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 (1 Hz) 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.

Distinctive Subcellular Inhibition of Cytokine-Induced Src by Salubrinal and Fluid Flow

“…During imaging, a unidirectional flow was applied to the cells grown in the µ-slide cell culture chamber (Ibidi) at 37°C [36], [37]. The chamber was perfused with HEPES-buffered (20 mM), phenol red-free DMEM without serum to maintain the pH at 7.4.…”

β-catenin as a regulator and therapeutic target for asthmatic airway remodeling

“…Additionally, FAK can activate JNK via Grb2 and Rac, which also activates b-catenin signaling [68]. A role for Rac1 in shear stress induced b-catenin activation has also been demonstrated in osteoblasts [69]. In the adherens junction, compressive stress can activate b-catenin signaling [70] and the activated b-catenin can team up with other mechanosensitive transcriptional regulators, such as TAZ, which play crucial roles in sensing stiffness of the extracellular environment [71].…”

Rac[e] to the pole

“…5 Mechanical loading of bone influences bone growth and is essential for maintenance, and Rac1 mediates force-dependent shear stress signaling in osteoblasts. 6 Rac1 is also activated in endothelial cells (ECs) as a result of fluid shear stress produced by blood flow, [7][8][9] and flow-induced Rac1 activation in ECs is critical for numerous vascular responses. The mechanisms and consequences of flow-mediated Rac1 activation in the endothelium will be the focus of this commentary.…”