Enhanced reparatory effect of EI1 on dental pulp via extracellular matrix remodeling by miR-181b-2-3p inhibitor.

Background/purpose: Extracellular matrix (ECM) is crucial for dental pulp repair. The aim of this paper is to investigate the ECM remodeling effect of miR-181b-2-3p (a microRNA) and to verify the reparatory effect of EI1 (an epigenetic drug) and miR-181b-2-3p inhibitor on dental pulp. Materials and methods: Levels of ECM-related factors in EI1-treated human dental pulp cells (hDPCs) were measured by qRT-PCR and Western blot. The anti-inflammation effect of EI1 was examined in Lipopolysaccharide-stimulated hDPCs. miR-181b-2-3p mimics or inhibitors were transfected into hDPCs and then the cells' functions were detected. A dual luciferase reporter assay was used to identify the targets of miR-181b-2-3p. Pulpotomy using miR-181b-2-3p antagomirs and EI1 as pulp capping materials was performed in male six-week-old Sprague-Dawley rats. Results: EI1 upregulated ECM-related genes expression in hDPCs, but failed to upregulate the collagen1A1 (COL1A1) protein level. Pro-inflammatory factors were downregulated by EI1 in Lipopolysaccharide-stimulated hDPCs. Overexpression of miR-181b-2-3p downregulated the expression of transforming growth factor-ß2 (TGF-ß2) and fibronectin type III domain-containing protein 5 precursor (FNDC5), while the inhibition had the opposite effect. Dual luciferase reporter assays demonstrated that miR-181b-2-3p targets TGF-ß2, FNDC5 and integrin alpha 4 protein (ITGA4). Compared to EI1 was used alone, EI1 combined with the inhibitor upregulated the protein levels of COL1A1, fibronectin (FN1) and TGF-ß2 in hDPCs, promoted hDPCs migration, and exhibited reparatory effects on inflamed rat pulp tissue. Conclusion: miR-181b-2-3p inhibitor could enhance the reparatory effect of EI1 via ECM remodeling in dental pulp both in vitro and in vivo.

EZH2 Might Affect Macrophage Chemotaxis and Anti-Inflammatory Factors by Regulating CCL2 in Dental Pulp Inflammation.

OBJECTIVES: We aimed to evaluate the effects of Enhancer of Zeste Homolog 2 (EZH2) on regulation of macrophage migration and expression of anti-inflammatory genes in pulpitis. METHODS: Dental pulp inflammation was verified by histology in rat pulpitis model induced by lipopolysaccharide (LPS). Immunohistochemistry staining was used to detect changes of the expression of EZH2 and tumor necrosis factor alpha (TNF-α) in dental pulp inflammation. The expression of EZH2, CCL2, and cluster of differentiation 68 (CD68: macrophage surface marker) was measured by immunofluorescence staining. The effect of EZH2 on microphage migration was assessed by cell migration assay. The expressions of anti-inflammatory cytokine interleukins (IL-4 and IL-10) and transforming growth factor-ß (TGF-ß) in HDPCs which were treated by EZH2 complex, CCL2 complex, and CCL2 antibody were examined by quantitative real-time polymerase chain reaction (q-PCR). RESULTS: The expression of TNF-α gradually increased in dental pulp inflammation. The expression of EZH2 in dental pulp decreased in 8 hours after LPS stimulation. However, the expression of EZH2 gradually increased in dental pulp after 1 day stimulation by LPS. The results of immunofluorescence staining showed that the expressions of EZH2, CCL2, and CD68 were significantly upregulated in dental pulp inflammation of rats. EZH2 could enhance macrophage migration. And the chemotactic activity of macrophages exposed to supernatants of EZH2-treated HDPCs could be inhibited by CCL2 inhibition. In addition, EZH2 suppressed the expression of anti-inflammatory genes, but CCL2 inhibition reversed the downregulation of anti-inflammatory factors, including IL-4 and TGF-ß in HDPCs. CONCLUSIONS: EZH2 might affect chemotaxis of macrophages and the expression of anti-inflammatory factors by regulating CCL2. EZH2 plays an important role in the development of dental pulp inflammation, and it might be as a target for treatment of pulpitis.

EZH2 Promotes Extracellular Matrix Degradation via Nuclear Factor-κB (NF-κB) and p38 Signaling Pathways in Pulpitis.

Pulpitis is a complicated chronic inflammatory process which can be in a dynamic balance between damage and repair. The extracellular matrix plays an important regulatory role in wound healing and tissue repair. The aim of this study was to explore the role of the epigenetic mark, enhancer of zeste homolog 2 (EZH2) on the degradation of extracellular matrix during pulpitis. Quantitative polymerase chain reaction was used to assess the expression of matrix metalloproteinases (MMPs) and type I collagen in human dental pulp cells (HDPCs) upon EZH2 and EI1 (EZH2 inhibitor) stimulation. The mechanism of EZH2 affecting extracellular matrix was explored through quantitative polymerase chain reaction and Western blot. A rat model of dental pulp inflammation was established, and the expression of type I collagen in dental pulp under EZH2 stimulation was detected by immunohistochemical staining. EZH2 upregulated the expression of MMP-1, MMP-3, MMP-8, and MMP-10 and decreased the production of type I collagen in HDPCs, while EI1 had the opposite effect. EZH2 activated the nuclear factor-kappa B (NF-κB) and p38 signaling pathways in HDPCs, the inhibition of which reversed the induction of MMPs and the suppression of type I collagen. EZH2 can downregulate the type I collagen levels in an experimental model of dental pulpitis in rats. EZH2 promotes extracellular matrix degradation via nuclear factor-κB (NF-κB) and P38 signaling pathways in pulpitis. EZH2 can decrease the type I collagen levels in vivo and in vitro.

Epigenetic regulation of matrix metalloproteinases in inflammatory diseases: a narrative review.

With the acceleration of urbanization and aging and the change of lifestyle, inflammatory diseases have become one of the important threats to the health of the global population. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are involved in the metabolism of extracellular matrix (ECM). They play a key role in inflammation-related diseases. Factors such as inflammation, oxidative stress and growth factors stimulate the production of MMPs with subsequent ECM remodeling. Recently, the studies of epigenetic regulation, including the ability to predict disease progression, important pathophysiological deficiencies as well as treatment methods have been extensively discussed. This article reviews the current studies on epigenetic alterations in MMPs during inflammatory response. It is likely to provide new insights into development of efficient medications of epigenetic therapy for inflammatory diseases.

Multi-lineage differentiation and clinical application of stem cells from exfoliated deciduous teeth.

Stem cells from human exfoliated deciduous teeth (SHED) have now been considered one of the most promising sources of stem cells for tissue engineering and stem cell therapies due to their stemness and potential to differentiate into other cell lines. The high proliferation rate, the differentiation capacity, the easy access and less ethical concerns make SHED a brilliant solution for many diseases. The purpose of this review is to describe current knowledge of SHED's capability of differentiation, applications and immune status and to draw attention to further research on the mechanism and the dependability of stem cell therapy with SHED.

Activation of ß-catenin signaling in aggrecan-expressing cells in temporomandibular joint causes osteoarthritis-like defects.

ß-Catenin plays a critical role in cartilage formation and development. To further understand the role of ß-catenin in osteoarthritis (OA) development in temporomandibular joint (TMJ), we have generated ß-catenin conditional activation mice (ß-cat(ex3) Agc1CreER ) by breeding Agc1-CreER mice with ß-cateninflox(ex3)/+ mice. Results of histologic analysis showed the progressive TMJ defects in 3- and 6-month-old ß-cat(ex3) Agc1CreER mice (tamoxifen induction was performed at 2 weeks of age), including decreased chondrocyte numbers in the superficial layer associated with less Alcian blue staining, increased numbers of hypertrophic chondrocytes in deep layers, and rough articular surface. Compared to the TMJ phenotype of ß-cat(ex3) Col2CreER mice, ß-cat(ex3) Agc1CreER mice showed much severe morphological defects in the superficial layer of TMJ. This may reflect that Agc1-CreER mice could efficiently target cells in the superficial layer of TMJ. Results of immunostaining showed significantly increased expression of MMP13, Col-X, Adamts4, and Adamts5 in TMJ of ß-cat(ex3) Agc1CreER mice. Results of proliferating cell nuclear antigen (PCNA), Ki67, and terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) staining further demonstrated that cell proliferation was decreased and cell apoptosis was increased in condylar cartilage of ß-cat(ex3) Agc1CreER mice. Our findings indicate that abnormal upregulation of ß-catenin in TMJ leads to defects assembling to OA-like phenotype, further demonstrating that ß-catenin plays a critical role in TMJ pathogenesis.

EZH2 regulates dental pulp inflammation by direct effect on inflammatory factors.

OBJECTIVE: Pulpitis is a multi-factorial disease that could be caused by complex interactions between genetics, epigenetics and environmental factors. We aimed to evaluate the role of Enhancer of Zeste Homolog 2 (EZH2) in the inflammatory response of human dental pulp cells (HDPCs) and dental pulp tissues. METHODS: The expressions of inflammatory cytokines in HDPCs treated by EZH2 complex or EZH2 siRNA with or without rhTNF-α were examined by quantitative real-time polymerase chain reaction (q-PCR). The levels of secreted inflammatory cytokines including IL-6, IL-8, IL-15, CCL2 and CXCL12 in culture supernatants were measured by Luminex assay. In rat pulpitis model, the effects of EZH2 on dental pulp tissues were verified by histology. We invested the mechanisms of the effect of EZH2 on the inflammatory factors by ChIP assay. RESULTS: EZH2 down-regulation inhibited the expression of inflammatory factors, including IL-6, IL-8, IL-15, CCL2 and CXCL12 in HDPCs. EZH2 complex promoted the expression and secretion of these inflammatory factors in HDPCs, while EZH2 silencing could attenuate the promotion of inflammatory factors that were induced by rhTNF-α. In pulpitis models of rats, EZH2 down-regulation inhibited the inflammatory process of dental pulp while EZH2 complex showed no significant facilitation of pulpal inflammation. In addition, EZH2 could bind on the promoters of IL-6, IL-8 and CCL2, but not IL-15 and CXCL12, to affect the transcription of these proinflammatory cytokines. CONCLUSIONS: In HDPCs, EZH2 could induce inflammation, while EZH2 down-regulation could attenuate the inflammatory responses. EZH2 plays an important role in this inflammatory process of dental pulp.

Wnt/ß-catenin signaling plays a key role in the development of spondyloarthritis.

Spondyloarthritis (SpA) is a group of diseases consisting of psoriatic arthritis (PsA), reactive arthritis, arthritis related to inflammatory bowel disease (a subgroup of juvenile idiopathic arthritis), and ankylosing spondylitis (the prototype of SpA). Axial bone formation and the combination of concurrent erosion and new bone formation are specific characteristics of SpA disease. The use of antiproinflammatory cytokines, such as inhibitors of tumor necrosis factor α (TNF-α), appears to be the greatest advance in the treatment of SpA over the past 20 years. However, TNF-α blockers do not halt new bone formation. Recent clinical observations and animal studies demonstrate that Wnt signaling proteins and natural Wnt inhibitors, such as DKK1 and sclerostin, are likely to play important roles in the process of ankylosis in SpA, and could potentially serve as therapeutic targets for the treatment of SpA.

Bone morphogenetic protein 2-induced human dental pulp cell differentiation involves p38 mitogen-activated protein kinase-activated canonical WNT pathway.

Both bone morphogenetic protein 2 (BMP2) and the wingless-type MMTV integration site (WNT)/ß-catenin signalling pathway play important roles in odontoblast differentiation and dentinogenesis. Cross-talk between BMP2 and WNT/ß-catenin in osteoblast differentiation and bone formation has been identified. However, the roles and mechanisms of the canonical WNT pathway in the regulation of BMP2 in dental pulp injury and repair remain largely unknown. Here, we demonstrate that BMP2 promotes the differentiation of human dental pulp cells (HDPCs) by activating WNT/ß-catenin signalling, which is further mediated by p38 mitogen-activated protein kinase (MAPK) in vitro. BMP2 stimulation upregulated the expression of ß-catenin in HDPCs, which was abolished by SB203580 but not by Noggin or LDN193189. Furthermore, BMP2 enhanced cell differentiation, which was not fully inhibited by Noggin or LDN193189. Instead, SB203580 partially blocked BMP2-induced ß-catenin expression and cell differentiation. Taken together, these data suggest a possible mechanism by which the elevation of ß-catenin resulting from BMP2 stimulation is mediated by the p38 MAPK pathway, which sheds light on the molecular mechanisms of BMP2-mediated pulp reparative dentin formation.

Epigenetic and microRNA regulation during osteoarthritis development.

Osteoarthritis (OA) is a common degenerative joint disease, the pathological mechanism of which is currently unknown. Genetic alteration is one of the key contributing factors for OA pathology. Recent evidence suggests that epigenetic and microRNA regulation of critical genes may contribute to OA development. In this article, we review the epigenetic and microRNA regulations of genes related to OA development. Potential therapeutic strategies may be developed on the basis of novel findings.

EZH2, a potential regulator of dental pulp inflammation and regeneration.

INTRODUCTION: Dental pulp has limited capability to regenerate, which happens in the early stage of pulpitis. An ambiguous relationship exists; inflammation may impair or support pulp regeneration. Epigenetics, which is involved in cell proliferation and inflammation, could regulate human dental pulp cell (HDPCs) regeneration. The aim of this study was to determine the role of the epigenetic mark, enhancer of zeste homolog 2 (EZH2), in the inflammation, proliferation, and regeneration of dental pulp. We used trimethylated histone H3 lysine 27(H3K27me3) and its lysine demethylase 6B (KDM6B) to monitor functional effects of altered EZH2 levels. METHODS: We detected epigenetic marks (EZH2, H3K27me3, and KDM6B) in pulp tissue by immunohistochemistry and immunofluorescence. EZH2 levels in HDPCs in inflammatory responses or differentiation were analyzed by quantitative polymerase chain reaction and Western blot. Quantitative polymerase chain reaction was used to assess the effects of EZH2 inhibition on interleukins in HDPCs upon tumor necrosis factor alpha stimulation. Cell proliferation was tested by cell counting kit-8, cell cycle, and apoptosis analysis. HDPC differentiation was investigated by quantitative polymerase chain reaction, alkaline phosphatase activity, and oil red O staining. RESULTS: EZH2 and H3K27me3 were decreased, whereas KDM6B was increased in infected pulp tissue and cells, which were similar to HDPC differentiation. EZH2 inhibition suppressed IL-1b, IL-6, and IL-8 messenger RNA (mRNA) in HDPCs upon inflammatory stimuli and impeded HDPC proliferation by decreasing cell number, arresting cell cycle, and increasing apoptosis. Suppressed EZH2 impaired adipogenesis, peroxisome proliferator-activated receptor r (PPAR-r), and CCAAT-enhancer binding protein a (CEBP/a) mRNA in adipogenic induction while enhancing alkaline phosphatase activity, Osx, and bone sialoprotein (BSP) mRNA in mineralization induction of HDPCs. CONCLUSIONS: EZH2 inhibited HDPC osteogenic differentiation while enhancing inflammatory response and proliferation, suggesting its role in pulp inflammation, proliferation, and regeneration.

Wnt5a promotes inflammatory responses via nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways in human dental pulp cells.

Wnt5a has been found recently to be involved in inflammation regulation through a mechanism that remains unclear. Immunohistochemical staining of infected human dental pulp and tissue from experimental dental pulpitis in rats showed that Wnt5a levels were increased. In vitro, Wnt5a was increased 8-fold in human dental pulp cells (HDPCs) after TNF-α stimulation compared with control cells. We then investigated the role of Wnt5a in HDPCs. In the presence of TNF-α, Wnt5a further increased the production of cytokines/chemokines, whereas Wnt5a knockdown markedly reduced cytokine/ chemokine production induced by TNF-α. In addition, in HDPCs, Wnt5a efficiently induced cytokine/chemokine expression and, in particular, expression of IL-8 (14.5-fold) and CCL2 (25.5-fold), as assessed by a Luminex assay. The cytokine subsets regulated by Wnt5a overlap partially with those induced by TNF-α. However, no TNF-α and IL-1ß was detected after Wnt5a treatment. We then found that Wnt5a alone and the supernatants of Wnt5a-treated HDPCs significantly increased macrophage migration, which supports a role for Wnt5a in macrophage recruitment and as an inflammatory mediator in human dental pulp inflammation. Finally, Wnt5a participates in dental pulp inflammation in a MAPK-dependent (p38-, JNK-, and ERK-dependent) and NF-κB-dependent manner. Our data suggest that Wnt5a, as an inflammatory mediator that drives the integration of cytokines and chemokines, acts downstream of TNF-α.