Role of Smad3, acting independently of transforming growth factor-beta, in the early induction of Wnt-beta-catenin signaling by parathyroid hormone in mouse osteoblastic cells.
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.
Inoue
Yoshifumi
Y
Division of Diabetes, Metabolism and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Chuo-ku, Kobe 650-0017, Japan.
Canaff
Lucie
L
Hendy
Geoffrey N
GN
Hisa
Itoko
I
Sugimoto
Toshitsugu
T
Chihara
Kazuo
K
Kaji
Hiroshi
H
D000818:Animals; D002460:Cell Line; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000378:metabolism*; D010641:Phenotype; D015398:Signal Transduction; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D051153:Wnt Proteins / Q000378:metabolism*; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*
D010281:Parathyroid Hormone; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D051153:Wnt Proteins; D051176:beta Catenin