Parathyroid Hormone-responsive Smad3-related Factor, Tmem119, Promotes Osteoblast Differentiation and Interacts with the Bone Morphogenetic Protein-Runx2 Pathway

Abstract

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...

Role of Osteoglycin in the Linkage between Muscle and Bone

“…Transient and Stable Transfection-Each vector was transfected into MC3T3-E1 or C2C12 cells with Lipofectamine (Invitrogen) as previously described (12). Six hours later, the cells were supplied with fresh ␣-MEM or DMEM containing 10% FBS.…”

Combinatorial effect of Si4+, Ca2+, and Mg2+ released from bioactive glasses on osteoblast osteocalcin expression and biomineralization

“…This early enhanced OCN gene expression was probably owed to early enhanced SMAD3 expression (day 2), which has been implicated in the promotion of mineralized tissue formation in MC3T3-E1 subclone 4 cells [23,24]. Since OCN binds extracellular matrix Ca 2+ to the developing bone matrix, a lack of OCN up-regulation would inhibit enhanced biomineralization.…”

New tools for studying microglia in the mouse and human CNS

“…Tmem119 is a type IA single-pass transmembrane protein with a reported role in osteoblast differentiation (23,24). Because no specific anti-mouse Tmem119 antibodies existed, we made two different monoclonal antibodies (mAbs) against the predicted extracellular (ECD) and intracellular (ICD) domains of mouse Tmem119 (SI Appendix, Fig.…”