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I
B
provides negative feedback to control NF-B oscillations, signaling dynamics, and inflammatory gene expression
Correspondence to Alexander Hoffmann: ahoffmann{at}ucsd.edu
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NF-
B signaling is known to be critically regulated by the NF-B–inducible inhibitor protein IB
. The resulting negative feedback has been shown to produce a propensity for oscillations in NF-B activity. We report integrated experimental and computational studies that demonstrate that another IB isoform, IB
, also provides negative feedback on NF-B activity, but with distinct functional consequences. Upon stimulation, NF-B–induced transcription of IB is delayed, relative to that of IB, rendering the two negative feedback loops to be in antiphase. As a result, IB has a role in dampening IB-mediated oscillations during long-lasting NF-B activity. Furthermore, we demonstrate the requirement of both of these distinct negative feedback regulators for the termination of NF-B activity and NF-B–mediated gene expression in response to transient stimulation. Our findings extend the capabilities of a computational model of IB–NF-B 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.
B kinase; MEF, murine embryonic fibroblast; RPA, RNase protection assay.
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Introduction |
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TopAbstractIntroductionResults and discussionMaterials and methodsReferences |
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B family of transcription factors controls diverse mammalian signaling responses that mediate cell survival, inflammation, and immune response (Gerondakis et al., 1999; Li and Verma, 2002; Hoffmann and Baltimore, 2006). Functional NF-B exists in a dimeric form that is composed of combinations of five proteins containing a Rel homology region, i.e., cRel, RelA, RelB, p50, and p52 (Ghosh et al., 1998; Hoffmann and Baltimore, 2006). In resting cells, the NF-B dimer is bound to inhibitor IB proteins, i.e., IB, -ß, and -, which inhibit NF-B DNA-binding activity and prevent its nuclear accumulation. Activation of the NF-B signaling pathway relies upon signal-dependent phosphorylation and degradation of the IB proteins that result in subsequent nuclear translocation of the NF-B dimer (Ghosh et al., 1998).
Termination of NF-B activity after cellular stimulation is critical, as deregulated inflammatory gene expression can be detrimental to the health of the organism, and several attenuation mechanisms have been described (Greten and Karin, 2004; Li et al., 2005). Importantly, IB, which is a target gene of NF-B, is induced by numerous NF-B–inducing stimuli, resulting in the termination of NF-B DNA-binding activity and nuclear localization (Scott et al., 1993; Ghosh et al., 1998).
Temporal control of NF-B activity has been shown to mediate stimulus-specific gene expression programs in response to different inflammatory stimuli (Werner et al., 2005), and understanding the dynamic regulation of NF-B by IB proteins is of critical importance. Negative feedback that is mediated by IB was shown to confer the propensity for oscillatory NF-B nuclear activity, both when examined biochemically in gene knockout cells containing only the IB isoform (Fig. 1 B; Hoffmann et al., 2002) and when examined by microscopy using transiently transfected IB and RelA proteins fused to fluorescent moieties (Fig. 1 D
; Nelson et al., 2004). The oscillations are not apparent in cells containing all three IB proteins at normal expression levels (Fig. 1, A and C; Hoffmann et al., 2002; Barken et al., 2005).
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Bß and/or - proteins play a role in dampening IB-mediated oscillations and determining the dynamics of NF-B activity. The mechanism that confers dampening of oscillations in NF-B activity was proposed to involve the nuclear accumulation of newly synthesized IBß that binds nuclear and promoter-bound NF-B and shields it from IB-mediated nuclear export (Suyang et al., 1996; Phillips and Ghosh, 1997). This mechanism was included in our computational model that recapitulates NF-B activation in response to TNF stimulation (Hoffmann et al., 2002), but our later studies were unable to observe the IBß effect in murine embryonic fibroblasts (MEFs), which are the cells for which the model was constructed (unpublished data). Removal of the mathematical term for this mechanism from the model resulted in highly oscillatory NF-B responses (Fig. 1, E and F). Although genetic evidence points to important roles for IBß and - in regulating the dynamics of NF-B signaling, the mechanisms by which they function remained unclear.
In this study, we investigated the dynamic behavior of all three canonical IB isoforms, especially by contrasting IB and -ß functions with that of IB. Our studies revealed that IB expression is, in fact, highly NF-B inducible, and that it mediates functional negative feedback on NF-B activity; however, it does so in antiphase to that of IB. Two antiphase negative feedbacks emerge as an important regulatory module that may be present for the dynamic control of signaling in other pathways as well.
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Results and discussion |
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TopAbstractIntroductionResults and discussionMaterials and methodsReferences |
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B is TNF inducible via NF-BB mRNAs to characterize the regulation of IB and -ß gene expression in response to stimulation. Analysis of the mRNA levels in MEFs that were stimulated with TNF confirmed that IB was strongly induced (Fig. 2 A
). IBß showed only weak induction, which suggests that it is not a strong NF-B–responsive gene in this cell type. Remarkably, IB transcription was highly induced by TNF stimulation, and quantitation of these results showed that IB was induced to a higher degree than IB (Fig. 2 B). Although the exact induction folds varied in replicate assays using separate MEF cell stocks (unpublished data), IB fold induction was consistently higher than that for IB. Furthermore, IB expression was also induced in response to LPS in MEFs, the macrophage cell line RAW264.7, and the B cell line 70Z (unpublished data), indicating that the dynamic control of its synthesis is not specific to TNF or to fibroblasts. Analysis of IB mRNA levels in MEF cells deficient in NF-B showed no induction of any of the three IBs (Fig. 2 A).
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B transcript induction during chronic TNF stimulation is well described (Scott et al., 1993). It shows rapid activation as early as 15 min, a peak within 1 h, and a slow attenuation over many hours. We observed a similar activation profile for IB induction, but were surprised to note a distinct 45-min onset delay (Fig. 2, A and B). This suggests that the IB promoter may involve a delay mechanism, such as a requirement for the activation of an NF-B–responsive transcription factor (a feed-forward regulation). However, the inhibition of protein synthesis by cycloheximide did not attenuate transcriptional activation of either IB or - (Fig. 2 D and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200510155/DC1) and, thus, does not support the notion of feed-forward regulation.
It has been previously shown that transient TNF stimulation leads to transient nuclear NF-B activity lasting only 
60 min (Hoffmann et al., 2002). To determine whether short stimulation would efficiently induce IB expression, an RPA was performed on MEF cells that were stimulated for 15 min with TNF. The results show that IB transcription is still activated with an onset delay and with an induction profile similar to that of chronically stimulated cells (Fig. 2 C and Fig. S1). Because NF-B activity is diminished when IB mRNA levels are still rising, and because cycloheximide treatment precludes a feed-forward mechanism, we suggest that the time delay in the activation of IB transcription occurs after NF-B recruitment to the promoter. Further study is required to elucidate the mechanism of this delay.
Computational modeling reveals dynamic control mechanisms
NF-B–responsive syntheses of IB and -ß were added to the mathematical model to explore the functional consequences imparted by these negative feedback regulators upon NF-B activity. To determine the kinetic parameter values for the inducible transcription of each isoform, the temporal responses of IB transcription in the model were constructed such that the mRNA induction profiles calculated by the model correlated with our RPA data (Fig. 3 A
). This required the fitting of parameters defining transcription and translation rates and mRNA stability. The new model also includes revised IB protein degradation parameters from earlier studies (unpublished data).
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B protein degradation immediately after IB kinase (IKK) activation and rapid synthesis in response to NF-B nuclear localization (Fig. 3, B and C). Chronic stimulation results in repeated IB protein degradation and synthesis (Fig. 3 B). In addition, the model shows delayed induction of IB and -ß protein syntheses. The low inducibility of IBß transcription results in very low IBß protein synthesis, whereas the high inducibility of IB transcription results in notable accumulation of IB protein (Fig. 3 B).
Earlier studies revealed oscillatory NF-B activity in cells lacking IBß and - (Hoffmann et al., 2002) and in cells in which NF-B–inducible IB was overexpressed (Nelson et al., 2004), whereas in wild-type cells late NF-B activity (beyond 2 h) was remarkably steady (Hoffmann et al., 2002). However, the underlying dampening mechanism that results in steadied late activity remained obscure. Because induced synthesis of IB is delayed, we reasoned that IB may mediate an antiphase negative feedback that provides effective dampening of IB-mediated oscillations. Indeed, our simulations of signaling modules lacking IB revealed oscillations in nuclear NF-B that persist with a higher amplitude than those that contain IB and represent wild-type cells (Fig. 3 D). In contrast, simulations of cells lacking IBß do not show such aberrant oscillations, whereas systems lacking both IB and -ß do.
We set out to examine these predictions experimentally, using nuclear extracts prepared from TNF-treated MEFs harboring genetic deficiencies for IB and/or -ß. We measured NF-B DNA-binding activity by electrophoretic mobility shift assay (EMSA; Fig. 3 E) and nuclear localization of the NF-B protein RelA by Western blot (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200510155/DC1). In both assays, all four cell types exhibited fast induction of nuclear NF-B in response to TNF stimulation by 15 and 30 min, a transient trough at 60–75 min, and subsequent recovery at 90–120 min. However, a second trough at 135–150 min was most pronounced in ib–/– and ibß–/––/– cells, as was a third trough at 225 min. These studies suggest that negative feedback, provided by IB in antiphase to that of IB, is the primary mechanism that dampens the propensity for oscillations in NF-B activity.
The regulatory motif consisting of two antiphase negative feedback systems may be present in other signaling pathways to control the dynamics of signal transduction, and variations in the relative strength of the two systems may provide for altered response dynamics to the same stimulus. A similar model has recently been proposed for two NF-B–inducing signaling pathways emanating from the TLR4 receptor (Covert et al., 2005). In contrast to the two interacting negative feedback mechanisms, this model depicts the coupling of two positive oscillatory signals in an antiphase relationship that produces stable NF-B activity in response to LPS stimulation in wild-type cells. As the temporal control of NF-B activity determines NF-B–responsive gene expression (Hoffmann et al., 2002; Nelson et al., 2004; Werner et al., 2005), the interaction of antiphase regulation by IB and - may contribute to the regulation of stimulus-specific and cell type–specific gene expression programs by modulating the dynamics of this transcription factor.
IB mediates postinduction repression of NF-B activity and inflammatory gene expression
We used the computational model to identify conditions in which NF-B–responsive IB expression would mediate negative feedback on stimulus-induced NF-B activity and found that the most significant role for IB was in systems with reduced IB. To model such conditions, we used computational simulations to study the temporal profile of nuclear NF-B in response to 15-min stimulation in systems lacking IB, -, or both (Fig. 4 A
). Systems containing all three IBs show rapid nuclear localization of NF-B followed by removal from the nucleus within 1 h, as previously shown (Hoffmann et al., 2002). However, in systems lacking IB, we predicted effective down-regulation of NF-B activity in the third hour and beyond. In this context, IB deficiency results in prolonged NF-B activity, whereas in systems containing high IB expression it does not have an effect.
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B-deficient MEFs as a model for cell types that have reduced IB expression. These MEFs showed NF-B activity to last 3 h in response to 15-min transient TNF stimulation, after which it was dramatically attenuated (Fig. 4 B). In contrast, cells that were deficient in both IB and - showed a pronounced delay in attenuation, with NF-B still present in the nucleus even at 6 h. Wild-type and IB-deficient cells are nearly indistinguishable, and both have strong NF-B accumulation at 30 min and attenuation within 1 h. Collectively, these data strongly suggest that IB is responsible for the removal of NF-B from the nucleus at late time points, allowing for dynamic functional interplay with the faster-acting feedback of IB.
Temporal control of NF-B localization by IB was shown to control NF-B–responsive gene expression not only quantitatively (Nelson et al., 2004) but also qualitatively (Hoffmann et al., 2002). To study the effects of IB-negative feedback on NF-B–dependent gene expression, the transcription of five NF-B–responsive genes was monitored by RPA after transient TNF stimulation in wild-type, ib–/–, and ib–/–ib–/– cells. The genes encoding TNF, G-CSF, and LIF are inducibly expressed in fibroblasts upon TNF stimulation, but mRNA levels return to baseline within 3 h in wild-type cells. In IB-deficient cells, these genes are attenuated within 4 h (Fig. 4 C). In this context, the loss of IB-negative feedback results in a further delay in attenuation and quantitative deregulation of TNF, G-CSF, and LIF expression. Interestingly, the loss of both IB- and --negative feedback has a dramatic qualitative effect for GM-CSF and MIP-2. Although these genes are not induced in wild-type or IB-deficient cells, both are strongly responsive to NF-B activation when both IB- and --negative feedbacks are absent. The data presented here demonstrate that IB-dependent negative feedback regulates the termination of NF-B–responsive gene expression in both a quantitative (in the cases of TNF, G-CSF, and LIF) and qualitative (in the cases of GM-CSF and MIP-2) manner.
The functional interplay between the antiphase IB- and --negative feedback responses may explain differences in NF-B–dependent gene expression profiles seen in various cell types. In MEFs, IB-mediated negative feedback appears to be secondary to that provided by IB in response to transient inflammatory stimuli, and it is therefore assumed that IB controls the bulk of the NF-B–responsive gene expression (Ghosh et al., 1998). However, the ratio of the abundance of IB in relation to IB is cell type–specific (Memet et al., 1999; Spiecker et al., 2000; Emmerich et al., 2003; Doerre et al., 2005), suggesting that IB may play a predominant role in NF-B–responsive gene expression in particular cell types. Indeed, in vivo studies have shown that a deficiency of functional IB has physiological consequences (Memet et al., 1999; Spiecker et al., 2000; Emmerich et al., 2003; Doerre et al., 2005) and, thus, emphasize the notion that no IB isoform functions on its own. To understand regulation of NF-B activity in different cell types and in response to diverse stimuli, the interplay of all IB isoforms within the IKK–IB–NF-B signaling module must be considered.
Our studies aimed to quantitatively characterize the temporal expression profiles of the three IB isoforms and to examine their functional consequences on NF-B regulation. Earlier studies showed inducible IB expression (Simeonidis et al., 1997; Whiteside et al., 1997). We have demonstrated that induction of IB is NF-B dependent and functions to attenuate NF-B activity and terminate NF-B–responsive gene expression. Based on these three criteria we conclude that IB mediates bona fide functional negative feedback regulation on NF-B activity. Importantly, our studies reveal that inducible expression of IB is delayed by 45 min with respect to that of IB, thus, creating a two–negative feedback regulatory module that critically controls the dynamics of NF-B activity. We suggest that the relative strength of the two feedback mechanisms and their temporal relationship to each other may account for cell type–specific dynamic regulation of NF-B activity.
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Materials and methods |
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TopAbstractIntroductionResults and discussionMaterials and methodsReferences |
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DNA-binding assays and Western blot
EMSAs were performed as previously described (Hoffmann et al., 2002). Western blots using whole-cell extracts were performed as previously described (Hoffmann et al., 2003). IB and -ß antibodies were obtained from Santa Cruz Biotechnology, Inc. (SC-371 and SC-945, respectively). An antigen-purified polyclonal mouse antiserum raised against recombinant full-length mouse protein was used for IB.
RPA
Total cellular RNA was isolated from confluent and serum-starved cells with Trizol reagent (Invitrogen). Transcript levels were monitored with -[32P]UTP–labeled probes using a RiboQuant kit (BD Biosciences) according to the manufacturer's instructions. Data was obtained using a storage phosphor screen (GE Healthcare) and a variable mode imager (Typhoon 9400; GE Healthcare). Data was quantitated using ImageQuant software version 5.2 (GE Healthcare) by normalization to L32 and/or glyceraldehyde- 3-phosphate dehydrogenase after local background subtraction. IB probes were designed to select for mature mRNA species by spanning exon–exon junctions. The following primer pairs were used to amplify fragments from reverse-transcribed RNA: 5'-TCGCTCTTGTTGAAATGTGG-3' and 5'TGGAGATTTTCCAGGGTCAG-3' (IB); 5'-GCCCTTAGTCTTTGGCTACG-3' and 5'-TCTCAGCCACCAACACTCCT-3' (IBß); and 5'-GGCAGACAGCTTTCTCATCC-3' and 5'-TGAGGTCGCAGTCTTCAATG-3' (IB). G-CSF, LIF, MIP-2, TNF, L32, and glyceraldehyde-3-phosphate dehydrogenase probes were obtained from RiboQuant sets (BD Biosciences).
In silico studies
We previously constructed a computational model to describe NF-B activation events in response to IKK activation by TNF (Hoffmann et al., 2002). This model comprises a singular NF-B species, three IB isoforms (IB, -ß, and -), and IKK. Synthesis and degradation of the IBs and cellular localization and interactions for all components were calculated using a system of ordinary differential equations. The model used in this study includes NF-B–induced IB and -ß transcription and was written in MatLab V7.0 (MathWorks) using previously described methods (Hoffmann et al., 2002). MatLab simulation files are available upon request.
Online supplemental material
Fig. S1 supports Fig. 2 C, with quantitation of IB and - gene induction profiles. Fig. S2 supports Fig. 3 E, with super-shift and oligonucleotide competition EMSAs, as well as nuclear westerns for RelA. Table S1 contains the computational model parameters and reactions.
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Acknowledgments |
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Bß, Andre Levchenko and Gouri Ghosh for discussions and critical reading of the manuscript, and Santa Cruz Biotechnology, Inc. for antibodies. This study was supported by National Institutes of Health grants GM071573 and GM08326.
Submitted: 28 October 2005
Accepted: 28 April 2006
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