TCIRG1-IP3R2-Calcium Oscillatio-NFATc1 Signaling Regulate Osteoclastogenesis

ObjectiveVacuolar proton pumping(ATPase,V-ATPase,Atp6)is a complex enzyme that is highly conserved and widely distributed in eukaryotic cells.V-ATPase is comprised of the proton translocation domain V0,which contains a,c,c',d and e subunits,as well as the ATP hydrolysis domain V1,which contains A-H subunits.The a subunit is the proton transport channel and has four isoforms:al-4.The a4 isoforms is specifically expressed in renal cells.The a3 isoform which encoded by Tcirgl(Atp6i)gene is expressed in osteoclasts 100 times more than other types of cells.More than 50%of human malignant infantile osteopetrosis is accounted for by Tcirgl mutations.Furthermore,Tcirgl-/-mice have small and dysfunctional osteoclasts.Any defects in Tcirgl gene lead to functional and genesis impairment of osteoclasts.functional defects of osteoclast are attributed to acidification dysfunction caused by v-ATpase damage.However,the molecular mechanisms of Tcirgl loss on osteoclast generation has not been widely discussed.The Nuclear factor of activated T-cells 1(NFATc1)is a key factor for osteoclastogenesis.Usually,NFATc1 located in the cytoplasm in a phosphorylated state.After treated by RANKL or TNF,through a series of signal cascades,inositol-1,4,5-trisphosphate receptor(IP3Rs)in the endoplasmic reticulum triggering Ca2+release.Subsequently,calcineurin causes the dephosphorylation of the serine residues in NFATcl,which then translocates into the nucleus to initiate osteoclastogenesis.Therefore,we hypothesized whether knockout of Tcirg1 gene could directly affect the expression of NFATc1 or inhibit the activation of NFATc1 by affecting calcium signaling.In the current study,we transfected mouse bone marrow-derived monocytes with control or Tcirgl-knockdown lentiviruses to further investigate the mechanisms of Tcirgl.Methods1.Knockdown of Tcirgl limits large-osteoclast genesis in mouse bone marrow monocytes.(1)BMMs were transduced with the Tcirgl-RNAi lentivirus,Empty lentivirus and untreated cells were used as control.(2)M-CSF and RANKL were used to induce the three groups of BMMs,and TRAP staining was used to observe osteoclastogeneration.(3)RT-PCR was used to detect the expression of osteoclast related genes.(4)Immunofluorescence was used to detect the location relationship of V-ATPase and lysosomes.2.Knockdown of Tcirg1 inhibits NFATcl nuclear translation by down-regulating IP3R2 expression.(1)Western blot was used to detect the expression of NFATcl and IP3R2 in total protein.(2)Cytoplasmic protein and nuclear protein were harvested respectively,and the expression of NFATcl in both were detected by western blot.(3)Immunohistochemistry was used to detect the distribution of NFATcl in osteoclasts.(4)The intracellular calcium oscillation was detected using a confocal microscopy.3.Chloriquine inhibits the expression of Tcirgl gene in mouse bone marrow monocytes.(1)Different concentrations of chloroquine(0?M,5?M,10?M)were added to osteoclast induction medium to induce BMMs,and TRAP staining was used to detect osteoclastogenesis.(2)RT-PCR was used to detect the expression of osteoclast related genes and V-ATPase related genes.(3)Immunofluorescence was used to detect the location relationship of V-ATPase and lysosomes.(4)Western blot was used to detect the expression of Tcirgl and NFATc1.(5)Immunohistochemistry was used to detect the location of NFATc1 in cells.(6)RT-PCR and Western blot were used to detect the expression of IP3Rs.Result1.knockdown of Tcirgl inhibits large-osteoclast(>100 ?m)generation.The expression of NFATc1,Cathespin k,Mmp9,Dc-stamp were decreased.The position relationship between V-ATpase and lysosome have no signigicantly changes.2.The expression of IP3R2 but not IP3R1 and IP3R3 reduced in Tcirgl knockdown osteoclasts.IP3R2 mediates persistent Ca2+oscillation,the decrease of IP3R2 ultimately leading to the restriction of NFATc1 nuclear translocation.3.Chloroquine not only reduce the expression of osteoclastogenesis genes and V-ATPase genes,but also the IP3R2 expression and NFATc1 nuclear translation.Different from Tcirgl knockdown cells,chloroquine also inhibited IP3R1 and IP3R3 expression.ConclusionThe current study shows that knock-down of Tcirgl gene not only reduce the expression of NFATc1,but also inhibit calcium oscillation by reducing the expression of IP3R2,thus limited the nuclear translation of NFATcl and leading to the expression inhibition of osteoclast related genes.These findings provide a mechanism to explain the effects of Tcirgl impairment,with potential implications for the development of therapies for osteopetrosis.