| As a disease closely related to aging,osteoporosis and its fractures have become a major public health problem which seriously endangers the quality of life of the elderly.Developing interventions that prevent the development of osteoporosis and thereby reduce the incidence of fractures is important to promote healthy aging and reduce social burdens.Osteoporosis is closely associated with osteoblast-mediated decline in bone formation and osteoclast mediated hyperabsorption of bone.Due to the failure of anti-osteoporosis drugs to improve bone integrity and the serious negative effects of osteoblast differentiation regulation and development of bone-promoting interventions is the focus of current research in osteoporosis prevention and treatment.Histone modification is an important epigenetic control mechanism,and dynamic histone methylation and acetylation modification play a key role in many biological processes,including chromosome assembly,gene transcription,and DNA repair.H3K9 and H3K27 trimethylation modifications mediated by histone methyltransferases Setdb1 and Ezh2,respectively,were associated with transcriptional silencing,while H3K4 trimethylation modifications mediated by COMPASS-like proteins were associated with transcriptional activation.A series of studies have shown that histone methylation modification influences osteoblast differentiation by modulating the expression of key transcription factors Runx2 and Sp7.As a regulator for H3K9 methylation,Setdb1 is implicated in osteoblast proliferation and differentiation.The activity and nucleus localization of Setdb1 are regulated by its binding partner Atf7 ip.However,it remains unclear whether Atf7 ip is involved in regulating osteoblast differentiation.Therefore,we investigated the role of Atf7 ip in osteoblasts differentiation in combination with overexpression,siRNA knockdown or conditional gene knockout models to explore potential regulatory mechanisms.Objective: To investigate the role of Atf7 ip in osteogenesis,and to further reveal its regulatory mechanisms,provide new evidence and clues to improve bone formation mechanisms.Methods: In vitro cell assays:(1)BMSC cells were treated with osteogenic differentiation media(50 mg/mL vitamin C and 10 mM β-phosphorylin)for 0,3,and 14 days,and Atf7 ip expression was observed by RT-RCR assays.C3H10t1/2 cells were treated with bone differentiation medium for 0,3,6,12,24,and 48 hours,and Atf7 ip expression was further observed by RT-PCR,Western blot,and so on.Expression of Atf7 ip was further validated by treatment of MC3T3-E1 cells with bone formation promoter 500 ng / mL PTH for 0,12,24,and 48 hours.(2)To construct the interference and overexpression model of Atf7 ip,the effect of interference with Atf7 ip on osteogenesis was observed by RT-PCR,Western blot,alkaline phosphatase staining and alizarin red staining.Animal experiments: Conditional knockout mouse models were constructed using the Osteocalcin-Cre tool to target the mature stage of osteogenesis and differentiation,and the effects of Atf7 ip on bone formation in vivo were investigated by micro-CT,Von Kossa staining,Calcein double labeling,and RNA sequencing.Mechanistic exploration: After Atf7 ip interference and overexpression,the effect of Atf7 ip on intracellular localization of Setdb1 was first detected by immunofluorescence.The expression of key osteogenic transcription factors Sp7 and Runx2 was further detected by RT-PCR and Western blot.After identifying the possible regulatory factors,the siRNA of Sp7 and Atf7 ip were co-treated with MC3T3-E1 cells,and the regulatory mechanism of Atf7 ip affecting osteogenic differentiation was explored by Western blot,alkaline phosphatase staining and alizarin red staining.Results: We found that Atf7 ip expression was upregulated in primary osteoblastic mesenchymal cells during osteogenic differentiation using osteoblast media,and mRNA was upregulated in osteoblast markers Alp,Runx2,Osteocalcin,and Col1a1,and further upregulated in C3H10t1/2 and MC3T3-E1 cells.On this basis,treatment of MC3T3-E1 cells with Atf7ip-siRNA revealed enhanced osteodifferentiation,increased mRNA expression of osteodifferentiation markers Alp,Osteocalcin and Col1a1,significantly increased alkaline phosphatase staining and cissin red staining positive cells,and increased ALP activity.In contrast,treatment of MC3T3-E1 cells with Atf7 ip overexpression vector revealed inhibition of osteogenesis,decreased mRNA expression of bone-differentiation markers Alp,Osteocalcin,and Col1a1,significant decrease in alkaline phosphatase staining and cissin red staining positive cells,and decreased alkaline phosphatase activity.Further,using a conditioned knockout model of osteoblast Atf7 ip mice,we found that mice knockout of osteoblast Atf7 ip showed a significant increase in bone formation and microstructure of the trabecular bone.Mechanistically,we first investigated the effect of Atf7 ip on intracellular localization of Setdb1 and found that osteoblast Atf7 ip influenced Setdb1 localization in the nucleus.We further observed the effect of Atf7 ip expression on the key bone-differentiation transcription factors Runx2 and Sp7,and found that Atf7ip-siRNA-treated cells showed elevated Sp7 expression and Atf7 ip overexpression inhibited Sp7 expression,but no significant change in mRNA and protein levels in Runx2.On this basis,treatment of Atf7ip-knockdown MC3T3-E1 cells with Sp7-siRNA revealed inhibition of Atf7ip-siRNA promoting bone differentiation.Alkaline phosphatase(ALP)staining and siderin red staining were significantly decreased and ALP activity was decreased.Conclusion:(1)Atf7ip expression was elevated in osteogenic differentiation of multiple myeloid cells;(2)Interference with Atf7 ip expression promoted MC3T3-E1 osteogenic differentiation;(3)Atf7ip overexpression inhibited MC3T3-E1 osteogenic differentiation;(4)conditionally deficient Atf7 ip mice showed higher bone formation;(5)inhibition of Sp7 expression attenuated the osteogenic differentiation-promoting effect of Atf7ip-siRNA. |
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