Activin Receptor Signaling In Bone Development and Regeneration

Bone and skeletal muscle mass are highly correlated in mammals, suggesting the existence of common signaling networks that serve to coordinate the development of these two anatomically adjacent tissues. Myostatin and its related activin ligands of the TGB-beta superfamily are known regulators of skeletal muscle mass and are also expressed in bone. Given the well-established role of myostatin in muscle, these ligands may serve a similar function in bone. To investigate the role of this pathway in bone, pharmacological and genetic approaches were employed to determine the role of activin receptor signaling in bone. To identify which components of the activin signaling pathway were expressed in bone, we used immunohistochemistry. ACVR2A and ACVR2B were localized to osteoblasts and osteocytes in both trabecular and cortical bone. Primary mouse calvarial osteoblasts also expressed the major activin receptor signaling components, including ACVR2A, ACVR2B, and ACVR1B (ALK4). Furthermore, upon exposure to activin ligands, osteoblasts demonstrated active signaling by increased levels of phosphorylated Smad2/3. Osteoblasts deficient in ACVR2A by Cre-mediated recombination exhibited accelerated differentiation, evidenced by increased alkaline phosphatase activity, mineral deposition, and increased transcriptional expression of Osterix, Osteocalcin, and Dmp1 compared to controls. To determine the importance of activin receptor signaling in bone in vivo, we analyzed the skeletal phenotypes of mice selectively deficient in ACVR2A and ACVR2B in osteoblasts and osteocytes (osteocalcin-Cre). Whereas ACVR2B deficient mice (Ob.ACVR2B-/-), had no significant changes in bone parameters, mice lacking ACVR2A (Ob.ACVR2A-/-) had significantly increased femoral trabecular bone volumes at six and twelve weeks of age. In addition, Ob.ACVR2A-/- mice also exhibited increases in cortical parameters at twelve weeks of age, which resulted in enhanced mechanical properties. Compound mutant mice lacking both ACVR2A and ACVR2B demonstrated increases in trabecular bone volumes similar to Ob.ACVR2A-/- mice. Taken together, these data indicate that activin receptor signaling in osteoblasts is predominantly mediated through ACVR2A and acts as a negative regulator of bone mass. We then evaluated activin-targeted biologics for their efficacy in muscle and bone healing. Therapeutic potential of soluble activin receptor administration was investigated utilizing a composite musculoskeletal injury model, consisting of a pin-stabilized femur osteotomy and an overlying volumetric muscle defect. With ACVR2B/Fc treatment, the bone fracture callus volume and mineral content were significantly increased as compared to vehicle controls while mechanical properties were unchanged between groups. Furthermore, muscle mass was significantly increased while injury fibrosis and fat infiltration were reduced with ACVR2B/Fc administration – supporting the possibility of using soluble activin receptors as a dual muscle and bone anabolic agent. In conclusion, the results from these studies demonstrate that the activin signaling pathway occurs primarily through ACVR2A in osteoblasts and acts to negatively regulate bone mass. Furthermore, inhibition of this pathway may act as a dual anabolic agent and is a potential therapeutic to augment composite muscle and bone healing