Depletion of EREG enhances the osteo/dentinogenic differentiation ability of dental pulp stem cells via the p38 MAPK and Erk pathways in an inflammatory microenvironment.

BACKGROUND: Epiregulin (EREG) is an important component of EGF and was demonstrated to promote the osteo/dentinogenic differentiation of stem cells from dental apical papilla (SCAPs). Whether EREG can stimulate the osteo/dentinogenic differentiation of dental pulp stem cells (DPSCs) in inflammatory environment is not clear. The purpose of the present study is to investigate the role of EREG on the osteo/dentinogenic differentiation ability of DPSCs in inflammatory environment. METHODS: DPSCs were isolated from human third molars. Short hairpin RNAs (shRNAs) were used to knock down EREG expression in DPSCs. Recombinant human EREG (rhEREG) protein was used in the rescue experiment. TNF-α was employed to mimic the inflammatory environment in vitro. Alkaline phosphatase (ALP) staining, Alizarin red staining, quantitative calcium analysis, and real-time RT-PCR were performed to detect osteo/dentinogenic differentiation markers and related signalling pathways under normal and inflammatory conditions. RESULTS: EREG depletion promoted the ALP activity and mineralization ability of DPSCs. The expression of BSP, DMP-1, and DSPP was also enhanced. Moreover, 50 ng/mL rhEREG treatment decreased the osteo/dentinogenic differentiation potential of DPSCs, while treatment with 10 ng/mL TNF-α for 4 h increased the expression of EREG in DPSCs. Conversely, EREG knockdown rescued the impaired osteo/dentinogenic differentiation ability caused by TNF-α treatment. Further mechanistic studies showed that EREG depletion activated the p38 MAPK and Erk signalling pathways in DPSCs under normal and inflammatory conditions. CONCLUSIONS: Our results demonstrated that EREG could inhibit the osteo/dentinogenic differentiation potential of DPSCs via the p38 MAPK and Erk signalling pathways. Under inflammatory environment, EREG depletion enhanced osteo/dentinogenic differentiation potential of DPSCs by improving the expression of p-p38 MAPK and p-Erk.

Specific RNA m6A modification sites in bone marrow mesenchymal stem cells from the jawbone marrow of type 2 diabetes patients with dental implant failure.

The failure rate of dental implantation in patients with well-controlled type 2 diabetes mellitus (T2DM) is higher than that in non-diabetic patients. This due, in part, to the impaired function of bone marrow mesenchymal stem cells (BMSCs) from the jawbone marrow of T2DM patients (DM-BMSCs), limiting implant osseointegration. RNA N6-methyladenine (m6A) is important for BMSC function and diabetes regulation. However, it remains unclear how to best regulate m6A modifications in DM-BMSCs to enhance function. Based on the "m6A site methylation stoichiometry" of m6A single nucleotide arrays, we identified 834 differential m6A-methylated genes in DM-BMSCs compared with normal-BMSCs (N-BMSCs), including 43 and 790 m6A hypermethylated and hypomethylated genes, respectively, and 1 gene containing hyper- and hypomethylated m6A sites. Differential m6A hypermethylated sites were primarily distributed in the coding sequence, while hypomethylated sites were mainly in the 3'-untranslated region. The largest and smallest proportions of m6A-methylated genes were on chromosome 1 and 21, respectively. MazF-PCR and real-time RT-PCR results for the validation of erythrocyte membrane protein band 4.1 like 3, activity-dependent neuroprotector homeobox (ADNP), growth differentiation factor 11 (GDF11), and regulator of G protein signalling 2 agree with m6A single nucleotide array results; ADNP and GDF11 mRNA expression decreased in DM-BMSCs. Furthermore, gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses suggested that most of these genes were enriched in metabolic processes. This study reveals the differential m6A sites of DM-BMSCs compared with N-BMSCs and identifies candidate target genes to enhance BMSC function and improve implantation success in T2DM patients.

The cannabinoid receptor I (CB1) enhanced the osteogenic differentiation of BMSCs by rescue impaired mitochondrial metabolism function under inflammatory condition.

BACKGROUND: Periodontitis is a chronic infectious disease leading to bone resorption and periodontal tissue disruption under inflammatory stimulation. The osteogenic differentiation ability of mesenchymal stem cells (MSCs) is impaired under the inflammatory environment, which limits the effect of treatment. The cannabinoid receptor I (CB1) is the main effector of the endogenous cannabinoid system (ECS), and our previous study verified that CB1 could enhance the osteo/dentinogenic differentiation of dental MSCs, which might be a target for alveolar bone regeneration. However, the effect of CB1 on the osteogenic differentiation of MSCs derived from bone remains unknown. In present study, we investigated the role and mechanism of CB1 on mitochondrial function and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) under inflammatory environment. METHODS: Alkaline phosphatase (ALP) activity, alizarin red staining, quantitative calcium analysis, and osteogenic markers were used to detect the osteogenic differentiation ability of BMSCs. Real-time RT-PCR and Western blot were used to detect the gene expression. Seahorse Cell Mito Stress Test was used to detect the oxygen consumption rate (OCR). JC-10 assay was used to determine the mitochondrial membrane potential (MMP). RESULTS: CB1 increased osteogenic differentiation potential and mitochondrial energy metabolism, including the OCR, MMP, and enhanced the expressions of Nrf1 and Nrf2 in hBMSCs without or with TNF-α or INF-γ stimulation. Then, the inhibitor of mitochondrial electron transport chain (ETC), rotenone (ROT), inhibited the osteogenic differentiation in hBMSCs, and CB1 could rescue ROT impaired osteogenic differentiation potentials of hBMSCs without or with TNF-α or INF-γ stimulation. Activation of ETC by Coenzyme Q10 (CoQ10) could restore the impaired osteogenic differentiation of hBMSCs by depletion of CB1 without or with TNF-α or INF-γ stimulation. Mechanismly, CB1 could activate the JNK signaling pathway, p38 MAPK signaling pathway, and inhibit the Erk1/2 signaling pathway. CONCLUSIONS: The activating of CB1 enhanced the osteogenic differentiation by rescuing the mitochondrial metabolism function in hBMSCs under the inflammatory environment, suggesting that CB1 is a potential target for enhancing bone regeneration under the inflammatory environment.

The role and mechanism of mitochondrial functions and energy metabolism in the function regulation of the mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent cells that show self-renewal, multi-directional differentiation, and paracrine and immune regulation. As a result of these properties, the MSCs have great clinical application prospects, especially in the regeneration of injured tissues, functional reconstruction, and cell therapy. However, the transplanted MSCs are prone to ageing and apoptosis and have a difficult to control direction differentiation. Therefore, it is necessary to effectively regulate the functions of the MSCs to promote their desired effects. In recent years, it has been found that mitochondria, the main organelles responsible for energy metabolism and adenosine triphosphate production in cells, play a key role in regulating different functions of the MSCs through various mechanisms. Thus, mitochondria could act as effective targets for regulating and promoting the functions of the MSCs. In this review, we discuss the research status and current understanding of the role and mechanism of mitochondrial energy metabolism, morphology, transfer modes, and dynamics on MSC functions.

CB1 enhanced the osteo/dentinogenic differentiation ability of periodontal ligament stem cells via p38 MAPK and JNK in an inflammatory environment.

OBJECTIVES: Periodontitis is an inflammatory immune disease that causes periodontal tissue loss. Inflammatory immunity and bone metabolism are closely related to periodontitis. The cannabinoid receptor I (CB1) is an important constituent of the endocannabinoid system and participates in bone metabolism and inflammation tissue healing. It is unclear whether CB1 affects the mesenchymal stem cell (MSC) function involved in periodontal tissue regeneration. In this study, we revealed the role and mechanism of CB1 in the osteo/dentinogenic differentiation of periodontal ligament stem cells (PDLSCs) in an inflammatory environment. MATERIALS AND METHODS: Alkaline phosphatase (ALP) activity, Alizarin Red staining, quantitative calcium analysis and osteo/dentinogenic markers were used to assess osteo/dentinogenic differentiation. Real-time RT-PCR and Western blotting were employed to detect gene expression. RESULTS: CB1 overexpression or CB1 agonist (10 µM R-1 Meth) promoted the osteo/dentinogenic differentiation of PDLSCs. Deletion of CB1 or the application of CB1 antagonist (10 µM AM251) repressed the osteo/dentinogenic differentiation of PDLSCs. The activation of CB1 enhanced the TNF-α- and INF-γ-impaired osteo/dentinogenic differentiation potential in PDLSCs. Moreover, CB1 activated p38 MAPK and JNK signalling and repressed PPAR-γ and Erk1/2 signalling. Inhibition of JNK signalling could block CB1-activated JNK and p38 MAPK signalling, while CB1 could activate p38 MAPK and JNK signalling, which was inhibited by TNF-α and INF-γ stimulation. CONCLUSIONS: CB1 was able to enhance the osteo/dentinogenic differentiation ability of PDLSCs via p38 MAPK and JNK signalling in an inflammatory environment, which might be a potential target for periodontitis treatment.