| BackgroundOsteoporosis(OP)is a systemic progressive bone disease characterised by low bone mass,damage to the microarchitecture of bone tissue leading to increased bone fragility and susceptibility to fracture.It is most commonly seen in post-menopausal women and men over the age of 50.Osteoporosis is a huge and growing public health problem with a complex pathogenesis involving a complex interplay between local and systemic regulation of skeletal cell function.At the cellular level,communication and coupling between osteoblasts and osteoclasts play a key role in the process of bone remodelling.Imbalances in the differentiation and functional coordination between these two types of cells underlie the pathogenesis of osteoporosis.In recent years,the mechanisms of energy production and utilisation in skeletal cells have also received increasing attention.Both osteoblasts and osteoclasts consume large amounts of energy to perform their biological functions in the body.When energy metabolism is impaired in skeletal cells,this can lead to disturbances in the balance of bone metabolism and the development of osteoporosis and other related diseases.Studies have shown that the energy metabolism of osteoblasts and osteoclasts is different,with glucose being the main source of energy for both types of cells.Glycolysis is the main metabolic pathway to meet the demand for adenosine triphosphate(ATP)during osteoblast differentiation.Lactate is the main end product of glucose metabolism in osteoblasts.The process of osteoblast fusion is energy intensive,with more mitochondria per surface area than almost any other cell.The differentiation of osteoblasts from circulating monocyte precursors and the process of fusion are primarily driven by oxidative phosphorylation.Coactivator-associated arginine methyltransferase 1(CARM1),also known as PRMT4,was identified as a co-regulator of transcription at the time of its discovery.Recent studies have identified novel roles for CARM1 in autophagy,metabolism and early development.In terms of metabolic regulation,it was shown that methylation of pyruvate kinase isoform M2(PKM2)by CARM1 shifts the metabolic balance of breast cancer cells from oxidative phosphorylation to aerobic glycolysis.However,whether CARM1 can regulate energy metabolism in osteoblasts or osteoclasts is unclear,and its role in the pathogenesis of osteoporosis has not been elucidated.Objectives(1)To investigate the changes in CARM1 expression in osteoporosis and its effects on the osteogenic and osteolytic differentiation process.(2)To investigate the mechanisms by which CARM1 regulates glucose metabolism in osteoblasts and osteoclasts.(3)To explore the effect of intervention of CARM1 expression on bone quality in mice with osteoporosis model and to add new research basis for clinical exploration of effective ways to alleviate osteoporosis.MethodsPart Ⅰ:Study of the effect of CARM1 on osteogenic differentiation and osteoclastic differentiation of cells and the effect of CARM1 on mice in osteoporosis models1.1 Analysis of database resources and validation using patient-derived specimens(1)Analysis of GSE156508 data(expression data in primary osteoblasts obtained from women with osteoporotic fractures or severe osteoarthritis)to clarify the changes in CARM1 expression.(2)Analysis of dataset GSE176265(gene expression profiling during RANKL-mediated osteoblast differentiation)to clarify changes in CARM1 expression.(3)RT-qPCR experiments to validate CARM1 expression in osteoporotic and control patient-derived bone specimens.1.2 Effect of CARM1 on osteogenic differentiation(1)Overexpression and knockdown of Carm1 in MC3T3-E1 cell line and osteogenic induction of differentiation were performed,and the level of osteogenic differentiation of cells was detected by alizarin red(ARS)staining and alkaline phosphatase(ALP)staining.(2)The expression changes of osteogenic-related genes in MC3T3-E1 cells were detected by RT-qPCR,Western blot and cellular immunohistochemistry assays.1.3 Effect of CARM1 on osteoblastic differentiation(1)Overexpression and knockdown of Carm1 in RAW264.7 cell line and osteoclast-induced differentiation were performed,and the level of osteoclast differentiation and maturation of cells was detected by anti-tartrate acid phosphatase(TRAP)staining and cellular F-actin staining.(2)Changes in the expression of osteoclast-related genes in RAW264.7 cells were detected by RT-qPCR,Western blot,and cellular immunohistochemistry assays.1.4 Effects of CARM1 on osteoporosis model mice(1)C57BL/6 wild-type female mice were divided into sham-operated,osteoporosis and treatment groups,and mice in the osteoporosis and treatment groups were subjected to bilateral ovariectomy.(2)The mice in the sham-operated group were injected with an equal volume of PBS into the femoral bone marrow cavity,the mice in the osteoporotic group were injected with the control lentivirus into the femoral bone marrow cavity,and the mice in the treated group were injected with Carml overexpressing lentivirus into the femoral bone marrow cavity.lentivirus infection was detected by bioluminescence assay after 4 weeks.(3)After 8 weeks,the mice were executed and the femurs were sampled and the expression of Carm1 in femoral bone tissue was measured by RT-qPCR.The expression of osteogenic and osteoclast-related genes was detected by micro-CT,osteogenic and osteoclast-related genes by immunohistochemistry,collagen formation by Masson staining,and osteoclast differentiation and maturation by TRAP staining.Part Ⅱ:CARM1 regulates glucose metabolism in osteoblasts and osteoclasts2.1 Effect of CARM1 on cellular glucose metabolism(1)Overexpression and knockdown of CARM1 in MC3T3-E1 and RAW264.7 cell lines for cellular glycolytic rate(ECAR)and mitochondrial stress(OCR)assays.(2)Detection of mitochondrial membrane potential by JC-10 assay.2.2 Specific pathways by which CARM1 affects glycolytic flux(1)Using metabolomic assays to clarify the types of differential metabolites following CARM1 overexpression in MC3T3-E1 and RAW264.7 cells.(2)The results of the metabolomics assay were verified by phosphofructokinase-1(PFK1)activity assay,and the effect of CARM1 on cellular osteogenic differentiation after inhibition of glycolysis was observed by applying the glycolysis inhibitor DCA and by ARS and ALP staining.(3)Application of PFK15,an inhibitor of PFK1,to observe the effect of CARM1 on osteogenic differentiation of cells after inhibition of PFK1 activity by ARS and ALP staining and Western blot assay.Part Ⅲ,Mechanistic studies on the regulation of PFK-1 activity by CARM13.1 Studies on the regulation of PFK1 activity by CARM1(1)By transcriptomic assay and KEGG analysis,the genes and signalling pathways regulated by CARM1 were clarified,and the upstream factors affecting PFK1 activity were searched for.(2)To detect changes in the phosphorylation levels of PFK1 and 6-phosphofructokinase-2(PFKFB3)in CARM1 overexpression and knockdown cells by Western blot assay.(3)Changes in AKT/AMPK phosphorylation levels in Carm1 overexpression and knockdown cells were detected by Western blot assay.3.2 AKT/AMPK phosphorylation mediates the regulation of PFK1 activity by CARM1(1)The AKT inhibitor MK2206 was applied to observe the role of AKT in the regulation of PFK1 activity by CARM1 through PFK1 activity assay.(2)Application of AKT inhibitor MK2206 and detection of osteogenic differentiation of CARM1 overexpression cells by ARS and ALP staining.The expression of osteogenic-related genes in Carml overexpressing cells was detected by Western blot assay.(3)Application of AKT activator SC-79 and the inhibitor of PFK1,PFK15,on top of this,to detect osteogenic differentiation of Carm1 knockdown cells by ARS and ALP staining.The expression of osteogenic-related genes in Carm1 knockout cells was detected by Western blot assay.(4)Application of AMPK inhibitor Compound C and detection of osteogenic differentiation in Carml overexpressing cells by TRAP staining.Part Ⅳ,Mechanistic studies on the regulation of AKT/AMPK phosphorylation levels by CARM14.1 Study on the interaction between CARM1 and PPP1CA(1)The upstream regulators of AKT/AMPK phosphorylation were clarified by analysing CARM1 GST-pulldown results.(2)The direct interaction of CARM1 with PPP1CA was clarified by immunoprecipitation,immunofluorescence and Biacore assays.4.2 Methylation site detection and validation of PPP1CA(1)The methylation sites of PPP1CA were detected by in vitro methylation assay and tandem mass spectrometry(MS/MS)analysis.(2)The major methylation sites of PPP1CA were clarified by in vitro methylation assay,in vitro transcription and translation assay,liquid scintillation counting,and radioautography assay.4.3 The role of PPP1CA-R23 methylation in CARM1 regulation of grape metabolism to regulate osteogenesis and osteoblastic differentiation(1)The role of PPP1CA-R23 methylation in the regulation of glycolysis by CARM1 was examined by ECAR assay.(2)The role of PPP1CA-R23 methylation in the regulation of AKT/AMPK phosphorylation by CARM1 was examined by Western blot assay.(3)The effect of PPP1CA-R23 methylation on the regulation of osteogenic differentiation by CARM 1 was detected by ARS and ALP staining,and the effect of PPP1CA-R23 methylation on the regulation of osteoclastic differentiation by CARM1 was detected by TRAP staining.The expression of osteogenic and osteolytic-related genes in CARM1 overexpression cells was examined by Western blot assay.Part Ⅴ.Mechanistic studies on the regulation of aerobic cellular glucose metabolism by CARM 15.1 Studies on the involvement of PDK3 in the regulation of aerobic cellular metabolism by CARM1(1)Analysis of transcriptomic data to identify downstream factors involved in CARM1 regulation of aerobic cellular metabolism.(2)To validate the expression changes of pyruvate dehydrogenase kinase(PDK)family members in bone specimens from osteoporosis patients.(3)To detect the expression of PDK3 in CARM1 overexpression/knockdown cells by Western blot assay.5.2 Mechanistic studies on the regulation of PDK3 expression by CARM1(1)Search for transcription factors of PDK3 by searching the Cistrome Data Browser database.(2)Validation of the transcription factors of PDK3 by chromatin immunoprecipitation assay.(3)Validation of the joint role of CARM1 and EP300 in regulating PDK3 expression by immunoprecipitation and immunofluorescence assays.5.3 Study of the role of PDK3 in CARM1 regulation of grape metabolism,regulation of osteogenesis and osteoblastic differentiation(1)The role of PDK3 in the regulation of aerobic glucose metabolism by CARM1 was detected by OCR assay and JC-10 staining.(2)The effects of PDK3 on the reactive oxygen levels in CARM1 overexpressing cells were detected by ROS level assay,NADP+/NADPH ratio and GSH concentration.The effect of PDK3 on the phosphorylation level of PDH in Carml overexpression cells was detected by Western blot assay.(3)The effect of PDK3 on osteolytic differentiation of Carml overexpressing cells was examined by TRAP staining.(4)Application of AMPK activator AICAR and transfection of siPdk3 on this basis,and detection of osteogenic differentiation of Carm1 knockdown cells by TRAP staining.Detection of osteoblast-related gene expression in Carm1 knockout cells by Western blot assayResultsPart Ⅰ:Study on the effect of CARM1 on osteogenic differentiation and osteoclastic differentiation of cells and the effect of CARM1 on osteoporosis model mice1.1 Expression changes of CARM1 in osteoporosis(1)GSE156508 data suggest that CARM1 expression is down-regulated in primary osteoblasts from patients with osteoporotic fractures.(2)GSE176265 data suggest that Carm1 expression is down-regulated in bone marrow-derived macrophages during osteoclastic differentiation in mice.(3)The RT-qPCR results suggested that CARM1 expression was down-regulated in patients with osteoporosis compared to controls.1.2 CARM1 promotes osteogenic differentiation in MC3T3-E1 cell line(1)RT-qPCR results suggested that osteogenic induction in MC3TE-E1 cells in the CARM1-OE group upregulated the expression of osteogenic-related genes,including osteocalcin(Ocn),bone bridging protein(Spp1)and type Ⅰ collagen α1(Colla1),in a time-dependent manner.(2)ALP staining and ARS staining showed that ALP activity and extracellular matrix mineralization were significantly elevated in Carml overexpressing MC3TE-E1 cells,with the opposite trend in results from Carm1 knockout cells.(3)Western blot and cellular immunohistochemistry results showed that Carml overexpression significantly increased the expression of osteogenesis-related genes(OCN,SPP1,COL1A1,ALP).1.3 CARM1 inhibits osteoblastic differentiation in RAW264.7 cell line(1)RT-qPCR results suggested that osteoblast-related genes,including histone k(Ctsk),tumour necrosis factor family member 11(Rankl)and activated T cell cytoplasmic nuclear factor 1(Nfatcl),were significantly down-regulated in Carm1-OE group cells with increasing induction time.(2)The results of TRAP staining and F-actin ring formation assay suggested that Carm1 overexpression inhibited osteoblast differentiation,while Carm1 knockdown significantly increased the number and maturation of osteoblasts.(3)Western blot and cellular immunohistochemistry results showed that Carm1 overexpression downregulated the expression of osteoclast-related genes(CTSK,RANKL,NFATC1,C-FOS).1.4 CARM1 supplementation improved bone quality in mice with osteoporosis model(1)Bioluminescence results showed that mice in the osteoporosis group and mice in the treatment group were infected with Carm1-NC and Carm1-OE lentivirus respectively after intra-femoral marrow injection.RT-qPCR results from femurs taken after execution of mice suggested that Carm1 expression was elevated in the femoral tissues of mice in the treatment group.(2)The results of micro-CT analysis indicated that the bone mineral density(BMD),cancellous bone volume/tissue volume(BV/TV),trabecular thickness(Tb.Th)and trabecular number(Tb.N)were higher in the treated mice than in the osteoporotic group;the trabecular separation(Tb.Sp)and cortical porosity(Ct.Po)were significantly lower than in the osteoporotic group.(3)Hematoxylin-eosin(H&E)staining and Masson staining showed that the bone trabeculae and collagen in the osteoporotic group were significantly less than those in the treated and sham-operated groups,and TRAP staining showed that the number of osteoclasts formed in the osteoporotic group was significantly more than those in the other two groups.(4)Immunohistochemical staining suggested that the expression of OCN and SPP1 in bone sections of mice in the treated group was elevated compared to that in osteoporosis.In contrast,the expression of osteoclast-associated proteins CTSK,RANKL and NFATC1 were significantly down-regulated.Part Ⅱ:CARM1 regulation of glucose metabolism in osteoblasts and osteoclasts2.1 CARM1 mediates reprogramming of glucose metabolism in osteoblasts and osteoclasts(1)CARM1 overexpression significantly increased the extracellular acidification rate(ECAR)in MC3T3-E1 and RAW264.7 cells.(2)In MC3T3-E1 and RAW264.7 cells,Carm1 knockdown significantly increased the cellular oxygen consumption rate(OCR)and decreased lactate production.(3)Measurements of mitochondrial membrane potential(ΔΨm)also showed that ΔΨm was elevated in the CARM1 knockdown group compared to the control group.2.2 CARM1 regulates glycolytic flux through upregulation of phosphofructokinase 1 activity(1)Metabolomic analysis suggested that fructose 1,6-bisphosphate was a significantly up-regulated metabolite in the MC3T3-E1 and RAW264.7 cell Carml overexpression groups.(2)The results of PFK1 activity assay showed that the cells in the Carml overexpression group had significantly higher PFK1 activity than the control group.(3)The osteogenic differentiation of cells in the MC3T3-E1 Carm1 overexpression group was attenuated after inhibition of glycolysis using DCA.Meanwhile,after application of PFK1 inhibitor PFK15,the ALP and ARS results suggested that the role of CARM1 in promoting osteogenic differentiation could be reversed by PFK15.Part Ⅲ,Mechanistic studies on the regulation of PFK-1 activity by CARM13.1 Overexpression of CARM1 upregulates the phosphorylation level of AKT/AMPK(1)Transcriptomic assays and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis revealed that the PI3K-AKT signalling pathway is one of the major enrichment pathways with differentially expressed genes(DEGs).(2)Western blot results showed that overexpression of Carm1 in MC3T3-E1 cells upregulated the phosphorylation levels of AKT,including Thr450,Thr308 and Ser473.(3)Western blot results showed that overexpression of Carm1 in RAW264.7 cells did not cause changes in AKT phosphorylation levels,but AMPK-Thr172 phosphorylation levels were elevated.3.2 Phosphorylation of AKT/AMPK mediates increased PFK-1 and PFKFB3 activity in Carml overexpression group cells(1)PFK activity assays showed that the regulation of PFK activity by CARM1 could be reversed by MK2206(AKT inhibitor)and Compound C(AMPK inhibitor)in MC3T3-E1 and RAW264.7 cells,respectively.(2)ALP staining and ARS staining showed that MK2206 prevented CARM1 from promoting osteoblast differentiation.SC79(AKT activator)rescued the inhibition of osteoblast differentiation caused by CARM1 knockdown,and PFK15 reversed this regulation again.western blot results showed that osteoblast-related genes regulated by CARM1(Ocn,Spp1,Colla1)expression was also affected by AKT inhibitors and activators.(3)Inhibition of osteoclast differentiation by CARM1 overexpression was rescued by Compound C during induction of osteoclast differentiation in RAW264.7 cells.Part Ⅳ,Mechanistic studies of CARM1 regulation of AKT/AMPK phosphorylation levels4.1 CARM1 interacts with PPP1CA(1)The immunoprecipitation results showed that CARM1 interacts with PPP1CA.(2)Biacore assay results showed that the binding strength of CARM1 and PPP1CA increased with the increase of protein concentration.(3)Confocal microscopy images show the distribution of CARM1 and PPP1CA in the cytoplasm and nucleus.4.2 CARM1 methylates PPP1CA at R23,R142 and R317(1)PPP1CA is highly conserved during evolution and the amino acid sequences of PPP1CA in mouse,rat and human are consistent.(2)Three arginine methylation sites of PPP1CA,R23,R142 and R317,were identified using in vitro methylation assays and liquid chromatography with tandem mass spectrometry(LC-MS/MS).4.3 R23 is the major methylation modification site of PPP1CA(1)The radioautography results suggest that mutation of the PPP1CA-R23 site results in significantly reduced methylation of 3H-SAM as a methyl donor in PPP1CA.(2)Liquid scintillation counting results showed that mutating all three sites(R23,R142 and R317)or mutating the R23 site resulted in a significant decrease in PPP1CA 3H count rate.the R23 mutation prevented CARM1-mediated PPP1CA methylation.4.4 PPP1CA-R23 methylation plays an important role in CARM1 regulation of grape metabolism,regulation of osteogenesis and osteoblastic differentiation(1)PFK activity assays showed that mutating all three sites(R23,R142 and R317)or mutating the R23 site significantly reduced PFK1 activity in peri-epithelial cells of Carm1 and decreased the rate of glycolysis in MC3T3-E1 and RAW264.7 cells.(2)Western blot results showed that full mutation(R23,R142 and R317)or mutation of the three PPP1CA loci in over-expressed cells of Carm1 down-regulated the phosphorylation levels of AKT and AMPK and caused down-regulation of osteogenesis-related gene(Ocn,Spp1,Colla1)expression in MC3T3-E1 cells and upregulation of osteoblast-related genes(Ctsk,Nfatc1,C-fos)in RAW264.7 cells.(3)ARS and ALP results showed that full mutation(R23,R142 and R317)or mutation of R23 in three loci of PPP1CA in peri-epitope cells of CARM 1 inhibited osteogenic differentiation of MC3T3-E1 cells and elevated osteolytic differentiation of RAW264.7 cells.Part Ⅴ.Mechanistic studies on the regulation of aerobic cellular glucose metabolism by CARM15.1 PDK3 is involved in CARM1-mediated reprogramming of glucose metabolism(1)Analysis of transcriptome data from MC3T3-E1 cells revealed that the expression levels of PDK family members were regulated by CARM1,with the expression of Pdk3 and Pdk4 being upregulated.(2)RT-qPCR examined the expression of PDK3 and PDK4 in osteoporosis patients and control patients.The expression of PDK3 and CARM1 maintained a consistent trend in the pathogenesis of osteoporosis.(3)Western blot detection of PDK3 expression in MC3T3-E1 and RAW264.7 cells showed increased expression of PDK3 in the CARM1-OE group.5.2 CARM1 as a transcriptional co-activator of EP300 regulates PDK3 expression(1)The results of chromatin immunoprecipitation(CHIP)assay showed that CARM1 is not a transcription factor of PDK3.(2)Cistrome Data Browser search results intersected with CARM1 mass spectrometry results and revealed that EP300 is a transcription factor for PDK3 and is also able to bind to CARM1.(3)Immunoprecipitation experiments and cellular immunofluorescence staining results indicated that EP300 interacts with CARM1.5.2 CARM1 regulates cellular oxidative phosphorylation flux through upregulation of PDK3 expression(1)OCR experiments showed that knockdown of Pdk3 in cells caused an increase in cellular OCR.By knocking down Pdk3,cellular ROS levels increased and the increase in ROS,NADP+/NADPH ratio was accompanied by a decrease in GSH concentration.(2)Knockdown of Pdk3 resulted in reduced PDH phosphorylation,increased PDH activity and partially reversed CARM1-induced metabolic changes,increased oxidative phosphorylation flux and increased cellular ΔΨm(3)Knockdown of Pdk3 increased osteolytic differentiation in RAW264.7 Carm1-OE group cells.Knockdown of Pdk3 reversed the inhibition of osteoclast differentiation induced by the AMPK agonist AICAR and upregulated the expression of osteoclast-related genes(Ctsk,Nfatc1,C-fos).Conclusions(1)CARM1 reprogrammed glucose metabolism in osteoblasts and osteoclasts from oxidative phosphorylation to aerobic glycolysis,thereby promoting osteogenic differentiation and inhibiting osteoclastic differentiation.CARM1 supplementation alleviated bone loss due to ovariectomy in an osteoporosis model mouse.(2)CARM1 methylates PPP1CA-R23,affects dephosphorylation of AKT-T450 and AMPK-T172,and increases phosphofructokinase-1(PFK1)and fructose-2,6-bisphosphatase 3(PFKFB3)activities,causing upregulation of glycolytic flux.(3)As a transcriptional cofactor,CARM1 upregulates the expression of pyruvate dehydrogenase kinase 3(PDK3),which inhibits pyruvate dehydrogenase(PDH)activity,leading to a downregulation of oxidative phosphorylation fluxes. |
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