A Quantitative Modular Modeling Approach Reveals the Effects of Different A20 Feedback Implementations for the NF-kB Signaling Dynamics.

Signaling pathways involve complex molecular interactions and are controled by non-linear regulatory mechanisms. If details of regulatory mechanisms are not fully elucidated, they can be implemented by different, equally reasonable mathematical representations in computational models. The study presented here focusses on NF-κB signaling, which is regulated by negative feedbacks via IκBα and A20. A20 inhibits NF-κB activation indirectly through interference with proteins that transduce the signal from the TNF receptor complex to activate the IκB kinase (IKK) complex. A number of pathway models has been developed implementing the A20 effect in different ways. We here focus on the question how different A20 feedback implementations impact the dynamics of NF-κB. To this end, we develop a modular modeling approach that allows combining previously published A20 modules with a common pathway core module. The resulting models are fitted to a published comprehensive experimental data set and therefore show quantitatively comparable NF-κB dynamics. Based on defined measures for the initial and long-term behavior we analyze the effects of a wide range of changes in the A20 feedback strength, the IκBα feedback strength and the TNFα stimulation strength on NF-κB dynamics. This shows similarities between the models but also model-specific differences. In particular, the A20 feedback strength and the TNFα stimulation strength affect initial and long-term NF-κB concentrations differently in the analyzed models. We validated our model predictions experimentally by varying TNFα concentrations applied to HeLa cells. These time course data indicate that only one of the A20 feedback models appropriately describes the impact of A20 on the NF-κB dynamics in this cell type.

RC3H1 post-transcriptionally regulates A20 mRNA and modulates the activity of the IKK/NF-κB pathway.

The RNA-binding protein RC3H1 (also known as ROQUIN) promotes TNFα mRNA decay via a 3'UTR constitutive decay element (CDE). Here we applied PAR-CLIP to human RC3H1 to identify ∼ 3,800 mRNA targets with >16,000 binding sites. A large number of sites are distinct from the consensus CDE and revealed a structure-sequence motif with U-rich sequences embedded in hairpins. RC3H1 binds preferentially short-lived and DNA damage-induced mRNAs, indicating a role of this RNA-binding protein in the post-transcriptional regulation of the DNA damage response. Intriguingly, RC3H1 affects expression of the NF-κB pathway regulators such as IκBα and A20. RC3H1 uses ROQ and Zn-finger domains to contact a binding site in the A20 3'UTR, demonstrating a not yet recognized mode of RC3H1 binding. Knockdown of RC3H1 resulted in increased A20 protein expression, thereby interfering with IκB kinase and NF-κB activities, demonstrating that RC3H1 can modulate the activity of the IKK/NF-κB pathway.

Sources of dynamic variability in NF-κB signal transduction: a mechanistic model.

The transcription factor NF-κB (p65/p50) plays a central role in the coordination of cellular responses by activating the transcription of numerous target genes. The precise role of the dynamics of NF-κB signalling in regulating gene expression is still an open question. Here, we show that besides external stimulation intracellular parameters can influence the dynamics of NF-κB. By applying mathematical modelling and bifurcation analyses, we show that NF-κB is capable of exhibiting different types of dynamics in response to the same stimulus. We identified the total NF-κB concentration and the IκBα transcription rate constant as two critical parameters that modulate the dynamics and the fold change of NF-κB. Both parameters might vary as a result of cell-to-cell variability. The regulation of the IκBα transcription rate constant, e.g. by co-factors, provides the possibility of regulating the NF-κB dynamics by crosstalk.