Teruhito Yamashita

Negative Regulation of BMP/Smad Signaling by Tob in Osteoblasts

Bone morphogenetic protein (BMP) controls osteoblast proliferation and differentiation through Smad proteins. Here we show that Tob, a member of the emerging family of antiproliferative proteins, is a negative regulator of BMP/Smad signaling in osteoblasts. Mice carrying a targeted deletion of the tob gene have a greater bone mass resulting from increased numbers of osteoblasts. Orthotopic bone formation in response to BMP2 is elevated in tob-deficient mice. Overproduction of Tob represses BMP2-induced, Smad-mediated transcriptional activation. Finally, Tob associates with receptor-regulated Smads (Smad1, 5, and 8) and colocalizes with these Smads in the nuclear bodies upon BMP2 stimulation. The results indicate that Tob negatively regulates osteoblast proliferation and differentiation by suppressing the activity of the receptor-regulated Smad proteins.

Resistance to Unloading-Induced Three-Dimensional Bone Loss in Osteopontin-Deficient Mice†

Recent development in three‐dimensional (3D) imaging of cancellous bone has made possible true 3D quantification of trabecular architecture. This provides a significant improvement in the measures available to study and understand the mechanical functions of cancellous bone. We recently reported that the presence of osteopontin (OPN) was required for the effects of mechanical stress on bone as OPN‐null (OPN−/−) mice showed neither enhancement of bone resorption nor suppression of bone formation when they were subjected to unloading by tail suspension. However, in this previous study, morphological analyses were limited to two‐dimensional (2D) evaluation. Although bone structure is 3D and thus stress effect should be evaluated based on 3D parameters, no such 3D morphological features underlying the phenomenon have been known. To elucidate the role of OPN in mediating mechanical stress effect based on true quantitative examination of bone, we evaluated 3D trabecular structures of hindlimb bones of OPN−/− mice after tail suspension. Tail suspension significantly reduced 3D parameters of bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and anisotropy and increased 3D parameters on trabecular separation (Tb.Sp) in wild‐type mice. In contrast, these 3D parameters were not altered after tail suspension in OPN−/− mice. These data provided evidence that OPN is required for unloading‐induced 3D bone loss.

Retardation in Bone Resorption after Bone Marrow Ablation in Klotho Mutant Mice

The klotho gene mutant mice exhibit both osteopetrotic phenotype, including elongation of trabeculae in the epiphyses of long bones and vertebral bodies, and osteopenic phenotype, such as thin cortical bones in the diaphyses of these bones. These diverse features raise the question of whether the klotho gene defect results in alteration in bone resorption in vivo. Therefore, we examined the effect of the klotho gene defect on bone resorption by using bone marrow ablation model. At 1 week after bone marrow ablation, trabecular bones were formed in the ablated marrow cavity to levels higher than those in unablated bones in both klotho mutant and wild-type mice. At 2 weeks postsurgery, newly formed trabecular bones were resorbed in wild-type mice to resume normal bone marrow and trabecular bone volume fraction as reported previously. In contrast, the newly formed trabecular bones in the ablated marrow in klotho mutant mice remained at levels similar to those at 1 week. The defect in the bone resorption phase...

Enhancing Effect of Tob Deficiency on Bone Formation Is Specific to Bone Morphogenetic Protein-Induced Osteogenesis†

Tob is a recently reported novel bone morphogenetic protein (BMP) inhibitor, which originally was identified by West‐Western procedure using ErbB2 as a probe and contains a nuclear localization signal. To further characterize the effects of Tob deficiency on BMP‐induced new bone (NB) formation, we examined microcomputed tomography (μCT) on the cross‐section of the bone induced by daily injection with BMP onto the calvariae of newborn mice. The calvariae of the saline‐injected Tob‐deficient (TD) mice were similar to those of the saline‐injected or untreated wild‐type (WT) mice. BMP injection locally produced NB on the calvaria in WT mice as known previously. In contrast to WT mice, BMP injection onto the calvariae of TD mice produced a calcified area in the cross‐section of NB, which was more than that produced by BMP in the WT calvariae. In addition, the horizontal width and the vertical height of the NB induced by BMP in TD mice were several‐fold more than those in WT mice. The effect of Tob deficiency on bone‐forming activity was selective to the response to the injection with BMP because the levels of injury‐induced NB formation examined by μCT 10 days after bone marrow ablation in the femora were similar between the TD and WT mice. These data indicate that Tob acts as a novel specific antagonist against bone formation induced by BMP treatment in bone.