Icariin, a prenylated flavonol glycoside isolated from the herb Epimedium, has been considered as a potential alternative therapy for osteoporosis. Previous research has shown that, unlike other flavonoids, icariin is unlikely to act via the estrogen receptor, but its exact mechanism of action is unknown. In this study, using rat calvarial osteoblast culture and rat bone growth models, we demonstrated that icariin promotes bone formation by activating the cAMP/protein kinase A (PKA)/cAMP response element–binding protein (CREB) pathway requiring functional primary cilia of osteoblasts. We found that icariin increases the peak bone mass attained by young rats and promotes the maturation and mineralization of rat calvarial osteoblasts. Icariin activated cAMP/PKA/CREB signaling of the osteoblasts by increasing intracellular cAMP levels and facilitating phosphorylation of both PKA and CREB. Blocking cAMP/PKA/CREB signaling with inhibitors of the cAMP-synthesizing adenylyl cyclase (AC) and PKA inhibitors significantly inhibited the osteogenic effect of icariin in the osteoblasts. Icariin-activated cAMP/PKA/CREB signaling was localized to primary cilia, as indicated by localization of soluble AC and phosphorylated PKA. Furthermore, blocking ciliogenesis via siRNA knockdown of a cilium assembly protein, IFT88, inhibited icariin-induced PKA and CREB phosphorylation and also abolished icariin's osteogenic effect. Finally, several of these outcomes were validated in icariin-treated rats. Together, these results provide new insights into icariin function and its mechanisms of action and strengthen existing ties between cAMP-mediated signaling and osteogenesis.

It is well documented that microgravity in space environment leads to bone loss in astronauts. These physiological changes have also been validated by human and animal studies and modeled in cell-based analogs. However, the underlying mechanisms are elusive. In the current study, we identified a novel phenomenon that primary cilia (key sensors and functioning organelles) of rat calvarial osteoblasts (ROBs) gradually shrank and disappeared almost completely after exposure to simulated microgravity generated by a random positioning machine (RPM). Along with the abrogation of primary cilia, the differentiation, maturation and mineralization of ROBs were inhibited. We also found that the disappearance of primary cilia was prevented by treating ROBs with cytochalasin D, but not with LiCl or dynein light chain Tctex-type 1 (Dynlt1) siRNA. The repression of the differentiation, maturation and mineralization of ROBs was effectively offset by cytochalasin D treatment in microgravity conditions. Blocking ciliogenesis using intraflagellar transport protein 88 (IFT88) siRNA knockdown inhibited the ability of cytochalasin D to counteract this reduction of osteogenesis. These results indicate that the abrogation of primary cilia may be responsible for the microgravity’s inhibition on osteogenesis. Reconstruction of primary cilia may become a potential strategy against bone loss induced by microgravity.