Inhibition Of MAT2A Suppresses Osteoclastogenesis And Prevents Ovariectomy-induced Bone Loss

Objective:Osteoclast-mediated function of bone resorption is involved in the process of bone turnover.The excessive number or overactive function of osteoclasts leads to the occurrence of various skeletal diseases,like the rheumatoid arthritis and osteoporosis.The intracellular ROS level is increased and inflammatory signaling pathways like the NF-κB and MAPK pathways are activated during the process of osteoclastogenesis.MAT2 A is the key enzyme to transform methionine and adenosine-triphosphate to SAM,based on this regulating methylation modifications in vitro.MAT2 A also participates in the activation of NF-κB pathway and the balance of ROS.Whether MAT2 A affects the process of osteoclastogenesis and the mechanism have not been reported.This project aims to investigate the effects on osteoclasts and the potential mechanism involved,and provide potential intervention target and new therapeutic strategy for the treatment of osteoporosis and other osteoclast-related diseases.Methods:1.The expression level of MAT2 A during the process of osteoclastogenesis was analyzed by western blotting and immunofluorescence imaging.AG-270,one of specific inhibitor of MAT2 A was used to intervene BMMs to investigate the effect on their proliferative capacity.2.AG-270 and sh RNA lentivirus targeting mouse MAT2 A were used to inhibit the activity of MAT2 A,TRAP staining,F-Actin staining and pit formation assay were conducted to evaluate the effects on the formation of osteoclasts and function of them.q PCR and western blotting analysis were performed to analyze the expression of important transcription factors involved in osteoclast differentiation and osteoclast-related marker genes,cellular ROS level was also detected,and content of Nrf2 pathway-related proteins and the activity of NF-κB and MAPK pathways were also detected by immunoblotting.3.Ovariectomy mouse model was established to mimic postmenopausal osteoporosis.According to the group design,the solvent or different concentrations of AG-270 were administered by intragastric injection administration every day after the surgery.6 weeks later,the femurs of each mouse were isolated and fixed for 3 days.Micro CT scanning and data analysis were performed to evaluate the content of cancellous bone of the distal femurs.TRAP staining and morphological analysis of tissue sections were performed to analyze the osteoclasts in the femurs.4.Quasi-targeted metabolomics analysis of the metabolic changes of osteoclast precursor cells under the stimulation of RANKL was conducted,and MAT2A-related metabolites were also analyzed to find out the potential mechanism.Inhibition MAT2 A and addition of SAM was performed to detect the effects on osteoclastogenesis and the function of osteoclasts,q PCR and western blotting were conducted to evaluate the expression of osteoclast-related crucial genes,detection of cellular ROS level was also performed,the expression of Nrf2 pathway-related proteins and activities of NF-κB and MAPK signaling pathways were analyzed by western blotting.Results:1.The content of MAT2 A in osteoclast precursor cells and mature osteoclasts was increased during the process of osteoclast differentiation,proving that MAT2 A contributes to osteoclastogenesis,and AG-270 had no obvious toxic effects on BMMs and the mice.2.AG-270 and sh RNA lentivirus targeting mouse MAT2 A treatments significantly inhibited the number and volume of osteoclasts,and suppressed the capacity of osteoclast-mediated bone resorption,the expressions of crucial genes involved in the process of osteoclastogenesis were also reduced,proving that MAT2 A plays an important role in the process of osteoclast differentiation.3.AG-270 administration by intragastric injection could reduce the ovariectomy-induced bone loss,and osteoclast formation in the bone tissue was significantly inhibited,indicating that AG-270 can inhibit the differentiation of osteoclast in vivo.4.Inhibition of MAT2 A significantly reduced the cellular ROS level and increased the activity of antioxidant-related pathways,and inhibited the phosphorylation of key proteins in NF-κ B and MAPK pathways.Suggesting that inhibition of MAT2 A can inhibit the intracellular ROS levels and the activty of NF-κB and MAPK signaling pathways.5.Osteoclast precursor cells revealed an active metabolism level with the stimulation of RANKL,and the content of MAT2A-related metabolites also increased.Inhibition of MAT2 A could reduce the content of SAM and other related metabolites,and the addition of SAM could partially rescue the suppressant effects caused by inhibition of MAT2 A on the intracellular ROS level and the activity of NF-κB and MAPK signaling pathways,demonstrating that MAT2 A affects the downstream pathways through SAM to participate in the process of osteoclast differentiation.Conclusion:In this study,we demonstrated that the inhibition of MAT2 A suppressed the formation of osteoclasts and osteoclast-mediated function of bone resorption,the content of intracellular MAT2A-related metabolites declined,which suppressed the intracellular ROS level,and the activity of NF-κB and MAPK pathways.AG-270,the specific inhibitor of MAT2 A could inhibit ovariectomy-induce bone loss in the mice model.Generally speaking,these findings revealed that MAT2 A is crucial for osteoclast differentiation and function,it may be a latent target for the therapy of skeletal diseases like the osteoporosis,which is caused by hyperactivity of osteoclast.