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B
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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)-
B. Treatment with dexamethasone effectively blocked the
loss of nuclear NF-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-B inhibits the
ability of dexamethasone to rescue HTC cells from apoptosis induced by
serum deprivation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 (m)
and generation of reactive oxygen species (12). This
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)-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-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-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-B.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
-fluoro-16-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 pCMVIB
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 pCMVIBN) (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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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. 1A). 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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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. 2A). 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. 2A). 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. 2B. 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. 2B).
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, 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. 3). Glucocorticoids are therefore the
only steroid/thyroid hormones capable of suppressing apoptosis of
serum-starved HTC cells.
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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. 4). 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. 4) 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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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 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. 5). 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 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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B function in HTC cellsB 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-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-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-B might explain the effect. Initially, Western analyses were
performed to determine if the amount of p65 (the 65 kDa subunit of
NF-B) increased in nuclei after DEX treatment (which would suggest
activation of NF-B). Figure 6 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-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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B, experiments were performed in which HTC
cells overexpressing a superrepressor of NF-B were starved for serum
in the presence or absence of DEX. This repressor is a truncated form
of the NF-B inhibitory protein IB- termed IBN (31).
Expression of this protein was achieved by transient transfection of
these cells with an appropriate vector encoding this protein. Figure 6B
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,
pCMVIBN, 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. 6C, DEX treatment significantly increased the survival of HTC
cells transfected with the control vector and starved for serum. In
contrast, cells transfected with pCMVIBN (and therefore
expressing the superrepressor form of IB) and starved for serum in
the presence of DEX did not show significantly enhanced survival. These
data indicate that inhibition of NF-B function by the superrepressor
is able to abrogate the antiapoptotic effect of DEX. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-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 (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-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-B has been inactivated die in utero
with massive apoptosis in the liver (57). We demonstrate that a
superrepressor of NF-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-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-B.
Received December 27, 1999.
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R. Roca, R. M. Kypta, and M. d. M. Vivanco Loss of p16INK4a results in increased glucocorticoid receptor activity during fibrosarcoma development PNAS, March 18, 2003; 100(6): 3113 - 3118. [Abstract] [Full Text] [PDF]
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M. L. L. Leong, A. C. Maiyar, B. Kim, B. A. O'Keeffe, and G. L. Firestone Expression of the Serum- and Glucocorticoid-inducible Protein Kinase, Sgk, Is a Cell Survival Response to Multiple Types of Environmental Stress Stimuli in Mammary Epithelial Cells J. Biol. Chem., February 14, 2003; 278(8): 5871 - 5882. [Abstract] [Full Text] [PDF]
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T. Kino and G. P. Chrousos Tumor Necrosis Factor alpha Receptor- and Fas-associated FLASH Inhibit Transcriptional Activity of the Glucocorticoid Receptor by Binding to and Interfering with Its Interaction with p160 Type Nuclear Receptor Coactivators J. Biol. Chem., January 24, 2003; 278(5): 3023 - 3029. [Abstract] [Full Text] [PDF]
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 C. Barazzone-Argiroffo, A. Pagano, C. Juge, I. Metrailler, A. Rochat, C. Vesin, and Y. Donati Glucocorticoids aggravate hyperoxia-induced lung injury through decreased nuclear factor-kappa B activity Am J Physiol Lung Cell Mol Physiol, January 1, 2003; 284(1): L197 - L204. [Abstract] [Full Text] [PDF]
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D. R. Brickley, C. A. Mikosz, C. R. Hagan, and S. D. Conzen Ubiquitin Modification of Serum and Glucocorticoid-induced Protein Kinase-1 (SGK-1) J. Biol. Chem., November 1, 2002; 277(45): 43064 - 43070. [Abstract] [Full Text] [PDF]
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 J. C. Webster, R. M. Huber, R. L. Hanson, P. M. Collier, T. F. Haws, J. K. Mills, T. C. Burn, and E. A. Allegretto 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 Endocrinology, October 1, 2002; 143(10): 3866 - 3874. [Abstract] [Full Text] [PDF]
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 J. Stankova, S. Turcotte, J. Harris, and M. Rola-Pleszczynski Modulation of Leukotriene B4 Receptor-1 Expression by Dexamethasone: Potential Mechanism for Enhanced Neutrophil Survival J. Immunol., April 1, 2002; 168(7): 3570 - 3576. [Abstract] [Full Text] [PDF]
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 C.-L. Hu, R. G. Cowan, R. M. Harman, D. A. Porter, and S. M. Quirk Apoptosis of Bovine Granulosa Cells After Serum Withdrawal Is Mediated by Fas Antigen (CD95) and Fas Ligand Biol Reprod, February 1, 2001; 64(2): 518 - 526. [Abstract] [Full Text]
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C. Widen, J. Zilliacus, J.-A. Gustafsson, and A.-C. Wikstrom Glucocorticoid Receptor Interaction with 14-3-3 and Raf-1, a Proposed Mechanism for Cross-talk of Two Signal Transduction Pathways J. Biol. Chem., December 8, 2000; 275(50): 39296 - 39301. [Abstract] [Full Text] [PDF]
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C. A. Mikosz, D. R. Brickley, M. S. Sharkey, T. W. Moran, and S. D. Conzen Glucocorticoid Receptor-mediated Protection from Apoptosis Is Associated with Induction of the Serine/Threonine Survival Kinase Gene, sgk-1 J. Biol. Chem., May 11, 2001; 276(20): 16649 - 16654. [Abstract] [Full Text] [PDF]
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F. Demarchi, R. Verardo, B. Varnum, C. Brancolini, and C. Schneider Gas6 Anti-apoptotic Signaling Requires NF-kappa B Activation J. Biol. Chem., August 17, 2001; 276(34): 31738 - 31744. [Abstract] [Full Text] [PDF]
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