The TrkA agonist gambogic amide augments skeletal adaptation to mechanical loading through actions on sensory nerves and osteoblasts

ABSTRACTThe periosteal and endosteal surfaces of mature bone are densely innervated by sensory nerves expressing TrkA, the high-affinity receptor for nerve growth factor (NGF). In previous work, we demonstrated that administration of exogenous NGF significantly increased load-induced bone formation through the activation of Wnt signaling. However, the translational potential of NGF is limited by the induction of substantial mechanical and thermal hyperalgesia in mice and humans. Here, we tested the effect of gambogic amide (GA), a recently identified robust small molecule agonist for TrkA, on hyperalgesia and load-induced bone formation. Behavioral analysis was used to assess pain up to one week after axial forelimb compression. Contrary to our expectations, GA treatment was not associated with diminished use of the loaded forelimb or sensitivity to thermal stimulus. Furthermore, dynamic histomorphometry revealed a significant increase in relative periosteal bone formation rate as compared to vehicle treatment. Additionally, we found that GA treatment was associated with an increase in the number of osteoblasts per bone surface in loaded limbs as well as a significant upregulation of Wnt1, Wnt7b, and Ngf in loaded bones. To determine if these effects were exclusively mediated by NGF-TrkA signaling in sensory nerves, we cultured MC3T3-E1 cells for 7 or 14 days in osteogenic differentiation media containing NGF (50 ng/mL), GA (5, 50, or 500 nM), or vehicle (DMSO). After 7 days of culture, we observed increases in osteoblastic differentiation markers Runx2, Bglap2, and Sp7 in response to GA, whereas treatment with NGF was not different than vehicle. Only cells treated with the highest dose of GA (500 nM) had significantly impaired cell proliferation. In conclusion, our study indicates GA may be useful for augmenting skeletal adaptation to mechanical forces without inducing hyperalgesia through actions on both sensory nerves and osteoblasts.

NGF-TrkA signaling in sensory nerves is required for skeletal adaptation to mechanical loads in mice

Sensory nerves emanating from the dorsal root extensively innervate the surfaces of mammalian bone, a privileged location for the regulation of biomechanical signaling. Here, we show that NGF-TrkA signaling in skeletal sensory nerves is an early response to mechanical loading of bone and is required to achieve maximal load-induced bone formation. First, the elimination of TrkA signaling in mice harboring mutant TrkAF592A alleles was found to greatly attenuate load-induced bone formation induced by axial forelimb compression. Next, both in vivo mechanical loading and in vitro mechanical stretch were shown to induce the profound up-regulation of NGF in osteoblasts within 1 h of loading. Furthermore, inhibition of TrkA signaling following axial forelimb compression was observed to reduce measures of Wnt/β-catenin activity in osteocytes in the loaded bone. Finally, the administration of exogenous NGF to wild-type mice was found to significantly increase load-induced bone formation and Wnt/β-catenin activity in osteocytes. In summary, these findings demonstrate that communication between osteoblasts and sensory nerves through NGF-TrkA signaling is essential for load-induced bone formation in mice.

Bone loss patterns in cortical, subcortical, and trabecular compartments during simulated microgravity

Disuse studies provide a useful model for bone adaptation. A direct comparison of these studies is, however, complicated by the different settings used for bone analysis. Through pooling and reanalysis of bone data from previous disuse studies, we determined bone loss and recovery in cortical, subcortical, and trabecular compartments and evaluated whether the study design modulated skeletal adaptation. Peripheral quantitative tomographic (pQCT) images from control groups of four disuse studies with a duration of 24, 35, 56, and 90 days were reanalyzed using a robust threshold-free segmentation algorithm. The pQCT data were available from 27 young healthy men at baseline, and at specified intervals over disuse and reambulation phases. The mean maximum absolute bone loss (mean ± 95% CI) was 6.1 ± 4.5 mg/mm in cortical, 2.4 ± 1.6 mg/mm in subcortical, and 9.8 ± 9.1 mg/mm in trabecular compartments, after 90 days of bed rest. The percentage changes in all bone compartments were, however, similar. During the first few weeks after onset of reambulation, the bone loss rate was systematically greater in the cortical than in the trabecular compartment ( P < 0.002), and this was observed in all studies except for the longest study. We conclude that disuse-induced bone losses follow similar patterns irrespective of study design, and the largest mean absolute bone loss occurs in the cortical compartment, but apparently only during the first 60 days. With longer study duration, trabecular loss may become more prominent.