The Roles And Mechanisms Of Primary Cilium In Bone Loss Induced By Microgravity

Along with the growing number of flights in space,astronauts will be exposed to a harsh space environment longer and more frequently.Space microgravity can cause serious bone loss and lead to the osteopenia of astronauts at vertebra,pelvis and femur,which is a major challenge in long-term spaceflight.While microgravity induced bone loss cannot yet be counteracted adequately since mechanisms are still elusive.Primary cilium is a solitary organelle that emanates from the surface of most mammalian cells,and has been reported to be a mechanosensor and chemosensor.It is now known that a variety of receptors,ion channels,and transporter proteins,as well as some of their downstream molecules have been localized to the cilium,which serves to translate extracellular stimulation to intracellular signals.Inspired by its established role as a mechanosensor,we hypothesize that primary cilium is a critical target of microgravity stimuli and plays a key role in microgravity induced bone loss.In here,rat calvarial osteoblasts(ROBs)were exposed in simulated microgravity produced by a random positioning machine(RPM),and the changes of primary cilium,cytoskeleton and cAMP/PKA/CREB signaling were analysed to identify the role and relationship between them in microgravity induced bone loss.We obtained results through these experiments and summarized in three hands as follows:First,we found that primary cilia of ROBs gradually shrank and disappeared almost completely after exposure to simulated microgravity.Along with the abrogation of primary cilia,the differentiation,maturation and mineralization of ROBs were significantly inhibited.After the elongation of primary cilium,we found that the disappearance of primary cilia was prevented by treating ROBs with cytochalasin D.The repression of the differentiation,maturation and mineralization of ROBs was effectively offset by cytochalasin D treatment in microgravityconditions.Blocking ciliogenesis using 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.Secondly,it was found that,along with the disappearance of primary cilia,the cytoskeleton was dramatically changed by microgravity,and there were intrinsic connections between cytoskeleton changes and primary cilia.We found that microgravity depolymerized the microtubule network of ROBs,but had no effects on the architecture of microfilament.Preventing primary cilium formation using siRNA-mediated depletion of the IFT88 or chloral hydrate treatment effectively offset the microtubule depolymerization,whereas stabilization of microtubule using pharmacological approach could not prevent the disappearance of primary cilium in microgravity condition.Furthermore,quantification of the number of microtubules emanating from the ciliary base body showed that microgravity significantly decreased the number of basal microtubules,which also depended on the existence of primary cilia.In addition,microgravity induced repression of the differentiation,maturation and mineralization of ROBs were effectively offset by stabilization of cytoplasmic microtubule.Together these dates suggest that primary cilia-dependent depolymerization of microtubule induced by microgravity is responsible for the microgravity’s inhibition on osteogenesis.Finally,we attempted to identified the cell signaling pathway between primary cilia and cytoskeleton in microgravity induced inhibition of osteogenesis.Microgravity activated cAMP signaling of the osteoblasts by increasing intracellular cAMP levels and facilitating phosphorylation of both PKA and CREB.Microgravity-activated cAMP signaling was localized to primary cilia,as indicated by localization of soluble AC and phosphorylated PKA.Therefore we think that cAMP signaling may be indispensable for microgravity’s inhibition on osteogenesis through primary cilia-dependent depolymerization of microtubule.Together,we found that along with the repression of osteogenesis,the primary cilia were abrogated and the microtubules were depolymerized in microgravity condition.Microgravity depolymerized the microtubule by disrupting microtubules emanating from the ciliary base through primary cilium.As a result,the bone formation process was inhibited and bone loss was induced.Besides,cAMP/PKA/CREB signaling might play a key role in the primary cilia-dependent depolymerization of microtubule and microgravity’s inhibition on osteogenesis.