The Histone Demethylase KDM3B Promotes Osteo-/Odontogenic Differentiation, Cell Proliferation, and Migration Potential of Stem Cells from the Apical Papilla

Abstract

Understanding the regulation mechanisms of mesenchymal stem cells (MSCs) can assist in tissue regeneration. The histone demethylase (KDM) family has a crucial role in differentiation and cell proliferation of MSCs, while the function of KDM3B in MSCs is not well understood. In this study, we used the stem cells from the apical papilla (SCAPs) to test whether KDM3B could regulate the function of MSCs. By an alkaline phosphatase (ALP) activity assay, Alizarin red staining, real-time RT-PCR, and western blot analysis, we found that KDM3B enhanced the ALP activity and mineralization of SCAPs and promoted the expression of runt-related transcription factor 2 (RUNX2), osterix (OSX), dentin sialophosphoprotein (DSPP), and osteocalcin (OCN). Additionally, the CFSE, CCK-8, and flow cytometry assays revealed that KDM3B improved cell proliferation by accelerating cell cycle transition from the G1 to S phase. Scratch and transwell migration assays displayed that KDM3B promoted the migration potential of SCAPs. Mechanically, microarray results displayed that 98 genes were upregulated, including STAT1, CCND1, and FGF5, and 48 genes were downregulated after KDM3B overexpression. Besides, we found that the Toll-like receptor and JAK-STAT signaling pathway may be involved in the regulating function of KDM3B in SCAPs. In brief, we discovered that KDM3B promoted the osteo-/odontogenic differentiation, cell proliferation, and migration potential of SCAPs and provided a novel target and theoretical basis for regenerative medicine.

Figure 1

KDM3B enhanced the osteo-/odontogenic differentiation potential of SCAPs. (a) The knockdown efficiency of KDM3B in SCAPs was tested by western blot. (b) KDM3B knockdown significantly depressed the ALP activity in SCAPs. (c) The Alizarin red staining and (d) the quantitative calcium analysis showed that KDM3B knockdown reduced the mineralization capacity of SCAPs compared with the control group. (e, f) Real-time RT-PCR analysis confirmed that KDM3B knockdown reduced the expression of (e) RUNX2 and (f) OSX in SCAPs. (g) Western blot analysis showed the expression of RUNX2 and OSX in the KDM3B knockdown group and the control group. Histone H3 served as an internal control. (h) Western blot analysis revealed that the expression of DSPP and OCN was decreased after KDM3B was knocked down. Histone H3 served as an internal control. (i) The KDM3B overexpression was tested by western blot. (j) KDM3B overexpression significantly enhanced the ALP activity in SCAPs. (k, l) The results of (k) the Alizarin red staining and (l) the quantitative calcium analysis revealed that KDM3B overexpression enhanced the mineralization capacity of SCAPs compared with the control group. (m, n) Real-time RT-PCR analysis revealed that KDM3B overexpression increased the expression of (m) RUNX2 and (n) OSX in SCAPs. (o) Western blot analysis showed the expression of RUNX2 and OSX in the KDM3B overexpression group and the control group. Histone H3 served as an internal control. (p) Western blot analysis revealed that the expression of OCN and DSPP was enhanced after KDM3B was overexpressed. Histone H3 served as an internal control. Statistical significance was determined using Student's t-test. All error bars represent SD (n = 3). P < 0.05. ^∗∗P ≤ 0.01.

Figure 2

KDM3B enhanced the cell proliferation of SCAPs and regulated the cell cycle. The (a) CFSE assay and (b) quantitative analysis results revealed that KDM3B knockdown decreased the cell numbers at 3 days in SCAPs. (c) The CCK-8 assay revealed that KDM3B knockdown reduced the cell numbers at 24 h and 48 h in SCAPs. (d) Flow cytometric cell cycle analysis revealed that KDM3B knockdown increased the proportion of cells in the G0/G1 phase and decreased the proportion of cells in the S phase. (e) Comparison of cell cycle distribution between the KDM3B knockout group and the control group. (f) The proliferation index between the KDM3B knockout group and the control group. The (g) CFSE assay and (h) quantitative analysis results revealed that KDM3B overexpression enhanced the cell numbers at 3 days in SCAPs. (i) The CCK-8 assay revealed that KDM3B overexpression increased the cell numbers at 24 h and 48 h in SCAPs. (j) Flow cytometric cell cycle analysis revealed that KDM3B overexpression decreased the percentage of cells in the G0/G1 phase and increased the proportion of cells in the S phase. (k) Comparison of cell cycle distribution between the KDM3B overexpression group and the control group. (l) The proliferation index between the KDM3B overexpression group and the control group. Statistical significance was determined using Student's t-test. All error bars represent SD (n = 3 or 6). P < 0.05. ^∗∗P ≤ 0.01.

Figure 3

KDM3B enhanced the migration and chemotaxis potential of SCAPs. The (a) scratch migration assay and (b) quantitative analysis results demonstrated that KDM3B knockdown led to a significant decrease in the migration ability of SCAPs at 24 h and 48 h. The (c) transwell chemotaxis assay and (d) quantitative analysis results showed that KDM3B knockdown led to a decrease in the chemotaxis ability of SCAPs at 24 h and 48 h. The (e) scratch migration assay and (f) quantitative analysis results demonstrated that KDM3B overexpression led to a significant increase in the migration ability of SCAPs at 24 h and 48 h. The (g) transwell chemotaxis assay and (h) quantitative analysis results showed that KDM3B overexpression led to an increase in the chemotaxis ability of SCAPs at 24 h and 48 h. Statistical significance was determined using Student's t-test. All error bars represent SD (n = 3 or 6). P < 0.05. ^∗∗P ≤ 0.01.

Figure 4

Significant gene ontology (GO) analyses of differentially expressed genes in KDM3B overexpression SCAPs compared with the control group. (a) The significant GO functions of upregulated genes during KDM3B overexpression. (b) The significant GO functions of downregulated genes during KDM3B overexpression. The y-axis represents the GO category, and the x-axis represents the negative logarithm of the P value (−LgP). A larger −LgP indicated a smaller P value for the difference.

Figure 5

Analysis of the interaction network of significant pathways (Path-net) in the KDM3B overexpression group compared with the control group. A pathway of a high degree means that it played an essential role in the signaling network. Red indicates upregulated pathways, blue indicates downregulated pathways, and yellow indicates up- and downregulated pathways. Circle nodes represent a pathway, and the lines indicate the interactions between two pathways.

Figure 6

Analysis of the interaction network of differentially expressed genes (Signal-net) in the KDM3B overexpression group compared with the control group. In the Signal-net, the intermediate centrality of genes represents the characteristics of genes. Red represents an important upregulated gene, and the degree of interaction between genes is indicated by the circle size and the interaction is indicated by the line.