Non-canonical Wnt signaling and N-cadherin related beta-catenin signaling play a role in mechanically induced osteogenic cell fate

Abstract

Background: Understanding how the mechanical microenvironment influences cell fate, and more importantly, by what molecular mechanisms, will enhance not only the knowledge of mesenchymal stem cell biology but also the field of regenerative medicine. Mechanical stimuli, specifically loading induced oscillatory fluid flow, plays a vital role in promoting healthy bone development, homeostasis and morphology. Recent studies suggest that such loading induced fluid flow has the potential to regulate osteogenic differentiation via the upregulation of multiple osteogenic genes; however, the molecular mechanisms involved in the transduction of a physical signal into altered cell fate have yet to be determined.

Methods and principal findings: Using immuno-staining, western blot analysis and luciferase assays, we demonstrate the oscillatory fluid flow regulates beta-catenin nuclear translocation and gene transcription. Additionally, real time RT-PCR analysis suggests that flow induces Wnt5a and Ror2 upregulation, both of which are essential for activating the small GTPase, RhoA, upon flow exposure. Furthermore, although beta-catenin phosphorylation is not altered by flow, its association with N-cadherin is, indicating that flow-induced beta-catenin signaling is initiated by adherens junction signaling.

Conclusion: We propose that the mechanical microenvironment of bone has the potential to regulate osteogenic differentiation by initiating multiple key molecular pathways that are essential for such lineage commitment. Specifically, non-canonical Wnt5a signaling involving Ror2 and RhoA as well as N-cadherin mediated beta-catenin signaling are necessary for mechanically induced osteogenic differentiation.

Figure 1. Oscillatory fluid flow induces β-catenin nuclear translocation and initiation of TCF/LEF associated gene transcription.

(A) Immuno-staining of control and cells exposed to oscillatory fluid flow illustrate that 35 minutes of mechanical stimulation results in an increase in β-catenin localization to the nucleus. (B) To validate β-catenin translocation with flow, nuclear protein was isolated and western blot analysis was used to demonstrate that there is a 3.5±0.9-fold (p<0.05) increase in nuclear β-catenin in mechanically stimulated cells versus control. (C) To determine β-catenin/TCF/LEF transcription, TOPflash transfected mouse L cells were exposed to 35 minutes of oscillatory fluid flow. A dual light luciferase assay was used to determine that mechanical stimulation induced a 2.0±0.4-fold increase (p<0.05) in luciferase activity indicating an increase in β-catenin/TCF/LEF transcription. Scale bar = 100 µm. (Error bars: SEM (n≥4)).

Figure 2. Non-canonical related Wnt proteins are regulated by oscillatory fluid flow.

To determine if Wnt expression was regulated by mechanical stimulation, MSCs were exposed to 15, 30 and 45 minutes of flow and lysed immediately. The canonical Wnt, Wnt3a was not affected by flow exposure. However, non-canonical associated Wnt5a and tyrosine kinase receptor, Ror2, were significantly upregulated with 15 and 30 minutes of flow. Wnt5a expression was increased 1.8±0.1-fold (p<0.01) in cells exposed to 15 minutes of flow versus control cells and was maintained after 30 minutes of flow to be 1.5±0.2-fold (p<0.05). Ror2 expression was increased 5.1±0.7-fold (p<0.01) in cells after 15 minutes of oscillatory fluid flow and 1.4±0.1-fold (p<0.05) after 30 minutes. C3H10T1/2 MSCs did not express canonical Wnt7b or Wnt10b. (Error bars: SEM (n≥10)).

Figure 3. Wnt5a and β-catenin signaling are both necessary for flow induced Runx2 upregulation.

(A) Flow induced Runx2 expression was upregulated 2.2±0.2-fold in scrambled siRNA treated cells exposed to flow verses scrambled siRNA control cells. This fold increase was significantly different (p<0.05) than Wnt5a siRNA treated cells, in which the flow induced Runx2 expression was abrogated. (B) The fold change in Runx2 expression with flow was significantly different between untreated cells and cells with inhibited β-catenin signaling via endostatin treatment (p<0.01). Untreated cells exposed to oscillatory fluid flow had a 2.8±0.5-fold increase in Runx2 expression over control cells; while endostatin treated cells resulted in no difference between flowed and control cells. (C) Western blot analysis demonstrates that there is a significant decrease in β-catenin levels after a 24 hour incubation with endostatin. (Error bars: SEM (n≥6)).

Figure 4. Wnt5a and Ror2 are necessary for flow-induced RhoA activation, but do not effect β-catenin translocation.

(A) Western blots of nuclear β-catenin indicate that both scrambled and Wnt5a siRNA treated cells maintained their potential to initiate β-catenin signaling with flow. (B) Analysis of the western blots demonstrates that scrambled and Wnt5a siRNA treated cells had a 1.9±0.2-fold and 1.8±0.3-fold increase in nuclear β-catenin with flow, respectively. (C) β-catenin/TCF/LEF transcription of downstream genes was also maintained in both scramble and Wnt5a treated cells with a 1.8±0.4-fold and 2.2±0.3-fold increase in luciferase activity, respectively, indicating that Wnt5a is not necessary for mechanically induced β-catenin signaling. (D) Western blots were used to assay Rho activation in response to flow in scrambled, Wnt5a and Ror2 siRNA treated cells. (E) Analysis of the blots indicates that the 1.7±0.1-fold increase observed in scrambled treated cells is significantly greater than Wnt5a (p<0.01) and Ror2 (p<0.05) siRNA treated cells, both of which lost flow-induced RhoA activation. (Error bars: SEM (n≥4)).

Figure 5. Flow-induced β-catenin signaling may be mediated by cadherin signaling rather than canonical Wnt signaling.

(A) Western blots were used to determine the level of phosphorylated β-catenin in cells exposed to oscillatory fluid flow versus controls. (B) Analysis of the western blot indicates that there is no significant difference between control and experimental cells in the level of phosphorylated β-catenin. (C) A western blot was also used to assay N-cadherin association with β-catenin as a function of mechanical stimulation. (D) Analysis of the western blot demonstrates that there is a significant 30% decrease in β-catenin/N-cadherin association with exposure to 35 minutes of oscillatory fluid flow (p<0.01). (Error bars: SEM (n≥4)).

Figure 6. A schematic diagram of potential signaling mechanisms involved in mechanically stimulated osteogenic differentiation.

Oscillatory fluid flow, a potent mechanical signal within the microenvironment of bone has the potential to regulate non-canonical Wnt5a and β-catenin signaling pathways in MSCs, both of which are essential for fluid flow induced osteogenic lineage commitment via Runx2 upregulation. Furthermore, Wnt5a signals through Ror2 to activate RhoA, a small GTPase that is necessary and sufficient for osteogenic differentiation. Finally, flow induced β-catenin signaling appears to be mediated by alterations in N-cadherin/β-catenin association indicating that adherens junctions may be involved in the transduction of a mechanical signaling into a cell fate decision.