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Endocrinology Vol. 143, No. 10 3866-3874
Copyright © 2002 by The Endocrine Society

ARTICLE

Dexamethasone and Tumor Necrosis Factor-{alpha} Act Together to Induce the Cellular Inhibitor of Apoptosis-2 Gene and Prevent Apoptosis in a Variety of Cell Types

Jeffrey C. Webster, Reid M. Huber1, Rebecca L. Hanson, Paul M. Collier1, Thomas F. Haws, Juliane K. Mills, Timothy C. Burn1 and Elizabeth A. Allegretto

Departments of Inflammatory Diseases Research (J.C.W., R.L.H., E.A.A.) and Biotechnology (R.M.H., P.M.C., J.K.M., T.C.B.), Bristol-Myers Squibb Pharma, Wilmington, Delaware 19880

Address all correspondence and requests for reprints to: Jeffrey C. Webster, Transtech Pharma, 4170 Mendehall Oaks Parkway, Suite 110, High Point, North Carolina 27265. E-mail: jwebster{at}ttpharma.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Using microarray technology, we analyzed 12,000 genes for regulation by TNF-{alpha} and the synthetic glucocorticoid, dexamethasone, in the human lung epithelial cell line, A549. Only one gene was induced by both agents, the cellular inhibitor of apoptosis 2 (c-IAP2), which was induced 17-fold and 5-fold by TNF-{alpha} at 2 h and 24 h, respectively, and increased 14-fold and 9-fold by dexamethasone at 2 h and 24 h, respectively. The combination of the two agents together led to an additive increase (34-fold) at 2 h and a more than additive effect (36-fold) at 24 h. The human c-IAP2 promoter contains two nuclear factor (NF)-{kappa}B sites that have been shown to be required for transcriptional activation by TNF-{alpha}. To test whether glucocorticoids regulate the c-IAP2 gene at the level of the promoter, a reporter vector containing 947 bases upstream of the start site of transcription of the human c-IAP2 promoter was linked to luciferase [IAP(-947–+54)-LUC] and transfected into A549 cells. Dexamethasone and TNF-{alpha} each induced reporter activity, whereas the combination of the two agents led to greater induction of luciferase than either one alone. Truncation of the promoter region containing a putative glucocorticoid response element (GRE) at -515 [IAP(-395–+54)-LUC] or mutation of the GRE in the context of the natural promoter [IAP(-947–+54mutGRE)-LUC] resulted in a loss of dexamethasone-mediated induction of reporter activity. Although the functional NF-{kappa}B sites were retained in the truncated and mutant c-IAP2 promoter constructs, dexamethasone did not inhibit the TNF-{alpha} induction of luciferase activity, indicating that GR repression through the NF-{kappa}B sites did not occur. Regulation of the c-IAP2 gene is therefore unique, as GR and NF-{kappa}B signaling pathways are usually mutually antagonistic, not cooperative. Treatment of A549 cells with TNF-{alpha} and/or dexamethasone had no effect on cell death, but the two agents were able to inhibit interferon-{gamma}/anti-FAS antibody-mediated apoptosis. In human glioblastoma A172 cells, TNF-{alpha} and dexamethasone together elicited a greater than additive increase in c-IAP2 mRNA levels and also inhibited anti-FAS antibody-mediated A172 cell apoptosis. In contrast, in human CEM-C7 leukemic T cells, whereas TNF-{alpha} and dexamethasone treatment also led to an increase in c-IAP2 mRNA, the two agents were able to induce apoptosis on their own. However, TNF-{alpha} and dexamethasone were also able to blunt anti-FAS-induced apoptosis in the T cells. These data indicate that the induction of the antiapoptotic protein, c-IAP2, by glucocorticoids and TNF-{alpha} correlates with the ability of these agents to inhibit apoptosis in a variety of cell types.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GLUCOCORTICOID RECEPTOR (GR) is a member of the intracellular receptor super family of ligand-activated transcription factors (1). Upon binding to ligand, GR translocates to the nucleus where the induction or repression of glucocorticoid-sensitive genes occurs by a variety of mechanisms (2, 3, 4, 5, 6). Glucocorticoids are essential for survival and are involved in variety of physiological processes including regulation of metabolism, immunity, and apoptosis (7).

Cellular apoptosis is a highly regulated process with a finely tuned balance between apoptotic mediators and antiapoptotic signals. Interestingly, a signal that leads to the induction of apoptosis in one cell type may not in other cell types. For example, glucocorticoids are potent inducers of apoptosis in resting T lymphocytes (8), but they protect against T cell activation-induced apoptosis (9) and serum depletion-induced apoptosis in T cells (10). Glucocorticoids also prevent induction of apoptosis in liver cells (11) and in glioma cells (12, 13). Another player in regulation of apoptosis is nuclear factor (NF)-{kappa}B (14). NF-{kappa}B activation results from signals such as TNF-{alpha} that bind to a membrane receptor allowing rel family members including NF-{kappa}B components such as p65 and p50, to translocate to the nucleus and subsequently modulate gene transcription (15, 16, 17). It is widely accepted that the activation of these rel family members leads to an antiapoptotic phenotype (14). Interestingly, GR and members of the rel family are mutual transcriptional antagonists of each other, as many genes induced by NF-{kappa}B activation are repressed by glucocorticoids and vice versa (7, 18).

Birnbaum et al. (19) and Crook et al. (20) identified a group of proteins from baculoviruses that could inhibit the apoptotic response of insect cells to viral infections. These investigators called this family of proteins the cellular inhibitor of apoptosis proteins (c-IAPs). A mammalian homolog of cIAP-2 protein was initially cloned as a protein that associates with the TNF receptor by interaction with TNF receptor-associated factors (TRAFs) 1 and 2 (21). Members of the IAP family are expressed in many adult and fetal tissues and expression of c-IAP2 message was found to be correlated with the ability of a cell to be protected from serum starvation-induced apoptosis (22). Uren et al. (23) demonstrated that expression of c-IAP2 in HeLa or CHO cells significantly reduced apoptosis mediated by the IL-1 converting enzyme-like proteases. These authors suggested that the interaction of the IAPs with TRAFs may inhibit apoptotic-signaling events by the interruption of activation of the caspase cascade necessary for apoptosis to occur.

TNF has been shown to increase c-IAP2 mRNA levels (24) and two NF-{kappa}B sites within the c-IAP2 promoter have been found to be required for its regulation by TNF (25). Dexamethasone, a synthetic glucocorticoid, was also previously observed to induce the expression of c-IAP2 message and prevent interferon (IFN)-{gamma} and FAS antibody-induced apoptosis in A549 cells; however, extremely high concentrations of dexamethasone (1 mM) were used in this study (26). To gain a better understanding of how these diverse signaling pathways may alter various genes, we performed a gene array analysis on the human lung cell A549. We report herein that dexamethasone and TNF-{alpha} acted together in a potent and more than additive manner to induce c-IAP2 mRNA expression in various cell types. This cooperativity between TNF and dexamethasone was also observed at the level of the human c-IAP2 promoter. Dexamethasone was found to stimulate transcription from the c-IAP2 promoter in a GR-dependent manner through a glucocorticoid response element (GRE) at -513 to -499, and did not repress TNF-induced promoter activity at the NF-{kappa}B sites. Furthermore, the induction c-IAP2 in lung, glioma, and T cells by dexamethasone and TNF-{alpha} correlated with their ability to inhibit apoptosis induced by anti-FAS antibody in these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Dexamethasone (9{alpha}-fluro-16{alpha}-methyl-11ß,17{alpha},21-trihydroxypregn-1,4-diene-3,20-dione) was purchased from Steraloids (Wilton, NH). TNF-{alpha} was purchased from R&D Systems (Minneapolis, MN). RU486 was purchased from Sigma (St. Louis, MO).

Cell culture
A549 human lung epithelial cells and A172 human glioblastoma cells were obtained from ATCC (Manassas, VA) and cultured in DMEM containing 10% FBS. (Life Technologies, Inc., Gaithersburg, MD) at 37 C with 5% CO2 in a humidified incubator. Human CEM-C7 T cells were kindly provided by Dr. John Cidlowski (NIEHS, NC) and were cultured in Roswell Park Memorial Institute 1640 media with 10% FBS, as previously described (27).

Microarray expression profiling
Sequence verified human EST cDNA clones enriched for annotated genes (plates 1–30, 55, 86–90, 95–98, 109, 110, 157, and 158) were purchased from Research Genetics, Inc. (Huntsville, AL). cDNA inserts from 4207 clones and 32 positive and negative controls were PCR amplified and purified using Qiaquick 96 PCR purification kits (QIAGEN, Valencia, CA). Single, homogeneous amplification products were verified for 96% (4070) of amplified clones by agarose gel electrophoresis. Purified products were lyophilized at 80 C and the resultant cDNA pellets were resuspended in 6 M NaSCN (final concentration of 150 ng/µl) and rearrayed into 384-well printing plates. Microarrays were printed in duplicate using a Generation III Array Spotter and slides (Molecular Dynamics, Inc., Sunnyvale, CA). After printing, microarrays were baked at 80 C for 24 h and stored vacuum desiccated. RNA was extracted from cells using RNeasy columns (QIAGEN). Fluorescently labeled cDNA was synthesized from 75–100 µg of total RNA by anchored oligo(deoxythymidine)-primed polymerization using Superscript II reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD). Final reaction concentrations were as follows: 1x first strand buffer, 10 mM dithiothreitol, 100 nM deoxy (d)ATP/dGTP/dTTP, 50 nM unlabeled dCTP, 50 nM Cy3- or Cy5-labeled dCTP (Amersham Pharmacia Biotech, Piscataway, NJ) and 10 U/µl Superscript II. Unincorporated nucleotides were removed by Qiaquick spin columns (QIAGEN) and dye incorporation was determined spectrophotometrically. Fluorescently labeled cDNAs were mixed in equal dye concentrations (50 pM each) and concentrated using Microcon-30 columns (Millipore Corp., Bedford, MA). Concentrated probes were added to hybridization solution (1x Version 2 Hybridization buffer (Amersham Pharmacia Biotech), 50% formamide, 75 ng/µl poly(A)80 and 75 ng/µl human CoT-1 DNA), denatured by boiling, and applied to isopropanol-washed, denatured microarrays under a 22 x 60-mm glass coverslip. Microarrays were incubated at 42 C for 16 h in humidified CMT hybridization chamber (Corning, Inc., Corning, NY). Following hybridizations, arrays were washed in 2x SSC/0.1% sodium dodecyl sulfate at 55 C for 5 min, 1x SSC/0.1% sodium dodecyl sulfate at 55 C for 5 min, 0.1x SSC at room temperature for 10 sec, and distilled H2O at room temperature for 10 sec. Arrays were dried under compressed air and scanned using Generation III Array Scanner (Molecular Dynamics, Inc.). All microarray hybridizations were performed in duplicate with fluorescent dye reversal.

Microarray data analysis
Cy3 and Cy5 TIFF images were analyzed using Autogene 2.0 (Biodiscovery, San Diego, CA). Tab-delimited text outputs were imported directly into Resolver 2.0 under an empirically-derived Autogene error model (Rosetta Biosoftware, Seattle, WA). This error model is based on a series of self vs. self control hybridizations that allow for the determination of the inherent variability within the Molecular Dynamics, Inc./Autogene system, and the identification of raw data parameters associated with that variability. Accordingly, the statistical significance (P value) of a given expression data point takes into account 1) the underlying error associated with the Autogene transcript abundance, and 2) the variability across multiple measurements within one sample (n = 4 per treatment group). The null hypothesis for this P value is that the transcript has a unity expression ratio. Clustering analysis was performed using an agglomerative hierarchical clustering algorithm where error-weighted log (ratio) correlation coefficients are used as similarity measurements.

Expression profiling by real-time PCR
Real-time PCR was performed essentially as described (28). The probe for c-IAP2 (5'-TTGGCATGTTGAACCCATGGATCATCT) was modified at the 5' end with 6-FAM and at the 3' end with TAMRA by Biosearch Technologies, Inc. (Novato, CA). The 18S rRNA probe (5'-TGCTGGCACCAGACTTGCCCTC) was modified at the 5' end with VIC and at the 3' end with TAMRA. Primers were as follows. c-IAP2: 5'-GGACTCAGGTGTTGGGAATCTG, 5'-CAAGTACTCACACCTTGGAAACCA; 18S rRNA: 5'-CGGCTACCACATCCAAGGAA, 5'-GCTGGAATTACCGCGGCT. Total RNA from various cell samples was prepared using the RNeasy purification system (QIAGEN). cDNA syntheses were performed using the Advantage RT-PCR kit (CLONTECH Laboratories, Inc., Palo Alto, CA). Briefly, 1 µg of total RNA from each tissue was treated with deoxyribonuclease I and reverse-transcribed using random hexamers and murine Moloney leukemia virus reverse transcriptase. For Taqman-based real-time PCR expression profiling, 25 ng of each cDNA was added to the Taqman Universal PCR Mater Mix along with 900 nM of each primer and 200 nM of probe according to the manufacturer’s (Applied Biosystems, Inc., Foster City, CA) instructions. Real-time fluorescence monitoring was done using a Perkin-Elmer 7700 instrument. Relative expression levels of the various transcripts were determined essentially as described (28). Briefly, standard curves were generated for each transcript using a serial dilution of human liver cDNA. Relative abundance was then determined by comparing the cycle threshold values for each reaction with this standard curve. Abundance levels calculated from negative control reactions performed in the absence of reverse transcriptase were then subtracted from experimental sample abundance. Variations in input cDNA mass were corrected by normalizing all data to 18S rRNA levels. All measurements were performed in duplicate in two independent experiments.

Promoter constructs
Luciferase reporter constructs were generated by PCR amplification of A549 cell genomic DNA using the following human c-IAP2 promoter (25) primers: -9475'-CTGGTTGGTAATTGTCTTTGAT, -3955'-GTGTATGGCGGATGGAGGGTGGA, and +545'-GCATGCACCAGCAAGGACAAGCC). The resulting PCR products were cloned into pcDNA2.1 Topo (Invitrogen, Carlsbad, CA), sequence confirmed, and subcloned into pGL2-basic-LUC (Promega Corp., Madison, WI) at the XhoI and HindIII sites, yielding IAP(-947–+54)-LUC and IAP(-395–+54)-LUC, respectively (see Fig. 1Go). The putative GRE at -513 to -499 was mutated by conversion of the G at -512 to T and the C at -509 to T using the QuikChange system (Stratagene, La Jolla, CA), yielding IAP(-947–+54mutGRE)-LUC (see Fig. 1Go). Identities of all constructs were confirmed by sequencing.



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  		Figure 1. Schematic representation of c-IAP2 promoter-reporter constructs. Human c-IAP2 promoter sequences (25 ) were cloned into the pGL2-luciferase basic vector as described in Materials and Methods. Mutations in the putative GRE at -513 to -499 are underlined.

 
Transfection and luciferase assays
A549 cells (106) were washed twice with PBS, once with OPTI-MEM reduced serum media (Life Technologies, Inc.) and then suspended in 60 µl of OPTI-MEM media. Plasmid DNA (1–2 µg) was added to 40 µl of OPTI-MEM media and the 60 µl cell suspension in an electroporation cuvette with gentle mixing. Electroporation was carried out at 200 V and 500 µF using a Bio-Rad Laboratories, Inc. (Hercules, CA) apparatus. The cells were allowed to stand for 10 min at room temperature and then were plated in 96-well tissue culture dishes in DMEM. Various concentrations of TNF-{alpha} and/or dexamethasone were added to the cells in fresh DMEM containing charcoal-activated serum for 24 h at 37 C. Cells were then washed with PBS and lysed in the plate wells by addition of 100 µl Luclite lysis buffer B/luciferin (Packard, Meriden, CT). The plate was allowed to stand at room temperature for 5 min and then the light output was measured for 20 sec using a Packard TopCount instrument.

Apoptosis assays
Cells were cultured in appropriate media containing 10% charcoal treated FBS and agents were added at the same time and allowed to incubate for 24 h at 37 C. The ADP/ATP ratio was measured using the ApoGlow adenylate nucleotide ratio assay from Lumitech (Nottingham, UK). Cells were processed following the manufacturer’s protocol. Microplates were read with a 20 sec/well interval. Caspase 8 activity was measured by the cleavage of the peptide IETD-pNA using the ApoAlert Caspase Assay kit (CLONTECH Laboratories, Inc.). The accumulation of the chromophore p-nitroaniline was measured at 405 nm using a microplate reader. Cellular viability was measured using trypan blue exclusion. An aliquot of cells was mixed with an equal volume of 0.1% trypan blue. Cells were then examined using a hemocytometer with four separate fields. All experiments were done in triplicate and SDs were calculated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of c-IAP2 mRNA by dexamethasone and TNF-{alpha} in A549 cells
Microarray analysis was performed on RNA samples from the GR-expressing human lung epithelial A549 cell line (29) treated with vehicle, TNF-{alpha} (1 ng/ml), dexamethasone (100 nM), or a combination of TNF-{alpha} and dexamethasone at 2 and 24 h. Genes that are known to be up-regulated by glucocorticoids, such as the metallothioneins and 11-ß-hydroxy-steroid dehydrogenase, were induced by dexamethasone in this microarray study. Also, TNF-{alpha}-sensitive genes such as TRAF family members were likewise stimulated by TNF-{alpha} treatment in this study (data not shown). Surprisingly, of the 12,000 genes examined, only one message was significantly (P = 0.01) increased in the presence of both TNF-{alpha} and dexamethasone. This gene, the cellular inhibitor of apoptosis 2 (c-IAP2, accession no. NM_001165), was induced by either TNF-{alpha} or dexamethasone alone, and to a greater extent with both agents together (Table 1Go). The levels of c-IAP2 RNA in the presence of TNF-{alpha} were increased 17.3-fold over control after 2 h and 4.6-fold after 24 h. Dexamethasone treatment led to a 13.6-fold increase in c-IAP2 message after 2 h and a 9.4-fold increase after 24 h. A549 cell treatment with both agents together led to an additive increase (33.9-fold) in c-IAP2 mRNA at the 2 h time point and a more than additive effect (36.2-fold) at 24 h. Real-time PCR analyses of independently treated A549 cell samples were performed and the observed fold increases in c-IAP2 RNA levels were in agreement with the results obtained with the microarray analysis (Table 1Go).


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  		Table 1. Induction of c-IAP2 mRNA in human lung epithelial A549 cells

 
The human c-IAP2 promoter contains a functional GRE
The human c-IAP2 promoter has been cloned previously and two of the three potential NF-{kappa}B sites (-210 and -147 relative to the start site of transcription) were found to be responsible for its transcriptional regulation by TNF (25). A reporter vector was constructed using the human c-IAP2 promoter sequence (-947 to +54) driving luciferase expression IAP(-947–+54)-LUC (Fig. 1Go). A549 cells were transiently transfected with IAP(-947–+54)-LUC and subsequently treated with dexamethasone alone or in combination with TNF-{alpha}. A dose-dependent increase in luciferase production was observed with increasing amounts of TNF-{alpha} or dexamethasone (Fig. 2AGo). The two agents together yielded additive or more than additive increases in luciferase activity (Fig. 2AGo). The EC50 value (~1 nM) for dexamethasone- mediated induction of luciferase activity was not altered by the addition of increasing concentrations of TNF-{alpha}. The GR antagonist RU486 was able to reverse the dexamethasone-stimulated increase in luciferase activity, indicating that the effect of dexamethasone to activate transcription from the c-IAP2 promoter was GR dependent (Fig. 2BGo). Similar effects of TNF-{alpha} and dexamethasone were also observed upon transfection of human embryonic kidney cells (HEK 293) with IAP(-947–+54)-LUC (data not shown).



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  		Figure 2. Dexamethasone and TNF-{alpha} act in an additive or more than additive manner to drive transcription from the human c-IAP2 promoter. A549 cells were transfected with IAP(-947–+54)-LUC and treated for 24 h with the indicated concentrations of dexamethasone (A and B), dexamethasone with RU486 (B), or dexamethasone with TNF-{alpha} (A, 0 ng/ml, closed square; 0.01 ng/ml, open circle; 0.1 ng/ml, inverted closed triangle; 1 ng/ml, open square, and 10 ng/ml, closed circle). Cells were lysed, and relative light units (RLU) were measured. Representative experiments from three separate assays are shown.

 
Truncation of the c-IAP2 promoter region containing a putative GRE at -513 to -499 [IAP(-395–+54)-LUC, see Fig. 1Go] resulted in a complete loss of glucocorticoid-induced luciferase activity (Fig. 3AGo). However, TNF-{alpha} induction of this c-IAP2 promoter construct was maintained (Fig. 3AGo). Interestingly, the addition of pharmacologic doses of dexamethasone up to 10 µM did not have an effect on TNF-{alpha}-mediated induction of IAP(-395–+54)-LUC activity, indicating that the commonly-observed ability of glucocorticoids and GR to repress transcription through NF-{kappa}B, was not observed with this gene. Upon mutation of the putative GRE to a nonfunctional binding site (30) in the context of the natural c-IAP2 promoter sequence [IAP(-947–+54mutGRE)-LUC], dexamethasone treatment of transfected A549 cells also did not result in induction of luciferase activity (Fig. 3BGo). Therefore, the sequence from -513 to -499 in the human c-IAP2 promoter represents a bona fide GRE. Dexamethasone also had no inhibitory effect on the induction of IAP(-947–+54mutGRE)-LUC by TNF, confirming that GR was unable to repress transcription through the NF-{kappa}B sites in the cIAP2 promoter (Fig. 3BGo).



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  		Figure 3. Human cIAP-2 promoter responsiveness to dexamethasone is conferred by a GRE at -513. A549 cells were transfected with IAP(-395–+54)-LUC (A) or IAP(-947–+54mutGRE)-LUC (B) and treated for 24 h with TNF-{alpha} (0 ng/ml, closed square; 0.01 ng/ml, closed triangle; 0.1 ng/ml, open circle; 1 ng/ml, inverted closed triangle; and 10 ng/ml, open square) and the indicated concentrations of dexamethasone. Cells were lysed, and relative light units (RLU) were measured. Representative experiments from three separate assays are shown.

 
Dexamethasone and TNF-{alpha} act together to suppress apoptosis in A549 lung cells
To determine if the increase in c-IAP2 mRNA observed upon treatment of A549 cells with TNF-{alpha} and dexamethasone might correlate with the ability of these agents to abrogate the induction of apoptosis in these cells, A549 cells were treated with IFN-{gamma} and anti-FAS antibody to initiate apoptosis (26). Treatment of the cells with dexamethasone or TNF-{alpha} was able to prevent A549 cell death induced by the IFN-{gamma}/anti-FAS stimuli, as assessed by a decrease in the ADP/ATP ratio (Fig. 4AGo). Another marker of apoptosis, caspase 8 (31), which has previously been shown to be directly inhibited by c-IAP1 and c-IAP2 (32), was also decreased by cell treatment with dexamethasone or TNF-{alpha} (Fig. 4BGo). Dexamethasone and TNF-{alpha} together were more effective inhibitors of apoptosis than either treatment alone (Fig. 4Go, A and B). Dexamethasone and/or TNF-{alpha} alone had no effect on A549 cell viability as assessed by trypan blue exclusion, whereas anti-FAS/IFN-{gamma} treatment led to 59% cell death (Table 2Go). Cotreatment of the cells with dexamethasone and the apoptotic stimuli decreased the number of dead cells from 59% to 22%. TNF treatment decreased the number of dead cells to 25% and a combination of dexamethasone and TNF led to only 4% nonviable cells (Table 2Go).



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  		Figure 4. Dexamethasone and TNF-{alpha} act together to abrogate apoptosis in human lung A549 cells. A549 cells were treated for 24 h with TNF-{alpha} (0 ng/ml, closed square; 0.01 ng/ml, closed triangle; 0.1 ng/ml, open circle; 1 ng/ml, inverted closed triangle; and 10 ng/ml, open square) and the indicated concentrations of dexamethasone. The ADP to ATP ratio was calculated and expressed as a percent (A), and caspase 8 activity was measured by absorbance of a cleaved colorimetric product at 405 nm (B). Data shown represent the average of three separate experiments.

 

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  		Table 2. Cell viability of various cell types in response to anti-FAS, IFN-{gamma}, dexamethasone, and TNF-{alpha}

 
Dexamethasone and TNF-{alpha} increase c-IAP2 levels and prevent apoptosis in a glioblastoma cell line
We next examined whether the induction of the c-IAP2 gene and subsequent rescue from apoptosis could occur in other cell types. Because dexamethasone is known to prevent apoptosis in certain brain cells (33), expression of c-IAP2 was examined in the GR-expressing glioblastoma cell line, A172 (34). A172 cell c-IAP2 mRNA was increased in a dose-dependent manner by treatment with either TNF-{alpha} or dexamethasone (Table 3Go). A 3.5-fold induction in A172 cell c-IAP2 message was observed with 100 nM dexamethasone and an 8.5-fold increase resulted with 1 ng/ml TNF-{alpha}. These increases were 2- to 4-fold less than those observed in A549 cells (Table 1Go). The induction of c-IAP2 message in A172 cells was increased in a greater than additive manner (18.9-fold) upon cell treatment with both 100 nM dexamethasone and 1 ng/ml TNF (Table 3Go).


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  		Table 3. Fold Induction of c-IAP2 mRNA in human brain A172 and T-lymphocyte CEM-C7 cells

 
A172 cells were next treated with anti-FAS antibody to stimulate apoptosis. Dexamethasone and TNF-{alpha}-were able to prevent A172 cell death as determined by a decreased ADP/ATP ratio (Fig. 5AGo) and by lowered caspase 8 activity (Fig. 5BGo). Dexamethasone and TNF-{alpha} treatment was also able to prevent A172 cell death induced by anti-FAS antibody as assessed by trypan blue exclusion (Table 2Go). Anti-FAS treatment led to 42% dead cells, whereas addition of TNF or dexamethasone led to less nonviable cells (20% and 21%, respectively). As was observed with the A549 lung cells, the combination of dexamethasone and TNF-{alpha} together was more effective in preventing cell death (4% dead cells) in A172 glioblastoma cells than either agent alone.



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  		Figure 5. Dexamethasone and TNF-{alpha} act together to abrogate apoptosis in human glioblastoma A172 cells. A172 cells were treated for 24 h with TNF-{alpha} (0 ng/ml, closed square; 0.01 ng/ml, closed triangle; 0.1 ng/ml, open circle; 1 ng/ml, inverted closed triangle; and 10 ng/ml, open square) and the indicated concentrations of dexamethasone. The ADP to ATP ratio was calculated and expressed as a percent (A), and caspase 8 activity was measured as absorbance of a cleaved colorimetric product at 405 nm (B). Data shown represent the average of three separate experiments.

 
Regulation of c-IAP2 mRNA and apoptosis in CEM-C7 T-lymphocytes
In contrast to brain or lung cells, glucocorticoids are known to stimulate T cell apoptosis (35, 36). However, they can also protect against T cell activation-induced apoptosis (9) and serum-depletion-induced T cell death (10). In the human T cell line, CEM-C7, dexamethasone and TNF-{alpha} have each been shown to elicit an apoptotic response (37). We next examined whether dexamethasone and/or TNF would regulate c-IAP2 mRNA levels in CEM-C7 cells. Increases in c-IAP2 mRNA were observed in the T cells with dexamethasone and TNF-{alpha}; however, the effects were less than those observed in the lung and brain cells (Tables 1Go and 3Go). A 2.1-fold increase in c-IAP2 mRNA was observed with 100 nM dexamethasone and a 3.2-fold increase resulted with 1 ng/ml TNF (Table 3Go). The combination of 100 nM dexamethasone and 1 ng/ml TNF led to a 7.2-fold increase in the c-IAP2 message.

TNF-{alpha} or dexamethasone treatment of CEM-C7 cells stimulated these cells to undergo apoptosis as assessed by an increased ADP/ATP ratio (Fig. 6AGo) and increased caspase 8 activity (Fig. 6BGo). The two agents together were more efficacious than either one alone. As expected, treatment of CEM-C7 cells with dexamethasone and/or TNF-{alpha} alone also led to decreased cell viability (Table 2Go). However, upon treatment of CEM-C7 cells with another inducer of apoptosis, anti-FAS antibody, addition of TNF and/or dexamethasone retarded the action of anti-FAS antibody. With anti-FAS antibody alone, 63% of the cells were dead, whereas cotreatment with dexamethasone resulted in 47% dead cells (Table 2Go). TNF-{alpha} also decreased the number of dead cells from 63% with anti-FAS antibody alone to 47%. The combination of TNF-{alpha} and dexamethasone administered with the anti-FAS antibody led to 35% nonviable cells. Therefore, whereas dexamethasone and TNF have the ability to kill T cells, they can also induce an inhibitor of apoptosis, c-IAP2, in these cells, and this may result in lessened killing by other apoptotic signals.



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  		Figure 6. Dexamethasone and TNF-{alpha} act together to induce apoptosis in human CEM-C7 T cells. CEM-C7 T cells were treated for 24 h with TNF-{alpha} (0 ng/ml, closed square; 0.01 ng/ml, closed triangle; 0.1 ng/ml, open circle; 1 ng/ml, inverted closed triangle; and 10 ng/ml, open square) and the indicated concentrations of dexamethasone. The ADP to ATP ratio was calculated and expressed as a percent (A), and caspase 8 activity was measured as absorbance of a cleaved colorimetric product at 405 nm (B). The data shown represent the average of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Apoptosis is an important and complex cellular process that is highly regulated. Several signaling pathways are involved in its coordination, and vary according to cell type. Two such apoptotic signaling pathways are driven by the transcription factors NF-{kappa}B and GR. Herein we have studied the effect of an NF-{kappa}B activator, TNF-{alpha}, and a GR agonist, dexamethasone, on one gene involved in the control of apoptosis, c-IAP2.

Glucocorticoids are known antiinflammatory agents that inhibit the expression of a variety of genes involved in the inflammation process, including cytokines and cellular adhesion molecules. Many of these genes are induced by activated NF-{kappa}B (38). There are a number of proposed mechanisms for the interference of NF-{kappa}B signaling by GR (39). One such mechanism involves the direct interaction of GR with members of the rel family (18, 38). One model for GR-NF-{kappa}B interaction involves tethering of GR with NF-{kappa}B at the NF-{kappa}B binding sites in regulatory regions of genes such as those of IL-8 and intracellular adhesion molecule (ICAM)-1 (40). Interestingly, the antagonism between GR and rel members can be mutual, in that rel family members are able to abolish the transactivation of glucocorticoid-sensitive genes, such as surfactant protein B (41, 42).

Here we have demonstrated that NF-{kappa}B and GR, two usually mutually antagonistic signals, can act together to induce the expression of the antiapoptotic gene, c-IAP2. This gene was previously shown to be induced by TNF-{alpha} (24) through two NF-{kappa}B sites within the c-IAP2 promoter (25). We have now established that the regulation of this promoter also occurs by glucocorticoids in a GR-dependent mechanism through a GRE at -513 to -499 within the promoter (Figs. 2Go and 3Go). Dose curves indicated that the EC50 values of dexamethasone induction from the c-IAP2 promoter were approximately 1–2 nM. Upon addition of TNF with dexamethasone, the promoter activity was increased in an additive or more than additive manner (Fig. 2Go). Increased TNF-{alpha} concentration did not cause a shift in the EC50 values for dexamethasone, indicating that these two signals seemed to be acting independently of one another. Truncation or mutation of the GRE within the c-IAP2 promoter-driven reporter vector led to a loss of luciferase induction by dexamethasone. Interestingly, there was also no inhibitory activity of dexamethasone through the NF-{kappa}B sites in the truncated or mutated promoter constructs (Fig. 3Go), indicating that the expected GR repression of TNF-signaling through NF-{kappa}B did not occur from this promoter.

Why certain NF-{kappa}B sites are targets for repression by GR and others are not has been an interesting question for some time. Nissen and Yamamoto (40) have shown that two genes that are repressed by GR at their NF-{kappa}B sites (ICAM-1 and IL-8) and one that is not (I{kappa}B{alpha}), each contain GR in a complex with NF-{kappa}B at their promoters. However, while GR interfered with phosphorylation of serine 2 of the associated RNA polymerase II carboxy-terminal domain at both the ICAM-1 and IL-8 promoters, it did not at the I{kappa}B{alpha} promoter. The hypothesized discrete differences between these promoters that may allow for this selectivity have not been identified; however, c-IAP2 may be similar to I{kappa}B{alpha} in that polymerase activity is not hindered by GR at the NF-{kappa}B sites within its promoter. Detailed comparative analyses of these promoters will be of interest and may elicit mechanistic insight into their differences.

The cooperative effects of TNF and dexamethasone that were observed on the induction of A549 lung cell, A172 glioma cell, and CEM-C7 T cell c-IAP2 message and on transcriptional activation from the c-IAP2 promoter in transfection experiments were also manifested in the ability of these two agents to prevent apoptosis in these cell lines. While TNF-{alpha} and dexamethasone are known inducers of apoptosis in CEM-C7 T cells, they were also able to induce c-IAP2 message in these cells, although not to the same extent as in A549 or A172 cells (Tables 1Go and 3Go). It should be noted that the basal levels of c-IAP2 message were similar among the three cell lines examined. While c-IAP2 induction in CEM-C7 T cells was not sufficient to prevent induction of apoptosis by dexamethasone and TNF, it may have been responsible for the inhibition of anti-FAS antibody-induced cell death in these cells by dexamethasone and TNF (Table 2Go). While it is possible that high enough levels of c-IAP2 were not achieved to lead to abrogation of apoptosis, it also clear that other genes that are induced or repressed by dexamethasone and TNF in this cell type contribute to the apoptotic phenotype (43, 44). These gene products may override the ability of c-IAP2 to prevent apoptosis. It is of interest that glucocorticoids appear to have a dual role in T cell apoptosis. While it is well known that glucocorticoids induce T cell apoptosis, this study indicates that under certain situations, these agents may also prevent T cell death.

The current study suggests a relationship between the ability of TNF and glucocorticoids to stimulate transcription from the c-IAP2 promoter in a more than additive manner and their ability to protect cells from anti-FAS-induced apoptosis. Furthermore, the c-IAP2 gene is one of very few genes (45, 46, 47) that are regulated in a permissive positive manner by both NF-{kappa}B and GR signaling pathways. It will be of interest to determine the detailed molecular mechanisms involved in the regulation of the c-IAP2 gene that prevents the usual mutual antagonism between these two transcription factors.


   Acknowledgments
 
We thank John Link for design of the real time PCR assay for c-IAP2.


   Footnotes
 
1 Current address: Incyte Genomics, Newark, Delaware. Back

Abbreviations: c-IAP2, Cellular inhibitor of apoptosis 2; d, deoxy; GR, glucocorticoid receptor; GRE, glucocorticoid response element; IAP, inhibitor of apoptosis protein; ICAM, intracellular adhesion molecule; IFN, interferon; NF, nuclear factor; TRAF, TNF receptor-associated factor.

Received February 14, 2002.

Accepted for publication June 25, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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