Smad4 controls bone homeostasis through regulation of osteoblast/osteocyte viability

Abstract

Regulation of osteoblast and osteocyte viability is essential for bone homeostasis. Smad4, a major transducer of bone morphogenetic protein and transforming growth factor-beta signaling pathways, regulates apoptosis in various cell types through a mitochondrial pathway. However, it remains poorly understood whether Smad4 is necessary for the regulation of osteoblast and osteocyte viability. In this study, we analyzed Smad4 Delta(Os) mice, in which Smad4 Delta(Os) was subjected to tissue-specific disruption under the control of the 2.3-kb Col1a1 promoter, to understand the functional significance of Smad4 Delta(Os) in regulating osteoblast/osteocyte viability during bone formation and remodeling. Smad4 Delta(Os).Os mice showed a significant increase in osteoblast number and osteocyte density in the trabecular and cortical regions of the femur, whereas osteoclast activity was significantly decreased. The proliferation of osteoblasts/osteocytes did not alter, as shown by measuring 5'-bromo-2' deoxyuridine incorporation. By contrast, the percentage of TUNEL-positive cells decreased, together with a decrease in the Bax/Bcl-2 ratio and in the proteolytic cleavage of caspase 3, in Smad4 Delta(Os).Os mice. Apoptosis in isolated calvaria cells from Smad4 Delta(Os).Os mice decreased after differentiation, which was consistent with the results of the TUNEL assay and western blotting in Smad4 Delta(Os).Os mice. Conversely, osteoblast cells overexpressing Smad4 Delta(Os) showed increased apoptosis. In an apoptosis induction model of Smad4 Delta(Os).Os mice, osteoblasts/osteocytes were more resistant to apoptosis than were control cells, and, consequently, bone remodeling was attenuated. These findings indicate that Smad4 Delta(Os) has a significant role in regulating osteoblast/osteocyte viability and therefore controls bone homeostasis.