Norlichexanthone,an Estrogen Receptor Ligand,Regulates Osteoblastogenesis And Osteoclastogenesis

Estrogen Receptor(ER),as a classical nuclear receptor,mediates various genot)ype and non-genotype functions in organisms.In addition to maintaining normal physiological functions,ER is closely related to many diseases and is an important drug target in the body.The activity of ER is regulated by its specific small molecule ligands.The discovery and study of new ER ligands will not only reveal novel biological activities and acting mechanisms of ER,but also hopefully provide new small molecule drugs targeting ER.This project is devoted to revealing a new ligand of ER,Norlichexanthone(NOR),and its related biological activities.We found that the natural product NOR selectively activated the transcriptional function of ERa and bound to ERa-LBD protein in vitro,revealing that NOR was a natural ligand of ER?,In view of the role of ERa and its ligands in regulating osteoblasts and osteoclasts,and thereby regulating bone turnover,we investigated the effects of NOR on osteoblastogenesis and osteoclastogenesis.We found that NOR significantly enhanced the activity of alkaline phosphatase(ALP)in 3T3-E1 pre-osteoblasts,and calcium deposition ability of 3T3-E1 cells.Thus,NOR had the ability of promotig osteoblastogenesis and the mineralization of osteoblasts.To investigate the role of NOR in osteoclastogenesis,we constructed osteoclast differentiation models using both mouse macrophage RAW264.7 and isolated primary monocytes.It was found that NOR had no proliferation inhibition effect on RAW264.7 cells in the absence of differentiation inductions.However,NOR significantly inhibited the proliferation of RAW264.7 cells in the presence of M-CSF and RANKL,the induction agents of osteoclastogenesis.Cell-specific resistance tartrate acid phosphatase(TRAP)staining and morphological studies indicated that NOR significantly inhibited the production of TRAP-positive and multinucleated osteoclasts induced by M-CSF and RANKL.Quantitative RT-PCR experiments also revealed that NOR inhibited the expressions of osteoclast marker genes.The above data indicated that NOR inhibited the production of osteoclasts.In order to find the mechanisms by which NOR inhibited osteoclastogenesis,we examined the effect of NOR on regulating RANKL signaling,an essential signaling pathway for osteoclast differentiation and survival.It was found that NOR significantly inhibited RANKL-induced phosphorylating activation of JNK,p38 and ERK,suggesting that NOR may inhibit osteoclastogenesis by inhibiting RANKL signaling.One of the significant side effects of estrogen is its strong hyperplasia induction of mammary gland and endometrium,thereby increasing the incidence of breast cancer and endometrial cancer.Accordingly,we compared the effect of NOR and estrogen E2 on inducing breast cancer cell proliferation.Although both E2 and NOR promoted the proliferation of ER-positive MCF-7 breast cancer cells,the effect of NOR was much weaker than that of E2,which was consistent with the weak induction of Cyclin D1 expression and AKT activation by NOR.Therefore,NOR may posses weaker estrogen-related side effects than E2.Nevertheless,NOR still needs to be further optimized to exclude or reduce its promoting effect on breast and endometrial proliferation.In conclusion,this study reveals that NOR,the natural ligand of ERa,induces osteoblastogenesis but inhibits osteoclastogenesis.Meanwhile,it reveals a possible mechanism of NOR inhibiting osteoclastogenesis.It also reveals that NOR has a weaker effect than E2 on promoting the proliferation of breast cancer cell.Therefore,this study provides a potential anti-osteoporosis lead compound and its possible mechanism of action,which are of certain theoretical significance and application values.