RhoA GTPase interacts with beta-catenin signaling in clinorotated osteoblasts

Bone is a dynamic tissue under constant remodeling in response to various signals including mechanical loading. A lack of proper mechanical loading induces disuse osteoporosis that reduces bone mass and structural integrity. The beta-catenin signaling together with a network of GTPases is known to play a primary role in load-driven bone formation, but little is known about potential interactions of beta-catenin signaling and GTPases in bone loss. In this study, we addressed a question: Does unloading suppress an activation level of RhoA GTPase and beta-catenin signaling in osteoblasts? If yes, what is the role of RhoA GTPase and actin filaments in osteoblasts in regulating beta-catenin signaling? Using a fluorescence resonance energy transfer (FRET) technique with a biosensor for RhoA together with a fluorescent T cell factor/lymphoid enhancer factor (TCF/LEF) reporter, we examined the effects of clinostat-driven simulated unloading. The results revealed that both RhoA activity and TCF/LEF activity were downregulated by unloading. Reduction in RhoA activity was correlated to a decrease in cytoskeletal organization of actin filaments. Inhibition of beta-catenin signaling blocked unloading-induced RhoA suppression, and dominant negative RhoA inhibited TCF/LEF suppression. On the other hand, a constitutively active RhoA enhanced unloading-induced reduction of TCF/LEF activity. The TCF/LEF suppression by unloading was enhanced by co-culture with osteocytes, but it was independent on the organization of actin filaments, myosin II activity, or a myosin light chain kinase. Collectively, the results suggest that beta-catenin signaling is required for unloading-driven regulation of RhoA, and RhoA, but not actin cytoskeleton or intracellular tension, mediates the responsiveness of beta-catenin signaling to unloading.