METTL3 promotes osteoblast ribosome biogenesis and alleviates periodontitis.

BACKGROUND: Periodontitis is a highly prevalent oral disease characterized by bacterium-induced periodontal inflammation and alveolar bone destruction. Osteoblast function is impaired in periodontitis with a global proteome change. METTL3 is the pivotal methyltransferase of N6-methyladenosine (m6A) that is recently proved to exert a crucial role in osteoblast differentiation. This study aims to investigate the role of METTL3 in osteoblast ribosome biogenesis in periodontitis progression. RESULTS: METTL3 was knocked down in osteoblasts, and the downregulated genes were enriched in ribosome and translation. METTL3 knockdown inhibited ribosome biogenesis and oxidative phosphorylation in LPS-stimulated osteoblasts, whereas METTL3 overexpression facilitated ribosomal and mitochondrial function. Mechanistically, METTL3 mediated osteoblast biological behaviors by activating Wnt/ß-catenin/c-Myc signaling. METTL3 depletion enhanced the mRNA expression and stability of Dkk3 and Sostdc1 via YTHDF2. In periodontitis mice, METTL3 inhibitor SAH promoted alveolar bone loss and local inflammatory status, which were partially rescued by Wnt/ß-catenin pathway activator CHIR-99021 HCl. CONCLUSIONS: METTL3 promoted ribosome biogenesis and oxidative phosphorylation by activating Wnt/ß-catenin/c-Myc signaling in LPS-treated osteoblasts and alleviated the inflammatory alveolar bone destruction in periodontitis mice.

METTL3 mediates osteoblast apoptosis by regulating endoplasmic reticulum stress during LPS-induced inflammation.

Osteoblast apoptosis is a prominent factor for disrupting skeletal homeostasis in multiple inflammatory bone diseases. METTL3, a key methyltransferase that catalyzes the N6-methyladenosine (m6A) modification of mRNA, has recently been shown to exert a critical role in osteogenic differentiation. However, the function of METTL3 in osteoblast apoptosis under inflammatory conditions remains elusive. In the present study, we observed that the total m6A level and METTL3 expression were upregulated in differentiated osteoblasts and downregulated after LPS stimulation. METTL3 knockdown induced a higher apoptotic rate in LPS-treated osteoblasts. The expression of the antiapoptotic protein BCL-2 decreased, and the apoptotic proteins cleaved Caspase-3, cleaved PARP-1 and cleaved Caspase-12 increased following METTL3 knockdown. Meanwhile, METTL3 silencing inhibited osteoblast proliferation and decreased osteogenic marker expression, ALP activity and mineralized nodules. RNA-seq analysis revealed that differentially expressed genes were significantly enriched in unfolded protein response pathways in METTL3-deficient cells. METTL3 depletion upregulated the expression of the ER stress-related markers, including p-PERK, p-eIF2α, p-IRE1α, GRP78, ATF4, CHOP and ATF6. Inhibition of ER stress by 4-PBA remarkably rescued METTL3 knockdown-induced apoptosis and promoted osteoblast proliferation and differentiation. Mechanistically, METTL3 depletion enhanced the expression and mRNA stability of Grp78, and similar results were observed after YTHDF2 knockdown. RIP-qPCR revealed that YTHDF2 directly interacted with Grp78 mRNA and that the interaction relied on METTL3. Taken together, our study demonstrated that METTL3 knockdown enhanced Grp78 expression through YTHDF2-mediated RNA degradation, which elicited ER stress, thereby promoting osteoblast apoptosis and inhibiting cell proliferation and differentiation under LPS-induced inflammatory condition.

METTL3 regulates LPS-induced inflammatory response via the NOD1 signaling pathway.

N6-methyladenosine (m6A) is a prevalent mRNA modification that plays a crucial function in multiple biological processes. Methyltransferase-like 3 (METTL3), an m6A methyltransferase, is essential for the m6A modification. Recently, the effect of METTL3 on the immune response has been reported. However, the effect is unclear, and the results are contradictory. In the present study, the total m6A and the expression of METTL3 decreased in LPS-stimulated macrophages. METTL3 knockdown significantly upregulated expression of proinflammatory cytokines, including TNF-α, IL-6 and NO. RNA sequencing analysis showed that the upregulated genes were enriched in inflammation-related signaling pathways and that the NOD-like receptor signaling pathway might be the target molecules of METTL3. METTL3 depletion resulted in upregulation of the NOD1 pathway without impacting NOD2. Moreover, the increase in proinflammatory cytokines induced by METTL3 knockdown was reversed by blocking the NOD1 pathway using specific inhibitors. Mechanistically, METTL3 knockdown promoted the mRNA expression and stability of NOD1 and RIPK2, and the same results were detected in m6A-binding protein YTHDF1- or YTHDF2-silenced cells. All findings suggested that METTL3 depletion inhibits the degradation of NOD1 and RIPK2 mRNA mediated by YTHDF1 and YTHDF2, which upregulate the NOD1 pathway and subsequently promote the LPS-induced inflammatory response in macrophages.

Preparation of coix seed oil bioactive delivery systems based on homologous polysaccharides and proteins.

Natural products belonging to a class of generally-recognized-as-safe biomaterials have exceptional biocompatibility and biodegradability and can be used as delivery vehicles for a variety of functional foods. Adlay (Coix lacryma-jobi), is a nutritious food, rich in various bioactive ingredients. Coix seed oil extract (CSO) is also bioactive but it is sensitive to oxidation. In this study, a bioactive delivery system based on homologous polysaccharides and proteins was developed to deliver coix seed oil. The results show that the CSO nanoparticles have high encapsulation efficiency, narrow particle size distribution, and good stability. Moreover, the fusion of the nanoparticles with the membrane enabled the transport of CSO through the Caco-2 cell monolayer and improved the intestinal permeability. These findings could provide useful information for designing homologous polysaccharide and protein-based delivery systems to increase the bioavailability of lipophilic nutraceuticals in the food industry.