title: RhoA-mediated signaling in mechanotransduction of osteoblasts.
abstract: Osteoblasts play a pivotal role in load-driven bone formation by activating Wnt signaling through a signal from osteocytes as a mechanosensor. Osteoblasts are also sensitive to mechanical stimulation, but the role of RhoA, a small GTPase involved in the regulation of cytoskeleton adhesion complexes, in mechanotransduction of osteoblasts is not completely understood. Using MC3T3-E1 osteoblast-like cells under 1 hr flow treatment at 10 dyn/cm(2), we examined a hypothesis that RhoA signaling mediates the cellular responses to flow-induced shear stress. To test the hypothesis, we conducted genome-wide pathway analysis and evaluated the role of RhoA in molecular signaling. Activity of RhoA was determined with a RhoA biosensor, which determined the activation state of RhoA based on a fluorescence resonance energy transfer between CFP and YFP fluorophores. A pathway analysis indicated that flow treatment activated phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling as well as a circadian regulatory pathway. Western blot analysis revealed that in response to flow treatment phosphorylation of Akt in PI3K signaling and phosphorylation of p38 and ERK1/2 in MAPK signaling were induced. FRET measurement showed that RhoA was activated by flow treatment, and an inhibitor to a Rho kinase significantly reduced flow-induced phosphorylation of p38, ERK1/2, and Akt as well as flow-driven elevation of the mRNA levels of osteopontin and cyclooxygenase-2. Collectively, the result demonstrates that in response to 1 hr flow treatment to MC3T3-E1 cells at 10 dyn/cm(2), RhoA plays a critical role in activating PI3K and MAPK signaling as well as modulating the circadian regulatory pathway.
authors.affiliation: Center for Craniofacial Molecular Biology, University of Southern California School of Dentistry, Los Angeles, CA, USA.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Hamamura
authors.forename: Gaurav
authors.initials: G
authors.lastname: Swarnkar
authors.forename: Nancy
authors.initials: N
authors.lastname: Tanjung
authors.forename: Eunhye
authors.initials: E
authors.lastname: Cho
authors.forename: Jiliang
authors.initials: J
authors.lastname: Li
authors.forename: Sungsoo
authors.initials: S
authors.lastname: Na
authors.forename: Hiroki
authors.initials: H
authors.lastname: Yokota
mesh_terms: D000577:Amides / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002940:Circadian Rhythm / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D040542:Mechanotransduction, Cellular / Q000187:drug effects; D051379:Mice; D008954:Models, Biological; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D012212:Rheology / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D000577:Amides; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C108830:Y 27632; D019869:Phosphatidylinositol 3-Kinases; D054460:rho-Associated Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020742:rhoA GTP-Binding Protein