Sphingosine-1-phosphate hinders the osteogenic differentiation of dental pulp stem cells in association with AKT signaling pathways

Abstract

Sphingosine-1-phosphate (S1P) is an important lipid mediator that regulates a diverse range of intracellular cell signaling pathways that are relevant to tissue engineering and regenerative medicine. However, the precise function of S1P in dental pulp stem cells (DPSCs) and its osteogenic differentiation remains unclear. We here investigated the function of S1P/S1P receptor (S1PR)-mediated cellular signaling in the osteogenic differentiation of DPSCs and clarified the fundamental signaling pathway. Our results showed that S1P-treated DPSCs exhibited a low rate of differentiation toward the osteogenic phenotype in association with a marked reduction in osteogenesis-related gene expression and AKT activation. Of note, both S1PR1/S1PR3 and S1PR2 agonists significantly downregulated the expression of osteogenic genes and suppressed AKT activation, resulting in an attenuated osteogenic capacity of DPSCs. Most importantly, an AKT activator completely abrogated the S1P-mediated downregulation of osteoblastic markers and partially prevented S1P-mediated attenuation effects during osteogenesis. Intriguingly, the pro-inflammatory TNF-α cytokine promoted the infiltration of macrophages toward DPSCs and induced S1P production in both DPSCs and macrophages. Our findings indicate that the elevation of S1P under inflammatory conditions suppresses the osteogenic capacity of the DPSCs responsible for regenerative endodontics.

The Ying and Yang of Sphingosine-1-Phosphate Signalling within the Bone

“…Matsuzaki et al first showed that S1P increased the protein expression and activation of PI3K/Akt/GSK-3β signalling at increasing concentrations of S1P (0.1–2 µM), resulting in increased ALP activity (p-nitrophenyl phosphate substrate levels) and increased Alizarin red-stained mineral [ 53 ]. Increased mineralisation in response to increasing concentrations of S1P (1–10 µmol-L −1 ) was replicated in MC3T3-E1 cells by Choi et al [ 47 ]. SaOS-2 cells, a human osteoblastic osteosarcoma cell line, also show increased PI3K/Akt/GSK-3β signalling in response to S1P stimulation, which resulted in enhanced ALP levels and mineralisation [ 53 ].…”

“…Additionally, S1P increased mineralisation (Alizarin red staining), suggesting it promotes MSC-like cells to differentiation towards an osteoblast lineage [ 50 , 51 ]. A recent study into the effect of S1P on murine dental pulp stem cells, which display a MSC-like phenotype and express S1PR1 and 2, showed that low levels of osteoblastic differentiation can be induced in response to S1P at various concentrations (1–10 µmol-L −1 ) [ 47 ]. These cells also displayed decreased expression of several osteoblast-specific genes (including ALP, RUNX2, and COL1A1), as well as decreased mineralisation following 3 weeks of culture with S1P [ 47 ].…”

“…A recent study into the effect of S1P on murine dental pulp stem cells, which display a MSC-like phenotype and express S1PR1 and 2, showed that low levels of osteoblastic differentiation can be induced in response to S1P at various concentrations (1–10 µmol-L −1 ) [ 47 ]. These cells also displayed decreased expression of several osteoblast-specific genes (including ALP, RUNX2, and COL1A1), as well as decreased mineralisation following 3 weeks of culture with S1P [ 47 ]. The differences between these two studies may stem from differences between the two cell types used, with the S1PR expression on C3H10T1/2 yet to be reported.…”

“…Studies have also looked at S1P in pre-osteoblasts and osteoblast-like cell lines [ 47 , 52 , 53 ]. Murine MC3T3-E1 cells are an immature osteoblast cell line that matures in response to S1P [ 47 , 53 ].…”

“…Studies have also looked at S1P in pre-osteoblasts and osteoblast-like cell lines [ 47 , 52 , 53 ]. Murine MC3T3-E1 cells are an immature osteoblast cell line that matures in response to S1P [ 47 , 53 ]. Matsuzaki et al first showed that S1P increased the protein expression and activation of PI3K/Akt/GSK-3β signalling at increasing concentrations of S1P (0.1–2 µM), resulting in increased ALP activity (p-nitrophenyl phosphate substrate levels) and increased Alizarin red-stained mineral [ 53 ].…”