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The Journal of Immunology, 2003, 171: 6014-6022.
Copyright © 2003 by The American Association of Immunologists

Inhibition of NF-{kappa}B Activation and Its Target Genes by Heparin-Binding Epidermal Growth Factor-Like Growth Factor 1

Veela B. Mehta and Gail E. Besner2

Department of Pediatric Surgery, Children’s Research Institute, and Ohio State University, Columbus, OH 43205


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Many cells upon injury mount extensive, compensatory responses that increase cell survival; however, the intracellular signals that regulate these responses are not completely understood. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been implicated as a cytoprotective agent. We have previously demonstrated that pretreatment of human intestinal epithelial cells with HB-EGF significantly decreased cytokine-induced activation of inducible NO synthase mRNA expression and NO production and protected the cells from apoptosis and necrosis. However, the mechanisms by which HB-EGF exerts these effects are not known. Here we show that cytokine exposure (IL-1{beta} and IFN-{gamma}) induced NF-{kappa}B activation and IL-8 and NO production in DLD-1 cells. Transient expression of a dominant negative form of I{kappa}B{alpha} decreased NO production, suggesting that the cytokines stimulated NO production in part through activation of NF-{kappa}B. HB-EGF dramatically suppressed NF-{kappa}B activity and IL-8 release and decreased NO production in cells pretreated with HB-EGF. HB-EGF blocked NF-{kappa}B activation by inhibiting I{kappa}B kinase activation and I{kappa}B phosphorylation and degradation, thus interfering with NF-{kappa}B nuclear translocation, DNA-binding activity, and NF-{kappa}B-dependent transcriptional activity. The data demonstrate that HB-EGF decreases inflammatory cytokine and NO production by interfering with the NF-{kappa}B signaling pathway. Inhibition of NF-{kappa}B may represent one of the mechanisms by which HB-EGF exerts its potent anti-inflammatory and cytoprotective effects.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Heparin-binding epidermal growth factor-like growth factor (HB-EGF)3 is a member of the EGF family that binds to and activates ErbB1 and ErbB4 EGF receptor subtypes. It also binds to heparin sulfate, which increases its biological activity (1, 2). HB-EGF was first identified from supernatants of cultured human macrophages in a soluble, secreted form (3). It originates from a 22-kDa membrane-anchored precursor that undergoes extracellular cleavage, releasing the soluble mature form of the growth factor. Various cell types, including epithelial cells, keratinocytes, monocytes, mesangial cells, lymphoid cells, and skeletal muscle cells, produce HB-EGF. It is a potent mitogen and chemotactic factor for epithelial cells, fibroblasts, and smooth muscle cells (1, 4).

During the course of infection or insult to the intestinal epithelium and in states of chronic inflammation such as inflammatory bowl disease (IBD), the expression and release of mediators of acute mucosal inflammation are markedly enhanced. In IBD, chemokines play a crucial role in the pathogenic infiltration of immunocytes. IL-8, a leukocyte chemoattractant, is rapidly induced in response to proinflammatory cytokines and is known to be transcriptionally regulated by NF-{kappa}B (5, 6).

Recent reports suggest that NO plays a regulatory role in gut barrier function. Sustained up-regulation of NO production in the intestine can lead to epithelial injury through the formation of peroxynitrite (7, 8).

NF-{kappa}B has been shown to mediate the induction of inducible NO synthase (iNOS) mRNA expression in response to LPS in mouse macrophages (9), by bacteria in IFN-{gamma}-primed DLD-1 cells (10) and in viral-infected glial cells (11). In a rat model of IBD, inhibition of NF-{kappa}B suppressed up-regulation of iNOS expression in the colon (12). IL-10 inhibited NF-{kappa}B activation and iNOS gene expression (11).

Despite the diverse strategies that various pathogens use to gain entry into intestinal epithelial cells, intracellular signaling converges to activate NF-{kappa}B and its target genes. Thus, NF-{kappa}B appears to be a key regulator of the intestinal epithelial cell innate immune response (13). NF-{kappa}B is a dimeric transcription factor composed of Rel proteins. Activation of NF-{kappa}B is tightly controlled by a group of inhibitory proteins, the I{kappa}Bs. In unstimulated cells, NF-{kappa}B is present in the cytosol in an inactive state, complexed with the inhibitory I{kappa}B proteins (14). Cell stimulation by inflammatory cytokines, growth factors and chemokines, results in activation of I{kappa}B kinase (15) that phosphorylates I{kappa}B{alpha} at serine 32 and 36. This leads to degradation of I{kappa}B{alpha} and the release and nuclear translocation of active NF-{kappa}B where it regulates gene transcription (16, 17). The activation of NF-{kappa}B initiates several regulatory events that result in autoregulation of inflammatory cytokines.

Recent observations from our laboratory and others suggest that HB-EGF promotes cell survival. Cytoprotective effects of HB-EGF have also been observed in several cell models. HB-EGF has been shown to promote the survival of rat dopaminergic neurons(18), renal epithelial cells (19), and hepatoma cells (20). We have shown that HB-EGF decreases iNOS expression and NO production in a colon carcinoma cell line (21) and in a rat model of ischemia/reperfusion (I/R) injury (22). HB-EGF also protected rat intestine from histologic damage (23), decreased reactive oxygen species production in polymorphonuclear leukocytes (24), and protected DLD-1 cells from proinflammatory cytokine-induced apoptosis (25). However, the molecular mechanisms of HB-EGF action have not yet been elucidated. In the present study we attempted to characterize the mechanism by which HB-EGF decreases IL-8 and NO production in colon carcinoma cells. We provide evidence that HB-EGF blocks cytokine-induced NF-{kappa}B activation and decreases NO production. Furthermore, we show that HB-EGF interferes with cytokine-induced I{kappa}B kinase activity, inhibits phosphorylation and degradation of I{kappa}B{alpha}, and suppresses the release of proinflammatory cytokine.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

DLD-1 human colon epithelial cells (American Type Culture Collection, Manassas, VA) were maintained in high glucose DMEM (BioWhittaker, Walkersville, MD) supplemented with 4 mM L-glutamine, 100 µg/ml penicillin-streptomycin, 1 mM sodium pyruvate, and 5% heat-inactivated FBS (Life Technologies, Gaithersburg, MD). HT29 colon carcinoma cells were cultured in McCoy’s 5A medium (Cellgro, Herndon, VA) containing 10% FBS. Cells were incubated at 37°C in an atmosphere of 7.5% CO2.

IL-8 and NO measurements

Cells were cultured and stimulated with cytokines as previously described (21). Briefly, 24 h after plating, some wells received HB-EGF (50 ng/ml) (26) for 48 h. Cells were then treated with HB-EGF alone, IL-1{beta} and IFN-{gamma}, or IL-1{beta}, IFN-{gamma}, and HB-EGF as indicated and incubated for an additional 24 h. Culture supernatants were collected for NO measurements. NO synthesis was determined using a NO analyzer (model 280; Sievers, Boulder, CO). NO concentrations were calculated from a standard curve derived from NaNO3. Human IL-8 levels were determined from cell supernatants using a commercially available ELISA kit (BioSource International, Camarillo, CA) according to the manufacturer’s instructions.

Western blot analysis

DLD-1 cells (7 x 105) were seeded in six-well plates and allowed to grow for 24 h. Cells were then serum-starved for 24 h in the presence or the absence of HB-EGF. As indicated, cells were then stimulated for 10 min. Monolayer cells were lysed in buffer containing 50 mM Tris-Cl, 0.2% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 10 mM {beta}-glycerophosphate, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 2 µg/ml protease inhibitors. Lysates were clarified by centrifugation at 14,000 x g for 15 min, and protein concentrations were determined using the Bio-Rad protein assay reagent (Hercules, CA). Cytosolic extracts containing 20 µg of protein were subjected to Western blot analysis. Membranes were incubated with a 1/1000 dilution of specific primary Abs overnight at 4°C. Protein bands were visualized with either ECL detection reagents (Amersham Pharmacia Biotech, Arlington Heights, IL) or 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium color substrates (Promega, Madison, WI). Abs for p65 NF-{kappa}B and total and phospho-I{kappa}B{alpha} were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-hemagglutinin (anti-HA) Ab was obtained from Clontech (Palo Alto, CA). To confirm equal protein loading, membranes were stripped and probed with a 1/5000 dilution of mouse anti-{beta}-actin mAb (Sigma-Aldrich, St. Louis, MO).

Preparation of cell lysates and EMSA

Nuclear and whole cell extracts were prepared using the method of Dignam et al. (27) with some modifications. For the preparation of total cell lysates, serum-starved cells were stimulated for 30 min and lysed in buffer A (10 mM HEPES (pH 7.9), 0.2 mM EDTA, 10 mM KCl, 0.2M NaCl, 2 mM MgCl2, 0.5 mM DTT, and 5 µg/ml protease inhibitors) containing 0.2% Nonidet P-40. Lysates were then incubated on ice for 15 min, freeze-thawed three times, and centrifuged at 14,000 x g for 15 min. Supernatants were diluted with 1 vol of buffer A containing 25% (v/v) glycerol without NaCl and were stored frozen at -70°C until use. For the preparation of nuclear extracts, cells were incubated in Nonidet P-40 and protease inhibitors containing buffer A, but without NaCl, and centrifuged at 350 x g for 5 min to remove cytoplasmic proteins. Nuclear pellets were suspended in buffer A containing 0.3 M NaCl and protease inhibitors, incubated on ice for 30 min, and clarified as described above. NF-{kappa}B DNA binding activity was determined by EMSA using the Promega gel shift assay system. The specificity of binding was determined by competition with cold probe and by supershift analysis with anti-p65 and anti-p50 (NF-{kappa}B) Abs. DNA-protein complexes were resolved on 4% nondenaturing polyacrylamide gels. The gels were dried and subjected to autoradiography.

Immunocytochemistry

DLD-1 cells were fixed in 3% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 10 min, washed, and blocked with 2% BSA and 2% goat serum in PBS for 1 h. Cells were incubated in a 1/600 dilution of anti-p65 Ab (NF-{kappa}B subunit; Santa Cruz Biotechnology) and then in biotinylated secondary Ab (1/200 dilution; Vector Laboratories, Burlingame, CA) for 1 h at room temperature. After five washes with 0.2% Triton X-100 in PBS, cells were incubated with rhodamine Red-X-conjugated strepavidin for 15 min, washed, mounted, and viewed under confocal microscopy.

Transfections and luciferase reporter assays

DLD-1 cells were transiently transfected with plasmids containing the HA-tagged, wild-type I{kappa}B{alpha} gene, the dominant negative (DN) I{kappa}B{alpha} (S32A/S36A) gene, or empty vector (provided by Dr. W. Green) using the Superfect reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Cells were allowed to recover for 24 h after transfection and were then serum-starved for 24 h before cytokine stimulation. Where indicated, cells received HB-EGF for 48 h. For luciferase assays, cells (1 x 105) were seeded in 12-well plates 24 h before transfections. A total of 2.7 µg of DNA (0.5 µg of 4xNF-{kappa}B-Luc reporter plasmid, 0.2 µg of pRL-null (Renilla-luciferase; Promega) reporter vector, and 2 µg of I{kappa}B{alpha} construct were cotransfected. The total amount of DNA used was adjusted with empty vector. Cells were lysed 10 h after cytokine exposure, and luciferase activity for both the NF-{kappa}B reporter and the Renilla reporter were measured in a TD 20/20 luminometer (Turner Design, Palo Alto, CA) using the Promega dual luciferase reporter assay system. Firefly luciferase activity of NF-{kappa}B was normalized to the corresponding Renilla-luciferase activity.

I{kappa}B kinase (IKK) assay

Whole cell extracts were immunoprecipitated with IKK-{alpha} Ab (BD PharMingen, San Diego, CA) and subjected to an IKK assay using GST-I{kappa}B{alpha}1–317 as a substrate essentially as previously described (28).

Statistical analysis

All values were expressed as the mean ± SEM of n observations. Statistical analyses were performed using a one-way ANOVA with Scheffé’s post hoc comparisons. A value of p <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HB-EGF inhibits cytokine-stimulated proinflammatory IL-8 and NO production in human intestinal epithelial cells

We initially examined the effect of HB-EGF on the release of IL-8 in IL-1{beta}- plus IFN-{gamma}-stimulated DLD-1 cells. HB-EGF treatment was given before cytokine exposure, simultaneously with cytokine exposure, or both before and with cytokine exposure. Unstimulated DLD-1 cells or cells stimulated with IFN-{gamma} alone or HB-EGF alone did not produce IL-8. Stimulation with either IL-1{beta} alone or IL-1{beta} plus IFN-{gamma} led to an increase in the release of IL-8, which was significantly inhibited (~65–75%) in cells that received HB-EGF pretreatment and cotreatment (Fig. 1A, bar 6; p < 0.001). HB-EGF given as either pretreatment or cotreatment with IL-1{beta} and IFN-{gamma} did not affect the release of IL-8 (Fig. 1A, bars 7 and 8).



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  		FIGURE 1. Effect of HB-EGF on cytokine-induced IL-8 and NO production in DLD-1 cells. A, HB-EGF suppresses cytokine-stimulated IL-8 production. DLD-1 cells (7 x 104) were cultured in 24-well plates, and 24 h later the pretreatment group received HB-EGF (50 ng/ml) for an additional 48 h. Cells were then exposed to various stimuli as indicated. Supernatants were collected 24 h after the addition of cytokines, and IL-8 levels were determined by ELISA. Pre, Pretreatment (received HB-EGF for the first 48 h only); co, cotreatment (HB-EGF added only during IL-1{beta} plus IFN-{gamma} treatment); pre+co, pretreatment and cotreatment. Data are shown as the mean ± SE. *, p < 0.001 (n = 4). B, HB-EGF pretreatment decreases cytokine-stimulated NO production. Supernatants were collected as described above, and NO measurements were performed using an NO analyzer (Sievers). Data represent the mean ± SE of duplicate measurements of four independent experiments. *, p < 0.001 (bars 6 and 7) compared with IL-1{beta}- and IFN-{gamma} only-treated cells (bar 5).

 
Next, we examined the effect of HB-EGF on NO production in IL-1{beta}- plus IFN-{gamma}-stimulated DLD-1 cells. Unstimulated DLD-1 cells or cells stimulated with either IL-1{beta} or IFN-{gamma} alone, did not produce significant levels of NO (Fig. 1B). Treatment of cells for 24 h with a combination of IL-1{beta} and IFN-{gamma} resulted in stimulation of NO production. This IL-1{beta}- plus IFN-{gamma}-activated NO production was decreased significantly in cells pre- and cotreated with HB-EGF (Fig. 1B, bar 6; p < 0.001). However, HB-EGF did not decrease NO production when given only during cytokine stimulation (cotreatment; Fig. 1B, bar 8). The NO inhibitory effect of HB-EGF was intermediate when cells received HB-EGF treatment only before IL-1{beta} and IFN-{gamma} treatment (pretreatment; Fig. 1B, bar 7; p < 0.001). HB-EGF alone had no effect on constitutive NO production (Fig. 1B, bar 2) (21).

Expression of a DN form of I{kappa}B{alpha} inhibits NF-{kappa}B-transcription and NF-{kappa}B DNA binding activity and decreases NO production

NF-{kappa}B has been shown to control iNOS gene transcription induced by LPS and cytokines (10, 29). To determine whether NF-{kappa}B plays a role in IL-1{beta}- and IFN-{gamma}-activated NO production, DLD-1 cells were transiently transfected with DN (S32A/S36A) HA-tagged I{kappa}B{alpha} constructs and then exposed to IL-1{beta} and IFN-{gamma} for 24 h. Due to serine to alanine substitutions at residues 32 and 36, DN-I{kappa}B{alpha} does not undergo phosphorylation and subsequent degradation (30), thus preventing the activation of NF-{kappa}B. IL-1{beta}- plus IFN-{gamma}-activated NO production was inhibited by ~40–50% in DN-I{kappa}B{alpha}-expressing cells compared with vector-transfected cells (Fig. 2A, bar 4).



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  		FIGURE 2. Effect of DN-I{kappa}B{alpha} on NO production and NF-{kappa}B transcriptional activity. A, Transient expression of super-repressor I{kappa}B{alpha} decreases NO production. DLD-1 cells (7 x 104) were transfected with 1 µg each of vector alone or DN-HA-I{kappa}B{alpha} (S32A/S36A). Twenty-four hours after cytokine stimulation, supernatants were analyzed for NO by chemiluminescence. Data were expressed as the mean of duplicates of three independent experiments. *, p < 0.015 (bar 4) compared with the value obtained for empty vector-transfected cells (bar 3). B, Expression of DN-I{kappa}B{alpha} blocks NF-{kappa}B transcriptional activity. DLD-1 cells were cotransfected with 4x-NF-{kappa}B-Luc, pRL-null (Renilla luciferase as an internal control), and HA-DN-I{kappa}B{alpha} or empty vector as indicated. Forty-eight hours post-transfection cells were stimulated as indicated. Ten hours later cells were lysed, and NF-{kappa}B-dependent luciferase reporter and Renilla luciferase activities were determined as described. The results represent the average of duplicate transfections of four independent experiments. Firefly luciferase activity of NF-{kappa}B reporter was normalized to the corresponding Renilla luciferase activity and presented as the fold induction (n = 4). Insets of A, B, and D, Detection of HA-I{kappa}B{alpha} expression by Western blot analysis. A, After collecting supernatants for NO measurements, cell monolayers were lysed in 1x Laemmli buffer and subjected to Western blot analysis using an anti-HA Ab to detect HA-I{kappa}B. B and D, lysates of luciferase and EMSAs were used for Western blot analysis using HA-tag Ab. For insets in A and B: lane 1, vector; lane 2, HA-DN-I{kappa}B{alpha}. C, DN-I{kappa}B{alpha} expression inhibits NF-{kappa}B DNA binding activity. DLD-1 cells (7 x 104) were transfected with 1 µg of vector alone or DN-I{kappa}B{alpha} cDNA constructs using Superfect reagent as described. Forty-eight hours after transfection, cells were exposed to cytokines (IL-1{beta}, 20 ng/ml; IFN-{gamma}, 10 ng/ml) for 30 min, and cell extracts were subjected to EMSA. Lane 1, Unstimulated control; lanes 2–6, IL-1{beta}- plus IFN-{gamma}-stimulated. E, Quantitative analysis of C. The autoradiograph was scanned and quantitated using the gel expert software from Nucleotech (Hayward, CA). Data are presented as a percentage of the total. An arbitrary value obtained for vector-transfected cells was set at 100%.

 
We then examined whether the decreased NO production in the presence of HA-DN-I{kappa}B{alpha} protein was due to impaired transcriptional activation of NF-{kappa}B. To address this question, cells were cotransfected with 4x-{kappa}B-Luc reporter, Renilla luciferase reporter construct, and HA-DN-I{kappa}B{alpha} expression plasmids, and luciferase reporter activity was determined. IL-1{beta} plus IFN-{gamma} induced transcriptional activity of NF-{kappa}B by ~25-fold. The expression of DN-I{kappa}B{alpha} completely abolished IL-1{beta}- plus IFN-{gamma}-induced NF-{kappa}B transcriptional activity (Fig. 2B, bar 5). This suppression of 4x-{kappa}B luciferase reporter activity was not due to cell death, since the Renilla luciferase reporter (internal control) displayed comparable activity to vector-transfected cells (data not shown). In addition, Western blot analysis with anti-HA Ab confirmed the expression of an HA-tagged fusion I{kappa}B{alpha} protein band (Fig. 2B, lane 2 of inset).

To determine whether the suppression of NF-{kappa}B{alpha} transcriptional activity correlated with decreased DNA binding activity, EMSA was performed. Cytokine-induced DNA binding activity of NF-{kappa}B was suppressed by as much as 80% in HA-DN-I{kappa}B{alpha}-expressing cells compared with empty vector-transfected cells (Fig. 2, C and E). HA-I{kappa}B{alpha} protein production was confirmed by Western blot analysis using HA Ab (Fig. 2, B and D). As expected these data demonstrate that in DLD-1 cells, IL-1{beta}- and IFN-{gamma}-activated NO production is in part mediated by NF-{kappa}B. It is worthy of note that although IL-1{beta} alone induced NF-{kappa}B activation (Fig. 3C) and IL-8 production (Fig. 1A), it alone did not induce iNOS mRNA expression (data not shown) or NO production. The induction of NO production required the simultaneous presence of IL-1{beta} and IFN-{gamma} (Fig. 1B).



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  		FIGURE 3. Effect of HB-EGF on cytokine-induced NF-{kappa}B DNA binding activity in DLD-1 and HT-29 cells. A, HB-EGF pretreatment blocks NF-{kappa}B activation in DLD-1 cells. Serum-starved DLD-1 cells were stimulated for 30 min as indicated, and nuclear extracts were prepared for EMSA. Where indicated, cells received HB-EGF pre- plus cotreatment (24-h pretreatment). NS, nonspecific signal. Lane 1 contains extracts from cells treated with vehicle. Competition with cold probe and supershifting by anti-p65- or anti-p50 NF-{kappa}B Ab showed that the retarded band was an NF-{kappa}B-DNA complex. Lane 7 contains a 26-mer nonspecific competitor, and lane 10 contains a goat IgG Ab control. These results are representative of four separate experiments. EMSA performed with total cell lysates showed similar results. C, HB-EGF pretreatment blocks IL-1{beta}-induced NF-{kappa}B activation. Serum-starved DLD-1 cells were stimulated with IL-1{beta} (20 ng/ml) with or without HB-EGF, and EMSA was performed as described in A. Lane 1, IL-1{beta} alone; lane 2, IL-1{beta} plus HB-EGF (cotreatment); lane 3, IL-1{beta} plus HB-EGF (pre- plus cotreatment). D, HB-EGF cotreatment does not block NF-{kappa}B activation. HB-EGF was added to DLD-1 cells simultaneously with IL-1{beta} and IFN-{gamma} (cotreatment; lane 2), and EMSA was performed as described above. E, HB-EGF pretreatment blocks NF-{kappa}B activation in HT-29 cells. EMSA was performed as described in A. The results shown are representative of two independent experiments. B and F, Quantitative analysis of DNA binding activity in A and E. Quantitative analysis of NF-{kappa}B DNA-protein complexes was performed on scanned images. The value shown for IL-{beta} plus IFN-{gamma} treatment is presented relative to that obtained for vehicle-treated control cells (medium alone; set at 1). The value shown for IL-{beta}, IFN-{gamma}, and HB-EGF cotreatment is presented relative to that for HB-EGF alone (set at 1) to show the effect of HB-EGF on IL-{beta}- plus IFN-{gamma}-stimulated NF-{kappa}B DNA binding activity. The data are expressed as the mean ± SE of two independent experiments for HT-29 cells and four independent experiments for DLD-1 cells (n = 5 gel shifts). G, HB-EGF pretreatment suppresses IL-8 release in HT-29 cells. IL-8 release was measured in culture supernatants as described in Fig. 1. The data are expressed as the mean ± SE of triplicate measurements of two independent experiments (n = 2).

 
HB-EGF pretreatment inhibits cytokine-induced NF-{kappa}B DNA binding activity

As stated above, NF-{kappa}B regulates cytokine-stimulated up-regulation of the IL-8 gene, iNOS mRNA expression, and NO production. We therefore tested whether the decreased IL-8 and NO accumulation in cells preconditioned with HB-EGF was due to impaired activation of NF-{kappa}B. Pretreatment of DLD-1 cells with HB-EGF before stimulation with IL-1{beta} and IFN-{gamma} blocked NF-{kappa}B activation (Fig. 3A, compare lanes 5 and 11; and Fig. 3B, bar 4). We also observed low levels of NF-{kappa}B DNA binding in cells that received only HB-EGF (Fig. 3A, lane 2). This NF-{kappa}B complex did not contain the active p50/p65 heterodimer, as the DNA binding was not supershifted with anti-p65 (Fig. 3A, lane 4). It appears that this complex contained p50, as it partially supershifted with p50 Ab (data not shown); however, by immunostaining, p50 was not detected in the nucleus (data not shown). Moreover, HB-EGF did not induce transcriptional activity of NF-{kappa}B (see below). As expected, IL-1{beta} and IFN-{gamma} (Fig. 3A, lane 5) induced NF-{kappa}B DNA binding activity. This activity was competed away with excess unlabeled NF-{kappa}B probe (Fig. 3A, lane 6) and was supershifted with p65 and p50 Abs (subunits of NF-{kappa}B; Fig. 3A, lanes 8 and 9). IL-1{beta}-activated NF-{kappa}B DNA binding activity was also blocked in cells pretreated with HB-EGF (Fig. 3C, lane 3). However HB-EGF cotreatment did not block NF-{kappa}B DNA binding activity (Fig. 3, C and D, lane 2). IFN-{gamma} alone did not activate NF-{kappa}B DNA binding activity (data not shown). These observations suggest that NF-{kappa}B is a component of a pathway leading to NO production in DLD-1, and that HB-EGF pretreatment can interfere with this pathway.

We also examined the effect of HB-EGF on cytokine-induced NF-{kappa}B DNA binding activity in HT-29 intestinal epithelial cells. Pretreatment of HT-29 cells with HB-EGF also suppressed IL-1{beta}- plus IFN-{gamma}-induced NF-{kappa}B DNA binding activity (Fig. 3E, lanes 4 and 8, and Fig. 3F, bars 3 and 4).

HB-EGF decreases IL-8 release in HT29 cells

We then tested whether decreased NF-{kappa}B DNA binding activity correlated with decreased IL-8 release. As shown in Fig. 3G, HB-EGF pretreatment decreased IL-1{beta}- plus IFN-{gamma}-induced IL-8 release in HT29 cells (Fig. 3G, bars 3 and 4).

HB-EGF pretreatment suppresses cytokine-stimulated NF-{kappa}B transcriptional activity

DNA binding alone does not reflect transcriptional activity of NF-{kappa}B. Therefore, to investigate whether suppression of NF-{kappa}B DNA binding activity in HB-EGF-pretreated cells correlated with NF-{kappa}B transcriptional activity, a transient transfection assay with an NF-{kappa}B-dependent reporter (NF-{kappa}B-Luc) was performed. HB-EGF alone did not stimulate NF-{kappa}B transcriptional activity (Fig. 4, bar 2). A 15- to 18-fold higher activity was observed in IL-1{beta}- plus IFN-{gamma}-stimulated cells compared with mock control cells. Pretreatment with HB-EGF markedly suppressed IL-1{beta}- plus IFN-{gamma}-stimulated NF-{kappa}B transcriptional activity (~70–80%; Fig. 4, bar 4). These results correlate with the level of NF-{kappa}B DNA binding activity observed in Fig. 3.



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  		FIGURE 4. Effect of HB-EGF on NF-{kappa}B transcriptional activity. DLD-1 cells were cotransfected with 4xNF-{kappa}B-Luc reporter and pRL-null plasmids, and the firefly luciferase activity of 4xNF-{kappa}B and Renilla luciferase activities were determined using the Promega dual luciferase assay system. The results represent the average of duplicate transfections of three independent experiments. The data for firefly luciferase were normalized to Renilla luciferase activity and plotted as the fold induction. *, p < 0.01 (n = 4).

 
HB-EGF pretreatment blocks nuclear translocation of NF-{kappa}B

To determine whether the decreased NF-{kappa}B trans-activational and DNA binding activities produced by HB-EGF were due to decreased nuclear translocation of NF-{kappa}B, we examined the cellular distribution of NF-{kappa}B. Confocal microscopy of DLD-1 cells stained with an Ab to the p65 subunit of NF-{kappa}B indicated that p65 was cytoplasmic in unstimulated and HB-EGF-stimulated cells (Fig. 5A, panels 1 and 2, respectively), but after 20 min of cytokine stimulation, most of the staining was nuclear (Fig. 5A, panel 3). Pre- and cotreatment of cells with HB-EGF before IL-1{beta} plus IFN-{gamma} stimulation resulted in cytosolic staining of p65 (Fig. 5A, panel 4). In the presence of HB-EGF neutralizing Ab, p65 staining was nuclear, confirming the specificity of the HB-EGF effect (Fig. 5A, panel 5). Similar results were obtained with anti-p50 Ab (data not shown). As expected, transient expression of DN-I{kappa}B{alpha} also led to retention of p65 in the cytoplasm (Fig. 5B, panel 2).



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  		FIGURE 5. Effect of HB-EGF on NF-{kappa}B nuclear translocation. A, HB-EGF pretreatment inhibits cytokine-activated nuclear translocation of NF-{kappa}B. Serum-starved DLD-1 cells were stimulated as described in Materials and Methods. The cellular distribution of NF-{kappa}B was identified by labeling with anti-p65 Ab. Panels 1–5, Confocal images; panel 6a, immunofluorescence; panel 6b, transmission image. B, Transient expression of DN-I{kappa}B{alpha} blocks the cytokine-induced nuclear translocation of NF-{kappa}B. DLD-1 cells were transfected as shown and stimulated with IL-1{beta} and IFN-{gamma} for 30 min. 1, Empty vector transfected; 2, DN-HA-I{kappa}B{alpha} transfected. All images shown represent typical microscopic fields of cells. Transfection efficiency varied from 80–95% between the experiments.

 
Thus, the results of the p65 staining confirm the NF-{kappa}B-Luc reporter activity and EMSA studies and suggest that HB-EGF inhibits DNA binding activity by blocking NF-{kappa}B nuclear translocation.

HB-EGF inhibits NF-{kappa}B activation by preventing cytokine-induced phosphorylation and degradation of I{kappa}B{alpha}

NF-{kappa}B activation usually occurs following degradation of I{kappa}B (16, 17). To determine whether HB-EGF blocks NF-{kappa}B nuclear translocation and DNA binding activity by inhibiting degradation of I{kappa}B{alpha}, we examined the effect of HB-EGF on phosphorylation and degradation of I{kappa}B{alpha} in IL-1{beta}- plus IFN-{gamma}-stimulated DLD-1 cells by Western blot analysis. The results showed significantly decreased phosphorylation (Fig. 6A, lane 4) and degradation (Fig. 6D, lane 4) of I{kappa}B{alpha} in cells that received pre- plus cotreatment with HB-EGF. HB-EGF inhibited I{kappa}B{alpha} phosphorylation by ~75% (Fig. 6C, bar 4) and I{kappa}B{alpha} degradation by ~65–80% (Fig. 6F, bar 4). We have also examined the effects of various stimuli on I{kappa}B{alpha} degradation with time. In IL-1{beta}- plus IFN-{gamma}-treated cells, I{kappa}B{alpha} protein levels decreased with time, approaching ~10% of the total in 30 min, after which the I{kappa}B{alpha} content increased. In cells that received pre- plus co-HB-EGF treatment, the maximum I{kappa}B{alpha} degradation observed was ~30% of the total in 30 min, after which the I{kappa}B{alpha} content increased (data not shown). HB-EGF cotreatment did not inhibit phosphorylation (Fig. 6A, lane 5) or degradation (Fig. 6D, lane 5) of I{kappa}B{alpha}. Densitometric scan revealed an ~15% increase in total I{kappa}B{alpha} content in HB-EGF-pretreated DLD-1 cells (Fig. 6F, lane 2). These data show that HB-EGF blocks cytokine-induced phosphorylation and degradation of I{kappa}B{alpha}.



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  		FIGURE 6. Effect of HB-EGF on phosphorylation and degradation of I{kappa}B{alpha}. A, HB-EGF inhibits phosphorylation of I{kappa}B{alpha}. DLD-1 cells were stimulated as indicated, and cytosolic extracts (20 µg) were subjected to SDS-PAGE and analyzed by Western blot analysis using phospho-I{kappa}B{alpha} Ab. The blot was developed using 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium color reagents (n = 3). B and E, {beta}-Actin Western blot analysis. Blots were stripped and immunoblotted with anti-{beta}-actin Ab to demonstrate equal protein loading in all lanes. Blots were developed using ECL reagents. C and F, Quantitative analysis of A and D. Immunoblots were scanned and quantified. Values shown are presented as a percentage of the total. C, Values for IL-1{beta} and IFN-{gamma} were set at 100%. F, Values for vehicle control (medium) were set at 100% (n = 3). D, HB-EGF pretreatment inhibits degradation of I{kappa}B{alpha}. Cells were stimulated for 30 min, and cytosolic extracts were immunoblotted with anti-I{kappa}B{alpha} Ab. Blots were developed with ECL reagents. This Ab detects both unphosphorylated and phosphorylated forms of I{kappa}B{alpha} (T-I{kappa}B{alpha}; n = 3).

 
HB-EGF blocks cytokine-stimulated IKK activation

The key regulatory step in the NF-{kappa}B activation pathway by IL-1{beta} and other proinflammatory cytokines involves activation of IKK. Therefore, to determine whether HB-EGF blocked NF-{kappa}B activation by inhibiting cytokine-stimulated IKK activation, we performed an in vitro IKK assay using GST-I{kappa}B{alpha} as a substrate. As shown in Fig. 7, IKK activity was significantly increased in cytokine-stimulated DLD-1 cells, and HB-EGF pretreatment significantly decreased the IKK activity. Again HB-EGF cotreatment had no effect on cytokine-stimulated IKK activity. These data demonstrate that HB-EGF abrogates NF-{kappa}B activation via inhibition of I{kappa}B kinase.



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  		FIGURE 7. Effect of HB-EGF on IKK activity. A, HB-EGF blocks cytokine-stimulated I{kappa}B kinase activity. DLD-1 cells were stimulated as indicated for 10 min, and IKK complexes were isolated from whole cell lysates by immunoprecipitation as described in Materials and Methods. The immunoprecipitates were subjected to an IKK assay using GST-I{kappa}B{alpha}1–317 as a substrate. B, Quantitative analysis of A. Autoradiographs were scanned and quantified. The data were presented as the fold induction of kinase activity compared with vehicle-treated control cells (lane 1; n = 2).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Studies from our laboratory and others have shown that HB-EGF acts as a cytoprotective agent. Endogenous HB-EGF protected mouse fibroblasts from H2O2-induced apoptosis (31) and promoted the survival of renal epithelial cells (19) and hepatoma cells (20). Recently, HB-EGF was found to be essential for normal heart function (32). We have shown that HB-EGF protects intestinal epithelial cells from cytokine-induced apoptosis (25). HB-EGF also down-regulated cytokine-induced iNOS expression and decreased NO production in DLD-1 cells (21). In this study we present data demonstrating that HB-EGF exerts an inhibitory effect on cytokine-induced IL-8 and NO production in intestinal epithelial cells (Fig. 1). These data suggest that HB-EGF may play an important role in the inflammatory responses elicited during intestinal injuries. The data also demonstrate that the anti-inflammatory effects of HB-EGF are mediated through its ability to block NF-{kappa}B activation, and this effect is not restricted to one cell line. Proinflammatory cytokines play an important role in the pathophysiology of inflammation in the intestine. Invasion of the intestinal mucosa by pathogenic bacteria led to the expression of a distinct array of proinflammatory cytokines that caused an acute mucosal inflammatory response (33). Our observations suggest an important role for HB-EGF in the modulation of acute inflammatory responses elicited by infection and injury. Thus, HB-EGF may hold promise for anti-inflammatory intervention.

Our studies show that HB-EGF suppresses the release of IL-8 and decreases NO production only in cells that received pretreatment with HB-EGF (Fig. 1B). It is therefore possible that HB-EGF may up-regulate the transcription of an inhibitor protein that regulates the I{kappa}B{alpha} signaling cascade. The analysis of gene expression profiles may shed light on this. Recently, it was shown that preconditioning neurons with erythropoietin led to large increases in steady state NF-{kappa}B activity that provided neuroprotection; however, acute increases in NF-{kappa}B activity contribute to an apoptotic signaling pathway (34). It is conceivable that HB-EGF may increase the steady state activity of an as of yet unknown factor that regulates I{kappa}B{alpha} activation.

In conditions of oxidative stress, such as I/R or chronic inflammation, NO and its metabolites, such as peroxinitrite, may be involved in intestinal epithelial barrier dysfunction. Large amounts of NO produced from iNOS may promote inflammation-induced tissue injury. Indeed, a specific inhibitor of iNOS given orally provided protection in a model of IBD (35). We have shown that administration of HB-EGF protects rat intestine from I/R injury; decreases post-I/R iNOS expression, NO production, and reactive oxygen species production; and decreases neutrophil infiltration and bacterial translocation (22, 23). We have not examined the effects of HB-EGF on endothelial NOS or neuronal NOS, but from the data presented in Fig. 1B it appears that HB-EGF does not alter constitutive NO production in DLD-1 cells. Therefore, HB-EGF could function as a specific inhibitor of iNOS and may be useful as a possible therapeutic agent.

It has been reported that the transcription factor NF-{kappa}B plays a central role in coordinating the production of a wide variety of genes that regulate immune responses. NF-{kappa}B also regulates iNOS gene expression and NO production in response to a variety of stimuli. In the present study we tested the hypothesis that HB-EGF decreases NO production by suppressing NF-{kappa}B activation in DLD-1 cells. The data presented here demonstrate that HB-EGF blocked IL-1{beta}- and IFN-{gamma}-induced NF-{kappa}B DNA binding activity. HB-EGF pretreatment inhibited IKK activation and I{kappa}B{alpha} phosphorylation, thus inhibiting the degradation of I{kappa}B{alpha} and subsequent NF-{kappa}B nuclear translocation, DNA binding, and NF-{kappa}B-dependent transcription. This inhibition of NF-{kappa}B activation was seen only in cells preconditioned with HB-EGF. The inhibition of NF-{kappa}B activation correlated with decreased NO production. Furthermore, a dominant negative form of I{kappa}B{alpha} decreased cytokine-activated NO production. Our data suggest that HB-EGF decreases NO production at least in part by interfering with the NF-{kappa}B signaling pathway.

HB-EGF and the I{kappa}B{alpha} super-repressor blocked cytokine-activated NF-{kappa}B DNA binding activity by as much as 90% (Fig. 3B) and ~80%, respectively (Fig. 2E), whereas the level of inhibition of NO production by both varied from 35–45% (Figs. 1 and 2A). This suggests that the combination of IL-1{beta} and IFN-{gamma} may use NF-{kappa}B-dependent and-independent mechanisms for the activation of NO. It is also possible that cytokines induce transcription factors that bypass the inhibition of iNOS expression and NO production by HB-EGF or other inhibitors of iNOS expression.

Various pharmacological factors, such as steroid hormones, protease inhibitors, and antioxidants, inhibit iNOS mRNA expression by interfering with NF-{kappa}B activation (36, 37, 38). IL-10 inhibited TNF-{alpha}-induced NF-{kappa}B activity (39). Recently, other subclasses of Erb1 receptor ligands, such as epiregulin 1 and TGF-{alpha}, have been implicated as survival factors in bladder cancer patients (40). EGF protected esophageal carcinoma cells from apoptosis (41).

In conclusion, we have demonstrated that HB-EGF inhibits NF-{kappa}B activation and production of IL-8 in intestinal epithelial cells. Additionally, HB-EGF decreased NO production at least in part through the inhibition of NF-{kappa}B activation. We have also elucidated the mechanism of inhibition of NF-{kappa}B activation by HB-EGF. Future experiments will further explore the signal transduction pathways required to inhibit IKK. It is worthy of note that HB-EGF pretreatment had no effect on EGF receptor phosphorylation (data not shown). The inhibition of NF-{kappa}B may represent one of the mechanisms by which HB-EGF exerts its anti-inflammatory and cytoprotective effects. Accumulated evidence shows that NF-{kappa}B is involved in regulation of the acute stress response and in the development of critical diseases in vivo (42, 43). Gene delivery of NF-{kappa}B inhibitors has been considered for the treatment of human diseases. For example, the therapeutic effects of antagonist IL-10 gene transfer were observed in an animal model of arthritis (44). Transfection of NF-{kappa}B decoy reduced the extent of I/R injury in a rat model of myocardial infarction (45). Our current findings support our belief that HB-EGF may hold clinical promise in the treatment of diseases associated with increased NF-{kappa}B activation, such as I/R injury and inflammatory bowel disease.


   Acknowledgments
 
We thank Dr. Warner Greene for the generous gift of I{kappa}B{alpha} constructs, Dr. David Brigstock for helpful suggestions, and Jason Zimmerer for technical help. We also thank Cynthia McAllister for confocal microscopy, Dr. John Hayes for statistics, Carrie Worthington for help with the manuscript, and Brad Hoehne for preparation of the figures.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (GM61193) and the Children’s Research Institute (292699; to G.E.B.) and from the Children’s Hospital Firefighters Endowment (to V.B.M.). Back

2 Address correspondence and reprint requests to Dr. Gail E. Besner, Department of Pediatric Surgery, Children’s Hospital, 700 Children’s Drive, Columbus, OH 43205. E-mail address: besnerg{at}chi.osu.edu Back

3 Abbreviations used in this paper: HB-EGF, heparin-binding epidermal growth factor-like growth factor; DN, dominant negative; HA, hemagglutinin; IBD, inflammatory bowel disease; IKK, I{kappa}B kinase; iNOS, inducible NO synthase; I/R, ischemia/reperfusion. Back

Received for publication June 9, 2003. Accepted for publication September 29, 2003.


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