Figure 7.
mTOR Activation Suppressed OC Differentiation, Fusion, and Resorption Activity, but Enhanced Proliferation
(A) Monocytes isolated from LysM-Cre; Tsc1f/f mice showed an increase in proliferation. The monocytes isolated from WT and LysM-Cre; Tsc1f/f mice were cultured for 3 days in the presence of M-CSF and RANKL. Scale bar, 50 μm. Right panel: quantitation data. N = 4 independent experiments.
(B) Ex vivo differentiation assays revealed that monocytes isolated from LysM-Cre; Tsc1f/f mice showed a decrease in the number TRAP-positive OCs. Monocytes were isolated from the bone marrow and were induced to differentiate by M-CSF and RANKL. The cell cultures were stained for TRAP 5 days later. Bottom panel: quantitation data. Scale bar, 100 μm. N = 4 independent experiments.
(C) Images of WT and Tsc1−/− OCs. Scale bar, 50 μm.
(D) qPCR analysis revealed that Tsc1 ablation in LysM+ monocytes showed a decrease in the expression of OC differentiation markers and genes regulating OC fusion. N = 4 independent experiments.
(E) Tsc1-deficient monocytes showed a decrease in bone resorption activity in vitro. Monocytes isolated from the LysM-Cre; Tsc1f/f mice were induced to differentiate by M-CSF and RANKL, and then plated onto dentine slices. After 5 days, the slices were stained with toluidine blue and the resorption areas were measured (bottom panel). Red arrow, resorption pit. N = 3 independent experiments.
(F) Tsc1−/− monocytes showed a decrease in NF-κB activation during OC differentiation.
(G) Ex vivo differentiation assays revealed that monocytes isolated from Ctsk-Cre; Tsc1f/f mice showed a slight increase in TRAP-positive OCs. Monocytes were isolated from the bone marrow and were induced to differentiate by M-CSF and RANKL. The cell cultures were stained for TRAP 5 days later. Bottom panel: quantitation data. Scale bar, 100 μm. N = 5 independent experiments.
(H) Tsc1-deficient OCs showed a decrease in bone resorption activity in vitro. Monocytes isolated from the Ctsk-Cre; Tsc1f/f mice were induced to differentiate by M-CSF and RANKL for 3 days, and then plated onto dentine slices. After 5 days, the slices were stained with toluidine blue and the resorption areas were measured (bottom panel). Red arrow, resorption pit. Scale bar, 50 μm. N = 3 independent experiments.
(I) CT images of TSC patients revealed sclerotic bone lesions (orange arrows) with roundish or irregular shape in the vertebral body and appendix of the cervical (a), thoracic (b), and lumbar (c) spine, as well as the sternum (d), rib (e), and pelvis (f).
(J) The TSC patient serum samples showed an increase in the levels of osteocalcin. N = 20.
(K) The TSC patient serum samples showed a decrease in the levels of CTX. N = 20.
Error bars represent the SD. ∗p < 0.05; ∗∗p < 0.01.