Spontaneous NF-κB Activation by Autocrine TNFα Signaling: A Computational Analysis

et al. (2013) Spontaneous NF-kB Activation by Autocrine TNFa Signaling: A Computational Analysis. PLoS ONE 8(11): e78887. doi:10.1371/journal.pone.0078887 Spontaneous NF-kB Activation by Autocrine TNFa Signaling: A Computational Analysis Jakub Pe kalski 0 Pawel J. Zuk 0 Marek Kochan czyk 0 Michael Junkin 0 Ryan Kellogg 0 Savas Tay 0 Tomasz Lipniacki 0 Jordi Garcia-Ojalvo, Universitat Politecnica de Catalunya, Spain 0 1 Institute of Fundamental Technological Research, Polish Academy of Sciences , Warsaw , Poland , 2 Institute of Physical Chemistry, Polish Academy of Sciences , Warsaw , Poland , 3 Institute of Theoretical Physics, Faculty of Physics, University of Warsaw , Warsaw , Poland , 4 Department of Biosystems Science and Engineering, ETH Zurich , Zurich , Switzerland , 5 Department of Statistics, Rice University , Houston, Texas , United States of America NF-kB is a key transcription factor that regulates innate immune response. Its activity is tightly controlled by numerous feedback loops, including two negative loops mediated by NF-kB inducible inhibitors, IkBa and A20, which assure oscillatory responses, and by positive feedback loops arising due to the paracrine and autocrine regulation via TNFa, IL-1 and other cytokines. We study the NF-kB system of interlinked negative and positive feedback loops, combining bifurcation analysis of the deterministic approximation with stochastic numerical modeling. Positive feedback assures the existence of limit cycle oscillations in unstimulated wild-type cells and introduces bistability in A20-deficient cells. We demonstrated that cells of significant autocrine potential, i.e., cells characterized by high secretion of TNFa and its receptor TNFR1, may exhibit sustained cytoplasmic-nuclear NF-kB oscillations which start spontaneously due to stochastic fluctuations. In A20-deficient cells even a small TNFa expression rate qualitatively influences system kinetics, leading to long-lasting NF-kB activation in response to a short-pulsed TNFa stimulation. As a consequence, cells with impaired A20 expression or increased TNFa secretion rate are expected to have elevated NF-kB activity even in the absence of stimulation. This may lead to chronic inflammation and promote cancer due to the persistent activation of antiapoptotic genes induced by NF-kB. There is growing evidence that A20 mutations correlate with several types of lymphomas and elevated TNFa secretion is characteristic of many cancers. Interestingly, A20 loss or dysfunction also leaves the organism vulnerable to septic shock and massive apoptosis triggered by the uncontrolled TNFa secretion, which at high levels overcomes the antiapoptotic action of NF-kB. It is thus tempting to speculate that some cancers of deregulated NF-kB signaling may be prone to the pathogeninduced apoptosis. - Funding: Foundation for Polish Science grant # Team 2009-3/6; Polish National Science Centre grant # 2011/03/B/NZ2/00281; Swiss National Science Foundation grant # 205321_141299. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. NF-kB Regulatory System Innate immunity forms the first line of defense against pathogens. In the first phase, cells detect pathogens with their membrane and cytoplasmic receptors. This leads to the activation of transcription factors from the NF-kB, IRF and AP-1 families. These factors jointly regulate the activity of several hundred genes responsible for inflammation, antiviral protection, proliferation and apoptosis. In particular, they induce the production of proinflammatory cytokines like IL-1, TNFa, as well as IFN-a and IFN-. Secretion of these cytokines leads to the second phase of the cellular innate immune response in cells that have not yet encountered the pathogen. The cytokine-activated cells may themselves produce and secrete the same cytokines leading to the spread of paracrine signaling [1,2] or to augmenting and stabilizing signaling in the secreting cells via autocrine regulation [3,4]. In the current study, the focus is on the analysis of TNFa autocrine regulation in the NF-kB pathway. NF-kB regulates numerous genes important for pathogen- or cytokine-induced inflammation, immune response, cell proliferation and survival (reviewed in [5,6]). Nuclear activity of NF-kB is tightly controlled by negative feedback loops mediated by NF-kBresponsive proteins: IkBa [79], IkBE [8,10,11] and A20 [1214]. These negative feedback loops lead to oscillatory responses, in which NF-kB circulates between the cytoplasm and nucleus with the period of about 100 min [8]. The primary inhibitors, IkBa and IkB, directly bind to NF-kB, inhibit its transcriptional activity and transport it back to the cytoplasm. Interestingly, expression of IkBE is delayed with respect to IkBa [11], which increases desynchronization of cells and leads to damping of oscillations observed at the population level, resulting in robust tissue responses [15]. A20 mediates the outer negative feedback loop by attenuating the catalytic activity of the IKK complex (consisting of IKKc, also called NEMO, IKKa and IKK). In A20-deficient cells the IKK activity remains at a high level preventing the accumulation of inhibitors IkBa and IkBE [14]. This leads, in turn, to the elevated NF-kB transcriptional activity and causes chronic inflammation. There are at least two levels of A20-mediated regulation of IKK complex activity: (1) A20 directly interacts with the IKK complex reducing its catalytic activity [1618] and (2) A20 primes TNF receptor interacting protein (RIP) for degradation, and thus attenuates TNF receptor downstream signaling [19]. Regarding the direct regulation mode, A20 binds to IKKc and speeds up further phosphorylation of active IKK kinase into the inactive form [16,20]. (IKK activation proceeds via phosphorylation at Ser-177 and Ser-181, but further phosphorylation at the C-terminal serine cluster inhibits its catalytic activity [20].) Later, it was found that A20 and ABIN-1 bind to the IKK complex, and A20 inhibits activation of NF-kB by de-ubiquitination of IKKc [17], reviewed recently in [21]. (Lys-63-linked ubiquitination of IKKc is an important step for the activation of IKK and NF-kB following various stimuli, including TNFa [22].) Interestingly, A20 itself is a putative substrate of IKK, which phosphorylates A20 on Ser-381, thereby increasing its ability to downregulate NF-kB in response to multiple stimuli [23]. Recently, Skaug et al. reported a direct non-catalytic mechanism of IKK inhibition by A20 showing that overexpressed A20 impaired IKK activation without reducing RIP1 ubiquitination [18]. Regarding the indirect IKK regulation mode, A20 acts as a ubiquitin editing protein: it removes Lys-63-linked ubiquitin chains from RIP and then functions as a ubiquitin ligase by polyubiquitinating RIP with Lys-48-linked (...truncated)