Min Lian

JAB1 accelerates odontogenic differentiation of dental pulp stem cells

Jun activation domain-binding protein 1 (JAB1) is a multifunctional protein that participates in the control of cell proliferation and the stability of multiple proteins. JAB1 regulates several key proteins, and thereby produces varied effects on cell cycle progression, genome stability and cell survival. Some studies have shown that the loss of JAB1 in osteochondral progenitor cells severely impairs embryonic limb development in mice. However, the biological significance of JAB1 activity in the odontogenic differentiation of dental pulp stem cells (DPSCs) remains unclear. This study aimed to determine the role of JAB1, a key player in tooth development, in reparative dentin formation, especially odontogenic differentiation. We found that increased expression of JAB1 promoted odontogenic differentiation of DPSCs via Wnt/β-catenin signaling. The role of JAB1 in the odontogenic differentiation of DPSCs was further confirmed by knocking down JAB1. Our findings provide novel insights on odontogenic differentiation of DPSCs.

AGS3 is involved in TNF-α medicated osteogenic differentiation of human dental pulp stem cells.

Dental pulp stem cells (DPSCs) are multipotent adult stem cells capable of differentiating along the osteoblast, adipocyte, and chondrocyte lineages. Regulating differentiation of DPSCs may be a useful tool for regenerative medicine and cell-based therapy in oral diseases. Multisignaling pathways are involved in osteogenic differentiation of DPSCs. Recent studies show that cAMP/PKA/CREB signaling could stimulate the expression of genes such as bone morphogenic proteins 2 (BMP2), inhibitor of DNA binding 2 (ID2), bone sialoprotein, osteocalcin, and type XXIV collagen, which have been implicated in osteogenesis and bone formation. Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, plays an important role in regulating the phosphorylation of cyclic AMP response element-binding protein (p-CREB). However, the involvement of AGS3 in osteogenic differentiation of DPSCs has not been explored. Our data indicated that increased expression of AGS3 would inhibit osteogenic differentiation of DPSCs exposed to inflammatory cytokine tumor necrosis factor α (TNF-α) via cAMP/PKA/CREB signaling. The negative role of AGS3 in osteogenic differentiation was further confirmed by knocking down and over expression of AGS3. Our findings may provide clinical implications for osteoporosis.

Expression of Peroxiredoxin 1 After Traumatic Spinal Cord Injury in Rats

Reactive astrogliosis and microgliosis after spinal cord injury (SCI) contribute to glial scar formation that impedes axonal regeneration. The mechanisms underlying reactive astrocyte and microglia proliferation upon injury remain partially understood. Peroxiredoxin 1 (PRDX1) is an antioxidant participating in cell proliferation, differentiation, and apoptosis. However, PRDX1 functions in SCI-induced astrocyte and microglia proliferation are unknown. In this study, we established an acute spinal cord contusion injury model in adult rats to investigate the potential role of PRDX1 during the pathological process of SCI. We found the palpable expression increase of PRDX1 after SCI by western blot and immunohistochemistry staining. Double immunofluorescence staining showed that PRDX1 expression mainly increased in astrocytes and microglia. In addition, PRDX1/proliferating cell nuclear antigen (PCNA) colocalized in astrocytes and microglia. Furthermore, PCNA expression also elevated after SCI, as well as was positively correlated with PRDX1 expression. In vitro, PRDX1 expression in primary rat spinal cord astrocytes and microglia changed in a concentration- and time-dependent manner according to LPS treatment. In addition, PRDX1 knockdown in astrocytes and microglia resulted in the decrease of PCNA expression after LPS stimulation, showing that PRDX1 promoted astrocyte and microglia proliferation after inflammation. Our results suggested that PRDX1 might play a crucial role in astrocyte and microglia proliferation after SCI.

RAC1 regulate tumor necrosis factor-α-mediated impaired osteogenic differentiation of dental pulp stem cells.

Human dental pulp contains a rapidly proliferative subpopulation of precursor cells termed dental pulp stem cells (DPSCs) that show self‐renewal and multilineage differentiation, including neurogenic, chondrogenic, osteogenic and adipogenic. We previously reported that tomuor necrosis factor‐α (TNF‐α) (10 ng/mL) triggered osteogenic differentiation of human DPSCs via the nuclear factor‐κB (NF‐κB) signaling pathway. While previous studies showed that cells treated with TNF‐α at higher concentrations showed decreased osteogenic differentiation capability. In this study we analyze the function of TNF‐α (100 ng/mL) on osteogenic differentiation of human DPSCs for the first time and identify the underlying molecule mechanisms. Our data revealed that TNF‐α with higher concentration significantly reduced mineralization and the expression of bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP) and runt‐related transcription factor 2 (RUNX2). Further, we revealed that TNF‐α could suppress the osteogenic differentiation of DPSCs via increasing the expression of RAC1, which could activate the Wnt/β‐catenin signaling pathway and liberate β‐catenin to translocate into the nucleus. Genetic silencing of RAC1 expression using siRNA restored osteogenic differentiation of DPSCs. Our findings may provide a potential approach to bone regeneration in inflammatory microenvironments.

Effects of Nerve Growth Factor and Basic Fibroblast Growth Factor Promote Human Dental Pulp Stem Cells to Neural Differentiation

Dental pulp stem cells (DPSCs) were the most widely used seed cells in the field of neural regeneration and bone tissue engineering, due to their easily isolation, lack of ethical controversy, low immunogenicity and low rates of transplantation rejection. The purpose of this study was to investigate the role of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on neural differentiation of DPSCs in vitro. DPSCs were cultured in neural differentiation medium containing NGF and bFGF alone or combination for 7 days. Then neural genes and protein markers were analyzed using western blot and RT-PCR. Our study revealed that bFGF and NGF increased neural differentiation of DPSCs synergistically, compared with bFGF and NGF alone. The levels of Nestin, MAP-2, βIII-tubulin and GFAP were the most highest in the DPSCs + bFGF + NGF group. Our results suggested that bFGF and NGF signifiantly up-regulated the levels of Sirt1. After treatment with Sirt1 inhibitor, western blot, RT-PCR and immunofluorescence staining showed that neural genes and protein markers had markedly decreased. Additionally, the ERK and AKT signaling pathway played a key role in the neural differentiation of DPSCs stimulated with bFGF + NGF. These results suggested that manipulation of the ERK and AKT signaling pathway may be associated with the differentiation of bFGF and NGF treated DPSCs. Our date provided theoretical basis for DPSCs to treat neurological diseases and repair neuronal damage.

CKIP-1 suppresses odontoblastic differentiation of dental pulp stem cells via BMP2 pathway and can interact with NRP1

ABSTRACT Aim: Casein kinase 2 interacting protein-1 (CKIP-1) is a recently discovered intracellular regulator of bone formation, muscle cell differentiation, and tumor cell proliferation. Our study aims to identify the inhibition of BMP2-Smad1/5 signaling by CKIP-1 in odontoblastic differentiation of human dental pulp stem cells (DPSCs). Materials and Methods: DPSCs infected CKIP-1 siRNA or transfected CKIP-1 full-length plasmid were cultured in odontoblastic differentiation medium or added noggin (200 ng/mL) for 21 days. We examined the effects of CKIP-1 on odontoblastic differentiation, mineralized nodules formation, and interaction by western blot, real-time polymerase chain reaction (RT-PCR), alkaline phosphatase (ALP) staining, alizarin red S staining, and immunoprecipitation. Results: Firstly, we have demonstrated that CKIP-1 expression markedly decreased time-dependently along with cell odontoblastic differentiation. Indeed, the silence of CKIP-1 upregulated odontoblastic differentiation via BMP2-Smad1/5 signaling, while CKIP-1 over-expression had a negative effect on odontoblastic differentiation of DPSCs. Furthermore, CKIP-1 could interact with Neuropilin-1 (NRP1). Conclusions: This work provides data that advocates a novel perception on odontoblastic differentiation of DPSCs. Therefore, inhibiting the expression of CKIP-1 may be of great significance to the development of dental caries.

Wedelolactone Enhances Odontoblast Differentiation by Promoting Wnt/β-Catenin Signaling Pathway and Suppressing NF-κB Signaling Pathway

Wedelolactone is a multitarget natural plant compound with many pharmacological activities, including anti-inflammatory, anticancer, and antiosteoporosis. In this study, dental pulp stem cells (DPSCs) were treated with or without wedelolactone. We found that wedelolactone stimulated odontoblast differentiation and mineralization. At the molecular level, wedelolactone directly promoted the nuclear accumulation of β-catenin, and thereafter stimulated the expression of odontoblast-related marker genes containing dentin matrix protein-1 (DMP1), dentin sialophosphoprotein (DSPP), and runt-related transcription factor 2 (Runx2). Furthermore, wedelolactone upregulated the expression of IκBα and inhibited phosphonation and nuclear migration of p65. As a result, wedelolactone remarkably induced odontoblast differentiation through semaphorin 3A (Sema3A)/neuropilin-1 (NRP1) pathway-mediated β-catenin activation and nuclear factor kappa B (NF-κB) pathway inhibition. Our findings provide novel perceptions on odontogenic differentiation of DPSCs.

The Forkhead Box C1, a Novel Negative Regulator of Osteogenesis, Plays a Crucial Role in Odontogenic Differentiation of Dental Pulp Stem Cells

The forkhead box C1 (Foxc1) protein, a member of the forkhead/winged helix transcription factor family, is required in stem cell developmental processes. Recently, multiple studies have indicated the crucial role of Foxc1 in mesenchymal stem cell differentiation, but the precise effects and mechanisms on dental pulp stem cells (DPSCs) remain unclear. In this study, we evaluate the role of Foxc1 on the odontogenic differentiation and proliferation of DPSCs. Our results show that Foxc1 decreases time dependently in odontogenic differentiation of DPSCs. Meanwhile, overexpression of Foxc1 could significantly inhibit the mineralization of DPSCs and the expression of odontogenic-related genes, such as runt-related transcription factor 2 (Runx2), dentin sialophosphoprote (DSPP), and dentin matrix acidic phosphoprotein 1 (DMP-1). Foxc1 overexpression does not significantly alter the proliferation of DPSCs. In addition, Foxc1 reduces the expression of p-Smad1/5, an important modulator of bone morphogenetic protein (BMP)/Smad signaling pathway, inhibiting BMP/Smad signaling pathway. In conclusion, our data demonstrated that Foxc1 inhibits odontogenic differentiation of DPSCs and odontogenic-related gene expression through the BMP/Smad signaling pathway which may be useful for the dental regeneration and repair.

The Role of Neuropilin-1–FYN Interaction in Odontoblast Differentiation of Dental Pulp Stem Cells

Abnormal odontoblast differentiation of dental pulp stem cells (DPSCs) caused by inflammation is closely related to the development of dental caries. Neuropilin-1 (NRP1) is one of the members of neuropilin family. It can combine with disparate ligands involved in regulating cell differentiation. FYN belongs to the protein-tyrosine kinase family, which has been implicated in the control of cell growth, and the effect can be further strengthened by inflammatory factors. In our studies, we verified that NRP1 can form complexes with FYN and have the correlation changes in odontoblast differentiation of DPSCs. Therefore, we surmise that in the progress of dental caries, NRP1 interacts with FYN, by expanding inflammation and inhibition of odontoblast differentiation of DPSCs through nuclear factor kappa B (NF-κB) signaling pathway. In this subject, we first investigated the expression and interaction of NRP1 and FYN in DPSCs. And then, we researched the effect of this complex controlling downstream signal pathway in normal or inflammation stimulated DPSCs. Finally, we analyzed the relationship between this role and odontoblast differentiation of DPSCs. This research will provide the molecular mechanism of inflammation factors of dental caries through activating NF-κB signal regulating odontoblast differentiation in DPSCs for finding new potential drug targets for the clinical treatment of dental caries.

Clinical Significance and Prognostic Value of the Expression of LAMP3 in Oral Squamous Cell Carcinoma

Recent studies demonstrated high expression of lysosome-associated membrane protein 3 (LAMP3) in a variety of malignancies including esophageal squamous cell carcinoma, gastrointestinal cancer, breast cancer, and cervical cancer and its involvement in several biological activities of tumor cells. However, the expression of LAMP3 and its value in oral squamous cell carcinoma (OSCC) remain unclear. In this study, we examined the expression of LAMP3 in OSCC tissue samples and investigated the relationship between LAMP3 and clinical characteristics of patients with OSCC. We examined mRNA and protein levels of LAMP3 in OSCC tissues and neighboring normal tissues using quantitative real-time polymerase chain reaction and immunohistochemistry analyses, respectively. Both the mRNA and protein levels of LAMP3 were significantly higher in OSCC tissues than in adjacent normal tissues. Chi-square analysis showed that the high LAMP3 expression was notably linked to the degree of tumor differentiation and advanced TNM stage. Univariate and multivariate analyses showed that the high LAMP3 expression was an independent prognostic marker in OSCC. Our results suggest that LAMP3 might act as a potential anticancer target and a prognostic marker in patients with OSCC.