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The Journal of Immunology, 2000, 165: 4290-4297.
Copyright © 2000 by The American Association of Immunologists

Arsenite Induces Apoptosis of Murine T Lymphocytes Through Membrane Raft-Linked Signaling for Activation of c-Jun Amino-Terminal Kinase1

Khaled Hossain, Anwarul A. Akhand, Masashi Kato, Jun Du, Kozue Takeda, Jianghong Wu, Kei Takeuchi, Wei Liu, Haruhiko Suzuki and Izumi Nakashima2

Department of Immunology, Nagoya University Graduate School of Medicine, Nagoya, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Because of its dual roles in acute toxicity and in therapeutic application in cancer treatment, arsenic has recently attracted a renewed attention. In this study, we report NaAsO2-induced signal cascades from the cell surface to the nucleus of murine thymic T lymphocytes that involve membrane rafts as an initial signal transducer. NaAsO2 induced apoptosis through fragmentation of DNA, activation of caspase, and reciprocal regulation of Bcl-2/Bax with the concomitant reduction of membrane potential. We demonstrated that NaAsO2-induced caspase activation is dependent on curcumin-sensitive c-Jun amino-terminal kinase and barely dependent on SB203580-sensitive p38 kinase or PD98059-sensitive extracellular signal-regulated kinase. Additionally, staurosporine, which severely inhibited the activation of mitogen-activated protein (MAP) family kinases and c-Jun, partially blocked the NaAsO2-mediated signal for poly(ADP-ribose) polymerase (PARP) degradation. Potentially as the initial cell surface event for intracellular signaling, NaAsO2 induced aggregation of GPI-anchored protein Thy-1 and superoxide production. This Thy-1 aggregation and subsequent activation of MAP family kinase and c-Jun and the degradation of PARP induced by NaAsO2 were all inhibited by DTT, suggesting the requirement of interaction between arsenic and protein sulfhydryl groups for those effects. ß cyclodextrin, which sequestrates cholesterol from the membrane rafts, inhibited NaAsO2-induced activation of protein tyrosine kinases and MAP family kinases, degradation of PARP, and production of superoxide. In addition, ß cyclodextrin dispersed NaAsO2-induced Thy-1 clustering. These results suggest that a membrane raft integrity-dependent cell surface event is a prerequisite for NaAsO2-induced protein tyrosine kinase/c-Jun amino-terminal kinase activation, superoxide production, and downstream caspase activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Arsenic, which has been used in agriculture and forestry as a component of pesticides and insecticides, is now known as a pollutant that causes an environmental tragedy in some areas of the world in which a large population is drinking arsenic-contaminated ground water. An alarming number of toxicity cases have been reported in these areas (1), in which arsenic has been found to cause immunotoxicity, immunosuppression, skin lesions, and increased risk of cancer (2, 3, 4, 5, 6). At the same time, renewed attention has been created due to the therapeutic application of arsenic in the treatment of lymphoid and hemopoietic neoplasmas such as acute promyelocytic leukemia (7). These two apparently opposite effects of arsenic on human life may share a common molecular mechanism.

Recently, arsenic compounds have been shown to be a potent inducer of apoptotic and necrotic death for both normal (8) and malignant cells (9, 10, 11, 12). A number of earlier studies have partially elucidated the arsenic-induced mechanism that may be linked to apoptotic death in leukemia cells. Caspase activation may (13, 14) or may not (15) be associated with arsenic-induced apoptosis of neoplastic cells. Arsenite has also been shown to induce activation of mitogen-activated protein kinase (MAPK)3 (2) family member proteins, some of which may play a key role in apoptosis induction in leukemia cells (10). These observations in studies using neoplastic cells, which have partially elucidated the mechanism of arsenite-induced apoptosis, have not, however, enabled clarification of the total signal cascade from the cell surface to the nucleus for arsenite-induced apoptosis of normal lymphocytes, which may be important for a better understanding of the arsenite-induced immunotoxicity and immunosuppression.

In recent years, there has been mounting evidence that rafts in the plasma membrane of mammalian cells, which are enriched with cholesterol and glycosphingolipids, play an important role in delivering a number of intracellular signals (16, 17, 18, 19, 20, 21, 22). This detergent-insoluble membrane integrity, which is implicated in a number of signaling molecules, has recently been shown to be essential for effective TCR signal transduction (23, 24, 25, 26). However, the role of membrane rafts in the chemically induced signaling event is still unknown.

In this study, we show for the first time that sodium arsenite induces caspase activation through membrane raft-linked promotion of superoxide production and activation of c-Jun amino-terminal kinase (JNK).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells, reagents, and Abs

Single cell suspensions of murine thymic T lymphocytes (thymocytes) in MEM were prepared from 6- to 8-wk-old C57BL/6 strain mice. Splenic T lymphocytes were prepared by passing through a nylon wool column, as described previously (27). The cells were incubated in the presence or absence of NaAsO2 (Sigma, St. Louis, MO) at 37°C before analysis. Herbimycin A, staurosporine, SB203580, curcumin, PD98059, ß cyclodextrin, nystatin, DTT, hydroethidine (HE), and carbamoyl cyanide m-chlorophenyl hydrazone (mClCCP) were purchased from Sigma, and 3,3'-dihexyloxacarbocyanine iodide (DiOC6) was obtained from Molecular Probes (Eugene, OR). Polyclonal Abs purchased from New England Biolabs (Beverly, MA) were anti-phospho-p44/42 MAPK, anti-phospho-p38 MAPK, anti-phospho-JNK, and anti-phospho-c-Jun (serine 73) Abs, and Abs purchased from Transduction Laboratories (Lexington, KY) were anti-pan extracellular signal-regulated kinase (ERK) (monoclonal) and anti-phosphotyrosine (polyclonal). Anti-Bcl-2 and anti-Bax (polyclonal) Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-poly(ADP-ribose) polymerase (PARP) polyclonal Ab was obtained from Upstate Biotechnology (Lake Placid, NY).

Analysis of DNA fragmentation by electrophoresis

Cells were lysed in 100 µl of hypotonic lysing buffer (50 mM Tris-HCl, 0.5% SDS, 10 mM EDTA), followed by the addition of 2 µl of proteinase K (20 mg/ml) and 6 µl of RNase (10 mg/ml). The resultant mixture was incubated at 55°C for 1 h. Each sample (10 µl) was mixed with 3 µl of 0.25% (w/v) bromophenol blue and 40% (w/v) sucrose and was run on 1.5% agarose gel with 0.1 µg/ml ethidium bromide (16).

SDS-PAGE and immunoblotting

SDS-PAGE and immunoblotting were performed as described elsewhere (16). In brief, cells were lysed by adding an equal volume of a 2-fold concentrated sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 10% 2-ME, 20% glycerol), and proteins thus obtained were subjected to SDS-PAGE on 8–12.5% gel. The proteins were then transferred to a polyvinylidene difluoride membrane. Subsequently, the membrane was stained with different Abs, followed by goat anti-rabbit or anti-mouse IgG to HRP (Tago, Burlingame, CA). The protein bands were visualized by Western blot chemiluminescence reagent (DuPont-NEN, Boston, MA), according to the instructions of the manufacturer. For reprobing, membranes were stripped (2% SDS, 62.5 mM Tris, pH 6.8, 100 mM 2-ME, 50°C, 30 min) and reprobed with corresponding Abs. The molecular sizes of the developed proteins were determined by comparison with prestained protein markers (New England Biolabs).

Cytofluorometric analysis of mitochondrial membrane potential ({Delta}{psi}) and superoxide anion

To evaluate mitochondrial transmembrane potential ({Delta}{psi}) and superoxide anion, cells were incubated for 15 min at 37°C in PBS containing 40 nM DiOC6 (28). Thereafter, cells were kept on ice until cytofluorometric analysis within 60 min. In control experiments, cells were labeled after preincubation with the uncoupler mClCCP (50 µM, 37°C, 30 min). Analysis was performed by flow cytometry (excitation, 488 nM; emission, 525 nm). Forward and side light scatter were gated on the major population of normal-sized lymphoid cells. Generation of superoxide anion was measured as described (28, 29) by incubating the cells with 2 µM HE for 15 min at 37°C.

Fluorescence Ab technique

Thymocytes (106/100 µl) were incubated with or without NaAsO2 at 37°C for 20 min and were fixed with 4% paraformaldehyde at room temperature for 30 min. They were then stained with FITC-labeled anti-Thy-1.2 mAb (Becton Dickinson, Mountain View, CA) and were mounted on a glass slide in the presence of p-phenylenediamine (1 mg/ml). The stained cells were observed under a fluorescence microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NaAsO2 induces an apoptotic signal in murine T lymphocytes

We first tested the effect of exposure of murine thymocytes to NaAsO2 on fragmentation of their chromosomal DNA. As shown in Fig. 1Go, agarose gel electrophoresis of DNA showed that 10 µM, but not 1 µM, NaAsO2 induced extensive fragmentation of DNA, and that 100 µM NaAsO2, which accompanied necrotic cell death (as demonstrated by a dye exclusion test), induced less extensive fragmentation than did 10 µM NaAsO2.



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  		FIGURE 1. NaAsO2 induces DNA fragmentation in murine thymic T lymphocytes. Thymocytes were incubated at 37°C with or without the indicated concentrations of NaAsO2 for 10 h. These cells were lysed in hypotonic lysing buffer, and DNAs were then analyzed by agarose gel electrophoresis. Representative results of three experiments with consistent results are shown.

 
We next examined whether the NaAsO2-induced DNA fragmentation involves activation of caspase, a known key enzyme to mediate DNA fragmentation. Thymocytes were incubated with 10 µM NaAsO2 for 4–16 h, and the cell lysates were examined by immunoblotting with an Ab specific to PARP as an intracellular substrate of caspase-3. After 8 h of incubation, most of the PARP molecules (116 kDa) were degraded to produce 85-kDa fragments, and nearly complete degradation was observed after 12 h (Fig. 2GoA). PARP degradation developed less extensively when 100 µM NaAsO2 was added to the culture (Fig. 2GoB, left). Treatment with 10–100 µM NaAsO2 for 12 h also induced PARP degradation in splenic T lymphocytes (Fig. 2GoB, right). This result indicated that NaAsO2 induces caspase activation independent of the maturation stage of T lymphocytes.



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  		FIGURE 2. NaAsO2 induces PARP degradation. Thymocytes (A, B) and splenic T lymphocytes (B) were incubated with the indicated concentrations of NaAsO2 at 37°C for 4–16 h (A) or 12 h (B). The cells were then lysed with sample buffer and subjected to immunoblot assay with anti-PARP Ab. The position of undegraded (116-kDa) and degraded (85-kDa) PARP molecules is shown on the right of both panels. Representative results of three experiments with consistent results are shown.

 
We then measured the mitochondrial membrane potential by staining cells with fluorochrome DiOC6 for analysis by flow cytometry. It was found in this study that incorporation of DiOC6 was reduced in NaAsO2-treated cells, indicating the reduction of membrane potential (Fig. 3Go). We next examined the effect of NaAsO2 treatment on the expression levels of Bcl-2 and Bax, which are known as negative (Bcl-2) or positive (Bax) regulators of cell death. As shown in Fig. 4Go, the NaAsO2-mediated cellular signal caused a gradual decrease in Bcl-2 expression as the incubation time was increased and an increase in Bax protein expression with a peak after 12-h incubation. These results confirmed that murine normal lymphocytes are subjected to NaAsO2-mediated signals for apoptosis induction through caspase activation that may be initiated by damage to the mitochondrial membrane and imbalance of Bcl-2/Bax protein expression.



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  		FIGURE 3. Assessment of mitochondrial transmembrane potential ({Delta}{psi}). Thymocytes were treated with or without 10 µM NaAsO2 for 7 h at 37°C, stained by incubation with DiOC6 (40 nM), and subjected to cytofluorometric analysis. To determine zero potential, cells were incubated with mClCCP, an uncoupler that completely abolished {Delta}{psi}. Representative results of three experiments with consistent results are shown.

 


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  		FIGURE 4. NaAsO2 induces reciprocal regulation of Bcl-2 and Bax protein expression. Thymocytes were incubated with or without NaAsO2 for the indicated times at 37°C. The cells were then lysed with sample buffer and subjected to immunoblot assay with anti Bcl-2 (A) or anti-Bax (B) Ab. Representative results of three experiments with consistent results are shown.

 
NaAsO2 induces tyrosine phosphorylation of multiple cellular proteins of thymocytes

We performed another experiment to try to understand the signal transduction cascade upstream of the demonstrated effector phase of NaAsO2-induced apoptosis in T lymphocytes. To examine whether promotion of tyrosine phosphorylation of cellular proteins is involved in the NaAsO2-mediated signaling as the initial event, murine thymocytes were exposed to different concentrations of NaAsO2 and the cellular proteins were analyzed by immunoblotting for phosphotyrosine. As shown in Fig. 5Go, both 10 and 100 µM NaAsO2 induced a dose-dependent increase in tyrosine phosphorylation of a number of cellular proteins after 10 min of incubation, and this increase lasted for at least 1 h (not shown). The apparent molecular masses of the proteins phosphorylated were 110–120, 90, 70, 56, and 40 kDa. These results suggested that the NaAsO2-mediated signal involves an elevated protein tyrosine kinase (PTK) activity at the initial stage.



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  		FIGURE 5. NaAsO2 induces tyrosine phosphorylation of cellular proteins. Thymocytes were incubated at 37°C with or without the indicated concentrations of NaAsO2 for 10 min. The cells were then lysed and subjected to immunoblot assay with anti-phosphotyrosine Ab. Positions of molecular mass markers (kDa) are shown on the left. Representative results of three experiments with consistent results are shown.

 
Phosphorylation of MAPK family members by NaAsO2

The members of the MAPK family that are activated by dual phosphorylation on both tyrosine and threonine residues have been implicated in the transduction of a wide variety of extracellular signals (30, 31). Therefore, we next examined whether NaAsO2 could promote phosphorylation of three classes of MAPK family proteins, ERK, JNK, and p38 kinase. As shown in Fig. 6GoA, a relatively high (100 µM), but not low (10 µM), concentration of NaAsO2 induced increases in phosphorylation of ERK1 (p44), ERK2 (p42) (upper panel, left), JNK (middle panel, left), and p38 MAPK (lower panel, left) within 10 min. The latter concentration (10 µM) of NaAsO2, however, induced heavy phosphorylation of JNK (Fig. 6GoB, upper panel), but not ERK (not shown) after 1 h of incubation, and the phosphorylation later decreased. As is also shown in Fig. 6GoB (lower panel), 10 µM of NaAsO2 induced an increase in phosphorylation of c-Jun as the substrate of JNK and the extent of phosphorylation corresponded well with that of JNK.



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  		FIGURE 6. NaAsO2 induces phosphorylation of MAP family kinases and c-Jun. Thymocytes were incubated with or without the indicated concentrations of NaAsO2 for 10 min (A) or 10 µM of NaAsO2 for the indicated times (B) at 37°C. Then the cells were lysed and subjected to immunoblot assay with anti-phospho-ERK, anti-phospho-JNK, anti-phospho-p38, or anti-phospho-c-Jun Ab. The membranes were stripped and reprobed with corresponding Abs specific to MAP family kinases (A, right). Positions of respective MAPK family members are shown between two panels (A) and on the right (B). Representative results of three experiments with consistent results are shown.

 
Characterization of the NaAsO2-induced apoptotic signal

We next investigated the causative relation between NaAsO2-induced increase in PTK activity and activation of MAPK family members. As shown in Fig. 7GoA, both herbimycin A and staurosporine as inhibitors of PTK (32, 33), which were added into the cell suspension before NaAsO2, had effectively inhibited the action of NaAsO2 to promote phosphorylation of ERKs (upper panel), JNKs (second from the upper panel), p38 kinase (third from the upper panel), and c-Jun (bottom panel). These results suggest that NaAsO2-mediated activation of MAPK family members, as well as c-Jun, is basically dependent on herbimycin A/staurosporine-sensitive activity of PTK.



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  		FIGURE 7. Effects of PTK, JNK, and p38 kinase inhibitors on NaAsO2-mediated activation of MAPKs and c-Jun. Thymocytes were incubated with or without 100 µM of NaAsO2 for 1 h at 37°C. In some groups, herbimycin A (HA, 10 µg/ml) was added 4 h before the addition of NaAsO2, or staurosporine (STS, 10 µg/ml) was added 5 min before the addition of NaAsO2 (A), and curcumin (Cur, 20 µM; B) and SB203580 (SB, 40 µM; C) were added 1 h before the addition of NaAsO2. The cells were then lysed and subjected to immunoblot assay with the indicated Abs. Positions of the respective proteins are shown on the right of each panel. Representative results of three experiments with consistent results are shown.

 
The possible roles of JNK and p38 MAPK in NaAsO2-induced c-Jun phosphorylation were then examined. For this purpose, we treated some cells with curcumin and SB203580 before adding NaAsO2. Curcumin, a known JNK pathway inhibitor (34) that inhibited JNK phosphorylation (Fig. 7GoB, second from the top panel), also blocked c-Jun phosphorylation (Fig. 7GoB, bottom panel), whereas SB203580, a p38 kinase-specific inhibitor (35), only partially did so (Fig. 7GoC). However, curcumin also inhibited NaAsO2-mediated ERK (Fig. 7GoB, upper panel) and p38 MAPK phosphorylation (Fig. 7GoB, third from the upper panel). These results suggest the possible involvement of a curcumin-sensitive upstream kinase that may be commonly recruited by NaAsO2 for the activation of all three members of MAPK family proteins. To determine the relationship between MAPK family members and PARP degradation, we further investigated whether curcumin, SB203580, and PD98059, a specific inhibitor of ERK could prevent PARP degradation induced by NaAsO2. As shown in Fig. 8GoA, curcumin completely inhibited NaAsO2-induced PARP degradation, whereas SB203580 and PD98059 did not (Fig. 8GoB). These results suggest that the signal cascade for the NaAsO2-induced caspase activation involves the JNK, but not the ERK or p38 MAPK signaling pathway, which is linked up with herbimycin A/staurosporine-sensitive PTK activities. Correspondingly, staurosporine partially but clearly inhibited the NaAsO2-induced caspase activation, although staurosporine and herbimycin A by themselves induced some degradation of PARP, and the inhibiting effect of herbimycin A on NaAsO2-induced PARP degradation was marginal (Fig. 8GoC). The above results showing inhibition of JNK and caspase by curcumin suggest that arsenite-induced caspase activation is downstream of JNK. For further confirmation, we treated some cells with Z-VAD, a specific inhibitor of caspase, followed by immunoblotting with anti-PARP and anti-phospho-JNK Abs. As shown in Fig. 8GoD, Z-VAD inhibited NaAsO2-mediated degradation of PARP, but failed to block the phosphorylation of JNK. This result confirmed our assumption that NaAsO2-induced PARP degradation is downstream of JNK activation.



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  		FIGURE 8. Effects of PTK, JNK, p38 kinase, and ERK inhibitors on NaAsO2-induced PARP degradation. Thymocytes were incubated with or without 10 µM of NaAsO2 for 12 h at 37°C. In some groups, curcumin (Cur, 20 µM, 1 h) or SB203580 (SB, 40 µM, 1 h; A), PD98059 (PD, 50 µM, 30 min; B), herbimycin A (HA, 10 µg/ml, 4 h) or staurosporine (STS, 10 µg/ml, 5 min; C), or Z-VAD (100 µM, 30 min; D) was added at the times indicated in parentheses before the addition of NaAsO2. The cells were then lysed and subjected to immunoblot assay with anti-PARP or anti-phospho-JNK Ab. Positions of undegraded (116-kDa) and degraded (85-kDa) PARP molecules, and JNK are shown on the right. Representative results of three experiments with consistent results are shown.

 
Cell surface chemical event for NaAsO2-induced intracellular signaling

All of the above results suggest that PTK-linked or -unlinked JNK activation occurs upstream of the NaAsO2-induced caspase activation in T lymphocytes. What could then be the initial event to start the signaling? Previously, we showed that exposure of thymocytes to sulfhydryl group (SH)-reactive Hg2+ induced aggregation of cell surface proteins, including GPI-anchored Thy-1 molecules, through an intermolecular S-Hg-S bond, and that this aggregation occurred in close association with Lck activation and extensive tyrosine phosphorylation of cellular proteins (36). Because arsenic is also highly reactive with the SH group (37, 38, 39), we tested whether NaAsO2 works in a similar way to as Hg2+. As shown in Fig. 9Go, treatment of thymocytes with NaAsO2 caused remarkable aggregation of Thy-1 compared with the untreated control cells. Addition of DTT, a reducing reagent, before the addition of NaAsO2 to the lymphocyte suspension evidently blocked NaAsO2-mediated Thy-1 aggregation (Fig. 9Go, right panel) and subsequent signaling for activation/phosphorylation of MAPK family kinases and c-Jun and for PARP degradation (Fig. 10Go). It is therefore likely that NaAsO2-induced signaling requires chemical interaction between arsenite and SH groups of cell surface proteins represented by Thy-1 for cross-linkage and potentially some redox-linked event downstream of the cell surface event.



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  		FIGURE 9. NaAsO2 induces aggregation of cell surface Thy-1. Thymocytes were incubated at 37°C with or without 1 mM NaAsO2 for 20 min. In one group of cells, DTT was added 5 min before the addition of NaAsO2. They were then fixed and were stained with FITC-labeled anti-Thy-1 mAb. Fluorescence was examined under a fluorescence microscope. Data shown are representative of more than 20 photographs for each picture (x400, upper panel; x1000, lower panel) obtained by four independent experiments. Note remarkable aggregation of Thy-1 on the cells in middle panel. Less-marked, but clear Thy-1 aggregation was also observed on the cells treated with 100 µM of NaAsO2 (not shown).

 


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  		FIGURE 10. DTT inhibits NaAsO2-induced activation of PTK, MAP family kinases, and c-Jun, and degradation of PARP. Thymocytes were incubated with or without 100 µM of NaAsO2 for 1 h (top four panels) or 10 µM of NaAsO2 for 10 h (bottom panel) at 37°C. In one group, DTT (500 µM) was added 1 h before the addition of NaAsO2. The cells were then lysed and subjected to immunoblot assay with the indicated Abs. The positions of MAPK family kinases, c-Jun, and PARP are shown on the right. Representative results of three experiments with consistent results are shown.

 
The role of the membrane raft structure in arsenite-mediated intracellular signaling

A growing body of evidence suggests that cholesterol-rich, detergent-resistant membrane raft integrity is a prerequisite for delivering signal into lymphocytes and many other cells (20, 21, 22, 23, 24, 25). Correspondingly, we previously suggested that GPI-anchored proteins are obligatorily involved in Hg2+-induced signaling in T lymphocytes (36, 40). To investigate the possible role of the detergent-insoluble membrane raft structure in protein tyrosine phosphorylation and subsequent MAPK family member activation by arsenite, we preincubated thymocytes with ß cyclodextrin, which disrupts cholesterol-rich microdomains in the membrane (23, 41), before adding NaAsO2. Interestingly, ß cyclodextrin inhibited NaAsO2-mediated protein tyrosine phosphorylation and phosphorylation of ERKs, JNKs, p38 kinase, and c-Jun (Fig. 11Go). Furthermore, ß cyclodextrin effectively blocked NaAsO2-induced PARP degradation (Fig. 11Go, bottom panel). Similar inhibition of NaAsO2-mediated signaling was observed in experiments in which another raft structure disrupter nystatin was used (23 , data not shown). Corresponding to these biochemical data, treatment of thymocytes with ß-cyclodextrin partially blocked the NaAsO2-induced Thy-1 aggregation (Fig. 12Go), indicating a disorganization of the raft structure after treatment. These results provide strong evidence of the involvement of an integrated raft structure in delivering NaAsO2-mediated apoptotic signals.



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  		FIGURE 11. Depletion of cholesterol from membrane rafts. ß cyclodextrin impairs NaAsO2-induced activation of PTK and MAP family kinases and degradation of PARP. Thymocytes were incubated with or without 100 µM of NaAsO2 for 1 h (top five panels) or 10 µM of NaAsO2 for 10 h (bottom panel) at 37°C. In one group, ß cyclodextrin (ß Cyd, 10 mM) was added 1 h before the addition of NaAsO2. The cells were then lysed and subjected to immunoblot assay with the indicated Abs. The positions of molecular mass marker proteins (top panel) are shown on the left, and those of MAPK family kinase, c-Jun, and PARP (bottom five panels) are shown on the right. Representative results of three experiments with consistent results are shown.

 


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  		FIGURE 12. ß cyclodextrin impairs NaAsO2-induced aggregation of cell surface Thy-1. Thymocytes were incubated at 37°C with or without 1 mM NaAsO2 for 20 min. In one group of cells, ß cyclodextrin (5 mM) was added 20 min before the addition of NaAsO2. They were then fixed and were stained with FITC-labeled anti-Thy-1 mAb. Fluorescence was examined under a fluorescence microscope. Data shown are representative of more than 20 photographs for each picture (x1000) obtained by four independent experiments.

 
Membrane raft structure-dependent signaling induces superoxide production in the cell

To further clarify the mechanism of membrane raft structure-dependent signaling for activation of PTK/MAPK family kinases, we investigated whether the signal cascade involves superoxide production. NaAsO2-treated cells and untreated cells were labeled with HE. HE is known to be oxidized by superoxide to ethidium (42), which emits red fluorescence. As shown in Fig. 13Go, the population of ethidium-containing cells clearly increased in NaAsO2-treated cells compared with the untreated controls, suggesting that NaAsO2 promoted the production of superoxide. Pretreatment of ß cyclodextrin before NaAsO2 prevented this superoxide production, and the population of ethidium-containing cells dropped to the control level.



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  		FIGURE 13. NaAsO2 induces superoxide production in a membrane raft structure-dependent pathway. Thymocytes were incubated with or without 100 µM of NaAsO2 for 1 h at 37°C. In one group, ß cyclodextrin (ß Cyd, 10 mM) was added 1 h before the addition of NaAsO2. The cells were then labeled with HE (2 µM) for 20 min at 37°C and subjected to cytofluorometric analysis. Representative results of three experiments with consistent results are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we characterized the cascade of NaAsO2-mediated signal transduction from the cell surface to the nucleus for inducing apoptosis of murine T lymphocytes. The cascade of signal transduction mainly with normal thymic T lymphocytes, described in this study, is probably common to other cell types as the potential target of arsenite for immunotoxicity and immunosuppression and for antitumor therapeutic effects.

Earlier studies have shown that NaAsO2 induces DNA fragmentation through caspase activation (13, 14), down-regulation of Bcl-2 (13), and activation of MAP family kinases (10) in various types of tumor cells. We have confirmed these previously reported observations in native T lymphocytes (Figs. 1Go, 2Go, and 4Go) and provided evidence that activation of JNK is upstream of caspase activation. Curcumin, which inhibits an upstream kinase common to MAP family kinases, including JNK, thus completely blocked the NaAsO2-mediated PARP degradation, whereas the p38 MAPK inhibitor SB203580 (Fig. 8GoA) and ERK inhibitor PD98059 failed to do so (Fig. 8GoB). Arsenite-mediated c-Jun phosphorylation was also completely inhibited by curcumin and partially by SB203580 (Fig. 7Go, B and C). The exact mechanism through which activation/phosphorylation of JNK leads to caspase activation still remains to be clarified. It could, however, be that activated JNK and downstream c-Jun play some roles in regulating expression levels of Bcl-2 and Bax, which were reversely modified by arsenic treatment (Fig. 4Go), for inducing reduction of mitochondrial membrane potential (Fig. 3Go).

The primary purpose of the present study was to elucidate the signal cascade upstream of arsenite-induced phosphorylation/activation of JNK and c-Jun that is responsible for caspase activation. In our experiments, both herbimycin A, as a PTK-specific inhibitor, and staurosporine, as a powerful PTK/PKC inhibitor of a broad spectrum of activity, extensively blocked the NaAsO2-mediated activation of all three classes of MAP family kinases and c-Jun (Fig. 7GoA), suggesting the involvement of PTK activation in the signal cascade upstream of JNK and c-Jun. These PTK inhibitors, however, barely or only partially inhibited the NaAsO2-mediated caspase activation (Fig. 8GoC). This result suggests that arsenite triggers two JNK-oriented subpathways for caspase activation that are either PTK dependent or independent. Furthermore, the caspase inhibitor Z-VAD blocked NaAsO2-induced PARP degradation, but failed to inhibit JNK phosphorylation (Fig. 8GoD), thereby providing strong evidence in support of our speculation that NaAsO2-induced caspase activation is downstream of JNK.

The most intriguing observation in this study is successful attenuation of NaAsO2-mediated protein tyrosine phosphorylation and subsequent activation of MAP family kinases by partial sequestration of cholesterol from the detergent-insoluble membrane raft by ß cyclodextrin. Recently, cholesterol-rich, detergent-resistant membrane microdomains, rafts, have come into focus for their crucial role in intracellular signal transduction. A number of signal-mediating molecules, including GPI-anchored proteins, selected transmembrane proteins such as CD4 and linker for activation of T cells (LAT), Src family PTKs such as Lck, and phosphatidylinositol phosphate 2, are known to reside in the cholesterol-rich rafts of the cell membrane (16, 17, 18, 19, 20). Xavier et al. (23) and Montixi et al. (24) recently showed that membrane raft integrity is a prerequisite for efficient T cell-mediated signaling and that T cell activation leads to strong compartmentation of TCRs and associated signal molecules in the raft, although the role of the raft structure in chemical stress-mediated signaling has not been reported before. Our present study demonstrates for the first time that perturbation of cholesterol in the raft makes the cell resistant to arsenite to trigger the PTK/MAP family kinase-dependent signal for apoptosis induction. In relation to this observation, we noticed that treatment of T lymphocytes with NaAsO2 induces clustering of raft-associated GPI-anchored proteins, including Thy-1, possibly through chemical reaction of arsenite with cysteine SH groups of cell surface proteins (Fig. 9Go), linked to the membrane raft integrity (Fig. 12Go). Therefore, it seems that the arsenite-mediated cell surface event represented by Thy-1 clustering, which requires membrane raft integrity, starts transduction of the PTK/MAP family kinase-dependent signal for caspase activation. Finally, we observed that arsenite treatment causes superoxide production (Fig. 13Go), confirming the previously reported results (14, 43, 44). Interestingly, this arsenite-mediated superoxide production was also shown to be inhibited by treatment with ß cyclodextrin. This suggests that the arsenite-triggered signal cascade, which requires an intact raft structure, includes production of superoxide, which may work as a second signal messenger. The superoxide produced in the cell might be involved in activation of apoptosis signal-regulated kinase 1 as MAPK/ERK kinase (MEKK) of JNK and p38 kinase (45) or Src family kinase (46, 47). DTT, which abolished all the arsenite-mediated signaling for apoptotic cell death (Fig. 10Go), might have actually scavenged both arsenite and the superoxide produced as the second messenger. The exact role of arsenite-induced superoxide in signal transduction remains to be clarified, but it could be related to one of or to both of the subpathways for caspase activation.

Taken together, our results demonstrate for the first time that the integrated membrane raft structure plays a crucial role in transducing NaAsO2-induced signals that activate JNK through herbimycin A/staurosporine-sensitive, PTK-dependent, or PTK-independent subpathways for caspase activation.


   Acknowledgments
 
We thank Y. Umeda and H. Saeki for their technical assistance.


   Footnotes
 
1 This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture, and the funds for Comprehensive Research on Aging and Health from the Ministry of Health and Welfare of Japan. A.A.A. is the recipient of a Grant from Research for the Future Program of the Japanese Society for the Promotion of Science. Back

2 Address correspondence and reprint requests to Dr. Izumi Nakashima, Department of Immunology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Back

3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; DiOC6, 3,3'-dihexiloxacarbocyanine iodide; ERK, extracellular signal-regulated kinase; HE, hydroethidine; JNK, c-Jun amino-terminal kinase; mClCCP, carbamoyl cyanide m-chlorophenyl hydrazone; MAP, mitogen-activated protein; PARP, poly(ADP-ribose) polymerase; PTK, protein tyrosine kinase; SH, sulfhydryl group. Back

Received for publication February 29, 2000. Accepted for publication July 25, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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