title: Rac1 and Cdc42 GTPases regulate shear stress-driven β-catenin signaling in osteoblasts.
abstract: Beta-catenin-dependent TCF/LEF (T-cell factor/lymphocyte enhancing factor) is known to be mechanosensitive and an important regulator for promoting bone formation. However, the functional connection between TCF/LEF activity and Rho family GTPases is not well understood in osteoblasts. Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1Hz) shear stress (10 dynes/cm2) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified. Our findings in this study demonstrate that both Rac1 and Cdc42 GTPases are critical regulators in shear stress-driven β-catenin signaling in osteoblasts.
authors.affiliation: Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA.
authors.forename: Qiaoqiao
authors.initials: Q
authors.lastname: Wan
authors.forename: Eunhye
authors.initials: E
authors.lastname: Cho
authors.forename: Hiroki
authors.initials: H
authors.lastname: Yokota
authors.forename: Sungsoo
authors.initials: S
authors.lastname: Na
mesh_terms: D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus; D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D003599:Cytoskeleton / Q000378:metabolism; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D020690:GTPase-Activating Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D040542:Mechanotransduction, Cellular; D051379:Mice; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction; D013314:Stress, Mechanical; D051822:T Cell Transcription Factor 1 / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein
chemical_list: D000634:Aminoquinolines; C115491:Arhgap31 protein, mouse; C495267:CTNNB1 protein, mouse; D020690:GTPase-Activating Proteins; C487513:NSC 23766; D009479:Neuropeptides; D011743:Pyrimidines; C484602:Rac1 protein, mouse; D051822:T Cell Transcription Factor 1; D051176:beta Catenin; D049452:Green Fluorescent Proteins; C526380:RhoA protein, mouse; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein