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Glucocorticoids Regulate Plasma Membrane Potential During Rat Thymocyte Apoptosis in Vivo and in Vitro

Molecular Endocrinology Group, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: John A. Cidlowski, P.O. Box 12233 MD F3–07, 111 Alexander Drive, Research Triangle Park, North Carolina 27709. E-mail: Cidlowski{at}niehs.nih.gov

	Abstract

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Glucocorticoids induce a series of profound biochemical changes in thymocytes that initiate apoptosis; however, the pathways beyond receptor transactivation that lead to this form of cell death are not fully understood. In this study, we report a novel site of action for glucocorticoids at the site of the plasma membrane. Specifically, we find that glucocorticoids induce the loss of plasma membrane potential both in vivo and in vitro. The glucocorticoid-induced loss of plasma membrane potential in cultured primary isolated rat thymocytes was both dose and time dependent. Other steroid hormones, including progesterone, estrogen, and testosterone, fail to alter the depolarization state of the thymocyte plasma membrane. Interestingly, other nonsteroid stimuli that also activate apoptosis in thymocytes also lead to cellular depolarization. In contrast, HeLa cells, which contain functional glucocorticoid receptors but do not die in response to hormone, do not alter their plasma membrane potential in response to glucocorticoids, indicating a strong association between depolarization and apoptosis. Furthermore, the ability of glucocorticoids to depolarize the plasma membrane of thymocytes required the interaction of glucocorticoids with their cognate receptor, because RU486 failed to depolarize thymocytes and antagonized the effect of glucocorticoids. Finally, experiments using inhibitors of transcription and translation indicated that the loss of plasma membrane potential in thymocytes following glucocorticoid treatment required de novo gene expression. The results of these studies establish that the loss of plasma membrane potential is an early important feature of glucocorticoid-induced apoptosis of thymocytes.

	Introduction

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HOMEOSTASIS of T cell number in the thymus is controlled by the interplay of proapoptotic and antiapoptotic factors that regulate the thymocyte population. The elimination of unwanted thymocytes by proapoptotic signals, including endocrine signals such as glucocorticoids, is crucial to maintaining the integrity of the immune system. Glucocorticoids induce a cell death program in primary isolated thymocytes that is biochemically and morphologically characterized by chromatin condensation, DNA fragmentation, caspase activation, and the loss of mitochondrial membrane potential (1, 2, 3, 4). These cellular alterations are conserved features of apoptosis that are characteristic of apoptosis in many lymphoid and other cell types (5, 6).

In addition to these cytoplasmic and nuclear components, the plasma membrane of the cell undergoes profound changes during apoptosis. Early observations of apoptotic death described the cell membrane as blebbing and pinching off late in apoptosis to form inclusions termed apoptotic bodies (7, 8). The reorientation of phosphatidyl serine residues to the exterior of the plasma membrane aids in the recognition of the apoptotic cell by macrophages (9). In addition, glucocorticoids have been shown to decrease transmembrane transport of amino acids, glucose, and nucleosides (10). Previous studies also showed that apoptotic cells shrink (11) and suggested that this is due to the loss of fluid and ions from the cell (12). More recent studies have demonstrated that apoptotic cell shrinkage occurs as a result of ionic efflux from the cell (13, 14). Furthermore, studies from our lab have demonstrated that cell shrinkage and potassium efflux from thymocytes is an early event in apoptosis that allows for the subsequent activation of caspases and nucleases (15).

A primary function of the plasma membrane is the maintenance of a potential difference by its ability to barricade the free passage of ions across the membrane. Normally, most cells maintain an electrical potential across the plasma membrane of -60 mV to -70 mV that renders the inside of the membrane more negative than the outside (16). This is due to the asymmetrical distribution of ions across the plasma membrane (17, 18). In some cellular processes, orchestrated changes in plasma membrane potential may be involved. For example, T cells depolarize during mitogenic activation (19, 20), and these potential changes were accompanied by changes in ion permeability, but the precise role of such changes in cellular homeostasis is poorly understood.

Until recently, the role of plasma membrane potential in apoptosis has been largely ignored; however, recent studies have suggested that the plasma membrane potential may be compromised during apoptosis of lymphocytes (21). For example, in Jurkat cells, three different stimuli that differ in their mode of action, anti-Fas antibody, A23187, and thapsigargin, all induced a loss of plasma membrane potential associated with apoptosis. In the present investigation, we wished to investigate whether primary isolated thymocytes undergo plasma membrane potential changes in response to treatment by glucocorticoids or other steroids and further, to help define the sequence of events that leads to the loss of plasma membrane potential in thymocytes. To accomplish this goal, we used an oxonal dye that has been previously shown to be selectively sensitive to plasma membrane potential changes in lymphocytes (21, 22). The data presented herein indicate that the loss of plasma membrane potential is a fundamental feature of thymocyte apoptosis and that the ability of glucocorticoids to induce a loss of plasma membrane potential is restricted to their ability to induce apoptosis in the target cell.

	Materials and Methods

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Reagents
FCS was purchased from Summit Biotechnology (Fort Collins, CO) and dexamethasone was purchased from Steraloids (Wilton, NH). DiBAC₄ (3) was purchased from Molecular Probes, Inc. (Eugene, OR). Fas-L was purchased from Kamiya Biomedical Co. (Seattle, WA). A23187 was purchased from Calbiochem (La Jolla, CA). Cycloheximide, actinomycin-D, propidium iodide, and thapsigargin were purchased from Sigma (St. Louis, MO). RU486 was a gift of Dr. R. Deraedt, Roussel-Uclaf (Romainville, France).

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 days before use and maintained under controlled conditions of temperature (25 C) and lighting and allowed free access to food and 0.85% saline. For each in vivo study, one rat was administered dexamethasone (5 mg/kg BW, DEX) by ip injection of the steroid resuspended in PBS by sonication. A control rat received PBS alone (CON). Three independent in vivo experiments were conducted. 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 Public Health Service. Animals were killed by decapitation and the thymus was surgically removed.

Thymocyte cultures
To expand our analysis of glucocorticoid-induced thymocyte depolarization, we turned to an in vitro model which we have described previously (4). Following surgical removal of the thymus from an untreated adrenalectomized rat, thymocytes were prepared according to previously published methods (4, 23). Thymocytes were dispersed by gentle homogenization in a Kontes no. 22 glass/glass homogenizer (Kontes Co., Vineland, NJ), filtered through 202 µM Nitex mesh (Tetko, New York, NY) washed in cold PBS and counted on a hemacytometer. Cells were cultured at 5 x 10⁶ cells/ml in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, 4 mM glutamine, 100 U/ml penicillin, and 75 U/ml streptomycin sulfate (prepared by the media facility at NIEHS). Cells were incubated at 37 C, 5% CO₂ for 0, 2, 4, and 6 h before harvest.

HeLa cell culture
HeLa S₃ cells were grown in suspension culture at 37 C in Joklik’s MEM, supplemented with 2% FCS, 2 mM glutamine, 75 U/ml penicillin, and 50 U/ml streptomycin sulfate (prepared by the media facility at NIEHS).

FACS analysis and analysis of plasma membrane potential
Plasma membrane potential was measured with the anionic oxonal dye, DiBAC₄ (3). Previous studies from our lab (21) as well as others (22) have established that DiBAC₄ (3) serves as an indicator for the plasma membrane potential in lymphoid cells. In the resting state, DiBAC₄ (3) is excluded from the cell. Upon plasma membrane depolarization, DiBAC₄ (3) enters the cell and can be detected with FACS analysis by the increase in fluorescence. For plasma membrane potential analyses, cells were incubated with 150 nM DiBAC₄ (3) (Molecular Probes, Inc.) for 30 min at 37 C, 5% CO₂. To exclude cells that had lost membrane integrity, propidium iodide was added to a concentration of 10 µg/ml (24). All fluorescence measurements were made with a Becton Dickinson and Co. (San Jose, CA) FACSort equipped with CellQuest software (Becton Dickinson and Co.). Propidium iodide fluorescence was measured on FL-3 (650 nm) to exclude nonviable cells. Cell size was monitored by alterations in the forward light-scattering properties of the cells as described previously (4). To determine membrane potential, the DiBAC₄ (3) fluorescence of 15,000 viable cells was measured on FL-1 (excitation at 488 nm, emission at 530 nm). An increase in DiBAC₄ (3) fluorescence indicated a decrease in plasma membrane potential. 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 were quantified for each treatment. The averages ± SEM for each treatment represent at least three independent experiments. Statistical analyses were performed using the Student’s t test with = 0.05. In the text, * indicates that the treatment is significantly different from freshly isolated cells (P < 0.05). ** indicates that the treatment is significantly differently from time-matched control cells (P < 0.05).

	Results

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In vivo plasma membrane depolarization induced by dexamethasone
In vivo administration of glucocorticoids causes pronounced thymic regression as a result of thymocyte apoptosis. Approximately 90% of the thymocytes are eliminated within 48–72 h of glucocorticoid treatment (25) via biochemical and morphological characteristics that are consistent with cells undergoing apoptosis (26). To determine whether glucocorticoids alter plasma membrane potential, thymocytes were isolated from rats 4 h after an ip injection of dexamethasone (5 mg/kg BW) as described in Materials and Methods. Freshly isolated thymocytes from control rats had low DiBAC₄ (3) fluorescence (11.0 ± 1.4%), indicating that the plasma membrane potential was intact and that the cells can exclude the dye (Fig. 1). In vivo treatment with dexamethasone produced a population of viable cells with increased DiBAC₄ (3) fluorescence that was significantly larger than the control population (25.9 ± 3.4% **). This increase in DiBAC₄ (3) fluorescence indicates that the cells have lost plasma membrane potential. Thus, these results show that in vivo glucocorticoid treatment results in an early depolarization of thymocytes before the loss of membrane integrity.

View larger version (23K): [in this window] [in a new window]   Figure 1. Glucocorticoids depolarize primary thymocytes in vivo. Adrenalectomized male Sprague-Dawley rats were injected with PBS (CON) or dexamethasone (5 mg/kg BW, DEX) 4 hours prior to isolation of primary thymocytes. Isolated thymocytes were stained with DiBAC4(3 ) as described in Materials and Methods. Prior to flow cytometric analysis, propidium was added to exclude non-viable cells. The figure shows representative histograms for DiBAC4(3 ) fluorescence in primary isolated thymocytes from control (CON) and dexamethasone-treated (DEX) rats. The histograms are representative of three independent experiments.

View larger version (35K): [in this window] [in a new window]   Figure 2. Time course of plasma membrane depolarization in primary isolated thymocytes. Thymocytes were isolated and placed in culture for 0, 2, 4, and 6 h in the presence or absence of dexamethasone (100 nM). Following the indicated times, cells were stained with DiBAC4(3 ) as described in Materials and Methods. Propdium iodide was added before flow cytometric analysis, and viable cells were analyzed by flow cytometry for DiBAC4(3 ) fluorescence. A, Representative histograms for DiBAC4(3 ) fluorescence in untreated thymocytes 0, 2, 4, and 6 h. B, Representative histograms for DiBAC4(3 ) fluorescence in dexamethasone-treated cells at 0, 2, 4, and 6 h.

View larger version (33K): [in this window] [in a new window]   Figure 3. Dose-response for glucocorticoid-induced thymocyte apoptosis. Primary isolated thymocytes were cultured for 6 h in the absence of dexamethasone (Control) or with increasing concentrations of dexamethasone. Cells were stained with DiBAC4(3 ) as described in Materials and Methods. Propdium iodide was added before flow cytometric analysis, and viable cells were analyzed by flow cytometry for DiBAC4(3 ) fluorescence. Representative histograms for DiBAC4(3 ) fluorescence are shown for each dose of dexamethasone.

View larger version (26K): [in this window] [in a new window]   Figure 4. Steroid specificity of plasma membrane depolarization in primary thymocytes. Primary thymocytes were cultured for 6 h in the presence of different steroid hormones to evaluate the steroid-specificity of plasma membrane depolarization in thymocytes. Cells were stained with DiBAC4(3 ) as described in Materials and Methods. Propdium iodide was added before flow cytometric analysis, and viable cells were analyzed by flow cytometry for DiBAC4(3 ) fluorescence. A, Representative DiBAC4(3 ) fluorescence histograms for thymocytes treated with 100 nM dexamethasone, 100 nM cortisol, or 100 nm corticosterone for 6 h. B, Representative DiBAC4(3 ) fluorescence histograms for thymocytes treated with 100 nM dexamethasone, 1 µM progesterone, 1 µm estradiol, or 1 µm testosterone for 6 h.

View larger version (32K): [in this window] [in a new window]   Figure 5. Other apoptotic stimuli induce plasma membrane depolarization in thymocytes. Primary thymocytes were cultured for 6 h in the presence of different apoptotic agents to evaluated their ability to depolarize thymocytes. Cells were stained with DiBAC4(3 ) as described in Materials and Methods. Propdium iodide was added before flow cytometric analysis, and viable cells were analyzed by flow cytometry for DiBAC4(3 ) fluorescence. The figure shows representative DiBAC4(3 ) fluorescence histograms for untreated thymocytes (gray line) or thymoctyes treated with 100 nM dexamethasone, 1 ng/ml fas-L, 2 µM A23 187, or 0.5 µm thapsigargin (solid black).

View larger version (20K): [in this window] [in a new window]   Figure 6. Glucocorticoids induce plasma membrane potential changes only in cells that undergo apoptosis. Plasma membrane potential was examined in primary thymocytes and HeLa cells following 6 h of dexamethasone treatment (100 nM). Cells were stained with DiBAC4(3 ) as described in Materials and Methods. Propdium iodide was added before flow cytometric analysis, and viable cells were analyzed by flow cytometry for DiBAC4(3 ) fluorescence. Representative histograms show DiBAC4(3 ) fluorescence in thymocytes and HeLa cells.

View larger version (32K): [in this window] [in a new window]   Figure 7. Glucocorticoid-induced apoptosis is dependent upon the interaction with the glucocorticoid receptor. Cell size and plasma membrane potential were examined simultaneously in primary thymocytes cultured alone (CON) or in the presence of 1 µM RU486, 100 nM dexamethasone, or both. Cells were stained with DiBAC4(3 ) as described in Materials and Methods. Propdium iodide was added before flow cytometric analysis, and viable cells were analyzed by flow cytometry. To compare DiBAC4(3 ) fluorescence vs. cell size, cells were analyzed on a representative DiBAC4(3 ) fluorescence vs. forward scatter plot.

Glucocorticoid-induced loss of plasma membrane potential is dependent upon de novo gene expression
It is well established that glucocorticoid-induced apoptosis of rat thymocytes is dependent upon de novo gene expression (37, 38). The manifestation of biochemical endpoints of apoptosis, including phosphatidylserine exposure, cell shrinkage, and caspase activation, are all dependent upon de novo gene expression (4). Spontaneous apoptosis of thymocytes however, occurs independently of de novo gene expression. To determine whether the loss of plasma membrane potential is dependent upon de novo gene expression in primary rat thymocytes, thymocytes were cultured with actinomycin-D to prevent RNA synthesis or cycloheximide to prevent protein synthesis. Spontaneously dying thymocytes showed a loss of plasma membrane potential independent of either RNA or protein synthesis (Fig. 8). Inhibition of protein synthesis by cycloheximide or inhibition of RNA synthesis by actinomycin-D significantly increased the percentage of depolarized cells over freshly isolated thymocytes to 18.7 ± 1.7% * and 14.6 ± 2.7% *, respectively. However, these values were not significantly different from spontaneously dying thymocytes, indicating that at this time point, inhibition of protein or RNA synthesis neither potentiates nor inhibits the depolarization of the plasma membrane in spontaneously dying cells. These data also confirmed that the failure of protein and RNA synthesis inhibition to affect spontaneous depolarization correlated with the inability to enhance or prevent spontaneous cell shrinkage.

View larger version (28K): [in this window] [in a new window]   Figure 8. Glucocorticoid-induced depolarization is dependent upon de novo gene expression. Cell size and plasma membrane potential were examined simultaneously in primary thymocytes cultured for 6 h alone (CON) or in the presence of cycloheximide (10 µM), actinomycin-D (1 µg/ml), or dexamethasone (100 nM). Cells were stained with DiBAC4(3 ) as described in Materials and Methods. Propdium iodide was added before flow cytometric analysis, and viable cells were analyzed by flow cytometry. To compare DiBAC4(3 ) fluorescence vs. cell size, cells were analyzed on a representative DiBAC4(3 ) fluorescence vs. scatter plot. To inhibit protein synthesis, primary thymocytes were cultured with cycloheximide (1 µM) in the presence or absence of dexamethasone for 6 h. To inhibit RNA synthesis, primary thymocytes were cultured with actinomycin-D (1 µg/ml) in the presence of absence of dexamethasone for 6 h.

	Discussion

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It is well documented that glucocorticoids affect the structure and function of the thymocyte plasma membrane during glucocorticoid-induced apoptosis by inducing membrane blebbing, reorientation of phosphatidylserine residues, and by altering the transmembrane transport of amino acids, glucose, and nucleosides (7, 8, 9, 10). In this study, we have established that physiological concentrations of glucocorticoids can also depolarize the plasma membrane of thymocytes and that this correlates with their ability to induce apoptosis in thymocytes. Our observations are supported by an earlier study that observed depolarization at pharmacological doses of methylprednisolone (39). The loss of plasma membrane potential precedes the loss of membrane integrity, as measured by the ability of the cells to exclude propidium iodide. The percentage of viable cells with a loss of plasma membrane potential increased as the length of time of exposure to the apoptotic stimulus increased. Glucocorticoids and other stimuli increased the number of depolarized cells, but not the magnitude of depolarization. This was observed both with survival factor withdrawal and glucocorticoid-induced apoptosis. A similar effect has been observed with other markers of apoptosis (4). The percentage of cells that have shrunken or have exposed phosphatidyl serine residues to the exterior of the cell, for example, increases with the duration of cell culture in both survival factor withdrawal and glucocorticoid-induced apoptosis. In addition, glucocorticoids increase the percentage of depolarized cells in a dose-dependent fashion that reflects the binding of glucocorticoids to the glucocorticoid receptor. We have also observed that Jurkat cells treated with increasing doses of anti-Fas antibody also show a dose-dependent increase in the percentage of depolarized cells (21). Thus, the percentage of depolarized thymocytes corresponds to the duration and intensity of exposure to glucocorticoids.

The ability of glucocorticoids to induce thymocyte apoptosis is unique to this class of steroids. We have previously shown that the activation of caspase-3-like activity, a fundamental feature of glucocorticoid-induced apoptosis in thymocytes, is unique to glucocorticoids (4). In the present study, the ability of glucocorticoids to induce a loss of plasma membrane potential was also unique to this class of steroids. Although cortisol and corticosterone depolarized the plasma membrane to a similar degree as dexamethasone, extremely high doses of other steroid hormones did not increase the percentage of depolarized cells above control levels. Although other steroid hormones could not depolarize thymocytes, we found that other apoptotic stimuli can. A nonsteroidal apoptotic agent, Fas-L induced a rapid loss of plasma membrane potential in the primary thymocytes, as did two other apoptotic agents, A23187 and thapsigargin. This supports previous findings from our laboratory which show that anti-Fas antibody, A23187, and thapsigargin can depolarize Jurkat cells (21). In addition, this observation suggests that the ability of glucocorticoids to depolarize thymocytes is related to their ability to induce apoptosis.

Glucocorticoids induce tissue specific changes depending on the target tissue. In the case of immune cells, glucocorticoids trigger apoptosis. This cell death program is characterized by the classical morphologic and biochemical changes traditionally associated with apoptosis such as cell shrinkage and internucleosomal DNA fragmentation. Although plasma membrane depolarization has been recently described in apoptosis, it was unknown whether the plasma membrane depolarization induced by glucocorticoids was a general effect regardless of the target cell, or whether it was a specific effect associated with apoptosis. HeLa cells, which display nuclear translocation of ligand-bound glucocorticoid receptor (32), but do not undergo apoptosis, failed to depolarize in response to glucocorticoid treatment. These data clearly show that the ability of glucocorticoids to induce plasma membrane depolarization in the target cell is directly related to their ability to induce apoptosis.

The fact that inhibition of glucocorticoid receptor binding and de novo gene expression did not block the spontaneous loss of plasma membrane potential demonstrates that in survival factor withdrawal-induced death, the glucocorticoid receptor and gene regulation are not required for plasma membrane depolarization. These data support our previous observation that the biochemical features of spontaneous cell death manifest independently of the glucocorticoid receptor and de novo gene expression (4). These experiments also demonstrated that the glucocorticoid receptor requires interaction with the agonist ligand to induce cellular depolarization. In fact, we also observe that the glucocorticoid antagonist RU486 can block the simultaneous depolarization and shrinkage caused by glucocorticoids, as can inhibition of gene expression. These results are consistent with previous studies, which show that the glucocorticoid receptor and subsequent gene regulation are required for glucocorticoid-induced cell shrinkage, phosphatidylserine flipping, and other endpoints of apoptosis. The fact that depolarization is limited to the shrunken population of cells and that agents that block cell shrinkage in thymocytes also block depolarization lends further proof that plasma membrane depolarization is an important component of the apoptotic pathway.

Plasma membrane depolarization is an essential component of many cellular processes in a variety of cell types. Stimulus-induced alterations to the electrical field across a membrane could effect the structure and function of charged, membrane-embedded macromolecules. Indeed, conformational changes in voltage-dependent ion channels of electrically excitable cells are mediated by changes in the plasma membrane potential (40, 41). Stimulus-secretion coupling in some nonexcitable cells is also mediated by ionic and electrical events at the plasma membrane (42). In lymphocytes, plasma membrane depolarization plays a central role in lymphocyte activation. Activation of T cells by antigen-dependent or antigen-independent stimuli results in plasma membrane depolarization, turnover of polyphosphoinositides, and an increase in free intracellular calcium (20). In the present study, we have described the effect of glucocorticoids on plasma membrane potential during thymocyte apoptosis and define a sequence of events that places the loss of plasma membrane potential early in the apoptotic pathway following gene expression. Currently, the specific mechanism that leads to apoptotic plasma membrane depolarization and the exact consequences of this depolarization are unclear. However, studies to identify specific targets involved in glucocorticoid-induced loss of plasma membrane potential are currently underway in our laboratory.

	Acknowledgments

 
We would like to thank Carl D. Bortner for his technical assistance and Sue Edelstein of Image Associates, Inc. for her assistance with graphic design.

Received June 8, 2000.

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