The mechanisms whereby the parathyroid hormone (PTH) exerts its anabolic action on bone are incompletely understood. We previously showed that inhibition of ERK1/2 enhanced Smad3-induced bone anabolic action in osteoblasts. These findings suggested the hypothesis that changes in gene expression associated with the altered Smad3-induced signaling brought about by an ERK1/2 inhibitor would identify novel bone anabolic factors in osteoblasts. We therefore performed a comparative DNA microarray analysis between empty vector-transfected mouse osteoblastic MC3T3-E1 cells and PD98059-treated stable Smad3-overexpressing MC3T3-E1 cells. Among the novel factors, Tmem119 was selected on the basis of its rapid induction by PTH independent of later increases in endogenous TGF-␤. The levels of Tmem119 increased with time in cultures of MC3T3-E1 cells and mouse mesenchymal ST-2 cells committed to the osteoblast lineage by BMP-2. PTH stimulated Tmem119 levels within 1 h as determined by Western blot analysis and immunocytochemistry in MC3T3-E1 cells. MC3T3-E1 cells stably overexpressing Tmem119 exhibited elevated levels of Runx2, osteocalcin, alkaline phosphatase, and ␤-catenin, whereas Tmem119 augmented BMP-2-induced Runx2 levels in mesenchymal cells. Tmem119 interacted with Runx2, Smad1, and Smad5 in C2C12 cells. In conclusion, we identified a Smad3-related factor, Tmem119, that is induced by PTH and promotes differentiation in mouse osteoblastic cells. Tmem119 is an important molecule in the pathway downstream of PTH and Smad3 signaling in osteoblasts.Bone remodeling is controlled by bone resorption and subsequent osteoblastic bone formation. Insights into the mechanisms of osteoclastic bone resorption have led to the development of reagents that potently suppress bone resorption for the treatment of osteoporosis. On the other hand, the impairment of osteoblastic bone formation is considered to be the main driving force in age-related, glucocorticoid-induced, and diabetes-related osteoporosis. However, many aspects of osteoblastic bone formation remain to be understood. Intermittent parathyroid hormone (PTH) 3 is one of the most potent anabolic agents; it stimulates de novo osteoblastic bone formation and is superior to bisphosphonates in the treatment of osteoporosis (1, 2). The anabolic action of PTH is exerted partly through local growth factors and transcriptional regulators as well by as an anti-apoptotic action in osteoblasts (3, 4). However, the precise mechanisms by which PTH exerts its anabolic action on bone are incompletely understood.We showed previously that Smad3, a crucial TGF-␤-signaling molecule, promotes alkaline phosphatase (ALP) activity in mouse osteoblastic MC3T3-E1 cells (5, 6), and Borton et al. (7) have found that mice with targeted disruption of Smad3 exhibit osteopenia caused by decreased bone formation, suggesting that the Smad3 molecule is a promoter of bone formation. Moreover, we demonstrated that TGF-␤-responsive ERK1/2 and c-Jun N-terminal kinase (JNK) cascades negatively regulate Smad3-ind...

Parathyroid hormone (PTH) exerts an anabolic action on bone but the mechanisms are incompletely understood. We showed previously that PTH interacts with the canonical Wnt-beta-catenin signaling pathway via the transforming growth factor (TGF)-beta signaling molecule, Smad3, to modulate osteoblast differentiation and apoptosis. Here, we examined which actions of Smad3 are TGF-beta-independent in stimulating the osteoblast phenotype and PTH-induced Wnt-beta-catenin signaling. For this, the TGF-beta receptor type 1 [activin receptor-like kinase (ALK5)] inhibitor (SB431542), and a Smad3 mutant in which the site normally phosphorylated by ALK5 is mutated from SSVS to AAVA, was used. PTH induced total beta-catenin and reduced phosphorylated beta-catenin levels at 1, 6, and 24 h in mouse osteoblastic MC3T3-E1 cells. Transient transfection of Smad3AAVA inhibited the PTH induction of total beta-catenin and reduction of phosphorylated beta-catenin levels at 6 and 24 h, but not at 1 h, indicating that the early effects occur independently of TGF-beta receptor signaling. On the other hand, MC3T3-E1 cell clones in which Smad3AAVA was stably expressed demonstrated elevated beta-catenin levels, although alkaline phosphatase (ALP) activity and mineralization were unaltered. In contrast, MC3T3-E1 cell clones in which wild-type Smad3 was stably expressed exhibited increased ALP activity and mineralization that were decreased by the ALK5 inhibitor, SB431542, although the beta-catenin levels induced in these cells were not modulated. In conclusion, the present study indicates that PTH induces osteoblast beta-catenin levels via Smad3 independently of, and dependently on, TGF-beta in the early and later induction phases, respectively.

Alendronate, an aminobisphosphonate, is an effective reagent to reduce fracture risk in osteoporotic patients. Although several studies suggest that bisphosphonates affect osteoblast differentiation, how they affect the genes relating to the mineralization step remains unknown. The present study was performed to clarify the effects of alendronate on mineralization and its related genes in mouse osteoblastic MC3T3-E1 cells. Alendronate at 10 (-8) and 10 (-7) M induced mineralization in MC3T3-E1 cells. As for the genes that suppress mineralization, alendronate enhanced the level of PC-1 mRNA in a dose-dependent manner in 7-day cultures in semiquantitative RT-PCR, although it reduced the levels of PC-1 mRNA in 21-day cultures. On the other hand, alendronate did not affect the levels of ANK, osteopontin and matrix Gla protein mRNA in both 7- and 21-day cultures. Moreover, alendronate reduced the level of osteocalcin mRNA at 10 (-7) and 10 (-6) M in 14-day cultures of these cells. As for the expression of alkaline phosphatase (ALP), an important positive regulator of mineralization in osteoblasts, alendronate enhanced the levels of ALP mRNA and protein at 10 (-7)-10 (-5) M. In conclusion, low-dose alendronate induced mineralization in mouse osteoblastic cells. The regulation of PC-1, osteocalcin and ALP by alendronate might play some role in these effects.

A 49-year-old

Bisphosphonate is an effective drug to reduce fracture risk in osteoporotic patients; however, factors affecting the efficacy of bisphosphonate treatment are not fully known, especially in Japanese patients. In the present study, we examined the relationships between an increase in lumbar spine bone mineral density (BMD) by bisphosphonates and several pretreatment parameters, including biochemical, bone/mineral, and body composition indices, in 85 postmenopausal osteoporotic patients treated with alendronate or risedronate. BMD increase was measured by dual-energy X-ray absorptiometry at the lumbar spine before and 2 years after treatment. BMD increase at the lumbar spine was observed as independent of age, height, weight, body mass index, and fat mass, although lean body mass seemed slightly related. On the other hand, fasting plasma glucose (FPG) levels were significantly and positively related to BMD increase at the lumbar spine. In multiple regression analysis, FPG levels were not significantly related to BMD increase at the lumbar spine when lean body mass was considered. As for bone/mineral parameters, BMD increase at the lumbar spine was not significantly related to serum levels of calcium, parathyroid hormone (PTH), and alkaline phosphatase or urinary levels of deoxypiridinoline and calcium excretion. As for BMD parameters, Z-scores of BMD at any site and bone geometry parameters obtained by forearm peripheral quantitative computed tomography were not significantly related to BMD increase at the lumbar spine. BMD increases at the lumbar spine were similar between groups with or without vertebral fractures. In conclusion, BMD increase at the lumbar spine by bisphosphonate treatment was not related to any pretreatment parameters, including body size, body composition, and bone/mineral metabolism in postmenopausal Japanese women with primary osteoporosis, although FPG correlated partly to BMD through lean body mass.