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). http://www.fasebj.org
Bone morphogenetic protein (BMP) 9 is one of the most osteogenic BMPs, but its mechanism of action has not been fully elucidated. Hes1, a transcriptional regulator with a basic helix‐loop‐helix domain, is a well‐known effector of Notch signaling. Here, we find that BMP9 induces periodic increases of Hes1 mRNA and protein expression in osteoblasts, presumably through an autocrine negative feedback mechanism. BMP9‐mediated Hes1 induction is significantly inhibited by an ALK inhibitor and overexpression of Smad7, an inhibitory Smad. Luciferase and ChIP assays revealed that two Smad‐binding sites in the 5′ upstream region of the mouse Hes1 gene are essential for transcriptional activation by BMP9. Thus, our data indicate that BMP9 induces Hes1 expression in osteoblasts via the Smad signaling pathway.
Periodontal ligament fibroblasts (PDLFs) have osteogenic capacity, producing bone matrix proteins. Application of bone morphogenic proteins (BMPs) to PDLFs is a promising approach for periodontal regeneration. However, in chronic bone metabolic disorders, such as periodontitis, proper control of accompanying inflammation is essential for optimizing the effects of BMPs on PDLFs. We have previously shown that low‐intensity pulsed ultrasound (LIPUS), a medical technology that induces mechanical stress using sound waves, significantly promotes osteogenesis in mesenchymal stem cells. Here, we demonstrate that LIPUS promotes the BMP9‐induced osteogenic differentiation of PDLFs. In contrast, BMP2‐induced osteogenic differentiation was not altered by LIPUS, probably due to the LIPUS‐induced secretion of Noggin, a BMP2 antagonist, from PDLFs. To examine if LIPUS affects inflammatory responses of PDLFs to lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (LPS‐PG), we also simultaneously treated PDLFs with LIPUS and LPS‐PG. Treatment with LIPUS significantly inhibited the phosphorylation of ERKs, TANK‐binding kinase 1, and interferon regulatory factor 3 in LPS‐PG‐stimulated PDLFs, in addition to inhibiting the degradation of IκB. Furthermore, LIPUS treatment reduced messenger RNA (mRNA) expression of interleukin‐1alpha (IL‐1alpha), IL‐1beta, IL‐6, IL‐8, C‐C motif chemokine ligand 2, C‐X‐C motif chemokine ligand 1 (CXCL1), CXCL10 and receptor activator of nuclear factor kappa‐B ligand, and also diminished IL‐1ß and tumor necrosis factor a (TNFa)‐induced inflammatory reactions. Phosphorylation of Rho‐associated kinase 1 (ROCK1) was induced by LIPUS, while ROCK1‐specific inhibitor prevented the promotive effects of LIPUS on p38 phosphorylation, mRNA expression of CXCL1 and Noggin, and osteogenesis. The suppressive effects of LIPUS on LPS‐PG‐stimulated inflammatory reactions were also prevented by ROCK1 inhibition. Moreover, LIPUS treatment blocked inhibitory effects of LPS‐PG and IL‐1ß on osteogenesis. These results indicate that LIPUS suppresses inflammatory effects of LPS‐PG, IL‐1ß, and TNFa and also promotes BMP9‐induced osteogenesis through ROCK1 in PDLFs.
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