Inflammatory NF-kappaB/RelA activation is mediated by the three canonical inhibitors, IkappaBalpha, -beta, and -epsilon. We report here the characterization of a fourth inhibitor, nfkappab2/p100, that forms two distinct inhibitory complexes with RelA, one of which mediates developmental NF-kappaB activation. Our genetic evidence confirms that p100 is required and sufficient as a fourth IkappaB protein for noncanonical NF-kappaB signaling downstream of NIK and IKK1. We develop a mathematical model of the four-IkappaB-containing NF-kappaB signaling module to account for NF-kappaB/RelA:p50 activation in response to inflammatory and developmental stimuli and find signaling crosstalk between them that determines gene-expression programs. Further combined computational and experimental studies reveal that mutant cells with altered balances between canonical and noncanonical IkappaB proteins may exhibit inappropriate inflammatory gene expression in response to developmental signals. Our results have important implications for physiological and pathological scenarios in which inflammatory and developmental signals converge.

TNF-induced NF-B activity shows complex temporal regulation whose different phases lead to distinct gene expression programs. Combining experimental studies and mathematical modeling, we identify two temporal amplification steps-one determined by the obligate negative feedback regulator IB␣-that define the duration of the first phase of NF-B activity. The second phase is defined by A20, whose inducible expression provides for a rheostat function by which other inflammatory stimuli can regulate TNF responses. Our results delineate the nonredundant functions implied by the knockout phenotypes of ib␣ and a20, and identify the latter as a signaling cross-talk mediator controlling inflammatory and developmental responses.[Keywords: NF-B signaling; negative feedback; computational modeling; temporal control; IB␣; A20] Supplemental material is available at http://www.genesdev.org.

NF-κB signaling is known to be critically regulated by the NF-κB–inducible inhibitor protein IκBα. The resulting negative feedback has been shown to produce a propensity for oscillations in NF-κB activity. We report integrated experimental and computational studies that demonstrate that another IκB isoform, IκBɛ, also provides negative feedback on NF-κB activity, but with distinct functional consequences. Upon stimulation, NF-κB–induced transcription of IκBɛ is delayed, relative to that of IκBα, rendering the two negative feedback loops to be in antiphase. As a result, IκBɛ has a role in dampening IκBα-mediated oscillations during long-lasting NF-κB activity. Furthermore, we demonstrate the requirement of both of these distinct negative feedback regulators for the termination of NF-κB activity and NF-κB–mediated gene expression in response to transient stimulation. Our findings extend the capabilities of a computational model of IκB–NF-κB signaling and reveal a novel regulatory module of two antiphase negative feedback loops that allows for the fine-tuning of the dynamics of a mammalian signaling pathway.

The NF-κB protein RelB controls dendritic cell (DC) maturation and may be targeted therapeutically to manipulate T cell responses in disease. Here we report that RelB promoted DC activation not as the expected RelB-p52 effector of the non-canonical NF-κB pathway, but as a RelB-p50 dimer regulated by canonical IκBs, IκBα and IκBε. IκB control of RelB minimized spontaneous maturation but enabled rapid pathogen-responsive maturation. Computational modeling of the NF-κB signaling module identified control points of this unexpected cell-type-specific regulation. Fibroblasts that were engineered accordingly showed DC-like RelB control. Canonical pathway control of RelB regulated pathogen-responsive gene expression programs. This work illustrates the potential utility of systems analyses in guiding the development of combination therapeutics for modulating DC-dependent T cell responses.

Cellular signal transduction pathways are usually studied following administration of an external stimulus. However, disease-associated aberrant activity of the pathway is often due to misregulation of the equilibrium state. The transcription factor NF-jB is typically described as being held inactive in the cytoplasm by binding its inhibitor, IjB, until an external stimulus triggers IjB degradation through an IjB kinase-dependent degradation pathway. Combining genetic, biochemical, and computational tools, we investigate steady-state regulation of the NF-jB signaling module and its impact on stimulus responsiveness. We present newly measured in vivo degradation rate constants for NF-jB-bound and -unbound IjB proteins that are critical for accurate computational predictions of steady-state IjB protein levels and basal NF-jB activity. Simulations reveal a homeostatic NF-jB signaling module in which differential degradation rates of free and bound pools of IjB represent a novel cross-regulation mechanism that imparts functional robustness to the signaling module.

Mammalian signaling networks contain an abundance of negative feedback regulators that may have overlapping (''fail-safe'') or specific functions. Within the NF-B signaling module, I B␣ is known as a negative feedback regulator, but the newly characterized inhibitor I B␦ is also inducibly expressed in response to inflammatory stimuli. To examine I B␦'s roles in inflammatory signaling, we mathematically modeled the 4-I B-containing NF-B signaling module and developed a computational phenotyping methodology of general applicability. We found that I B␦, like I B␣, can provide negative feedback, but each functions stimulusspecifically. Whereas I B␦ attenuates persistent, pathogen-triggered signals mediated by TLRs, the more prominent I B␣ does not. Instead, I B␣, which functions more rapidly, is primarily involved in determining the temporal profile of NF-B signaling in response to cytokines that serve intercellular communication. Indeed, when removing the inducing cytokine stimulus by compound deficiency of the tnf gene, we found that the lethality of i b␣ ؊/؊ mouse was rescued. Finally, we found that I B␦ provides signaling memory owing to its long half-life; it integrates the inflammatory history of the cell to dampen NF-B responsiveness during sequential stimulation events.inflammation ͉ mathematical modeling ͉ NF-kappaB ͉ pathogen

Inflammatory activation of NF-kappaB involves the stimulus-induced degradation of the NF-kappaB-bound inhibitor IkappaB via the IkappaB kinase (IKK). In response to UV irradiation, however, the mechanism and function of NF-kappaB activation remain unclear. Using a combined biochemical, genetic, and computational modeling approach, we delineate a dual requirement for constitutive IKK-dependent and IKK-independent IkappaB degradation pathways in conjunction with UV-induced translational inhibition. Interestingly, we find that the high homeostatic turnover of IkappaB in resting cells renders the NF-kappaB system remarkably resistant to metabolic stresses, but the two degradation pathways critically and differentially tune NF-kappaB responsiveness to UV. Indeed, in the context of low chronic inflammation that accelerates NF-kappaB-bound IkappaB degradation, UV irradiation results in dramatic NF-kappaB activation. Our work suggests that the human health relevance of NF-kappaB activation by UV lies with cellular homeostatic states that are associated with pathology rather than with healthy physiology.

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