Transforming growth factor-β1 promotes early odontoblastic differentiation of dental pulp stem cells via activating AKT, Erk1/2 and p38 MAPK pathways

Abstract

Background/purpose: TGF-β1 (Transforming growth factor-β1) plays an important role in the regeneration and repair of pulp-dentin complex. However, the biological function of TGF-β1 on odontoblastic differentiation remains unclear, mainly due to the processes of differentiation were controlled by complex signaling pathways. This study aimed to investigate the signaling pathways involved in regulating the early differentiation of dental pulp stem cells (DPSCs) by TGF-β1 and their functional role.

Materials and methods: DPSCs were treated with 1 ng/mL TGF-β1 and Western blotting was conducted to examine the activation of protein kinase B (AKT), small mothers against decapentaplegic 3 (Smad3), p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (Erk1/2). DPSCs were exposed to mineralization medium contained TGF-β1 with/without the specific signaling pathway inhibitors, and early odontogenic differentiation was evaluated by assessing the expression of alkaline phosphatase (ALP), collagen type 1 alpha 1 (COL1A), dentin matrix protein 1 (DMP-1) and runt-related transcription factor 2 (Runx2).

Results: TGF-β1 stimulated AKT, Smad3, p38 MAPK, Erk1/2 and JNK phosphorylation in DPSCs within 120 min. TGF-β1 enhanced ALP activity and elevated levels of COL1A, DMP-1 and Runx2. LY294002, U0126 and SB203580 attenuated the effect of TGF-β1 on DPSCs, however, the SIS3 and SP600125 treated groups had no significant effect.

Conclusion: TGF-β1 promotes the early stage of odontoblastic differentiation in DPSCs by activating AKT, Erk1/2 and p38 MAPK signaling pathways, but not by Smad3 and JNK.

Keywords: AKT; Dental pulp stem cells; MAPK; Odontoblastic differentiation; Smad3; Transforming growth factor-β1.

Figure 1

Characterization of DPSCs. (A) Primary DPSCs cultures on day 5 (40×). (B) DPSCs at passage 3 exhibit a spindle-shaped morphology (40×). (C) Calcified nodules stained with Alizarin red S (50×). D: Oil red O staining for lipid droplets (200×). E: Flow cytometry showed that cells were positive for CD29, CD90, and CD146, but negative for CD34 and CD45. Scale bars are shown. DPSCs, dental pulp stem cells.

Figure 2

TGF-β1 actives AKT, Smad3 and MAPK pathways in DPSCs. (A) Western blot analysis of p-AKT, AKT, p-p38 MAPK, p38 MAPK, p-Smad3, Smad3, p-JNK, JNK, p-Erk and Erk from DPSCs treated with 1 ng/mL TGF-β1 in a time course. (B) Western blot analysis of p-AKT, p-p38, p-Smad3, p-JNK, p-Erk in DPSCs after treatment with pathways inhibitor respectively for 1 h. Error bars = means ± standard deviation (SD), n = 3, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. AKT, protein kinase B; DPSCs, dental pulp stem cells; Erk 1/2, extracellular signal-regulated kinase 1/2; JNK, c-Jun N-terminal kinase and; p38 MAPK, p38 mitogen-activated protein kinase; Smad3, small mothers against decapentaplegic 3; TGF-β1, Transforming growth factor-β1.

Figure 3

Involvement of p38 MAPK, Erk1/2 and AKT signaling in the TGF-β1-induced early odontoblastic differentiation of DPSCs. (A) ALP activities and ALP staining in the control group, TGF-β1 treated groups with or without pathways inhibitors at day 7(50×). (B) The protein expressions of DMP-1, COL1A, and Runx2 in different groups at day 7. Scale bars are shown. Error bars = means ± standard deviation (SD), n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. NS P > 0.5. COL1A, collagen type 1 alpha 1; DPSCs, dental pulp stem cells; DMP-1, dentin matrix protein 1; Runx2, runt-related transcription factor 2; TGF-β1, Transforming growth factor-β1.

Figure 4

Schematic illustration showing the molecular mechanism of TGF-β1 on early odontoblastic differentiation of DPSCs. AKT, protein kinase B; DPSCs, dental pulp stem cells; Erk 1/2, extracellular signal-regulated kinase 1/2; JNK, c-Jun N-terminal kinase and; p38 MAPK, p38 mitogen-activated protein kinase; Smad3, small mothers against decapentaplegic 3; TGF-β1, Transforming growth factor-β1.