Vanadium-induced nuclear factor of activated T cells activation through hydrogen peroxide.

The present study investigated the role of reactive oxygen species (ROS) in activation of nuclear factor of activated T cells (NFAT), a pivotal transcription factor responsible for regulation of cytokines, by vanadium in mouse embryo fibroblast PW cells or mouse epidermal Cl 41 cells. Exposure of cells to vanadium led to the transactivation of NFAT in a time- and dose-dependent manner. Scavenging of vanadium-induced H(2)O(2) with N-acety-L-cyteine (a general antioxidant) or catalase (a specific H(2)O(2) inhibitor) or the chelation of vanadate with deferoxamine, resulted in inhibition of NFAT activation. In contrast, an increase in H(2)O(2) generation by the addition of superoxide dismutase or NADPH enhanced vanadium-induced NFAT activation. This vanadate-mediated H(2)O(2) generation was verified by both electron spin resonance and fluorescence staining assay. These results demonstrate that H(2)O(2) plays an important role in vanadium-induced NFAT transactivation in two different cell types. Furthermore, pretreatment of cells with nifedipine, a calcium channel blocker, inhibited vanadium-induced NFAT activation, whereas and ionomycin, two calcium ionophores, had synergistic effects with vanadium for NFAT induction. Incubation of cells with cyclosporin A (CsA), a pharmacological inhibitor of the phosphatase calcineurin, blocked vanadium-induced NFAT activation. All data show that vanadium induces NFAT activation not only through a calcium-dependent and CsA-sensitive pathway but also involved H(2)O(2) generation, suggesting that H(2)O(2) may be involved in activation of calcium-calcineurin pathways for NFAT activation caused by vanadium exposure.

Vanadium is a transition metal widely distributed in the environment. Occupational exposure to vanadium is common in oil-fired electrical generating plants and the petrochemical, steel, and mining industries (1). The available data from the literature provides evidence that in mammalian cell culture systems vanadium(V) is biologically more active than vanadium(IV) in inducing toxic effects (2). It has been reported that the cell can protect itself against vanadate toxicity by reduced glutathione-mediated conversion of vanadium(V) to vanadi-um(IV), whereas vanadate(IV) does not undergo any change in valency state in BALB/3T3 cells (2). It has been found that vanadium-containing compounds exert potent toxic and carcinogenic effects, such as DNA damage and cell transformation (3)(4)(5). In animal studies, vanadium compounds or vanadiumcontaining air pollution particles have been shown to induce inflammation in the respiratory tract (6,7). It has been reported that vanadium associated with air pollution particles, such as residual oil fly ash, can induce the synthesis and expression of inflammatory cytokines, such as IL-6, 1 IL-8, and TNF-␣ (6 -9). Vanadium exposure in vitro was also found to cause the production of IL-1, TNF-␣, and prostaglandin E2 (6,10).
The nuclear factor of activated T cells (NFAT) was originally described as a transcriptional factor expressed in activated but not resting T cells (11)(12)(13)(14). The induction of NFAT in T cells required a calcium-activated signaling pathway and was blocked by cyclosporin A (CsA) and FK506 (15)(16)(17)(18)(19)(20)(21). Over the last decade, studies from several laboratories have indicated that the pre-existing/cytoplasmic component of NFAT was a mixture of proteins belonging to a novel family of transcription factors (22)(23)(24). The first member of this family (NFATp, later renamed NFAT 1 ) was purified from cytoplasmic extracts of a murine T cell cloned by affinity chromatography using the distal NFAT site of murine IL-2 promoter (19,25) and cloned from murine (Ar-5) and human (Jurkat) T cell cDNA libraries (25,26). Other distinct proteins belonging to the same family, such as NFATc, NFAT 3 , and NFAT 4 , were also isolated and cloned (27)(28)(29)(30). There are three functional domains in NFAT family proteins: the Rel-similarity domain, which is responsible for DNA-binding activity and interaction with AP-1; the NFAT-homology region, which regulates intracellular localization; and the transcriptional activation domain (31). The activation of NFAT in T cells includes dephosphorylation, nuclear translocation, and an increase in affinity for DNA binding (15). Stimuli that elicit calcium mobilization result in rapid dephosphorylation of NFAT proteins and their translocation to the nucleus. These dephosphorylated proteins show increased affinity for DNA binding (15).
Growing evidence indicates that NFAT is not only a T cellspecific transcriptional factor but also is expressed in a variety of lymphoid cells and in non-lymphoid tissue (15,32,33) , NFAT  involvement is reasonably well established for the production  of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-18,  TNF-␣, interferon ␥, and granulocyte macrophage-colony-stimulating factor in a variety of cell types (15). In addition, previous studies from our laboratory and other groups have shown that TNF-␣, IL-6, and IL-8 are involved in vanadium-induced inflammation (6 -10). Therefore, the objective of the present study was to determine if activation of NFAT occurred in response to vanadium and if so to investigate the molecular mechanism, by which lead to NFAT activation.
Generation of Stable Co-transfectants-PW cells were cultured in a 6-well plate until they reached 85-90% confluence. 1 g of CMV-neo vector, with or without 12 g of NFAT-luciferase reporter plasmid DNA and 15 l of LipofectAMINE reagent, was used to transfect each well in the absence of serum. After 10 -12 h, the medium was replaced with 5% FBS MEM. Approximately 30 -36 h after the beginning of the transfection, the cells were digested with 0.033% trypsin and cell suspensions were plated onto 75-ml culture flasks and cultured for 24 -28 days with G418 selection (800 g/ml). The stable transfectants were identified by measuring basal level of luciferase activity. Stable transfectant PW NFAT mass 1 were established and cultured in G418-free MEM for at least two passages before each experiment.
Assay for NFAT Activity in Vitro-Confluent monolayers of PW NFAT mass 1 or Cl 41 NFAT mass 1 were trypsinized, and 5 ϫ 10 3 viable cells were suspended in 100 l of medium were added into each well of a 96-well plate. Plates were incubated at 37°C in a humidified atmosphere of 5% CO 2 and 95% air. The cells were then exposed to vanadium for the indicated times and dosages. The cells were extracted with lysis buffer, and luciferase activity was measured as previously described (39). The results were expressed as NFAT activity relative to controls or relative luciferase unit (RLU) (33 Electron Spin Resonance (ESR) Measurements-ESR spin trapping was used to detect short-lived free radical intermediates. This technique involves the addition-type reaction of a short-lived radical with a diamagnetic compound (spin trap) to form a relatively long-lived free radical product, the so-called spin adduct, which can be observed by conventional ESR. The intensity of the spin adduct signal corresponds to the amount of short-lived radicals trapped, and the hyperfine splittings of the spin adduct are generally characteristic of the trapped radical. ESR measurements were carried out using a Varian E9 ESR spectrometer and a flat cell assembly. Hyperfine couplings were measured (0.1 G) directly from magnetic field separation using potassium tetraperoxochromate (K 3 CrO 8 ) and 1,1-diphenyl-2-picrylhydrazyl as reference standards. PW cells (1 ϫ 10 6 ) were mixed with 100 mM DMPO, 100 M NADPH, and 1 mM vanadate. The reaction mixture was then transferred to a flat cell for ESR measurement as described previously (2,41).

Induction of NFAT Transactivation in PW Cells and Cl 41
Cells by Vanadium-To study the regulation of NFAT transcription activity by vanadium in cells, we generated a NFATluciferase reporter stable transfectant by co-transfecting the NFAT-luciferase reporter plasmid and CMV-neo plasmid into mouse fibroblast cells, PW NFAT mass 1 . The results observed from this stable transfectant show that NFAT activities were markedly induced by exposure of PW cells to either vanadium(V) (increasing RLU from 3837 Ϯ 183 to 42838 Ϯ 1920) or vanadium(IV) (increasing RLU from 3837 Ϯ 183 to 38321 Ϯ 2839) (p Ͻ 0.05) (Fig. 1a). The activation of NFAT by vanadium appears to be time-and dose-dependent manner (Fig. 1, c and  d). The maximum induction of NFAT activity by vanadium occurred between 36 and 48 h after cells were exposed to vanadium (Fig. 1d). To test whether vanadium-induced NFAT activation is cell-type-specific, we also exposed the Cl 41 NFAT mass 1 to vanadium. The results show that exposure of Cl 41 cells to vanadium resulted in significant activation of NFAT After being cultured at 37°C overnight, the cells were treated with: a, vanadium (100 M) for 24 h; b and c, for a dose-response study, the cells were exposed to different concentrations of vanadium as indicated for 24 h; d, for a time course study, the cells were exposed to 100 M vanadium for various times as indicated. Then, the luciferase activity was measured. Each bar indicates the mean and standard deviation of four repeat assay wells. The results are presented as NFAT-dependent transcription activity relative to control. The asterisk indicates a significant increase from control (p Ͻ 0.05). activity at 25 and 100 M (p Ͻ 0.05) (Fig. 1b). These results demonstrate that vanadium is a stimulus for NFAT transactivation. This activation is not limited to a single cell type but appears to be a more generalized reaction to vanadium treatment.
It is well known that, in T cells, transactivation of NFAT is regulated tightly in response to elevations of both intracellular calcium ion (Ca 2ϩ ) and diacylglycerol following activation of phospholipase C (15). Increased intracellular calcium stimulates the activation of calmodulin (15). It is believed that binding of calmodulin to a region near the C terminus of calcineurin displaces the auto-inhibiting domain and exposes the calcineurin active site (15). Activated calcineurin subsequently dephosphorylates the cytoplasmic NFAT proteins, leading to NFAT nuclear translocation (16,48). NFAT then forms a heteromeric transcriptional co-activator complex with AP-1 that co-induces NFAT-dependent transactivation (15). Because our previous data indicated that vanadium exposure also led to transactivation of AP-1 (3), the data shown above can not rule out the involvement of AP-1 in vanadium-induced NFAT transactivation even though the NFAT-luciferase reporter only contains the firefly luciferase under the control of three NFAT binding sites. To test this, we used a chemical inhibitor, SB202190, that can inhibit AP-1 activation by inhibition of AP-1 upstream kinase, p38 kinase. We find that pretreatment of cells with SB202190 does not show any inhibitory effects on vanadate-induced NFAT activation ( Fig. 2A), whereas it markedly inhibits AP-1 activation induced by vanadate (Fig. 2B). These results suggest that vanadium-induced NFAT transactivation in PW cells is only dependent on NFAT, but not AP-1.
Reactive Oxygen Species Are Involved in NFAT Activation by Vanadium-Our previous studies have indicated that reactive oxygen species (ROS) are involved in vanadate-induced biological activities (42). If NFAT activation is responsible for some of the biological effects caused by vanadium, ROS generation may play a role in vanadium-induced NFAT activation. To test this possibility, ROS generation was determined in PW cells exposed to vanadium by both dye staining and ESR. The results show that PW cells alone did not generate any detectable amount of free radicals (Fig. 3A), whereas PW cells exposed to vanadate generated a strong ESR signal (Fig. 3B). The spectrum consists of a 1:2:2:1 quartet with hyperfine splittings of a H ϭ a N ϭ 14.9 G, where a N and a H denote hyperfine splittings of the nitroxyl nitrogens and ␣-hydrogen, respectively. Based on these splittings and the 1:2:2:1 line shape, the spectrum was assigned to the DMPO-OH adduct, which is evidence of ⅐ OH radical generation. Time course studies show that maximum generation of free radical is between 6.3 and 11.2 min after addition of vanadate (Fig. 4). Addition of catalase, a scavenger of H 2 O 2 , inhibited ⅐ OH radical generation (Fig. 3C), indicating that H 2 O 2 was produced in the vanadate-treated cells and involved in ⅐ OH generation. Addition of sodium formate, an ⅐ OH radical scavenger, decreased the signal intensity (Fig. 3D), further supporting the ⅐ OH radical generation. Incubation of the mixture with deferoxamine, a metal chelator, dramatically decreased the signal intensity (Fig. 3E) (increasing percentage of positive cells from 28.1% to 79.6%) and H 2 O 2 (increasing percentage of positive cells from 34.6% to 87.5%) (Fig. 5). To investigate the possible role of ROS in NFAT activation by vanadium, the effects of specific modifiers of ROS on vanadium-induced NFAT activation were determined. The results show that pretreatment of cells with deferoxamine, NAC, or catalase caused inhibition of vanadium-induced NFAT activation (p Ͻ 0.05) (Fig. 6, a, b, and e), whereas addition of NADPH, which promotes ROS generation, or SOD, whose function is to scavenge O 2 . and generate H 2 O 2 , enhanced vanadiuminduced NFAT activation (p Ͻ 0.05) (Fig. 6, c and d). In contrast, treatment of cells with sodium formate did not inhibit vanadium-induced NFAT activation, suggesting that ⅐ OH may not play a significant role in vanadium-induced NFAT activation (p Ͼ 0.05) (Fig. 6f). These data support the hypothesis that H 2 O 2 generation by vanadium is required for its activation of NFAT. We note that the time gap exists between the kinetics of ROS generation and NFAT induction. ROS generation reaches the peak between 6.3 and 11.2 min after exposure of cells to vanadate, whereas the maximum induction of NFAT activity is between 36 and 48 h post vanadate exposure. Because the NFAT activation is measured by using NFAT-luciferase reporter gene, so the time discrepancy not only includes the time for transmitting the signaling from extracellular stimulation to nuclear but also the luciferase gene transcription, translation as well as luciferase protein modifications. This explanation is supported by the observation that the maximum NFAT induction by H 2 O 2 also needs 36 -48 h after H 2 O 2 exposure (data not shown). Synergistic Effect of A23187 and Ionomycin on Vanadiuminduced NFAT Activation-Ionomycin reportedly has a synergistic effect with TPA for NFAT transactivation in T and B cells, whereas ionomycin or TPA alone does not induce NFAT transactivation (14). Our previous studies have demonstrated that A23187 exhibited a synergistic effect with UV radiation for NFAT induction (33). To test the effect of Ca 2ϩ ionophore on vanadium-induced NFAT activation in PW cells, we incubated PW NFAT mass 1 with either A23187 or ionomycin. The results show that both A23187 and ionomycin exhibited a high synergistic augmentation of vanadium-induced NFAT activation (p Ͻ 0.05) (Fig. 7, a and b), whereas administration of A23187 or ionomycin alone did not show induction of NFAT activity in PW cells (Fig. 7, a and b). These synergistic effects appear to be dose-dependent (Fig. 7, c and d). These data suggest that elevation of intracellular calcium in PW cells plays a role in the activation of NFAT in response to vanadium.

Vanadium-induced NFAT Activation Is Dependent on Calcium Mobilization in PW Cells-
To determine the role of calciumdependent pathways in vanadium-induced NFAT activation, we investigated the effect of nifedipine, a calcium channel blocker, on vanadium-induced NFAT activation. Blocking calcium channel activity by pretreatment of PW cells with nifedipine resulted in inhibition of vanadium-induced NFAT activation (p Ͻ 0.05) (Fig. 8). These data suggest that activation of NFAT by vanadate and the synergistic effect of A23187 and ionomycin on vanadium-induced activation of NFAT both occur through calcium-dependent pathways.
Inhibition of Vanadium-induced NFAT Transactivation by Cyclosporin A (CsA)-Previous studies demonstrated that in T cells the major NFAT activation pathway appears to involve a calcium/calmodulin-dependent phosphatase, calcineurin (15,16). To test the role of calcineurin in vanadate-induced NFATdependent transcription activity in PW cells and Cl 41 cells, cyclosporin A (CsA), a widely used pharmacological inhibitor of the phosphatase calcineurin, was used. Pretreatment of cells with CsA resulted in a dramatic inhibition of NFAT transactivation induced by vanadium (p Ͻ 0.05) in both PW (Fig. 9) and Cl 41 cells (date not shown). These data suggest that activation of calcineurin is required for vanadium-induced NFAT activation and that vanadium activates the NFAT transcription ac- tivity in mouse embryo fibroblasts and epidermal cells through the pathway that is similar to that in T cells.

DISCUSSION
The antigen-regulated, cyclosporin A-sensitive nuclear factor of activated T cells (NFAT) is not only expressed in lymphoid cells as once thought but also expressed in other cells and organs, such as the skin and lung (15,32). The stimuli and regulation of NFAT in cells other than lymphoid cells, however, remains unknown. In this report, we investigated the possible involvement of H 2 O 2 in NFAT activation of fibroblasts and epidermal cells in response to vanadium. Exposure of cells to vanadium caused marked increases in NFAT-dependent transcription in a time-and dose-dependent manner. Co-stimulation studies show that A23187 or ionomycin augments the NFAT-mediated transcription synergistically in response to vanadium. Pretreatment of cells with nifedipine or CsA results in impairment of NFAT transactivation induced by vanadium. These results strongly suggest that vanadium is able to induce NFAT activation. Furthermore, scavenging of vanadium-induced H 2 O 2 by NAC or catalase, or the chelation of vanadate by deferoxamine resulted in inhibition of NFAT activation, whereas an increase in H 2 O 2 generation in response to SOD or NADPH enhanced vanadium-induced NFAT activation. These results suggest that vanadium induces NFAT transactivation is dependent on H 2 O 2 generation caused by vanadium.
NFAT isoforms are expressed in different tissues. It has been reported that NFAT 1 and NFAT 2 mRNAs have been detected in brain, heart, skeletal muscle, testis, placenta, pancreas, small intestine, prostate, colon, skin tumors, as well as in lung (15,32). NFAT expression or NFAT-derived transactivation has also been described in several types of nonlymphoid cells, including mast (43), endothetial (44), neuronal (45), vascular smooth muscle (46), and liver-derived Chang cells (47). In this study, we have found that exposure of either mouse embryo fibroblasts (PW cells) or mouse epidermal cells (Cl 41) resulted in marked activation of NFAT-dependent transcription. These results are not only consistent with previous findings that NFAT mRNA is detectable in skin (32) but also for the first time provide the evidence that NFAT is expressed in embryo FIG. 6. Effects of free radical modifiers on NFAT activation by vanadium. PW NFAT mass 1 cells suspended in 10% FBS DMEM were added to each well of 96-well plates and cultured overnight. The cells were pretreated with different free radical modifiers at the concentrations indicated. The cells were then exposed to vanadium (100 M) for 24 h. The NFAT activity was determined by the luciferase activity assay. The results are presented as relative NFAT activity. Each column and bar indicates the mean and S.D. from triplicate assays. The asterisk indicates a significant increase (c and d) and a significant decrease (e) from vanadium alone (p Ͻ 0.05). For the dose-response study, the cells were exposed to vanadium (100 M) plus different concentrations of A23187 (c) or ionomycin (d). The cells were harvested after being cultured for 48 h. The NFAT activity was measured by luciferase activity assay. The results are presented as relative NFAT-dependent transcription activity. Each bar indicates the mean and standard deviation of assays from triplicate wells. The asterisk indicates a significant increase from vanadium alone (p Ͻ 0.05).
fibroblasts, suggesting that NFAT may play some role in embryonic development. The activation of NFAT includes dephosphorylation, nuclear translocation, and an increase in affinity for DNA binding (15). Stimuli that elicit calcium mobilization result in rapid dephosphorylation of NFAT proteins and their translocation to the nucleus. NFAT then binds to the promoter region of its target genes and initiates the transcription of these target genes, in turn leading to translation (15). Therefore, the translocation of NFAT occurs within 3 h (33), whereas its target gene proteins could be detected between 24 and 48 h after cells exposed to stimulus (15). The system used in this study is NFATluciferase reporter cDNA, which includes the firefly luciferase cDNA under the control of NFAT regulatory element (promoter region of mouse IL-2, a typical target gene of NFAT). Thus, the NFAT activation by vanadium appears at 12 h and reaches peak around 36 -48 h after cells exposed to vanadium.
In T cells, transactivation of NFAT is regulated tightly in response to elevations of both intracellular calcium ion (Ca 2ϩ ) and diacylglycerol following activation of phospholipase C (15). Increased intracellular calcium stimulates the activation of calmodulin (15). It is believed that binding of calmodulin to a region near the C terminus of calcineurin displaces the autoinhibiting domain and exposes the calcineurin active site (15). Activated calcineurin subsequently dephosphorylates the cytoplasmic NFAT proteins, leading to NFAT nuclear translocation (15,16,48). NFAT forms a heteromeric transcriptional coactivator complex with AP-1 that co-induces NFAT-dependent transactivation (15). It has also been reported that phosphorylation of NFAT is regulated by several protein kinases, including GSK 3 and JNK 2 (15, 49 -51). Other than lymphocytes, NFAT activation has been demonstrated in only a few cell types, such as vascular smooth muscle cells following induction with platelet-derived growth factor and liver-derived Chang cells following exposure to hepatitis B virus ϫ protein (46,47). The results obtained from the present investigation show that vanadium alone could induce an increase of NFAT activity of more than 10-fold. It appears that vanadium alone is the strongest NFAT inducer reported thus far. Recently, we reported (33) that UV irradiation resulted in activation of NFAT-mediated transcription through a calcium-dependent pathway in mouse epidermal JB6 cells as well as in mouse skin. The present study found that A23187 or ionomycin augmented the NFAT-mediated transcription synergistically in combination with vanadium. Pretreatment of cells with nifedipine or CsA resulted in impairment of NFAT transactivation induced by vanadium. Therefore, the vanadium-induced NFAT transactivation appears to require an increase in free intracellular calcium and calcineurin activation. Vanadium-containing compounds exert potent toxic and carcinogenic effects, such as cell transformation (3)(4)(5). It has been demonstrated that vanadium-mediated generation of reactive oxygen species (ROS) is responsible for most biological effects caused by vanadium (41,42). It is well accepted that extracellular stimuli trigger signals through a cascade of protein-protein interactions (52)(53)(54). It is generally believed that these extracellular stimuli generate and/or require reactive free radicals or derived oxidant species to successfully transmit their signals to the nucleus (53,55). In addition to inducing cellular injury, such as DNA damage and lipid peroxidation, ROS also function as intracellular messengers (2,53,56). Accumulating data suggest a vital role of ROS in mediating cellular responses by various extracellular stimuli (2, 40 -42, 53, 56). It has been reported that generation of H 2 O 2 is required for platelet-derived growth factor signal transduction (57). Involvement of ROS in NFAT activation in T lymphocytes was first demonstrated by the observation that treatment of T cells with dithiocarbamate resulted in inhibition of NFAT activation and NFAT-mediated cytokine gene expression (58,59 8. Inhibition of vanadium-induced NFAT activity by pretreatment of cells with nifedipine. 8 ϫ 10 3 PW NFAT mass 1 cells suspended in 10% FBS DMEM were added to each well of 96-well plates. After being cultured at 37°C overnight, the cells were first treated with 40 M nifedipine for 30 min and sequentially were exposed to vanadium (100 M). After being cultured for 48 h, the cells were harvested, and the NFAT activity was measured by luciferase activity assay. The results are presented as relative NFAT-dependent transcription activity. Each bar indicates the mean and standard deviation of assays from triplicate wells. The asterisk indicates a significant decrease from vanadium alone (p Ͻ 0.05).
FIG. 9. Blocking vanadium-induced NFAT transactivation by cyclosporin A. PW NFAT mass 1 were seeded to each well of 96-well plates and cultured until 90% confluent. The cells were then treated with cyclosporin A at (a) 0.5 M or (b) at the concentration as indicated for 30 min and sequentially were exposed to 100 M vanadium. After being cultured for 48 h, the cells were harvested and the NFAT activity was measured by luciferase activity assay. Each bar indicates the mean and standard deviation of assays from triplicate wells. The asterisk indicates a significant decrease from vanadium alone (p Ͻ 0.05).
um-induced NFAT activation, we speculate that H 2 O 2 may be an initiator for calcium-calcineurin pathway for NFAT activation (Fig. 10). This hypothesis was strongly supported by the previous findings that exposure of cells to H 2 O 2 induces early release of Ca 2ϩ from mitochondria into cytosol, which was mediated by annexin VI translocation and inactivation of plasma membrane Ca 2ϩ -ATPase (60).
NFAT is a transcription factor, which has been reported to play an essential role in IL-2 gene expression (14). Binding sites for NFAT have also been found within the promoter regions of cytokines, including granulocyte macrophage-colonystimulating factor, TNF-␣, IL-1, IL-3, IL-4, IL-5, and IL-8 (15,47,50). Previous studies have indicated that expression of IL-8, TNF-␣, and other cytokines is associated with the initiation and control of effective immune and inflammatory responses and may be related to cancer development (47,50). Therefore, we suggest that NFAT is involved in vanadium-induced inflammation and may subsequently be involved in the carcinogenic effects of vanadium. This idea is supported by previous findings that CsA and FK506, the two most commonly used inhibitors for NFAT activation, exhibit strong anti-tumor promotion activity by specifically only targeting and blocking the activation of a Ca 2ϩ -dependent phosphatase, calcineurin (61,62).
In conclusion, we demonstrate that NFAT is expressed in embryonic cells and vanadium is a strong inducer of NFAT activation. Vanadium induces NFAT activation through CsAsensitive and calcium-dependent signal transduction pathways, which required H 2 O 2 generation caused by vanadium. Transactivation of NFAT in cells other than lymphocytes supports the hypothesis that NFAT activation may play an important role in vanadium-induced biological effects, such as inflammation and tumor promotion, possibly through NFAT-mediated expression of inflammatory cytokines, such as IL-1, IL-2, IL-6, IL-8, and TNF-␣.