METTL3 potentiates osteogenic differentiation of bone marrow mesenchymal stem cells via IGF2BP1/m6A/RUNX2.

OBJECTIVE: Maxillofacial bone defect is a critical obstacle for maxillofacial tumors and periodontal diseases. The osteogenic differentiation of bone marrow mesenchymal stem cells BMSCs is critical for maxillofacial osteogenesis and functional reconstruction. Here, our study focused on the functions and mechanism of N6 -methyladenosine during BMSCs osteogenic differentiation BMSCs. SUBJECT AND METHODS: Biofunctions of BMSCs were detected using ALP activity and alizarin red S staining assays. The molecular interaction within RNA/protein was identified by RNA immunoprecipitation and/or methylation immunoprecipitation. RESULTS: Results indicated that m6 A 'writer' METTL3 upregulated during the osteogenic differentiation of BMSCs upon osteogenic induction. Functionally, assays' results revealed that METTL3 overexpression promoted the osteogenic differentiation of BMSC, while METTL3 knockdown repressed the osteogenic differentiation. Mechanistically, results revealed that RUNX2 mRNA was a m6 A-methylated target by METTL3 at its 3'-UTR. Moreover, m6 A reader IGF2BP1 recognized the m6 A site on RUNX2 mRNA to enhance its stability. CONCLUSION: In conclusion, our findings revealed the novel roles of METTL3 in BMSCs osteogenic differentiation via the IGF2BP1/m6 A/RUNX2 signaling axis of m6 A-dependent manner, providing a potential therapeutic target for maxillofacial bone defects treatment.

Associations between osteoporosis and risk of periodontitis: A pooled analysis of observational studies.

BACKGROUND: Periodontitis and osteoporosis are most popular among aging population and both conditions might be linked, even though, this suggestion still until now debated. OBJECTIVES: A meta-analysis on previous investigations has been used to evaluate the correlation between periodontitis and osteoporosis to determine whether osteoporosis is a local indicator of bone loss, or whether it is depending on or related to periodontitis causes. METHODS: The literature database, including but not excluding, The Cochrane Central Register of Controlled Trials, MEDLINE, EMBASE, CINAHL, and Science Citation Index Expanded, was searched in this work during Feb, 2020. We conducted the investigations contain cohort studies, cross-sectional studies, as well as case-control studies with relative risk (RR) or odds ratio (OR) and 95% confidence intervals (CIs). Subgroup and Sensitivity analysis were also applied to identify heterogeneity sources. RESULTS: 23 observational studies with 12 cohorts, 7 cross-sectional and 4 case-control studies, were included, together with 2,157,037 participants. Osteoporosis patients were more exposed to periodontitis (OR, 1.96; 95% CI, 1.50-2.54). Subgroup analyses showed that the higher risk of osteoporosis in periodontitis patients exists in both cross-sectional studies (OR, 2.17; 95% CI, 1.80-2.61) and case-control studies (OR 2.63; 95% CI, 1.69-4.09), and marginally in cohort studies (OR, 1.70; 95% CI, 1.16-2.49). CONCLUSION: Review analyses have shown that osteoporosis is closely related to the increased risk of periodontitis in the future. Dental specialists better to understand the potential association between periodontitis and osteoporosis.

RCOR1 directly binds to MED28 and weakens its inducing effect on cancer stem cell-like activity of oral cavity squamous cell carcinoma cells.

BACKGROUND: Mediator is a multiprotein complex that acts as an essential transcriptional coactivator in eukaryotic cells for successful transcription. In this study, we aimed to explore the expression profile of 33 mediator subunit genes in oral cavity squamous cell carcinoma (OCSCC) and the functional role of MED28 in cellular behaviors of OCSCC cells. METHODS: Single-cell (sc)RNA-seq data from OCSCC cells (Puram 2017's dataset) and bulk-seq data of the OCSCC subgroup of TCGA-head and neck squamous cell carcinoma (HNSC) were used for bioinformatic analysis. SCC9 cells were used in in-vitro and in-vivo analysis. RESULTS: Among the 33 genes subjected to screening, MED28 showed the best prognostic value and its upregulation might independently predict shorter OS (HR: 3.699, 95% CI: 1.383-9.892, P = .009) and PFS (HR: 2.769, 95% CI: 1.462-5.244, P = .002). MED28 expression was positively correlated with cancer stem cell (CSC)-like properties of SCC9 cells, including colony/sphere formation, and the expression of CSC markers (CD44, KLF4, NANOG, and OCT4). RCOR1 could suppress the CSC-like properties of SCC9 cells and had direct interaction with MED28. Its overexpression partly abrogated MED28-induced expression of CSC markers. RCOR1 expression was associated with promoter hypermethylation, while MED28 expression was positively correlated with its MED28 copy number (Pearson's r = .44) in OCSCC tissues. CONCLUSION: Among the mediator complex subunits, MED28 might serve as a potential biomarker of unfavorable survival. Its overexpression increased CSC-like activity of OCSCC cells, the effect of which could be abrogated by RCOR1 via direct interaction.

Associations Between Poor Oral Health and Risk of Squamous Cell Carcinoma of the Head and Neck: A Meta-Analysis of Observational Studies.

PURPOSE: Many epidemiologic studies have reported an association of poor oral health, especially periodontal disease (PD) and tooth loss, with the risk of squamous cell carcinoma of the head and neck (SCCHN). However, these studies have yielded inconsistent results. Therefore, the present study investigated whether poor oral health is an independent predictor of SCCHN through a meta-analysis of observational studies. MATERIALS AND METHODS: The PubMed, EMBASE, and Cochrane Library databases were systematically searched for relevant observational studies of the association between oral health and risk of SCCHN conducted up to October 2017. The meta-analysis was conducted using STATA 12.0 (StataCorp, College Station, TX). A fixed- or random-effects model was applied to evaluate pooled risk estimates, and sensitivity and subgroup analyses were performed to identify sources of heterogeneity and pooled estimation. Publication bias was assessed using the Begg test, the Egger test, and funnel plots. RESULTS: Twenty-seven relevant observational studies were identified, consisting of 24 case-and-control studies, 2 prospective studies, and 1 cross-sectional study, with 26,750 participants. Notably, oral health correlated meaningfully with SCCHN (odds ratio [OR] = 2.24; 95% confidence interval [CI], 1.77-2.82). In subgroup analyses, participants with PD (OR = 2.52; 95% CI, 1.43-4.44) had a higher risk of developing SCCHN than those with tooth loss (OR = 2.13; 95% CI, 1.63-2.78). The risk estimates exhibited substantial heterogeneity. Evidence of publication bias was limited. CONCLUSIONS: The results of this meta-analysis suggest that patients with tooth loss or PD might face a substantial and independent risk of SCCHN, even after adjusting for smoking and alcohol consumption. However, the pooled estimates from observational studies could not establish a causative relation among PD, tooth loss, and SCCHN. Additional investigations of this correlation are warranted.

Adrenomedullin promotes the odontogenic differentiation of dental pulp stem cells through CREB/BMP2 signaling pathway.

Adrenomedullin (AM) could promote the proliferation, the odontogenic differentiation and inhibit the apoptosis of dental pulp stem cells (DPSCs). AM in combination with DPSCs may be an effective strategy for pulp repair. However, there was no report on the mechanisms of AM in the odontogenic differentiation of DPSCs. The aim of this study is to investigate the molecular mechanisms through which AM promotes the odontogenic differentiation of DPSCs. Freshly extracted wisdom teeth were obtained from 27 patients. Cells at passage 3 to passage 5 were used in this study. DPSCs were treated with or without 10-7 M AM in Dulbecco's modified Eagle's medium culture, and then the accumulated calcium deposition was analyzed after 21 days by using alizarin red S staining. Odontogenic differentiation markers were determined by western blot analysis and quantitative real-time PCR. Western blot analysis results showed that AM had the capability of promoting the odontogenic differentiation of DPSCs and AM could enhance the phosphorylation of CREB and up-regulate the expression of BMP2. H89 is a CREB inhibitor which can inhibit the odontogenic differentiation of DPSCs through inhibiting the phosphorylation of CREB. Noggin could inhibit the odontogenic differentiation of DPSCs through inhibiting the activity of BMP2. These results indicated that AM could promote the odontogenic differentiation of DPSCs by upregulating the expression of BMP2 through the CREB signaling pathway.

How Is Third Molar Status Associated With the Occurrence of Mandibular Angle and Condyle Fractures?

PURPOSE: Third molars (M3s) have been hypothesized to be associated with the risk of mandibular angle fracture and mandibular condylar fracture. The authors systematically estimated the relative risk (RR) of M3 status for the development of mandibular angle fracture and mandibular condylar fracture through a meta-analysis of cohort studies. MATERIALS AND METHODS: In this systematic review, the PubMed, EMBASE, and Cochrane Library databases were searched from inception to October 2016. The predictor of risk was the presence or absence of M3s. The primary outcome was the RR of mandibular angle or condylar fracture. A fixed- or a random-effects model was applied to evaluate the pooled risk estimates. Sensitivity analysis also was performed to identify the potential sources of heterogeneity. Publication bias was assessed by the Begg and Egger tests. RESULTS: Overall, 13 retrospective cohort studies were included. Of these, 13 reported the association between M3s and mandibular angle fracture, and 5 reported the association with mandibular condylar fracture. Patients with M3s had an increased risk of mandibular angle fractures (RR = 2.63; 95% confidence interval [CI], 2.15-3.21) but a decreased risk of mandibular condylar fractures (RR = 0.47; 95% CI, 0.25-0.86). Substantial heterogeneity in the risk estimates was found. No evidence of publication bias was found. CONCLUSION: The present meta-analysis provides further evidence associating the presence of M3s with an increased risk of mandibular angle fractures and a simultaneously decreased risk of mandibular condylar fracture. Because of potentially more serious complications associated with condylar fracture, clinicians should carefully consider the decision to remove M3s to decrease the risk of mandibular angle fracture.

Connexin43-containing gap junctions potentiate extracellular Ca²âº-induced odontoblastic differentiation of human dental pulp stem cells via Erk1/2.

Extracellular Ca(2+) can promote dentin sialophosphoprotein (DSPP) expression and odontoblastic differentiation of dental pulp stem cells (DPSCs). Gap junctions mediated by connexin43 (Cx43) allow diffusion of small molecules (such as Ca(2+)) among cells to regulate cell-to-cell communications. However, it is unclear whether Cx43 is required for the Ca(2+)-induced cell differentiation. Here, we found that the influx of extracellular Ca(2+) through L-type Ca(2+) channels increases intracellular free Ca(2+) levels to promote DSPP expression. Cx43 overexpression potentiated the extracellular Ca(2+)-induced DSPP expression via Erk1/2. Flow cytometry analyses showed that Cx43 increased the percentage of p-Erk1/2 positive cells in response to Ca(2+), indicating that Cx43 in DPSCs possibly acts as a traditional gap junction channel, which permits the sharing of signals among coupled cells to make more DPSCs respond to Ca(2+). Furthermore, inhibition of Cx43 function and gap junction communication decreased Ca(2+)-induced the expression of DSPP, suggesting that cell-to-cell contacts are required for Cx43 to promote the Ca(2+)-induced cell differentiation. Similarly, the study performed on DPSCs cultured at low-density and high-density revealed that intercellular contacts are required to potentiate Erk1/2 activity and DSPP expression. In total, this study indicates that Cx43 increases Ca(2+)-induced DSPP expression and odontoblastic differentiation of DPSCs via Erk1/2 through gap junction-mediated cell-to-cell contacts.

Extracellular Ca2+ Promotes Odontoblastic Differentiation of Dental Pulp Stem Cells via BMP2-Mediated Smad1/5/8 and Erk1/2 Pathways.

Ca(2+) is the main element of many pulp capping materials that are used to promote the regeneration of tertiary dentin, but the underlying molecular mechanism is not clear. In this study, we found that Ca(2+) increased the expression of the odontoblastic differentiation marker gene DSPP and promoted odontoblastic differentiation and mineralization of DPSCs, but inhibited ALP activity. Ca(2+) increases the expression of endogenous BMP2, which activates the Smad1/5/8 pathway and promotes the Smad1-Runx2 and Runx2-DSPP interaction in DPSCs. Inhibition of Smad1/5/8 with dorsomorphin partially blocked Runx2 activity; however, inhibition of the BMP2 receptor with Noggin nearly fully suppressed Runx2 activity. These results indicate that Ca(2+) promotes cell differentiation mainly via BMP2-mediated Smad-dependent and Smad-independent pathways. We then determined that the phosphorylation level of Erk1/2, but not JNK or p38, was significantly increased as a result of Ca(2+) stimulation. Blockage of Erk1/2 was found to inhibit Runx2 activity, indicating that Ca(2+) triggers the Erk1/2 pathway, which subsequently regulates Runx2 activity. In addition, inhibition of Erk1/2 differentially attenuated the phosphorylation levels of Smad1/5/8 and Smad2/3. Collectively, this study demonstrates that Ca(2+) activates the BMP2-mediated Smad1/5/8 and Erk1/2 pathways in DPSCs and that Smad1/5/8 and Erk1/2 signaling converge at Runx2 to control the odontoblastic differentiation of DPSCs.

The influence of RAMP1 overexpression on CGRP-induced osteogenic differentiation in MG-63 cells in vitro: an experimental study.

The aim of this study was to elucidate the influence of receptor activity modifying protein 1 (RAMP1) overexpression on the expression and distribution of calcitonin receptor-like receptor (CRLR) in MG-63 cells. Our research also focused on whether RAMP1 overexpression enhanced the promoting effect of exogenous CGRP on osteogenic differentiation in MG-63 cells. We first constructed a eukaryotic expression vector containing human RAMP1 and stably transfected it into MG-63 cells. Real-time PCR and Western blotting were used to determine the expression levels of RAMP1 and CRLR mRNA and protein, respectively. Immunofluorescence analysis was employed to compare the distribution of CRLR in transfected cells. After treatment with CGRP, the extent of osteogenic differentiation was evaluated by simultaneous monitoring of alkaline phosphatase activity, the expression patterns of osteoblastic markers and mineralisation staining. We found that RAMP1 was more highly expressed in the transfected group compared with the control groups (P < 0.01). The CRLR expression was significantly higher than that in the control groups (P < 0.05). In addition, after 7 days of CGRP treatment to induce osteogenic differentiation, the expression of collagen I mRNA was markedly increased in the transfected group (P < 0.05). The transfected group exhibited more granular precipitation in the cytoplasm with alkaline phosphatase staining after 7 and 14 days of differentiation. When stained with Alizarin Red, cells overexpressing RAMP1 were darker and formed many mineralised nodules with clear boundaries and calcium deposition typical of mineralised bone matrix structures at 28 days post-induction of differentiation. The CGRP-induced ALP activity in the RAMP1 overexpression group was significantly higher 3, 6 and 9 days after induction than that in the two control groups (P < 0.05). RAMP1 overexpression promotes CRLR expression, localisation on the cell membrane and enhanced CGRP-mediated differentiation of MG-63 cells. This study contributes to a better understanding of the molecular mechanisms governing CGRP-induced MG-63 differentiation.

Calcitonin gene-related peptide stimulates BMP-2 expression and the differentiation of human osteoblast-like cells in vitro.

AIM: To investigate whether bone morphogenic protein-2 (BMP-2) expression was involved in calcitonin gene-related peptide (CGRP)-induced osteogenesis in human osteoblast-like cells in vitro. METHODS: MG-63 osteogenic human osteosarcoma cells were treated with CGRP (10-8 mol/L) for 48 h. Cell cycle phases were determined using flow cytometry assay. The protein levels of BMP-2, ALP, Osteocalcin, ColIa1, CREB, and pCREB were measured with Western blotting, while the mRNA level of BMP-2 was measured with qR-T PCR. The expression of ALP in MG-63 cells was also studied using immunofluorescence staining. The level of cAMP was measured with ELISA assay. RESULTS: CGRP treatment significantly stimulated proliferation of MG-63 cells, and increased the expression of BMP-2 and the osteogenic proteins ALP, Osteocalcin and ColIa1. Pretreatment with the BMP signaling inhibitor Noggin (100 ng/mL) did not affect CGRP-stimulated proliferation and BMP-2 expression, but abolished the CGRP-induced increases of the osteogenic proteins ALP, Osteocalcin and ColIa1. Furthermore, CGRP treatment markedly increased cAMP level in MG-63 cells, whereas pretreatment with the cAMP pathway inhibitor H89 (5 µmol/L) abolished the CGRP-induced increases of cAMP level and BMP-2 expression. CONCLUSION: In MG-63 cells, the BMP pathway is involved in CGRP-induced osteogenic differentiation but not in proliferation, whereas the cAMP/pCREB pathway is involved in the expression of BMP-2.

miR-141-3p Targeted SIRT1 to Inhibit Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells.

Purpose: To explore the expression of miR-141-3p during the osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs) and its regulatory effect. Methods: Differentiation of BMSCs was induced by dexamethasone. The mRNA expression of miR-141-3p, ALP, RUNX2, and OCN was measured using RT-qPCR. The protein expression was detected via western blot. The target of miR-141-3p was predicted through the TargetScan website and confirmed using luciferase reporter assay. Results: miR-141-3p expression declined during osteogenic differentiation. The relative ALP activities and the mRNA expression of ALP, RUNX2, and OCN were markedly reduced in the miR-141-3p mimic group while increased in the inhibitor group. Cell viability was suppressed in the miR-141-3p mimic group and promoted in the inhibitor group. SIRT1 was predicted to be a downstream gene of miR-141-3p, and this prediction was confirmed via the luciferase reporter assay. The results of the western blot assay demonstrated that SIRT1 expression was decreased in the miR-141-3p mimic group. SIRT1 reversed the inhibitory influence of miR-141-3p on the osteogenic differentiation ability of BMSCs. Conclusion: miR-141-3p targeted SIRT1 to inhibit osteogenic differentiation of BMSCs via the Wnt/ß-catenin signaling pathway.

Cytochrome P450 1A1 Ile462Val polymorphism and oral carcinoma risk: an updated meta-analysis including 1,515 cases and 2,233 controls.

Cytochrome P450 (CYP) 1A1 Ile462Val (exon7) polymorphism has been suggested to be a risk factor for several cancers. Published data on its association with oral cancer risk have generated conflicting results. Our previous meta-analysis containing data from prior to Jan 2008 regarding this issue failed to find a significant association between CYP1A1 Ile462Val variation and oral cancer susceptibility. An updated meta-analysis with eligible studies for the period up to May 2012 was conducted. Separate analyses on ethnicity and source of controls were also performed. A total of 13 case-control studies comprising 1,515 cases and 2,233 controls were lastly selected for analysis. Compared with the previous meta-analysis, the overall data also failed to indicate a significant association of CYP1A1 Ile462Val polymorphism with oral cancer risk (Val/Val vs. Ile/Ile--OR = 1.46; 95 % CI = 0.96-2.24; dominant model--OR = 1.01; 95 % CI = 0.81-1.25; and recessive model--OR = 1.46; 95 % CI = 0.96-2.23). However, in the subgroup analysis by ethnicity, increased cancer risk was observed among Asians under the additive and recessive models (Val/Val vs. Ile/Ile--OR = 1.74; 95 % CI = 1.04-2.90 and recessive model-OR = 1.73; 95 % CI = 1.04-2.87), inconsistent with the previous meta-analysis. Collectively, the data of the present study suggest that CYP1A1 variant Val/Val alleles might modify the susceptibility to oral cancer among Asians. Further well-designed investigations with large sample sizes are required to confirm this conclusion.

Polymorphisms of MTHFR C677T and A1298C association with oral carcinoma risk: a meta-analysis.

Investigations regarding association of MTHFR polymorphisms with oral carcinoma risk have yielded inconclusive results. Thus, meta-analyses were performed. Results showed that no associations of C677T polymorphisms with oral carcinoma were observed for the overall data. In subgroup analyses by drinking status, homozygous TT alleles exhibited elevated oral cancer susceptibility in heavy drinkers. For A1298C polymorphism, CC alleles presented a possible preventive role for oral cancer. Collectively, results suggest that MTHFR 677TT polymorphism might be a low-penetrant risk factor for oral carcinoma only in heavy drinkers. Conversely, 1298CC alleles might play a preventive role for oral cancer.

Quantitative assessment of CYP1A1*2A variations with oral carcinoma susceptibility: evidence from 1,438 cases and 2,086 controls.

Published evidence concerning association of CYP1A1*2A (MspI) variation with oral carcinoma risk has generated controversial results. Our previous meta-analysis in 2008 failed to reveal a marked association between CYP1A1*2A polymorphism and oral cancer susceptibility. Therefore, updated meta-analyses were conducted. Results suggested significant associations between CYP1A1*2A polymorphisms and oral carcinoma risk for the overall data, inconsistent with our previous meta-analysis. In subgroup analyses by ethnicity, variant C allele might elevate oral cancer susceptibility among Asians but not Caucasians. In conclusion, results suggest that CYP1A1*2A polymorphism might be a low-penetrant risk factor for oral carcinoma, particularly among Asians.

NAT2 polymorphisms with oral carcinoma susceptibility: a meta-analysis.

Published data have implicated NAT2 polymorphisms as risk factors for various cancers. A number of studies have focused on the association of NAT2 polymorphisms with susceptibility to oral carcinoma and have yielded inconclusive results. The aim of the present study was to derive a more precise estimation of the relationship. We first carried out a deliberate search in the databases without a language limitation, covering all papers published up to Dec 2011. A total of seven case-control studies including 1,379 cases and 1,868 controls were selected and the relevant data were extracted for systematic meta-analyses. No significant association was found for the overall data (OR: 1.04, 95 % CI: 0.79-1.39). In subgroup analyses according to ethnicity, slow acetylators might increase oral cancer risk among Asians (OR: 1.38, 95 % CI: 1.04-1.82) but not Caucasians or Mixed races. The data suggested that NAT2 polymorphisms might be a low-penetrant risk factor for oral carcinoma in Asians.

D609 inhibition of phosphatidylcholine-specific phospholipase C attenuates prolonged insulin stimulation-mediated GLUT4 downregulation in 3T3-L1 adipocytes.

Glucose uptake is stimulated by insulin via stimulation of glucose transporter 4 (GLUT4) translocation to the plasma membrane from intracellular compartments in adipose tissue and muscles. Insulin stimulation for prolonged periods depletes GLUT4 protein, particularly in highly insulin-responsive GLUT4 storage vesicles. This depletion mainly occurs via H2O2-mediated retromer inhibition. However, the post-receptor mechanism of insulin activation of oxidative stress remains unknown. Here, we show that phosphatidylcholine-specific phospholipase C (PC-PLC) plays an important role in insulin-mediated downregulation of GLUT4. In the study, 3T3-L1 adipocytes were exposed to a PC-PLC inhibitor, tricyclodecan-9-yl-xanthogenate (D609), for 30 min prior to the stimulation with 500 nM insulin for 4 h, weakening the depletion of GLUT4. D609 also prevents insulin-driven H2O2 generation in 3T3-L1 adipocytes. Exogenous PC-PLC and its product, phosphocholine (PCho), also caused GLUT4 depletion and promoted H2O2 generation in 3T3-L1 adipocytes. Furthermore, insulin-mediated the increase in the cellular membrane PC-PLC activity was observed in Amplex Red assays. These results suggested that PC-PLC plays an important role in insulin-mediated downregulation of GLUT4 and that PCho may serve as a signaling molecule.

Preparation of BMP-2/PDA-BCP Bioceramic Scaffold by DLP 3D Printing and its Ability for Inducing Continuous Bone Formation.

Digital light processing (DLP)-based 3D printing is suitable to fabricate bone scaffolds with small size and high precision. However, the published literature mainly deals with the fabrication procedure and parameters of DLP printed bioceramic scaffold, but lacks the subsequent systematic biological evaluations for bone regeneration application. In this work, a biphasic calcium phosphate (BCP) macroporous scaffold was constructed by DLP-based 3D printing technique. Furthermore, bone morphogenetic protein-2 (BMP-2) was facilely incorporated into this scaffold through a facile polydopamine (PDA) modification process. The resultant scaffold presents an interconnected porous structure with pore size of ∼570 µm, compressive strength (∼3.6 MPa), and the self-assembly Ca-P/PDA nanocoating exhibited excellent sustained-release property for BMP-2. Notably, this BMP-2/PDA-BCP scaffold presents favorable effects on the adhesion, proliferation, osteogenic differentiation, and mineralization of bone marrow stromal cells (BMSCs). Furthermore, in vivo experiments conducted on rats demonstrated that the scaffolds could induce cell layer aggregation adjacent to the scaffolds and continuous new bone generation within the scaffold. Collectively, this work demonstrated that the BMP-2/PDA-BCP scaffold is of immense potential to treat small craniofacial bone defects in demand of high accuracy.

TRK-fused gene (TFG) regulates ULK1 stability via TRAF3-mediated ubiquitination and protects macrophages from LPS-induced pyroptosis.

TRK-fused gene (TFG) is known to be involved in protein secretion and plays essential roles in an antiviral innate immune response. However, its function in LPS-induced inflammation and pyroptotic cell death is still unknown. Here, we reported that TFG promotes the stabilization of Unc-51 like autophagy activating kinase (ULK1) and participates in LPS plus nigericin (Ng) induced pyroptotic cell death. Our results showed that TFG-deficient THP-1 macrophages exhibit higher mitochondrial ROS production. LPS/Ng stimulation triggers a much higher level of ROS and induces pyroptotic cell death. ULK1 undergoes a rapid turnover in TFG-deficient THP-1 cells. TFG forms complex with an E3 ligase, tumor necrosis factor receptor-associated factor 3 (TRAF3), and stabilizes ULK1 via disturbing ULK1-TRAF3 interaction. Knockdown of TFG facilitates the interaction of ULK1 with TRAF3 and subsequent K48-linked ULK1 ubiquitination and proteasome degradation. Rescue of ULK1 expression blocks LPS/Ng-induced cell death in TFG-deficient THP-1 macrophages. Taken together, TFG plays an essential role in LPS/Ng-induced pyroptotic cell death via regulating K48-linked ULK1 ubiquitination in macrophages.

Amelogenin promotes odontoblast-like MDPC-23 cell differentiation via activation of ERK1/2 and p38 MAPK.

Amelogenin (AMG) is a highly conserved protein secreted by ameloblasts. Some research indicates that AMG might induce the differentiation and maturation of odontoblasts. The aim of this study was to clarify the function of AMG during the differentiation of odontoblast-like MDPC-23 cells. The results revealed that the alkaline phosphatase activity and the number of mineralized nodules were significantly enhanced in AMG-overexpressing MDPC-23 cells during the mineralization process. Tissue-specific markers such as dentin matrix protein 1 and dentin sialophosphoprotein also elevated significantly, indicating the cell differentiation and maturation process. Furthermore, AMG could upregulate the phosphorylation levels of ERK1/2 and p38 MAPK. However, JNK, another MAPK pathway molecule, didn't change the activity at all. And the differentiation induced by AMG was abrogated when the MDPC-23 cells were treated with U0126 and SB203580, the inhibitors of ERK1/2 and p38, respectively. Taken together, our present results showed that AMG could promote the differentiation of odontoblast-like MDPC-23 cells via ERK1/2 and p38 MAPK pathways.