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.

Fig. 1

Effect of oscillatory fluid flow shear stress on TCF/LEF activity, β-catenin nuclear localization, and Rho family GTPase activity. (A) TCF/LEF activity under shear stress. Dashed lines indicate cell boundaries obtained from the corresponding DIC images (inlets). Fluorescence intensities of the TCF/LEF reporter were normalized to those at 0 min. n = 7 (10 dyn/cm², black line), and 6 (XAV939 + 10 dyn/cm², gray line). (B) β-catenin nuclear localization under flow. Color bar represents fluorescence intensity of the EGFP-β-catenin probe. Bar graphs represent relative fluorescence intensities in the nucleus. * p < 0.05. n = 7. (C–E) Cells were transfected with the GTPase biosensors and subjected to shear stress. Color bars represent YFP/CFP emission ratios of the biosensors. The time courses of the emission ratios were normalized to time 0. (C) Rac1 activity. n = 14. (D) Cdc42 activity. n = 6. (E) RhoA activity. n = 8. Scale bars = 10 μm.

Fig. 2

Rac1 and Cdc42 are required for shear stress-induced TCF/LEF activity. (A–C) TCF/LEF activity in cells expressing Rac1-N17 (n = 16), Cdc42-N17 (n = 14), or RhoA-N19 (n = 8). Cells were co-transfected with a TCF/LEF reporter and one of Rac1-N17, Cdc42-N17, or RhoA-N19. (D–E) TCF/LEF activity in cells pretreated with Rac1 inhibitor (NSC23766, n = 7) or Cdc42 inhibitor (ML141, n = 12). (F) Basal levels of TCF/LEF activity. Data were normalized and compared to a control group. * p < 0.05, ** p < 0.01, *** p < 0.001. (G–H) Cells were transfected with Rac1 or Cdc42 biosensor and treated with XAV939. During imaging, the cells were subjected to shear stress. (G) Rac1 activity and its basal levels. n = 7. ** p < 0.01 compared to control. (H) Cdc42 activity and its basal levels. n = 7. Scale bars = 10 μm.

Fig. 3

Elevated activation of Rac1 or Cdc42 enhances shear stress-induced TCF/LEF activity. (A–B) TCF/LEF activity in cells expressing Rac1-L61 (n = 9) or Cdc42-L61 (n = 7). Cells were co-transfected with a TCF/LEF reporter and one of Rac1-L61 or Cdc42-L61. (C) TCF/LEF activity in cells pretreated with Rac/Cdc42 activator (n = 9). (D) Basal levels of TCF/LEF activity. Data were normalized and compared to a control group. * p < 0.05, ** p < 0.01.

Fig. 4

Cytoskeletal integrity and its contractile activity do not effectively control shear stress-induced TCF/LEF activity. (A–C) TCF/LEF activity in cells treated with cytochalasin D (CytoD, n = 6), nocodazole (n = 5), or blebbistatin (Bleb, n = 6). (D) Basal levels of TCF/LEF activities in the drug-treated cells.