Adenylyl cyclases and TRPV4 mediate Ca²⁺/cAMP dynamics to enhance fluid flow-induced osteogenesis in osteocytes

Bone adapts to physical forces and this process is dependent on osteocyte mechanotransduction. One way osteocytes sense mechanical stimulation is through the primary cilium, a sensory organelle that triggers intracellular signaling cascades in response to fluid shear. Our lab previously determined that flow-induced ciliary Ca²⁺ influx and changes in cytosolic cAMP levels are critical for osteogenesis. We also identified two proteins important for osteocyte mechanotransduction: transient receptor potential vanilloid 4 (TRPV4) and adenylyl cyclase 6 (AC6). Interestingly, disrupting the Ca²⁺-binding ability of these proteins results in loss of function. Although knockdowns of TRPV4 and AC6 disrupt osteogenesis, there is no definitive evidence linking them to Ca²⁺/cAMP dynamics that facilitate osteocyte mechanotransduction. We therefore transfected MLO-Y4 osteocytes with AC3/6 and TRPV4 overexpression plasmids that fail to interact with Ca²⁺ and observed the response to fluid shear. Indeed, mutant groups exhibited adverse changes in cAMP and lower mRNA expression of an osteogenic marker, COX-2, at the onset of flow. This pattern persisted for AC3 and TRPV4 but we detected no difference in AC6 at longer exposure to flow. These results suggest TRPV4 and ACs mediate Ca²⁺/cAMP dynamics that are important for osteocyte mechanotransduction. These mechanisms are potential targets for therapeutics to combat bone loss and should be investigated further.