Effects And Underlying Mechanisms Of Human Fc?RIIa On Osteoclastogenesis In A Transgenic Mouse Model

Bone homeostasis depends on a dynamic balance of bone formation and resorption,which are mediated by osteoblasts and osteoclasts,respectively.Tipping the balance in favour of osteoclasts leads to diseases characterized by a low bone mass,including osteoporosis.Osteoclasts originate from bone marrow-derived monocyte/macrophage lineage cells(BMMs)and their differentiation is mediated by many of the same regulators utilized in the immune system.Receptor activator of NF-?B ligand(RANKL)and macrophage colony-stimulating factor(M-CSF)are key cytokines in osteoclastogenesis.h Fc?RIIa is a 40KDa membrane-bound protein and widely expressed in all myeloid cells.Its intracellular part contains Immunoreceptor Tyrosine-based Activation Motifs(ITAM).h Fc?RIIa receptor binds to Fc segment of Ig G and activates ITAM signal.Under the process of osteoclast differentiation,ITAM as a costimulatory signal can promote osteoclast differentiation.At present,most studies about the relationship between ITAM signal pathway with osteoclastogenesis are mainly focused on DAP12 and Fc R?.However,the specific role of h Fc?RIIa in osteoclastogenesis is still unclear.In this study,Micro-CT analysis of trabecular areas of the femurs from h Fc?RIIa-Tg and WT mice revealed markedly decreased total bone volume in the h Fc?RIIa-Tg mice compared with WT mice.Histomorphometric analysis confirmed that the OC surface was significantly increased in h Fc?RIIa-Tg mice compared with WT mice.Bone volume/total bone volume,trabecular number,trabecular thickness and trabecular spacing were decreased significantly in h Fc?RIIa-Tg mice.Serum biochemical makers of bone turnover(TRAP5b?Ca²⁺?CTX-1)showed increased in h Fc?RIIa-Tg mice compared with WT mice.Then,bone marrow from WT and h Fc?RIIa-Tg mice were cultured in the presence of M-CSF and RANKL.TRAP⁺mononuclear cells were increased significantly in macrophage cultures from h Fc?RIIa-Tg mice compared with WT mice during the early phases(days 2 to 3)of culture.Bone marrow cultures from h Fc?RIIa-Tg mice expressed relatively higher levels of osteoclast specific makerscompared with WT mice on days 3 after addition of RANKL.Functional analysis showed significantly higher bone resorption by RANKL-differentiated osteoclasts from h Fc?RIIa-Tg mice compared with WT mice on the osteo assay stripwell plate.A rapid bone loss model by administration of RANKL to mice revealed markedly higher bone loss in the h Fc?RIIa-Tg mice compared with WT mice by Micro-CT analysis,histomorphometric analysis and measurement of serum biochemical makers.These results showed that h Fc?RIIa can promote osteoclastogenisis in vivo and in vitro.In order to study the mechanism of the phenomenon,we checked expression of surface makers on osteoclast precursors of osteoclast precursors,and they were no difference.Then,adding related signal pathway inhibitors in the culture system and Western-blot found that h Fc?RIIa-Tg mice had self-activated p-Syk,which activated m TOR-p70S6K and promoted the formation of osteoclast.We further studied the effect of coating anti-human CD32 Antibody(IV.3)on osteoclastogenesis by cross-linking h Fc?RIIa.We found that c-IV.3 could inhibit the formation of osteoclasts and differentiate into small fusion cells.Western-blot results showed that A20,c-Abl and STAT5 signal pathway were activated,which were the possible mechanisms.In conclusion,our results showed that h Fc?RIIa can stimulate osteoclastogenesis through the co-stimulatory signals of p-Syk/m TOR in the presence of M-CSF and RANKL.While,cross-linking h Fc?RIIa by c-IV.3 could inhibit the formation of osteoclasts through activating A20 and differentiate into small fusion cells through activating c-Abl/STAT5.These results clarified the effect of h Fc?RIIa on osteoclastogenesis under different conditions and provided a theoretical basis for immune related osteoporosis.