MSC contributions to loading-induced bone formation: a role for adenylyl cyclase 6

Physical loading is a potent regulator of bone anabolism, yet the cellular mechanisms by which this occurs are poorly understood. Due to the finite lifespan of bone forming osteoblasts, it is hypothesized that these cells must be replenished from a bone marrow stem cell (MSC) population in response to loading, as is seen in response to injury. It is hypothesized that the application of a mechanical stimulus may directly stimulate MSCs within the marrow to undergo osteogenic differentiation, as has been shown in vitro. However, how MSCs may sense this loading or whether loading actually regulates MSC differentiation in vivo is unknown. In this thesis, we first demonstrate that MSCs utilize cAMP as a 2nd messenger in mechanotransduction, that is required for flow mediated increases in osteogenic gene expression. Furthermore, we demonstrate that this mechanosignalling is dependent on the primary cilium and the ciliary localised adenylyl cyclase 6. Lastly, in this initial study we also demonstrate that this mechanotransduction mechanism can be targeted therapeutically to enhance cAMP concentrations and early osteogenic signalling, mimicking the beneficial effect of physical loading. In the second study of this thesis, we demonstrate that Gpr161 is a mechanoresponsive G-Protein Couple Receptor (GPCR), that localises to the cilium, and is required for fluid shear induced increases in cAMP and osteogenesis. This Gpr161 mediated mechanotransduction is dependent on the primary cilium but acts upstream of cilia localised adenylyl cyclase 6 (AC6), suggesting that Gpr161 may act through AC6 to regulate cAMP and MSC osteogenesis. Moreover, we demonstrate that Hedgehog (Hh) signalling is positively correlated with osteogenesis and demonstrate that Hh signalling is mechanically regulated and required for loading-induced MSC osteogenesis through a primary cilium-Gpr161-AC6-cAMP mechanism. Taking the first two studies together, we have delineated a molecular mechanism of MSC mechanotransduction which occurs at the primary cilium and can be targeted therapeutically, demonstrating a potential mechanotherapeutic for bone loss diseases such as osteoporosis. Finally, we utilized Leptin Receptor to identify and trace the contribution of bone marrow stem cells to bone mechanoadaptation in vivo. Lepr-Cre; tdTomato+ mice were subjected to compressive tibia loading and histological analysis revealed that Lepr-Cre; tdTomato+ stem cells arise perinatally around blood vessels and increase within the marrow following tibial compressive loading. Mechanical loading induces an increase in bone formation parameters yet loading does not result in an increase in Lepr-Cre; tdTomato+ osteoblasts or osteocytes. Moreover, mice with a LepR specific deletion of AC6 have an attenuated response to compressive tibia loading, nor is there a change in the percentage of Lepr-Cre; tdTomato+ cells. This therefore demonstrates that LepR+ cells on the bone surface directly contribute to bone formation and that adenylyl cyclase 6 is required for activation of these cells in response to loading, highlighting an activation of the resident bone lining cells via AC6 as the predominant mechanism mediating short term loading-induced bone formation.