Download Glucocorticoid-Induced Plasma Membrane Depolarization during

0013-7227/01/$03.00/0
Printed in U.S.A.
Endocrinology 142(12):5059 –5068
Copyright © 2001 by The Endocrine Society
Glucocorticoid-Induced Plasma Membrane
Depolarization during Thymocyte Apoptosis: Association
with Cell Shrinkage and Degradation of the Naⴙ/KⴙAdenosine Triphosphatase
CYNTHIA L. MANN, CARL D. BORTNER, CHRISTINE M. JEWELL,
AND
JOHN A. CIDLOWSKI
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health
(C.L.M., C.D.B., J.A.C.), Research Triangle Park, North Carolina 27709; and Curriculum in Toxicology, University of North
Carolina (C.L.M.), Chapel Hill, North Carolina 27599
Multiple signaling pathways are known to induce apoptosis in
thymocytes through mechanisms that include the loss of mitochondrial membrane potential, cell shrinkage, caspase activation, and DNA degradation but little is known about the
consequences of apoptosis on the properties of the plasma
membrane. We have previously shown that apoptotic signals,
including survival factor withdrawal and glucocorticoids, induce plasma membrane depolarization during rat thymocyte
apoptosis, but the mechanisms involved in this process are
unknown. We report here that inhibition of the Naⴙ/Kⴙ-adenosine triphosphatase (Naⴙ/Kⴙ-ATPase) with ouabain similarly
depolarized control thymocytes and enhanced glucocorticoid-induced membrane depolarization, suggesting a link between Naⴙ/Kⴙ-ATPase and plasma membrane depolarization
of thymocytes. To determine whether repression of Naⴙ/KⴙATPase levels within cells can account for the loss of plasma
E
ARLY STUDIES OF the effects of glucocorticoids on the
immune system showed that glucocorticoids caused a
profound reduction in thymic mass and volume due primarily to thymocyte apoptosis (1, 2). Glucocorticoids induce
a cell death program in thymocytes characterized by the loss
of mitochondrial membrane potential, cell shrinkage,
caspase activation, and DNA degradation (3–5). The structure and function of the plasma membrane are also altered
by glucocorticoids during apoptosis, as is evidenced by alterations in the transport of glucose and amino acids (6) and
the distribution of ions across the membrane (7–10). Phosphatidylserine residues also reorient to the exterior of the cell during apoptosis (11). Finally, late in apoptosis, the plasma membrane blebs and pinches off to form apoptotic bodies (12, 13).
The loss of water and ions from the cell during apoptosis,
particularly potassium (7, 10), results in cell shrinkage, a
characteristic feature of apoptosis (1, 13). In thymocytes,
glucocorticoids induce cell shrinkage through a receptordependent and gene expression-dependent pathway (5, 14).
In addition, studies with the pan-caspase inhibitor z-VAD
showed that glucocorticoid-induced cell shrinkage is depenAbbreviations: AEVD-fmk, Z-Ala-Glu (Ome)-Val-Asp (Ome)-FMK;
DEVD-fmk, Z-Asp-Glu-Val-Asp-fluoromethylketone; IETD-fmk, Z-IleGlu-Thr-Asp-fluoromethylketone; Na⫹/K⫹-ATPase, Na⫹/K⫹-adenosine triphosphatase; PI, propidium iodide; TBS, Tris-buffered saline;
z-VAD-fmk, Z-Val-Ala-Asp-fluoromethylketone.
membrane potential, we assessed protein levels of the Naⴙ/
Kⴙ-ATPase in apoptotic thymocytes. Spontaneously dying
thymocytes had decreased levels of both catalytic and regulatory subunits of Naⴙ/Kⴙ-ATPase, and glucocorticoid treatment enhanced the loss of Naⴙ/Kⴙ-ATPase protein. The pan
caspase inhibitor (z-VAD) blocked both cellular depolarization and repression of Naⴙ/Kⴙ-ATPase in both spontaneously
dying and glucocorticoid-treated thymocytes; however, specific inhibitors of caspase 8, 9, and caspase 3 did not. Interestingly, glucocorticoid treatment simultaneously induced
cell shrinkage and depolarization. Furthermore, depolarization and the loss of Naⴙ/Kⴙ-ATPase protein were limited to the
shrunken population of cells. The data indicate an important
role for Naⴙ/Kⴙ-ATPase in both spontaneous and glucocorticoid-induced apoptosis of rat thymocytes. (Endocrinology 142:
5059 –5068, 2001)
dent upon activation of the caspase cascade (5, 15). Interestingly, the loss of potassium has been shown to occur only in
the shrunken population of cells (10, 16), and the hypotonic
intracellular environment caused by the loss of potassium is
required for subsequent activation of caspase 3-like enzymes
and DNA degradation (15–17).
Recent studies from our laboratory have shown that antiFas antibody treatment of human Jurkat T cells led to sustained depolarization of the plasma membrane and other
characteristics of apoptosis (18). We have also observed that
glucocorticoids induce receptor-dependent depolarization of
the plasma membrane of primary isolated rat thymocytes
both in vivo and in vitro (19). The ability of glucocorticoids to
depolarize the membrane correlated with their ability to
induce apoptosis in the target cell. Interestingly, apoptotic
stimuli, such as survival factor withdrawal, also lead to depolarization of thymocytes and subsequent detection of apoptotic characteristics, but the mechanisms defining this cellular apoptosis are largely unknown.
The plasma membrane potential of lymphocytes is maintained predominantly by the electrogenic action of Na⫹/K⫹adenosine triphosphatase (Na⫹/K⫹-ATPase) (20, 21). The
Na⫹/K⫹-ATPase is an ATP-dependent membrane enzyme
that exchanges 3Na⫹ for 2K⫹ against an electrochemical gradient (22) and thus maintains high potassium and low sodium levels within the cell (23, 24). Inhibition of Na⫹/K⫹ATPase leads to depolarization of the plasma membrane in
5059
5060
Endocrinology, December 2001, 142(12):5059 –5068
a variety of cells, including Jurkat cells (25). Furthermore,
inhibition of Na⫹/K⫹-ATPase enhances the sensitivity of
Jurkat cells to the death-inducing actions of anti-Fas antibody. However, it is unknown whether Na⫹/K⫹-ATPase is
regulated during spontaneous or glucocorticoid-induced apoptosis of primary rat thymocytes. Additionally, it is unclear
whther caspases, which are known to be activated in both
spontaneous and glucocorticoid-induced apoptosis (5), are
involved in modulation of cellular Na⫹/K⫹-ATPase.
The present study examines the role of Na⫹/K⫹-ATPase
in glucocorticoid-mediated apoptosis of primary thymocytes. We report that thymocytes depolarize during spontaneous and glucocorticoid-induced cell death and that
inhibition of Na⫹/K⫹-ATPase enhances both cellular depolarization and apoptosis. Interestingly, although nonselective inhibition of the caspase cascade blocks cellular depolarization in response to both apoptotic stimuli, caspase
specific inhibitors do not. We show that Na⫹/K⫹-ATPase
protein levels selectively decrease in both spontaneous and
glucocorticoid-induced death. Finally, examination of the
relationship between cell shrinkage, depolarization, and
Na⫹/K⫹-ATPase repression reveals that depolarization and
repression of Na⫹/K⫹-ATPase protein levels occur in the
shrunken population of cells.
Materials and Methods
Reagents
FCS was purchased from Summit Biotechnology (Fort Collins, CO),
and dexamethasone was purchased from Steraloids (Wilton, NH). Z-IleGlu-Thr-Asp-fluoromethylketone (IETD-fmk), Z-Ala-Glu (Ome)-ValAsp (Ome)-FMK (AEVD-fmk), Z-Asp-Glu-Val-Asp-fluoromethylketone
(DEVD-fmk), and Z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk)
were purchased from Kamiya Biomedical Co. (Seattle, WA). DiBAC4(3)
was purchased from Molecular Probes, Inc. (Eugene, OR). Ouabain and
propidium iodide (PI) were purchased from Sigma (St. Louis, MO).
Animals
Male Sprague Dawley rats (2–3 months of age) were used in all
experiments. The animals were bilaterally adrenalectomized by the provider at least 5 d before use, maintained under controlled conditions of
temperature (25 C) and lighting, and allowed free access to food and
0.85% saline. All experimental protocols were approved by the animal
review committee at the institute and were performed in accordance
with the guidelines set forth in the NIH Guide for the Care and Use of
Laboratory Animals published by the USPHS. Animals were killed by
decapitation, and the thymus was surgically removed.
Thymocyte cultures
Thymocytes were prepared from freshly isolated thymus as previously described (16, 26, 27). Briefly, thymocytes were dispersed by gentle
homogenization in a glass/glass homogenizer (no. 22, Kontes Co., Vineland, NJ), filtered, washed in cold PBS, and counted on a hemocytometer. Cells were cultured (5 ⫻ 106 cells/ml) at 37 C in 5% CO2, in RPMI
1640 medium supplemented with 10% heat-inactivated FCS, 4 mm glutamine, 100 U/ml penicillin, and 75 U/ml streptomycin sulfate (prepared in house).
Mann et al. • Glucocorticoids and Plasma Membrane Depolarization
to exclude nonviable cells (28). Cells were examined as changes in their
plasma membrane potential and cell size by flow cytometry using a
FACSort (Becton Dickinson and Co., Mountain View, CA) equipped
with an argon (488-nm) laser. Fifteen thousand cells were examined
under each condition, and all flow cytometric analysis was accomplished using CellQuest software. Cell size was monitored by alterations
in the forward light-scattering properties of the cells as described previously (5). Fluorescent emission of DiBAC4(3) and PI were detected at
530 and 650 nm, respectively. An increase in DiBAC4(3) fluorescence at
530 nm indicates cellular depolarization. The percentage of cells with
increased DiBAC4(3) fluorescence was determined by gating on the
fresh, viable population of cells. Cells with DiBAC4(3) fluorescence
greater that that for the fresh population of cells were quantified for each
treatment. The average ⫾ sem for each treatment represent at least three
independent experiments. Statistical analyses were performed using t
test with ␣ ⫽ 0.05.
Protein extraction and immunoblot analysis
Total cellular protein was recovered by lysis in high detergent buffer
[20 mm Tris (pH 7.5), 2 mm EDTA, 150 mm NaCl, 0.5% Triton X-100, 0.1%
SDS, and 0.5% sodium deoxycholate] with protease inhibitors (Roche,
Indianapolis, IN). Protein was quantified by the method of Bradford
(Bio-Rad Laboratories, Inc., Hercules, CA) and denatured in 5 ⫻ Laemmli
buffer [250 mm Tris (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50%
glycerol, and 100 mm ␤-mercaptoethanol] at 100 C for 5 min and then stored
at ⫺70 C. Proteins were resolved by SDS-PAGE through 4–20% gradient
gels (Novex, San Diego, CA) and transferred to nitrocellulose in Tris-glycine
buffer [12 mm Tris-HCl (pH 8.3), 75 mm glycine, and 20% methanol].
Membranes were stained with Ponceau S (0.5% in 1% acetic acid) to confirm
equal loading and to evaluate transfer efficiency. Next, membranes were
preincubated overnight at 4 C in Tris-buffered saline [TBS; 10 mm Tris-HCl
(pH 7.4), 150 mm NaCl, and 0.05% Tween 20] with 10% nonfat dry milk.
The blots were then washed with TBS and incubated overnight at 4 C with
a 1:250 dilution of either antirabbit Na⫹/K⫹-ATPase-␣ monoclonal antibody or antirabbit Na⫹/K⫹-ATPase-␤ monoclonal antibody (Upstate Biotechnology, Inc., Saranac Lake, NY). To assess GR levels, blots were incubated with a 1:1,000 dilution of an antipeptide GR antibody (29). Actin levels
were determined by incubation with a 1:10,000 dilution of a mouse antiactin
antibody (Chemicon, Temecula, CA). Membranes were then washed according to the manufacturer’s instructions and incubated with a horseradish peroxidase-labeled goat antimouse or goat antirabbit secondary antibody (1:10,000 in TBS; Amersham Pharmacia Biotech, Arlington Heights,
IL), washed again, and reacted with chemiluminescent reagents for autoradiography (Amersham Pharmacia Biotech).
Cell sorting
To analyze Na⫹/K⫹-ATPase protein levels in normal vs. shrunken
cells, thymocytes were treated with dexamethasone for 3 h to induce cell
shrinkage. After this time the cells were stained with PI to eliminate from
further analysis cells that had lost their membrane integrity. Simultaneous sorting of both normal and shrunken populations of cells was
accomplished using a Becton Dickinson and Co. FACSVantage SE
equipped with CellQuest software. A gate was set on a forward scatter
vs. PI fluorescence plot based on an untreated control sample to isolate
the normal population of cells. A second gate has then set to the left of
this normal cell population denoting cells that had a decreased ability
to scatter light the forward direction, indicating cells that were of smaller
or shrunken cell size. Sorted cells were maintained at 4 C to maintain
their integrity after the sort. Eight million cells were immediately processed for protein analysis by immunoblot analysis as described above.
FACS analysis
Results
Plasma membrane depolarizes during spontaneous and
glucocorticoid-induced thymocyte apoptosis
The plasma membrane of isolated thymocytes was measured with the
anionic oxonal dye DiBAC4(3) (Molecular Probes, Inc.) as described
previously (18). DiBAC4(3) was prepared in dimethylsulfoxide according to the manufacturer’s instructions. Briefly, cells were incubated with
150 nm DiBAC4(3) for 30 min at 37 C in 5% CO2. PI was added to a final
concentration of 10 ␮g/ml immediately before flow cytometric analysis
We have recently shown that in rat thymocytes, glucocorticoids depolarize the plasma membrane in a dose-dependent manner that is dependent upon the interaction of glucocorticoids with the GR and subsequent gene expression
(19). These studies were conducted with the plasma mem-
Mann et al. • Glucocorticoids and Plasma Membrane Depolarization
brane potential-sensitive dye DiBAC4(3), an oxonal anionic
dye that responds to changes in plasma membrane potential
(18, 30). Figure 1 provides an example of DiBAC4(3) fluorescence in freshly isolated thymocytes, spontaneously dying thymocytes, and thymocytes treated with dexamethasone for 6 h. Freshly isolated thymocytes have low DiBAC4(3)
fluorescence, indicating that plasma membrane potential is
intact. Furthermore, addition of high extracellular potassium
to the media in these cells as well as human lymphocytes
results in cellular depolarization (18), indicating that this dye
is reliable for measurement of plasma membrane potential.
After 6 h in culture, there is a population of cells with increased DiBAC4(3) fluorescence (19.5 ⫾ 1.8% vs. 9.1 ⫾ 1.7%
in the fresh population; P ⬍ 0.05 vs. freshly isolated cells).
These data are consistent with our previous observations that
primary thymocytes undergo spontaneous death in the absence of an apoptotic stimulus (19). Glucocorticoid treatment
exacerbates this effect by dramatically increasing the percentage of depolarized cells after 6 h (41.2 ⫾ 6.2%; P ⬍ 0.05
vs. time-matched control cells). It is important to note that
glucocorticoids do not increase the magnitude of the depolarization, only the percentage of cells that have a depolarized plasma membrane, suggesting that apoptosis is stochastic in this model system. Similarly, examples of cellular
depolarization have been observed in Jurkat cells treated
with other apoptotic stimuli (18). These data in combination
with our previous observations demonstrate that rat thymocytes depolarize their plasma membrane during both spontaneous and glucocorticoid-induced apoptosis, but the
mechanisms underlying this effect and the role of caspases
in this process are currently unknown.
Inhibition of Na⫹/K⫹-ATPase depolarizes thymocytes and
potentiates glucocorticoid-induced loss of plasma
membrane potential
In thymocytes and lymphocytes, in general, Na⫹/K⫹-ATPase is the primary pump that maintains the electrochemical
FIG. 1. Dexamethasone depolarizes rat
thymocytes. Fresh thymocytes or thymocytes cultured for 6 h in the presence
or absence of 100 nM dexamethasone
were incubated with DiBAC4(3) to evaluate plasma membrane potential. Before flow cytometric analysis, PI was
added to exclude nonviable cells. Ten
thousand viable cells for each treatment were evaluated for DiBAC4(3) fluorescence as described in Materials and
Methods. The figure shows representative histograms for DiBAC4(3) fluorescence in freshly isolated thymocytes
(FRESH) or thymocytes cultured for 6 h
alone (CON) and with dexamethasone
(DEX).
Endocrinology, December 2001, 142(12):5059 –5068 5061
gradient that is responsible for establishment of plasma
membrane potential (20 –22). Disruption of Na⫹/K⫹-ATPase
by ouabain is well known to lead to depolarization the
plasma membrane (18). To determine whether Na⫹/K⫹-ATPase may be involved in the loss of plasma membrane potential during glucocorticoid-induced apoptosis of thymocytes, we blocked the activity of Na⫹/K⫹-ATPase with
ouabain, a specific inhibitor of Na⫹/K⫹-ATPase in rats as
well as other species (31). As shown in Fig. 2, ouabain treatment alone depolarizes primary thymocytes, as evidenced by
the increase in the number of cells with high DiBAC4(3)
fluorescence (47.9 ⫾ 5.3%; P ⬍ 0.05 vs. time-matched control
cells) vs. the control cells (19.5 ⫾ 1.8%; fresh, 9.1 ⫾ 1.7%; P ⬍
0.05 vs. freshly isolated cells). Additionally, ouabain potentiated the effects of glucocorticoids alone (38.5 ⫾ 4.7%; P ⬍
0.05 vs. time-matched control cells) by significantly increasing the number of cells with high DiBAC4(3) fluorescence
(60.0 ⫾ 1.6%; P ⬍ 0.05 vs. time-matched control cells). These
results demonstrate that inhibition of Na⫹/K⫹-ATPase potentiates glucocorticoid-induced depolarization and suggests a potential role for Na⫹/K⫹-ATPase in the depolarization of thymocytes during apoptosis.
Glucocorticoids repress Na⫹/K⫹-ATPase protein levels
during apoptosis in thymocytes
The fact that inhibition of Na⫹/K⫹-ATPase resulted in the
loss of plasma membrane potential suggested that Na⫹/K⫹ATPase might be a target for inhibition or perhaps degradation during glucocorticoid-induced apoptosis. In models
of Fas-induced apoptosis of Jurkat cells it has been shown
that decreased levels of both catalytic (␣) and regulatory (␤)
subunits of Na⫹/K⫹-ATPase occur (18). To determine
whether glucocorticoid treatment alters the protein levels of
Na⫹/K⫹-ATPase in primary thymocytes, we examined the
protein levels of both catalytic and regulatory subunits of
Na⫹/K⫹-ATPase (Fig. 3). Interestingly, we similarly found
that the protein levels of both the catalytic and regulatory
5062
Endocrinology, December 2001, 142(12):5059 –5068
FIG. 2. Inhibition of Na⫹/K⫹-ATPase potentiates glucocorticoidinduced loss of plasma membrane potential. Primary isolated thymocytes were cultured with ouabain (10 mM) in the presence or absence of dexamethasone (100 nM) for 6 h. After treatment, cells were
incubated with DiBAC4(3). PI was added before flow cytometric analysis to exclude nonviable cells. Ten thousand viable cells for each
treatment were evaluated for DiBAC4(3) fluorescence as described in
Materials and Methods. Representative DiBAC4(3) fluorescence histograms are shown for each treatment.
subunits decreased in untreated cells after 6 h compared with
freshly isolated cells, findings consistent with the fact that all
of these isolated cells will eventually depolarize and undergo
spontaneous apoptosis. Glucocorticoid treatment augmented the loss of Na⫹/K⫹-ATPase protein to negligibly
detectable levels. In contrast, under the same experimental
conditions, GR protein decreased only minimally under
these in vitro incubation conditions, and cellular actin levels
did not change. These results demonstrate that Na⫹/K⫹ATPase protein levels are selectively decreased during cell
death and are consistent with the observed depolarization of
the plasma membrane in both spontaneous and glucocorticoid-induced thymocyte apoptosis. The extent of decrease in
Na⫹/K⫹-ATPase protein levels largely reflects the number of
cells in spontaneous and glucocorticoid-induced death that
depolarize their plasma membrane, but the assays we have
used do not directly measure activity.
Glucocorticoid-induced loss of plasma membrane potential
is modulated by caspases
Our observation that plasma membrane depolarization
and the loss of Na⫹/K⫹-ATPase protein are observed in both
Mann et al. • Glucocorticoids and Plasma Membrane Depolarization
spontaneously dying thymocytes and glucocorticoid-treated
thymocytes suggests that a common pathway is activated
during both signaling cascades necessary for the induction of
apoptosis. We have previously shown that activation of the
caspase cascade is a common feature of both spontaneous
and glucocorticoid-induced apoptosis (5). Furthermore, we
have shown that z-VAD, a pan-caspase inhibitor, modulates
both spontaneous cell shrinkage and blocks glucocorticoidinduced cell shrinkage in thymocytes (5). In addition, cell
shrinkage and depolarization appeared to be associated in
glucocorticoid-induced apoptosis (19). To determine whether
caspases are involved in glucocorticoid-induced membrane depolarization, we next evaluated the effects of caspase inhibition
on glucocorticoid-induced membrane depolarization and cell
shrinkage. After 6 h in culture, 16.2 ⫾ 1.4% (P ⬍ 0.05 vs. freshly
isolated cells) of thymocytes undergoing spontaneous cell
death were depolarized, and 44.8 ⫾ 4.3% (P ⬍ 0.05 vs. timematched control cells) of glucocorticoid-treated thymocytes
were depolarized compared with freshly isolated thymocytes
(8.6 ⫾ 2.0%; Fig. 4). Cellular depolarization was accompanied
by concomitant cell shrinkage, indicating that thymocyte depolarization and shrinkage are tightly coupled. The pancaspase inhibitor z-VAD-fmk inhibited cell shrinkage and
blocked plasma membrane depolarization (11.4 ⫾ 2.3%), such
that a statistically significant difference could not be observed
between the percentage of depolarized cells in freshly isolated
thymocytes or thymocytes treated with z-VAD. Glucocorticoidinduced depolarization of thymocytes was also blocked by zVAD (14.9 ⫾ 3.2%) as was glucocorticoid-induced cell shrinkage. Interestingly, IETD-fmk, a specific inhibitor of caspase
8-like enzymes, did not block cell shrinkage or the spontaneous
depolarization of thymocytes (15.2 ⫾ 0.9%; P ⬍ 0.05 vs. freshly
isolated cells). Inhibition of caspase 8-like activity also did not
block the glucocorticoid-induced cell shrinkage or the loss of
plasma membrane potential (37.0 ⫾ 4.5%; P ⬍ 0.05 vs. timematched control cells). Inhibition of caspase 9-like activity by
the specific inhibitor AEVD also was ineffective in blocking
spontaneous (16.5 ⫾ 0.5%; P ⬍ 0.05 vs. freshly isolated cells) or
glucocorticoid-induced apoptosis (33.4 ⫾ 1.0%; P ⬍ 0.05 vs.
time-matched control cells). Furthermore, DEVD, a more specific inhibitor of caspase 3-like enzymes, was even less effective
at blocking cell shrinkage and the loss of plasma membrane
potential in spontaneous (25.0 ⫾ 3.2%; P ⬍ 0.05 vs. timematched control cells) or glucocorticoid-induced (39.5 ⫾ 7.2%;
P ⬍ 0.05 vs. time-matched control cells) cell death. These results
suggest that the individual activation of caspase 8-like enzymes,
caspase 9-like enzymes, and caspase 3-like enzymes is not sufficient for cell shrinkage or membrane depolarization in spontaneous or glucocorticoid-induced death. These specific caspase
inhibitor studies suggest that either an unusual caspase is mediating membrane depolarization or perhaps z-VAD-fmk may
inhibit proteases other than caspases, which are involved in the
generation of a cellular depolarized state.
Inhibition of caspase activity blocks the decrease in Na⫹/
K⫹-ATPase protein levels during thymocyte apoptosis
To determine the effect of caspases on Na⫹/K⫹-ATPase
protein levels during apoptosis, we examined the effect of
z-VAD-fmk on the loss of Na⫹/K⫹-ATPase protein levels
during both spontaneous and glucocorticoid-induced apoptosis. As shown earlier (Fig. 3), the catalytic and regulatory
subunits of Na⫹/K⫹-ATPase were both decreased in spon-
Mann et al. • Glucocorticoids and Plasma Membrane Depolarization
Endocrinology, December 2001, 142(12):5059 –5068 5063
FIG. 3. Glucocorticoids repress Na⫹/
K⫹-ATPase protein levels in primary
thymocytes. Total cellular protein was
extracted from fresh thymocytes
(FRESH) or thymocytes cultured for 6 h
in the presence (DEX) or absence (CON)
of 100 nM dexamethasone. Twenty-five
micrograms of total cellular protein was
resolved by SDS-PAGE. Protein levels
of the ␣1- and ␤1-subunits of Na⫹/K⫹ATPase as well as levels of GR and actin
were evaluated by immunoblot analysis
as described in Materials and Methods.
taneously dying and glucocorticoid-treated thymocytes
compared with freshly isolated thymocytes (Fig. 5). Inhibition of the caspase cascade by the pan-caspase inhibitor zVAD blocked the loss of in Na⫹/K⫹-ATPase levels in both
spontaneous and glucocorticoid-induced apoptosis (Fig. 5)
without affecting the level of cellular actin. These results are
consistent with the observation that z-VAD blocked plasma
membrane depolarization and suggest that a common pathway, perhaps mediated by novel caspases, is activated
in both spontaneously dying and glucocorticoid-treated
thymocytes.
Cells with a loss of plasma membrane potential comprise
the shrunken population of cells
The experiments described thus far were performed in a
population of cells that included both shrunken, apoptotic
cells and normal cells. We have previously shown that
shrinkage is a defining feature of apoptosis that discriminates committed and noncommitted cells, and in Fas-treated
Jurkat cells, depolarization precedes cell shrinkage (18). To
determine whether depolarization precedes cell shrinkage in
glucocorticoid-treated thymocytes, we analyzed thymocytes
by flow cytometry to simultaneously determine cell size and
relative plasma membrane potential (Fig. 6). Freshly isolated
cells form a population with a uniform size distribution that
has low DiBAC4(3) fluorescence, indicating that the plasma
membrane potential is intact. Spontaneous cell death results
in the appearance of a small population of shrunken cells,
which is consistent with our previous observation that spontaneously dying thymocytes shrink (5). In spontaneous apoptosis, the population of cells that are still of normal size has
low DiBAC4(3) fluorescence, indicating that these cells have
not lost their plasma membrane potential. However, the
shrunken population of cells has high DiBAC4(3) fluorescence, indicating that all of these cells have lost plasma membrane potential. Glucocorticoids dramatically increase the
shrunken population of cells, as we have reported previously
(5). However, there remains a subpopulation with a normal
cell volume. This population, similar to normal cells in the
fresh and spontaneously dying samples, still has low
DiBAC4(3) fluorescence. All of the shrunken cells, however,
have high DiBAC4(3) fluorescence, indicating they have lost
their plasma membrane potential. These results demonstrate
that in spontaneously dying and glucocorticoid-treated thymocytes, plasma membrane depolarization is limited to the
shrunken population of cells.
The decrease in Na⫹/K⫹-ATPase protein levels is limited to
the shrunken, depolarized population of cells
The results presented thus far suggest that cell shrinkage
and depolarization correlate with a decrease in Na⫹/K⫹ATPase protein levels. However, the analyses of Na⫹/K⫹ATPase protein levels were only conducted on the entire
population of cells, which contains a mixture of polarized
normal cells and depolarized shrunken cells. Therefore, we
were interested in determining whether the repression of
Na⫹/K⫹-ATPase protein levels was restricted to either the
normal or shrunken population of apoptotic cells. To accomplish this goal, glucocorticoid-treated cells were physically
sorted into shrunken and normal populations and evaluated
for changes in Na⫹/K⫹-ATPase levels. ATPase levels of the
catalytic and regulatory subunits of Na⫹/K⫹-ATPase were
also evaluated in freshly isolated thymocytes and in the
entire population of glucocorticoid-treated thymocytes (Fig.
7). Cells sorted into normal and shrunken populations
showed a dramatic difference in protein levels of the Na⫹/
K⫹-ATPase. In the normal population, Na⫹/K⫹-ATPase protein levels were the same as those for freshly isolated thymocytes. In contrast, the shrunken population showed
dramatically diminished levels of both catalytic and regulatory subunits of Na⫹/K⫹-ATPase, whereas no significant
difference in the cellular levels of actin was seen between the
normal and shrunken populations of cells. These results
demonstrate that the repression of Na⫹/K⫹-ATPase protein
levels, like depolarization, is limited to the shrunken population of cells.
5064
Endocrinology, December 2001, 142(12):5059 –5068
Mann et al. • Glucocorticoids and Plasma Membrane Depolarization
FIG. 4. Caspases modulate glucocorticoid-induced plasma membrane depolarization. Primary thymocytes were
cultured with 100 ␮M z-VAD-fmk, 100
␮M IETD-fmk, or 100 ␮M DEVD-fmk in
the presence or absence of 100 nM dexamethasone for 6 h. After treatment,
cells were incubated with DiBAC4(3). PI
was added before flow cytometric analysis to exclude nonviable cells.
DiBAC4(3) fluorescence was evaluated
in 10,000 viable cells as described in
Materials and Methods. To compare
DiBAC4(3) fluorescence vs. cell size,
cells were analyzed on a representative
DiBAC4(3) fluorescence vs. forward
scatter contour plot.
Discussion
Research over the last several years has established that
cell shrinkage and the movement of ions play important roles
in apoptosis (8 –10, 16, 32). The movement of ions and the
resulting alterations in the electrical field across the membrane lead to cellular depolarization, which is an important
step in many cellular processes ranging from stimulussecretion coupling (33) to mitogenic T cell activation (34).
Mann et al. • Glucocorticoids and Plasma Membrane Depolarization
Endocrinology, December 2001, 142(12):5059 –5068 5065
FIG. 5. z-VAD blocks glucocorticoid-induced repression of Na⫹/K⫹-ATPase protein levels. Total cellular protein was extracted from fresh
thymocytes or thymocytes cultured with 100 ␮M z-VAD-fmk in the presence or absence of 100 nM dexamethasone for 6 h. Twenty-five micrograms
of total cellular protein were resolved by SDS-PAGE. Protein levels of the ␣1- and ␤1-subunits of Na⫹/K⫹-ATPase and actin were evaluated by
immunoblot analysis as described in Materials and Methods.
FIG. 6. Simultaneous depolarization and shrinkage in rat thymocytes. Fresh thymocytes (FRESH) or thymocytes cultured in the presence (DEX)
or absence (CON) of dexamethasone for 6 h were analyzed for their light-scattering properties and DiBAC4(3) fluorescence. The center column
shows forward scatter vs. side scatter dot plots for each treatment. Gates were set based on the distribution of the normal population in the
freshly isolated cells (right gate). Shrunken cells have a decrease in forward scatter and a concomitant increase in side scatter and are shown
in the left gate. The left column shows the DiBAC4(3) fluorescence for the shrunken population. The right column shows the DiBAC4(3)
fluorescence for the normal population.
Work from our laboratory (18, 19) as well as others (35) has
suggested that plasma membrane depolarization may be an
important component of the apoptotic process. In addition,
we recently reported that Na⫹/K⫹-ATPase might play a key
role in anti-Fas-induced depolarization of Jurkat cells (18).
This study has extended these observations to define a role
for Na⫹/K⫹-ATPase in the depolarization of primary thymocytes during both spontaneous and glucocorticoidinduced apoptosis.
Na⫹/K⫹-ATPase is the primary determinant for setting
plasma membrane potential in lymphocytes (20 –22). The
present study shows that inhibition of Na⫹/K⫹-ATPase by
5066
Endocrinology, December 2001, 142(12):5059 –5068
Mann et al. • Glucocorticoids and Plasma Membrane Depolarization
FIG. 7. Na⫹/K⫹-ATPase protein levels
decrease in shrunken rat thymocytes.
Thymocytes were treated with dexamethasone for 3 h to induce cell shrinkage. After the incubation, thymocytes
were physically sorted into normal and
shrunken populations, as described in
Materials and Methods. Total cellular
protein was extracted from freshly isolated thymocytes (FRESH), thymocytes
treated with dexamethasone for 3 h just
before sorting (PRESORT), normal thymocytes (NORM), and shrunken thymocytes (SHRUNK). Twenty-five micrograms of total cellular protein were
resolved by SDS-PAGE. Protein levels
of the ␣1- and ␤1-subunits of Na⫹/K⫹ATPase as well as actin were evaluated
by immunoblot analysis as described in
Materials and Methods.
ouabain depolarizes thymocytes and potentiates glucocorticoid-induced depolarization of primary thymocytes. These
data support our previous observation that inhibition of
Na⫹/K⫹-ATPase by ouabain potentiates Fas-induced depolarization in Jurkat cells and suggests a role for Na⫹/K⫹ATPase in thymocyte depolarization and apoptosis. An important role for Na⫹/K⫹-ATPase in lymphocyte function is
also supported by the fact that Na⫹/K⫹-ATPase activity
increases during mitogenic T cell activation (36, 37). Furthermore, inhibition of Na⫹/K⫹-ATPase activity by ouabain
has been shown to block mitogenic T cell activation (38).
Together, these observations implicate Na⫹/K⫹-ATPase as
an important moderator of lymphocyte survival.
The plasma membrane depolarization induced during
both spontaneous and glucocorticoid-induced apoptosis was
associated with a selective decrease in Na⫹/K⫹-ATPase protein levels within the cell. The fact that we observed Na⫹/
K⫹-ATPase degradation in both spontaneous and glucocorticoid-induced death suggests that the repression of Na⫹/
K⫹-ATPase levels is a shared feature of a common apoptotic
pathway induced by disparate signals. Indeed, we also observed that Na⫹/K⫹-ATPase levels are decreased during
Fas-induced apoptosis of Jurkat cells, and inhibition of this
depolarization via activation of PKC blocked apoptotic cell
death (18). These results suggested that a pathway common
to these diverse apoptotic-signaling pathways results in the
repression of Na⫹/K⫹-ATPase protein levels.
The activation of the caspase cascade is a common feature
to these divergent forms of apoptosis (5, 15, 39 – 42). We have
previously shown that caspases are activated during spontaneous and glucocorticoid-induced apoptosis, and that
caspases mediate glucocorticoid-induced and spontaneous
cell shrinkage (5, 15). In this study we found that the pancaspase inhibitor z-VAD-fmk blocked the loss of plasma
membrane potential in both spontaneous and glucocorticoid-induced apoptosis, which suggests that the caspase cascade plays a role in cell membrane depolarization during
apoptosis. Furthermore, the inhibition of depolarization by
z-VAD occurred simultaneously with the inhibition of cell
shrinkage, suggesting a close relation between the two processes, as we have noted previously (18). Inhibition of the
caspase cascade by the pan-caspase inhibitor z-VAD-fmk
also blocked the repression of Na⫹/K⫹-ATPase protein levels in both spontaneous and glucocorticoid-induced apoptosis of thymocytes. These data suggest the involvement of the
caspase cascade in the repression of Na⫹/K⫹-ATPase levels.
However, selective inhibition of caspase 8-like enzymes,
caspase 9-like enzymes, or caspase 3-like enzymes did not
block spontaneous or glucocorticoid-induced depolarization
in primary thymocytes, suggesting that the activation of
these specific enzymes is not required or downstream of
plasma membrane depolarization in thymocytes. In anti-Fastreated Jurkat T cells, other characteristics, such as cell
shrinkage, potassium loss, and the loss of mitochondrial
membrane potential, were all shown to be independent of
caspase 9-like and caspase 3-like enzymes (17). However, the
nonspecific caspase inhibitor z-VAD-fmk was able to prevent
these characteristics of apoptosis in anti-Fas-treated Jurkat
cells. Therefore, either different caspases may be involved in
the pathway that leads to plasma membrane depolarization
or z-VAD-fmk may be a less specific inhibitor of proteases
than was previously thought, perhaps acting outside the
caspase pathway.
Activation of the caspase cascade and cell shrinkage are
Mann et al. • Glucocorticoids and Plasma Membrane Depolarization
tightly linked. In anti-Fas-treated Jurkat cells, DNA fragmentation, decreased potassium content, and active caspase
3-like enzymes are limited to the shrunken population of
cells (10). In fact, the activation of caspase 3-like enzymes is
inhibited by physiological concentrations of potassium, and
caspase 3-like and nuclease activity are observed only in cells
with decreased potassium content (16). The data presented
in the present study show that plasma membrane depolarization in thymocytes is limited to the shrunken population
of cells in both spontaneously dying cells and cells treated
with glucocorticoids. These results indicate that in thymocytes, two stimuli that induce apoptosis through different
signaling pathways (5) simultaneously trigger cell shrinkage
and depolarization. In contrast, we have observed that Jurkat
cells treated with anti-Fas depolarize before the loss of cell
volume (18). One possible explanation for this difference is
that thymocytes are much smaller than Jurkat cells and respond rapidly to glucocorticoid treatment, which may make
it difficult to temporally distinguish sequential activation of
depolarization and shrinkage in these cells. Additionally, the
difference in cellular responsiveness could be due to differences in the cell types. Whereas thymocytes are a primary cell
line and die spontaneously in culture, Jurkat cells are a transformed cell line that has been selected to grow in culture.
Alternatively, the difference between Jurkat cells and thymocytes could be due to differences in the pathways that
regulate depolarization and shrinkage in these cells.
Although levels of Na⫹/K⫹-ATPase decrease during
spontaneous and glucocorticoid-induced apoptosis, the protein levels were initially evaluated in a mixed population of
cells that included both normal and shrunken cells. When
these two populations of cells were subsequently physically
separated, we observed that the normal population retains
Na⫹/K⫹-ATPase expression levels equivalent to freshly isolated cells. The shrunken population, however, had a dramatic decrease in Na⫹/K⫹-ATPase protein levels, which
demonstrates that the repression of Na⫹/K⫹-ATPase protein
levels is limited to the shrunken population of cells and
supports our previous finding that Na⫹/K⫹-ATPase protein
levels of both the catalytic and regulatory subunits are repressed in anti-Fas-treated Jurkat cells.
The observation that ion fluxes and depolarization occur
early in apoptosis induced by disparate signals in different
cell types suggests that these are conserved features of apoptosis that are also important events in other cellular processes. Na⫹/K⫹-ATPase plays a central role in maintaining
this ionic balance and is conserved across species (43). Given
that the potential difference across the plasma membrane
results from the asymmetric distribution of ions and is maintained by the electrogenic action of Na⫹/K⫹-ATPase (22), it
stands to reason that Na⫹/K⫹-ATPase may play an important role in apoptosis as it does in other cellular processes.
The importance of Na⫹/K⫹-ATPase in apoptosis was first
demonstrated in Jurkat cells treated with anti-Fas. We have
now extended these studies to primary thymocytes and have
observed that two disparate apoptotic signals, survival factor
withdrawal and glucocorticoids, repress Na⫹/K⫹-ATPase
protein levels during thymocyte apoptosis. Additionally, we
have shown that both depolarization and the repression of
Na⫹/K⫹-ATPase protein levels are mediated by the caspase
Endocrinology, December 2001, 142(12):5059 –5068 5067
cascade and are limited to the shrunken population of cells.
Future work in this area will likely show that Na⫹/K⫹ATPase, which is essential to the maintenance of cellular
homeostasis, is an important target in other forms of apoptosis as well.
Acknowledgments
Received March 12, 2001. Accepted July 9, 2001.
Address all correspondence and requests for reprints to: Dr. John A.
Cidlowski, P.O. Box 12233, MD F3-07, 111 Alexander Drive, Research
Triangle Park, North Carolina 27709. E-mail: [email protected].
References
1. Wyllie A 1980 Glucocorticoid-induced thymocyte apoptosis is associated with
endogenous endonuclease activation. Nature 284:555–556
2. Compton M, Cidlowski J 1986 Rapid in vivo effects of glucocorticoids on the
integrity of rat lymphocyte genomic deoxyribonucleic acid. Endocrinology
118:38 – 45
3. Schwartzman RA, Cidlowski JA 1993 Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocr Rev 14:133–151
4. Schwartzman RA, Cidlowski JA 1994 Glucocorticoid-induced apoptosis of
lymphoid cells. Int Arch Allergy Immunol 105:347–354
5. Mann C, Hughes Jr FM, Cidlowski JA 2000 Delineation of the signaling
pathways involved in glucocorticoid-induced and spontaneous apoptosis of
rat thymocytes. Endocrinology 141:528 –538
6. Hallahan C, Young DA, Munck A 1973 Timecourse of early events in the
action of glucocorticoids on rat thymus cells in vitro: synthesis and turnover
of a hypothetical cortisol-induced protein inhibitor of glucose metabolism and
of a presumed RNA. J Biol Chem 248:2922–2927
7. Lockshin R, Beaulaton J 1981 Cell death: questions for histochemists concerning the causes of the various cytological changes. Histochemistry J 13:
659 – 666
8. Bortner CD, Hughes Jr FM, Cidlowski JA 1997 Cell volume regulation, ions,
and apoptosis. In: Shi YB, ed. Programmed cell death. New York: Plenum
Press; 63–70
9. Bortner CD, Cidlowski JA 1997 Cell volume regulation and the movement of
ions during apoptosis. In: Tilly J, Tenniswood M, eds. Cell death in reproductive physiology. New York: Springer-Verlag; 230 –248
10. Bortner CD, Hughes Jr FM, Cidlowski JA 1997 A primary role for K⫹ and Na⫹
efflux in the activation of apoptosis. J Biol Chem 272:32436 –32442
11. Fadock VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM
1992 Exposure of phosphatidylserine on the surface of apoptotic lymphocytes
triggers specific recognition and removal by macrophages. J Immunol 148:
2207–2216
12. Kerr JFR 1971 Shrinkage necrosis: a distinct mode of cellular death. J Pathol
105:13–20
13. Kerr JFR, Wyllie AH, Currie AR 1972 Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br J Cancer 26:239 –257
14. Wyllie AH, Morris RG, Smith AL, Dunlop D 1984 Chromatin cleavage in
apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol 142:67–77
15. Hughes Jr FM, Cidlowski JA 1998 Glucocorticoid-induced thymocyte apoptosis: protease-dependent activation of cell shrinkage and DNA degradation.
J Steroid Biochem Mol Biol 65:207–217
16. Hughes Jr FM, Bortner CD, Purdy GD, Cidlowski JA 1997 Intracellular K⫹
suppresses the activation of apoptosis in lymphocytes. J Biol Chem 272:30567–
30576
17. Bortner CD, Cidlowski JA 1999 Caspase independent/dependent regulation
of K(⫹), cell shrinkage, and mitochondrial membrane potential during lymphocyte apoptosis. J Biol Chem 274:21953–21962
18. Bortner CD, Gómez-Angelats M, Cidlowski JA 2001 Plasma membrane depolarization without repolarization is an early molecular event in apoptosis
mediated by the inactivation of the Na⫹/K⫹-ATPase. J Biol Chem 276:4304 –
4314
19. Mann C, Cidlowski J 2001 Glucocorticoids regulate the plasma membrane
potential of primary thymocytes in vivo and in vitro. Endocrinology 142:
421– 429
20. Flaciola, J, Volet B, Anner RM, Moosmayer M, Lacotte D, Anner BM 1994
Role of cell membrane Na,K-ATPase for survival of human lymphocytes in
vitro. Biosci Rep 14:189 –204
21. Felber SM, Brand MD 1982 Factors determining the plasma-membrane potential of lymphocytes. Biochem J 204:577–585
22. Garrahan PJ, Glynn IM 1967 The stoichiometry of the sodium pump. J Physiol
192:217–235
23. Al-Habori M 1994 Cell volume and ion transport regulation. Int J Biochem
26:319 –334
5068 Endocrinology, December 2001, 142(12):5059 –5068
24. Hoffmann E 1987 Volume regulation in cultured cells. Curr Top Membr
Transport 30:125–180
25. Gusovsky F, Daly JW 1988 Formation of second messengers in response to
activation of ion channels in excitable cells. Cell Mol Neurobiol 8:157–169
26. Compton MM, Haskill JS, Cidlowski JA 1988 Analysis of glucocorticoid
actions on rat thymocyte deoxyribonucleic acid by fluorescence-activated flow
cytometry. Endocrinology 122:2158 –2163
27. Cidlowski J 1982 Glucocorticoids stimulate ribonucleic acid degradation in
isolated rat thymic lymphocytes in vitro. Endocrinology 111:184 –190
28. Darzynkiewicz Z, Li X, Gong J 1994 Assays of cell viability. Discrimination
of cells dying by apoptosis. Methods Cell Biol 41:15–38
29. Cidlowski J, Bellingham D, Lubahn FPO, Sar M 1990 Novel antipeptide
antibodies to the human glucocorticoid receptor: recognition of multiple forms
in vitro and distinct localization of cytoplasmic and nuclear receptors. Mol
Endocrinol 4:1427–1437
30. Wilson HA, Chused TM 1985 Lymphocyte membrane potential and Ca2⫹sensitive potassium channels described by oxonol dye fluorescence measurements. J Cell Physiol 125:72– 81
31. Lichtman MA, Jackson AH, Peck WA 1972 Lymphocyte monovalent cation
metabolism: cell volume, cation content, and cation transport. J Cell Physiol
80:383–396
32. Bortner CD, Cidlowski JA 1996 Absence of volume regulatory mechanisms
contributes to the rapid activation of apoptosis in thymocytes. Am J Physiol
271:C950 –C961
33. Henquin JC 1987 Regulation of insulin release by ionic and electrical events
in B cells. Horm Res 27:168 –178
34. Gupta S 1989 Mechanisms of transmembrane signaling in human T cell activation. Mol Cell Biochem 91:45–50
35. Deckers CL, Lyons AB, Samuel K, Sanderson A, Maddy AH 1993 Alternative
Mann et al. • Glucocorticoids and Plasma Membrane Depolarization
36.
37.
38.
39.
40.
41.
42.
43.
pathways of apoptosis induced by methylprednisolone and valinomycin analyzed by flow cytometry. Exp Cell Res 208:362–370
Marakhova I, Vereninov A, Toropova F, Vinogradova T 1998 Na,K-ATPase
pump in activated human lymphocytes: on the mechanisms of rapid and
long-term increase in K influxes during the initiation of phytohemagglutinininduced proliferation. Biochim Biophys Acta 1368:61–72
Tandon N, Davidson L, Titus E 1983 Changes in (Na⫹ ⫹ K⫹)-ATPase activity
associated with stimulation of thymocytes by concanavalin A. J Biol Chem
258:9850 –9855
Kaplan JG 1978 Membrane cation transport and the control of proliferation of
mammalian cells. Annu Rev Physiol 40:19 – 41
Chandra J, Gilbreath J, Freireich EJ, Kliche KO, Andreeff M, Keating M,
McConkey DJ 1997 Protease activation is required for glucocorticoid-induced
apoptosis in chronic lymphocytic leukemic lymphocytes. Blood 90:3673–3681
McColl KS, He H, Zhong H, Whitacre CM, Berger NA, Distelhorst CW 1998
Apoptosis induction by the glucocorticoid hormone dexamethasone and the
calcium-ATPase inhibitor thapsigargin involves Bc1–2 regulated caspase activation. Mol Cell Endocrinol 139:229 –238
Varfolomeev EE, Schuchmann M, Luria V, Chiannilkulchai N, Beckmann JS,
Mett IL, Rebrikov D, Brodianski VM, Kemper OC, Kollet O, Lapidot T,
Soffer D, Sobe T, Avraham KB, Goncharov T, Holtmann H, Lonai P, Wallach
D 1998 Targeted disruption of the mouse Caspase 8 gene ablates cell death
induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally.
Immunity 9:267–276
Longthorne VL, Williams GT 1997 Caspase activity is required for commitment to Fas-mediated apoptosis. EMBO J 16:3805–3812
Takeyasu K, Tamkun M, Renaud K, Fambrough D 1988 Ouabain-sensitive
Na⫹/K⫹-ATPase activity expressed in mouse L cells by transfection with DNA
encoding the ␣-subunit of an avian sodium pump. J Biol Chem 263:4347– 4354