摘要
目的 本研究旨在鉴定与骨形态发生蛋白9(BMP9)有协同促成骨作用的新因子,以期为骨质疏松症的治疗提供新的分子靶点和实验依据。方法 为系统性评价钙释放激活钙调节蛋白2(ORAI2)在成骨分化中的功能,本研究构建了ORAI2过表达及沉默的细胞模型,在机制研究中,我们通过RNA测序(RNA-seq),利用实时定量PCR(RT-qPCR)和蛋白质印迹(Western blotting)验证关键分子的表达变化;借助碱性磷酸酶(ALP)染色及活性分析评估细胞成骨状态。结果 研究发现,ORAI2在BMP9诱导的鼠胚胎成纤维细胞(MEFs)成骨分化过程中的表达显著上调,而在骨质疏松(卵巢切除)大鼠模型中的表达下调。体外过表达ORAI2能有效促进成骨分化,相反,沉默ORAI2则显著抑制成骨分化。RNAseq及京都基因与基因组百科全书(KEGG)富集分析显示,PI3K/Akt是介导ORAI2功能的关键信号通路。此外,通过蛋白质谱分析鉴定出含IQ基序GTP酶激活蛋白1(IQGAP1)是ORAI2的结合蛋白,ORAI2通过与IQGAP1结合特异性地激活了MAPK/ERK1/2信号通路。结论 ORAI2通过与BMP9协同,经PI3K/Akt和MAPK/ERK1/2通路促进成骨分化,是骨质疏松症的潜在治疗靶点。
Objective To identify novel factors that act synergistically with bone morphogenetic protein 9(BMP9)to promote osteogenesis,with the goal of providing new molecular targets and experimental evidence for the treatment of osteoporosis.Methods We determined the effect of calcium release-activated calcium modulator 2(ORAI2)on osteogenic differentiation by using overexpressing or knocking-down ORAI2.In the mechanism study,we validated the expression changes of key molecules through RNA-seq,RT-qPCR,and Western blotting,and to evaluate the osteogenic status of cells using alkaline phosphatase(ALP)staining and activity analysis.Results ORAI2 was upregulated in BMP9-induced osteogenic differentiation in mouse embryomic fibroblasts(MEFs)and downregulated in the bone tissue of ovariectomy rats.Exogenous ORAI2 expression promoted osteogenic differentiation in vitro whereas silencing ORAI2 inhibited them.Mechanistically,we conducted an RNA-seq,and KEGG analysis revealed that the effects of ORAI2 may involve the PI3K/Akt signaling pathway.Furthermore,mass spectrometry results suggested that ORAI2 may bind to IQ motif-containing GTPase-activating protein 1(IQGAP1),notably,ORAI2 could interact with IQGAP1 and then facilitate mitogen-activated protein kinase/ERK1/2 signaling.Conclusion ORAI2,in synergy with BMP9,dually promotes osteogenic differentiation via the PI3K/Akt and the MAPK/ERK1/2 pathway,representing a potential therapeutic target for osteoporosis.
作者
任轶凡
兰钰
彭鑫亮
杨星宇
高翔
杜宇
Ren Yifan;Lan Yu;Peng Xinliang;Yang Xingyu;Gao Xiang;Du Yu(Department of Orthopedics,The Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China)
出处
《骨科》
2025年第4期289-297,共9页
ORTHOPAEDICS
基金
重庆市自然科学基金面上项目(CSTB2022NSCQ-MSX0066)
重庆市科卫联合医学科研项目(2025MSXM088)。
