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Endocrinology Vol. 141, No. 5 1854-1862
Copyright © 2000 by The Endocrine Society

ARTICLES

Delineation of an Antiapoptotic Action of Glucocorticoids in Hepatoma Cells: The Role of Nuclear Factor-{kappa}B

Rosemary B. Evans-Storms and John A. Cidlowski

Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709

Address all correspondence and requests for reprints to: John A. Cidlowski, P.O. Box 12233, MD E2–02, Research Triangle Park, North Carolina 27709. E-mail: Cidlowski{at}niehs.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoids are primarily recognized for their profound antiinflammatory actions and their ability to induce lymphocyte apoptosis. We report here that, in contrast to their effect on cells of the immune system, glucocorticoids suppress serum deprivation induced apoptosis of rat hepatoma (HTC) cells. Suppression of apoptosis in these cells occurs at physiological concentrations of glucocorticoid and is abrogated by the glucocorticoid antagonist RU486. Although HTC cells also express receptors for progesterone, estrogen, and thyroid hormone, ligands for these receptors fail to rescue these cells from programmed cell death. Because the sensitivity of cells to apoptotic stimuli is often regulated by the ratio of antiapoptotic to proapoptotic Bcl-2 family members, we analyzed the influence of glucocorticoids and induction of apoptosis by serum starvation on the expression of these proteins. Bcl-2, Bcl-xL, Bad, Bak, and Bax levels were not altered by either treatment. Mitochondrial function has recently been implicated as a critical early regulator of apoptosis in many cells including hepatocytes. Dexamethasone treatment blocked a decrease in this potential ({triangleup}{Psi}m) during serum deprivation induced apoptosis in HTC cells, indicating an action of this hormone upstream of mitochondria. We also show that the induction of apoptosis in HTC cells is associated with a decrease in nuclear factor (NF)-{kappa}B. Treatment with dexamethasone effectively blocked the loss of nuclear NF-{kappa}B, suggesting that this hormone acts to suppress apoptosis of HTC cells via regulation of this nuclear transcription factor. This hypothesis was confirmed by transfection experiments that show that expression of a superrepressor of NF-{kappa}B inhibits the ability of dexamethasone to rescue HTC cells from apoptosis induced by serum deprivation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOCORTICOIDS ARE BEST known for their antiinflammatory and immunomodulatory effects (1). These hormones also possess antipyretic activity, promote lung maturation in infants, act as antiemetics during chemotherapy, and induce apoptosis (programmed cell death) in many lymphocytes (2, 3, 4). This latter characteristic has made them useful as chemotherapeutic agents in the treatment of various leukemias, and as a model for the study of the regulation of apoptosis in lymphoid cells (4, 5). In contrast, glucocorticoids have been reported to suppress apoptosis in various cell types including cell death induced by transforming growth factor (TGF)-ß in certain rat hepatoma cell lines (6, 7, 8, 9). However, the underlying mechanisms responsible for this repression of programmed cell death are poorly understood.

The unique cellular characteristics originally described for apoptotic cells include blebbing of the cell membrane and internucleosomal degradation of DNA (10, 11). Additionally, many studies have shown that apoptotic cells also undergo mitochondrial perturbations including loss of the mitochondrial membrane potential ({triangleup}{Psi}m) and generation of reactive oxygen species (12). This {triangleup}{Psi}m is believed to be caused by the opening of a permeability transition pore (13). Cytochrome c is then released into the cytosol from the inner mitochondrial space, and acts in concert with additional cytosolic factors to activate caspases involved in the execution steps of apoptosis (14). Glucocorticoids are well known to have profound effects on mitochondria. These hormones promote fusion of mitochondria in certain cells, and treatment of rats with dexamethasone increases oxidative phosphorylation (15, 16). Dexamethasone has previously been shown to maintain mitochondrial function of rat hepatocytes in serum-free medium (17).

Whether cells survive or die is determined by a careful balance of proapoptotic and antiapoptotic signals that includes many transcription factors. Both glucocorticoid receptor and the ubiquitous transcription factor nuclear factor (NF)-{kappa}B are able to affect apoptosis in a positive or negative manner depending on the cell type. While glucocorticoids induce apoptosis in lymphoid cells, they suppress this process in neutrophils, mouse fibroblasts, the mouse mammary gland, and hepatocytes and hepatoma cells (6, 7, 9, 18, 19, 20). NF-{kappa}B is able to induce apoptosis in endothelial cells and some lymphoid cells, but suppresses it in hepatocytes and some B lymphoma cells (21, 22, 23, 24). The glucocorticoid receptor and NF-{kappa}B are able to physically interact and mutually antagonize the function of one another in some model systems (25). In this manuscript, we present evidence for an antiapoptotic effect of glucocorticoids that is mediated in part through modulation of nuclear NF-{kappa}B.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and analysis of proliferation and viability
The HTC rat hepatoma cell line (originally derived from the Morris 7288c hepatoma) was the generous gift of Dr. Stoney Simons (NIDDK, NIH) and was grown in DMEM supplemented with 5% heat-inactivated FBS (Irvine Scientific, Santa Ana, CA), 2 mM glutamine, 100 U/ml penicillin, and 75 U/ml streptomycin in 5% CO2 at 37 C. Tissue culture flasks and multiwell plates were purchased from Costar or Becton Dickinson and Co. (Cambridge, MA, and Franklin Lakes, NJ). Proliferation of cells was assessed by determining the increase in the total cell number (counted on a hemocytometer) over time after trypsinization to remove cells from the tissue culture wells/flasks. Cell viability was determined by dye exclusion after staining for 5 min at room temperature with 0.1% trypan blue dissolved in PBS.

Treatment of cells with hormones
For experiments requiring pretreatment with hormones, cells were plated at 2500/cm2 and grown for 24 h, and then fresh medium containing 5% FCS and hormones at the concentrations indicated in the text was added. 4-pregnen-3,30-diene (progesterone—dissolved in ethanol), 1,4-pregnadien-9{alpha}-fluoro-16{alpha}-methyl, 21-triol-3,20-dione (dexamethasone—dissolved in PBS), and 1,3,5 (10)-estratrien-3,17ß-diol (estrogen - dissolved in ethanol) were obtained from Steraloids, Inc., Wilton, NH., and 3,3',5-triiodo-L-thyronine (dissolved in 4 mM NaOH) was obtained from Sigma (St. Louis, MO). RU486 was the kind gift of Roussel-UCLAF (Romainville, France). After 18 h of exposure to hormone, the medium was removed and replaced with fresh media containing FCS and hormone, or alternatively, cells were washed twice with serum-free media and then starved in serum-free media containing hormone.

Flow cytometric analyses
Flow cytometry was performed by exciting cells at 488 nm with an argon laser on a Becton Dickinson and Co. FACSort (Becton Dickinson and Co. Immunocytometry Systems, San Jose, CA). All data were derived from analysis of 10,000 cells using CELLQuest software (Becton Dickinson and Co. Immunocytometry Systems). HTC cells were grown for 24 h in serum-free or 5% FCS supplemented media with or without hormone. DNA integrity was then determined by fixing cells in 70% ethanol for at least 30 min, washing once with PBS, staining at 2 x 106 cells/ml with 20 µg/ml propidium iodide containing 1 mg/ml RNase A (both from Sigma), and generating histograms of cell number vs. DNA content. Orientation of phosphatidylserine in the cell membrane, and cell size were determined for unfixed cells using reagents provided in the TACS Annexin V-FITC kit (Trevigen, Inc., Gaithersburg, MD). Cells were stained at 5 x 105 cells/ml with 1 µl each of propidium iodide and the annexin-FITC conjugate provided according to the directions enclosed. Histograms of propidium iodide vs. annexin-FITC fluorescence were then generated. These same unfixed samples were also used to analyze cell size by generating histograms of forward-scattered vs. side-scattered light.

For flow cytometric analysis of mitochondrial function, cells were removed from the tissue culture flasks by trypsinizing, washed once in room temperature PBS, and then resuspended in PBS at a concentration of 5 x 105 cells/ml. Cells were then added to a tube containing the dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1, Molecular Probes, Inc., Eugene, OR, dissolved at 10 mM in dimethylsulfoxide) at a final concentration of 10 µM (26). Cells were stained for 30 min at room temperature in the dark, and were then analyzed by flow cytometry as described above.

Subcellular fractionation
Cells were swollen on ice for 10 min in nuclear isolation buffer (150 mM MgCl2, 10 mM KCl, and 10 mM Tris-HCl, pH 6.7) containing protease inhibitors (0.1 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, 1 µM leupeptin) and then the plasma membrane was broken with 80–100 strokes in a Dounce homogenizer. Sucrose was added to 250 mM, and then nuclei were pelleted by centrifugation at 12,000 r.p.m. for 2 min and the cytosol removed. Nuclei were then washed once in 1 mM MgCl2 containing 11% sucrose and 1% NP-40, and then once in 1 mM MgCl2 containing 11% sucrose. Nuclei were lysed in cold lysis buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 150 mM NaCl, 0.5% Triton X-100) containing protease inhibitors (0.1 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, 1 µM leupeptin) by Dounce homogenization. The protein concentration of the lysates was determined by the method of Bradford using the Bio-Rad Laboratories, Inc. (Richmond, CA) protein microassay, and samples were diluted in Laemmli gel loading buffer to a final concentration of 50 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromophenol blue, 10% glycerol, and 100 mM dithiothreitol (27, 28). Samples were heated to 100 C for 5 min before electrophoresis.

Electrophoresis and Western analysis
Washed cells were lysed by homogenization in cold lysis buffer containing protease inhibitors. Determination of protein concentrations and preparation for electrophoresis were as described above. Proteins were separated by SDS-PAGE and transferred electrophoretically to 0.1 µm nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, NH) in Tris-glycine buffer (25 mM Tris-HCl, pH 8.3, 150 mM glycine, 15% methanol) overnight at 35 V with cooling (27, 29). Following staining with Ponceau S (0.5% in 1% acetic acid) to verify loading equivalency and transfer efficiency, membranes were treated with Tris-buffered saline with detergent (TBS-T–10 mM Tris-HCl, 154 mM NaCl, 0.05% Tween 20, pH 7.4) containing 10% nonfat dry milk for 1 h at room temperature with mixing (30). Membranes were then reacted with a 1:1000 dilution of rabbit antisera specific for Bcl-xL/S (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, L-19), Bcl-2 (PharMingen, San Diego, CA, 13456E), Bak (PharMingen, 65606E), Bad (Santa Cruz Biotechnology, Inc., SC-7869), Bax (Santa Cruz Biotechnology, Inc., SC-6236), or p65 (Santa Cruz Biotechnology, Inc., SC-372) in TBS-T for 1 h at room temperature with mixing. Membranes were then washed with TBS-T, reacted with horseradish peroxidase linked donkey antirabbit immunoglobulin (1:15,000) in TBS-T for 1 h at room temperature with mixing, washed with TBS-T, and reacted with detection reagents as described in directions enclosed with the ECL reagents (Amersham Pharmacia Biotech, Arlington Heights, IL). Autoradiography was then performed on the membranes using hyperfilm-ECL (Amersham Pharmacia Biotech). Specific peptides were used to block reactivity of the antisera against Bcl-xL and Bax and thereby verify the identity of these proteins in Western blots (data not shown). Such peptides were not available for use with the other antisera. In some cases, nitrocellulose membranes were reprobed with various antisera after stripping and reblocking the blots as described in instructions enclosed with the ECL reagents.

Transfection and analysis of cells
HTC cells were plated in 24-well plates at a density of 1.85 x 104 cells/well and allowed to recover for 24 h. These cells were then transfected with 1 µg of plasmid DNA per well using 6 µl of DMRIE-C (Life Technologies, Inc., Gaithersburg, MD) for 4 h in Optimem media (Life Technologies, Inc.) containing 5% FCS but no antibiotics (after a 45 min precipitation of DNA with the DMRIE-C in the absence of serum). The media was then exchanged for Optimem plus 5% FCS. After allowing cells to recover for 24 h, serum starvation was begun in the presence or absence of DEX. Viability of starved cells was determined after 72 h as previously described. The plasmids used were pCMVI{kappa}B{alpha}{Delta}N (a generous gift of Dr. D. W. Ballard, Vanderbilt University School of Medicine, Nashville, TN) and pCMV5 (differing only in the multiple cloning site from the pCMV4 backbone of pCMVI{kappa}B{alpha}{Delta}N) (31). Control cells transfected with pCMV-ß (CLONTECH Laboratories, Inc. Palo Alto, CA) were fixed and stained for ß-galactosidase expression to determine the percent transfection for each experiment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The effects of glucocorticoids on viability and proliferation of hepatoma cells
Glucocorticoids are well known for their ability to induce apoptosis in certain lymphoid cells (4). However, these same hormones can suppress the death of many cell types after a variety of stimuli (6, 7, 19). We have used rat hepatoma (HTC) cells induced to die by withdrawal of serum as a model in which to study the effects of glucocorticoids on the proliferation and viability of hepatic cells. The viability of HTC cells starved for serum (0% FCS) decreased in a time dependent manner (Fig. 1AGo). Approximately 42% of cells died after 72 h of serum starvation (as assessed by trypan blue exclusion). When HTC cells were pretreated for 18 h with 10 nM DEX and starved for serum in the presence of this same concentration of hormone, greater than 90% of cells were viable after 72 h (Fig. 1AGo). A similar effect was seen when DEX was added at the beginning of the starvation, but the rescue was less profound (data not shown). In contrast, the viability of cells starved for serum in the presence of DEX and the glucocorticoid antagonist RU486 at a concentration of 1000 nM was comparable to that of HTC cells starved in the absence of DEX. Treatment of HTC cells with RU486 alone had no effect on death induced by serum starvation (data not shown). These data demonstrate that DEX is able to rescue HTC cells from death induced by serum starvation in a glucocorticoid receptor-dependent manner.



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  		Figure 1. Viability and proliferation of HTC cells after serum starvation. Cells were maintained and then plated for experiments as described in Materials and Methods, and then grown for 72 h in 5% FCS, 0% FCS, 0% FCS + 10 nM DEX after pretreatment with hormone for 18 h, or 0% FCS + 10 nM DEX and 1000 nM RU486 after pretreatment with hormone plus antagonist for 18 h. A, The percentage of viable cells present was determined at 24, 48, and 72 h after starvation by subtracting the number of cells that stained with trypan blue from 100%. B, Total cell number was determined at 0, 24, 48, and 72 h starvation by counting on a hemocytometer.

 
In addition to their ability to modulate cell death, glucocorticoids have been reported to have profound inhibitory effects on the proliferation of many cell types including fibroblasts, muscle cells, and certain lymphocytes and hepatoma cells (32, 33, 34, 35). In contrast, these hormones can enhance proliferation of osteoprogenitors and adipocyte progenitors (36). We therefore examined whether the effect of dexamethasone on the viability of HTC cells might be associated with a proproliferative function. Figure 1BGo shows that HTC cells starved for serum undergo a rapid and complete cessation of proliferation. HTC cells that were pretreated with DEX and starved for serum in the presence of this hormone as described above underwent comparable growth arrest. DEX had no effect on proliferation of HTC cells grown in 5% serum (data not shown). Thus, although glucocorticoids have been reported to be able to affect both proliferation and apoptosis in various cell types, the ability of DEX to suppress apoptosis of HTC cells is independent of any effect on proliferation.

Characterization of cell death after serum starvation of HTC cells
Cells can die by either apoptosis or necrosis (37). These two modes of cell death can be distinguished by the unique alterations of several cellular parameters in apoptotic cells including cleavage of DNA between nucleosomes, cell shrinkage, and reorientation of phosphatidylserine to the outer face of the cell membrane (38, 39). These cellular characteristics were therefore analyzed to determine if HTC cells die by apoptosis after serum starvation. To assess the integrity of the DNA, cells were fixed with ethanol, stained with propidium iodide (which intercalates into the DNA of all fixed cells), and analyzed by flow cytometry (40). Cell cycle histograms from cells grown in 5% FCS with DEX (after an 18 h pretreatment with hormone) for 24 h show an increase in the number of cells in G2/M when compared with control cells grown in 5% serum in the absence of hormone (Fig. 2AGo). The meaning of this is unclear because DEX had no apparent effect on proliferation of cells grown in 5% FCS (assessed by the accumulation of cells over 72 h—data not shown). Cells that had been starved for serum (0% FCS) for 24 h show a distinct subdiploid peak of DNA that is characteristically found in apoptotic cells. This peak is not present in cells starved for serum in the presence of DEX after an 18 h pretreatment with hormone (Fig. 2AGo). Unlike cells grown in 5% FCS with DEX, no shift of cells into G2/M is seen in cells grown in 0% FCS in the presence of this hormone. Cell size was analyzed by flow cytometry of unfixed HTC cells (41). The amount of light scattered in a forward direction during flow cytometry is a function of cell size. Thus, the higher the forward scatter, the larger the cell. Serum-starved HTC cells had begun to shrink by 24 h after the start of serum starvation, as shown by the appearance of a population of cells shifted left on the x-axis in Fig. 2BGo. This loss of cell volume is inhibited in cells starved for serum in the presence of DEX. This hormone had no effect on the size of cells grown in 5% FCS (Fig. 2BGo).



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  		Figure 2. Analysis of DNA integrity, cell size, and phosphatidylserine orientation by flow cytometry. In all cases, cells were grown for 24 h with various treatments. These data are representative of at least three experiments. A, Analysis of DNA integrity. Cells were prepared for cell cycle analysis and analyzed by flow cytometry as described in Materials and Methods. Histograms of cell number vs. DNA content (which is measured by propidium iodide fluorescence) were generated after growth in 5% FCS, 5% FCS + 10 nM DEX (after 18 h pretreatment), 0% FCS, or 0% FCS + 10 nM DEX (after 18 h pretreatment). The subdiploid peak of DNA in serum-starved cells is marked with an asterisk. B, Analysis of cell size. The light scattering properties of unfixed cells were analyzed as described in Materials and Methods. C, Analysis of phosphatidylserine orientation. Cells were stained with annexin-FITC and propidium iodide and analyzed by flow cytometry as described in Materials and Methods. Growth was in 5% FCS, 5% FCS + 10 nM DEX (after 18 h pretreatment), 0% FCS, or 0% FCS + 10 nM DEX (after 18 h pretreatment).

 
Annexin V binds phosphatidylserine with high affinity, and can therefore be used in flow cytometry to detect the presence of this phospholipid on the outer cell membrane (42). We analyzed the orientation of phosphatidylserine in unfixed cells by staining with propidium iodide (P.I.) and fluorescein-conjugated annexin V. In contrast to fixed cells, only unfixed cells in which plasma membrane integrity has been compromised will stain with P.I. As shown in Fig. 2CGo, HTC cells grown in 0% serum for 24 h contain more cells stained with annexin (upper and lower right quadrants) than cells grown in 5% FCS (with or without DEX). Serum starvation of cells in the presence of DEX (after an 18 h pretreatment) reduced the number of cells that stained with annexin. The appearance of a subdiploid peak of DNA, reorientation of phosphatidylserine to the outside of the cell membrane, and shrinkage of the cells indicates that HTC cells die by apoptosis after serum starvation. Treatment of these cells with DEX inhibited alteration of all of these cellular characteristics, suggesting that the action of this hormone may be early, perhaps blocking the initiation of cell death rather than delaying the progression of the process.

Specificity of the suppression of apoptosis in HTC cells by glucocorticoids
Ligands for receptors of the steroid/thyroid receptor superfamily other than the glucocorticoid receptor can negatively regulate apoptosis. Progesterone and estrogen can inhibit apoptosis in uterine epithelial and breast cancer cells, respectively, while the latter can also suppress apoptosis in ovarian granulosa cells (43, 44, 45). In addition, thyroid hormone has been reported to inhibit apoptosis in cerebellar granule neurons (46). Because HTC cells express progesterone, estrogen, and thyroid hormone receptors in addition to glucocorticoid receptor, we examined whether these hormones might also suppress apoptosis of this cell type (47, 48, 49). Cells pretreated with estradiol, thyroxine (both at 10 nM), or progesterone (100 nM), followed by starvation in the presence of hormone underwent apoptosis at a level comparable to cells starved in the absence of any hormone treatment (Fig. 3Go). Glucocorticoids are therefore the only steroid/thyroid hormones capable of suppressing apoptosis of serum-starved HTC cells.



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  		Figure 3. The effect of estrogen, progesterone, and thyroxine on apoptosis of HTC cells. Cells were grown as previously described and pretreated for 18 h with 10 nM DEX, 10 nM triiodothyroxine, 100 nM progesterone, or 10 nM estrogen. Cells were then starved in the presence of hormone for 72 h and the percentage of apoptotic cells present was defined as the number of cells that stained with trypan blue per 100 cells counted.

 
Expression of proteins of the Bcl-2 family in HTC cells
The Bcl-2 family is comprised of both proapoptotic and antiapoptotic proteins whose tissue specific expression and/or association with one another regulates apoptosis in many cell types (50). Thus, alteration of the expression of a particular family member can alter the sensitivity of cells to induction of apoptosis. We previously demonstrated that HTC cells do not normally express the antiapoptotic protein Bcl-2 (51). However, experiments were now performed to determine if treatment with DEX is able to induce expression of this protein. The representive Western hybridization shown in Fig. 4Go demonstrates that Bcl-2 is not expressed in HTC cells grown in the presence or absence of FCS with or without DEX. Expression of Bcl-xL (another antiapoptotic protein of the Bcl-2 family) is increased by DEX treatment of McA-RH7777 and McA-RH8994 rat hepatoma cell lines (6). These cells were originally derived from a Morris hepatoma, as were HTC cells. Therefore, it seemed likely that DEX could induce expression of this protein in HTC cells. However, Western analysis performed with antisera specific for Bcl-xL/S shows that Bcl-xL is expressed at exceedingly low levels in HTC cells which are not altered by DEX treatment (Fig. 4Go). The proapoptotic Bcl-xs protein encoded by a splice variant of the bcl-x gene was not detected in HTC cells with this antisera (data not shown). Dexamethasone is able to suppress expression of a number of genes, either by binding to negative glucocorticoid regulatory elements in their promoters or through association with and subsequent antagonism of other transcription factors involved in positive regulation of these genes. Therefore, it was possible that DEX was protecting HTC cells from apoptosis by suppressing expression of proapoptotic Bcl-2 family members. Western analyses performed using antisera specific for Bad, Bak, and Bax (Fig. 4Go) show that while these proapoptotic proteins are expressed in HTC cells, their levels are not altered by treatment with DEX. Therefore, in serum-starved HTC cells, these Bcl-2 family members do not appear to play a role in regulation of apoptosis by DEX, or in induction of apoptosis by serum starvation.



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  		Figure 4. Western analysis of Bcl-2 family members. HTC cells were grown as previously described for 72 h in 5% FCS, 5% FCS + 10 nM DEX (after 18 h pretreatment), 0% FCS, or 0% FCS + 10 nM DEX (after 18 h pretreatment) and then lysed. The protein content of the clarified cell lysates was determined, and protein samples were prepared in Laemmli buffer. 100 µg of protein (or 60 µg for lanes 2 and 3 of the Bad Western blot) of total protein were separated by SDS-PAGE (using a 10% resolving gel for Bcl-2 and Bcl-xL, and a 12% resolving gel for Bad, Bak, and Bax). Protein was then transferred to nitrocellulose, and specific proteins detected by reaction with antisera as described in Materials and Methods. Normal rat thymus was used as the positive control for all proteins.

 
Mitochondrial function in glucocorticoid treated HTC cells
Glucocorticoids have previously been shown to have profound effects on mitochondrial function including increasing oxidative phosphorylation and the uptake of Mg2+, K+, and adenine nucleotides, and alteration of ultrastructure leading to fusion of these organelles (15, 16). In addition, data suggests that glucocorticoid receptor may actually directly regulate gene expression from the mitochondrial genome (52). An alteration of mitochondrial function characterized by a decrease in the mitochondrial membrane potential ({triangleup}{Psi}m) is a common phenomenon during apoptosis (12). This change in depolarization status is frequently associated with the release of cytochrome c from the inner mitochondrial space (14). Cytochrome c in concert with other cytosolic components then activates the cysteine protease caspase-9 (14). This protease subsequently cleaves and thereby activates other caspases, which then cleave various cellular substrates (14). We measured the {triangleup}{Psi}m in HTC cells grown with or without serum in the presence or absence of DEX to determine if this hormone affected mitochondrial function during serum starvation. These studies were performed by flow cytometric analysis of the fluorescent characteristics of the dye JC-1, which only accumulates in mitochondria (26, 53). Aggregrates of this dye form when the mitochondrial membrane potential is high, and fluoresce with a peak of 590 nm (red) after excitation at 488 nm. These aggregates dissociate into monomers which fluoresce at 527 nm (green) as the mitochondrial membrane potential decreases. The change in red fluorescence vs. cell number was measured by flow cytometry after 72 h of serum starvation. A decrease in the amount of aggregated JC-1 dye is seen in serum-starved cells, demonstrating that the mitochondrial membrane potential is lower in serum-starved cells compared with cells maintained in 5% FCS (Fig. 5Go). Treatment with DEX significantly reduced the effect of serum starvation on the mitochondrial membrane potential of HTC cells. These data demonstrate that DEX is able to inhibit the {triangleup}{Psi}m normally seen during apoptosis induced by serum starvation of HTC cells, and therefore suggest that DEX affects signal transduction at or above the level of the mitochondrian.



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  		Figure 5. Analysis of mitochondrial membrane potential by flow cytometry. HTC cells were grown as described in Materials and Methods in 5% FCS or 0% FCS for 72 h with or without DEX (after an 18 h pretreatment). They were then loaded with JC-1 and analyzed by flow cytometry. Contour plots of cell number vs. JC-1 aggregates (red fluorescence) were then generated.

 
The effect of glucocorticoids on NF-{kappa}B function in HTC cells
Activation of the transcription factor NF-{kappa}B induces apoptosis in a number of cell types including human coronary artery endothelial cells and certain cells of the lymphoid lineage (54, 55). However, NF-{kappa}B has been shown to suppress apoptosis of murine hepatocytes and certain murine B cell lymphoma cell lines (22, 56). In addition, mice deficient in the p65 subunit of NF-{kappa}B undergo massive apoptosis in the liver and die before birth (57). Because alteration of the expression of members of the Bcl-2 family is not associated with suppression of apoptosis of serum-starved HTC cells by DEX, we determined if an increase in the level or nuclear translocation of NF-{kappa}B might explain the effect. Initially, Western analyses were performed to determine if the amount of p65 (the 65 kDa subunit of NF-{kappa}B) increased in nuclei after DEX treatment (which would suggest activation of NF-{kappa}B). Figure 6Go shows a representative immunoblot performed using antisera specific for p65 on cytosolic and nuclear proteins from HTC cells after various treatments. A slight increase in both cytosolic and nuclear p65 is induced by DEX in HTC cells grown in 5% FCS. HTC cells that were starved for serum had very little nuclear p65, thus implying that a loss of NF-{kappa}B is associated with apoptosis in these cells. However, the amount of p65 present in nuclei of serum starved HTC cells treated with DEX is comparable to cells grown in 5% FCS, suggesting that DEX prevents the loss of p65 that occurs during apoptosis in this cell type.



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  		Figure 6. Analysis of NF-{kappa}B function during apoptosis of HTC cells. A, Subcellular fractionation and analysis of the p65 subunit of NF-{kappa}B in HTC cells. Cells were grown for 24 h in 5% FCS, 5% FCS + DEX (with an 18 h pretreatment), 0% FCS, or 0% FCS + DEX (with an 18 h pretreatment) and then preparations of cytosol and nucleoplasm were prepared as described in Materials and Methods. Total protein (50 µg) was separated by SDS-PAGE (10% resolving gel), transferred to nitrocellulose, and p65 was detected immunologically. B, Expression of I{kappa}B{alpha}{Delta}N in HTC cells. A representative immunoblot of cell lysates of transfected HTC cells is shown, demonstrating that the truncated I{kappa}B superrepressor was expressed in these cells from the vector. C, The effect of overexpression of a superrepressor of NF-{kappa}B on apoptosis of serum starved HTC cells. HTC cells were transfected with pCMV5 or pCMVI{kappa}B{alpha}{Delta}N as described in Materials and Methods. They were then starved for serum for 72 h in the presence or absence of DEX (no pretreatment was feasible in this experiment), and the relative percent surviving cells (shown on the x-axis) determined by staining with trypan blue.

 
To confirm that the suppression of apoptosis of serum-starved HTC cells by DEX is mediated by NF-{kappa}B, experiments were performed in which HTC cells overexpressing a superrepressor of NF-{kappa}B were starved for serum in the presence or absence of DEX. This repressor is a truncated form of the NF-{kappa}B inhibitory protein I{kappa}B-{alpha} termed I{kappa}B{alpha}{Delta}N (31). Expression of this protein was achieved by transient transfection of these cells with an appropriate vector encoding this protein. Figure 6BGo confirms that the superrepressor is expressed in HTC cells. A similar vector (pCMV5—only differing in the multiple cloning site) with no insert was transfected as a negative control. Representative cells were also transfected with a vector containing the ß-galactosidase complementary DNA (pCMV-ß) to normalize for transfection efficiency during each experiment. Cells that had been transfected with pCMV5, pCMVI{kappa}B{alpha}{Delta}N, or pCMV-ß were then allowed to recover for 24 h, after which they were starved for serum in the presence or absence of DEX. Pretreatment with DEX was not performed because of the risk that expression from the vectors would begin to decrease before the experiments could be completed. After 72 h of starvation, the percentage of cells transfected in each experiment was determined by staining a representative set of cells that had been transfected with pCMV-ß for ß-galactosidase activity. The number of cells staining with trypan blue (and therefore presumed to be apoptotic based on data previously shown) was then determined and normalized for 100% transfection. The percent of apoptotic cells in control samples (cells transfected with pCMV5 and starved for serum in the absence of DEX) was then set to 100 (so all controls were now comparable) and the number of apoptotic cells in the other wells was adjusted accordingly. As shown in Fig. 6CGo, DEX treatment significantly increased the survival of HTC cells transfected with the control vector and starved for serum. In contrast, cells transfected with pCMVI{kappa}B{alpha}{Delta}N (and therefore expressing the superrepressor form of I{kappa}B) and starved for serum in the presence of DEX did not show significantly enhanced survival. These data indicate that inhibition of NF-{kappa}B function by the superrepressor is able to abrogate the antiapoptotic effect of DEX.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Glucocorticoids are some of the most widely prescribed compounds in medical practice today. They are invaluable in the treatment of inflammatory disorders and pulmonary insufficiency in infants (1, 2). These hormones also induce apoptosis in many lymphocytes and are therefore used as chemotherapeutic agents against many leukemias (5, 58). This latter characteristic has also made them useful for studying induction of programmed cell death. In contrast, these hormones suppress spontaneous apoptosis of neutrophils, TNF-{alpha} mediated programmed cell death of mouse L929 mouse fibroblasts, and involution of the mouse mammary gland (8, 9, 20). In addition, they have profound suppressive effects on apoptosis of cells derived from the liver, and previous data suggested they might have a similar effect on HTC rat hepatoma cells (6, 7, 51). Early research on internucleosomal cleavage of DNA during apoptosis showed that administration of the synthetic glucocorticoid dexamethasone (DEX) to rats reduced the amount of endonuclease activity in liver cells (59). These hormones also protect normal rat hepatocytes from the effects of inhibition of electron transport and suppress apoptosis of K2 hepatoma cells induced by tamoxifen or TGF-ß, and spontaneous and TGF-ß1-induced apoptosis of McA-RH7777 and McA-RH8994 rat hepatoma cells (6, 7, 19). Thus, it is not surprising that apoptosis of serum starved HTC rat hepatoma cells is suppressed by DEX. This effect is glucocorticoid receptor dependent and is not due to a proproliferative effect of this hormone.

The manner by which glucocorticoids regulate apoptosis is poorly understood. These hormones can affect gene transcription in a positive or negative manner depending on the presence of specific elements in the promoter of a gene (60). The glucocorticoid receptor can also modulate the transcriptional activation function of the transcription factors NF-{kappa}B, CREB, Oct-1, and AP-1 (60). Thus, why glucocorticoids induce apoptosis in one cell type and suppress it in another probably depends on a combination of these transcriptional effects and on expression of other transcription factors in a given cell type. Glucocorticoids have been reported to suppress apoptosis in hepatocytes and different hepatoma cell lines by acting on several apoptotic signal transduction pathways. Protection of hepatocytes from inhibitors of electron transport and K2 hepatoma cells from tamoxifen and TGF-ß was associated with a decrease in the release of arachidonic acid (7, 19). Liberation of arachidonic acid in K2 cells led to increased production of prostaglandins via the cyclo- oxygenase pathway. Inhibition of this pathway with indomethacin suppressed apoptosis in these cells, however, this compound had no effect on apoptosis in serum-starved HTC cells (data not shown).

Members of the Bcl-2 family are extremely important for modulation of apoptosis in many cell types (50). In McA-RH7777 and McA-RH8994 rat hepatoma cells, protection by DEX correlated with increased expression of the antiapoptotic protein Bcl-xL (6). Since HTC cells are derived from a Morris hepatoma, as are McA-RH7777 and McA-RH8994 cells, it was quite possible that DEX could suppress apoptosis in them by increasing expression of Bcl-xL. However, this was not the case. DEX also had no effect on expression of the proapoptotic family members Bax, Bad, and Bak, (all of which have been shown to be expressed in normal liver) (61, 62). Although a large number of Bcl-2 related proteins have been identified whose expression was not examined in these experiments, most of these are not expressed in normal hepatocytes. Nonetheless, it remains possible that such a protein could still be responsible for the suppression of apoptosis in HTC cells by DEX.

Glucocorticoids have previously been shown to have profound effects on mitochondrial function (15, 16, 17). A decrease in the mitochondrial membrane potential ({triangleup}{Psi}m) is an invariant observation in apoptotic cells. DEX effectively inhibited this alteration in HTC cells starved for serum, suggesting that this hormone acts at or above the level of the mitochondria in these cells. Several models can be envisioned to explain how DEX acts on this organelle. Perhaps DEX increases expression of a protein that acts to decrease activity of a caspase that lies upstream of the mitochondria in the apoptotic cascade, or decreases expression of such a caspase (either directly or indirectly). Another attractive idea is that DEX might act to inhibit the decline in energy and the increase in the release of oxygen radicals observed during apoptosis by affecting energy metabolism in the mitochondria because DEX has been shown to directly act on some mitochondrial genes (52).

Our lab and others have shown that the glucocorticoid receptor can interact with and antagonize the function of NF-{kappa}B (63, 64, 65). However, a number of groups have reported that this transcription factor can suppress apoptosis in murine hepatocytes, and mouse fibroblasts and macrophages (21, 22, 66). In addition, mice in which the gene encoding the p65 subunit of NF-{kappa}B has been inactivated die in utero with massive apoptosis in the liver (57). We demonstrate that a superrepressor of NF-{kappa}B is able to abrogate the ability of DEX to rescue HTC cells from apoptosis induced by serum starvation, strongly suggesting that an increase in NF-{kappa}B activity by DEX is at least partially responsible for this rescue.

The data presented herein demonstrate that glucocorticoids suppress apoptosis induced by serum-starvation of HTC rat hepatoma cells in a glucocorticoid receptor dependent manner. This effect is specific for glucocorticoids (progesterone, estrogen, and thyroid hormone do not have a similar effect). Glucocorticoids effectively inhibit the loss of mitochondrial membrane potential associated with apoptosis, and appear to function via activation of the transcription factor NF-{kappa}B.

Received December 27, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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