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

Abstract

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.

Hepatitis B virus X protein amplifies TGF-β promotion on HCC motility through down-regulating PPM1a

“…Upon TGF-β stimulation, PPM1a level is upregulated, suggesting the feedback between TGF-β and PPM1a [29]. Our study here clearly showed that HBx destroyed this feedback.…”

Transcriptional and Post-Transcriptional Regulation of Thrombospondin-1 Expression: A Computational Model

“…TGFβ pathways have been reported to play profound roles in cancer and cardiovascular diseases; in both situations, the anti-angiogenic effect of the downstream target TSP-1 can be harnessed therapeutically [33–35]. Established models of TGFβ signaling are available in the literature and they cover a wide range of biological details including TGFβ receptors, SMADs and phosphatases in different cellular compartments [36–38]. Due to model complexity concerns, the TGFβ signaling pathway in our model is an adapted version of the work by Nicklas and Saiz, where they included receptor binding, trafficking, SMAD activation, shuttling and feedback [39].…”

Cell‐specific responses to the cytokine TGF β are determined by variability in protein levels

“…In the nucleus, phosphatases such as PPM1A revert the phosphorylation of SMAD2/3 and facilitate their export to the cytoplasm (Lin et al, 2006). Finally, transcriptional feedbacks acting at multiple levels including receptor deactivation (Valdimarsdottir et al, 2006;Wegner et al, 2012) or SMAD dephosphorylation (Wang et al, 2014a) contribute to signal termination.…”