INTRODUCTION

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The enveloped negative-sense RNA virus respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in infants and young children (20). In addition to producing persistent airway hyperreactivity in previously healthy children, RSV infection is a major cause of morbidity in children with preexisting pulmonary or heart disease (18, 28, 48). RSV replicates primarily in the respiratory epithelial cell. The infected airway epithelial cell is an important initiator in the response to RSV infection, by synthesizing and secreting potent cytokines that are involved in the immune and inflammatory responses in the airway mucosa (16, 41).

Recent studies have demonstrated that the cytokines interleukin-1 (IL-1), IL-6, IL-8, IL-11, RANTES, macrophage inflammatory protein-1, monocyte chemotactic protein-1, granulocyte/macrophage stimulatory factor, tumor necrosis factor alpha (TNF-), and the soluble TNF receptor are produced by RSV-infected respiratory epithelial cells and may play important roles in the development of inflammation in vivo (2, 4, 16, 17, 30, 38). Of particular relevance to mononuclear inflammation, IL-8 gene expression is activated at the level of transcription through the effects of RSV-inducible transcription factors nuclear factor-B (NF-B) and NF-IL6 (4, 13, 17, 25, 29). From these studies, NF-B emerges as the primary activator of IL-8 transcription whose binding is absolutely required for inducible expression (5, 13). Indeed, NF-B translocation appears to be essential for activity not only of IL-8 but also of a genetic network including the cytokines IL-1, IL-6, and IL-11 in the RSV-infected epithelium (4). NF-B constitutes a family of cytokine-inducible transcription factors that include the potent RelA (p65) transactivator, as well as the RelB, c-Rel, NF-B1 (p50), and NF-B2 (p52) (the last two being encoded by the proteolytically processed precursors p105 and p100, respectively [40]) subunits. Inducible NF-B subunits interact with cytoplasmic inhibitors, collectively known as IBs, through motifs contained within a conserved NH₂-terminal Rel homology domain (3). IB subunits, responsible for cytoplasmic retention and inactivation of NF-B DNA-binding activity, consist predominantly of four isoforms, IB, IB, IB, and the NF-B1 precursor, p105, that are expressed and regulated in a cell specific fashion (21, 23, 44).

NF-B-inducing signals control its cytoplasmic-nuclear abundance by a mechanism involving proteolytic degradation of the IB inhibitor. Once liberated, free cytoplasmic NF-B passes through the nuclear pore complex and enters the nucleus (translocates), to bind and activate target genes. Stimulation of cells with the prototypical activator TNF- results in rapid serine phosphorylation of the IB NH₂ terminus (6, 45), an event coupled to the rapid polyubiquitination and proteolysis of phospho-IB (IB^P) through the 26S proteasome (1, 10). The role of the 26S proteasome has been implicated by the ability of the proteasome-specific calpain inhibitor I, Z-LLF-CHO, or MG-132 to block IB proteolysis in TNF--stimulated cells (1, 5, 10, 12, 37, 46).

Although we have shown that pRSV infection increases the nuclear abundance of NF-B (RelA) in A549 cells, its mechanism of activation is unexplored. Therefore, we systematically investigated the mechanism and kinetics of NF-B activation in pRSV-infected airway epithelial cells in comparison to those of TNF-. We show herein that in contrast to the rapid activation of RelA produced by TNF-, pRSV infection produces a gradual increase in RelA binding peaking at 24 h. For both activators TNF- and pRSV, IB and IB proteolysis occurred in parallel with the increases in RelA DNA-binding activity. Although specific proteasome inhibitors significantly block 26S protease activity and IB proteolysis by TNF-, they do not completely prevent IB proteolysis by pRSV infection. These data indicate that a major aspect of IB proteolysis occurs via a proteasome-independent mechanism in virus-infected epithelium.

	MATERIALS AND METHODS

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Cell culture and treatment. Human A549 pulmonary type II epithelial cells (American Type Culture Collection, Manassas, Va.) were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum as previously described (17, 25). Logarithmically growing A549 cells were either infected with sucrose gradient-purified Long strain A2 RSV (pRSV) at a multiplicity of infection (MOI) of 1 (17, 25) or stimulated with 20 ng of recombinant human TNF- (rhTNF-; Calbiochem) per ml and incubated for various times at 37°C. After stimulation, cell pellets were washed in phosphate-buffered saline and fractionated into cytoplasmic and nuclear extracts by using hypotonic lysis and high-salt extraction (17, 25). The protein contents of cellular extracts were measured by a spectrophotometric (Bradford) assay using bovine serum albumin as a standard (Bio-Rad protein detection reagent). Where indicated, the protein synthesis inhibitor cycloheximide (CHX) was added to culture medium at a final concentration of 50 µg/ml. The proteasome inhibitors lactacystin (a gift of E. J. Corey, Harvard University), carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG-132; Sigma Aldrich, St. Louis, Mo.), and benzyloxycarbonyl-Leu-Leu-phenylalaninal (Z-LLF-CHO; a gift of M. Suto, Signal Pharmaceuticals) were added directly to the culture medium at the indicated concentrations.

EMSA. Electrophoretic mobility shift assay (EMSA) was performed with a duplex oligonucleotide corresponding to nucleotides 96 to 69 of the human IL-8 gene promoter: 5'-GATCCATCAGTTGCAAATCGTGGAATTTCCTCTCTA-3' 3'-    GTAGTCAACGTTTAGCACCTTAAAGGAGAGATCTAG-5'

Western immunoblotting and coimmunoprecipitation. For Western immunoblotting, indicated amounts of nuclear or cytoplasmic extracts were mixed with Laemmli buffer, boiled at 100°C for 5 min, fractionated on a sodium dodecyl sulfate (SDS)-10% polyacrylamide gel, and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, Mass.) as described elsewhere (5, 17, 25). Membranes were washed, blocked in 8% nonfat dry milk, and probed with affinity-purified rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif.) for RelA, c-Rel, and NF-B1 (in nuclear fractions) or IB, IB, and IB (in cytoplasmic fractions). After incubation with the secondary donkey anti-rabbit immunoglobulin G antibody-horseradish peroxidase conjugate (Amersham International), immunocomplexes were detected by enhanced chemiluminescence (ECL) reaction (ECL kit; Amersham) as specified by the manufacturer. For the two-step coimmunoprecipitation-Western immunoblot assay, A549 whole-cell extract (WCE) was made by adding radioimmunoprecipitation assay buffer (150 mM NaCl-1.0% Nonidet P-40-0.5% deoxycholate-0.1% SDS-50 mM Tris-Cl [pH 8.0] containing 1 µg of pepstatin A, 1 µg of leupeptin, and 10 µg of aprotinin per ml, 1 mM dithiothreitol, and 1 mM PMSF) to A549 monolayers. Equal amounts of WCE (2.5 mg of protein) were incubated with 12 µl of affinity-purified RelA antibody (Santa Cruz Biotechnology) in 1 ml of TST buffer (50 mM Tris-HCl [pH 7.5], 5 mM EDTA, 150 mM NaCl 0.05% Triton X-100) for 2 h at 4°C. Then protein A-agarose was added, and the mixture was incubated for another 2 h. The protein A-agarose immune complex was precipitated by centrifugation. After five washes with TST buffer, the precipitates were boiled in Laemmli buffer and immunoblotted with indicated antibodies as described above. Where indicated, anti-RelA and IB antibodies were preadsorbed by incubating them with 20-fold excess peptide at 4°C overnight.

Northern blotting. Forty micrograms total cellular RNA was extracted from A549 cells by acid guanidinium-phenol extraction (RNAzol B; Tel-Test Inc., Friendswood, Tex.), fractionated by electrophoresis on a 1.2% agarose-formaldehyde gel, capillary transferred to a nitrocellulose membrane (MSI, Westboro, Mass.), and prehybridized as described previously (47). The membrane was hybridized with 10⁶ cpm of ³²P-IB cDNA probe per ml at 50°C overnight in 5% SDS hybridization buffer (47). The membrane was washed with a buffer containing 5% SDS and 1× SSC (0.15 M NaCl, 0.015 M sodium citrate) for 20 min at room temperature followed by 30 min at 50°C. The membrane was exposed to XAR film (Kodak) for 24 to 48 h. For -actin Northern blotting, the same membrane was stripped and probed with ³²P-labeled -actin cDNA. Bands in the autoradiogram were quantitated by exposure to a Molecular Dynamics PhosphorImager cassette.

26S proteasome assay. Proteasome activity in fresh cytoplasmic extracts was measured as described previously (37). Two hundred micrograms of cytoplasmic protein was added to assay buffer (20 mM Tris-Cl [pH 8.0], 1 mM ATP, and 2 mM MgCl₂) in the presence of the synthetic fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amido-4-methyl coumarin (final concentration, 60 µM; Sigma Aldrich, St. Louis, Mo.) in a final volume of 1 ml. The tubes were incubated at 30°C for 30 min, after which the reaction was terminated by the addition of 1 ml of cold ethanol and the lysate was spun at 12,000 × g for 10 min at 4°C. Fluorescence in the supernatant was measured in a fluorometer at 440-nm emission (I_ex, 380 nm). For each assay, a standard curve was measured with known dilutions of 7-amino-4 methylcoumarin (AMC; Sigma Aldrich).

	RESULTS

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Comparative kinetics of RelA activation. Previously we have shown that both RSV and TNF- induce the binding of two sequence-specific complexes in EMSA, C1 and C2, to the IL-8-inducible enhancer (located at nucleotides 96 to 69 relative to the cap site [17]). C1 and C2 both contain the RelA transactivator, as indicated by a strong supershift produced in the presence of specific NH₂- and COOH-directed RelA antibodies (17), and are due to a heterotypic association with c-Rel and NF-B1 transcription factors, producing detectable differences in their migration on nondenaturing PAGE (5). We therefore used the presence of C1 and C2 in EMSA to compare the kinetics of RelA activation in response to pRSV infection to that produced by rhTNF- treatment. pRSV infection induced C1/C2 complex binding in a time-dependent manner; binding was first faintly detected 3 h after the addition of pRSV and peaked at 2.6-fold 24 h postinfection, with C2 predominating (Fig. 1A, top panel). At 36 h, an apparent decrease in C1/C2 complex binding was observed (at a time when the cells are fragile and beginning to lose viability). In contrast, the addition of 20 ng of rhTNF- per ml produced a much more rapid activation of C1/C2 DNA binding; maximal C1/C2 induction occurred at 15 min (at an 8.12-fold activation), with the C1 complex predominating. C1/C2 binding progressively declined thereafter (even in the continued presence of ligand), and in data not shown, returned to nearly control levels after 8 h of rhTNF- treatment.

View larger version (34K): [in this window] [in a new window]   FIG. 1.   NF-B activation and translocation in RSV-infected and rhTNF--stimulated A549 cells. (A) NF-B binding to the IL-8 promoter in EMSA. A549 cells were infected with pRSV (MOI of 1) or stimulated with 20 ng of rhTNF- per ml for different periods of time. Shown is an autoradiogram of EMSA using a nuclear extract binding the IL-8 TNF-responsive element (nucleotides 96 to 69; see Materials and Methods), with the autoradiographic exposure of the pRSV time course 1.5 times longer than that for the rhTNF- time course (to demonstrate relative C1/C2 induction patterns). C1 and C2 are the inducible NF-B complexes predominantly containing RelA (17). For pRSV, activation relative to control binding is 1.23-fold (3 h), 1.74-fold (6 h), 1.9-fold (12 h), 2.6-fold (24 h), or 1-fold (36 h). For rhTNF-, C1/C2 activation is 8.12-fold (0.25 h), 7.9-fold (0.5 h), 7.3-fold (1 h), 6.3-fold (2 h), or 5.2-fold (4 h). (B) Nuclear abundance of NF-B family members measured by Western immunoblotting. Nuclear extracts (216 µg) from control, pRSV-infected (24 h), and rhTNF--treated (15 min) cells were fractionated and probed with anti-RelA, -NF-B1, and -c-Rel antibodies. The levels of the 65-kDa RelA, 50-kDa NF-B1, and 75-kDa c-Rel proteins were increased following either pRSV or rhTNF- treatment. *, nonspecific protein detected by c-Rel antibody, confirming equivalent loading.

IB isoform expression in type II pulmonary epithelial cells and inducible changes in cytoplasmic abundance. In resting cells, NF-B is inactivated by complexing with cell-type-restricted IB, IB, IB, and the p105 NF-B1 inhibitors (21, 44). Western immunoblots of A549 cytoplasmic extracts (Fig. 2) revealed expression of 37-kDa IB, 49-kDa IB, and the p105 NF-B1 precursor, as assessed by determining the appropriate molecular weight and ability to block staining upon preadsorption of the antibody with the appropriate peptide (reference 21 and data not shown]. We noted that a greater amount of cytoplasmic lysate was needed to detect these latter subunits, probably indicating either that the IB isoform is more abundantly expressed or that the antibodies have significant differences in affinity for the target antigen (see Discussion). We were unable to detect the alternatively spliced NF-B1 COOH terminus, IB, even though its precursor, p105, was expressed. To determine the effect of RSV infection on IB abundance, A549 cells were infected with pRSV at an MOI of 1 for various times prior to cytoplasmic extraction and Western immunoblotting (Fig. 2A). A faintly detectable decrease in IB could be discerned at 3 h, and the level continued to decrease to 24 h, when 45% of the control signal remained. A similar pattern of proteolysis was observed for the IB isoform. Finally, a transient decrement of p105 could be detected at 6 h, which gradually returned to control values by 24 h. The decline in IB and -, but not p105, abundance exactly parallels the pattern of pRSV-induced nuclear C1/C2 DNA binding to the IL-8-inducible enhancer in EMSA (cf. Fig. 1) and is consistent with the requirement for IB proteolysis in NF-B translocation.

View larger version (36K): [in this window] [in a new window]   FIG. 2.   Expression and regulation of IB isoforms. (A) Kinetics of IB proteolysis following pRSV infection. A549 cells were infected with pRSV for 0 to 36 h, and cytoplasmic extracts were prepared. Results of Western immunoblotting using specific antibodies for IB, IB, and p105 are shown. For IB, 200 µg of cytoplasmic protein was loaded for each time point; for IB, 750 µg was loaded; and for p105, 500 µg was loaded. Relative to control levels, IB declined to 93% ± 3.4% (3 h), 84% ± 6% (6 h), 82% ± 3.6% (12 h), 46% ± 3.3% (24 h), or 44% ± 10.2% (36 h). No IB isoform was detectable. (B) Kinetics of IB proteolysis following rhTNF- stimulation. A549 cells were stimulated with rhTNF- for 0 to 4 h, and IB isoforms were detected by Western immunoblotting. Proteins were normalized by using the Bio-Rad protein detection reagent, and equivalent loading was confirmed by staining a parallel SDS-PAG with Coomassie brilliant blue. A phosphorylated form of IB (IBP), migrating more slowly, was produced by rhTNF- stimulation. *, an 85-kDa protein that specifically cross-reacts with anti-p105 antibody whose presence occurred in late passage A549 cells and probably represents p84NF-B1, an alternatively spliced product of the p105 gene (19). Relative to control values, IB decreased 26% ± 2.6% (0.25 h), 54% ± 4.6% (0.5 h), 73% ± 3% (1 h), 80% ± 4% (2 h), and 85% ± 5% (4 h).

pRSV infection induces changes in abundance of RelA-associated IB. These data indicated that proteolysis of IB subunits was temporally linked to RelA DNA-binding activation. To demonstrate whether IB associates with RelA and, if so, whether this association was lost as a result of pRSV infection, we used a two-step immunoprecipitation-Western immunoblot assay (21). In this assay, uninfected and infected A549 whole-cell lysate was prepared and RelA was immunoprecipitated under native conditions. RelA-associated IB was then detected in the immunocomplexes by Western immunoblotting using anti-IB antibody (Fig. 3A). IB immunostaining was not observed when preadsorbed RelA antibody was used in the primary immunoprecipitation or when either preadsorbed IB or preimmune serum was used in the secondary Western immunoblot analysis. Only when both anti-RelA (in the primary immunoprecipitation) and anti-IB (in the Western immunoblot analysis) antibodies were used sequentially could IB be detected. Thus, RelA is associated with IB in uninfected A549 cells.

View larger version (29K): [in this window] [in a new window]   FIG. 3.   Effect of pRSV infection on RelA-associated IB. (A) Determination of coimmunoprecipitation assay specificity. RSV-infected (0 or 24 h) WCEs were immunoprecipitated (IP) either with anti-RelA antibody or with preadsorbed RelA antibody (pre-RelA), and the immunoprecipitate was used for the detection of IB by Western blotting (IB) using anti-IB antibody, preimmune serum (PRS), or preadsorbed IB antibody (pre-IB). IgG, immunoglobulin G. (B) Degradation of RelA-associated IB by RSV infection. Uninfected or pRSV-infected A549 WCE (for 0, 12, or 24 h) was immunoprecipitated with RelA antibody. The immunoprecipitate was subjected to Western immunoblotting with both RelA and IB antibodies (Materials and Methods). After 24 h of pRSV infection, RelA-associated IB was significantly decreased. For the different time points, the IB/RelA ratio was 1.63 (0 h), 1.2 (12 h), and 0.9 (24 h), indicating that IB association with RelA falls following pRSV infection.

Quantitation of IB protein t_1/2 after pRSV infection. To more precisely demonstrate that the decrease in IB abundance after pRSV infection occurred as a consequence of proteolysis, we directly measured the IB half-life (t_1/2) in control, rhTNF--treated and 24-h pRSV-infected A549 cells. For this, changes in the abundance of IB were measured in the presence of the protein synthesis inhibitor CHX, a standard technique for determination of rapid IB turnover (26, 27). Relative changes in IB abundance were determined by comparing Western immunoblot signals by using identical amounts of cytoplasmic proteins under conditions where the changes in signal were linear to input protein (Fig. 4A). The time course of IB disappearance in the absence of new protein synthesis is shown for control, pRSV-infected, and rhTNF--treated cells (Fig. 4B; quantitation in Fig. 4C). In control cells, IB was stable, with a t_1/2 that exceeded 120 min. By contrast, the turnover of IB was very rapid in rhTNF--stimulated cells, with an estimated t_1/2 of less than 5 min. At 24 h of pRSV infection, the IB t_1/2 was 90 min, an intermediate value that was shorter than control measurements yet markedly longer than the t_1/2 seen after rhTNF- treatment. To exclude the effects of potential artifacts of CHX on IB turnover, relative differences in IB t_1/2 were confirmed by using [³⁵S]methionine pulse-chase-labeled cells (not shown). These data provide direct evidence that pRSV infection produces IB proteolysis and this is, in part, the mechanism for its disappearance during pRSV infection.

View larger version (22K): [in this window] [in a new window]   FIG. 4.   Turnover rates of IB. (A) Quantitation of Western immunoblot analysis. Indicated concentrations of cytoplasmic lysates (Cyto. Ext.; from control cells) were fractionated by SDS-PAGE, and levels of IB were detected by Western blotting using ECL. IB bands were quantitated by scanning densitometry (in arbitrary units [AU]) as 102,667 AU (400 µg of extract), 81,071 AU (200 µg), 54,839 AU (100 µg), 35,708 AU (50 µg), 7,503 AU (25 µg), and 3,891 AU (12.5 µg). Between 12.5 and 200 µg of protein, a linear dose-response profile was obtained (r2 = 0.93). (B) pRSV- and rhTNF--induced changes in IB levels. CHX-treated uninfected (control), pRSV-infected, and rhTNF--treated A549 cells were harvested at the indicated times. The autoradiogram shows a Western blot of IB. For pRSV-infected cells, cells infected for 24 h were used. (C) Quantitation of t1/2 (graphical representation of IB degradation taken from Fig. 5A). IB signals were scanned, quantitated, and expressed as percentage of initial signal (0-h cycloheximide). Each point in the curve is mean ± standard error of three independent experiments.

pRSV infection produces a delayed (and blunted) RelA:IB positive feedback loop. Infection with pRSV produced a disappearance of IB in A549 cells without appreciable resynthesis, in spite of only weakly inducing IB turnover (Fig. 2A). This was somewhat surprising because others have shown that in response to nuclear RelA translocation, a rapid activation of the ib gene expression results in IB protein resynthesis and termination of nuclear RelA activation (21, 43). Indeed, this phenomenon occurred in A549 cells upon rhTNF- administration because 30 min following rhTNF- treatment, cytoplasmic IB reaccumulated; this IB resynthesis temporally coincided with termination of nuclear C1/C2 DNA binding (Fig. 1 and 2B). To determine whether the RelA:IB positive feedback loop was operative in pRSV-infected cells, we compared the magnitude and kinetics of expression of IB mRNA to those produced by rhTNF- (Fig. 5). Upon pRSV infection, IB mRNA abundance increased 3-fold at 24 h and 5.5-fold at 36 h relative to control (uninfected) cells. By contrast, rhTNF- produced a 25-fold increase in IB mRNA by 2 h, which subsequently fell to a 13-fold increase at 8 h (the IB mRNA increase preceded increases in cytoplasmic IB protein abundance [cf. Fig. 2B and 5B]). Notably, levels induced by pRSV were significantly less than those produced by rhTNF- treatment. These data indicate that the RelA:IB positive feedback loop was less strongly activated by pRSV infection and is inadequate to produce significant reaccumulation of the protein.

View larger version (59K): [in this window] [in a new window]   FIG. 5.   RelA:IB positive feedback loop. (A) Induction of IB mRNA by pRSV infection. IB and -actin mRNA levels were detected by Northern blotting in pRSV-infected cells for indicated times between 0 and 36 h (top). IB cDNA hybridized with 1.8- and 2.7-kb (*) mRNA species (the latter representing a form containing a longer 3' untranslated region), both of which were increased in parallel by RSV infection. Compared to its level at 0 h, IB mRNA was increased 2.5-fold at 12 h, 3-fold at 24 h, and 5.5-fold at 36 h. (B) Induction of IB mRNA by rhTNF-. A549 cells were stimulated with rhTNF- for indicated times between 0 and 8 h (top). Relative to the control, in rhTNF--treated cells, IB mRNA increased 13-fold (1 h), 25-fold (2 h), 20-fold (4 h), and 13.2-fold (8 h).

Distinct mechanisms for intracellular IB proteolysis. A large body of work has indicated that the ubiquitin-26S proteasome pathway is a major, if not the sole, mechanism for inducible proteolysis of the IB inhibitor (1, 12, 33). However, our data indirectly argued for differences in the proteolytic pathway between rhTNF- stimulation and viral infection. The distinct kinetics of proteolysis and the absence of phosphorylated IB isoforms in cells infected with pRSV (Fig. 2) prompted us to further examine the proteolytic mechanism(s) involved. We therefore examined whether selective 26S proteasome inhibitors could block IB proteolysis in pRSV-infected A549 cells. These inhibitors included lactacystin, a Streptomyces metabolite that is a potent and irreversible inhibitor of the mammalian subunit X (MB1) of the proteasome and has been shown to be a highly selective cell permeable proteasome inhibitor without measurable inhibition of protein kinase C, thrombin, plasminogen activator, or cytoplasmic calpains (references 12 and 31 and our unpublished data), and the peptide aldehydes Z-LLF-CHO and MG-132, which are highly potent inhibitors of proteasome activity in cells (10, 38).

View larger version (26K): [in this window] [in a new window]   FIG. 6.   Effect of 26S proteasome inhibitors on IB proteolysis. (A) Experimental strategy showing time line of sequence of CHX or protease inhibitor addition and cell harvest. Due to the slower turnover of IB protein in pRSV-infected cells, 3 h of CHX treatment was used to detect proteolysis. To control for potential lability of the inhibitor, the rhTNF- experiments were designed so that protease inhibitors were added for the same length of time. (B) Western immunoblot of IB proteolysis. The upper section shows the effect of proteasome inhibitors on pRSV-induced IB proteolysis. Cell infected with pRSV for 24 h were incubated with the indicated proteasome inhibitors (INHIB) (10 µM lactacystin [Lacta], 10 µM Z-LLF-CHO [Z-LLF], and 25 µM MG-132) for 1.5 h prior to adding CHX (50 µg/ml). Cytoplasmic IB was detected by Western blotting. Relative to uninfected cells (lane 1, taken as 100%), pRSV-infected CHX treatment reduced IB to 37.5% ± 7.9% of the initial signal; the addition of lactacystin (lane 4; 36.9% ± 4.5% of control values) or Z-LLF (lane 5; 32.7% ± 6.5% of control) had no effect in three separate experiments (not significant); the addition of MG-132 (lane 6) also had no effect (38.2% ± 5.9% of control values, n = 3, not significant). The lower section shows the effect of proteasome inhibitors on rhTNF--induced IB proteolysis. To control for potential lability of proteasome inhibitors, in this experiment, before the brief rhTNF- treatment (0.75 h), cells were preincubated with the inhibitors for 3.75 h (the same duration as for the pRSV treatment). Cytoplasmic IB was detected by Western blotting. Relative to control cells (lane 1, taken as 100%), rhTNF--CHX treatment reduced IB to 17.5% ± 1.0%, lactacystin produced an increase in IB abundance to 60% ± 1.4% of the control value (n = 3, P < 0.0003), Z-LLF also increased IB abundance to 38.7% ± 0.14% of the control value (n = 3, P < 0.0103), and MG-132 increased IB abundance to 45.7% ± 0.17 (n = 3, P < 0.0024). Similar results were obtained with proteasome inhibitors added immediately prior to rhTNF- stimulation. (C) Dose response of IB proteolysis to increasing concentrations of proteasome inhibitor. Increasing concentrations of lactacystin (indicated at the top) were used to block pRSV-inducible IB proteolysis. Relative to control cells, IB abundance decreased 41% (0 µM lactacystin), 54% (10 µM lactacystin), 51% (20 µM lactacystin), and 67% (40 µM lactacystin).

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View larger version (29K): [in this window] [in a new window]   FIG. 7.   26S proteasome activity after treatment and in the presence of proteasome inhibitors. Control, pRSV-infected, or rhTNF--treated A549 cells were incubated in the absence or presence of 10 µM lactacystin (Lacta), 10 µM Z-LLF-CHO (Z-LLF), or 25 µM MG-132 for 4 h. After 3.5 h of preincubation, cells were stimulated with rhTNF- for 0.5 h. Proteasome activities were measured in the cytoplasmic extracts, using the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC (37). Results are expressed as the amount of AMC formed by the enzymatic cleavage of substrate. The inset represents a standard curve for AMC drawn by measuring the fluorescence of known quantities of AMC.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The airway epithelium, forming a physical barrier between the extracellular environment and the intracellular milieu, represents the major target for RSV replication. Through its ability to activate the expression and subcellular abundance of transcription factors controlling cytokine gene expression, the virally infected epithelium initiates a cytokine cascade responsible for initiating the host response, including recruitment of inflammatory mononuclear cells into the peribronchial mucosa. Although a number of studies have reported the activation of NF-B family members in respiratory epithelial cells following pRSV infection in vitro (4, 13, 17, 29), a systematic evaluation of the expression and regulation of tissue-specific IB isoforms has not been reported. In this report, we show that the IB, IB, and p105 are expressed and differentially regulated by ligand (TNF-) and infectious (pRSV) NF-B inducers. Surprisingly, although the proteasome is responsible for IB proteolysis in many cell lines, including TNF--mediated activation in A549 cells, we provide evidence for an independent proteolytic pathway involved in IB turnover following RSV infection.

Kinetics and magnitude of NF-B activation following pRSV infection. We and others have demonstrated an increase in NF-B DNA-binding activity following pRSV infection (4, 13, 17, 29) and a simultaneous increase in the nuclear abundance of the potent transcriptional activator RelA (17). In this study, using side-by-side comparisons with the effects of rhTNF-, a slower (and lower level of) NF-B activation is seen following pRSV infection, where a 2.2-fold increase in NF-B DNA-binding activity occurs over a 3- to 24-h period postinfection. Moreover, subtle differences in C1/C2 complex binding can also be seen, indicating quantitative effects in the NF-B induction profile. Other investigators, using nonpurified viral preparations, have shown a biphasic effect for NF-B activation (14), where an initial transient, replication-independent increase in NF-B binding is seen 30 to 90 min postinfection, followed by a nadir and a gradual reaccumulation of NF-B after 24 h. As a marker for NF-B activation, the early 2-h peak is associated with a burst of IL-8 gene expression (14). Taking equivalent time points (in data not shown), we do not see a consistent biphasic NF-B activation pattern, nor is there a biphasic profile of IL-8 expression in highly purified preparations of RSV (this study and reference 17). It should be emphasized that the highly purified RSV preparations lack any measurable NF-B-activating cytokine such as IL-1, known to be produced in abundance in pRSV-infected A549 cells (34), which may explain the discrepancy in our observations. We recognize, of course, that during RSV infection in vivo, both cytokine and direct viral effects act in concert and are likely to be operative in NF-B induction. Nevertheless, our studies on RelA activation and IB proteolysis in vitro are relevant only for the later, viral replication-dependent, phase of NF-B activation.

Expression and regulation of distinct IB isoforms. We have previously shown the expression of the IB, -, and - but not p105 isoforms in hepatocytes (21), and in this study, IB, IB, and p105, but not IB, were expressed in airway epithelial cells. Encoded by separate genes and containing differences in the number of ankyrin repeat domains, IB and - are widely expressed, binding to and inactivating the NF-B members RelA and c-Rel with slightly different affinities (8, 11, 22, 44). In our detection system, the level of IB expression is so low that RelA-associated IB is not detectable by coimmunoprecipitation, leading us to conclude that IB abundance is likely to be the major determinant of RelA subcellular localization in these cells. It still remains formally possible that there is a significant difference in the affinities of the two IB antibodies; additional studies will be required to unambiguously clarify this issue. Similarly, p105, the unprocessed precursor of the 50-kDa NF-B1, also contains ankyrin repeats and can function in cytoplasmic retention/inactivation of RelA. However, p105 is also expressed at low levels and is largely inert to stimulation, indicating that 105 processing is also irrelevant to the mechanism of NF-B activation by these agents.

The RelA:IB positive feedback loop in pRSV infection. Hormonal stimuli producing IB proteolysis and RelA translocation activate an autoregulatory pathway (RelA:IB positive feedback loop [24, 42]). In this pathway, nuclear RelA binds reiterated NF-B sites to activate robust expression of the IB gene promoter; IB resynthesis then recaptures RelA and terminates its activity. We show that in rhTNF--treated A549 cells, the IB gene is robustly stimulated, resulting in a 25-fold increase in steady-state levels of mRNA (Fig. 4B). Therefore, the IB gene is highly inducible in A549 cells. By contrast, pRSV infection stimulates only a 5.5-fold increase in IB mRNA, an amount insufficient to replace proteolyzed IB. This phenomenon may be due to the relatively weak NF-B activation induced by pRSV infection, since the rate of IB proteolysis is much less than that produced by rhTNF-, and thus a greater turnover rate of IB protein cannot account for the insufficient RelA:IB positive feedback loop. We do not think that insufficient IB gene expression can be ascribed to a nonspecific toxic effect of viral replication because (i) -actin mRNA continues to be expressed (Fig. 5A); (ii) increases in IB, as well as other proteins (25), continue to be observed in cells 24 to 36 h postinfection (Fig. 5A); and (iii) IL-8 mRNA continues to increase in abundance during this time (17).

Nonproteasome pathway involved in pRSV-induced IB proteolysis. The multicatalytic proteinase complex (proteasomes) are high-molecular-mass (~700-kDa) proteases involved in turnover of unstable cellular proteins, cell cycle progression, and major histocompatibility complex class I processing and have more recently been implicated in the inducible degradation and processing of NF-B family members including p105 and IB (1, 12, 33, 37; reviewed in reference 32). TNF-induced IB proteolysis is mediated through a two-step process whereby the IB NH₂ terminus is inducibly phosphorylated at serine residues 32 and 36 by a ubiquitously expressed kinase complex, targeting the newly phosphorylated IB (IB^P) for polyubiquitination and proteolysis (7, 36). That the proteasome pathway mediates IB^P degradation comes from the use of selective proteasome inhibitors such as lactacystin, Z-LLF-CHO, and MG-132, all of which are potent inhibitors of its inducible proteolysis (10, 12, 33). Our studies show that addition of proteasome inhibitors results in IB^P accumulation (Fig. 6) and significant inhibition of TNF--inducible proteolysis under conditions where we demonstrate directly a nearly complete inhibition of proteasome activity. It is important to note that total proteasome activity is unaffected by rhTNF- treatment. This observation strongly argues that a prior step, e.g., phosphorylation-ubiquitination, is the rate-limiting event in IB proteolysis.