title: Low intensity pulsed ultrasound (LIPUS) influences the multilineage differentiation of mesenchymal stem and progenitor cell lines through ROCK-Cot/Tpl2-MEK-ERK signaling pathway.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Dentistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan.
authors.forename: Joji
authors.initials: J
authors.lastname: Kusuyama
authors.affiliation: Department of Biochemistry and Molecular Dentistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan.
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Bandow
authors.affiliation: Department of Biochemistry and Molecular Dentistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Shamoto
authors.affiliation: Department of Biochemistry and Molecular Dentistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Kakimoto
authors.affiliation: Department of Biochemistry and Molecular Dentistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan.
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Ohnishi
authors.affiliation: Department of Biochemistry and Molecular Dentistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. Electronic address: tmatsugu@dent.kagoshima-u.ac.jp.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Matsuguchi
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology; D000818:Animals; D000880:Anthraquinones; D001391:Azo Compounds; D002454:Cell Differentiation; D019070:Cell Lineage; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D017102:Fracture Healing; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D051379:Mice; D010012:Osteogenesis; D010024:Osteoporosis / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D034622:RNA Interference; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology*; D014465:Ultrasonics; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: D000880:Anthraquinones; D001391:Azo Compounds; D011518:Proto-Oncogene Proteins; C004468:Alizarin Red S; D054460:rho-Associated Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases; C069937:MAP3K8 protein, human; C011049:oil red O
keywords: Adipogenesis; Cell Differentiation; LIPUS; MAP Kinases (MAPKs); Mechanotransduction; Mesenchymal Stem Cells; Osteogenesis