Notch signaling plays critical roles in various cell types by regulating cell fate determination and differentiation. Here, we investigated the ability to control differentiation of human periodontal ligament derived mesenchymal stem cells using modified surfaces containing the affinity immobilized Notch ligand, Jagged-1. After seeding human periodontal ligament derived mesenchymal stem cells (HPDLs) on Jagged-1 modified surfaces, expression of Notch signaling target genes, Hes-1 and Hey-1, was higher than those exposed to soluble Jagged-1 or control surfaces. Upregulation of Notch signaling target genes was attenuated after treatment with the γ secretase inhibitor. Upon seeding the cells on Jagged-1 immobilized surface and maintained in osteogenic medium, alkaline phosphatase enzymatic activity and mineralization as well as mRNA expression of alkaline phosphatase (ALP), collagen type I (COL I) and osteopontin (OPN) were significantly increased compared to those of controls. However, osteocalcin (OCN) mRNA expression level was decreased when cells were exposed to Jagged-1 modified surfaces. HPDLs on Jagged-1 modified surfaces expressed lower TWIST2 mRNA levels than the control, suggesting that the mechanism whereby Jagged-1 enhances osteogenic differentiation of HPDLs may occur through Notch signaling and TWIST regulation. In summary, an alteration of biomaterial interface using Notch ligands illustrates a promising system to control HPDLs differentiation toward osteogenic lineage.

Basic fibroblast growth factor (bFGF) and Notch signaling play critical roles in various cell behaviors. Here, we investigated the influence of bFGF and Notch signaling in alkaline phosphatase (ALP) expression and mineralization process in human periodontal ligament-derived mesenchymal stem cells (PDLSCs) and stem cells isolated from human exfoliated deciduous teeth (SHEDs). PDLSCs and SHEDs were cultured in osteogenic medium supplemented with bFGF or on the immobilized Notch ligands, JAGGED1. The ALP mRNA and protein expression were measured by quantitative reverse transcriptase polymerase chain reaction and enzymatic activity assay, respectively. Mineral deposition was determined using alizarin red S staining. The results showed that the addition of bFGF resulted in the decrease of ALP mRNA expression and enzymatic activity. In addition, the attenuation of mineralization was noted. These phenomenons were blocked by the addition of a fibroblast growth factor receptor inhibitor (SU5402) or a MEK inhibitor (PD98059). Interestingly, bFGF supplementation also decreased the Notch signaling component mRNA levels. Thus, to evaluate effect of Notch signaling in mineralization process, PDLSCs and SHEDs were exposed to JAGGED1 modified surface. The ALP mRNA and protein expression were significantly upregulated and the mineral deposition was markedly increased. These results could be reversed by the addition of a γ-secretase inhibitor. In addition, bFGF could attenuate the Notch-signaling-induced mineralization in both PDLSCs and SHEDs. These results suggest that mineralization was enhanced by Notch signaling but attenuated by bFGF signaling. This knowledge can be further utilized to control PDLSCs and SHEDs mineralization for tissue regeneration purpose.

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.

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The aim of this study was to investigate the response of human periodontal ligament (hPDL) fibroblasts to an intermittent compressive force and its effect on the expression of SOST, POSTN, and TGFB1. A computerized cell compressive force loading apparatus was introduced, and hPDL cells were subjected to intermittent compressive force. The changes in messenger RNA (mRNA) and protein expression were monitored by real-time polymerase chain reaction and Western blot analysis, respectively. An increased expression of SOST, POSTN, and TGFB1 was observed in a time-dependent fashion. Addition of cycloheximide, a transforming growth factor (TGF)-β inhibitor (SB431542), or a neutralizing antibody against TGF-β1 attenuated the force-induced expression of SOST and POSTN as well as sclerostin and periostin, indicating a role of TGF-β1 in the pressure-induced expression of these proteins. Enzyme-linked immunosorbent assay analysis revealed an increased level of TGF-β1 in the cell extracts but not in the medium, suggesting that intermittent compressive force promoted the accumulation of TGF-β1 in the cells or their surrounding matrix. In conclusion, an intermittent compressive force regulates SOST/POSTN expression by hPDL cells via the TGF-β1 signaling pathway. Since these proteins play important roles in the homeostasis of the periodontal tissue, our results indicate the importance of masticatory forces in this process.

Mechanical force regulates periodontal ligament cell (PDL) behavior. However, different force types lead to distinct PDL responses. Here, we report that pretreatment with an intermittent compressive force (ICF), but not a continuous compressive force (CCF), promoted human PDL (hPDL) osteogenic differentiation as determined by osteogenic marker gene expression and mineral deposition in vitro. ICF-induced osterix (OSX) expression was inhibited by cycloheximide and monensin. Although CCF and ICF significantly increased extracellular adenosine triphosphate (ATP) levels, pretreatment with exogenous ATP did not affect hPDL osteogenic differentiation. Gene-expression profiling of hPDLs subjected to CCF or ICF revealed that extracellular matrix (ECM)-receptor interaction, focal adhesion, and transforming growth factor beta (TGF-β) signaling pathway genes were commonly upregulated, while calcium signaling pathway genes were downregulated in both CCF-and ICF-treated hPDLs. The TGFB1 mRNA level was significantly increased, while those of TGFB2 and TGFB3 were decreased by ICF treatment. In contrast, CCF did not modify TGFB1 expression. Inhibiting TGF-β receptor type I or adding a TGF-β1 neutralizing antibody attenuated the ICF-induced OSX expression. Exogenous TGF-β1 pretreatment promoted hPDL osteogenic marker gene expression and mineral deposition. Additionally, pretreatment with ICF in the presence of TGF-β receptor type I inhibitor attenuated the ICF-induced mineralization. In conclusion, this study reveals the effects of ICF on osteogenic differentiation in hPDLs and implicates TGF-β signaling as one of its regulatory mechanisms.

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Notch signaling is regulated by mechanical stimuli in various cell types. It has previously been reported that intermittent compressive stimuli enhanced sclerostin (SOST) expression in human periodontal ligament cells (hPDLs) by regulating transforming growth factor-β (TGF-β) expression. The aim of the present study was to determine the involvement of Notch signaling in the TGF-β-induced SOST expression in hPDLs. Cells were treated with intermittent compressive stress in a computer-controlled apparatus for 24 h. The mRNA and protein expression of the cells were determined by real-time polymerase chain reaction and Western blot analysis, respectively. In some experiments, the target signaling pathway was impeded by the addition of a TGF-β receptor kinase inhibitor (SB431542) or a γ-secretase inhibitor (DAPT). The results demonstrated that hPDLs under intermittent compressive stress exhibited significantly higher NOTCH2, NOTCH3, HES1, and HEY1 mRNA expression compared with control, indicating that mechanical stress induced Notch signaling. DAPT pretreatment markedly reduced the intermittent stress-induced SOST expression. The expression of NOTCH2, NOTCH3, HES1, and HEY1 mRNA under compressive stress was significantly reduced after pretreatment with SB431542, coinciding with a reduction in SOST expression. Recombinant human TGF-β1 enhanced SOST, Notch receptor, and target gene expression in hPDLs. Further, DAPT treatment attenuated rhTGF-β1-induced SOST expression. In summary, intermittent compressive stress regulates Notch receptor and target gene expression via the TGF-β signaling pathway. In addition, Notch signaling participates in TGF-β-induced SOST expression in hPDLs. J. Cell. Physiol. 232: 2221-2230, 2017. © 2016 Wiley Periodicals, Inc.