Mesenchymal stem cells (MSCs) are pluripotent cells that can differentiate into multi-lineage 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 multi-lineage cell differentiation was affected. We found that LIPUS suppressed adipogenic differentiation of both cell types, represented by impaired lipid droplet appearance and decreased gene expressions of peroxisome proliferator activated receptor gamma2 (PPARγ2) and fatty acid binding protein 4 (Fabp4). LIPUS also down-regulated the phosphorylation level of PPARγ2 protein, inhibiting its transcriptional activity. In contrast, LIPUS promoted osteogenic differentiation of 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 protein 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 (ROCK). Taken together, these results indicate that mechanical stimuli with LIPUS suppress adipogenesis and promote osteogenesis of MSCs through ROCK-Cot/Tpl2-MEK-ERK signaling pathway.
KEYWORDS:
Adipogenesis, Cell differentiation, LIPUS, MAP kinases (MAPKs), Mechanotransduction, Mesenchymal stem cells, Osteogenesis

PMID: 24550383

Low-Intensity Pulsed Ultrasound (LIPUS) Influences the Multi-Lineage Differentiation of Mesenchymal Stem and Progenitor Cell Lines through ROCK-Cot/Tpl2-MEK-ERK Signaling Pathway.