Studies On The Molecular Mechanism Of ATP6V1H In Bone Formation

Vacuolar type H+ ATPase?V-ATPase?is an important enzyme in organisms,which regulates pH to maintain acid-base balance as a proton pump.Because of its special biological functions,V-ATPase plays significant roles in multiple tissues and organs,especially in bone.V-ATPase consists of multiple subunits,some of which are responsible for bone homostasis,such as B2,C1,a3,and d2.Subunit H is a recently found small subunit that takes part in the regulations of V-ATPase.However,researches on subunit H are relatively less,so it is unclear whether H subunit plays the key role in bone development.It is known that bone homostasis is maintained by cooperation of bone resorption mediated by osteoclasts and bone formation mediated by osteoblasts.As a result,the imbalance between bone resorption and bone formation will lead to bone related diseases,such as osteoporosis.Therefore,this research aims at exploring the impacts of Atp6v1 h deficiency on bone formation process and its molecular mechanism,which will help us further understand the function of H subunit in bone homostasis and related bone diseases,as well as find new therapeutic strategies.The studies content as follows:I.Generation,identification and raising of Atp6v1 h knockout mice1.Cyagen Transgenic Animal Center generated the Atp6v1 h knockout mice for us using CRISPR/Cas9 technique.2.The 2-3mm tails of 14-days-old mice were treated with the lysate,and then the DNA was used to conduct the PCR.Finally,the PCR products were sequenced and the gene type was determined according to sequencing results.3.All the mice were raised in the SPF rooms of our school's Lab Animal Centre.II.The in vivo studies on the bone formation characteristics of Atp6v1 h deficiency mice1.Femur samples from 3-months-old littlemate male wild-type and Atp6v1h^+/-mice were collected and performed paraffin sections,and then went through with Hematoxylin & Eosin staining,modified Ponceau red staining and ALP staining,to compare the cartilage formation,the bone volume,the trabecular thickness and ALP+ osteoblasts in bone surface.2.The 3-weeks-old littlemate male wild-type and Atp6v1h^+/-mice were injected calcein on day 1 and xylenol orange on day 8 respectively,and were killed on day 10.Then femurs were collected and made into plastic sections.Images were captured by confocal microscope,and the mineral apposition rate,the bone formation rate and the mineral surface were measured.3.Serum were collected from 3-weeks-old or 4-weeks-old littlemate male wild-type and Atp6v1h^+/-mice after overnight fasting.Then several items were measured including the concentrations of calcium?Ca2+?,phosphate ions?Pi?,alkaline phosphatase and osteocalcin in serum.4.The mRNA of calvarial bone tissues were collected.After RT-PCR,Q-PCR was performed to measure the osteogenic genes Col1a1,Alp,Bsp,Ocn,and Opg.III.The in vitro studies on ATP6V1 H involving in osteoblast biological properties1.Calvarial osteoblasts from newborn mice were cultured,and the cells of the third passage were grown in osteogenic induction medium.Then the osteogenic activity and calcification nodule were assessed by ALP staining and alizarin red staining respectively.2.The impacts of ATP6V1 H on the expression of osteogenic genes.?1?The mRNA of the primary osteoblasts were collected and performed RT-PCR.Then Q-PCR was performed to measure the osteogenic markers Col1a1,Alp,Bsp,Ocn,and Opg.?2?The wild-type osteoblasts of the third passage were treated with Atp6v1 h siRNA,and 72 hours later,the mRNA was collected.Then Q-PCR was performed to measure the osteogenic genes Col1a1,Alp,Bsp,Ocn,and Opg.?3?MC3T3-E1 cells were treated with Atp6v1 h siRNA,and 72 hours later,the mRNA was collected.Then Q-PCR was performed to measure the osteogenic genes Col1a1,Alp,Bsp,Ocn,and Opg.IV.The studies on the mechanism of ATP6V1 H involving in the interaction between osteoblasts and osteoclasts1.Calvarial osteoblasts from wild-type and Atp6v1h^+/-mice were cultured in high glucose DMEM medium in the presence of 10% FBS.The monocytes from wild-type and Atp6v1h^+/-mice were seeded on the osteoblasts with a ratio of 1:200 in the presence of 10-8 M 1?,25?OH?2D3 and 10-6 M PGE2.After 9 days,TRAP staining was performed and TRAP+ osteoclasts were calculated.2.Collect the protein of the primary osteoblasts,and then Western-blot was performed to detect the expression of RANKL and OPG.Collect the supernatant of primary osteoblasts and the serum of 3-months-old mice,respectively,and then ELISA was performed to detect the expression of RANKL and OPG.3.The protein of the primary osteoblasts was collected,and then Western-blot was performed to detect the expression of TGF-?1.The supernatant of primary osteoblasts and the serum of 3-months-old mice were collected,respectively,and then ELISA was performed to detect the expression of TGF-?1.The results as follows:I.Generation,identification and raising of Atp6v1 h knockout miceAll the homozygous Atp6v1h-/-mice embryos died before embryonic day 10,so all the results were from the comparation between wild-type and heterozygous mice.According to the sequencing results,there were a 5 bp?CGAGG?deletion and one base replacement?T>G?in Atp6v1 h gene of heterozygous mice.II.The in vivo studies on the bone formation characteristics of Atp6v1 h deficiency mice1.Hematoxylin & eosin staining and modified Ponceau red staining showed Atp6v1h^+/-mice exhibited less cartilage and bone formation,also the bone volume and trabecular thickness were decreased.In Atp6v1h^+/-mice,ALP stainning was weak and uneven,also the number of ALP+ osteoblasts were decreased.2.The decreased mineral apposition rate?MAR?and bone formation rate?BFR?were detected.3.In serum,the concentration of alkaline phosphatase and calcium?Ca2+?was increased,whereas the concentration of phosphate ions?Pi?was declined.4.No signifcant differences in osteogenic genes were found between the two types of calvarial bone tissues.III.The in vitro studies on ATP6V1 H involving in osteoblast biological properties1.ALP staining and alizarin red staining showed no significant difference between heterozygous and wild-type mice and both of which had the normal ability of carcified node formation.2.The impacts of ATP6V1 H on the expression of osteogenic genes:?1?In Atp6v1h^+/-osteoblasts,only Col1a1,Alp,Bsp were decreased,but others did not change.?2?After treated by Atp6v1 h siRNA,no signifcant changes of osteogenic genes were found in osteoblasts.?3?After treated by Atp6v1 h siRNA,only Ocn and Opg in MC3T3-E1 cells were changed,but others did not change.IV.The studies on the mechanism of ATP6V1 H involving in the interaction between osteoblasts and osteoclasts1.Different combinations of monocytes and osteoblasts from wild-type and heterozygous mice were used to establish the co-culture system.The co-culture of wild-type osteoblasts with wild-type and Atp6v1h^+/-monocytes induced the same amount of TRAP-positive osteoclasts,whereas Atp6v1h^+/-osteoblasts induced fewer osteoclasts from Atp6v1h^+/-monocytes.2.The Western-blot showed that in Atp6v1h^+/-osteoblasts,the expression of RANKL was decreased,but the OPG did not change.The ELISA showed that both in the Atp6v1h^+/-serum and induced supernatant of primary osteoblasts,the RANKL was decreased,but the OPG was increased.3.The Western-blot showed that in Atp6v1h^+/-osteoblasts,the expression of TGF-?1 did not change.The ELISA showed that in Atp6v1h^+/-serum,the expression of TGF-?1 was decreased,but in induced supernatant of primary Atp6v1h^+/-osteoblasts,it was increased.We found that deficiency of Atp6v1 h inhibits in vivo bone formation in mice,which could only cause several changes in osteogenic genes,but has no effect on bone mineralization.Thus the inhibition could impact the interaction between osteoblasts and osteoclasts,and probably through the TGF-?1 pathway.