Transient IκB Kinase Activity Mediates Temporal NF-κB Dynamics in Response to a Wide Range of Tumor Necrosis Factor-α Doses

Abstract

Dynamic properties of signaling pathways control their behavior and function. We undertook an iterative computational and experimental investigation of the dynamic properties of tumor necrosis factor (TNF)␣-mediated activation of the transcription factor NF-B. Surprisingly, we found that the temporal profile of the NF-B activity is invariant to the TNF␣ dose. We reverse engineered a computational model of the signaling pathway to identify mechanisms that impart this important response characteristic, thus predicting that the IKK activity profile must transiently peak at all TNF␣ doses to generate the observed NF-B dynamics. Experimental confirmation of this prediction emphasizes the importance of mechanisms that rapidly down-regulate IKK following TNF␣ activation. A refined computational model further revealed signaling characteristics that ensure robust TNF␣-mediated cell-cell communication over considerable distances, allowing for fidelity of cellular inflammatory responses in infected tissue.The transcription factor NF-B 3 is a key mediator of physiologic processes such as inflammation and adaptive immunity and has been implicated in numerous pathologic states such as cancer, rheumatoid arthritis, and sepsis (1). Consequently, understanding the mechanisms of NF-B activation and regulation is of prime importance. One major activator of NF-B is the potent inflammatory cytokine TNF␣. TNF␣ binds to and trimerizes its receptor, TNFR1, which leads to a receptorassociated signalosome that activates the kinase IKK (2). IKK phosphorylates IB proteins, which normally sequester NF-B in the cytoplasm; phosphorylated IBs are rapidly polyubiquitinated and proteasomally degraded, releasing free NF-B, which translocates to the nucleus and modulates gene expression (2).Detailed biochemical and genetic analyses over the past 25 years have helped elucidate the components that connect TNF␣ to NF-B. However, relatively little is known about how these molecular players act together as a signaling system, whose complex dynamics control the time-variable activity of NF-B and subsequent gene expression (3-6).Recently, it has become apparent that analysis of the systems properties of complex biochemical pathways can benefit from an integrated approach combining systematic experimental perturbations with an associated computational analysis of molecular interactions (5, 7, 8, 10 -13). This type of analysis applied to TNF␣-induced NF-B activity demonstrated that the ␣, ␤, and ⑀ isoforms of IB cooperate to produce a biphasic NF-B response (5). Varying the duration of the TNF␣ stimulus had no effect on the duration of the initial response, thus ensuring expression of some NF-B-regulated genes even in response to very short stimuli (5). This analysis, however, did not address the question of how other types of signaling inputs are processed.In this study, we analyze in detail a different type of inputs, constant stimulations at different TNF␣ doses, and experimentally and computationally analyze the resulting pathway characteristics. Surp...

Transcriptional regulation via the NF-κB signaling module

“…Subsequent experimental studies have supported these predictions. Although a TNF dose-response study showed that even 0.1 ng/ml, about 100-fold lower than commonly used concentration of this cytokine, can produce strong NF-kB activity (Cheong et al, 2006), persistent or pulse stimulation with TNF produce remarkably different gene expression programs (Hoffmann et al, 2002).…”

“…Typical representatives TNF-R1 and TLR4, which mediate TNF and LPS signaling, respectively, were found to produce remarkably different IKK induction profiles (Werner et al, 2005), which in the case of TNF are independent of the stimulus concentration (Cheong et al, 2006). A current research focus is the elucidation of the mechanisms that produce stimulus-specific IKK activities.…”

Thresholds in transient dynamics of signal transduction pathways

“…This can easily be verified by removing the equation for x 3 from Eqs. (5). The resulting two-dimensional model leads to two monotonously decreasing FTLs.…”

“…While steady state processes are most important in metabolic systems [1], transient dynamics play an important role in cell communication [2,3,4,5,6,7].…”

“…The EGFR stimulated MAPK cascade in the PC12 cell line shows a transient activation of ERK with a peak at 5 min and a return to its basal level after 30 min, which leads to cellular proliferation [4]. Using mathematical modelling Cheong et al [5] have predicted that the dynamic profile of the IKK signal must transiently peak at all TNFα doses in order to generate the observed NFκB activity which they could experimentally validate.…”