Research On A Potential Role Of TRPV6 As A Regulator In Murine Bone Metabolism And Osteoclast Formation

Background:Osteoporosis is a metabolic disease caused by the decrease of bone density and bone mass.Currently,there are about 200 million patients with osteoporosis in the world,and more than 90 million in China.Every year,there are about 8.9 million patients with fractures caused by osteoporosis,and the annual medical expense for osteoporosis treatment is about 25 billion yuan,which brings severe pain to patients and heavy burden to families and society.Therefore,the prevention and treatment of osteoporosis is an urgent medical problem.The disorder of bone metabolism and the abnormal function of osteoclast bone resorption are the main pathologic basis of osteoporosis,the study of related factors and signaling pathways regulating osteoclast bone resorption function is conducive to the in-depth understanding of the pathogenesis of osteoporosis,which has important scientific significance and medical value for the prevention and treatment of osteoporosis.Transient Receptor Potential Vanilloid（TRPV）receptors are one type of calcium ion transport proteins,there are six members in the family,including TRPV1,TRPV2,TRPV3,TRVP4,TRPV5 and TRPV6.TRPV5/TRPV6 is the only known two highly selective calcium ion channels in the TRPV subfamily.Recent studies have found that TRPV6 is an important calcium ion channel on osteoclasts,which may participate in the regulation of bone metabolism function by regulating intracellular calcium ion concentration.However,the regulation effects of TRPV6 on bone metabolism are still controversial and little is known about the involvement of TRPV6 in RANKL-induced osteoclastogenesis.Objective and Methods:In the early stage,our research group successfully established the animal model of TRPV6 gene knockout mice,which provided favorable conditions for further study of this project.In this study,micro-ct scanning and reconstruction,tetracycline double-label and TRAP staining,elisa and other methods were used in vivo experiments to clarify the effect of calcium channel protein TRPV6 on bone metabolism.Emphatically from the cellular and molecular levels,useing the TRAP staining,bone resorption lacunae experiment,cell immunofluorescence,slow virus transfection,western blot,real time quantitative PCR and relevant experimental methods,revealed the distribution and regulation fuction of TRPV6in osteoclast,explored the mechanism of bone resorption and related signaling pathway of TRPV6 in osteoclast formation.Results:In vivo study,we found that 3-month old TRPV6^-/-mice could occur osteoporosis,the bone density of 3-month old TRPV6^-/-mice was significantly lower than that of the control group（wild-type mice of the same age）,and the bone mass of TRPV6^-/-mice was significantly lower than that of the control group by Micro-CT scanning of the femur samples.There was no significant difference between the two groups in tetracycline double-standard staining in the femoral section of the mice,and Micro-CT scanning was performed to analyze the indicators of bone formation activity of the two groups,indicating that the osteogenic ability of TRPV6^-/-mice was not significantly different from that of the control group.TRPV6^-/-mice TRAP staining area was significantly higher than that of the control group,and ELISA detection showed that the serum c-terminal peptide of type I collagen cross-linking in TRPV6^-/-mice was significantly higher than that of the control group,suggesting that TRPV6 mainly affected bone metabolism by acting on bone resorption.Vitro studies shown that osteoclasts can express TRPV6 protein,the expression level of TRPV6 becomes lower and lower along with the process of differentiation and maturation.By immunocytochemical fluorescence method,it was found that TRPV6 was mainly distributed on the cell membrane of osteoclasts,with only a small distribution of cytoplasm and nucleus.Took TRPV6^-/-mice sources of osteoclasts and RNAi technology silence osteoclast TRPV6 genes in mice,by morphological observation,the TRAP staining and bone resorption pit experiments,QT-PCR detection methods,such as research on the osteoclast formation TRPV6,regulatory role of bone absorption,the results found TRPV6can negative regulation of osteoclast formation,inhibit bone absorption.In order to further clarify the mechanism of TRPV6 regulated osteoclast formation,we found silence TRPV6 expression does not affect osteoclast Ca²⁺oscillations,the result revealed that TRPV6 inhibiting osteoclast differentiation by non-Ca²⁺oscillation/CaN dependent pathways.As a non-Ca²⁺oscillatory signaling pathway,IGF signaling pathway plays an important role in osteoclast differentiation.Therefore,the research group speculated that IGF might mediated the regulation of TRPV6 on osteoclast formation and bone resorption.The results confirmed that the expression levels of osteoclasts IGF1R and IGFBP1 protein and mRNA were significantly increased after the expression of TRPV6was silenced.However,blocking the IGF signaling pathway can significantly inhibit the effect of TRPV6.PI3K-AKT pathway is an independent signal pathways downstream of IGF signal pathway.The results confirmed that TRPV6 could inhibit the phosphorylation levels of the PI3K-AKT pathway P85/p-P85,PDK1/p-PDK1 and AKT/p-AKT,respectively.These results suggest that IGF1R-PI3K-AKT pathways is involved in the negative regulation of TRPV6 on osteoclast formation.Conclusions:To sum up,We confirm that the main cause of osteoporosis in mice with TRPV6 gene knockout is abnormal bone resorption function,and TRPV6 is an important regulator of osteoclasts,which negatively regulates osteoclast formation and bone resorption.TRPV6inhibits osteoclast formation mainly through non-Ca²⁺oscillatory/caN pathway,and negatively regulates osteoclast formation and bone resorption by inhibiting the IGF1R-PI3K-AKT signaling pathways.