Jeeranan Manokawinchoke

Surface‐bound orientated Jagged‐1 enhances osteogenic differentiation of human periodontal ligament‐derived mesenchymal stem cells

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.

Notch Signaling Is Involved in Neurogenic Commitment of Human Periodontal Ligament-Derived Mesenchymal Stem Cells

Notch signaling plays critical roles in stem cells by regulating cell fate determination and differentiation. The aim of this study was to evaluate the participation of Notch signaling in neurogenic commitment of human periodontal ligament-derived mesenchymal stem cells (hPDLSCs) and to examine the ability to control differentiation of these cells using modified surfaces containing affinity immobilized Notch ligands. Neurogenic induction of hPDLSCs was performed via neurosphere formation. Cells were aggregated and form spheres as early 1 day in culture. In addition, the induced cells exhibited increased mRNA and protein expression of neuronal markers that is, β3-tubulin and neurofilament. During neuronal differentiation, a significant increase of Hes1 and Hey1 mRNA expression was noted. Using pharmacological inhibition (γ-secretase inhibitor) or genetic manipulation (overexpression of dominant negative mastermind-like transcription co-activators), neurosphere formation was attenuated and a marked decrease in neurogenic mRNA expression was observed. To confirm the role of Notch signaling in neuronal differentiation of hPDLSCs, the Notch ligand, Jagged-1, is bound to the surface using an affinity immobilization technique. The hPDLSC cultured on a Jagged-1-modified surface had increased expression of Notch signaling target genes, Hes-1 and Hey-1, confirming the activity and potency of surface-bound Jagged-1. Further, hPDLSC on surface-bound Jagged-1 under serum-free conditions showed multiple long and thin neurite-like extensions, and an increase in the expression of neurogenic mRNA markers was observed. Pretreatment of the cells with γ-secretase inhibitor, DAPT, before seeding on the Jagged-1-modified surface blocked development of the neurite-like morphology. Together, the results in this study suggest the involvement of Notch signaling in neurogenic commitment of hPDLSCs.

bFGF and JAGGED1 regulate alkaline phosphatase expression and mineralization in dental tissue‐derived mesenchymal stem cells

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. J. Cell. Biochem. 114: 2551–2561, 2013.

Iloprost Up-regulates Vascular Endothelial Growth Factor Expression in Human Dental Pulp Cells In Vitro and Enhances Pulpal Blood Flow In Vivo

INTRODUCTION Prostacyclin (PGI2) is a biomolecule capable of enhancing angiogenesis and cellular proliferation. METHODS We investigated the influence of a PGI2 analogue (iloprost) on dental pulp revascularization in vitro and in vivo by using human dental pulp cells (HDPCs) and a rat tooth injury model, respectively. Iloprost stimulated the human dental pulp cell mRNA expression of vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and platelet-derived growth factor (PDGF) in a significant dose-dependent manner. This mRNA up-regulation was significantly inhibited by pretreatment with a PGI2 receptor antagonist and forskolin (a protein kinase A activator). In contrast, a protein kinase A inhibitor significantly enhanced the iloprost-induced mRNA expression of VEGF, FGF-2, and PDGF. Pretreatment with a fibroblast growth factor receptor inhibitor attenuated the VEGF, FGF-2, and PDGF mRNA expression, indicating opposing regulatory mechanisms. RESULTS The effect of iloprost on the dental pulp was investigated in vivo by using a rat molar pulp injury model. The iloprost-treated group exhibited a significant increase in pulpal blood flow at 72 hours compared with control. CONCLUSIONS The present study indicates that iloprost may be a candidate agent to promote neovascularization in dental pulp tissue, suggesting the potential clinical use of iloprost in vital pulp therapy.

Fibronectin supports TNF-α-induced osteopontin expression through β1 integrin and ERK in HN-22 cells

The extracellular matrix (ECM), in collaboration with intracellular signal, plays a critical role in the modulation of cellular behavior and function. Herein, we investigated the influence of fibronectin (FN) and tumor necrosis factor-alpha (TNF-alpha) on OPN expression in HN-22, a human head and neck squamous cell carcinoma (HNSCC) cell line. The data showed that TNF-alpha significantly increased OPN expression only in the FN-coated condition. Application of function-blocking antibody directed against beta1 integrin abolished this OPN induction. Moreover, TNF-alpha when added together with activating beta1 integrin antibody is sufficient to induced OPN expression. The combination effect of FN and TNF-alpha was significantly deteriorated by a MEK inhibitor, but not NF-kappaB inhibitor. We further demonstrated that the phosphorylation of ERK1/2 was strongly enhanced by TNF-alpha and FN compared to the application of either one alone. Synergistic effect on ERK1/2 phosphorylation was also detected by TNF-alpha and activating beta1 integrin antibody, whereas inhibitory antibody to beta1 integrin attenuated FN and TNF-alpha-induced phosphorylation of ERK1/2. Our results indicate that FN coordinates TNF-alpha-mediated OPN induction via beta1 integrin-dependent signaling mechanism that activates ERK. The results suggest the critical role of tumor micro-environment signaling networks on the regulation of cytokine expression profiles during tumor progression.

Indirect immobilized Jagged1 suppresses cell cycle progression and induces odonto/osteogenic differentiation in human dental pulp cells

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.

TNF-α stimulates MMP-3 production via PGE2 signalling through the NF-kB and p38 MAPK pathway in a murine cementoblast cell line

BACKGROUND Cementoblasts are considered to play an important role in the homeostasis of periodontal tissues under both physiologic and pathologic conditions. Matrix metalloproteinases (MMPs) is the key family of enzymes participating in extracellular matrix remodelling. In the present study, the effects and regulatory mechanisms of tumour necrosis factor (TNF)-α on the expression of MMPs and their inhibitors (tissue inhibitor of metalloproteinases; TIMPs) were investigated. MATERIALS AND METHODS OCCM-30, an immortalised murine cementoblast cell line, was stimulated with TNF-α at 1 and 10ng/ml for 24h. The expression of Mmp-2, Mmp-3, Mmp-13, Mmp-14, Timp-1, and Timp-2 as well as PGE2 was determined. Inhibitors of MAPKs, PI3K/Akt, NF-kB and Cox-2 were employed to reveal possible TNF-α induced regulatory signalling pathway(s). The mRNA and protein expression were analysed by (semi)quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS TNF-α dose-dependently stimulated MMP-3 expression by cementoblasts. This was found for mRNA as well as protein expression. No significant differences were found in the mRNA expression of Mmp-2, Mmp-13, Mmp-14, Timp-1, and Timp-2 upon TNF-α stimulation. The level of PGE2, however, was significantly increased along with MMP-3. Treatment with a selective Cox-2 inhibitor resulted in partial suppression of TNF-α-induced Mmp-3 mRNA expression. Addition of PGE2 enhanced Mmp-3 mRNA in a dose dependent manner, suggesting an inductive effect of TNF-α partly via PGE2. The up-regulation of Mmp-3 by TNF-α was completely suppressed by a combination of NF-kB and p38 MAPK inhibitors, while partial suppression was found with each inhibitor. The effect of PGE2 on Mmp-3 expression was abolished by treating cells with an NF-kB inhibitor; a p38 MAPK inhibitor had only a small effect. CONCLUSIONS The present study indicates that cementoblasts respond to TNF-α by increasing MMP-3 production partially via PGE2 and signalling through the NF-kB and p38 MAPK pathway. MMP-3 may participate in periodontal tissue degradation/remodelling.

Characterization and cytological effects of a novel glycated gelatine substrate.

Hyperglycemia in diabetes results in the glycation of long-lived proteins. Protein glycation leads to the formation of advanced glycation end products (AGEs), which are implicated in delayed wound healing and other diabetes-associated pathologies, one of which is periodontal disease. Research into the mechanisms by which glycated long-lived proteins such as collagen exert their effects can allow for the understanding of diabetic pathologies and the development of appropriate treatments. However, the high cost of purified protein can be a limitation for many laboratories around the world. The objective of this study was to develop a low-cost in vitro model of glycated gelatine as an alternative to the glycated collagen model. We investigated the glycation of gelatine type A, a denatured form of collagen, which is low-cost and abundantly available. In this study, gelatine was incubated for 7 days with ribose or methylglyoxal (MG). Cross-linking, autofluorescence and UV-Vis spectrophotometry assays were performed and indicated a dose-dependent linear increase in cross-linking and autofluorescence of gelatine by ribose and MG. MG produced more cross-linking compared to ribose at the same concentrations. The UV-Vis spectra of the glycated gelatines confirmed the presence of AGE fluorophores. Because diabetes is a risk factor for periodontal disease, the effect of the glycated substrates on the basic behaviour of human periodontal ligament (HPDL) cells was evaluated. Glycation dose dependently reduced HPDL attachment and cell spreading, indicating that the novel glycated gelatine substrate affects cell behaviour. These results show that gelatine glycated with ribose or MG can be used as low-cost in vitro models to study the effects of protein glycation on cell behaviour in diabetes and ageing.

Jagged1 inhibits osteoprotegerin expression by human periodontal ligament cells.

BACKGROUND AND OBJECTIVE Notch signaling regulates bone homeostasis. The present study investigated the effect of Jagged1 on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) expression in human periodontal ligament stromal (hPDL) cells. MATERIAL AND METHODS hPDL cells were seeded on to indirect immobilized Jagged1 surfaces. OPG expression was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Lentiviral small hairpin RNA particles against NOTCH2 were employed to inhibit NOTCH2 expression. Osteoclast formation was evaluated using RAW264.7 cells. An influence of exogenous OPG on osteogenic differentiation was determined by real-time polymerase chain reaction and Alizarin Red S staining. RESULTS Jagged1 significantly enhanced HES1 and HEY1mRNA expression in a dose-dependent manner. Furthermore, OPG mRNA and protein levels dramatically decreased upon exposing hPDL cells to Jagged1. However, RANKL mRNA levels were not significantly different. There was also no difference in M-CSF and MCP-1mRNA expression. A γ-secretase inhibitor and cycloheximide treatment rescued Jagged1-attenuated OPG expression. Furthermore, shNOTCH2 overexpressing hPDL cells did not exhibit a decrease in OPG expression upon exposure to Jagged1, implying the involvement of NOTCH2 in the regulatory mechanism. Culturing RAW264.7 cells with conditioned medium from Jagged1-treated hPDL cells enhanced osteoclast formation compared with those cultured with conditioned medium of the control group. Lastly, OPG treatment did not influence osteogenic differentiation by hPDL cells. CONCLUSION These results suggest that Jagged1 activates Notch signaling in hPDL cells, leading to decreased OPG expression. This may imply an indirect role of Jagged1 on the regulation of osteoclast differentiation via hPDL cells.

Notch Signaling Participates in TGF-β-Induced SOST Expression Under Intermittent Compressive Stress: NOTCH INVOLVEMENT IN TGF-β INDUCED SOST

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.