinformatic tool to identify related affected pathways. The diagrams demonstrate the upregulated (A) and downregulated (B) pathways.

Figure 4

Differential gene expression analysis of indirect immobilized Jagged1 treated hDPs. Cells were seeded on Jagged1 immobilized surfaces for 24 h. RNA was extracted and subjected to RNA sequencing analysis for differential gene expression. KEGG pathway database enrichment analysis for the upregulated (A) and downregulated (B) genes was performed by WebGestalt. To validate the differential gene expression in Jagged1 treated hDPs, cells were plated on Jagged1 immobilized surfaces for 24 h. The differential gene expression of selected genes was confirmed using real-time polymerase chain reaction (C–J). Bars indicate a significant difference between groups (p < 0.05).

Figure 5

Indirect immobilized Jagged1 inhibited hDP cell proliferation and cell cycle progression. hDPs were plated on Jagged1 immobilized surfaces for 24 h. In the Jagged1 + DAPT group, the cells were pretreated with a γ-secretase inhibitor (DAPT) for 30 min prior to Jagged1 exposure. The mRNA expression of selected genes related to DNA replication and the cell cycle was evaluated using real-time polymerase chain reaction (A–I). For the colony forming unit assay, hDPs were maintained in growth medium for 14 days. Colonies were stained using methylene blue (J). The staining was solubilized and the absorbance was determined (K). Cell proliferation was identified using the MTT assay at day 1, 3, and 7 (L). Flow cytometry analysis of the cell cycle was performed at day 3 after exposing hDPs to Jagged1 (M). The percentage of the cell population in the cell cycle (N) is shown. Bars indicate a significant difference between groups (p < 0.05). Black dot (•) indicates an outlier data point.

Figure 6

Indirect immobilized Jagged1 enhanced TGF-β mRNA expression in hDPs. hDPs were seeded on Jagged1 immobilized surfaces for 24 h in growth medium. In the Jagged1 + DAPT group, cells were pretreated with a γ-secretase inhibitor (DAPT) for 30 min prior to Jagged1 exposure. The mRNA expression was determined using real-time polymerase chain reaction (A–C). Bars indicate a significant difference between groups (p < 0.05).

Figure 7

Indirect immobilized Jagged1 promoted osteogenic differentiation in hDPs. hDPs were seeded on indirect immobilized Jagged1 and maintained in osteogenic medium for 14 days. Cells on hFc immobilized surfaces were used as the control. Mineral deposition was determined using Alizarin Red S staining (A). For odonto/osteogenic marker gene expression, cells were seeded on indirect immobilized Jagged1 and maintained in osteogenic medium for 3 and 7 days. The osteogenic related gene expression was evaluated using real-time polymerase chain reaction (B–L). For scanning electron microscope analysis, hDPs were seeded on hFc control surfaces (M and N) or indirect immobilized Jagged1 surfaces (O,P) for 21 days in osteogenic medium. Mineral crystal and cell morphology were observed by SEM. Surface chemical composition was evaluated using EDX (Q,R). Bars indicate a significant difference between groups (p < 0.05).

Figure 8

Indirect immobilized Jagged1 promoted osteogenic differentiation in hDPs. hDPs were seeded on indirect immobilized Jagged1 and maintained in osteogenic medium for 3 or 7 days. Protein expression of osteogenic differentiation marker (OPN, COL1, RUNX2) was evaluated by immunofluorescence staining. DAPI was used to counterstain the nucleus.

Figure 9

γ-secretase inhibitor abolished Jagged1-induced ALP activity and mineral deposition. hDPs were seeded on indirect immobilized Jagged1 surfaces and maintained in osteogenic medium for 3 days. Some cells were pretreated with DAPT, a γ-secretase inhibitor, 30 min prior to Jagged1 exposure. The mRNA levels of HES1 (A), HEY1 (B), and ALP (C) were measured using real-time polymerase chain reaction. ALP enzymatic activity was evaluated (D). Mineral deposition was determined using Alizarin Red S staining after culturing for 7 d in osteogenic medium (E and F). Bars indicate a significant difference between groups (p < 0.05). Black dot (•) indicates an outlier data point.

Figure 10

NOTCH2 participated in Jagged1 induced odonto/osteogenic differentiation by hDPs. The shNOTCH2 and shControl transduced hDPs were seeded on indirect immobilized Jagged1 or the hFc control surfaces and maintained in osteogenic medium for 3 and 7 days. The mRNA expression of Notch target genes and osteogenic related genes was determined using real-time polymerase chain reaction (A–F). ALP enzymatic activity was evaluated (G,H). Mineral deposition was stained with Alizarin Red S dye at day 14 (I,J). Bars indicate a significant difference between groups (p < 0.05). Black dot (•) indicates an outlier data point.