The role of PKMYT1 in glucocorticoid-induced osteoblast apoptosis
10.3969/j.issn.1000-6710.2025.05.005
- VernacularTitle:PKMYT1在糖皮质激素诱导成骨细胞凋亡中的作用
- Author:
Chengyou YANG
1
;
Hong LUO
;
Tao WANG
;
Zhihong XIE
;
Liang LIANG
;
Fanchao LI
;
Jianhua WU
;
Fei ZHANG
;
Wuxun PENG
Author Information
1. 贵州医科大学临床医学院(贵阳 550004)
- Publication Type:Journal Article
- Keywords:
steroid-induced avascular necrosis of the femoral head;
PKMYT1;
osteoblasts;
apopto-sis;
proliferation
- From:
Chinese Journal of Sports Medicine
2025;44(5):381-393
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the role of membrane-associated tyrosine/threonine-protein ki-nase 1(PKMYT1)in glucocorticoid(GC)-induced osteoblast(OB)apoptosis,providing a theoretical basis and potential therapeutic targets for early-stage steroid-induced avascular necrosis of the femoral head(SANFH).Methods(1)Mouse calvarial osteoblastic cells(MC3T3-E1)were selected for the study.The control group was cultured in standard medium,while the experimental group was subject-ed to osteogenic induction culture,with osteogenic capacity verified by alkaline phosphatase(ALP)and Alizarin Red S(ARS)staining.Then,mouse osteoblasts(mOB)were treated with different con-centrations of GC.After that,apoptosis was detected by using Annexin V-FITC/PI double staining as-say,while cell proliferation was assessed by using Cell Counting Kit-8(CCK8).Moreover,the expres-sions of anti-apoptotic protein B-cell lymphoma/leukemia-2(BCL-2),pro-apoptotic proteins cleaved caspase-3andcleavedcaspase-9(cleavedcaspase 3/9)weredetectedbyusing Westernblotting(WB).Meanwhile,proteomic analysis was employed to identify molecules potentially regulating GC-in-duced apoptosis in mOBs.What's more,quantitative real-time PCR(qPCR)and WB were used to further analyze PKMYT1 expression.(2)mOBs were treated with PKMYT1 inhibitor GSK-1520489A of different concentrations to screen the optimal one,and all subjects were then further divided into a control,a GC,a GSK-1520489A,and a GC+GSK-1520489A group.Later,the expression of PK-MYT1 and apoptosis-related proteins BCL-2 and cleaved caspase 3/9 of all groups were detected us-ing WB,and cell viability and cytotoxicity were evaluated by CCK8 assay,with cell proliferation by using 5-ethynyl-2'-deoxyuridine(EDU)assay and apoptosis by cell live/dead staining and Annexin V-FITC/PI double staining.(3)mOBs were infected with PKMYT1 overexpression lentiviral vectors,and its efficiency was verified by using immunofluorescence,qPCR,and WB.After successful overexpres-sion of PKMYT1,all cells were divided into the control,GC,PKMYT1 overexpression(OE),and OE+GC groups,whose cell proliferation was detected by EDU assay,and apoptosis was assessed by Annexin V-FITC/PI double staining and cell live/dead staining.(4)To verify the changes in PKMYT1 expression in human osteoblasts(hOB),hOBs extracted from human femoral heads of healthy individu-als were chosen into the control group,while those from patients with hormone-induced avascular ne-crosis of the femoral head(hSANFH)were selected into the hSANFH group.Then,PKMYT1 expres-sion in both groups was detected by using qPCR and WB.Results(1)After inducing the differentia-tion of mouse calvarial osteoblastic cells(MC3T3-E1)into mature osteoblasts,under the action of GC,compared with the control group,with the increase of GC concentration,the experimental group showed increased mOB apoptosis(P<0.01)and expression of cleaved caspase 3/9(P<0.01),but de-creased cell viability(P<0.01)and expressionof apoptosis-relatedprotein BCL-2(P<0.01).More-over,according to the proteomic sequencing,significant decrease was observed in the PKMYT1 expres-sion in mature mOBs treated with GC.(2)As to treatment of mOBs with different concentrations of PKMYT1 inhibitor GSK-1520489A,with the increase of concentration,cell viability decreased and cy-totoxicity increased(P<0.001).Moreover,compared with the control group,mOBs proliferation de-creased(P<0.001)and apoptosis increased(P<0.001)in the GSK-1520489A group.Meanwhile,com-pared with the GC group,mOB proliferation decreased(P<0.05)and apoptosis increased significantly(P<0.01)in the GC+GSK-1520489A group.(3)After overexpression of PKMYT1,in comparison with the control group,mOB proliferation increased(P<0.001)but apoptosis did not increase significantly(P>0.05)in the OE group.Moreover,compared with the GC group,mOB proliferation increased(P<0.001)but apoptosis decreased(P<0.001)significantly in the OE+GC group.(4)In hOBs extracted from human femoral head tissues,qPCR and WB results showed that PKMYT1 expression of the hSANFH group was significantly lower than the control group(P<0.001).Conclusion Down regulation of PKMYT1 expression promotes GC-induced apoptosis of mOBs.Conversely,over expression of PK-MYT1 inhibits GC-induced apoptosis of mOBs.Therefore,PKMYT1 may serve as a potential target for the early treatment of SANFH.
