NF-{kappa}B-mediated IAP expression induces resistance of intestinal epithelial cells to apoptosis after polyamine depletion

Tongtong Zou,1,3 Jaladanki N. Rao,1,3 Xin Guo,1,3 Lan Liu,1,3 Huifang M. Zhang,1,3 Eric D. Strauch,1 Barbara L. Bass,1,3 and Jian-Ying Wang1,2,3

Departments of 1Surgery and 2Pathology, University of Maryland School of Medicine and 3Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201

Submitted 3 November 2003 ; accepted in final form 10 December 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis plays a crucial role in maintenance of intestinal epithelial integrity and is highly regulated by numerous factors, including cellular polyamines. We recently showed that polyamines regulate nuclear factor (NF)-{kappa}B activity in normal intestinal epithelial (IEC-6) cells and that polyamine depletion activates NF-{kappa}B and promotes resistance to apoptosis. The current study went further to determine whether the inhibitors of apoptosis (IAP) family of proteins, c-IAP2 and XIAP, are downstream targets of activated NF-{kappa}B and play a role in antiapoptotic activity of polyamine depletion in IEC-6 cells. Depletion of cellular polyamines by {alpha}-difluoromethylornithine not only activated NF-{kappa}B activity but also increased expression of c-IAP2 and XIAP. Specific inhibition of NF-{kappa}B by the recombinant adenoviral vector containing I{kappa}B{alpha} superrepressor (AdI{kappa}BSR) prevented the induction of c-IAP2 and XIAP in polyamine-deficient cells. Decreased levels of c-IAP2 and XIAP proteins by inactivation of NF-{kappa}B through AdI{kappa}BSR infection or treatment with the specific inhibitor Smac also overcame the resistance of polyamine-depleted cells to apoptosis induced by the combination of tumor necrosis factor (TNF)-{alpha} and cycloheximide (CHX). Although polyamine depletion did not alter levels of procaspase-3 protein, it inhibited formation of the active caspase-3. Decreased levels of c-IAP2 and XIAP by Smac prevented the inhibitory effect of polyamine depletion on the cleavage of procaspase-3 to the active caspase-3. These results indicate that polyamine depletion increases expression of c-IAP2 and XIAP by activating NF-{kappa}B in intestinal epithelial cells. Increased c-IAP2 and XIAP after polyamine depletion induce the resistance to TNF-{alpha}/CHX-induced apoptosis, at least partially, through inhibition of the caspase-3 activity.

programmed cell death; growth arrest; ornithine decarboxylase; I{kappa}B; caspase-3; {alpha}-difluoromethylornithine; intestinal epithelium


APOPTOSIS IS A CENTRAL REGULATOR of tissue homeostasis in multicellular organisms (15, 39, 46). The mammalian intestinal epithelium is continuously renewed and has the most rapid turnover rate of any tissue in the body (17, 26). Epithelial cells replicate in the proliferative zone within crypts and differentiate as they migrate up the luminal surface of intestine to replace lost cells (18, 34). To maintain mucosal homeostasis, the rates of epithelial cell proliferation and cell death must be tightly regulated. Increasing evidence indicates that apoptosis, rather than simple exfoliation of enterocytes, accounts for the majority of cell loss at the luminal surface of intestine (10, 12, 35). Apoptosis also occurs in the crypt area, where this process maintains the balance in cell number between newly divided and surviving cells (12, 33, 35). Maintenance of normal intestinal mucosal epithelial integrity depends on a dynamic balance among cell proliferation, growth arrest, and apoptosis (17, 26, 33, 35, 47). Changing this balance in any direction alters intestinal mucosal homeostasis and has significant pathological consequences. For example, mucosal hyperplasia may result from situations in which the rate of cell proliferation exceeds apoptosis or the rate of cell death falls below the rate of cell production. In contrast, mucosal atrophy may occur if the rate of cell renewal is reduced below the rate of apoptosis or the rate of cell death is increased beyond the rate of cell proliferation.

Normal intestinal mucosal growth requires cellular polyamines that regulate expression of various genes involved in multiple signaling pathways driving different epithelial cell functions (22, 29). Polyamines have been implicated recently in the control of the apoptotic response, but the role of polyamines in apoptotic pathways has been rather controversial, depending on the cell type and death stimulus (11, 24, 36). In normal intestinal epithelial cells, polyamine depletion by inhibition of ornithine decarboxylase, the first rate-limiting step in polyamine synthesis, with {alpha}-difluoromethylornithine (DFMO) does not directly induce cell death but alters susceptibility to apoptotic stimuli (21, 23, 41). Our previous studies (23) and others (32) have shown that polyamine depletion induces nuclear factor (NF)-{kappa}B activation, which is associated with the increased resistance of intestinal epithelial cells to tumor necrosis factor (TNF)-{alpha}/cycloheximide (CHX)-induced apoptosis. Inhibition of NF-{kappa}B activity by its chemical inhibitors sulfasalazine and MG-132 prevents this increased resistance to TNF-{alpha}/CHX-induced apoptosis (23). NF-{kappa}B is an inducible transcription factor and is thought to be the central regulator of transcription of genes involved in apoptosis (3, 14, 16). Under nonstress conditions, NF-{kappa}B is sequestered in the cytoplasm by binding to inhibitory I{kappa}B proteins (3, 30). In response to a host of stimuli, I{kappa}B proteins are phosphorylated and then degraded, allowing free NF-{kappa}B to translocate to the nucleus to activate transcription of specific genes (4, 16). However, the exact downstream targets of activated NF-{kappa}B following polyamine depletion are still unknown in intestinal epithelial cells.

The inhibitor of apoptosis (IAP) family of proteins is a potent natural suppressor of apoptosis and functions by directly inhibiting the activity of caspases, the principal effectors of apoptotic cell death (20, 58). The IAP proteins are defined by a domain of ~70 amino acids named as the baculovirus IAP repeat (BIR), which binds directly to caspases and is absolutely required for the ability of IAPs to suppress apoptosis. In mammalian cells, the IAP family of proteins includes c-IAP1, c-IAP2, X-linked IAP (XIAP), neuronal apoptosis inhibitory protein (NAIP), survivin, BRUCE, and ML-IAP (20). Of these, c-IAP1, c-IAP2, and XIAP exhibit the most structural homology, containing three tandem BIR repeats and a COOH-terminal RING finger domain (20, 58). Recently, it has been shown that expression of c-IAP2 and XIAP is regulated by NF-{kappa}B and that the NF-{kappa}B-mediated IAPs are involved in protecting endothelial cells from TNF-{alpha}/CHX-induced apoptosis (52). The current study tested the hypothesis that c-IAP2 and XIAP are downstream targets of activated NF-{kappa}B following polyamine depletion and play an important role in the observed resistance to TNF-{alpha}/CHX-induced apoptosis in normal intestinal epithelial cells (IEC-6 line). First, we sought to determine whether polyamine depletion increased expression of c-IAP2 and XIAP. Second, we examined whether the observed increase in IAPs was NF-{kappa}B dependent in polyamine-deficient cells. Third, we wanted to define whether increased IAPs induce the resistance to TNF-{alpha}/CHX-induced apoptosis by inhibiting caspase-3 activity. Some of these data have been published previously in abstract form (61).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and supplies. Disposable culture ware was purchased from Corning Glass Works (Corning, NY). Tissue culture media and dialyzed fetal bovine serum (dFBS) were obtained from Invitrogen (Carlsbad, CA), and biochemicals were obtained from Sigma (St. Loius, MO). The double-stranded oligonucleotides used in electromobility shift assay and antibodies against I{kappa}B{alpha}, c-IAP2, and caspase-3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal antibodies against NF-{kappa}B (p65) and XIAP proteins were purchased from BD Biosciences Clontech (Palo Alto, CA), and DFMO was purchased from Ilex Oncology (San Antonio, TX). [{gamma}-32P]ATP (3,000 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). The caspase-3 colorimetric assay kit was purchased from R&D Systems (Minneapolis, MN).

Recombinant adenovirus construction and infection. The recombinant adenoviral vector expressing I{kappa}B{alpha}-superrepressor (mutant I{kappa}B{alpha}) was constructed by using the Adeno-X Expression system (Clontech) according to the protocol recommended by the manufacturer. Briefly, the I{kappa}B{alpha}-superrepressor (I{kappa}BSR) cDNA (S32A/S36A) (37, 60) was cloned into the pShuttle by digesting pCMV-I{kappa}B{alpha}M with BamHI/HindIII and ligating the resulting fragments into the XbaI site of the pShuttle vector. pAdeno-X/I{kappa}BSR (AdI{kappa}BSR) was constructed by digesting pShuttle constructs with PI-SceI/I-CeuI and ligating the resulting fragments into the PI-SceI/I-CeuI sites of the pAdeno-X adenoviral vector. Recombinant adenoviral plasmids were packaged into infectious adenoviral particles by transfecting human embryonic kidney (HEK)-293 cells by using LipofectAMINE PLUS reagent. The adenoviral particles were propagated in HEK-293 cells and purified on CsCl ultracentrifugation. Titers of the adenoviral stock were determined by standard plaque assay. Recombinant adenoviruses were screened for expression of the introduced gene by Western blot analysis using the specific anti-I{kappa}B{alpha} antibody. pAdeno-X, which was the recombinant replication-incompetent adenovirus carrying no I{kappa}BSR cDNA insert, was grown and purified as described above and served as a control adenovirus. Cells were infected by various concentrations of the AdI{kappa}BSR or control vector, and cell samples were collected for various measurements 48 h after the infection.

Cell cultures and general experimental protocols. The IEC-6 cell line was purchased from American Type Culture Collection at passage 13. The cell line was derived from normal rat intestinal crypt cells and was developed and characterized by Quaroni et al. (38). Stock cells were maintained in T-150 flasks in DMEM supplemented with 5% heated-inactivated FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin. Flasks were incubated at 37°C in a humidified atmosphere of 90% air-10% CO2, and passages 15–20 were used in experiments. There were no significant changes of biological function and characterization of IEC-6 cells at passages 15–20 (31, 56).

Experimental design. The first series of studies were to determine whether polyamine depletion was paralleled by increased expression of c-IAP2 and XIAP proteins in IEC-6 cells. The general protocol of the experiments and methods were similar to those described previously (21, 23). Briefly, IEC-6 cells were plated at 6.25 x 104 cells/cm2 and grown in control medium (DMEM + 5% dialyzed FBS + 10 µg/ml insulin and 50 µg/ml gentamicin sulfate) or the DMEM medium containing 5 mM DFMO or DFMO plus 10 µM putrescine for 4 and 6 days. The dishes were placed on ice, and the monolayers were washed three times with ice-cold Dulbecco's phosphate-buffered saline (D-PBS). Levels of c-IAP2 and XIAP proteins were measured by Western blot analysis.

The second series of studies were to determine whether observed increase in c-IAP2 and XIAP protein expression resulted from the NF-{kappa}B activation following polyamine depletion. The increased NF-{kappa}B activity in polyamine-deficient cells was specifically prevented by ectopic expression of I{kappa}B{alpha} superrepressor through the infection with the AdI{kappa}BSR vector, and apoptosis was induced by TNF-{alpha} in combination with CHX. The NF-{kappa}B binding activity and levels of c-IAP2 and XIAP proteins were measured in cells grown in the DMEM medium containing DFMO for 6 days with the infection with AdI{kappa}BSR or control vector during the last 48 h.

The third series of studies were to define the relationship between increased expression of IAPs and the resistance to TNF-{alpha}/CHX-induced apoptosis in polyamine-deficient cells. Functions of IAP proteins were examined by using the specific IAP inhibitor Smac (2, 27). Cells were initially grown in the medium containing DFMO for 5 days, exposed to Smac for 24 h, and then treated with TNF-{alpha} plus CHX. Apoptosis was measured 4 h after administration of TNF-{alpha} and CHX. In addition, the involvement of caspase-3 in the observed role of IAPs in the resistance to apoptosis was also investigated. Levels of procaspase-3, caspase-3 proteins, and caspase-3 activity were measured in polyamine-deficient cells in the presence or absence of Smac.

Western blot analysis. Cell samples, dissolved in ice-cold RIPA buffer (50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA, 1.0% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 2 µg/ml leupeptin, and 2 mM sodium orthovanadate), were sonicated and centrifuged at 14,000 rpm for 15 min at 4°C. The protein concentration of the supernatant was measured by the methods described by Bradford (6), and each lane was loaded with 20 µg of protein equivalent. The supernatant was boiled for 5 min and then subjected to electrophoresis on 10% acrylamide gels according to Laemmli (19). Briefly, after the transfer of protein onto nitrocellulose filters, the filters were incubated for 1 h in 5% nonfat dry milk in 1x TBS-T buffer (Tris-buffered saline, pH 7.4, with 0.1% Tween 20). Immunologic evaluation was then performed overnight at 4°C in 5% nonfat dry milk/TBS-T buffer containing specific antibodies against NF-{kappa}B (p65), I{kappa}B{alpha}, c-IAP2, XIAP, and caspase-3 proteins. The filters were subsequently washed with 1x TBS-T and incubated with the secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. The immunocomplexes on the filters were reacted for 1 min with chemiluminiscence reagent (NEL-100; DuPont NEN).

Preparation of nuclear protein and electrophoretic mobility shift assays. Nuclear proteins were prepared via the procedure described previously (23, 59), and the protein contents in nuclear preparation were determined as described by Bradford (6). The double-stranded oligonucleotides used in these experiments included 5'-AGTTGAGGGGACTTTCCCAGGC-3', which contains a consensus NF-{kappa}B binding site (underlined). These oligonucleotides were radioactively end-labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase (Promega, Madison, WI). For mobility shift assays, 0.035 pmol of 32P-labeled oligonucleotides (~30,000 cpm) and 15 µg of nuclear protein were incubated in a total volume of 25 µl in the presence of 10 mM Tris·Hcl (pH 7.5), 50 mM Na Cl, 1 mM EDTA, 1 mM DTT, 5% glycerol, and 1 µl of poly(dI-dC). The binding reactions were allowed to proceed at room temperature for 20 min. Thereafter, 2 µl of bromphenol blue (0.1% in water) were added, and protein-DNA complexes were resolved by electrophoresis on nondenaturing 5% polyacrylamide gels and visualized by autoradiography. The specificity of binding interactions was assessed by competition with an excess of unlabeled double-stranded oligonucleotide of identical sequence.

Measurement of the caspase-3 activity. The caspase-3 activity was measured by using the caspase-3 colorimetric assay kit (R&D Systems) and performed according to the protocol recommended by the manufacturer. Briefly, cells were treated with TNF-{alpha} and CHX for 4 h, washed with ice-cold D-PBS, and scraped from the dishes. The collected cells were washed with D-PBS and then lysed in ice-cold cell lysis buffer [50 mM HEPES, pH 7.4, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 1 mM DTT, 0.1 mM EDTA, and 0.1% Nonidet P-40]. The assay for caspase-3 activity was carried out in a 96-well plate. In each well, there were 50 µl of cell lysate (~150 µg of total proteins), 50 µl of reaction buffer (50 mM HEPES, pH 7.4, 0.1% CHAPS, 100 mM NaCl, 10 mM DTT, and 1 mM EDTA), 5 µl of caspase-3 colorimetric substrate, and a caspase-specific peptide that is conjugated to a chromogen, p-nitroanilide (p-NA). The 96-well plate was incubated at 37°C for 90 min, during which the caspase-3 in the sample presumably cleaved the chromophore p-NA from the substrate molecule. Absorbency readings at 405 nm were made after the incubation, with the caspase-3 activity being directly proportional to the color reaction. Protein levels of each sample were determined by the method described by Bradford (6).

Assessment of morphology and annexin V staining. After various experimental treatments, cells were photographed with a Nikon inverted microscope before fixation. Annexin V staining of apoptosis was carried out by using a commercial apoptosis kit (Clontech) and performed according to the protocol recommended by the manufacturer. Briefly, cells were rinsed with 1x binding buffer and resuspended in 200 µl of 1x binding buffer. Annexin V (5 µl) was added on the slide and incubated at room temperature for 10 min in the dark. Annexin-stained cells were visualized and photographed under fluorescence microscope with the use of a dual filter set for FITC and rhodamine, and the percentage of "apoptotic" cells was determined.

Assays for DNA fragmentation. DNA from treated cells was assayed by using a modification of the method described by Armstrong et al. (1). Briefly, cells were lysed with 1.0 ml of digestion buffer and incubated at 50°C for 18 h. Samples were extracted twice with 1 volume of phenol-chloroform-isoamyl alcohol, precipitated with 7.5 M ammonium acetate and 100% ethanol, and resuspended in 10 mM Tris·HCl. Samples were then treated with RNase (40 µg/ml) in the presence of 0.1% SDS for 1 h at 37°C. Samples were reextracted, precipitated, and resuspended a second time as described above. DNA (2 µg) were loaded into each well and electrophoresed in 1.5% agarose gel. Gels were visualized by UV fluorescence and photographed with a Polaroid camera system.

Statistics. Values are means ± SE from three to six samples. Autoradiographic results were repeated three times. The significance of the difference between means was determined by ANOVA. The level of significance was determined by using Duncan's multiple-range test (13).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Changes in expression of c-IAP2 and XIAP proteins after polyamine depletion. Our previous studies showed that depletion of cellular polyamines by treatment with DFMO increased the basal levels of NF-{kappa}B proteins, induced NF-{kappa}B nuclear translocation, and activated its sequence-specific DNA binding activity in intestinal epithelial cells (23). To further define downstream targets of activated NF-{kappa}B following polyamine depletion, we focused the current study to determine the involvement of c-IAP2 and XIAP proteins in this process. Consistent with our previous results (23, 31), exposure of IEC-6 cells to 5 mM DFMO for 4 and 6 days almost completely depleted cellular polyamines putrescine, spermidine, and spermine (data not shown). As shown in Fig. 1, basal levels of c-IAP2 and XIAP proteins increased significantly in DFMO-treated cells. The induction of protein levels for c-IAP2 and XIAP occurred at 4 days and remained elevated at 6 days after exposure to DFMO. The levels of c-IAP2 protein in cells treated with DFMO for 4 and 6 days were ~2.4 and ~2.3 times the normal values (without DFMO), respectively (Fig. 1Ba). Although basal expression of XIAP protein was slightly lower than that of c-IAP2 in IEC-6 cells, levels of XIAP also increased significantly and were more than twice the normal values at 4 and 6 days after DFMO treatment (Fig. 1Bb). Putrescine (10 µM) given together with DFMO completely prevented the increased levels of c-IAP2 and XIAP proteins. Spermidine (5 µM) had an identical effect on c-IAP2 and XIAP protein expression when it was added to cultures that contained DFMO (data not shown).



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Fig. 1. Changes in expression of cellular inhibitor of apoptosis protein-2 (c-IAP2) and X-chromosome-linked IAP (XIAP) in control IEC-6 cells and cells treated with either {alpha}-difluoromethylornithine (DFMO) alone or DFMO + putrescine (PUT). Cells were grown in DMEM containing 5% dialyzed FBS in the presence or absence of DFMO (5 mM) or DFMO + PUT (10 µM) for 4 and 6 days. A: representative autoradiograms of Western immunoblots. Whole cell lysates were harvested, applied to each lane (20 µg) equally, and subjected to electrophoresis on 10% acrylamide gel. Levels of c-IAP2 (~75 kDa) and XIAP (~57 kDa) were identified by probing nitrocellulose with the specific antibodies. After the blot was stripped, actin (~42 kDa) immunoblotting was performed as an internal control for equal loading. B: quantitative analysis of Western blots by densitometry from cells described in A. a: c-IAP2; b: XIAP. Values are means ± SE of data from 3 separate experiments; relative levels of c-IAP2 and XIAP were corrected for loading as measured by densitometry of actin. *P < 0.05 compared with control and DFMO + PUT.

 

Inactivation of NF-{kappa}B prevents increased levels of c-IAP2 and XIAP proteins in polyamine-deficient cells. To determine the relationship between activated NF-{kappa}B and increased expression of IAPs following polyamine depletion, we examined the effect of inactivation of NF-{kappa}B by ectopic expression of the I{kappa}B{alpha} superrepressor on levels of c-IAP2 and XIAP proteins in DFMO-treated cells. The adenoviral vector encoding nondegradable I{kappa}B{alpha} mutant (I{kappa}B{alpha} superrepressor) cDNA under the control of the human cytomegalovirus immediate-early gene promoter (AdI{kappa}BSR) was constructed. It has been shown that this mutant I{kappa}B{alpha} protein, whose serines at 32 and 36 are substituted with alanines, does not undergo signal-induced phosphorylation and subsequent degradation (60). The reason we chose adenoviral vector (rendered replication incompetent by deletion of E1 sequences) over other methods of transfection is that adenoviral vectors have been shown to infect a variety of cultured rat and human intestinal epithelial cells with nearly 100% efficiency (25, 40). We have demonstrated that >95% of IEC-6 cells were positive when they were infected with the adenoviral vector encoding GFP that served as the marker for 24 h.

Results presented in Fig. 2A show that I{kappa}B{alpha}-SR protein was expressed in amounts increasing with the AdI{kappa}BSR load in IEC-6 cells. Levels of I{kappa}B{alpha}-SR protein were ~10, ~18, and ~20 times the control values when the AdI{kappa}BSR at concentrations ranging from 10 to 20 to 50 pfu/cell was used, respectively. An adenovirus that lacked exogenous I{kappa}B{alpha} mutant cDNA was used as negative control in this experiment and did not induce I{kappa}B{alpha}-SR protein levels (data not shown). To confirm the function of increased I{kappa}B{alpha}-SR protein in IEC-6 cells, we examined the effect of ectopic expression of I{kappa}B{alpha}-SR protein on the stimulation of NF-{kappa}B nuclear translocation in cells treated with TNF-{alpha} (20 ng/ml). As shown in Fig. 2B, increased I{kappa}B{alpha}-SR by infection with the AdI{kappa}BSR at a concentration of 50 pfu/cell completely prevented TNF-{alpha}-induced nuclear translocation of NF-{kappa}B in IEC-6 cells. Furthermore, the effect of overexpression of I{kappa}B{alpha}-SR on induced NF-{kappa}B sequence-specific DNA binding activity was examined in polyamine-deficient cells. Infection with the AdI{kappa}BSR totally prevented polyamine depletion-induced NF-{kappa}B binding activity in cells treated with DFMO for 6 days (Fig. 2C). In contrast, infection of the control vector at the same concentration did not affect NF-{kappa}B binding activity in DFMO-treated cells.



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Fig. 2. Effect of ectopic expression of the I{kappa}B{alpha} superrepressor (I{kappa}B{alpha}-SR) gene on levels of nuclear NF-{kappa}B protein (p65) and sequence-specific NF-{kappa}B binding activity in control and polyamine-deficient cells. A: representative Western immunoblots of I{kappa}B{alpha}-SR. IEC-6 cells were infected with the recombinant adenoviral vector encoding human I{kappa}B{alpha}-SR cDNA (AdI{kappa}BSR) or an adenoviral vector lacking I{kappa}B{alpha}-SR cDNA at a multiplicity of infection of 1–50 plaque-forming units per cell (pfu/cell). Levels of I{kappa}B{alpha}-SR protein were analyzed by Western blot analysis using the specific monoclonal anti-I{kappa}B{alpha} antibody 48 h after the infection. Actin immunoblotting was performed as an internal control for equal loading. Three separate experiments were carried out that showed similar results. B: effect of overexpression of I{kappa}B{alpha}-SR on levels of nuclear NF-{kappa}B protein (p65) in IEC-6 cells treated with TNF-{alpha}. a: I{kappa}B{alpha}-SR levels; b: nuclear NF-{kappa}B protein (p65) levels. Cells were infected with either the AdI{kappa}BSR or control vector (50 pfu/cell) for 48 h and then exposed to TNF-{alpha} (20 ng /ml). Total and nuclear proteins were prepared 4 h after the treatment with TNF-{alpha}, and levels of cellular I{kappa}B{alpha}-SR protein and nuclear NF-{kappa}B (p65) protein were assayed by Western blot analysis. C: effect of overexpression of I{kappa}B{alpha}-SR on sequence-specific NF-{kappa}B binding activity in polyamine-deficient cells. a: representative autoradiograms of NF-{kappa}B binding. Cells were grown in the cultures containing 5 mM DFMO for 4 days and then incubated with either the AdI{kappa}BSR or control vector (50 pfu/cell) for 48 h in the presence of DFMO. Nuclear extracts were prepared, and electrophoretic mobility shift assay (EMSA) was performed by using 10 µg of unclear proteins and 0.035 pmol of 32P-end-labeled oligonucleotides containing a single NF-{kappa}B binding site. Position of the specifically bound DNA-protein complex is indicated. b: quantitative analysis of EMSA by densitometry from cell described in Ca. Values are means ± SE from 3 separate experiments. *P < 0.05 compared with control and DFMO + AdI{kappa}BSR.

 

Importantly, inactivation of NF-{kappa}B activity by infection with the AdI{kappa}BSR prevented the increase in c-IAP2 and XIAP protein expression in polyamine-deficient cells (Fig. 3). Cells were grown in the cultures containing DFMO for 4 days and then infected with the AdI{kappa}BSR or control vector. Levels of c-IAP2 and XIAP proteins were measured 48 h after the infection in the presence of DFMO. Inactivation of NF-{kappa}B by the infection with the AdI{kappa}BSR significantly blocked the increase in c-IAP2 and XIAP proteins in DFMO-treated cells. In cells infected with the AdI{kappa}BSR, levels of c-IAP2 protein were decreased by ~60% at 10 pfu/cell and ~85% at 50 pfu/cell, respectively (Fig. 3A). Levels of XIAP protein were decreased by ~50% when cells were infected with the AdI{kappa}BSR at the concentration of 50 pfu/cell (Fig. 3B). There were no significant changes in levels of c-IAP2 and XIAP proteins in DFMO-treated cells infected with the control vector.



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Fig. 3. Effect of ectopic expression of I{kappa}B{alpha} superre-pressor on levels of c-IAP2 and XIAP in polyamine-deficient cells. A: changes in c-IAP2 protein. a: representative Western immunoblots of c-IAP2. Cells were grown in the cultures containing 5 mM DFMO for 4 days and then infected with either the AdI{kappa}BSR or control vector at different concentrations for 48 h in the presence of DFMO. Levels of c-IAP2 protein were identified by probing nitrocellulose with the specific antibody. b: quantitative analysis of Western blots by densitometry from cells described in Aa. Values are means ± SE from 3 separate experiments. *P < 0.05 compared with controls. B: changes in XIAP protein in cells described in A. a: representative Western immunoblots of XIAP protein. b: quantitative analysis of Western blots by densitometry from cells described in Ba. Values are means ± SE from 3 separate experiments. *P < 0.05 compared with control.

 

Effect of decreased IAPs by NF-{kappa}B inactivation on TNF-{alpha}/CHX-induced apoptosis. To investigate the involvement of NF-{kappa}B-mediated IAP proteins in the antiapoptotic effect of polyamine depletion, we carried out TNF-{alpha}/CHX-induced apoptosis in control and DFMO-treated IEC-6 cells. As shown in Fig. 4, polyamine depletion by DFMO significantly induced the resistance to TNF-{alpha}/CHX-induced apoptosis. When control cells (without DFMO) were exposed to TNF-{alpha} (20 ng/ml) plus CHX (25 µg/ml) for 4 h, typical morphological features of programmed cell death were identified (Fig. 4A, a vs. b). Annexin V staining showed a significant amount of phosphatidylserine (PS) in the cell membrane, an indicator of apoptotic cells (Fig. 4B, a vs. b). Morphological assessments of apoptosis were confirmed by measurement of internucleosomal DNA fragmentation (Fig. 4C). The classic "ladder" of DNA fragmentation was observed in control cells exposed to TNF-{alpha} plus CHX for 4 h. In polyamine-depleted cells, treatment with the same doses of TNF-{alpha} and CHX caused no significant apoptosis as indicated by morphological features (Fig. 4A, a vs. b), annexin V staining (Fig. 4B, a vs. b), and measurement of DNA fragmentation (Fig. 4C). On the other hand, decreased levels of c-IAP2 and XIAP proteins by inactivation of NF-{kappa}B through infection with the AdI{kappa}BSR blocked the resistance of DFMO-treated cells to TNF-{alpha}/CHX-induced apoptosis. Morphological features (Fig. 4, A and B) and internucleosomal DNA levels (Fig. 4C) in DFMO-treated cells infected with the AdI{kappa}BSR were indistinguishable from those in control cells exposed to TNF-{alpha} plus CHX. The percentage of apoptotic cells was ~27% in DFMO-treated cells infected with the AdI{kappa}BSR compared with 2% in DFMO-treated cells infected with control vector after exposure to TNF-{alpha} plus CHX (Fig. 4C).



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Fig. 4. Apoptosis response of IEC-6 cells to TNF-{alpha} in combination with cycloheximide (CHX) in the presence or absence of cellular polyamines. Cells were grown in control medium or in medium containing 5 mM DFMO for 6 days and then exposed to TNF-{alpha} (20 ng/ml) + CHX (25 mg/ml) for an additional 4 h. In studies dealing with ectopic expression of I{kappa}B{alpha}-SR, cells were treated with DFMO for 4 days, infected with either the AdI{kappa}BSR or control vector at the concentration of 50 pfu/cell for 48 h, and then exposed to TNF-{alpha} + CHX. A: TNF-{alpha}-induced apoptosis in control cells and in cells pretreated with DFMO alone or DFMO-treated cells infected with the AdI{kappa}BSR (DFMO + AdI{kappa}BSR). a: cells treated without TNF-{alpha} + CHX (no-TNF-{alpha}); b: cells treated with TNF-{alpha} + CHX for 4 h (TNF-{alpha}). Original magnification, x150. B: images of apoptotic cells detected by ApoAlert annexin V assays from cells described in A. a: no-TNF-{alpha}; b: TNF-{alpha}. Original magnification, x350. C: induction of DNA fragmentation induced by the combination of TNF-{alpha} and CHX in cells described in A. Genomic DNA was isolated from cells exposed to TNF-{alpha} + CHX for 4 h and analyzed on 1.5% agarose gel. Three experiments showed similar results. D: percentage of apoptotic cells after different treatments. Values are means + SE of data from 3 experiments. *P < 0.05 compared with no-TNF-{alpha}.

 

Effect of the IAP inhibitor Smac on TNF-{alpha}/CHX-induced apoptosis in polyamine-depleted cells. To further define the role of NF-{kappa}B-mediated IAP proteins in the process of apoptosis, we used the specific IAP inhibitor Smac (2, 27) in this study. As shown in Fig. 5, exposure of polyamine-deficient cells to Smac significantly decreased levels of c-IAP2 and XIAP proteins. When Smac at the concentration of 1 µg/ml was added to the medium for 24 h, levels of c-IAP2 and XIAP proteins in DFMO-treated cells were decreased by ~60 and ~50%, respectively. This inhibitory effect of Smac on IAP protein expression is specific because levels of E-cadherin protein were not affected (Fig. 5A). In addition, there was no apparent loss of cell viability in cells treated with DFMO alone or DFMO plus Smac for 5 or 24 h (data not shown).



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Fig. 5. Effect of treatment with Smac on levels of c-IAP2 and XIAP proteins in polyamine-deficient IEC-6 cells. Cells were grown in the presence of DFMO for 6 days, and Smac at the concentration of 1 µg/ml was given during the last 5 or 24 h. A: representative autoradiograms of Western immunoblots for c-IAP2, XIAP, and E-cadherin. Levels of c-IAP2, XIAP, and E-cadherin proteins were identified by Western blot analysis using specific antibodies, and actin immunoblotting was performed as an internal control for equal loading. B: quantitative analysis of c-IAP2 (a) and XIAP (b) immunoblots by densitometry from cells described as in A. Values are means + SE from 3 separate experiments. *P < 0.05 compared with DFMO alone.

 

Inhibition of IAP proteins by treatment with Smac not only enhanced programmed cell death in control cells but also prevented the resistance of polyamine-deficient cells to apoptosis after exposure to TNF-{alpha} plus CHX (Fig. 6). In control cells (without DFMO), typical morphological features of apoptosis increased markedly when cells were pretreated with Smac at the concentration of 1 µg/ml for 24 h (Fig. 6A, b vs. c). The percentage of TNF-{alpha}/CHX-induced apoptotic cells was increased from ~48% in cells without Smac treatment to ~61% in cells pretreated with Smac (Fig. 6B). In DFMO-treated cells, the percentage of TNF-{alpha}/CHX-induced apoptotic cells was increased from ~2% in cells without Smac pretreatment to ~23% in cells pretreated with Smac for 24 h. These findings strongly suggest that NF-{kappa}B-mediated IAP proteins after polyamine depletion play a critical role in the regulation of susceptibility of intestinal epithelial cells to TNF-{alpha}/CHX-induced apoptosis.



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Fig. 6. Effect of treatment with Smac on TNF-{alpha}-induced apoptosis in control and polyamine-deficient cells. A: TNF-{alpha}-induced apoptosis after various treatments. IEC-6 cells were grown in the absence (control) or presence of DFMO for 6 days, and Smac at the concentration of 1 µg/ml was given during the last 24 h. Apoptosis was measured 4 h after exposure to TNF-{alpha} + CHX. a: cells treated without TNF-{alpha}; b: cells exposed to TNF-{alpha} and CHX for 4 h; c: cells treated with Smac for 24 h and then exposed to TNF-{alpha} and CHX. Original magnification, x150. B: percentage of apoptotic cells after various treatments. Values are means ± SE from 3 separate experiments. *P < 0.05 compared with no-TNF-{alpha}. +P < 0.05 compared cells treated with TNF-{alpha} + CHX.

 

Increased IAP proteins inhibit caspase-3 activation in polyamine-deficient cells. Exposure of control cells to TNF-{alpha} and CHX significantly induced caspase-3 activation as indicated by increases in active caspase-3 protein level (Fig. 7A) and enzyme activity (Fig. 7B). In contrast, increased levels of IAP proteins following polyamine depletion were associated with a remarkable inhibition of caspase-3 activation. There was only procaspase-3 detectable in DFMO-treated cells after administration of TNF-{alpha} and CHX, and levels of caspase-3 activity were decreased by >90% in the absence of cellular polyamines. Putrescine given together with DFMO not only prevented the increased IAP proteins but also returned caspase-3 activation to near-normal levels. To further determine the relationship between NF-{kappa}B-mediated IAP expression and caspase-3 activation, we used Smac to specifically block IAPs in polyamine-deficient cells. Results presented in Fig. 8 show that treatment of polyamine-depleted cells with Smac for 24 h dose-dependently restored caspase-3 activation. When polyamine-deficient cells were pretreated with Smac at the concentrations of 0.1 and 1 µg/ml, levels of active caspase-3 protein were increased by ~3.9 and ~4.6 times, and levels of caspase-3 activity were ~1.7 and ~2 times the value of DFMO-treated cells, respectively. These results indicate that inhibition of caspase-3 activation by induced IAP proteins is crucial for the resistance of polyamine-depleted cells to TNF-{alpha}/CHX-induced apoptosis.



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Fig. 7. Changes in caspase-3 protein expression and caspase activity in control IEC-6 cells and cells treated with DFMO alone or DFMO + PUT. A: effect of polyamine depletion on caspase-3 protein expression. a: representative autoradiograms of Western immunoblots. Cells were grown in DMEM containing 5% dialyzed FBS in the presence or absence of DFMO (5 mM) or DFMO + PUT (10 µM) for 4 and 6 days and then exposed to TNF-{alpha} and CHX for 4 h. Whole cell lysates were harvested and applied to each lane (20 µg) equally, and levels of procaspase-3 (~32 kDa) and caspase-3 (~17 kDa) were identified by probing nitrocellulose with the specific anti-caspase-3 antibody. Actin (~42 kDa) immunoblotting was performed as an internal control for equal loading. b: quantitative analysis of caspase-3 immunoblots by densitometry from cells described in Aa. Values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with control and DFMO + PUT. B: changes in caspase-3 activity in cells described in A. Values are means ± SE from 6 samples. *P < 0.05 compared with controls and DFMO + PUT.

 


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Fig. 8. Effect of inhibition of IAPs by treatment with Smac on levels of caspase-3 protein and its enzyme activity in polyamine-deficient cells. A: caspase-3 protein expression. a: representative autoradiograms of Western immunoblots. IEC-6 cells were grown in the presence of DFMO for 6 days, exposed to Smac (0.1 or 1 µg/ml) during the last 24 h, and then treated with TNF-{alpha} plus CHX. Caspase-3 protein was measured 4 h after treatment with TNF-{alpha} plus CHX. b: quantitative analysis of Western blots by densitometry from cells described in Aa. Values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with cells treated with DFMO alone. B: changes in caspase-3 activity in cells described in A. Values are means ± SE from 6 samples. *P < 0.05 compared with the cells treated with DFMO alone.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We (23) and others (32) have recently reported that depletion of cellular polyamines induces NF-{kappa}B activation and promotes resistance to TNF-{alpha}/CHX-induced apoptosis. Inactivation of NF-{kappa}B by its pharmaceutical inhibitors sulfasalazine or MG-132 prevents this antiapoptotic effect in polyamine-deficient cells. The present studies further confirm our previous observations (23) that activated NF-{kappa}B is critical for the resistance of intestinal epithelial cells to apoptosis by demonstrating that specific inhibition of NF-{kappa}B through use of the recombinant adenoviral vector containing I{kappa}B{alpha} superrepressor (AdI{kappa}BSR) blocks the antiapoptotic effect of polyamine depletion on TNF-{alpha}/CHX-induced cell death. The most significant of the new findings reported in this study, however, is that IAP proteins are the downstream targets of activated NF-{kappa}B and play an important role in the increased resistance to TNF-{alpha}/CHX-induced apoptosis. Activation of NF-{kappa}B was associated with an increased expression of c-IAP2 and XIAP proteins in polyamine-deficient cells (Fig. 1), whereas specific inhibition of NF-{kappa}B by infection with the AdI{kappa}BSR prevented the induction of c-IAP2 and XIAP proteins (Fig. 3). Furthermore, decreased c-IAP2 and XIAP proteins by inactivation of NF-{kappa}B through the AdI{kappa}BSR infection (Fig. 4) or treatment with the specific IAP inhibitor Smac (Figs. 5 and 6) decreased the resistance of polyamine-deficient cells to TNF-{alpha}/CHX-induced apoptosis.

Normal physiological regulators and pathological stimuli are shown to induce apoptosis via different signal transduction cascades (10, 12, 18, 34, 35, 53). NF-{kappa}B is the dimeric transcription factor made from monomers that have ~300 amino acid Rel regions that bind to DNA, interact with each other, and bind the I{kappa}B inhibitors (3, 30). In mammalian cells, the NF-{kappa}B family consists of five different subunits, including p50, p52, p65/Rel A, and c-Rel-B (3). On one hand, NF-{kappa}B activation is induced by >150 different stimuli (3, 30). On the other hand, active NF-{kappa}B, in turn, participates in the control of transcription of >150 target genes. Many of the genes that are involved in apoptosis are target genes of NF-{kappa}B. Activated NF-{kappa}B turns on or off transcription of its downstream target genes, interacts with other cellular effectors, and plays distinct roles in regulating apoptosis (3, 30, 54). The natural polyamines spermidine, spermine, and their precursor, putrescine, are cations in eukaryotic cells and regulate expression of various genes, including NF-{kappa}B. In polyamine-deficient IEC-6 cells, activation of NF-{kappa}B is associated with both increased susceptibility to staurosporine (STS)-induced apoptosis and the tolerance to TNF-{alpha}/CHX-induced cell death (23). Inhibition of NF-{kappa}B activity not only prevents the increased susceptibility to STS-induced apoptosis but also blocks the resistance to cell death induced by TNF-{alpha}/CHX. Clearly, the NF-{kappa}B-mediated alteration in the tolerance or the sensitization to apoptosis after polyamine depletion depends on its downstream targets and the pathway engaged by the death stimulus.

The data from the current studies provide new evidence that polyamine depletion-induced NF-{kappa}B stimulates expression of IAPs in normal intestinal epithelial cells. Induced NF-{kappa}B activity was paralleled by increased levels of c-IAP2 and XIAP proteins in polyamine-deficient cells, and inhibition of NF-{kappa}B by infection with the AdI{kappa}BSR prevented the induction of c-IAP2 and XIAP expression. Although the exact mechanisms involved in transcriptional regulation of the IAP genes are still unclear, several studies have demonstrated that NF-{kappa}B plays a critical role in the stimulation of IAP expression in various types of cells. It has been shown that TNF-{alpha}, through either interferon-{alpha} or TNF-{alpha}-related apoptosis-inducing ligand (TRAIL), increases expression of the c-IAP2 and XIAP genes through activation of NF-{kappa}B in both ovarian granulose cells (57) and endothelial cells (52, 55). Similarly, exposure of Jurkat leukemic T cells to the phorbol ester phorbol 12-myristate 13-acetate (PMA) activates NF-{kappa}B and induces expression of IAPs (7). Focal adhesion kinase (FAK) has been shown to activate the phosphatidylinositol 3-kinase (PI3K)/Akt survival pathway and NF-{kappa}B, resulting in upregulation of c-IAP1, c-IAP2, and XIAP in HL 60 cells (49). NF-{kappa}B also interacts with the cAMP signaling pathway and regulates transcription of IAP expression through the cAMP-responsive elements that are present in the promoter of IAP genes (9, 28).

The NF-{kappa}B-mediated IAP expression plays an important role in the regulation of apoptosis in normal intestinal epithelial cells. Decreased IAP proteins by Smac not only enhanced programmed cell death in control cells (without DFMO) but also blocked the increased resistance of polyamine-deficient cells to apoptosis after exposure to TNF-{alpha} and CHX (Fig. 6). These findings are consistent with results from others (20, 42, 45), who have demonstrated that IAP proteins suppress apoptosis induced by a variety of stimuli. The products of the IAP genes are shown to play a role in TNF-{alpha}/CHX-induced apoptosis, and different IAP proteins appear to interfere with the cell death-triggering cascade at various levels. For example, hIAP1 and hIAP2 are shown to bind to the TNF receptor-associated factor-2 (TRAF2), a molecule that is associated with the cytoplasmic part of the TNF receptor complex and is necessary for the activation of other cell survival factors (43, 48). In contrast, XIAP protects embryonic kidney HEK-293 cells from Bax-triggered apoptosis by inhibiting caspase-3 and caspase-7, but it has not been found to be associated with members of the TRAF family (8, 44).

The observations from the current study also indicate that NF-{kappa}B-mediated IAP expression induces the resistance of polyamine-deficient IEC-6 cells to TNF-{alpha}/CHX-induced apoptosis, at least partially, through inhibition of the caspase activity. Caspases are the most extensively investigated activators of apoptosis and directly execute the death program (20, 39). To date, 14 different caspases ranging in size from 32 to 55 kDa have been identified in mammals (46). The caspase-3 is a short proarm caspase that is mostly activated through the action of initiator caspases to result in irreversible cell damage leading to cell death. As shown in Fig. 7, increased expression of IAPs following polyamine depletion was associated with a decrease in caspase-3 activation, which was prevented by treatment with Smac (Fig. 8). Consistent with our findings, Stefanelli et al. (50, 51) have reported that polyamines are necessary for the caspase activation through stimulation of ERK phosphorylation in transformed mouse fibroblasts. Bhattacharya et al. (5) have recently shown that NH2-terminal c-Jun kinase (JNK) is involved in the activation of caspases in response to TNF-{alpha}/CHX, and polyamine depletion prevents the activation of JNK and of caspases-3, -6, -8, and -9. The current studies further demonstrate that induction of NF-{kappa}B-mediated IAP expression also contributes to inhibition of caspase activation in polyamine-deficient cells.

Taken together, the current findings and our previous studies (21, 23) strongly suggest that polyamines are the negative regulators for NF-{kappa}B activation, whereas IAPs are the down-stream intracellular effectors of activated NF-{kappa}B. Decreased polyamines activate NF-{kappa}B and stimulate expression of the IAP genes, leading to the accumulation of IAP proteins. The resultant increase in IAPs in polyamine-deficient cells inhibits the activation of caspases and protects cells from TNF-{alpha}/CHX-induced apoptosis. In contrast, excessive polyamine levels favor TNF-{alpha}/CHX-induced apoptosis by enhancing caspase activation through inhibition of NF-{kappa}B-mediated IAP expression. Because TNF-{alpha} is a biological apoptotic inducer and because the induction of TNF-{alpha} synthesis and release occurs under various physiological conditions, NF-{kappa}B-mediated IAP expression plays a crucial role in the control of intestinal mucosal homeostasis. Polyamines are required for maintenance of intestinal epithelial integrity in vivo in association with their ability to modulate NF-{kappa}B/IAP survival pathway.


    ACKNOWLEDGMENTS
 
GRANTS

This work was supported by Merit Review Grants from the Department of Veterans Affairs (to J.-Y. Wang and B. L. Bass) and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-57819 and DK-61972 (to J.-Y. Wang). J.-Y. Wang is a Research Career Scientist, Medical Research Service, U.S. Department of Veterans Affairs.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J.-Y. Wang, Dept. of Surgery, Baltimore Veterans Affairs Medical Center, 10 North Greene St., Baltimore, MD 21201 (E-mail: jwang{at}smail.umaryland.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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