Intracellular Signaling Pathways Mediating Muscle-bone Crosstalk

Objectives: Osteocytes are regarded as responsible for mechanotransduction in bone response to loading. It has been shown that factors secreted by C2C12 myotubes prevent glucocorticoid-induced death of osteocytes through the activation of the Wnt/β-catenin pathway (Jahn et al, 2012). The mTOR signaling pathway is known to interact with Wnt/β-catenin signaling and be involved in cell differentiation. We examined the role of mTOR signaling in the production of the factor and response of osteocytes to myotube conditioned media (MT-CM). Our objective is to determine the role that the mTOR pathway plays in the production of this muscle-derived factor and the synergistic activation of the β-catenin pathway in osteocytes.
Methods: C2C12 Myotubes were cultured and treated with 10µM of the mTOR inhibitor (Ku-0063794) on d5 of differentiation for 24h. MT-CM was collected on d5-8. Osteocyte cells were subsequently incubated with 10% of MT-CM (± 10ng/mL Wnt3a) for 24h. Separately, osteocyte cells were cultured and treated with 0.5µM Ku-0063794 and d6 MT-CM for 24hrs. Triplicate determinations were measured with each experiment, n=5. Quantitation of active β-catenin was performed using the osteocyte reporter cell line, TOPflash-MLO-Y4, which expresses luciferase when β-catenin signaling is activated (Lara & Johnson, 2017).
Results: Treatment with MT-CM from d6-8 significantly increased in β-catenin activation in osteocytes. Treatment of myotubes with Ku-treated C2C12 myotubes blocked this activation in osteocytes. The production of the critical factor partially recovered after the mTOR inhibitor was removed. The direct inhibition of the mTOR pathway in osteocytes also significantly blocked levels of β-catenin activation.
Conclusions: The mTOR pathway plays an important role in the production of the myotube-factor that activates the Wnt/β-catenin pathway in osteocytes and the synergistic effect of this factor with Wnt3a also involves mTOR signaling. Identification of this factor(s) may provide therapeutic approaches for increasing bone mass. Supported by UMKC SOD Summer Scholars program.