LIM Mineralization Protein-1 Enhances the Committed Differentiation of Dental Pulp Stem Cells through the ERK1/2 and p38 MAPK Pathways and BMP Signaling

Abstract

Tissue regeneration is the preferred treatment for dentin and bone tissue defects. Dental pulp stem cells (DPSCs) have been extensively studied for their use in tissue regeneration, including the regeneration of dentin and bone tissue. LIM mineralization protein-1 (LMP-1) is an intracellular non-secretory protein that plays a positive regulatory role in the mineralization process. In this study, an LMP-1-induced DPSCs model was used to explore the effect of LMP-1 on the proliferation and odonto/osteogenic differentiation of DPSCs, as well as the underlying mechanisms. As indicated by the cell counting kit-8 assay, the results showed that LMP-1 did not affect the proliferation of DPSCs. Overexpression of LMP-1 significantly promoted the committed differentiation of DPSCs and vice versa, as shown by alkaline phosphatase activity assay, alizarin red staining, western blot assay, quantitative real-time polymerase chain reaction assay, and in vivo mineralized tissue formation assay. Furthermore, inhibiting the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) pathways using specific pathway inhibitors showed that the ERK1/2 and p38 MAPK pathways attenuated the differentiation of DPSCs. Besides, the expression of BMP signaling pathway components were also determined, which suggested that LMP-1 could activate BMP-2/Smad1/5 signaling pathway. Our results not only indicated the underlying mechanism of LMP-1 treated DPSCs but also provided valuable insight into therapeutic strategies in regenerative medicine.

Keywords: Dental pulp stem cells; Differentiation; LIM mineralization protein-1; Mitogen-activated protein kinase pathway; Tissue regeneration.

Figure 1

Characterization of DPSCs and LMP-1 expression in transfected DPSCs. (A) Flow cytometry demonstrated that the expression of CD29, CD90, CD105, CD146, and STRO-1 were positive in DPSCS, which were negative against CD34 and CD45. (B) Relative mRNA expression of LMP-1 in LV-scramble group, LV-shLMP-1 group, LV-vector group and LV-LMP-1 group after 3 days transfected in DPSCs, respectively (*P < 0.05, **P < 0.01). (C) Western blot analysis of LMP-1 protein expression in LV-scramble group, LV-shLMP-1 group, LV-vector group, and LV-LMP-1 group after 3 days transfected in DPSCs, respectively. (D) Grayscale analysis of (C) by ImageJ software (**P < 0.01).

Figure 2

Effects of LMP-1 on the proliferation of DPSCs. CCK-8 assay showed no effect when LMP-1 low-expression or overexpression at day 1, 3, 5, 7, and 9, respectively.

Figure 3

Effects of LMP-1 on ALP activity and mineralized formation capacity of DPSCs. (A) ALP activity of LMP-1 transfected DPSCs in LV-scramble group, LV-shLMP-1 group, LV-vector group, and LV-LMP-1 group after 7 days cultured in osteogenic medium (*P < 0.05). (B) The formation of mineralized nodules measured by alizarin red staining in four groups after transfected DPSCs cultured in growth medium (GM) or osteogenic medium (OM) for 14 days. (C) Quantification of alizarin red staining in four groups by CPC assay (*P < 0.05, **P < 0.01).

Figure 4

Effects of LMP-1 on the odonto/osteogenic differentiation of DPSCs. (A) Relative mRNA expression of DPSS, DMP-1, OCN and RUNX2 in LV-vector group and LV-LMP-1 group at day 1, 3 and 7, respectively (*P < 0.05, **P < 0.01). (B, C) Western blot analysis of LMP-1 protein expression in LV-vector group and LV-LMP-1 group at day 1, 3, and 7, respectively (*P < 0.05, **P < 0.01). (D) Relative mRNA expression of DPSS, DMP-1, OCN and RUNX2 in LV-scramble group and LV-shLMP-1 group at day 1, 3 and 7, respectively (*P < 0.05, **P < 0.01). (E, F) Western blot analysis of LMP-1 protein expression in LV-scramble group and LV-shLMP-1 group at day 1, 3, and 7, respectively (*P < 0.05, **P < 0.01).

Figure 5

Histological analysis of LMP-1 on the odonto/osteogenesis of LMP-1 in vivo. (A) H&E staining and Masson staining in LV-scramble group, LV-shLMP-1 group, LV-vector group, and LV-LMP-1 group after 8 weeks growth. (B) CVF of LV-scramble group, LV-shLMP-1 group, LV-vector group, and LV-LMP-1 group after 8 weeks growth (**P < 0.01). Bd Bone/dentin-like tissue, S Surrounding scaffold. Scale Bar = 50 µm.

Figure 6

Immunohistochemical observation of LMP-1 on the odonto/osteogenesis of LMP-1 in vivo. (A) Immunohistochemical staining of DSPP and OCN in LV-scramble group, LV-shLMP-1 group, LV-vector group, and LV-LMP-1 group after 8 weeks growth. (B) MOD of DSPP and OCN in LV-scramble group, LV-shLMP-1 group, LV-vector group, and LV-LMP-1 group after 8 weeks growth (*P < 0.05, **P < 0.01). Scale Bar = 50 µm.

Figure 7

Effects of LMP-1 on regulating the odonto/osteogenic differentiation of DPSCs through the MAPK pathway. Western blot analysis of p-ERK1/2 (A), p-p38 MAPK (C), p-JNK (E), and protein levels of the odonto/osteogenic markers (DSPP, DMP-1, OCN, and RUNX2) (A, C, E) in DPSCs after being treated with ERK1/2 pathway inhibitor U0126 (20 µM), p38 MAPK pathway inhibitor SB203580 (10 µM), JNK pathway inhibitor SP600125 (25 µM), respectively. GAPDH served as an internal control. Quantitative analysis for the ratios of p-ERK/ERK (B), p-p38/p38 (D) and p-JNK/JNK (F) (**P < 0.01).

Figure 8

The expression of BMP signaling pathway components of DPSCs with LMP-1 induction. (A) Western blot analysis of BMP-2, BMPRIA, BMPRII, p-Smad1/5, and Smad5 in DPSCs after LMP-1 induced, respectively. GAPDH served as an internal control. (B) Quantitative analysis for the BMP-2, BMPRIA and BMPRII (*P < 0.05, **P < 0.01). (C) Quantitative analysis for the ratios of p-Smad1/5/Smad5 (*P < 0.05).