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Osteopontin (OPN) is an osteoblast-derived secretory protein that plays a role in bone remodeling, osteoblast responsiveness, and inflammation. We recently found that osteoblast differentiation is type-specific, with conditions of JNK inactivation inducing osteoblasts that preferentially express OPN (OPN-type). Since OPN-type osteoblasts highly express osteogenesis-inhibiting proteins and Rankl, an important inducer of osteoclastogenesis, an increased appearance of OPN-type osteoblasts may be associated with inefficient and poor-quality bone regeneration. However, whether specific osteogenic inducers can modulate OPN-type osteoblast differentiation is completely unknown. Here, we demonstrate that bone morphogenic protein 9 (BMP9) prevents induction of OPN-type osteoblast differentiation under conditions of JNK inhibition. Although JNK inactivation suppressed both BMP2- and BMP9-induced matrix mineralization and osteocalcin expression, the expression of Rankl and specific cytokines such as Gpha2, Esm1, and Sfrp1 under similar conditions was increased in all cells except those treated with BMP9. Increased expression of Id4, a critical transcriptional regulator of OPN-type osteoblast differentiation, was similarly prevented only in BMP9-treated cells. We also found that BMP9 specifically induces the expression of Hey1, a bHLH transcriptional repressor, and that Id4 inhibits the suppressive effects of Hey1 on Opn promoter activity by forming Id4-Hey1 complexes in osteoblasts. Using site-direct mutagenesis, ChIP, and immunoprecipitation, we elucidated that BMP9-induced overexpression of Hey1 can overcome the effects of Id4 and suppress OPN expression. We further found that p38 activation and JNK inactivation are involved in BMP9-induced Hey1 expression. Collectively, these data suggest that BMP9 is a unique osteogenic inducer that regulates OPN-type osteoblast differentiation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mesenchymal stem cells (MSCs) are a powerful tool for cell-based, clinical therapies like bone regeneration. Therapeutic use of cell transplantation requires many cells, however, the expansion process needed to produce large quantities of cells reduces the differentiation potential of MSCs. Here, we examined the protective effects of low intensity pulsed ultrasound (LIPUS) on the maintenance of osteogenic potency. Primary osteoblastic cells were serially passaged between 2 and 12 times with daily LIPUS treatment. We found that LIPUS stimulation maintains osteogenic differentiation capacity in serially passaged cells, as characterized by improved matrix mineralization and Osteocalcin mRNA expression. Decreased expression of Nanog, Sox2, and Msx2, and increased expression of Pparg2 from serial passaging was recovered in LIPUS-stimulated cells. We found that LIPUS stimulation not only increased but also sustained expression of Nanog in primary osteoblasts and ST2 cells, a mouse mesenchymal stromal cell line. Nanog overexpression in serially passaged cells mimicked the recuperative effects of LIPUS on osteogenic potency, highlighting the important role of Nanog in LIPUS stimulation. Additionally, we found that spleen tyrosine kinase (Syk) is an important signaling molecule to induce Nanog expression in LIPUS-stimulated cells. Syk activation was regulated by both Rho-associated kinase 1 (ROCK1) and extracellular ATP in a paracrine manner. Interestingly, the LIPUS-induced increase in Nanog mRNA expression was regulated by ATP-P2X4-Syk Y323 activation, while the improvement of Nanog protein stability was controlled by the ROCK1-Syk Y525/526 pathway. Taken together, these results indicate that LIPUS stimulation recovers and maintains the osteogenic potency of serially passaged cells through a Syk-Nanog axis., (Copyright © 2019. Published by Elsevier Inc.)

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., (© 2019 Wiley Periodicals, Inc.)

Osteoblasts are versatile cells involved in multiple whole-body processes, including bone formation and immune response. Secretory amounts and patterns of osteoblast-derived proteins such as osteopontin (OPN) and osteocalcin (OCN) modulate osteoblast function. However, the regulatory mechanism of OPN and OCN expression remains unknown. Here, we demonstrate that p54/p46 c-jun N-terminal kinase (JNK) inhibition suppresses matrix mineralization and OCN expression but increases OPN expression in MC3T3-E1 cells and primary osteoblasts treated with differentiation inducers, including ascorbic acid, bone morphogenic protein-2, or fibroblast growth factor 2. Preinhibition of JNK before the onset of differentiation increased the number of osteoblasts that highly express OPN but not OCN (OPN-OBs), indicating that JNK affects OPN secretory phenotype at the early stage of osteogenic differentiation. Additionally, we identified JNK2 isoform as being critically involved in OPN-OB differentiation. Microarray analysis revealed that OPN-OBs express characteristic transcription factors, cell surface markers, and cytokines, including glycoprotein hormone α2 and endothelial cell-specific molecule 1. Moreover, we found that inhibitor of DNA binding 4 is an important regulator of OPN-OB differentiation and that dual-specificity phosphatase 16, a JNK-specific phosphatase, functions as an endogenous regulator of OPN-OB induction. OPN-OB phenotype was also observed following LPS from Porphyromonas gingivalis stimulation during osteogenic differentiation. Collectively, these results suggest that the JNK-Id4 signaling axis is crucial in the control of OPN and OCN expression during osteoblastic differentiation.-Kusuyama, J., Amir, M. S., Albertson, B. G., Bandow, K., Ohnishi, T., Nakamura, T., Noguchi, K., Shima, K., Semba, I., Matsuguchi, T. JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

Bone marrow stromal cells (BMSCs) are multipotent cells that can differentiate into adipocytes and osteoblasts. Inadequate BMSC differentiation is occasionally implicated in chronic bone metabolic disorders. However, specific signaling pathways directing BMSC differentiation have not been elucidated. Here, we explored the roles of spleen tyrosine kinase (Syk) in BMSC differentiation into adipocytes and osteoblasts. We found that Syk phosphorylation was increased in the early stage, whereas its protein expression was gradually decreased during the adipogenic and osteogenic differentiation of two mouse mesenchymal stromal cell lines, ST2 and 10T(1/2), and a human BMSC line, UE6E-7-16. Syk inactivation with either a pharmacological inhibitor or Syk-specific siRNA suppressed adipogenic differentiation, characterized by decreased lipid droplet appearance and the gene expression of fatty acid protein 4 (Fabp4), peroxisome proliferator-activated receptor γ2 (Pparg2), CCAAT/enhancer binding proteins α (C/EBPα), and C/EBPβ. In contrast, Syk inhibition promoted osteogenic differentiation, represented by increase in matrix mineralization and alkaline phosphatase (ALP) activity, as well as the expression levels of osteocalcin, runt-related transcription factor 2 (Runx2), and distal-less homeobox 5 (Dlx5) mRNAs. We also found that Syk-induced signals are mediated by phospholipase C γ1 (PLCγ1) in osteogenesis and PLCγ2 in adipogenesis. Notably, Syk-activated PLCγ2 signaling was partly modulated through B-cell linker protein (BLNK) in adipogenic differentiation. On the other hand, growth factor receptor-binding protein 2 (Grb2) was involved in Syk-PLCγ1 axis in osteogenic differentiation. Taken together, these results indicate that Syk-PLCγ signaling has a dual role in regulating the initial stage of adipogenic and osteogenic differentiation of BMSCs., (© 2017 Wiley Periodicals, Inc.)

Osteoblasts express Toll like receptor (TLR) 4 and produce osteoclast-activating cytokines in response to the stimulation by lipopolysaccharide (LPS). It has recently been reported that LPS exerts an inhibitory effect on osteoblast differentiation into osteocytes. However, the molecular mechanisms of this inhibitory effect remain ambiguous. The downstream signals of TLR4 are mediated by adaptor molecules including myeloid differentiation factor 88 (MyD88), leading to the activation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases (ERKs), whose activation by LPS requires the upstream serine/threonine kinase, Cot/Tpl2. To determine the signal molecules responsible for the inhibitory effects of LPS on osteoblast differentiation, we examined the in vitro differentiation of the primary osteoblasts from myd88(-/-) and cot/tpl2(-/-) mice. The matrix mineralization by the wild-type and cot/tpl2(-/-) osteoblasts was significantly inhibited by LPS, whereas that of myd88(-/-) was not affected. During differentiation, LPS suppressed the mRNA expression of runt related transcription factor 2 (Runx2), osterix (Sp7), and activating transcription factor 4 (ATF4) in the wild-type, but not in the myd88(-/-) osteoblasts. The inhibitory effect of LPS on the mRNA expression of these transcription factors was absent in the early phase but partially impaired in the late phase of differentiation in the cot/tpl2(-/-) osteoblasts. Thus, the inhibitory effect of LPS on osteoblast differentiation is Myd88-dependent, whereas the degree of its requirement for Cot/Tpl2 varies depending on the differentiation phase., (Copyright © 2010 Elsevier Inc. All rights reserved.)