HDL Impair Osteoclastogenesis And Induce Osteoclast Apoptosis Via Up Regulate The Expression Of ABCG1 In Vitro

Objective: Cholesterol is one of the major components of biological membranes, and have important function when osteoclast forming and survival. Cholesterol de novo biosynthesis does not function in osteoclast lineage, so the intracellular cholesterol homeostasis will more likely to unbalance and its formation and survival will be affected. Study found that HDL promotes cholesterol efflux from osteoclasts induces its apoptosis, but we still don’t know the mechanism of HDL promote cholesterol efflux from osteoclast and the effect of HDL acting to osteoclast formation. To ascertain the effect and mechanism of HDL, which promote cholesterol efflux, acting to osteoclast formation and survival, we use RAW264.7 cell as preosteoclast, adding RANKL and M-CSF to stimulate it form osteoclast.Methods: RAW264.7 cell were culture in DMEM/ 10% FBS /M-CSF/s RANKL with HDL(800ng/ml), observe the number, size and pyknotic nuclei of TRAP-positive multinucleated cells at different times. Cultured RAW264.7 cell in DMEM/ 10% FBS /M-CSF/s RANKL 3 days with HDL with a series of concentration, test the cholesterol efflux from osteoclast with liquid scintillation counter. RAW264.7 cell were culture in DMEM/ 10% FBS /M-CSF/s RANKL with HDL(800ng/ml), test the cholesterol efflux from osteoclast at different times. Cultured RAW264.7 cell in DMEM/ 10% FBS /M-CSF/s RANKL 3 days with HDL3, HDL2 and Aop A1, test the cholesterol efflux from osteoclast and observe the number, size and pyknotic nuclei of TRAP-positive multinucleated cells. Down-regulate the expression of ABCG1, test the cholesterol efflux, intracellular cholesterol level with liquid scintillation counter, detect m RNA level and protein level of ABCG1, SR-B1 and Cav1 with RT-PCR and WB, respectively.Results: Our results have shown that the maximum diameter and fusion index of osteoclasts were declining and the ratio of osteoclasts with pyknotic nuclei were increasing when cells exposed with HDL(800ng/ml). HDL enhanced cellular cholesterol efflux from osteoclast in a dose- and time- dependent manner. The ability of HDL3 stimulate cholesterol efflux was more powerful than nature HDL, HDL2, andApo A1. The expression of ABCG1 decrease when osteoclast formation, but the decrease was rescued when osteoclasts were exposure to HDL. The expression of SR-B1 of osteoclasts decrease when osteoclasts were exposure to HDL. The cholesterol efflux reduce and the osteoclast formation was rescued when treat with abcg1 si RNA. Finally, HDL promote sphingomyelin efflux from osteoclast, and the expression of Cav-1 was declining when treat with HDL.Conclusions: HDL can up regulate ABCG1 expression and promote cholesterol efflux from osteoclast, and then lead to intracellular cholesterol homeostasis unbalance and impair osteoclastogenesis, induce osteoclast apoptosis.