RESUMEN
Inflammatory environments can trigger endoplasmic reticulum (ER) stress and lead to pyroptosis in various tissues and cells, including liver, brain, and immune cells. As a key factor of ER stress, DNA damage-inducible transcript 3 (DDIT3)/CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is upregulated in osteoblasts during inflammatory stimulation. DDIT3/CHOP may therefore regulate osteoblast pyroptosis in inflammatory conditions. During this investigation, we found that lipopolysaccharides (LPS)/adenosine 5'-triphosphate (ATP) stimulation in vitro induced osteoblasts to undergo pyroptosis, and the expression of DDIT3/CHOP was increased during this process. The overexpression of DDIT3/CHOP further promoted osteoblast pyroptosis as evidenced by the increased expression of the inflammasome NLR family pyrin domain containing 3 (NLRP3) and ratios of caspase-1 p20/caspase-1 and cleaved gasdermin D (GSDMD)/GSDMD. To explore the specific mechanism of this effect, we found through fluorescence imaging and Western blot analysis that LPS/ATP stimulation promoted PTEN-induced kinase 1 (PINK1)/E3 ubiquitin-protein ligase parkin (Parkin)-mediated mitophagy in osteoblasts, and this alteration was suppressed by the DDIT3/CHOP overexpression, resulting in increased ratio of pyroptosis compared with the control groups. The impact of DDIT3/CHOP on pyroptosis in osteoblasts was reversed by the application of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a specific mitophagy agonist. Therefore, our data demonstrated that DDIT3/CHOP promotes osteoblast pyroptosis by inhibiting PINK1/Parkin-mediated mitophagy in an inflammatory environment.
Asunto(s)
Lipopolisacáridos , Piroptosis , Lipopolisacáridos/farmacología , Mitofagia , Caspasa 1/metabolismo , Caspasa 1/farmacología , Adenosina Trifosfato/metabolismo , Osteoblastos/metabolismo , Proteínas Quinasas , Ubiquitina-Proteína Ligasas/farmacologíaRESUMEN
The coordination of osteoblasts and osteoclasts is essential for bone remodeling. DNA damage inducible script 3 (DDIT3) is an important regulator of bone and participates in cell differentiation, proliferation, autophagy, and apoptosis. However, its role in bone remodeling remains unexplored. Here, we found that Ddit3 knockout (Ddit3-KO) enhanced both bone formation and resorption. The increased new bone formation and woven bone resorption, i.e., enhanced bone remodeling capacity, was found to accelerate bone defect healing in Ddit3-KO mice. In vitro experiments showed that DDIT3 inhibited both osteoblast differentiation and Raw264.7 cell differentiation by regulating autophagy. Cell coculture assay showed that Ddit3-KO decreased the ratio of receptor activator of nuclear factor-κß ligand (RANKL) to osteoprotegerin (OPG) in osteoblasts, and Ddit3-KO osteoblasts inhibited osteoclast differentiation. Meanwhile, DDIT3 knockdown (DDIT3-sh) increased receptor activator of nuclear factor-κß (RANK) expression in Raw264.7 cells, and DDIT3-sh Raw264.7 cells promoted osteoblast differentiation, whereas, DDIT3 overexpression had the opposite effect. Mechanistically, DDIT3 promoted autophagy partly by increasing ULK1 phosphorylation at serine555 (pULK1-S555) and decreasing ULK1 phosphorylation at serine757 (pULK1-S757) in osteoblasts, thereby inhibiting osteoblast differentiation. DDIT3 inhibited autophagy partly by decreasing pULK1-S555 in Raw264.7 cells, thereby suppressing osteoclastic differentiation. Taken together, our data indicate that DDIT3 is one of the elements regulating bone remodeling and bone healing, which may become a potential target in bone defect treatment.
Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia , Remodelación Ósea , Osteoblastos , Osteoclastos , Factor de Transcripción CHOP , Animales , Ratones , Autofagia , Resorción Ósea/metabolismo , Diferenciación Celular/fisiología , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoprotegerina/metabolismo , Ligando RANK/metabolismo , Factor de Transcripción CHOP/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismoRESUMEN
DNA damage-inducible transcript 3 (DDIT3), a stress response gene, engages in the physiological and pathological processes of organisms, whereas its impact on pulpitis has not been defined yet. It has been demonstrated that macrophage polarization has a significant impact on inflammation. This research intends to investigate the effect of DDIT3 on the inflammation of pulpitis and macrophage polarization. C57BL/6J mice were used to model experimental pulpitis at 6, 12, 24, and 72 h after pulp exposure, with untreated mice as the control. The progression of pulpitis was visible histologically, and DDIT3 showed a trend of initially upward and downward later. Compared with wild-type mice, inflammatory cytokines and M1 macrophages were reduced, while M2 macrophages were increased in DDIT3 knockout mice. In RAW264.7 cells and bone borrow-derived macrophages, DDIT3 was found to enhance M1 polarization while impair M2 polarization. Targeted knockdown of early growth response 1 (EGR1) could rescue the blocking effect of DDIT3 deletion on M1 polarization. In conclusion, our results indicated that DDIT3 could exacerbate inflammation of pulpitis through the regulation of macrophage polarization, and DDIT3 could promote M1 polarization by inhibiting EGR1. It provides a new target for pulpitis treatment and tissue regeneration in the future.
Asunto(s)
Pulpitis , Animales , Ratones , Inflamación , Macrófagos , Ratones Endogámicos C57BL , Células RAW 264.7RESUMEN
BACKGROUND AND OBJECTIVE: Periodontitis is a multifactorial inflammatory disease that leads to the destruction of supporting structures of the teeth. DNA damage-inducible transcript 3 (DDIT3) plays crucial roles in cell survival and differentiation. DDIT3 regulates bone mass and osteoclastogenesis in femur. However, the role of DDIT3 in periodontitis has not been elucidated. This research aimed to explore the role and mechanisms of DDIT3 in periodontitis. METHODS: DDIT3 gene knockout (KO) mice were generated using a CRISPR/Cas9 system. Experimental periodontitis models were established to explore the role of DDIT3 in periodontitis. The expression of DDIT3 in periodontal tissues was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). The alveolar bone phenotypes were observed by micro-CT and stereomicroscopy. The inflammation levels and osteoclast activity were examined by histological staining, immunostaining, and qRT-PCR. Bone marrow-derived macrophages (BMMs) were isolated to confirm the effects of DDIT3 on osteoclast formation and function in vitro. RESULTS: The increased expression of DDIT3 in murine inflamed periodontal tissues was detected. DDIT3 knockout aggravated alveolar bone loss and enhanced expression levels of inflammatory cytokines in murine periodontitis models. Increased osteoclast formation and higher expression levels of osteoclast-specific markers were observed in the inflamed periodontal tissues of KO mice. In vitro, DDIT3 deficiency promoted the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts and the bone resorption activity of mature osteoclasts. CONCLUSIONS: Our results demonstrate that DDIT3 deletion aggravated alveolar bone loss in experimental periodontitis through enhanced inflammatory reactions and osteoclastogenesis. The anti-inflammation and the inhibition of bone loss by DDIT3 in murine periodontitis provides a potential novel therapeutic strategy for periodontitis.
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Pérdida de Hueso Alveolar , Resorción Ósea , Periodontitis , Animales , Ratones , Pérdida de Hueso Alveolar/patología , Daño del ADN , Inflamación/patología , Osteoclastos/metabolismo , Periodontitis/tratamiento farmacológico , Ligando RANK/metabolismoRESUMEN
As the main cells in endochondral osteogenesis, chondrocytes have limited self-repair ability due to weak proliferation activity, low density, and dedifferentiation tendency. Here, a thorough inquiry about the effect and underlying mechanisms of methyltransferase like-3 (Mettl3) on chondrocytes was made. Functionally, it was indicated that Mettl3 promoted the proliferation and hypertrophic differentiation of chondrocytes. Mechanically, Dmp1 (dentin matrix protein 1) was proved to be the downstream direct target of Mettl3 for m6A modification to regulate the differentiation of chondrocytes through bioinformatics analysis and correlated experiments. The Reader protein Ythdf1 mediated Dmp1 mRNA catalyzed by Mettl3. In vivo, the formation of subcutaneous ectopic cartilage-like tissue further supported the in vitro results. In conclusion, the gene regulation of Mettl3/m6A/Ythdf1/Dmp1 axis in hypertrophic differentiation of chondrocytes for the development of endochondral osteogenesis may supply a promising treatment strategy for the repair and regeneration of bone defects.
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Condrocitos , Metiltransferasas , Diferenciación Celular/genética , Condrocitos/metabolismo , Proteínas de la Matriz Extracelular , Humanos , Hipertrofia , Metiltransferasas/genética , Metiltransferasas/metabolismo , Osteogénesis/genética , Fosfoproteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
DNA damage-inducible transcript 3 (DDIT3), a member of the CCAAT/enhancer-binding protein (C/EBP) family, is involved in cellular apoptosis and differentiation. DDIT3 participates in the regulation of adipogenesis and osteogenesis in vitro and in vivo. However, the role of DDIT3 in osteoclastogenesis is not yet known. In this study, the involvement of DDIT3 in osteoclast differentiation and function was reported for the first time. CRISPR/Cas9-mediated DDIT3 knockout (KO) mice were generated for functional assessment. Tartrate-resistant acid phosphatase (TRAP) staining of distal femurs showed increased positive cells in DDIT3 KO mice. DDIT3 expression was downregulated during the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation of bone marrow-derived macrophages (BMMs). The loss of DDIT3 increased the expression of osteoclast-specific markers, including nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), TRAP, cathepsin K (CTSK), and dendritic cell-specific transmembrane protein (DC-STAMP) and promoted the formation of TRAP-positive multinucleated osteoclasts. The actin ring number and resorption area of bone slices were also increased in DDIT3 KO BMMs. Lentivirus-mediated DDIT3 overexpression significantly inhibited the osteoclast differentiation of RAW264.7 cells. In the tumor necrosis factor-α-induced osteolysis model, DDIT3 deficiency enhanced osteoclast formation and aggravated bone resorption. DDIT3 inhibited osteoclast differentiation by regulating the C/EBPα-CTSK axis. Furthermore, DDIT3 KO intensified the RANKL-triggered activation of the MAPKs and Akt signaling pathways. Taken together, the results revealed the essential role of DDIT3 in osteoclastogenesis in vitro and in vivo and its close relationship with osteoclast-associated transcription factors and pathways.
Asunto(s)
Resorción Ósea , Osteólisis , Animales , Resorción Ósea/genética , Diferenciación Celular , Daño del ADN , Ratones , Factores de Transcripción NFATC , Osteoclastos , Osteogénesis , Ligando RANKRESUMEN
OBJECTIVES: The proliferation, migration capacity, and expression of activation-related proteins of NHGFs+Cal27-exo were determined by coculturing Cal27 exosome (Cal27-exo) with normal human gingival fibroblasts (NHGFs) to explore the effects of Cal27-exo on the activation and biological behavior of NHGFs. METHODS: Cal27-exo was extracted using supercentrifugation, and exosomes were identified using Western blot, transmission electron microscopy (TEM), and particle size detection. Cal27-exo was cocultured with NHGFs to detect the uptake of Cal27-exo by NHGFs, and the proliferation and migration capacity of NHGFs+Cal27-exo were detected using CCK8 and wound healing tests, respectively. The expression levels of NHGF activation-related proteins, i.e., matrix metalloproteinase-9 (MMP-9), fibroblast-activating protein (FAP), alpha smooth muscle actin (αSMA), and transforming growth factor-ß (TGF-ß), were detected using real-time quantitative polymerase chain reaction (qRT-PCR). RESULTS: Cal27-exo was extracted u-sing supercentrifugation, and Western blot showed the positive expression levels of Alix and CD63. TEM showed that Cal27-exo had a circular double-layer vesicle. The particle size was between 30 and 150 nm. Cal27-exo labeled with PKH67 entered NHGFs after the coculture method. The wound healing test showed that the migration capacity of NHGFs+Cal27-exo was stronger after the scratch compared with that of NHGFs. CCK8 results showed that the proliferation activity of NHGFs+Cal27-exo was enhanced. qRT-PCR results showed that the MMP-9 levels of NHGFs+Cal27-exo were upregulated, whereas the TGF-ß and αSMA mRNA levels of NHGFs+Cal27-exo were downregulated (P<0.05). CONCLUSIONS: The proliferation and migration ability of NHGFs+Cal27-exo are enhanced, and the mRNA expression of related proteins is changed. Cal27-exo can activate NHGFs, which suggests that Cal27-exo has potential significance in tumor invasion and metastasis.
Asunto(s)
Exosomas , Proliferación Celular , Fibroblastos , Encía , Humanos , Metaloproteinasa 9 de la MatrizRESUMEN
Oral squamous cell carcinoma (OSCC) is the most prevalent form of malignant tumour in the oral cavity and its early detection is critical for improving the prognosis of affected patients. The present study aimed to isolate and confirm exosomes derived from the supernatant of the OSCC cell line CAL-27 and human oral epithelial cells (HOECs), analyze long non-coding RNA (lncRNA) expression profiles and determine the diagnostic value based on bioinformatics analyses. The results indicated that the particles isolated from the supernatant of CAL-27 and HOECs were either round or oval, had a size range of 30-150 nm and were enriched with ALG-2 interacting protein X (ALIX) and tumour susceptibility 101 proteins (TSG101). These characteristics confirmed that these particles were exosomes. Three lncRNAs (NR-026892.1, NR-126435.1 and NR-036586.1) were selected as potential diagnostic biomarkers for OSCC. The expression levels of the selected lncRNAs were significantly different in CAL-27-exo vs. HOEC-exo, as well as in whole cells (CAL-27 vs. HOECs) (P<0.001). The expression levels of the three lncRNAs confirmed by quantitative PCR were consistent with the sequencing data. In conclusion, various lncRNAs were aberrantly expressed between cancerous and non-cancerous exosomes, suggesting that they may serve as biomarkers for cancer.
RESUMEN
The key of the root canal therapy is to eliminate the micro-organism infection, fill the root canal tightly and reduce the stimulation to the periapical tissues. However, it is quite difficult to meet all the conditions due to the defect of the material. Here we develop a novel root canal sealer (MZOE), in which zinc oxide eugenol (ZOE) were fabricated with polyhexamethylene guanidine (PHMG), and the PHMG's concentration is 0.8, 1.0, 1.2 and 1.4%. Our investigation tested its physical properties, antibacterial effect to E. faecalis, C. albicans, E. coli, S. aureus and cytotoxicity to human periodontal ligament fibroblasts (HPDLFs). The physical properties of the MZOE conformed to the ISO 6876:2001, and its antibacterial effect was stronger than ZOE (p<0.05), the RGR of HPDLFs was tested between 1 to 24%, belonging to moderate cytotoxicity. It was suggested that MZOE had good physical properties, high antibacterial effect, and moderate cytotoxicity.