HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 10, 8058-8064, March 7, 2003

This Article Abstract Full Text (PDF) All Versions of this Article: 278/10/8058    most recent M207781200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by He, Y.-Y. Articles by Chignell, C. F. Search for Related Content PubMed PubMed Citation Articles by He, Y.-Y. Articles by Chignell, C. F. Social Bookmarking                      What's this?

Role of Reduced Glutathione Efflux in Apoptosis of Immortalized Human Keratinocytes Induced by UVA^*

Yu-Ying He, Jian-Li Huang, Dario C. Ramirez, and Colin F. Chignell

From the Laboratory of Pharmacology and Chemistry, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received for publication, August 1, 2002, and in revised form, December 26, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have investigated the role played by GSH efflux in apoptosis of human HaCaT keratinocytes induced by UVA irradiation. UVA irradiation of HaCaT cells caused a rapid rise in GSH efflux across the intact cell membrane, followed by an increase in apoptosis. GSH efflux was stimulated by glucose and was reduced by the addition of exogenous GSH and intracellular GSH depletion by buthionine sulfoximine, suggesting that GSH transport is active and is influenced by the GSH concentration gradient across the cell membrane. Verapamil and cyclosporin A, blockers of the multidrug resistance-associated protein, decreased UVA-induced GSH efflux. GSH efflux occurred within 2 h of UVA irradiation, suggesting that the stimulation of GSH efflux is due to an increase in the activity of pre-existing multidrug resistance-associated protein transporter carrier. Although inhibition of GSH efflux did not affect caspase activation and DNA fragmentation, it delayed the gradual increase in plasma membrane permeability and reduced phosphatidylserine translocation in HaCaT cells. It is therefore likely that upon UVA irradiation, GSH efflux increased the intracellular oxidative stress without intervention of reactive oxygen species, thus resulting in more phosphatidylserine externalization and membrane rearrangement. These provide targets for macrophage recognition and phagocytosis and thus minimize the potential to invoke inflammation or neoplastic transformation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Solar UV irradiation reaching the surface of the earth, consisting mainly of UVB (280-315 nm) and UVA (315-400 nm), presents a major environmental challenge to the skin, contributing not only to photo-aging but also to carcinogenesis (1-3). UVA has long been considered less of a causative factor in skin carcinogenesis than UVB due to its negligible absorption by DNA. However, the greater abundance of UVA in solar UV irradiation and deeper penetration of UVA into the actively dividing basal layer of the skin increases the relative importance of UVA as compared with UVB (4). UVA has been shown to be a risk factor for melanoma in fish (5) and could be also in humans (6-8).

Fortunately, the carcinogenic effects of UV irradiation may be decreased by apoptosis, programmed cell death, which eliminates DNA-damaged or potentially mutated cells. Much more is known about UVB-induced apoptosis than about apoptosis induced by UVA in both normal keratinocytes and immortalized keratinocytes (9-19). However, Godar and co-workers (20-22) have reported that UVA-induced apoptosis in lymphoma and Jurkat cells involves mechanisms that differ from those seen with UVB. Oxidative stress is presently considered to be involved in UVA-induced apoptosis (3). In support of this involvement, a protective role is played by tea polyphenols against both cytotoxicity and apoptosis induced by UVA in rat keratinocytes; this protection correlates well with the ability of the polyphenols to quench reactive oxygen species (23). Nevertheless, the mechanisms involved in UVA-induced apoptosis of human keratinocytes are still poorly understood.

There is evidence that protection of human skin cells against a wide range of solar UV radiation damage, including UVA, involves endogenous glutathione (24). This thiol is involved in many biological processes, including the regulation of gene expression, apoptosis, and membrane transport (25-27). Glutathione is considered to be the most prevalent and most important intracellular non-protein thiol-disulfide redox buffer in mammalian cells (25-27). The higher glutathione content in immortalized HaCaT cells is expected to confer resistance to UVA irradiation as compared with normal keratinocytes (28).

In apoptosis, the role of glutathione is controversial and dependent on cell types and pro-apoptotic stimuli. (i) High intracellular reduced GSH levels have been found to prevent Fas-induced cell apoptosis in the Fas-resistant variant CEM2D1R (29), whereas decreased GSH levels enhance Fas-induced apoptosis in the Fas-resistant variant CEM2D1R and the human hepatoma cell line HepG (2, 29, 30). (ii) Low intracellular GSH levels have also been shown to prevent apoptosis by compromising caspase activation in mouse hepatocytes (31, 32). Depletion of intracellular GSH prevented CD95-triggered apoptosis upstream of caspase-8 activation in T and B cells (33). Furthermore, cells undergoing apoptosis also appear to export GSH into the extracellular space (34-37). However, neither the mechanism involved in the transport of GSH nor the functional benefit of this process is known.

In this study we have found that in immortalized HaCaT cells, UVA irradiation induces the active efflux of GSH. We have surveyed the mechanisms involved in this efflux, focusing on the possible roles of MRP¹ transporter protein. We also found that GSH export is closely associated with UVA-induced apoptosis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Reduced GSH, GSSG, verapamil (VP), cyclosporin A (CsA), 5-sulfosalicylic acid glutathione reductase, and buthionine sulfoximine (BSO) were purchased from Sigma. 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB), 2-vinylpyridine, EDTA, glucose, NADPH, and triethanolamine were purchased from Aldrich.

Cell Culture-- The spontaneously immortalized human keratinocyte cell line HaCaT (38), obtained from Prof. N. Fusenig (German Cancer Research Center, Heidelberg, Germany), was maintained in monolayer culture in 95% air, 5% CO₂ at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 31 µg/ml penicillin, and 50 µg/ml streptomycin. For experiments, HaCaT keratinocytes were grown in 12-well plates or plastic Petri dishes (100 mm) for 24-48 h. 4 h prior to UVA treatment, subconfluent cells were given fresh Dulbecco's modified Eagle's medium containing 1% fetal bovine serum. For GSH depletion, BSO (50 µM) was added to the culture medium, and the cells were incubated for 18 h prior to UVA exposure (39); this treatment had no effect on cell viability.

UVA Treatment-- The medium was removed, and cells were washed once with sterile PBS (PBS-CMF, calcium/magnesium-free). After the addition of sterile PBS or PBS containing 10 mM of glucose, the cells were irradiated with fluorescent lamps (Houvalite F20T12BL-HO PUVA, National Biological Corp., Twinsburg, OH) with the dish lid on. The UVA dose was monitored with a Goldilux UV meter equipped with a UVA detector (Oriel Instruments, Stratford, CT). Control samples were kept in the dark under the same conditions. After treatment, the supernatant was removed or collected as indicated, and the cells were washed with PBS. For the apoptosis assay, fresh medium containing 1% fetal bovine serum was added after exposure, and the cells were incubated at 37 °C. At predetermined time points, attached and floating cells were harvested and subjected to apoptosis analysis. In selected experiments, cells were pretreated with GSH or inhibitors for multidrug resistance-associated protein (MRP) at 37 °C for 15 min prior to irradiation.

Determination of Intracellular and Extracellular Glutathione-- GSH and GSSG were measured using a modified method for glutathione determination in microtiter plates (40, 41). Briefly, after removal of the supernatant, cells in 12-well plates were washed with PBS and then treated with 500 µl of 10 mM hydrochloric acid and stored at 20 °C until analyzed. Cells were scraped and sonicated three times for 5 s and then centrifuged at 10,000 × g for 10 min at 4 °C. Aliquots (350 µl) of the supernatant were collected, of which 50 µl were transferred to separate microtubes for BCA protein assay (Pierce). To the remaining aliquot (300 µl), 300 µl of 5% 5-sulfosalicylic acid was added, and the mixture was vortexed and then kept on ice for 5 min to precipitate protein. After centrifugation at 10,000 × g for 10 min at 4 °C, 500 µl of supernatant were collected, neutralized by the addition of 4 M triethanolamine solution, and divided into 2 aliquots for measurement of GSSG and total glutathione. To conjugate GSH, 2-vinylpyridine was added to one of the aliquots to a final concentration of 2% (v/v).

The microtiter plate was prepared by pipetting 50 µl of standards or samples per well. Immediately, 100 µl of freshly prepared assay mix (0.49 ml of 6 mM DTNB, 3.75 ml of 1 mM NADPH, 8.15 ml of phosphate/EDTA buffer, and 20 units of glutathione reductase) was pipetted into each well. The plate was placed into the microplate reader (Tecan US SPECTRAFluor Plus, Research Triangle Park, NC) and the absorption at 405 nm monitored. The GSH or GSSG levels were expressed as nmol/mg protein or ratio as compared with respective controls without UVA treatment. For extracellular GSH and GSSG, cell supernatants were treated similarly, with or without 2-vinylpyridine followed by microtiter detection using the DTNB/NADPH/glutathione reductase assay mix.

DNA Fragmentation-- The pattern of DNA cleavage was analyzed by agarose gel electrophoresis. Briefly, cell pellets were resuspended in lysis buffer (5 mM Tris-HCl, pH 8.0; 20 mM EDTA; 0.5% Triton X-100) and incubated on ice at 4 °C overnight. After incubation at 56 °C for 1 h with RNase A (100 µg/ml) and then 1 h with proteinase K (200 µg/ml), the cell lysate was extracted with phenol/chloroform/isopropyl alcohol (25:24:1, v/v). DNA was precipitated with ethanol and subsequently washed with 70% ethanol. DNA samples, dissolved in 1× TE buffer, were separated by horizontal electrophoresis on 1.5 or 1.8% agarose gels, stained with ethidium bromide, and visualized under UV light.

Caspase Activity-- Caspases were assayed using ApoAlert Caspase Fluorescent Assay Kits (Clontech, Palo Alto, CA). Briefly, cells were extracted in lysis buffer, and the cell lysate was incubated for 1 h at 37 °C with assay buffer containing one of the following fluorescent caspase substrates: Ac-DEVD-AFC for caspase-3, Ac-IETD-AFC for caspase-8, and Ac-LEHD-AFC for caspase-9. The fluorometric detection of cleaved AFC product was performed on a plate reader (excitation 400 nm and emission 505 nm). For preparation of the AFC calibration curve, 80 µM free AFC was diluted in the caspase assay buffer without substrate to give 0.5, 1, 2, and 4 µM of free AFC. The results were expressed as ratio of the treated samples to respective control samples.

Lactate Dehydrogenase (LDH) Release-- LDH, a stable cytosolic enzyme that is released upon the increase in plasma membrane permeability, is determined by CytoTox96 non-radioactive cytotoxicity assay kit (Promega, Madison, WI). Briefly, after treatment, 50 µl of medium was taken at different time points to the enzymatic assay plate. After all samples were taken, 50 µl of substrate mix was added to each well of the enzymatic assay plate, and the plate was incubated at room temperature for 30 min in the dark. The reaction was stopped by adding 50 µl of stop solution, and LDH was determined by recording the absorbance at 492 nm by a plate reader. The maximum LDH release control was done by adding lysis solution to the control cells. The results were expressed as LDH released (%) = (treated  control)/(maximum  control) × 100.

Flow Cytometry-- Annexin V staining was used to determine the translocation of phosphatidylserine (PS) in UVA-induced apoptosis. After incubation, control or treated cells including attached and floating cells were harvested and collected by centrifugation at 300 × g for 5 min at room temperature. Cells were washed with cold PBS and stained with TACS^TM annexin V kits according to the manufacturer's instructions (Trevigen, Gaithersburg, MD). Cells positive for annexin V-fluorescein isothiocyanate were quantified by flow cytometry using a BD FACSort (BD Biosciences).

Statistics-- Data are presented as mean ± S.E. of three to six experiments. The Student's t test was used for comparisons between experimental groups (n = 3-6). A value of p < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

UVA-Induced GSH Efflux in HaCaT Cells-- In order to determine the effect of UVA on the efflux of GSH, we irradiated subconfluent HaCaT cells in PBS with UVA, with and without glucose. We found that in the presence of glucose to provide energy, UVA (25 J/cm²) induced significant efflux of GSH (Fig. 1).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   UVA induced GSH efflux in the presence of glucose in HaCaT cells. Intracellular (A) and extracellular (B) GSH, GSSG, and total glutathione levels immediately after the HaCaT cells were switched from normal culture medium to PBS or 10 mM glucose PBS and then sham-irradiated or UVA-irradiated (25 J/cm2). Results are the mean ± S.E. of three to six experiments (**, p < 0.01 as compared with dark control samples). C, the cells were treated as in A and B with different doses of UVA. The extracellular glutathione ratios of UVA-irradiated samples to their respective control samples were determined. D, cells were irradiated with 25 J/cm2 UVA in the presence of different glucose concentrations. The extracellular glutathione ratios of UVA-irradiated samples to their respective control samples were determined. The GSH efflux depended on the concentration of glucose. Cells were seeded in 12-well plates and used after 48 h of culture. After washing with PBS once, medium was replaced by an equivalent quantity of PBS or glucose PBS, and the plates were irradiated with UVA. Control samples were kept in the dark. For intracellular glutathione determination, cells were harvested after treatment and prepared for total glutathione and GSSG assay by DTNB method as described under "Experimental Procedures." The supernatants were collected for extracellular total glutathione and GSSG assay by the same method.

The total glutathione level in HaCaT cells in normal culture is 62.6 nmol/mg protein, of which more than 98% is in the reduced form (GSH). As compared with normal cells cultured in medium, intracellular total glutathione, GSH + GSSG, of cells incubated in the dark decreased to 41.9 and 40.6 nmol/mg protein for PBS and glucose PBS solutions, respectively (Fig. 1A). GSH levels also decreased, whereas levels of the oxidized form, GSSG, increased ~3-fold. When HaCaT cells were irradiated with UVA at a dose of 25 J/cm² in the presence of glucose (10 mM), intracellular total glutathione levels and GSH decreased significantly from dark controls, whereas GSSG levels remained unchanged (Fig. 1A). Thus the decrease in total glutathione appears to be due to a reduction in GSH.

To determine whether GSH was exported into the extracellular medium, supernatants were collected, and GSH, GSSG, and total glutathione levels were determined (Fig. 1B). In the dark, the presence of glucose had no effect on the levels of extracellular GSH, GSSG, or total glutathione. When the sum of the intracellular and extracellular total glutathione was compared with intracellular total glutathione in normal cultured cells, the loss of intracellular total glutathione was recovered completely in the supernatant. Efflux of GSH caused a decrease in intracellular GSH. In the absence of glucose, however, there was no GSH efflux, and the intracellular GSH depletion was due to the oxidation to GSSG (Fig. 1, A and B).

When HaCaT cells were irradiated with different doses of UVA (Fig. 1C), GSH efflux increased with the UVA dose. There was no GSH efflux below 10 J/cm², but GSH efflux increased up to 25 J/cm² and remained unchanged. Interestingly, consistent with Fig. 1, A and B, no GSH efflux was observed under different doses of UVA irradiation in the absence of glucose. To confirm the requirement of glucose, GSH efflux was determined in the presence of different concentrations of glucose (Fig. 1D). Under UVA irradiation (25 J/cm²), GSH was exported in a glucose concentration-dependent manner. These results suggest that glucose is required for GSH efflux induced by UVA irradiation.

Effect of Exogenous GSH Addition and GSH Depletion on GSH Efflux-- To determine whether the efflux of GSH under UVA irradiation is influenced by GSH levels, we used exogenous GSH to increase extracellular GSH levels or BSO, a known GSH-depleting agent, to decrease intracellular GSH. Each agent was added and incubated with cells prior to UVA irradiation.

In the dark, exogenous GSH (1 mM) incubated with cells for 15 min at 37 °C did not affect intracellular GSH or GSSG even in the presence of glucose (Fig. 2A). As observed previously, when cells were irradiated with 25 J/cm² of UVA in the presence of glucose, GSH was exported into the extracellular medium (see above). However, the addition of exogenous GSH reduced the loss in intracellular GSH and thus the UVA-induced efflux significantly. The GSSG level did not change significantly with or without exogenous GSH (Fig. 2A).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effect of the addition of GSH and GSH depletion by BSO on UVA-induced GSH efflux. Intracellular (A) and extracellular (B) GSH, GSSG, and total glutathione levels when the HaCaT cells were pretreated with GSH (1 mM) or BSO and then kept in the dark or UVA-irradiated (30 J/cm2). For GSH treatment, after 48 h of culture cells were washed once with PBS, after which glucose PBS (10 mM) containing 1 mM of GSH was added, and cells were incubated at 37 °C for 15 min prior to irradiation. After irradiation, cells were washed twice with PBS to eliminate the effect of exogenous GSH on the determination of intracellular GSH levels. For BSO treatment, BSO (50 µM) was added 18 h before irradiation; cells were then washed with PBS twice, after which glucose PBS was added prior to irradiation. For intracellular glutathione determination, cells were harvested after irradiation and prepared for intracellular GSH and GSSG assay by the DTNB method. The supernatants were collected for extracellular GSH and GSSG assay by the same method. Results are the mean ± S.E. of three to six experiments. (*, p < 0.05; **, p < 0.01 as compared with the UVA-irradiated samples without GSH or BSO).

When BSO (50 µM) was added to the cell culture 18 h prior to UVA exposure, intracellular total glutathione decreased dramatically; no cytotoxicity was detected (data not shown). As was the case with non-BSO-pretreated cells, the UVA-induced decrease in total intracellular glutathione in BSO-pretreated cells was recovered from the extracellular medium (Fig. 2B). Although GSH efflux was induced by UVA irradiation after GSH depletion by BSO incubation, the efflux process slowed down due to the dramatic decrease in intracellular GSH.

Effect of MRP1 Inhibitors on UVA-induced GSH Efflux-- To identify the possible transport carrier involved in UVA-induced GSH efflux, inhibitors for multidrug resistance-associated protein 1 (MRP1) were preincubated with cells prior to irradiation. In the dark, VP (10 or 20 µM) and CsA (5 or 10 µM) caused no significant change (data not shown). When cells pretreated with verapamil or CsA were irradiated with UVA, UVA in the presence of either inhibitor caused less of a GSH efflux as compared with UVA alone (Fig. 3A). Higher concentrations of CsA or VP exhibited more inhibition on GSH efflux. These results indicated that verapamil and CsA both inhibited the efflux of GSH, with CsA being somewhat more effective. The much lower inhibition by verapamil could be due to the fact that verapamil inhibits MRP activity non-competitively and may exhibit lower inhibition on the binding of GSH with MRP1, as compared with cyclosporin A, a competitive inhibitor of MRP1-substrate binding.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   CsA and VP inhibited UVA-induced GSH efflux in HaCaT cells. A, cells were incubated with CsA (5 or 10 µM) or VP (10 or 20 µM) at 37 °C for 15 min and then irradiated with UVA (25 J/cm2) in the presence of 10 mM glucose. Immediately after irradiation, supernatants were taken to measure the extracellular glutathione. The results are expressed as the ratio of treated levels to respective control levels. B, after UVA irradiation (25 J/cm2), cells were given fresh medium. At predetermined time points, medium was taken to determine the extracellular glutathione levels. Results are the mean ± S.E. of three to six experiments. (*, p < 0.05; **, p < 0.01 as compared with the UVA-irradiated samples without CsA or VP).

However, after irradiation, UVA did not cause further GSH efflux as compared with the control samples (Fig. 3B). Neither verapamil nor cyclosporin A affected the GSH efflux. This suggests that UVA-induced GSH efflux occurs during the irradiation but not after irradiation.

Role of GSH Efflux in UVA-induced Apoptosis-- To evaluate the physiological role of UVA-induced GSH efflux, we examined the influence of GSH efflux inhibition on UVA-induced apoptosis. HaCaT cells were first treated with or without cyclosporin A, verapamil, or GSH and then subjected to UVA irradiation. DNA fragmentation was monitored by electrophoresis; the activities of caspases-3, -8, and -9 were measured with their respective fluorescent substrates; the plasma membrane permeability was determined by LDH release; and the externalization of PS on the cell surface was monitored by flow cytometry in combination with annexin V staining.

As shown in Fig. 4, A and B, UVA irradiation caused a dose- and time-dependent increase in DNA fragmentation in HaCaT cells. However, UVA-induced DNA fragmentation was not markedly inhibited by prior treatment of HaCaT cells with cyclosporin A, verapamil, or GSH (Fig. 4C). UVA irradiation activated caspases-3, -8, and -9 in a time-dependent manner (Fig. 5A). After UVA irradiation (25 J/cm²), caspases-3, -8, and -9 were activated as early as 30 min. However, pretreatment with cyclosporin A, verapamil, or GSH did not affect the activation of these caspases at 1 (Fig. 5B) or 6 h (Fig. 5C). Furthermore, the pretreatment of the cells with Ac-DEVD-CHO, the caspase-3 inhibitor, had no effect on UVA-induced GSH efflux (Fig. 5D). There was no caspase-3 activation (data not shown) while dramatic GSH efflux occurred (Fig. 1) immediately after UVA exposure, suggesting that GSH efflux induced by UVA precedes the caspase-3 activation.

View larger version (67K): [in this window] [in a new window]   Fig. 4.   Inhibition of GSH efflux has no effect on UVA-caused DNA fragmentation. A, cells were irradiated with different doses of UVA (0, 10, 20, 25, 30, and 40 J/cm2) in the presence of 10 mM glucose and then incubated for 15 h. DNA was extracted and separated on 1.5% agarose gel. B, cells were irradiated with 25 J/cm2 UVA and incubated for different times (3, 6, 12, 15, 18, and 24 h). Extracted DNA was subjected to 1.5% agarose gel electrophoresis. C, cells were preincubated with CsA (5 or 10 µM) or VP (10 or 20 µM) at 37 °C for 15 min and them irradiated with UVA (25 J/cm2) in the presence of 10 mM glucose. After incubation for 15 h, DNA from treated and untreated samples was separated on 1.8% agarose gel. Cells were seeded in 100-mm Petri dishes and used at a 50-70 confluence. Results are representative of three independent experiments.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Inhibition of GSH efflux has no effect on the activation of caspases-3, -8, and -9 by UVA. A, after UVA exposure (25 J/cm2), the cells were incubated for different times. The caspase activity was determined in cytosolic extracts prepared at the time point given. B, in a parallel experiment, the cells were pretreated with CsA (10 µM), VP (20 µM), or GSH (1 mM) and then irradiated with UVA (25 J/cm2). The caspase activity was determined 1 h after UVA treatment. C, the cells were treated the same as in B, and the caspase activity was determined 6 h after UVA irradiation. D, the cells were pretreated with caspase-3 inhibitor, Ac-DEVD-CHO (10 and 20 µM). After UVA irradiation (25 J/cm2), the supernatants were taken, and glutathione levels were determined. Results are the mean ± S.E. of four to six experiments.

UVA also caused a gradual increase in LDH release in HaCaT cells in a dose- and time-dependent manner (Fig. 6A). The presence of cyclosporin A (2.5, 5, or 10 µM) (Fig. 6B), verapamil (5, 10, or 20 µM) (Fig. 6C), or GSH (0.2 or 1 mM) (Fig. 6D) inhibited the LDH release. It is evident that GSH is more efficient in inhibiting UVA-caused LDH release than CsA or VP. These results suggest that GSH efflux induced by UVA is involved in the increased in plasma membrane permeability. Cell lysis especially occurs in the late stage of apoptosis when macrophages are absent.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Inhibition of GSH efflux decreased the LDH release induced by UVA. A, the cells were irradiated with different doses of UVA (10, 20, 25, 30, and 40 J/cm2) as indicated. Supernatant was taken at different time points to determine the LDH release. In a parallel experiment, the cells were pretreated with different concentrations of CsA (B), VP (C), or GSH (D) as indicated and then irradiated with UVA (25 J/cm2). At different time points given, supernatant was taken to determine the LDH release. Results are the mean ± S.E. of four to six experiments. (*, p < 0.05 as compared with the UVA-irradiated samples without CsA, VP, or GSH).

In order to test whether GSH efflux is involved in the translocation of PS, a membrane lipid rearrangement occurring in the early or intermediate stage of apoptosis and the triggering event in the recognition of apoptotic cells by the scavenger receptors of macrophages (42), annexin V was used to identify PS exposed on the external membrane surface following rearrangement of the lipid bilayer. As shown in Fig. 7, UVA induced a dramatic increase in PS translocation as early as 4 h after exposure. However, the pretreatment with CsA (10 µM), VP (20 µM), or GSH (1 mM) significantly inhibited PS translocation at both 4 and 18 h after UVA irradiation. These findings suggest that GSH efflux contributes to UVA-induced PS translocation in HaCaT cells.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Inhibition of GSH efflux decreased the translocation of PS in UVA-induced apoptosis. Cells were preincubated with CsA (10 µM), VP (20 µM), or GSH (1 mM) at 37 °C for 15 min and then irradiated with UVA (25 J/cm2). After incubation for 4 or 18 h, the cells were harvested to determine the binding of annexin V to the externalized PS on the cell surface (55, 60). Results are the mean ± S.E. of four to six experiments. (**, p < 0.01; *, p < 0.05 as compared with UVA-irradiated samples without CsA, VP, or GSH at 4 and 18 h after UVA exposure, respectively).

It is noteworthy that inhibition of MRP1 is not the only effect of verapamil and cyclosporin A. Verapamil is known to be a calcium channel blocker and protect methoxyacetic acid-induced spermatocyte apoptosis in cultured rat seminiferous tubules (43). Cyclosporin A increased K⁺-induced calcium influx (44) and mitochondria calcium storage (45). Although the presence of verapamil or cyclosporin A might influence calcium homeostasis, no effect on DNA fragmentation or caspase activation, the two hallmarks for apoptosis, was observed in our study. This is the same with the addition of exogenous GSH. On the other hand, similar effect was observed by the presence of verapamil, cyclosporin A with exogenous GSH, which all inhibited GSH efflux and PS translocation. Therefore, we proposed that the effect of CsA and verapamil on UVA-triggered apoptosis was at least partially due to the inhibition of MRP1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recent studies have revealed that several types of cells undergoing apoptosis induced by pro-apoptotic stimuli, such as diphenyleneiodonium, puromycin, ricin, and anti-Fas/APO-1 antibody, appeared to release GSH rapidly and selectively into the extracellular space (34-37). However, neither the mechanisms involved in the transport of GSH nor the biological functional significance of this cellular GSH release is known. Prior to this study the involvement of GSH efflux in apoptosis of epidermal keratinocytes induced by UVA irradiation, an important environmental stress factor, was also unknown.

In the present study, we have tested the putative mechanisms involved in GSH efflux and apoptosis of HaCaT cells induced by UVA. We have shown that exposure of HaCaT cells to UVA irradiation increased the transport of reduced GSH into the extracellular medium and that UVA-induced apoptosis was closely associated with this efflux.

It is evident that UVA irradiation caused significant glutathione efflux in HaCaT cells considering the decreased intracellular total glutathione and increased extracellular total glutathione, provided that an energy source (glucose) was present. For cells kept in the dark, the efflux of intracellular GSH was much smaller, probably resulting from cystine withdrawal when the cells were switched from normal culture medium to cystine-free PBS or glucose PBS solution (35, 46).

We found that UVA-induced GSH efflux depended on the presence of glucose. Thus the transport of GSH into the extracellular space is an active process and energy-dependent (35, 47) and is not due to passive leakage as a result of the loss of membrane integrity. Immediately after UVA irradiation, only about 2% of cells were propidium iodide-positive and had lost their membrane integrity (data not shown). It seems unlikely that these damaged cells could be responsible for an increase in GSH efflux of almost 70%. Without glucose, UVA irradiation did not induce a greater GSH efflux than in dark controls, suggesting that GSH efflux under these conditions is due to cystine withdrawal following substitution of PBS for medium. Evidently the presence of glucose not only provides the energy for active transport of GSH but also maintains lower intracellular GSSG levels and higher GSH levels under UVA stress by providing a source of NADPH that is essential for the recycling of GSSG to GSH (48, 49).

In previous studies, under UVA irradiation in the absence of glucose, the oxidation of both intracellular and extracellular GSH to GSSG was found to be the predominant reaction for glutathione, causing the depletion of intracellular GSH (24, 50). No significant GSH efflux was observed, as was the case in our studies. The efflux of GSH was inhibited by exogenous addition of GSH and slowed down by GSH depletion (80%) with BSO. This suggests that the transport process is affected by the concentration gradient of GSH across the cell membrane.

As far as the mechanisms and carriers involved in GSH transport are concerned, it is known that the multidrug resistance-associated proteins, MRP1 and MRP2, are responsible for GSH efflux in the liver (47, 51, 52). Because the expression of MRP2 is restricted to liver, kidney, and gut, whereas MRP1 is found in many tissues (53), it is reasonable to suppose that in HaCaT keratinocytes, MRP1 may be the transporter protein that is activated by UVA irradiation to cause GSH efflux. Furthermore, the rapid efflux of GSH during UVA irradiation (within 2 h) suggests that this active transport process may be due to the activation of pre-existing transporter proteins by UVA in the presence of the energy provider glucose, rather than the induction of transporter proteins by UVA irradiation or oxidative stress (54). Verapamil and CsA, inhibitors for MRP1, prevent the efflux of GSH induced by UVA irradiation, which implies the participation of MRP1 in the transport process of GSH in HaCaT cells.

The rapid and active efflux of GSH induced by UVA irradiation in HaCaT cells could be an important biological response of keratinocytes to UVA-induced stress. Although little is known about the biological significance of GSH efflux in keratinocytes, we have found that apoptosis of HaCaT cells that occurred after GSH efflux is closely related to the specific GSH export process. GSH efflux has no effect on caspase activation and DNA fragmentation in our study. However, the increases in plasma membrane permeability and PS translocation during apoptosis of HaCaT cells are associated with GSH efflux. Inhibition of GSH efflux by the MRP1 inhibitors verapamil and CsA also prevents the loss of plasma membrane integrity and PS externalization. The presence of exogenous GSH has a similar effect to CsA and verapamil. Previous studies (56, 57) have shown that PS translocation is independent of both nuclear activity (55) and caspase activation. It seems most likely that in HaCaT cells GSH efflux is involved in membrane rearrangement rather than caspase activation or DNA fragmentation in UVA-induced apoptosis. As a matter of fact, in apoptosis of Jurkat lymphocytes induced by anti-Fas/APO-1, GSH efflux was observed during apoptosis, but inhibition of this process did not affect DNA fragmentation (35). Inhibition of GSH efflux has been shown to protect cells from apoptosis induced by puromycin in HepG2 cells but not in U937 cells by inhibiting DNA fragmentation (34). As in the case of puromycin-treated HepG2 cells (34), UVA-induced GSH efflux in HaCaT cells precedes DNA fragmentation; however, DNA fragmentation is not affected by inhibition of GSH efflux.

It appears that cells stimulated by UVA to undergo apoptosis get rid of their GSH during the irradiation in order to optimize the functioning of the overall process. GSH efflux preceding apoptosis provides a novel mechanism to deplete GSH in the cells rather than oxidizing GSH to GSSG, which enhances the oxidative tonus without intervention of reactive oxygen species (34, 35). The increased oxidative stress has been shown to result in the selective phosphatidylserine oxidation that precedes phosphatidylserine externalization in oxidant-induced apoptosis (58, 59). Because externalized phosphatidylserine serves as an important signal for targeting recognition and elimination of apoptotic cells by macrophages (42), the earlier phosphatidylserine translocation increased the macrophage recognition and phagocytosis of the apoptotic cells in vivo and thus minimized their potential to invoke inflammation or form neoplastic transformation. These changes may be important for skin health and the prevention of UVA-induced skin cancer.

	ACKNOWLEDGEMENTS

We thank Dr. David Miller and Dr. Jie Liu for their helpful discussions about the MRP transporter system. We are grateful to Dr. Carl Bortner for help with fluorescence-activated cell sorter measurements. We also thank Dr. Ann Motten for reading this manuscript.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 919-541-4751; Fax: 919-541-5750; E-mail: he3@niehs.nih.gov.

Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.M207781200

	ABBREVIATIONS

The abbreviations used are: MRP, multidrug resistance-associated protein; BSO, buthionine sulfoximine; CsA, cyclosporin A; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); LDH, lactate dehydrogenase; PS, phosphatidylserine; VP, verapamil; PBS, phosphate-buffered saline; AFC, 7-amino-4-trifluoromethyl coumarin.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				R. Franco, W. I. DeHaven, M. I. Sifre, C. D. Bortner, and J. A. Cidlowski Glutathione Depletion and Disruption of Intracellular Ionic Homeostasis Regulate Lymphoid Cell Apoptosis J. Biol. Chem., December 26, 2008; 283(52): 36071 - 36087. [Abstract] [Full Text] [PDF]

				Y.-Y. He, S. E. Council, L. Feng, and C. F. Chignell UVA-Induced Cell Cycle Progression Is Mediated by a Disintegrin and Metalloprotease/Epidermal Growth Factor Receptor/AKT/Cyclin D1 Pathways in Keratinocytes Cancer Res., May 15, 2008; 68(10): 3752 - 3758. [Abstract] [Full Text] [PDF]

				R. Franco, M. I. Panayiotidis, and J. A. Cidlowski Glutathione Depletion Is Necessary for Apoptosis in Lymphoid Cells Independent of Reactive Oxygen Species Formation J. Biol. Chem., October 19, 2007; 282(42): 30452 - 30465. [Abstract] [Full Text] [PDF]

				C. L. Hammond, R. Marchan, S. M. Krance, and N. Ballatori Glutathione Export during Apoptosis Requires Functional Multidrug Resistance-associated Proteins J. Biol. Chem., May 11, 2007; 282(19): 14337 - 14347. [Abstract] [Full Text] [PDF]

				A. Campalans, R. Amouroux, A. Bravard, B. Epe, and J. P. Radicella UVA irradiation induces relocalisation of the DNA repair protein hOGG1 to nuclear speckles J. Cell Sci., January 1, 2007; 120(1): 23 - 32. [Abstract] [Full Text] [PDF]

				R. Franco and J. A. Cidlowski SLCO/OATP-like Transport of Glutathione in FasL-induced Apoptosis: GLUTATHIONE EFFLUX IS COUPLED TO AN ORGANIC ANION EXCHANGE AND IS NECESSARY FOR THE PROGRESSION OF THE EXECUTION PHASE OF APOPTOSIS J. Biol. Chem., October 6, 2006; 281(40): 29542 - 29557. [Abstract] [Full Text] [PDF]

				A. Rothnie, R. Callaghan, R. G. Deeley, and S. P. C. Cole Role of GSH in Estrone Sulfate Binding and Translocation by the Multidrug Resistance Protein 1 (MRP1/ABCC1) J. Biol. Chem., May 19, 2006; 281(20): 13906 - 13914. [Abstract] [Full Text] [PDF]

				R. N. Watts, C. Hawkins, P. Ponka, and D. R. Richardson Nitrogen monoxide (NO)-mediated iron release from cells is linked to NO-induced glutathione efflux via multidrug resistance-associated protein 1 PNAS, May 16, 2006; 103(20): 7670 - 7675. [Abstract] [Full Text] [PDF]

				Y.-Y. He, J.-L. Huang, and C. F. Chignell Delayed and Sustained Activation of Extracellular Signal-regulated Kinase in Human Keratinocytes by UVA: IMPLICATIONS IN CARCINOGENESIS J. Biol. Chem., December 17, 2004; 279(51): 53867 - 53874. [Abstract] [Full Text] [PDF]

				M. A. Bachelor and G. T. Bowden Ultraviolet A-induced Modulation of Bcl-XL by p38 MAPK in Human Keratinocytes: POST-TRANSCRIPTIONAL REGULATION THROUGH THE 3'-UNTRANSLATED REGION J. Biol. Chem., October 8, 2004; 279(41): 42658 - 42668. [Abstract] [Full Text] [PDF]

				D. Trompier, X.-B. Chang, R. Barattin, A. d. M. d'Hardemare, A. Di Pietro, and H. Baubichon-Cortay Verapamil and Its Derivative Trigger Apoptosis through Glutathione Extrusion by Multidrug Resistance Protein MRP1 Cancer Res., July 15, 2004; 64(14): 4950 - 4956. [Abstract] [Full Text] [PDF]

				Y.-Y. He, J.-L. Huang, J. B. Gentry, and C. F. Chignell Epidermal Growth Factor Receptor Down-regulation Induced by UVA in Human Keratinocytes Does Not Require the Receptor Kinase Activity J. Biol. Chem., October 24, 2003; 278(43): 42457 - 42465. [Abstract] [Full Text] [PDF]

				Y. Yang, A. Sharma, R. Sharma, B. Patrick, S. S. Singhal, P. Zimniak, S. Awasthi, and Y. C. Awasthi Cells Preconditioned with Mild, Transient UVA Irradiation Acquire Resistance to Oxidative Stress and UVA-induced Apoptosis: ROLE OF 4-HYDROXYNONENAL IN UVA-MEDIATED SIGNALING FOR APOPTOSIS J. Biol. Chem., October 17, 2003; 278(42): 41380 - 41388. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 278/10/8058    most recent M207781200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by He, Y.-Y. Articles by Chignell, C. F. Search for Related Content PubMed PubMed Citation Articles by He, Y.-Y. Articles by Chignell, C. F. Social Bookmarking                      What's this?