METTL3 Regulates Osteoclast Biological Behaviors via iNOS/NO-Mediated Mitochondrial Dysfunction in Inflammatory Conditions

Abstract

Excessive differentiation of osteoclasts contributes to the disruption of bone homeostasis in inflammatory bone diseases. Methyltransferase-like 3 (METTL3), the core methyltransferase that installs an N6-methyladenosine (m6A) modification on RNA, has been reported to participate in bone pathophysiology. However, whether METTL3-mediated m6A affects osteoclast differentiation in inflammatory conditions remains unelucidated. In this study, we observed that the total m6A content and METTL3 expression decreased during LPS-induced osteoclastogenesis. After knocking down METTL3, we found reduced levels of the number of osteoclasts, osteoclast-related gene expression and bone resorption area. A METTL3 deficiency increased osteoclast apoptosis and pro-apoptotic protein expression. RNA sequencing analysis showed that differentially expressed genes in METTL3-deficient cells were mainly associated with the mitochondrial function. The expression of the mitochondrial function-related genes, ATP production and mitochondrial membrane potential decreased after METTL3 knockdown. Moreover, the most obviously upregulated gene in RNA-Seq was Nos2, which encoded the iNOS protein to induce nitric oxide (NO) synthesis. METTL3 knockdown increased the levels of Nos2 mRNA, iNOS protein and NO content. NOS inhibitor L-NAME rescued the inhibited mitochondrial function and osteoclast formation while suppressing osteoclast apoptosis in METTL3-silenced cells. Mechanistically, a METTL3 deficiency promoted the stability and expression of Nos2 mRNA, and similar results were observed after m6A-binding protein YTHDF1 knockdown. Further in vivo evidence revealed that METTL3 knockdown attenuated the inflammatory osteolysis of the murine calvaria and suppressed osteoclast formation. In conclusion, these data suggested that METTL3 knockdown exacerbated iNOS/NO-mediated mitochondrial dysfunction by promoting a Nos2 mRNA stability in a YTHDF1-dependent manner and further inhibited osteoclast differentiation and increased osteoclast apoptosis in inflammatory conditions.

Harnessing osteoimmunity to treat peri-implant inflammatory osteolysis

“…74 Methyltransferase-like 3 (METTL3), the core methyltransferase that installs an N 6-methyladenosine (m6A) modification on RNA, stabilizes the mitochondrial function by reducing the iNOS/NO expression, thereby inhibiting osteolysis under inflammatory conditions. 86 The above studies suggest that epigenetic modifications are feasible for the treatment of inflammatory osteolysis.…”

N6-Methyladenosine in Cell-Fate Determination of BMSCs: From Mechanism to Applications

“…STM2457 pretreatment down-regulated the expression of MyD88 in bone marrow-derived macrophages and alleviated the symptoms of osteomyelitis in mice [ 154 ]. Meanwhile, METTL3 knockdown could inhibit osteoclast differentiation and raise osteoclast apoptosis in inflammatory bone disease by promoting NOS2 mRNA stability in a YTHDF1-dependent manner [ 155 ]. Nonetheless, it should be highlighted that the blocking of METTL3, on the one hand, avoids the progression of inflammatory osteolysis and destruction and, on the other hand, impedes the osteogenesis of BMSCs and encourages the survival and proliferation of colonized bacteria [ 154 ].…”

N6-Methyladenosine (m6A) Modification in Natural Immune Cell-Mediated Inflammatory Diseases

“…Its overexpression promoted IL-6 expression and secretion of inducible NO synthase (iNOS) in M1 macrophages by increasing the m6A modification of dual specificity phosphatase 14 ( Dusp14 ) and histone deacetylase 5 ( Hdac5 ) [43]. However, METTL3 inhibited iNOS mRNA stability via a YTHDF1-dependent mechanism during inflammation, reducing the mitochondrial dysfunction mediated by iNOS/NO [44]. Additionally, extracellular vesicles derived from human umbilical cord mesenchymal stem cells exerted anti-inflammatory effects by downregulating macrophage METTL3, resulting in enhanced chondrocyte proliferation/migration and reduced NLRP3-induced inflammation which alleviated osteoarthritis [45].…”