Circadian rhythm disruption upregulating Per1 in mandibular condylar chondrocytes mediating temporomandibular joint osteoarthritis via GSK3ß/ß-CATENIN pathway.

BACKGROUND: Temporomandibular joint osteoarthritis (TMJOA) has a high incidence rate, but its pathogenesis remains unclear. Circadian rhythm is an important oscillation in the human body and influences various biological activities. However, it is still unclear whether circadian rhythm affects the onset and development of TMJOA. METHODS: We disrupted the normal rhythm of rats and examined the expression of core clock genes in the mandibular condylar cartilage of the jaw and histological changes in condyles. After isolating rat mandibular condylar chondrocytes, we upregulated or downregulated the clock gene Per1, examined the expression of cartilage matrix-degrading enzymes, tested the activation of the GSK3ß/ß-CATENIN pathway and verified it using agonists and inhibitors. Finally, after downregulating the expression of Per1 in the mandibular condylar cartilage of rats with jet lag, we examined the expression of cartilage matrix-degrading enzymes and histological changes in condyles. RESULTS: Jet lag led to TMJOA-like lesions in the rat mandibular condyles, and the expression of the clock gene Per1 and cartilage matrix-degrading enzymes increased in the condylar cartilage of rats. When Per1 was downregulated or upregulated in mandibular condylar chondrocytes, the GSK3ß/ß-CATENIN pathway was inhibited or activated, and the expression of cartilage matrix-degrading enzymes decreased or increased, which can be rescued by activator and inhibitor of the GSK3ß/ß-CATENIN pathway. Moreover, after down-regulation of Per1 in mandibular condylar cartilage in vivo, significant alleviation of cartilage degradation, cartilage loss, subchondral bone loss induced by jet lag, and inhibition of the GSK3ß/ß-CATENIN signaling pathway were observed. Circadian rhythm disruption can lead to TMJOA. The clock gene Per1 can promote the occurrence of TMJOA by activating the GSK3ß/ß-CATENIN pathway and promoting the expression of cartilage matrix-degrading enzymes. The clock gene Per1 is a target for the prevention and treatment of TMJOA.

Alteration of circular RNA expression in rat dental follicle cells during osteogenic differentiation.

Circular RNAs (circRNAs) are novel noncoding RNAs and play crucial roles in various biological processes. However, little is known about the functions of circRNAs in osteogenic differentiation. The current study aimed to investigate the differential expression of circRNAs in rat dental follicle cells (rDFCs) during osteogenic differentiation, identified by RNA high-throughput sequencing and quantitative real-time polymerase chain reaction. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to further explore the biofunctions of circRNA biofunctions. Two hundred sixty-six differentially-expressed circRNAs that are involved in several important signaling pathways, including mitogen-activated protein kinases (MAPK) and transforming growth factor-ß (TGF-ß) signaling pathways were revealed. Among these, circFgfr2 and its predicted downstream targets, miR-133 and BMP6 (bone morphogenetic protein-6), were identified both in vivo and in vitro. For further validation, circFgfr2 was overexpressed in rDFCs, the results showed that the expression of miR-133 was downregulated and the expression of BMP6 was upregulated. Taken together, the results revealed the circRNA expression profiles and indicated the importance of circRNAs of rDFCs. In addition, circFgfr2 might promote osteogenesis by controlling miR-133/BMP6, which is a potential new target for the manipulation of tooth regeneration and bone formation.

Dopamine promotes osteogenic differentiation of PDLSCs by activating DRD1 and DRD2 during orthodontic tooth movement via ERK1/2 signaling pathway.

Introduction: Orthodontic tooth movement (OTM) involves complex interactions between mechanical forces and periodontal tissue adaptation, mainly mediated by periodontal ligament cells, including periodontal ligament stem cells (PDLSCs), osteoblasts, and osteoclasts. Dopamine (DA), a neurotransmitter known for its critical role in bone metabolism, is investigated in this study for its potential to enhance osteogenic differentiation in PDLSCs, which are pivotal in OTM. This study examined the potential of DA to facilitate OTM by binding to DA receptors (D1R and D2R) and activating the ERK1/2 signaling pathway. We propose that DA's interaction with these receptors on PDLSCs could enhance osteogenic differentiation, thereby accelerating bone remodeling and reducing the duration of orthodontic treatments, which offering a novel approach to improve clinical outcomes in orthodontic care. Methods: This study utilized a rat OTM model, micro-CT, histological analyses, and in vitro assays to investigate dopamine's effect on osteogenesis. PDLSCs were cultured and treated with DA, and cytotoxicity, osteogenic differentiation, gene and protein expression assessed. Results: Dopamine administration significantly increased trabecular bone density and osteogenic marker expression in an OTM rat model. In vitro, DA at 10 nM optimally promoted human PDLSCs osteogenesis without affecting proliferation. Blocking DA receptors or inhibiting the ERK1/2 pathway attenuated these effects, underscoring the importance of dopaminergic signaling in tension-induced osteogenesis during OTM. Conclusion: Taken together, our study reveals that local dopamine administration at a concentration of 10 nM not only enhances tension-induced osteogenesis in vivo but also significantly promotes osteogenic differentiation of PDLSCs in vitro through D1 and D2 receptor-mediated ERK1/2 signaling pathway activation.

Alendronate sodium induces G1 phase arrest and apoptosis in human umbilical vein endothelial cells by inhibiting ROS-mediated ERK1/2 signaling.

Bisphosphonates are potent bone resorption inhibitors, among which alendronate sodium (ALN) is commonly prescribed for most osteoporosis patients, but long-term application of ALN can cause bisphosphonate-related osteonecrosis of jaw (BRONJ), the pathogenesis of which remains unclear. Previous studies have suggested that bisphosphonates cause jaw ischemia by affecting the biological behavior of vascular endothelial cells, leading to BRONJ. However, the impacts of ALN on vascular endothelial cells and its mechanism remain unclear. The purpose of this work is to assess the influence of ALN on human umbilical vein endothelial cells (HUVECs) and clarify the molecular pathways involved. We found that high concentration of ALN induced G1 phase arrest in HUVECs, demonstrated by downregulation of Cyclin D1 and Cyclin D3. Moreover, high concentration of ALN treatment showed pro-apoptotic effect on HUVECs, demonstrated by increased levels of the cleaved caspase-3, the cleaved PARP and Bax, along with decreased levels of anti-apoptotic protein Bcl-2. Further experiments showed that ERK1/2 phosphorylation was decreased. Additionally, ALN provoked the build-up of reactive oxygen species (ROS) in HUVECs, leading to ERK1/2 pathway suppression. N-acetyl-L-cysteine (NAC), a ROS scavenger, efficiently promoted the ERK1/2 phosphorylation and mitigated the G1 phase arrest and apoptosis triggered by ALN in HUVECs. PD0325901, an inhibitor of ERK1/2 that diminishes the ERK1/2 phosphorylation enhanced the ALN-induced G1 phase arrest and apoptosis in HUVECs. These findings show that ALN induces G1 phase arrest and apoptosis through ROS-mediated ERK1/2 pathway inhibition in HUVECs, providing novel insights into the pathogenic process, prevention and treatment of BRONJ in individuals receiving extended use of ALN.

Infusion of GMSCs relieves autoimmune arthritis by suppressing the externalization of neutrophil extracellular traps via PGE2-PKA-ERK axis.

INTRODUCTION: Rheumatoid arthritis (RA) is a systemic autoimmune disease with limited treatment success, characterized by chronic inflammation and progressive cartilage and bone destruction. Accumulating evidence has shown that neutrophil extracellular traps (NETs) released by activated neutrophils are important for initiating and perpetuating synovial inflammation and thereby could be a promising therapeutic target for RA. K/B × N serum transfer-induced arthritis (STIA) is a rapidly developed joint inflammatory model that somehow mimics the inflammatory response in patients with RA. Human gingival-derived mesenchymal stem cells (GMSCs) have been previously shown to possess immunosuppressive effects in arthritis and humanized animal models. However, it is unknown whether GMSCs can manage neutrophils in autoimmune arthritis. OBJECTIVES: To evaluate whether infusion of GMSCs can alleviate RA by regulating neutrophils and NETs formation. If this is so, we will explore the underlying mechanism(s) in an animal model of inflammatory arthritis. METHODS: The effects of GMSCs on RA were assessed by comparing the symptoms of the K/B × N serum transfer-induced arthritis (STIA) model administered either with GMSCs or with control cells. Phenotypes examined included clinical scores, rear ankle thickness, paw swelling, inflammation, synovial cell proliferation, and immune cell frequency. The regulation of GMSCs on NETs was examined through immunofluorescence and immunoblotting in GMSCs-infused STIA mice and in an in vitro co-culture system of neutrophils with GMSCs. The molecular mechanism(s) by which GMSCs regulate NETs was explored both in vitro and in vivo by silencing experiments. RESULTS: We found in this study that adoptive transfer of GMSCs into STIA mice significantly ameliorated experimental arthritis and reduced neutrophil infiltration and NET formation. In vitro studies also showed that GMSCs inhibited the generation of NETs in neutrophils. Subsequent investigations revealed that GMSCs secreted prostaglandin E2 (PGE2) to activate protein kinase A (PKA), which ultimately inhibited the downstream extracellular signal-regulated kinase (ERK) pathway that is essential for NET formation. CONCLUSION: Our results demonstrate that infusion of GMSCs can ameliorate inflammatory arthritis mainly by suppressing NET formation via the PGE2-PKA-ERK signaling pathway. These findings further support the notion that the manipulation of GMSCs is a promising stem cell-based therapy for patients with RA and other autoimmune and inflammatory diseases.

Role of Interleukin-17A in the Pathomechanisms of Periodontitis and Related Systemic Chronic Inflammatory Diseases.

Periodontitis is a chronic inflammatory and destructive disease caused by periodontal microbial infection and mediated by host immune response. As the main cause of loosening and loss of teeth in adults, it is considered to be one of the most common and serious oral diseases in the world. The co-existence of periodontitis and systemic chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, inflammatory bowel disease, diabetes and so on is very common. It has been found that interleukin-17A (IL-17A) secreted by various innate and adaptive immune cells can activate a series of inflammatory cascade reactions, which mediates the occurrence and development of periodontitis and related systemic chronic inflammatory diseases. In this work, we review the role of IL-17A in the pathomechanisms of periodontitis and related systemic chronic inflammatory diseases, and briefly discuss the therapeutic potential of cytokine targeted agents that modulate the IL-17A signaling. A deep understanding of the possible molecular mechanisms in the relationship between periodontitis and systemic diseases will help dentists and physicians update their clinical diagnosis and treatment ideas.

TGF-ß-induced CD4+ FoxP3+ regulatory T cell-derived extracellular vesicles modulate Notch1 signaling through miR-449a and prevent collagen-induced arthritis in a murine model.

CD4+FOXP3+ Treg cells are central to the maintenance of self-tolerance and can be defective in autoimmunity. In autoimmune rheumatic diseases, dysfunctional self-tolerance, is to a large extent, caused by insufficient Treg-cell activity. Although nTregs have therapeutic effects in vivo, their relative scarcity and slow rate of in vitro expansion hinder the application of nTreg therapy. It was previously reported that EVs contribute significantly to the suppressive function of FOXP3+ Treg cells. Considering that the stability and plasticity of nTregs remain major challenges in vivo, we established EVs derived from in vitro TGF-ß-induced Treg cells (iTreg-EVs) and assessed their functions in a murine model of autoimmune arthritis. The results demonstrated that iTreg-EVs preferentially homed to the pathological joint and efficiently prevented the imbalance in Th17/Treg cells in arthritic mice. Furthermore, we found that miR-449a-5p mediated Notch1 expression modulation and that miR-449a-5p knockdown abolished the effects of iTreg-EVs on effector T cells and regulatory T cells in vitro and in vivo. Taken together, our results show that iTreg-EVs control the inflammatory responses of recipient T cells through miR-449a-5p-dependent modulation of Notch1 and ameliorate the development and severity of arthritis, which may provide a potential cell-free strategy based on manipulating iTreg-EVs to prevent autoimmune arthritis.

[Corrigendum] Nkd2 promotes the differentiation of dental follicle stem/progenitor cells into osteoblasts.

After the publication of the article, the authors realized that the surname of the author listed second on the paper was spelt incorrectly as 'Zhan' instead of 'Zhang'; the corrected name is now featured above. The authors regret that this error was not corrected prior to the publication of their paper, and apologize for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 42: 2403­2414, 2018; DOI: 10.3892/ijmm.2018.3822].

Nkd2 promotes the differentiation of dental follicle stem/progenitor cells into osteoblasts.

Dental follicle stem/progenitor cells have the potential to undergo osteogenesis. naked cuticle homolog 2 (Nkd2) is a signal­inducible feedback antagonist of the canonical Wnt signaling pathway. The purpose of the present study was to investigate the function of Nkd2 in the differentiation of dental follicle stem/progenitor cells (DFSCs) into osteoblasts. Immunohistochemistry, reverse transcription­quantitative polymerase chain reaction and western blotting were employed to detect Nkd2 expression in rat DFSCs. In addition, rat DFSCs (rDFSCs) were transfected with small interfering RNAs to examine the effect of Nkd2 on the differentiation of these cells into osteoblasts. Furthermore, the function of Nkd2 in the Wnt/ß­catenin pathway in rDFSCs was investigated using ß­catenin/T­cell factor luciferase activity assays and western blotting. It was revealed that the expression of Nkd2 was upregulated during the differentiation of rDFSCs into osteoblasts. Furthermore, osteoblast differentiation ability and Wnt/ß­catenin pathway activity were significantly decreased in Nkd2­silenced rDFSCs compared with the si­NC group (P<0.05 and P<0.001, respectively). The results suggest that Nkd2 promotes the differentiation of rDFSCs into osteoblasts through Wnt/ß­catenin signaling.

Characterization of different osteoclast phenotypes in the progression of bone invasion by oral squamous cell carcinoma.

The present study aimed to characterize different phenotypes of osteoclasts in the progression of bone invasion by oral squamous cell carcinoma (OSCC). A local bone invasion model of OSCC was established by injecting SCC25 human OSCC cells into the center of calvariae in nude mice, and all mice were found to have a typical bone resorption area. Staining for tartrate-resistant acid phosphatase (TRAP) revealed various types of giant osteoclasts in the tumour-bone interface. Bone marrow cells (BMCs) were isolated from the nude mice for primary osteoclast culture, but only a few giant osteoclasts were generated. Additionally, special blood centrifuge tubes were utilized to obtain large numbers of peripheral blood mononuclear cells (PBMCs). Using magnetic activated cell sorting (MACS) and the cytokines colony-stimulating factor (CSF) and receptor activator of nuclear factor-κb ligand (RANKL), we differentiated human osteoclasts from CD14+ monocytes of PBMCs. Bone resorption was further confirmed by a bone resorption assay. Finally, Transwell inserts were used for indirect cell co-culture of SCC25 cells and CD14+ monocytes. Expression of specific osteoclast markers was detected by real-time PCR and western blotting. After co-culture for 3 and 6 days, conditioned medium (CM) of SCC25 cells stimulated the expression of osteoclast markers, and additional osteoclasts were detected through staining of TRAP and F-actin. In the present study distinct osteoclast phenotypes were observed in the established bone invasion animal model, and were confirmed using various primary osteoclast cultures. CM of OSCC cells may promote the expression of osteoclast markers and induce the differentiation of monocytes to mature osteoclasts, which can resorb adjacent bone tissue.

Utilization of E-cadherin by monocytes from tumour cells plays key roles in the progression of bone invasion by oral squamous cell carcinoma.

E-cadherin (E-cad) is recently reported to be expressed in early stages of osteoclastogenesis, and blocking E-cad with neutralizing antibodies decreases osteoclast differentiation. Since our previous research demonstrates the loss of E-cad protein in the bone invasion by oral squamous cell carcinoma (OSCC), we hypothesize that E-cad may be utilized by monocytes to fuse and differentiate into osteoclasts. Two research models are used in the present study to explore our hypothesis. On one hand, we use OSCC cells of SCC25 to establish an animal model of bone invasion by OSCC, and investigate whether E-cad protein disappears in vivo; on the other hand, we use the indirect co-culture model of SCC25 and RAW 264.7 cells, with the treatment of transforming growth factor-ß1 (TGF-ß1), and observe whether the decreased E-cad protein is 'hijacked' in vitro. Results showed the animal model of OSCC with bone invasion was successfully established. Immunohistochemistry (IHC) found similar changes of E-cad protein, which was weakly stained by tumour cells. By using 5 ng/ml of TGF-ß1, we confirmed the artificial epithelial-mesenchymal transition (EMT) of SCC25 cells, with changes of EMT marker expression and cell morphology. Real-time PCR showed E-cad mRNA decreased in SCC25 while increased in RAW 264.7 of the indirect cell co-culture model, and immunofluoresence (IF) observed the evident switch of E-cad staining from SCC25 to RAW 264.7. With the supplement of receptor activator of NF-κB ligand (RANKL), tartrate-resistant acid phosphatase (TRAP) and F-actin staining confirmed the increased number of osteoclasts. Taken together, our study found the switch of E-cad protein in the progression of bone invasion by OSCC. The loss of E-cad in tumour cells may be utilized by monocytes to differentiate into osteoclasts, thus further explaining the underlying mechanisms of bone invasion by OSCC, which may supply clues for future molecular biotherapies.