Research On Function And Mechanism Of The M~6A Demethylase ALKBH5 In The Mechanical Signal Transduction Of Osteocytes

The structure and strength of the skeletal system are regulated by external mechanical stimulation.Mechanical loading is crucial for maintaining the health and stability of the skeletal system.Physiological mechanical loading（such as running and impedance exercise）can increase bone mass,improve bone microstructure and enhance mechanical properties.Lack of mechanical stimulation leads to bone degeneration and bone loss,and then causes osteoporosis（OP）,which is common in patients with long-term bed rest due to diseases and astronauts in the microgravity environment.Mechanical loading plays an important role in regulating the dynamic balance between bone resorption and bone formation.Therefore,it is of great significance to understand the mechanism of skeletal adaptation under physiological stress and microgravity environments.The skeletal system mainly includes osteoblasts（OB）,osteoclasts（OC）and osteocytes（OCY）.OB and OC located on the surface of bone tissue regulate bone formation and bone resorption,respectively.OCY located in the bone mineralized matrix is differentiated from OB and is the most abundant cell type in the bone.OCY senses external mechanical stimulation and secretes many cytokines to regulate bone formation and bone resorption,such as receptor activator of nuclear factorКappa-B ligand（RANKL）,Sclerostin,osteoprotegerin（OPG）.Therefore,OCY is not only the key mechano-sensor of bone,but also the master cell in bone remodeling.The cytokines secreted by OCY,such as RANKL,Sclerostin,DKK1,are potential new drug targets for OP,so OCY is a novel target cell for the prevention and treatment of OP.However,the mechanism of mechanical responses and signal transduction of OCY under physiologic mechanical stimulation and microgravity is still unclear.N6-methyladenosine6（m⁶A）is one of the most common chemical modifications on mRNA of various eukaryotes,which affects mRNA splicing,stability,transport and translation.Studies have shown that m⁶A methylation plays a key role in bone development such as osteogenic differentiation and cartilage differentiation,and the occurrence and development of osteosarcoma and osteoporosis are related to m⁶A methylation modification.The level of m⁶A modification is regulated by methyltransferases,demethylases and binding proteins.Does m⁶A play an important role in the mechanosensation and transduction of bone and osteocytes?Are m⁶A methylases involved in regulating the signal transduction of OCY under physiologic mechanical stimulation and microgravity?What is the core mechanism of m⁶A methylation affecting bone mechanosensitivity?These questions are still unclear.This study aims to explore the key m⁶A methylase that affects the mechanosensation and transduction of OCY,and systematically explore the effects of m⁶A methylase on the activity and function of OCY under physiologic mechanical stimulation and microgravity,which is expected to provide theoretical and experimental basis for seeking therapeutic approaches to bone diseases based on m⁶A modification.This study is mainly divided into the following five parts:Part Ⅰ:Effects of physiological loading and microgravity on the expression ofALKBH5 and total m⁶A in osteocytesBackgrounds:The health and stability of the skeletal system are regulated by the external mechanical environment.OCY is the mechanosensor of bone and secretes cytokines to regulate bone remodeling in response to mechanical stimulation.m⁶A,as the most prevalent modification in eukaryotic mRNAs,plays a key role in many physiological and pathological processes.Whether m⁶A methylation is involved in the regulation of bone mechanosensitivity,and what role m⁶A methylase plays in the mechanosensation and transduction of OCY are still unclear.Methods:The oscillatory fluid shear stress（OFSS）mode controller was developed to construct an experimental platform in vitro,and then cell and animal models under physiological loading and microgravity were constructed.The total m⁶A level of OCY was detected by dot blotting,and the gene and protein expressions of key m⁶A methylases（METTL3,METTL14,ALKBH5 and FTO）under physiological stress and SMG environment were analyzed by qRT-PCR and Western Blotting.Results:The total m⁶A levels of OCY were significantly decreased under in vitro OFSS stimulation and in vivo physiological mechanical loading,while the m⁶A modifications in OCY were increased both in vitro and in vivo under simulated microgravity（SMG）.OFSS stimulation significantly increased the mRNA and protein expressions of demethylase ALKBH5 in OCY,while SMG treatment decreased the mRNA and protein levels of ALKBH5 in OCY.However,OFSS stimulation and SMG environment had no significant effect on the expression of METTL3,METTL14 and FTO in OCY in vitro.Physiological mechanical loading significantly upregulated the mRNA and protein expressions of ALKBH5 in tibial OCY of mice,while hindlimb suspension significantly decreased the expression of ALKBH5 in tibial OCY.Consistent with the results of in vitro experiments,mechanical loading and hindlimb disuse did not affect the expression of METTL3,METTL14 and FTO in OCY in vivo.Conclusion:Physiologic mechanical stimulation increases the expression of demethylase ALKBH5 in OCY in vitro and in vivo,and reduces the levels of m⁶A methylation.The expression of ALKBH5 in OCY in vitro and in vivo under simulated microgravity environment decreases and then inhibits demethylase activity,resulting in the increase of m⁶A levels.This part of the study showes that the levels of m⁶A methylation in OCY change under physiological loading and microgravity,which is mainly caused by the change of ALKBH5 expression in OCY.Part Ⅱ:Effects of ALKBH5 on the activity and function of OCY under physiological loading and microgravityBackgrounds:m⁶A modification is a dynamic and reversible process.Methyltransferase catalyzes m⁶A methylation of RNA,and demethylase mediates m⁶A demethylation.Demethylase ALKBH5 plays a key role in reproductive development,tumorigenesis and cardiovascular repair.However,the influence of ALKBH5 on the activity and function of OCY under physiological loading and microgravity has not been reported.Methods:Firstly,ALKBH5 overexpression/knockdown stable cells were constructed by lentivirus infections,and then the ALKBH5 overexpressing OCY was treated with SMG,and the ALKBH5 knockdown OCY was subjected to physiological OFSS stimulation.The activity,apoptosis,gene and protein expression of ALKBH5 overexpressing/knockdown OCY under physiological loading and microgravity were detected by CCK-8,Annexin V-FITC/PI assay,qRT-PCR and Western Blotting.Results:Under SMG environment,the cell viability of Lv Ctrl group was decreased,and the apoptosis was increased,and the mRNA and protein levels ofβ-catenin,OPG and OPG/RANKL secreted by OCY were decreased,and the expression of DKK1 and RANKL were increased.After overexpressing ALKBH5,the SMG environment had no significant effect on the cell viability,apoptosis and gene and protein expression of key cytokines（β-catenin,OPG,RANKL,DKK1 and OPG/RANKL）of Lv ALKBH5 group.In addition,physiological OFSS stimulation significantly improved the cell viability,inhibit OCY apoptosis,upregulated the gene and protein expression ofβ-catenin,OPG and OPG/RANKL in normal OCY,and downregulated the expression of DKK1 and RANKL of sh Ctrl group.However,after ALKBH5 knockdown,OFSS stimulation had no significant influence on the cell viability,apoptosis and the expression ofβ-catenin,OPG,RANKL,DKK1 and OPG/RANKL in sh ALKBH5-1 and sh ALKBH5-2 groups.Conclusion:Overexpression of ALKBH5 can resist SMG-induced adverse effects on cell viability,apoptosis and the function of inhibiting bone formation and promoting bone resorption of OCY.ALKBH5 knockdown inhibits the positive regulation of physiological OFSS stimulation on the activity,apoptosis and secretion of cytokines regulating bone remodeling of OCY.ALKBH5 plays an important role in regulating the activity and function of OCY under physiological loading and microgravity.This study provides important evidence for ALKBH5 to be a potential new target for bone mechanical sensitization and disuse OP.Part Ⅲ:Effects of ALKBH5 in OCY on OB function under physiological loading and microgravityBackgrounds:OCY senses external mechanical signals and converts them into intracellular biochemical signals,and secretes cytokines such as RANKL,Sclerostin and OPG to regulate the activity of OB and OC,thereby regulating bone formation and bone resorption.Under the conditions of normal loading and microgravity,the effects of ALKBH5 knockdown or overexpression in OCY on OB function have not been fully elucidated.Methods:Firstly,ALKBH5 overexpressing OCY was exposed to SMG,and physiological OFSS was applied to ALKBH5 knockdown OCY,and then conditioned medium was collected respectively.The conditioned medium was added to primary OB,and the differentiation,mineralization and the expression of osteogenic genes and proteins were detected.Results:After adding conditioned medium of the SMG＿Lv Ctrl group of OCY,the ALP-stained area,mineralized nodules,and the gene and protein expression of Col1a1,Runx2,OCN and Osx in OB were significantly decreased;however,the above parameters did not change significantly after adding conditioned medium of the SMG＿Lv ALKBH5group.Adding conditioned medium of the OFSS＿sh Ctrl group of OCY significantly increased the ALP-stained area,mineralized nodules and the expression of Col1a1,Runx2,OCN and Osx in primary OB;adding conditioned medium of the OFSS＿Lv ALKBH5-1group and OFSS＿Lv ALKBH5-2 group,the differentiation ability,mineralization ability and the expression of osteogenic genes and proteins of OB did not change significantly.Conclusion:Overexpressing ALKBH5 in OCY alleviates the negative regulation of OCY on the differentiation,mineralization and the secretion of osteogenic cytokines of OB under SMG.The positive effects of OFSS-stimulated OCY on the osteogenic function of OB are significantly inhibited after knocking down ALKBH5.This study shows that ALKBH5 plays a key role in the mechanoresponse of OCY,which is indispensable for OCY to respond to external stress,regulate OB function,and affect bone remodeling.Part ⅠV:Research on the bone mechanosensitivity in ALKBH5 knockout mice under physiological loadingBackgrounds:Moderate mechanical stimulation,such as running and aerobic exercise,effectively promotes the structure and quality of bone tissue,thus maintaining bone health.The in vitro experiments in Part ⅠI and Part Ⅲ revealed that ALKBH5 is a key regulator of OCY activity and bone metabolism under physiological loading and microgravity.However,the regulatory effects of ALKBH5 in OCY on bone adaptation and bone remodeling under mechanical loading need to be further clarified.Methods:Firstly,the OCY-specific ALKBH5 knockout mice were constructed.Then,the right tibiae of ALKBH5^flox/floxmice and ALKBH5 cKO mice were subjected to physiological compressive loading for 2 weeks,and the left tibiae were served as unloaded control.Parameters of bone phenotype and bone metabolism between the loaded and unloaded tibiae were analyzed by Micro-CT scanning,dynamic bone histomorphometry and bone histological analyses.Results:Cyclic axial compression significantly improved the bone mass,bone microstructure and bone formation rate of trabecular and cortical bone in the loaded tibiae of ALKBH5^flox/floxmice,promoted the formation of OB and reduced the number of OC on the bone surface.In addition,physiological loading also significantly improved the structure of the lacunar-canalicular system of tibial OCY in ALKBH5^flox/floxmice,inhibited OCY apoptosis,reduced the secretion of Sclerostin and RANKL,and increased the expression of OPG in OCY.However,mechanical stimulation did not significantly promote the bone mass,bone remodeling,cell viability of OCY and the expression of important cytokines in OCY in ALKBH5 cKO mice.Conclusion:The bone mechanosensitivity of ALKBH5 cKO mice is significantly reduced,the mechanoresponse of tibial OCY in regulating OB and OC is weakened,and the effects of mechanical stimulation on bone anabolism and metabolism are declined.This study further enriches the mechanism of ALKBH5 in regulating bone mechano-adaptation.Part V:The Mechanism of ALKBH5 regulating bone mechanosensitivity under the conditions of physiological loading and microgravityBackgrounds:ALKBH5 regulates mRNA metabolism by m⁶A demethylation,including mRNA synthesis,splicing,modification and transport.The previous four parts revealed that ALKBH5 plays an important role in the activity of OCY under physiological loading and simulated microgravity.This part of the study aims to further explore the potential molecular mechanism of ALKBH5 in regulating bone mechanosensitivity.Methods:MeRIP-Seq was performed after ALKBH5 knockdown to detect the changes in gene expression and m⁶A methylation in OCY.Changes in the gene and protein expression of Lars2 in OCY after overexpression/knockdown of ALKBH5 under physiological loading and SMG were further detected by qRT-PCR and Western Blotting.Plasmid/si RNA was used to overexpress/knockdown Lars2 in OCY,and then Ca²⁺signaling and the expression of key cytokines of osteocyte to mechanical loading were detected.Results:After ALKBH5 knockdown,the gene expression of Lars2 in OCY was decreased,and the m⁶A level was increased.The expression of Lars2 in GO entries of aminoacyl-tRNA editing activity,ATP binding,and leucine-tRNA ligase activity was significantly decreased.Overexpression of ALKBH5 increased the expression of Lars2and alleviated the decrease of Lars2 expression in OCY under SMG.Knockdown of ALKBH5 decreased the expression of Lars2 in OCY,and OFSS-induced promotion on the expression of Lars2 was inhibited.SMG inhibited the calcium response and the expression ofβ-catenin and OPG in the Vector group,and increased the expression of RANKL and DKK1,but had no significant effect on the calcium oscillation and the cytokines of bone metabolism in the oe-Lars2 group.After OFSS stimulation,OCY generated stronger calcium oscillation in response to mechanical reloading,and the expression of genes and proteins promoting bone formation and inhibiting bone resorption increased,while the expression of cytokines inhibiting bone formation and promoting bone resorption decreased.The expression ofβ-catenin,OPG,RANKL and DKK1 did not change significantly after ALKBH5 knockdown.Conclusion:Knockdown of ALKBH5 gene in OCY significantly affects the gene expression and m⁶A methylation of downstream Lars2,and affects the activity of aminoacyl-tRNA ligase,which adversely affects the binding of leucine to tRNA and the process of aminoacylation.The change of ALKBH5 expression under physiological loading and simulated microgravity environment influences the expression of Lars2,and then affects the calcium response and function of OCY.This part of the study enriches the the mechanosensation and transduction of OCY,and has important scientific research value and clinical application prospects.