Regulation Of Bone Homeostasis By Notch Signaling In Osteocytes

ObjectiveIn this study,we used mouse Cre-LoxP technology to specifically activate Notch1-NICD,a key regulatory molecule of Notch signaling pathway,in osteocytes to explore the regulation of Notch signaling in osteocytes on bone homeostasis in mice and provide a theoretical basis for the development of osteocyte-targeted anti-osteoporosis drugs.Methods1.By crossing Notch1-NICD^flox/flox mice,where a lox P-flanked STOP cassette is placed between the Rosa26 promoter and Notch1 intracellular domain sequences,with DMP1-8kb-Cre mice which express Cre recombinase in osteocytes,mutant mice(named daNICD^Ot)and control littermates（ctrl）were identified by genotyping.2.Alizarin red staining was performed to analyze the skeletal development of embryonic mice.3.The growth and development of mice were continuously monitored to calculate the survival rate.4.The bone mineral density of one-month-old mice were detected using X-ray.5.HE staining and TRAP staining were performed on the tibias of mice,and static bone histomorphology was used to analyze the cancellous bone mass,osteoblast and osteoclast number of one-month-old mice.6.qPCR was used to detect Notch signaling target genes Hes1,Hes5,Hes7,Hey1,Hey2 and HeyL;osteoblast marker genes Alp,Runx2,Osx and Ocn;osteoclast marker genes Rank L/Opg,M-CSF,cathepsin K,calcitonin receptor and RANK;Wnt signaling target genes Bmp4,Smad6,Lef1,Axin2 and Cyclin D1.7.ELISA was used to detect the levels of serumal P1NP and CTX in one-month-old mice.Results1.DMP1-Cre^+/-;Notch1-NICD^fl/+mice(daNICD^Ot)were identified by genotyping as mutant mice with activated osteocyte Notch1-NICD,and DMP1-Cre^-/-;Notch1-NICD^fl/+mice（ctrl）were identified as control mice.2.daNICD^Ot mice displayed no gross skeletal abnormalities at birth.3.Mutant mice with activated osteocyte Notch1-NICD showed severe growth and development disorders,premature death of mice,and zero mouse survival rate at 35 days.4.X-Ray results showed that the bone mineral density of daNICD^Otmice was significantly reduced（P<0.05）.5.Static bone histomorphological analysis results showed that daNICD^Ot mice had decreased cancellous bone mass,decreased osteoblast number,increased osteoclast number,and increased cortical bone porosity（P<0.05）.6.Notch signaling target genes（Hes1,Hey1,HeyL）expression was increased in daNICD^Ot mice（P<0.05）.Osteoblast marker genes（Alp,Runx2,Osx,Ocn）expression was decreased（P<0.05）,and osteoclast marker genes（M-CSF,cathepsin K,RANK）expression was increased,and Rank L/Opg ratio was increased（P<0.05）.The expression of Wnt signaling target genes（Bmp4,Smad6,Lef1,Axin2）was decreased（P<0.05）.7.ELISA results showed that the serumal P1NP level was decreased and the CTX level was increased（P<0.05）.ConclusionActivating Notch signaling in osteocytes reduces osteoblast and decreases osteoblast differentiation;increases osteoclasts and enhances bone resorption.