The Self-Limiting Dynamics of TGF-β Signaling In Silico and In Vitro, with Negative Feedback through PPM1A Upregulation

The TGF-β/Smad signaling system decreases its activity through strong negative regulation. Several molecular mechanisms of negative regulation have been published, but the relative impact of each mechanism on the overall system is unknown. In this work, we used computational and experimental methods to assess multiple negative regulatory effects on Smad signaling in HaCaT cells. Previously reported negative regulatory effects were classified by time-scale: degradation of phosphorylated R-Smad and I-Smad-induced receptor degradation were slow-mode effects, and dephosphorylation of R-Smad was a fast-mode effect. We modeled combinations of these effects, but found no combination capable of explaining the observed dynamics of TGF-β/Smad signaling. We then proposed a negative feedback loop with upregulation of the phosphatase PPM1A. The resulting model was able to explain the dynamics of Smad signaling, under both short and long exposures to TGF-β. Consistent with this model, immuno-blots showed PPM1A levels to be significantly increased within 30 min after TGF-β stimulation. Lastly, our model was able to resolve an apparent contradiction in the published literature, concerning the dynamics of phosphorylated R-Smad degradation. We conclude that the dynamics of Smad negative regulation cannot be explained by the negative regulatory effects that had previously been modeled, and we provide evidence for a new negative feedback loop through PPM1A upregulation. This work shows that tight coupling of computational and experiments approaches can yield improved understanding of complex pathways.Author Summary: TGF-β signaling pathway regulates a variety of cellular responses, such as differentiation, migration and apoptosis. Phosphorylated R-Smad, the central signaling protein in this pathway, exhibits self-limiting behaviors: it not only decreases quickly after TGF-β is removed, but it also decreases slowly when TGF-β remains abundant. These two self-limiting behaviors are important to understand clearly because diseases such as cancer and fibrosis might benefit from treatments to decrease Smad signaling. Several negative regulatory effects have been reported previously, and we studied the dynamics of these effects with computational modeling. Analyzing the timing of negative regulation revealed that the three most widely accepted effects were not sufficient to explain the observed declines. After considering and excluding several alternative models, we arrived at a model in which TGF-β upregulated the phosphatase PPM1A. We tested for PPM1A upregulation in cell culture experiments. In addition, our model was able to explain why different durations of TGF-β exposure could cause seemingly opposite results about the importance of Smad degradation.