Bone morphogenetic protein (BMP)9 has been reported to be the most potent BMP to induce bone formation. However, the details of BMP9-transduced intracellular signaling remain ambiguous. Here, we have investigated signal transduction mechanisms of BMP9 in comparison to BMP2, another potent inducer of bone formation, in osteoblasts. In a mouse osteoblast cell line, BMP9 induced higher mRNA levels of alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2) than BMP2 within 2 h. Unlike BMP2, BMP9 induced rapid phosphorylation of glycogen synthase kinase 3-β (GSK3-β) and protein kinase B (Akt) and increased the cellular protein content of β-catenin. BMP9 moderately increased mRNA levels of several canonical Wingless-related integration site to lower degrees than BMP2. Furthermore, BMP9-induced GSK3-β phosphorylation was not inhibited by pretreatment with actinomycin D, cycloheximide, or Brefeldin A, indicating it is independent of Wnt protein secretion. BMP9-induced GSK3-β phosphorylation was abrogated by Akt or class I PI3K-specific inhibitors. Moreover, inactivation of GSK3-β by LiCl did not further promote ALP and Runx2 mRNA induction by BMP9 as significantly as that by BMP2. Notably, BMP9-induced GSK3-β phosphorylation was inhibited by small interfering RNA against endoglin and GIPC PDZ domain-containing family, member 1. Taken together, our present findings have indicated that BMP9 directly activates GSK3β-β-catenin signaling pathway through class I PI3K-Akt Axis in osteoblasts, which may be essential for the potent osteoinductive activity of BMP9.—Eiraku, N., Chiba, N., Nakamura, T., Amir, M. S., Seong, C.-H., Ohnishi, T., Kusuyama, J., Noguchi, K., Matsuguchi, T. BMP9 directly induces rapid GSK3-β phosphorylation in a Wnt-independent manner through class I PI3K-Akt axis in osteoblasts. FASEB J. 33, 12124-12134 (2019). www.fasebj.org.
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