Mesenchymal stem cells (MSCs) are pluripotent cells that can differentiate into multilineage cell types, including adipocytes and osteoblasts. Mechanical stimulus is one of the crucial factors in regulating MSC differentiation. However, it remains unknown how mechanical stimulus affects the balance between adipogenesis and osteogenesis. Low intensity pulsed ultrasound (LIPUS) therapy is a clinical application of mechanical stimulus and facilitates bone fracture healing. Here, we applied LIPUS to adipogenic progenitor cell and MSC lines to analyze how multilineage cell differentiation was affected. We found that LIPUS suppressed adipogenic differentiation of both cell types, represented by impaired lipid droplet appearance and decreased gene expression of peroxisome proliferator-activated receptor γ2 (Pparg2) and fatty acid-binding protein 4 (Fabp4). LIPUS also down-regulated the phosphorylation level of peroxisome proliferator-activated receptor γ2 protein, inhibiting its transcriptional activity. In contrast, LIPUS promoted osteogenic differentiation of the MSC line, characterized by increased cell calcification as well as inductions of runt-related transcription factor 2 (Runx2) and Osteocalcin mRNAs. LIPUS induced phosphorylation of cancer Osaka thyroid oncogene/tumor progression locus 2 (Cot/Tpl2) kinase, which was essential for the phosphorylation of mitogen-activated kinase kinase 1 (MEK1) and p44/p42 extracellular signal-regulated kinases (ERKs). Notably, effects of LIPUS on both adipogenesis and osteogenesis were prevented by a Cot/Tpl2-specific inhibitor. Furthermore, effects of LIPUS on MSC differentiation as well as Cot/Tpl2 phosphorylation were attenuated by the inhibition of Rho-associated kinase. Taken together, these results indicate that mechanical stimulus with LIPUS suppresses adipogenesis and promotes osteogenesis of MSCs through Rho-associated kinase-Cot/Tpl2-MEK-ERK signaling pathway.

Chemokines are a family of cytokines inducing cell migration and inflammation. Recent reports have implicated the roles of chemokines in cell differentiation. However, little is known about the functional roles of chemokines in adipocytes. Here, we explored gene expression levels of chemokines and chemokine receptors during adipogenic differentiation. We have found that two chemokines, chemokine (C-X-C motif) ligand 3 (CXCL3) and CXCL13, as well as CXC chemokine receptor 2 (CXCR2), a CXCL3 receptor, are highly expressed in mature adipocytes. When 3T3-L1 cells and ST2 cells were induced to differentiate, both the number of lipid droplets and the expression levels of adipogenic markers were significantly promoted by the addition of CXCL3, but not CXCL13. Conversely, gene knockdown of either CXCL3 or CXCR2 by specific siRNA effectively inhibited the course of adipogenic differentiation. CXCL3 treatment of 3T3-L1 cells significantly induced the phosphorylation of ERK and JNK. Furthermore, CXCL3-induced C/EBPβ and δ expression was suppressed by both ERK and JNK-specific inhibitors. Furthermore, Chromatin immunoprecipitation assay revealed functional binding of PPARγ2 within cxcl3 promoter region. Taken together, these results have indicated that CXCL3 is a novel adipokine which facilitates adipogenesis in an autocrine and/or a paracrine manner through induction of c/ebpb and c/ebpd.

Osteopontin (OPN) is an osteogenic marker protein. Osteoblast functions are affected by inflammatory cytokines and pathological conditions. OPN is highly expressed in bone lesions such as those in rheumatoid arthritis. However, local regulatory effects of OPN on osteoblasts remain ambiguous. Here we examined how OPN influences osteoblast responses to mechanical stress and growth factors. Expression of NO synthase 1 (Nos1) and Nos2 was increased by low-intensity pulsed ultrasound (LIPUS) in MC3T3-E1 cells and primary osteoblasts. The increase of Nos1/2 expression was abrogated by both exogenous OPN overexpression and recombinant OPN treatment, whereas it was promoted by OPN-specific siRNA and OPN antibody. Moreover, LIPUS-induced phosphorylation of focal adhesion kinase (FAK), a crucial regulator of mechanoresponses, was down-regulated by OPN treatments. OPN also attenuated hepatocyte growth factor–induced vitamin D receptor (Vdr) expression and platelet-derived growth factor–induced cell mobility through the repression of FAK activity. Of note, the expression of low–molecular weight protein tyrosine phosphatase (LMW-PTP), a FAK phosphatase, was increased in both OPN-treated and differentiated osteoblasts. CD44 was a specific OPN receptor for LWW-PTP induction. Consistently, the suppressive influence of OPN on osteoblast responsiveness was abrogated by LMW-PTP knockdown. Taken together, these results reveal novel functions of OPN in osteoblast physiology.

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