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J. Biol. Chem., Vol. 283, Issue 13, 8687-8698, March 28, 2008

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Identification of a Ligand-induced Transient Refractory Period in Nuclear Factor-B Signaling^*

Britney L. Moss¹², Shimon Gross¹, Seth T. Gammon, Anant Vinjamoori, and David Piwnica-Worms³

From the Molecular Imaging Center, Mallinckrodt Institute of Radiology, and the Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, August 16, 2007 , and in revised form, January 8, 2008.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In response to a variety of extracellular ligands, nuclear factor-B (NF-B) signaling regulates inflammation, cell proliferation, and apoptosis. It is likely that cells are not continuously exposed to stimulating ligands in vivo but rather experience transient pulses. To study the temporal regulation of NF-B and its major regulator, inhibitor of NF-B (IB), in real time, we utilized a novel transcriptionally coupled IB-firefly luciferase fusion reporter and characterized the dynamics and responsiveness of IB processing upon a short 30-s pulse of tumor necrosis factor (TNF) or a continuous challenge of TNF following a 30-s preconditioning pulse. Strikingly, a 30-s pulse of TNF robustly activated inhibitor of NF-B kinase (IKK), leading to IB degradation, NF-B nuclear translocation, and strong transcriptional up-regulation of IB. Furthermore, we identified a transient refractory period (lasting up to 120 min) following preconditioning, during which the cells were not able to fully degrade IB upon a second TNF challenge. Kinase assays of IKK activity revealed that regulation of IKK activity correlated in part with this transient refractory period. In contrast, experiments involving sequential exposure to TNF and interleukin-1β indicated that receptor dynamics could not explain this phenomenon. Utilizing a well accepted computational model of NF-B dynamics, we further identified an additional layer of regulation, downstream of IKK, that may govern the temporal capacity of cells to respond to a second proinflammatory insult. Overall, the data suggested that nuclear export of NF-B·IB complexes represented another rate-limiting step that may impact this refractory period, thereby providing an additional regulatory mechanism.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Adequate resolution of an inflammatory reaction is as equally important as initiation. Persistent or fulminant responses can cause detrimental consequences both locally and systemically (1), and resolution of inflammation is important for both termination of an acute response as well as for prevention of destructive chronic responses. It is therefore not surprising that mechanisms aimed at rapid and specific initiation of proinflammatory reactions have co-evolved with mechanisms that provide timely termination of such processes. From a systems biology perspective, such "switchability" can be achieved by intracellular feedback loops that permit ligand-induced desensitization and resensitization of proinflammatory signaling cascades (2).

In this regard, recent studies have shown that nuclear factor-B (NF-B)⁴ signaling plays a critical role in both initiation and resolution of inflammation (2, 3). The transcription factor NF-B is a key regulator of innate and adaptive immune responses as well as a mediator of cell survival and proliferation (4). Improper regulation of NF-B contributes to induction and progression of a wide range of human disorders, including a variety of pathological inflammatory conditions, neurodegenerative diseases as well as many types of cancer (5, 6). In resting cells, inactive NF-B is sequestered in the cytoplasm by binding to members of the inhibitor of NF-B(IB) family. Canonical activation of NF-B depends on IB kinase (IKK)-regulated proteasomal degradation of IB, an event that frees NF-B for nuclear translocation within minutes (4, 7). Upon nuclear transport, NF-B regulates the transcription of a few hundred genes (8-10) that can be divided into four major families (10, 11): 1) proinflammatory genes (e.g. cyclooxygenase 2, interleukin-1 (IL-1), tumor necrosis factor (TNF), inducible nitricoxide synthase, intercellular adhesion molecule-1, E-selectin, etc.), 2) proproliferative genes (e.g. cyclin D, and cellular myelocytomatosis), 3) antiapoptotic genes (B-cell leukemia/lymphoma 2, B-cell leukemia/lymphoma extra long, X-linked inhibitors of apoptosis protein, and cellular inhibitors of apoptosis protein), and 4) autoinhibitory genes (e.g. A20, cylindro-matosis, suppressor of cytokine signaling 1, and IB).

With respect to the last, other transcriptionally independent processes, aimed at autoinhibition of NF-B activity, do exist. Such mechanisms down-regulate NF-B signaling on a much shorter time frame (seconds to minutes). These include homologous receptor desensitization (12, 13), asymmetric heterologous receptor desensitization (13, 14), autocatalytic C-terminal IKK hyperphosphorylation (15), and protein phosphatase 2C-dependent dephosphorylation of IKK (16).

Considering the complex nature of the inflammatory milieu, one would expect that stationary tissue-residing cells are exposed to a myriad of temporally distinct NF-B-stimulating cues. For instance, cells can be directly stimulated by pathogen-derived products (e.g. lipopolysaccharide through TLR4 (toll-like receptor 4) receptors (17)), exposed to numerous soluble proinflammatory stimuli produced by circulating effector cells (e.g. cytokines, chemokines, etc.), and/or experience inflammation-induced oxidative stress (18). These signals can occur simultaneously or sequentially to one another. For example, systemic administration of bacterial lipopolysaccharide to mice was shown to induce transient production of TNF (serum levels peaking at 1.5 h and quickly returning to base line), but IL-1β production was delayed and prolonged (first detected at 2 h, but lasting >5-6 h) (19). Thus, cells co-expressing TLR4, IL-1, and TNF receptors would sequentially interrogate signals arising from lipopolysaccharide, TNF, and IL-1β, each of which could independently activate NF-B.

Central to any signaling desensitization mechanism is a refractory period during which cells cannot fully respond to a second insult (autologous or heterologous desensitization). Therefore, consideration of the dynamic pattern of stimulus exposure described above begs the immediate question of whether cells can instantly initiate an NF-B response to a second activating stimulus, and if not, when will such cells be able to remount a full response again? Specifically, are ligand-preconditioned cells capable of eliciting NF-B activation to the same extent as naive cells?

Little is known about the capacity of cells to activate NF-B in response to a second activating challenge, since the highly dynamic nature of this process presents many technical difficulties. These include low temporal resolution of conventional transcriptionally dependent NF-B reporter gene assays, low throughput, inability to acquire longitudinal data, and the semiquantitative nature of traditional biochemical assays (e.g. electrophoretic mobility shift assay, immunoblotting, etc.). Such limitations render these assays incapable of accurate analysis of the early, ligand-induced dynamic changes in the capacity of cells to elicit a response to a second challenge.

To efficiently address this question, we generated an improved, transcriptionally coupled version of a previously published genetically encoded IB-firefly luciferase (IB-FLuc) fusion reporter (20) in conjunction with dynamic, live cell bioluminescence imaging of cultured cells. We chose to focus on HepG2 human hepatoma cells as a model system, because 1) NF-B signaling has been extensively studied in these cells, 2) HepG2 cells have been shown to activate NF-B in response to a variety of proinflammatory ligands (21), 3) these cells can be easily transfected with readily available reagents, and, most importantly, 4) the pivotal role that NF-B signaling plays in hepatocytes to regulate inflammation, apoptosis, and carcinogenesis (22).

Using bioluminescence imaging of live cells in conjunction with a variety of biochemical assays, we demonstrate herein that a 30-s preconditioning exposure to TNF is sufficient to robustly activate IKK, culminating in IB degradation, NF-B nuclear translocation, and strong transcriptional up-regulation of IB. Furthermore, the capacity of preconditioned cells to degrade IB in response to a second TNF challenge is transiently refractory, regaining full responsiveness 120 min later. Finally, both IKK regulation and possibly NF-B nuclear export, but not receptor dynamics, govern this transient refractory period. This study highlights the interlocking layers of NF-B regulation, ensuring efficient and timely propagation as well as termination of proinflammatory signals.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—D-Luciferin (potassium salt) was from Biosynth (Naperville, IL). Human TNF and IL-1β were from R&D Systems (Minneapolis, MN). Complete protease inhibitor mixture was from Roche Applied Science (Basel, Switzerland). [-³²P]ATP was from PerkinElmer Life Sciences. Carbenicillin, isopropyl β-D-1-thiogalactopyranoside, ampicillin, kanamycin, glutathione S-transferase (GST), β-glycerolphosphate, NaCl, NaF, Na₃VO₄, KOH, MgCl₂, EDTA, phenylmethylsulfonyl fluoride, Nonidet P-40, Tween 20, Triton X-100, ATP, dithiothreitol, paraformaldehyde, cycloheximide, and HEPES were from Sigma.

Plasmids—pB₅FLuc (Stratagene, La Jolla, CA) contains five repeats of a B motif upstream of a minimal TATA box controlling expression of firefly luciferase. pB₅IB-FLuc was produced by cloning an EcoRI/HpaI (blunt) fragment from pCMVIB-FLuc (20) into the EcoRI and EcoRV (blunt) sites of pB₅FLuc. pB₅FLuc, pCMVIB-FLuc, and pB₅IB-FLuc were propagated in TOP10 electrocompetent Escherichia coli (Invitrogen) and purified using Qiagen HiSpeed Maxi Kits (Qiagen, Valencia, CA). pGST-IBN (encoding for GST fused to the N-terminal fragment of human IB-(1-54)) was a kind gift from Prof. Alexander Hoffmann (University of California San Diego). pGST-IBN was propagated in BL21 codon⁺ E. coli cells (Stratagene).

Cells and Transfections—HepG2 human hepatoma cells were from the American Type Culture Collection (Manassas, VA). Cells were cultured in DMEM supplemented with heat-inactivated fetal bovine serum (10%) and L-glutamine (2 mM). Cell cultures were grown at 37 °C in a humidified atmosphere of 5% CO₂. HepG2 cells (10⁵) were transiently transfected (Fugene 6; Roche Applied Science) with pB₅IB-FLuc (200 ng/well) and plated in black-coated 24-well plates (In Vitro Systems GmbH, Gottingen, Germany). Cells were then allowed to recover for 48 h prior to imaging.

Dynamic Bioluminescence Live Cell Imaging—Prior to imaging, cells were washed with prewarmed phosphate-buffered saline (PBS, pH 7.4) and placed into 900 µl of colorless HEPES-buffered DMEM, supplemented as above and with D-luciferin (150 µg/ml). Cells were allowed to equilibrate for 1 h (37 °C) before proceeding with ligand stimulation and imaging. Four different stimulation regimens were included in this study. 1) For continuous TNF (C), TNF (final concentration 20 ng/ml) or vehicle (colorless DMEM) was added (100 µl) to D-luciferin-containing DMEM, and imaging was performed before and at the indicated time points after the addition of TNF.2) For TNF pulse (30 s, P), cells were pulsed for 30 s with TNF (20 ng/ml) or vehicle, washed with PBS, returned to D-luciferin-containing DMEM, and imaged before and at the indicated time points after the pulse of TNF. 3) For TNF preconditioning (30-s pulse) followed by continuous TNF challenge (P + C), at t₀ cells were pulsed for 30 s with TNF (20 ng/ml) or vehicle, washed with PBS, returned to D-luciferin-containing DMEM (900 µl), and imaged before and at the indicated time points after the pulse of TNF. At t_x, TNF (final concentration 20 ng/ml) or vehicle (colorless DMEM) were again added (100 µl), and imaging was performed before and at the indicated time points after the addition of TNF.4) TNF preconditioning (30-s pulse) followed by continuous IL-1β challenge (P + C) was as in method 3, but continuous challenge was performed with IL-1β (10 ng/ml).

TNF or IL-1β challenge was performed at the following time points: t_x = 0 (no preconditioning) and 30, 60, 120, and 240 min postpreconditioning. Assay plates were imaged using an IVIS100 imaging system (Xenogen Caliper, Alameda, CA). Acquisition parameters were as follows: acquisition time, 60 s; binning, 4; field of view, 10 cm; f/stop, 1; filter, open; image-image interval, 5 min; number of acquisitions, 73 (360 min).

To analyze ligand-induced regulation of de novo reporter resynthesis, cells were pretreated with cycloheximide (100 µg/ml) for 1 h before continuous stimulation with TNF and bioluminescence imaging (as above).

IB Responsiveness Assays—HepG2 cells, transfected with pB₅IB-FLuc (as above) or HeLa cells, stably expressing pCMVIB-FLuc (20), were plated in 4 wells of a 6-well plate (one plate per time point) and grown for 48 h. At t₀, all wells were washed with prewarmed PBS, pulsed for 30 s with TNF (20 ng/ml, 1 ml) or vehicle (PBS), washed again with PBS, returned to regular medium (1 ml), and placed in a 37 °C incubator. This procedure was defined as TNF preconditioning (P). At t_x, two wells were treated (continuously) with TNF (20 ng/ml), and two wells were treated with vehicle only (PBS). This procedure was defined as TNF challenge (C). Following this TNF challenge, cells were returned to the incubator. At t_{x + 25 min} (time of maximal IB degradation (20); see Fig. 2A for schematic timeline), cells were harvested (by scraping) in reporter lysis buffer (Promega, Madison, WI). Cell lysates were normalized for protein content by a BCA protein assay (Promega), aliquoted, and frozen (-80 °C) for in vitro bioluminescence and Western blot analyses (see below). For in vitro bioluminescence assays, lysates (10 µl, in triplicate) were mixed with luciferase assay buffer (190 µl; 25 mM HEPES, 154 mM NaCl, 5.4 mM MgSO₄, 10 mM dithiothreitol, 5 mM ATP, 150 µg/ml D-luciferin, pH 8.0) in a 96-well plate immediately prior to imaging. Assay plates were imaged using the IVIS 100 bioluminescence imager (acquisition time, 10 s; binning, 4; field of view, 10 cm; f/stop, 1; filter, open).

Western Blot Analyses—Whole cell lysates were resolved by 10 or 7.5% SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed for the indicated proteins using standard immunoblotting techniques. Primary antibodies against total human IB, β-actin, and IKK were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phospho-IB (Ser-32/36) was from Cell Signaling Technologies (Danvers, MA). Secondary horseradish peroxidase-labeled anti-mouse and anti-rabbit IgG antibodies were from GE Healthcare Biosciences.

IKK Kinase Assay (IKK-KA)—IKK-KA reactions were carried out as per Werner et al. (23) and quantified in a medium throughput manner as per Hastie et al. (24). Briefly, HepG2 cells were grown in 10 cm tissue culture dishes to confluence. Cells were then washed in PBS (once) and treated with 20 ng/ml TNF using three different treatment regimens: P, C or P + C (see above). To capture the full IKK activity profiles of cells treated with continuous (C) or pulse (P) regimens, cytosolic extracts were prepared at t = 0 (before) or 5, 10, 15, 30, 60, 120, or 240 min post-TNF treatment. To capture maximal IKK activity of P + C-treated cells, cytosolic extracts were prepared 10 min post-TNF challenge (given at 10, 30, 60, 120, and 240 min post-preconditioning). Cells were harvested by removing media, washing in ice-cold PBS + EDTA (1 mM), scraping, and pelleting at 2000 x g. To prepare cytosolic extracts, cell pellets were resuspended in 200 µl of cytosolic extract buffer (10 mM HEPES-KOH, pH 7.9, 250 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 0.2% Tween 20, 2 mM dithiothreitol, 20 mM β-glycerophosphate, 10 mM NaF, and 0.1 mM Na₃VO₄ supplemented with complete protease inhibitor mixture), incubated on ice (2 min), vortexed (1 min), and pelleted at 2000 x g. Supernatants were collected, normalized for protein content by a Bradford assay (Pierce), and stored at -80 °C. To immunoprecipitate IKK complexes, cytoplasmic extracts (100 µl) were incubated with anti-IKK antibody (15 µl, overnight, 4 °C with rotation) and then with Protein G 4FF bead slurry (20 µl, 50% (v/v)). Beads were pelleted at 4600 rpm and washed twice with cytosolic extract buffer (500 µl) and once with Kinase Buffer (500 µl; 20 mM HEPES, pH 7.7, 20 mM β-glycerophosphate, 100 mM NaCl, 0.1 mM Na₃VO₄, 10 mM MgCl₂, 2 mM dithiothreitol supplemented with complete protease inhibitor mixture). For the IKK kinase reaction, beads were incubated for 30 min at 30 °C in Kinase Buffer (20 µl) containing 20 µM ATP, 10 µCi of [³²P]ATP, and 0.5 µg of GST-IB-(1-54). Beads were removed by centrifugation (4600 rpm), and 15 µl of each reaction supernatant was spotted onto a 1-cm² square of P81 phosphocellulose paper (Millipore, Billerica, MA) and immediately immersed in phosphoric acid (75 mM) for 5 min. Phosphoric acid washes were performed two more times, and papers were rinsed in acetone and then allowed to dry. Each paper was transferred to a scintillation vial, and radioactivity was determined on a β counter (Beckman Coulter, Fullerton, CA). Blank and no-lysate controls were subtracted from the experimental samples. Data were represented as fold-initial (untreated controls).

Calculating Ligand-dependent IKK Responsiveness—IKK responsiveness profiles (i.e. the net kinase capacity of IKK in response to a second challenge of TNF, as a function of time after initial 30-s preconditioning) were calculated numerically from IKK-KA data using the following formula,

where PC_{x + 10} is IKK activity of preconditioned plus challenged cells, as recorded 10 min postchallenge. P_{x + 10} is the residual IKK activity of preconditioned but unchallenged cells at this exact time point. C₀ and PC₀ are initial IKK activities of challenged but unpreconditioned and fully preconditioned and challenged cells, respectively. C₁₀ is the maximal IKK activity of challenged but unpreconditioned cells (recorded 10 min postchallenge). Note that although all parameter units in the nominator and denominator are in counts/min, IKK responsiveness is dimensionless, similar to IB responsiveness.

Computational Simulations—To simulate the dynamics of major regulators on the IKK-NF-B axis, we used a well established computational model generated by Hoffmann et al. (25) and refined by Werner et al. (23). Briefly, an experimentally or hypothetically derived IKK activity profile was fed into the program as an input. Embedded in the model were 24 components, 70 reactions, and 70 parameters or rate constants for these reactions. Differential equations were solved numerically using Matlab 7.0 (Mathworks, Natick, MA) with subroutine Ode15. Interpolated and extrapolated (0-360 min at 5-min intervals) IKK activity profiles were calculated (Origin version 7.5, OriginLab, Northampton, MA) from experimental IKK-KA data (see above). To fit the model, initial steady-state IKK activity (i.e. intracellular concentration of active IKK) was set to be 1 nM. To computationally simulate IB dynamics of cells challenged at different times after initial preconditioning, when assuming no upstream IKK or receptor regulation, we used hypothetical IKK activity profiles as inputs, derived from superimposing experimentally acquired IKK activity profiles of 30-s pulsed and continuously treated cells at increasing intervals (30, 60, 120, and 240 min; see Fig. 4, black lines).

Immunofluorescence Microscopy—HepG2 cells were seeded into 35-mm glass bottom culture dishes (MatTek Corp., Ashland, MA) and grown to 40% confluence. Cells were pulsed for 30 s with TNF as above and fixed at the indicated time points (by washing once with PBS, followed by fixation (4% paraformaldehyde for at least 15 min) and permeabilization (ice-cold methanol, 10 min at -20 °C)). Cells were washed in PBS, blocked in 5% normal goat serum in 0.3% Triton X-100, PBS (1 h), and then incubated with anti-p65 antibody (Santa Cruz Biotechnology; 1:200 in 0.3% Triton X-100, PBS at 4 °C, overnight with rocking). Cells were next incubated with Alexa-Fluor 635-conjugated goat anti-rabbit antibody (Invitrogen; 1:200 in 0.3% Triton X-100, PBS, 90 min, at room temperature with rocking). Cells were washed three times with PBS before being mounted with VECTASHIELD Mounting medium (Vector Laboratories; Burlingame, CA). Confocal images were captured using the x40 objective (water immersion) on a Zeiss Axiovert 200 (Zeiss, Thornwood, NY) laser-scanning microscope equipped with the appropriate filter sets and analyzed using a Zeiss LSM Image Browser and Adobe Photoshop CS2.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Real Time Bioluminescence Imaging of pB₅IB-FLuc-expressing Cells Recapitulated IKK-induced Dynamics of Endogenous IB—To monitor ligand-induced IB rapid dynamics as well as physiologic transcriptionally coupled behavior, we modified our previous IB-FLuc fusion reporter (20) to be driven by a synthetic promoter composed of five tandem B response elements (TGGGGACTTTCCGC) followed by a minimal TATA-box. We hypothesized that this reporter would allow quantitative measurements of IKK-induced degradation as well as NF-B-induced resynthesis and post-translational stabilization of IB from intact living cells (Fig. 1A). To validate use of this reporter, HepG2 cells were transiently transfected with a plasmid encoding the reporter and allowed to recover for 2 days before stimulation with a continuous or 30-s pulse of TNF (20 ng/ml) to induce IKK activation. Upon the addition of TNF, a rapid and dramatic decrease in bioluminescence was observed when readouts were normalized to untreated controls (20) under both continuous (C) and 30-s pulse (P) regimens (Fig. 1, B and C). This decrease in normalized bioluminescence, reflecting IKK-induced reporter degradation was followed by a sharp increase in bioluminescence, reflecting NF-B-dependent reporter resynthesis, reaching maximum values at 120 min and then gradually declining toward base line. Note that the rate at which IB levels return to base line is steeper under continuous TNF treatment compared with the 30-s pulse, providing evidence for reactivation of ligand-induced IB degradation during continuous stimulation (23). The magnitude of the initial decrease in bioluminescence was greater in continuously treated cells than in 30-s pulsed cells (70% versus 40% of initial decrease, respectively), indicating that a 30-s pulse of TNF leads to 50% depletion of the IB·NF-B pool compared with continuous TNF exposure (Fig. 1C, 120 min). These data suggested that 1) this reporter construct could report on both IKK-induced IB degradation and successive resynthesis of IB, 2) a 30-s pulse of TNF at a saturating concentration (20 mg/ml) elicited robust IKK activity, culminating in IB degradation and a full IB transcriptional up-regulation, and 3) with the current B₅ synthetic promoter system, there was a nonlinear relationship between IB degradation and NF-B-dependent resynthesis of IB (i.e. saturation of IB resynthesis even at submaximal IB degradation levels).

Strikingly, Western blot analysis revealed that endogenous IB behaved exactly as the reporter under both C and P conditions, recapitulating the degree of degradation, recovery, and return to base line (Fig. 1D). Pretreating pB₅IB-FLuc-expressing HepG2 cells with cycloheximide did not affect degradation of IB-FLuc but abolished signal recovery, indicating that this phase was totally dependent upon transcription and translation of new IB-FLuc (Fig. 1E).

TNF Preconditioning Induces a Transient Refractory Period of IB Processing—Upon a proinflammatory insult in vivo, effector cells (e.g. circulating macrophages) release TNF and other activating cytokines in a temporally and spatially discrete manner. As a consequence, stationary target cells (e.g. epithelial cells, endothelial cells, hepatocytes, etc.) will sense a stochastic rise in the levels of such proinflammatory ligands. In such a dynamic environment, as ligand-secreting cells continuously migrate to sites of inflammation, it is anticipated that over time, target cells will experience multiple pulses of activating ligands.

We therefore aimed to elucidate the effects of such ligand pulses on the capacity of hepatocytes to respond to a subsequent challenge of the same ligand. Having shown that 1) pB₅IB-FLuc provided an accurate readout of IB processing in intact cells and that 2) a 30-s pulse was sufficient to induce robust IKK activity, we next sought to investigate whether a short 30-s preconditioning pulse with TNF had a substantial effect on the capacity of cells to process IB upon a subsequent continuous TNF challenge.

View larger version (29K): [in this window] [in a new window]   FIGURE 1. pB5IB-FLuc, a transcriptionally coupled reporter for monitoring IB dynamics in live cells. A, a schematic representing ligand-induced degradation and transcriptionally coupled resynthesis of the reporter. B, raw bioluminescence images of HepG2 cells transiently expressing pB5IB-FLuc treated with TNF (20 ng/ml) continuously (C) or as a 30-s pulse (P) or with vehicle only (V) and imaged for 360 min. Images show pseudocolor-coded photon flux maps superimposed on black-and-white photographs of the assay plate. C, graphical representation of the changes in photon flux from B as a function of time after TNF addition. Data are plotted as fold-initial, fold-vehicle treated (n = 3 for all points; S.E. 5%; representative of three independent experiments). D, Western blot analysis of endogenous IB from HepG2 cell lysates prepared at the indicated times after a 30-s pulse or continuous treatment with TNF (20 ng/ml). E, pretreatment with cycloheximide (CHX;1h, 100 µg/ml) totally abrogated TNF-induced IB-FLuc resynthesis but had no effect on reporter degradation.

We observed that challenging preconditioned cells with a continuous exposure to TNF near the time that they had achieved maximal degradation from the preconditioning pulse (i.e. 30 min postpreconditioning) resulted in a small amount of additional IB degradation. As the interval between preconditioning and challenge increased, the magnitude of challenge-induced IB degradation also increased. These data suggested that the TNF-NF-B system possessed a built-in refractory period following TNF treatment that prevented cells from fully responding to a second exposure to ligand. To quantify this phenomenon independent of confounding factors that may affect dynamic bioluminescence readouts (e.g. D-luciferin, ATP, O₂, or pH dynamics) and to verify its existence for endogenous IB, we performed a similar experiment, but instead of live cell imaging, we harvested whole cell lysates at t_{x + 25 min} (time of maximal IB degradation after a ligand challenge given at t_x (Fig. 1C); for a schematic timeline, see Fig. 2B). IB-FLuc reporter levels in these lysates were analyzed by bioluminescence imaging (upon the addition of saturating D-luciferin and ATP), and endogenous IB levels were determined by Western blot analysis and semiquantitative densitometric analysis (Fig. 2C). From these data, we were then able to calculate responsiveness levels for both IB and IB-FLuc as a function of time after TNF preconditioning. Responsiveness at each challenge time was calculated by determining the magnitude of IB degradation induced by TNF challenge divided by the magnitude of IB degradation in unpreconditioned cells from the same plate. Specifically, the ratio at t_{x + 25 min} of IB in preconditioned cells challenged with TNF over preconditioned cells challenged with vehicle was divided by the ratio at t_{x + 25 min} of IB in unpreconditioned cells challenged with TNF over unpreconditioned cells challenged with vehicle, the latter ratio representing the maximal possible response. We observed a strong correlation (r = 0.95) between levels of responsiveness for endogenous IB and IB-FLuc (Table 1). Consistent with our earlier observations derived from live cell dynamic bioluminescence imaging experiments (Fig. 2A), we observed that at 30 min postpreconditioning, cells were approximately half as responsive as naive (i.e. unpreconditioned) cells to a TNF challenge and had gained full responsiveness by 120 min. Thus, a transient refractory period seemed to exist from 30 to 120 min post-TNF preconditioning that rendered the cells unable to fully respond (as measured via IB degradation) to a second challenge of TNF, and beyond this period, the cells were able to mount a full response to a second TNF challenge. Notably, similar experiments performed with HeLa cells stably expressing pCMVIB-FLuc (HeLa^IB-FLuc (20)) yielded almost identical results (data not shown), suggesting that 1) the TNF-induced transient refractory period was not limited to hepatocytes and 2) this effect was independent of both NF-B-induced IB transcription and the initial levels of IB-FLuc (substantially higher in HeLa^IB-FLuc (20)).

View this table: [in this window] [in a new window]   TABLE 1 Percentage responsiveness of IB processing Quantification of IB-FLuc and IB responsiveness to a second continuous challenge of TNF at the indicated interval following a 30-s preconditioning pulse of TNF was determined from the bioluminescence imaging and Western blot data shown in Fig. 2C. Responsiveness at each challenge time was calculated by determining the percentage of challenge-specific IB degradation divided by the percentage of IB degradation in unpreconditioned cells from the same plate. The responsiveness of IKK was determined by an IKK kinase assay.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. TNF induced a transient refractory period for IB processing. A, dynamic live cell bioluminescence imaging profiles of IB-FLuc from TNF preconditioning plus challenge experiments. The black arrows denote 30-s preconditioning pulse; red arrows denote the beginning of continuous TNF challenge; black profiles represent cells preconditioned and then challenged at the indicated time points; red profiles represent cells treated at time 0 with continuous TNF (denoting the maximal possible degradation response of IB upon continuous TNF treatment). Data are presented as -fold initial, -fold TNF-untreated. B, schematic representation of the experimental timeline as used in C. Cells were preconditioned with TNF for 30 s and then, at increasing intervals (0-240 min), were continuously challenged with TNF. The arrowheads represent when cells were harvested and lysates were prepared (25 min post-challenge for quantitative bioluminescence imaging and Western blot analysis). C, IB-Fluc and endogenous IB levels, 25 min post-TNF or vehicle challenge, as measured by bioluminescence imaging and Western blot, respectively.

To determine the extent of receptor dynamics in governing the observed loss and regain of IB processing, we took advantage of a discovery, made 20 years ago (14), that IL-1β induces transient down-regulation of TNF receptors but not vice versa (i.e. TNF has no effect on either the affinity or the number of IL-1β surface receptors), as tested in a variety of cell lines and primary cells. Hence, we aimed to determine IB responsiveness to an IL-1β challenge as a function of time after TNF preconditioning in HepG2 cells. Cells expressing pB₅IB-FLuc were treated with a 30-s pulse of TNF (20 ng/ml), followed by a continuous challenge with IL-1β, initiated at increasing P-C intervals (0-240 min). IB processing was analyzed by live cell dynamic bioluminescence imaging (Fig. 3A). Using this experimental setup, we again observed a transient refractory period (from 30 to 120 min post-TNF preconditioning) during which HepG2 cells exhibited decreased IB responsiveness. The magnitude of the ligand-induced degradation increased as the interval to the IL-1β challenge increased, becoming fully responsive again by 120 min (Fig. 3A). These data suggested that even in the absence of ligand-induced receptor desensitization or cross-regulation, the capacity of cells to process IB was compromised within the first 2 h after a short TNF stimulation.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. Impact of receptor dynamics and IKK regulation on IB responsiveness. A, dynamic live cell bioluminescence imaging profiles of IB-FLuc from TNF preconditioning, IL-1β challenge experiments. The black arrows denote the 30-s preconditioning pulse of TNF; blue arrows denote the beginning of continuous IL-1β (10 ng/ml) challenge; black profiles represent cells preconditioned and then challenged with IL-1β at the indicated time points; blue profiles represent cells treated at time 0 with continuous IL-1β (denoting the maximal possible degradation response of IB upon continuous IL-1β treatment). Data are presented as -fold initial. B, IKK kinase activity was measured at the indicated time points after initiation of a continuous (blue curve) or a 30-s pulse (red curve) of TNF (20 ng/ml). Results are presented as background-normalized, -fold initial (untreated) controls. C, Western blot analysis of IKK in cytoplasmic fractions, used as inputs for immunoprecipitation and kinase reactions presented in B.

IKK-KA data were also collected from preconditioned cells, 10 min post-challenge (at the time of maximal IKK activity; see Fig. 3B) at increasing P-C intervals (0-240 min). Using these data together with the IKK activity profiles generated for 30-s pulse and continuous TNF treatment regimens (Fig. 3B), we were able to calculate the net capacity of IKK to phosphorylate IB as a function of time after TNF preconditioning (i.e. IKK responsiveness (Table 1); see "Experimental Procedures" for details on this calculation). Based on this calculation, we noted that the capacity of IKK to respond to a second challenge of TNF was significantly compromised at 30 min post-TNF preconditioning and then gradually increased, reaching 75% responsiveness by 120 min. Up to 240 min, IKK activity did not fully recover to initial levels, consistent with other reports indicating that upon TNF stimulation, IKK activity rapidly and transiently declines due to autocatalytic C-terminal hyperphosphorylation (15) and protein phosphatase 2C-dependent dephosphorylation (16), followed by late NF-B-dependent down-regulation, a process attributed, in part, to A20, an IKK-inhibitory protein (29). Hence, these data suggested that 1) the observed ligand-induced transient refractory period of IB processing (Figs. 2 and 3 and Table 1) correlated only in part with ligand-induced transient down-regulation of IKK activity and that 2) the level to which cells are able to degrade IB was not linear with the capacity of IKK to phosphorylate IB (i.e. full IB responsiveness was observed as soon as 120 min post-TNF preconditioning (Figs. 2 and 3A), a time point where IKK responsiveness was still compromised (Table 1)). These data indicated that either submaximal IKK activity could now fully support ligand-induced IB degradation following the refractory period or that additional ligand-responsive elements existed that converged on IB to induce a full response.

Computational Modeling of NF-B Signaling Suggested an Additional Layer of Regulation, Downstream of IKK, Governing the Observed Refractory Period for IB Processing—The NF-B pathway provides an excellent example of a complex signaling system employing numerous temporally distinct autoregulatory mechanisms and negative feedback loops. IKK enzymatic activity, which is both endogenously and exogenously regulated, controls the degradation of its own substrate (IB), which is later strongly up-regulated in an NF-B-dependent manner (Fig. 1A). Rapid changes in substrate availability, conformation, and subcellular localization imply that alternative mechanisms of regulation might exist other than changes in enzymatic activity. Although a ligand-induced transient refractory period of IB processing could be explained in part by down-regulation of IKK activity, we were intrigued to examine whether an alternative regulatory mechanism, based on substrate (IB) dynamics, might exist to complement or "back up" IKK regulation. Obviously, inhibition of IKK was not a viable option for analyzing downstream regulation, since such inhibition will result in complete loss of responsiveness in the absence or presence of preconditioning. We therefore decided to undertake a computational approach and explore IB dynamics in silico, assuming no down-regulation of IKK activity. We used a well accepted computational model that used experimentally or hypothetically driven IKK activity profiles as inputs and, in return, calculated ligand-induced dynamics of 24 different subpopulations of mediators on the IKK-NF-B axis.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. Computational simulation of IB responsiveness in the absence of upstream receptor or IKK regulation. A, interpolated and extrapolated (0-360 min, at 5-min intervals) IKK activity profiles (right) of cells treated continuously (C, green curve) or by a 30-s pulse (P, blue curve) of TNF (20 ng/ml) were used as inputs to computationally simulate total IB dynamics (right). B-E, left panels, hypothetical IKK activity profiles of preconditioned cells, challenged at the indicated times (denoted by black arrowheads) with a second, continuous dose of TNF were generated by superimposing the continuous TNF-induced IKK profiles at increasing intervals after the 30-s pulse TNF-induced IKK profile. For generating these hypothetical profiles, we assume no preconditioning-induced receptor or IKK regulation. Right panels, the hypothetical IKK profiles were used as inputs into the model to predict IB dynamics. Note that challenge-induced IB degradation (initiated at the red arrowhead) is recovered in a time-dependent manner.

We next generated hypothetical IKK profiles representing IKK activities from preconditioned/challenged cells, assuming no upstream receptor or IKK regulation (i.e. experimentally derived challenge-specific IKK activity was overlaid on top of experimentally derived precondition-specific residual IKK activity). These hypothetical IKK activity profiles (Fig. 4, B-E, left, each generated with a different P-C interval) were used as inputs for computing total IB dynamics (Fig. 4, B-E, right). Surprisingly, the computational model predicted that even in the absence of receptor dynamics or IKK regulation, IB processing would be transiently compromised (compare, for example, the second, challenge-induced degradation phase at 120 or 240 min with the ones at 30 or 60 min). These data suggested that although IKK down-regulation partially correlated with the ligand-induced transient refractory period for IB processing, an additional regulatory mechanism was present down-stream of IKK. Importantly, IB availability per se was not sufficient to explain changes in IB responsiveness, because, as confirmed experimentally and computationally, at 60 min post-preconditioning, the IB concentration had already recovered, whereas degradation potential was still low (compare Fig. 2, A and C, Table 1, and Fig. 4C).

Nuclear Export of IB—NF-B complexes may also control the capacity of cells to process IB. Having demonstrated experimentally the phenomenon of a ligand-induced transient refractory period for IB processing and after dissecting biochemically and computationally the origins of this observation, we next sought to more closely examine the components of the computational model in order to identify candidates, down-stream of IKK, capable of regulating IB responsiveness. While examining the rate constants of a variety of reactions used by the model, we noticed that free versus NF-B-bound IB differed tremendously in their capacity to associate with IKK (1.35 versus 11.1 µM^-1 min^-1, respectively) and to be degraded in an IKK-dependant manner (0.12 versus 0.00006 min^-1, respectively). These differences in IKK association and ligand-induced degradation were experimentally established by Zandi et al. (30).

This led us to put forward the following model (Fig. 5A). 1) free IB and NF-B-bound IB represent "protected" and "unprotected" populations with respect to ligand-induced, IKK-dependent proteasomal degradation. 2) Under steady-state conditions, there is a stoichiometric excess of IB over NF-B in the cytoplasm (0.7 NF-B per IB according to the model). This may explain our observations that even at saturating concentrations of TNF or IL-1β, IB degradation never exceeded 70-80% of initial level (e.g. Fig. 1C). 3) Upon ligand stimulation, NF-B-bound IB is degraded, NF-B translocates to the nucleus, and IB is resynthesized. 4) At this point, although IB is highly abundant, its capacity to be degraded in response to a second stimulus is still severely compromised, because NF-B is in the nucleus. 5) IB can freely shuttle between the cytoplasm and the nucleus, pulling NF-B molecules (that lack nuclear export signals (31)) back to the cytoplasm. This step may be the rate-limiting step for acquisition of full responsiveness. 6) Newly synthesized IB molecules uncomplexed with NF-B are rapidly degraded (32), and only after all NF-B molecules are recovered back to the cytosol and the NF-B-bound IB over free IB ratio returns to prestimulation levels (0.7) are cells able to mount a full response again.

To experimentally examine the nuclear export hypothesis, we sought to analyze ligand-induced changes in cytoplasmic IB·NF-B complexes. However, the computational model predicted that ligand-induced changes of cytoplasmic IB·NF-B and total cytoplasmic NF-B were essentially the same (i.e. at any given time, virtually all cytoplasmic NF-B was bound to IB; Fig. 5B), suggesting that monitoring cytoplasmic total NF-B was an excellent approximation for following cytoplasmic IB·NF-B complexes. We therefore pulsed HepG2 cells for 30 s with TNF (20 ng/ml), and at various times after stimulation, we fixed, permeabilized, and immunostained the cells for p65 NF-B (Fig. 5C). We found that upon a 30-s TNF pulse, p65 rapidly translocated to the nucleus (maximal by 30 min) but by 60-120 min was back in the cytoplasm. The excellent temporal correlation between the levels of cytoplasmic NF-B (as derived computationally or experimentally; Fig. 5, B and C, respectively) and the competence of cells to degrade IB in response to a proinflammatory ligand (i.e. Table 1) strongly suggested that nuclear transport of NF-B provided a potential alternative mechanism to transiently desensitize IB processing (refractory period), in addition to the mechanism of IKK down-regulation (Fig. 3, B and C, and Table 1).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Ligand-induced desensitization is a common theme in many biological systems (13), thereby allowing cells to mount an appropriate response independently of ligand exposure time. Thus, prolonged exposures will not result in excessive responses, but instead, cells are enabled to build up a downstream response while being unable to perceive a second activating cue. Desensitization and resensitization are traditionally perceived to be linked to receptor dynamics (internalization, shedding, and recycling); however, any mediator or regulator along a signaling pathway can be hypothetically desensitized, therefore transiently blocking signal transduction (13).

In this work, we demonstrated that although cells can efficiently activate NF-B in response to a TNF exposure as short as 30 s, such stimulation was followed by a refractory period during which the capacity of cells to respond to a second homologous or heterologous stimulus was severely compromised. We further found that this transient refractory period correlated in part with a temporal down-regulation of IKK activity but not with receptor desensitization. Computational modeling enabled us to identify an additional layer of regulation, downstream of IKK, controlling the capacity of cells to respond to a second challenge. Ligand-induced dynamic changes in substrate (IB) availability, conformation, and subcellular localization form the basis for this mechanism. Further analysis led us to conclude that nuclear export of NF-B may be a rate-limiting step in controlling IB homeostatic metabolism, a term recently coined by O'Dea et al. (33).

Our study highlights the multifaceted regulation of NF-B signaling (Fig. 6) and sheds light on the refractory nature of IB processing as a route to transiently desensitize NF-B activity upon subsequent rounds of stimulation. Rapid and transient deactivation of IKK activity as well as temporal reduction in its capacity to respond to a subsequent challenge (IKK responsiveness) seems to play a crucial role in this process. Previous studies indicated that both the amplitude and the timing of IKK activation affect not only the intensity of NF-B-dependent transcription but also the specificity of the transcriptional response (23, 34). This indicated that besides resolution of the inflammatory response and induction of a refractory period (temporally preventing subsequent rounds of IB degradation upon restimulation), rapid down-regulation of IKK activity (28) plays a pivotal role in determining the type of elicited transcriptional program.

In addition to IKK regulation, our work demonstrated that nuclear export of IB·NF-B complexes may have also regulated IB responsiveness (Figs. 4 and 5). This suggested that NF-B positively controls IB both transcriptionally and post-translationally. Such double-layered feedback regulation ensures that NF-B transcriptional activity will fully resume only after reconstitution of the cytosolic pool of NF-B. Two other IB isoforms, IBβ and IB, are degraded more slowly under both TNF-induced and unstimulated conditions (23, 25) and have been implicated in dampening IB-mediated oscillations of NF-B activity (25, 35, 36). IB has been shown to be highly NF-B-inducible in mouse embryo fibroblasts and to contribute to nuclear export of NF-B but only at times greater than 3 h poststimulation (37). Thus, it is seems unlikely that IB contributes substantially to the export of NF-B over the 2 h of the refractory period observed in the present study. It may be interesting to determine whether similar transient refractory periods exist for processing of other IB isoforms.

View larger version (41K): [in this window] [in a new window]   FIGURE 5. Nuclear export of NF-B may regulate IB sensitivity to ligand-induced degradation. A, a schematic illustrating subcellular localization and levels of free IB, free NF-B, and NF-B-bound IB in response to a pulse of TNF. B, computationally predicted profile of all cytoplasmic populations of NF-B following a pulse of TNF. Note the exceedingly small free NF-B population. C, HepG2 cells were stimulated with a 30-s TNF pulse. At the indicated time points, cells were fixed, permeabilized, and immunostained for p65 NF-B. Shown are representative immunofluorescence confocal photomicrographs.

View larger version (93K): [in this window] [in a new window]   FIGURE 6. Refractory period in NF-B signaling. Shown is a schematic representation of the different ligand-induced autoregulatory mechanisms that control responsiveness in the NF-B signaling pathway.

In the present and previous studies (20), we demonstrated that dynamic bioluminescence imaging of IB-FLuc reporters in live cells provides robust and accurate readouts of ligand-induced IB dynamics. In effect, real time bioluminescence imaging was equivalent to performing continuous on-line Western blots of IB at 5-min intervals. An analogous transcriptionally coupled reporter (kB₅IB-EGFP) was generated by Nelson et al. (35) for monitoring IB dynamics in single cells by live cell fluorescence microscopy. Although such a system provides the means to monitor ligand-induced translocations and oscillations in IB levels, temporal resolution of this reporter is limited by the long maturation time of EGFP (>1 h) (38, 39). This notion and the fact that Nelson et al. (35, 36) co-overexpressed p65-red fluorescent protein may explain the vast difference between the observed period of IB-EGFP oscillations (300 min) and the period of endogenous IB oscillations, as predicted computationally (90-120 min) (25). Although longer term IB oscillatory behavior was not the focus of the present study, we did observe single oscillations within 150-180 min. Because FLuc is active immediately upon translation, our reporter should afford greater temporal resolution, enabling accurate readouts of IB dynamics and oscillations in live cells for such studies as well as the multistimulation protocols as described herein.

Of note, a previous study aimed at analysis of IB stabilization indicated a role for p38 in IB stabilization and, in some cell lines, in prevention of sequential degradation of IB upon concurrent exposure to TNF following continuous pretreatment with IL-1β (40). However, since IL-1β has been shown to induce rapid and dramatic down-regulation of TNF receptors (but not vice versa) (14), inhibition of TNF-induced IB processing, as observed by Place et al. (40), could be attributed directly to receptor dynamics rather than IB stabilization. This confounding factor highlights the importance of asymmetric receptor cross-desensitization, a phenomenon that remains poorly understood but has far reaching physiological consequences.

In conclusion, TNF preconditioning protocols and dynamic imaging revealed a transient suppression of the capacity of cells to process IB. This refractory period for IB processing was controlled both by IKK activity and NF-B distribution. In particular, the data suggested that nuclear export of NF-B may provide additional rate-limiting regulation governing the refractory period machinery. These regulatory mechanisms provide a "molecular timer" controlling the amplitude, timing, and specificity of the NF-B-mediated transcriptional program.

	FOOTNOTES

 
^* This work was supported in part by National Institutes of Health Molecular Imaging Center Grant P50 CA94056. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Supported in part by a National Science Foundation graduate research fellowship grant.

³ To whom correspondence should be addressed: Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510 S. Kingshighway Blvd., Box 8225, St. Louis, MO 63110. Tel.: 314-362-9359; Fax: 314-362-0152; E-mail: piwnica-wormsd{at}mir.wustl.edu.

⁴ The abbreviations used are: NF-B, nuclear factor-B; IB, inhibitor of NF-B; IKK, IB kinase; IL-1, interleukin-1; TNF, tumor necrosis factor ; FLuc, firefly luciferase; GST, glutathione S-transferase; PBS, phosphate-buffered saline; DMEM, Dulbecco/Vogt modified Eagle's minimal essential medium; IKK-KA, IKK kinase assay; C, continuous; P, pulse; P + C, pulse followed by continuous; EGFP, enhanced green fluorescent protein.

	ACKNOWLEDGMENTS

 
We thank Prof. Alexander Hoffmann, Shannon Werner, and Derren Barken (University of California, San Diego) for providing the computational model software. We also thank Dr. Yun-Feng Feng and Prof. Greg Longmore (Washington University) and Prof. Phillip Cohen (University of Dundee) for advice with the IKK kinase assays and Dr. Dustin Maxwell (Washington University) for assistance with immunofluorescence microscopy.

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