The Role Of TET3-5hmC And Akt Signaling In Steroid-associated Osteonecrosis

[Objective]To investigate the relationship between TET protein-mediated abnormal epigenetic hydroxymethylation modification and steroid-associated osteonecrosis(SAON);To clarify the role of TET1,TET2 and TET3 in the pathogenesis of SAON;To explore the changes of TET-5hmC downstream signaling pathway induced by glucocorticoid;To verify the effect of TET3 on the apoptosis of osteocytes and Akt pathway changes induced by glucocorticoid;To observe the effect of knockdown TET3 on the development of SAON in vivo.[Method]Part I: 6 cases of SAON and 6 cases of femoral neck fractures samples were obtained with basic information and imaging data.The mRNA level of TET1,TET2 and TET3 was detected by real-time quantitative PCR.The expression of TET3 protein was detected by Western Blotting.Dot Blotting was used to detect the changes of 5hmC enrichment.Part II: Different doses or time of dexamethasone was adminstrated in MLO-Y4 cells.TUNEL staining was used to detect of the cell apoptosis;CCK8 was used to detect of the number of viable cells;trypan blue staining was used to evaluate cell death;Edu staining was used to observe the cell proliferation.The expressions of Bax,Bcl-2,Cleaved Caspase-3 and TET1,TET2 and TET3 were detected by Western Blotting.The changes of TET3 in the nuclear protein were detected by subcellular isolation.Immunofluorescence staining and Dot Blotting were usded to evaluate the 5hmC enrichment level.The hMeDIP-chip analysis was performed and the Akt signaling pathway was found to be closely associated with the epigenetic regulation induced by dexamethasone.MLO-Y4 cells were pretreated with Akt signaling activator bpV(phen).Then the cell apoptosis and the expression of Bcl-2,Bax and Cleaved Caspase-3 were evaluated.The expression of Akt signaling and apoptosis-associated proteins were further verified in clinical specimens of SAON.Then,the expression of TET1,TET2 and TET3 were knocked down by siRNA in vitro.The effect of TET3 on the apoptosis of osteocytes induced by dexamethasone was analyzed.The changes of TET3 in total protein and nucleoprotein,the 5hmC content and Akt signaling were measured after knock down TET3.Part III: Methylprednisolone sodium succinate(21mg/kg·d)was used to establish SAON in SD rats.TET3 siRNA transfection complex was injected into the medullary cavity of distal femur on day 1 and day 15.After establishment of the model,the magnetic resonance imaging was performed and T1 and T2-weighted image signal changes within femoral head were observed(n=6).Anesthetized rats were sacrificed to remove bilateral femoral head.Femoral head trabecular micstructure was analyzed by Micro-CT scan of the left femoral head(n=6).TUNEL apoptosis was then used to evaluate the apoptosis of osteocytes.The bone histomorphological changes were observed by HE staining(n=6).The right femoral head was used to detected the change of 5hmC level(n=4)and expression of TET1,TET2 and TET3(n=4).[Results]Part I: 6 patients with SAON were aged 66.2 ± 6.6 years(59-77 years),4 males and 2 females;6 patients with femoral neck fractures were 76.2 ± 5.5 years(68-85 years),3 males and 3 females.The results of TUNEL staining showed that the osteocytes apoptosis rate of SAON group was 30.14 ± 3.73%(20-46.67%),while the apoptosis rate of femoral neck fracture group was 9.47 ± 1.21%(5.26-11.76%).Realtime quantitative PCR showed that the levels of TET1 and TET2 were unchanged,whereas the expression of TET3 was increased in SAON tissues,which was confirmed by Western blotting.Dot blotting indicated strong enrichment of 5hmC in SAON gorup.Part II: Dexamethasone induced MLO-Y4 cells apoptosis in a time-dependent and dose-dependent manner.10-6M dexamethasone significantly promoted MLO-Y4 cells apoptosis at 8h and 12 h,while decreased viable cells,increased dead cells and reduced cell proliferation and nucleic acid replication.But the effect of 10-6M dexamethasone on MLO-Y4 cells was not significant at 2h and 4h,expect cell proliferation was significantly inhibited at 4h.10-6M and 10-5M dexamethasone increased apoptotic cells,reduced viable cells,increased dead cells and inhibited cell proliferation significantly,but 10-8M and 10-7M dexamethasone had no significant effect.Dexamethasone promoted the expression of Cleaved Caspase-3 by up-regulating the ratio of Bax/Bcl-2 protein,thus induced the apoptosis of MLO-Y4 cells.To further explore the epigenetic modification effect of dexamethasone on MLO-Y4 cells,5hmC was found to be increased by dexamethasone.The expression of TET1 and TET3,especially TET3,was up-regulated.Subcellular isolation western blotting indicated that dexamethasone can induce TET3 into the nucleus,suggesting that TET3 may be involved in the regulation of dexamethasone-induced 5hmC modification and subsequent osteocyte apoptosis.The hMeDIP-chip results indicated 5hmC dynamic change with dexamethasone treatment.GO analysis revealed significant accumulation of different 5hmC-enriched genes(DEG)involved in several important cellular processes,such as development,localization,metabolism,and differentiation.KEGG signal pathway analysis showed that signaling pathways,including the PI3K-Akt pathway,Notch pathway,apoptosis pathway,and Wnt pathway,exhibited dynamic changes in 5hmC levels.The PI3K-Akt was the signaling pathway showed the most significant 5hmC upregulation.Eleven genes associated with the PI3K-Akt signaling pathway was quantified.The mRNA level of Ppp2r5 d,PTEN and JAK3 were increased,while PIK3r5,PIK3 cd,TCL1 and PDK2 were decreased.Meanwhile,the protein level of PTEN was significantly increased after dexamethasone intervention,but the level of p-Akt was decreased.It was significant that Akt signaling activator bpV(phen)significantly inhibited dexamethasone-induced MLO-Y4 cells apopotosis.In addition,the expression of Bcl-2,Bax and Cleaved Capase-3 were reversed.Significantly,the expression of Akt signaling-related proteins were changed in SAON tissues.PTEN was up-regulated and p-PI3 K and p-Akt were down-regualted.The ratio of Bax/Bcl-2 and the level of Cleaved Caspase-3 were significantly increased.Knock down expression of TET3 significantly inhibited dexamethasone-induced MLO-Y4 cells apoptosis,but decreased TET1 and TET2 had no obvious effect.Meanwhile,the 5hmC level induced by dexamethasone was significantly inhibited after knockdown of TET3,and the expression of PTEN in Akt signal pathway was significantly decreased,while the level of p-Akt was significantly increased.Part III: Intrmedullary injection of TET3 siRNA transfection complex significantly inhibited TET3 expression in vivo,but the expression of TET1 and TET2 was not affected.At the same time,the 5hmC level in femoral head was also significantly decreased,which indicated that TET3 siRNA could significantly inhibit glucocorticoid-induced abnormal 5hmC modification in vivo.The HE staining showed obvious empty bone lacunae and adipocytes in the bone marrow of the SAON group.These effects were rescued by siTET3 treatment.TUNEL analysis showed that the osteocytes embedded in the trabeculae at the proximal epiphysis of the femoral head displayed intense staining in the SAON group,whereas few osteocytes displayed positive staining with siTET3 treatment.Micro-CT results showed that si TET3 treatment inhibited bone trabecular decline.Moreover,MRI scan indicated that the abnormal signal of femoral head significantly decreased in siTET3 group.[Conclusions]1.TET1,TET2 and TET3 were all expressed in bone tissue.TET3 was significantly increased in SAON tissues,accompanied by upregulation of epigenetic marker 5hmC.2.TET3-5hmC signaling plays an important role in dexamethasone-induced MLOY4 cells apoptosis.3.The hMeDIP-chip analysis showed that dexamethasone induced 5hmC dynamic change,which was closely associated with PI3K-Akt signaling.Knock down expression of TET3 significantly rescued Akt signaling and the apoptosis of MLO-Y4 cells.These indicated that TET3-mediated 5hmC modification can regulate osteocytes apoptosis through modulating Akt signaling activity.This signaling cascade was critical for SAON pathogenesis.4.Knock down expression of TET3 in vivo could significantly inhibit osteocytes apoptosis,bone histological abnormal changes,trabecular microstructure decline and aberrant signals in MRI scan.Thses indicated that TET3 might be a new target for SAON treatment.