Study On Regulation Of Osteoclast Function By ATP6V1H

Vacuolar ATPase(V-ATPase)is an important molecule of osteoclasts which can provide an acidic environment for bone resorption.ATP6V1 H gene is one of the essential protein subunits of the multivariate protein complex V-ATPase.We found two missense mutations in ATP6V1 H in a pedigree with skeletal phenotypes.Therefore we speculate that ATP6V1 H is involved in the regulation of osteoclast function and affects the balance of bone metabolism.However,there is no report about the relationship between ATP6V1 H and osteoclast function.To clarify the above question,we observed the formation of osteoclast in Atp6v1 h knockout mice generated by the CRISPR/Cas9 technique,and explored the mechanism of ATP6V1 H regulating osteoclast function.Methods:1.The femurs of three-month-old male mice were taken and fixed in 4% paraffin.The adjacent paraffin sections were used for ATP6V1 H immunohistochemistry and osteoclast-specific TRAP staining to observe the localization of ATP6V1 H in osteoclasts.2.The femoral were scanned with Micro-CT and 3D image were reconstructed.Differences of femoral density between the two genotype mice were analyzed.3.RANKL with different concentrations were used to observe the dose-dependent effect of RANKL on the induction of osteoclasts.Both OC of the two genotype mice were able to respond to RANKL induction and had a dose effect,but Atp6v1h+/-mice responded relatively weakly to RANKL.M-CSF and RANKL were used to induce osteoclasts formation of Atp6v1h+/+ and Atp6v1h+/-mice.TRAP staining was used to observe the cellular morphology and other general biological characteristics of the osteoclasts.Bone slice experiments were executed to verify the differences of bone resorption capacity between the two genotype mice.4.Immunohistochemical staining was used to determine the localization of ATP6V1 H in mouse pancreas.The mice were fed with high fat diet in order to observe the difference of blood glucose and serum insulin concentrations in Atp6v1h+/+ and Atp6v1h+/-mice.The effects of glucose and insulin concentration on the number of osteoclasts produced by Atp6v1h+/-mice were observed by in vitro cell experiments.Results:1.Immunohistochemistry showed that ATP6V1 H location was consistent with TRAP-stained osteoclasts,indicating that ATP6V1 H was specifically expressed in osteoclasts.2.The bone density of the femur in mice was significantly decreased when Atp6v1 h is insufficient.BV/TV,Tb.Th,and Ct.Th were respectively decreased and BS/TV was increased in Atp6v1h+/-mice compared with wild type mice(n=8)(P <0.01).3.Experiment results shows the optimum concentration of RANKL to introduce osteoclast formation is 100 ng/ml.And the number of induced osteoclasts in Atp6v1h+/-mice were notably reduced by 51.8%(P<0.01).Bone resorption experiments showed that the bone resorption ability of Atp6v1h+/-mice was significantly decreased,and the area of bone resorption lacunae of Atp6v1h+/-mice was reduced by 24.7%(P <0.01).4.ATP6V1 H is specifically expressed in pancreatic islets in mice,while the expression level of ATP6V1 H in islets of Atp6v1h+/-mice was decreased.There was no significant difference in fasting blood glucose levels between Atp6v1h+/+ and Atp6v1h+/-mice.However,the serum insulin concentration in Atp6v1h+/-mice was 10.2% lower than that in wild type and the glucose tolerance was significantly decreased.After high fat diet feeding,fasting blood glucose in Atp6v1h+/-mice was 21.5% higher than wild type mice,serum insulin concentration was decreased 30.5% compared with wild type(P<0.01),and glucose tolerance was also decreased.5.In the low concentration of insulin(0 nmol/L,1 nmo/L,2 nmol/L,5 nmol/L,10 nmol/L),the number of osteoclasts in wild type mice increased significantly with the increase of insulin concentration while the number of osteoclasts in heterozygous mice did not changed with the different insulin concentration when cultured with hige glucose medium.In the low glucose medium,the osteoclasts of this two genotype mice were not affected by insulin;Either in low-sugar or high-sugar culture medium,high concentrations of insulin(5 nmol/L,10 nmol/L)have a significant inhibitory effect on the production of osteoclasts.Conclusions:1.ATP6V1 H was expressed in osteoclasts of mice,and bone density was reduced in Atp6v1 h heterozygous knockout mice.2.Atp6v1 h can affect the formation and bone resorptive ability of osteoclasts in vitro.3.ATP6V1 H located in the islets and might be involved in the secretion of insulin.4.Atp6v1 h deficiency could impair the insulin sensitivity of osteoclasts.Our studies demonstrated the localization of ATP6V1 H in osteoclast,confirming that ATP6V1 H participates in the regulation of osteoclastic bone resorption function and found that ATP6V1 H was specifically expressed in islet tissue and might be involved in the secretion of insulin in mice.We provide an experimental basis for the mechanism of ATP6V1 H involved in the regulation of osteoclast bone resorption function.Our studies also set a new ?target? for clinical treatment and drug design while the mechanism of bone diseases related to BMD changes is gradually visible.