Abstract
BackgroundRhoA is a master regulator of cytoskeletal contractility, while nitric oxide (NO) is a master regulator of relaxation, e.g., vasodilation. There are multiple forms of cross-talk between the RhoA/ROCK pathway and the eNOS/NO/cGMP pathway, but previous work has not studied their interplay at a systems level. Literature review suggests that the majority of their cross-talk interactions are antagonistic, which motivates us to ask whether the RhoA and NO pathways exhibit mutual antagonism in vitro, and if so, to seek the theoretical implications of their mutual antagonism.ResultsExperiments found mutual antagonism between RhoA and NO in epithelial cells. Since mutual antagonism is a common motif for bistability, we sought to explore through theoretical simulations whether the RhoA-NO network is capable of bistability. Qualitative modeling showed that there are parameters that can cause bistable switching in the RhoA-NO network, and that the robustness of the bistability would be increased by positive feedback between RhoA and mechanical tension.ConclusionsWe conclude that the RhoA-NO bistability is robust enough in silico to warrant the investment of further experimental testing. Tension-dependent bistability has the potential to create sharp concentration gradients, which could contribute to the localization and self-organization of signaling domains during cytoskeletal remodeling and cell migration.
Highlights
RhoA is a master regulator of cytoskeletal contractility, while nitric oxide (NO) is a master regulator of relaxation, e.g., vasodilation
Our first objective was to confirm that both directions of RhoA-NO mutual antagonism occur in the same cells
For this verification we selected an epithelial cell line: Madin-Darby Canine Kidney (MDCK) cells stimulated with Hepatocyte Growth Factor (HGF)
Summary
Our first objective was to confirm that both directions of RhoA-NO mutual antagonism occur in the same cells. Western blot results (Fig. 1C) showed that treatment with ROCK inhibitor, Y-27632 increased Akt and eNOS phosphorylation This effect was seen both after initial stimulation with HGF and in unstimulated cells. (See figure on previous page.) Fig. 2 Network diagram and simulations of the initial model of HGF-activated RhoA-NO network, including known biochemical effects of mutual antagonism. Computing bifurcation diagrams for the extended model (Fig. 3C and D) showed that bistability was maintained over a wider range (five-fold increase) of the antagonism parameters. The bistability was still sensitive to three parameters: Kmcgmp (the rate constant for cGMP in the Hill equation), k12 and kdeg (the rate constants for synthesis and degradation of ROCK) These three parameters belong to the “RhoA-side” of the model, suggesting that the effects of tension on RhoA, and generally the feedback loop between RhoA and tension, would be important topics for future study, as they have disproportionate impact on the feasibility of bistability. Because our system retained bistabliity for 30% perturbation in 23 out of 36 parameters, we conclude that bistability is credible enough to begin experimental testing
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