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Previous Article | Next Article 
Molecular and Cellular Biology, October 2006, p. 7372-7387, Vol. 26, No. 20
0270-7306/06/$08.00+0 doi:10.1128/MCB.00580-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Department of Molecular Pharmacology,1 Department of Physiology and Biophysics,2 Department of Cell Biology,3 Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461,4 Department of Developmental Biology and Department of Pathology, Howard Hughes Medical Institute, Stanford University Medical School, Stanford, California 943055
Received 3 April 2006/ Returned for modification 16 May 2006/ Accepted 2 August 2006
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ABSTRACT |
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INTRODUCTION |
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Obesity is also correlated with various degrees of inflammation (21, 53). In addition to the proposed role in insulin and glucose homeostasis, a variety of adipocyte-secreted proteins, such as leptin, adiponectin, and resistin, also play a role in the inflammatory response. For example, infiltration of macrophage into adipose tissue is correlated with the level of inflammatory cytokines in the obese state. Thus, signaling cascades and transcription effectors that contribute to immune function can modulate obesity.
The NFAT group of transcription factors was first identified as a critical component in cytokine gene expression upon T-cell activation (7, 19). Subsequent studies demonstrate that NFAT also plays an important role in nonimmune cells. In addition to its established role in immune cells (38, 39, 43, 45, 48, 52, 57), targeted disruption of the calcineurin-regulated NFAT members has further illuminated the role of NFAT in multiple biological processes, including cardiac morphogenesis (5, 8, 34) and neural pathfinding (14). Whether NFAT contributes to obesity, however, has yet to be established.
Recent studies have indicated that NFAT also plays a role in adipocyte differentiation (16, 58, 60, 61). NFAT interacts with transcription factor CCAAT/enhancer binding protein (C/EBP) to form a composite element to regulate the peroxisome proliferator-activated receptor 
2 (PPAR2) gene. Given that NFAT regulates cytokine gene expression in immune cells, NFAT may also modulate adipokine gene expression and contribute to glucose and insulin homeostasis.
The purpose of this study was to examine the role of NFAT in glucose homeostasis and insulin sensitivity. We find that expression of NFATc2 and NFATc4 is induced upon adipogenesis and in obesity. In addition, aged Nfatc2–/– Nfatc4–/– mice exhibit a defect in fat accumulation and remain lean. Nfatc2–/– Nfatc4–/– mice are also resistant to diet-induced obesity. Ablation of NFATc2 and NFATc4 increases insulin sensitivity, in part, by sustained activation of the insulin signaling pathway. Analogous to its role in cytokine gene expression in immune cells, NFAT regulates resistin adipokine gene transcription. Together, these results demonstrate that NFAT contributes to glucose and insulin homeostasis.
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MATERIALS AND METHODS |
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Reagents.
The resistin promoter was amplified from mouse genomic DNA and subcloned into the pGL3-luciferase reporter plasmid using NheI and XhoI restriction sites. Antibody for NFATc1, (sc7294), NFATc2 (sc7296), NFATc3 (sc8321), NFATc4 (sc13036), PPAR (sc7273), C/EBP
(sc61), insulin receptor (IR) (sc711), Akt (sc1618), extracellular signal-regulated kinase (ERK) (sc1647), S6K (sc230), phospho-Akt (P-Akt; sc16646r), and phospho-ERK (P-ERK; sc7383) were obtained from Santa Cruz Biotech, while phospho-Akt (P-Akt) (no. 9271), phospho-S6K (P-S6K) (no. 9204 and no. 9205), AMP-dependent protein kinase (AMPK) (no. 2532), and phospho-AMPK (P-AMPK) (no. 2535) were obtained from Cell Signaling. Antibody for fatty acid binding protein aP2 is from Abcam (Ab654). Monoclonal ß-actin antibody, triglyceride determination kit, and 2-deoxy-D-[1,2-3H]glucose (2-DOG) were purchased from Sigma. Cholesterol and free fatty acid (FFA) determination kits were obtained from WAKO and Roche, respectively. Phospho-Tyr monoclonal antibody 4G-10 and anti-acetyl histone H3 (no. 06-599) were obtained from Upstate Biotech. The adipokine level in serum was determined by using the Lincoplex assay kit. The level of adiponectin and insulin in serum was determined by radioimmunoassay (LINCO).
Cell culture. 3T3/L1, HepG2, and COS cells were cultured in Dulbecco modified Eagle medium. BHK cells were cultured in minimal essential medium. All media were supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml) (Invitrogen). Cells were transfected by using Lipofectamine (Invitrogen). 3T3/L1 cells were differentiated into lipid-laden adipocytes as described previously (60) using an insulin (5 µg/ml), dexamethasone (1 µM), and phosphodiesterase inhibitor isobutylmethylxanthine (0.5 mM) cocktail. Primary hepatocytes were isolated by collagenase digestion, cultured in Dulbecco modified Eagle medium, and used within 48 h after isolation (36). Thioglycolate-elicited peritoneal macrophages were cultured in RPMI 1640 and stimulated within 24 h after isolation.
Metabolic analyses. A bolus of glucose (2.5 g/kg of body weight) or insulin (0.75 unit/kg) was administered orally or by intraperitoneal injection, respectively, into mice. Serum glucose was monitored from the tail vein using a glucometer (TrueTrack Smart System) at various times. Insulin collected at the various times was measured by radioimmunoassay. To determine oxygen consumption (VO2), carbon dioxide production (VCO2), heat release, and locomotor activity, mice were individually housed in respiratory chambers and were allowed to acclimate to the respiratory chambers for 1 day before the gas exchange measurements began (37). Data on gas exchanges were recorded for 2 to 3 days. Indirect calorimetry was performed with a computer-controlled open-circuit calorimetry system (Oxymax; Columbus Instruments) composed of four respiratory chambers. Oxygen consumption, CO2 production, heat release, and locomotor activity were measured for each mouse at 15- or 20-min intervals and presented.
Signaling analysis. Tissues from insulin-injected mice were collected at the times indicated. Tissue extracts prepared were immunoprecipitated with insulin receptor antibody and probed with the phospho-Tyr antibody 4G-10 to assay insulin receptor activation. Activation of insulin receptor downstream signaling components, such as the Akt protein kinase and ribosomal S6K protein kinase, was determined by phospho-specific antibodies. Activation of AMPK in epididymal fat in vivo was determined after overnight fasting. Activation of AMPK in vitro was elicited by administration of AICAR (500 µM) or sorbitol (500 mM) and assayed by using phospho-specific antibodies.
Gel mobility shift assays.
Nuclear extracts were prepared from cultured cells as described previously (60). Double-stranded oligonucleotides for gel mobility shift assays were labeled with [-32P]dCTP. Sequences for the resistin NFAT elements are as follows: –700 bp, 5'-TGTTGAGAAAGAGGGATTTCCAAAGGGACA-3'; –2,000 bp, 5'-GATTTTATGATAATATTTCCATAACTTTCCTTT-3'). The binding reactions were carried out at room temperature in gel shift buffer [1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES (pH 7.9), 50 mM NaCl, 15 mM ß-mercaptoethanol, 10% glycerol, 0.1 mg/ml bovine serum albumin, and 1 mg/ml poly(dI:dC)] for 30 min. The protein:DNA complexes were separated in 5% nondenaturing polyacrylamide gels in Tris-glycine-EDTA buffer (25 mM Tris, 200 mM glycine, and 1 mM EDTA) and were visualized by autoradiography.
Chromatin immunoprecipitations. Nuclear factors that were associated with chromatin in differentiated and undifferentiated 3T3/L1 cells were cross-linked to DNA using formaldehyde (1%). The Epididymal fat depot of control mice was stimulated or not with insulin for 60 min, isolated, and minced, and nuclear factors were cross-linked to DNA using 1% formaldehyde. Cross-linked chromatin was sheared by sonication, and isolated cell lysate was immunoprecipitated using NFAT or isotype-matching immunoglobulin G (IgG) antibodies. After reversed cross-linking and proteinase K digestion, DNA present in the immunoprecipitated chromatin was isolated and PCR was performed to examine the presence of the resistin gene promoter (5'-TGTCCCTTTGGAAATCCCTCTTTC-3' and 5'-TGCTGAAGAGGAAAGAGACAAATCTT-GCAC-3'). The presence of the glyceraldehyde phosphate-3-dehydrogenase (GAPDH) promoter (5'-GGCTCTCTGCTCCTCCCTGTTCC-3' and 5'-TCAATGAAGGGGTCGTTGATGGC-3') was also examined.
Semiquantitative RT-PCR.
Total RNA was isolated from epididymal fat using TRIZOL reagents (Invitrogen). Isolated RNA (1 µg) was reverse transcribed with Superscript II reverse transcriptase (Invitrogen), and cDNA prepared was amplified by PCR, separated, and visualized by agarose gel electrophoresis. At least two amplifications with different cycles were performed. Primers for PCR amplification are the following: PPAR, 5'-CACAGGCCGAGAAGGAGAAGC-3' and 5'-AGGGAGGCCAGCATCGTGTAG-3'; NFATc1, 5'-GGGTCAGTGTGACCGAAGAT-3' and 5'-GGAAGTCAGAAGTGGGTGGA-3'; NFATc2, 5'-CTGCTCATTATTCCCCCAGA-3' and 5'-GCATCCATGAGAACAGCAGA-3'; NFATc3, 5'-TGGATCTCAGTATCCTTTAA-3' and 5'-CACACGAAATACAAGTCGGA-3'; NFATc4, 5'-ACCCTCCGGTACAGAGGACT-3' and 5'-GGCTGCCCTCAGTCTCATAG-3'; GAPDH, 5'-CTGACGTGCCGCCTGGAGAAA-3' and 5'-TTGGGGGCCGAGTTGGGATAG-3'.
Glucose uptake assays. In vivo glucose uptake in various tissues were determined during a glucose tolerance test (3). In brief, overnight-fasted mice were injected intraperitoneally with a glucose solution (2.5g/kg) containing 2-DOG (0.2 µCi/g). After 60 min, mice were sacrificed and tissues harvested were homogenized in water. Ice-cold perchloric acid (7%) was added to the homogenate (1:1 [vol:vol]) and centrifuged to remove precipitates. The homogenate was further neutralized with 2.2 mol/liter of KHCO3 and centrifuged, and the 3H remaining in the supernatant was determined by a scintillation counter. The amount of radioactivity was normalized to the total protein concentration and presented.
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RESULTS |
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and C/EBP during adipocyte differentiation was used as a control. Expression of ß-actin was used as a loading control. These data indicate that expression of NFATc2 and NFATc4 is induced upon adipocyte differentiation.
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Recent gene targeting studies have revealed important roles of NFAT members in immune and nonimmune systems. For example, disruption of the NFATc1 gene causes early embryonic lethality that is associated with impaired development of heart valves and septa (8, 44). Mice deficient in expression of the NFATc2 and/or NFATc3 gene are viable and appear to have dysregulated adaptive immune responses (17, 52, 57). Although disruption of the NFATc4 gene has been reported, no readily obvious phenotypes are detected (12). NFATc4 is primarily expressed in nonimmune tissues (e.g., muscle, fat, brain) (18), and the lack of a phenotype for NFATc4 null mice suggests that other NFAT members are likely compensating for the loss. Recent studies confirmed the redundancy of the NFATc3 and NFATc4 genes, and the combined null alleles cause embryonic lethality due to vasculature defects (4, 12). Since the expression of NFATc2 and NFATc4 was induced upon adipocyte differentiation and in obese mice, combined disruption of these NFAT members may affect glucose and insulin homeostasis.
We first determined the expression level of NFAT in adipose tissues of Nfatc2–/– Nfatc4–/– mice to ascertain whether there would be compensation by the remaining NFATc1 and NFATc3 members (Fig. 1D and E). Expression of NFATc2 and NFATc4 was only found in Nfatc2–/+ Nfatc4–/+ control mice. The expression level of NFATc1 and NFATc3 was similar in Nfatc2–/+ Nfatc4–/+ control mice and Nfatc2–/– Nfatc4–/– mice. The expression level of adipocyte markers, including PPAR, C/EBP, and fatty acid binding protein aP2, in Nfatc2–/+ Nfatc4–/+ control mice and Nfatc2–/– Nfatc4–/– mice was also similar. The lack of compensation by NFATc1 or NFATc3 was corroborated by our previous finding that similar levels of NFATc1 and NFATc3 were found in primary fibroblasts prepared from Nfatc2–/– Nfatc4–/– mice (61). Together, these data indicate that Nfatc2–/– Nfatc4–/– mice provide an in vivo model for examining the role of NFAT in glucose homeostasis and insulin sensitivity.
Nfatc2–/– Nfatc4–/– mice are resistant to high-fat-diet-induced obesity. Next, we investigated the physiological consequence of targeted disruption of NFATc2 and NFATc4. Age-matched Nfatc2–/– Nfatc4–/– mice and Nfatc2–/+ Nfatc4–/+ control mice were fed ad libitum with regular chow or a high-fat diet for 20 weeks. Control mice fed a high-fat diet exhibited an increase in body weight gain compared to their littermates fed with regular chow (Fig. 2A). Nfatc2–/– Nfatc4–/– mice, however, exhibited similar body weight gains in both diets. The food intake of Nfatc2–/– Nfatc4–/– mice and that of control mice were similar (Fig. 2B). Increased body weight in control mice was, in part, due to increased adipose content in both white and brown adipose depots, whereas Nfatc2–/– Nfatc4–/– mice exhibited only modest changes (Fig. 2C). Sizes of liver, spleen, heart, and kidney, however, were similar in Nfatc2–/– Nfatc4–/– mice and control mice (Fig. 2D). These data demonstrate that Nfatc2–/– Nfatc4–/– mice are resistant to high-fat-diet-induced obesity.
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Altered metabolic parameters in Nfatc2–/– Nfatc4–/– mice. After overnight fasting, the levels of glucose and insulin for Nfatc2–/– Nfatc4–/– mice were lower than those for control mice (Fig. 4A and B). Control mice fed with a high-fat diet exhibited an increased level of glucose and insulin at the fasting state. Nfatc2–/– Nfatc4–/– mice, however, showed minimal changes. The levels of cholesterol, triglyceride, and FFA, however, were affected to a similar extent in both Nfatc2–/– Nfatc4–/– and control mice fed with normal chow (Fig. 4C to E). These data suggest that Nfatc2–/– Nfatc4–/– mice may have altered metabolic parameters.
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Increased glucose uptake in Nfatc2–/– Nfatc4–/– mice. We also performed glucose uptake assays to ascertain the increase in glucose handling in Nfatc2–/– Nfatc4–/– mice (Fig. 7). 2-DOG was used as a nonhydrolyzable tracer and coinjected with a glucose solution during the glucose tolerance test to determine glucose uptake in vivo. In addition to that in skeletal muscle, the extent of glucose uptake in the heart, liver, lung, and spleen was also determined. The amount of 2-DOG accumulated in the skeletal muscle of Nfatc2–/– Nfatc4–/– mice was significantly elevated over that for control mice (Fig. 7A). The amount of 2-DOG accumulated in the heart, liver, lung, or spleen, however, was similar (Fig. 7B to 7E). These data demonstrate that heightened glucose handling in Nfatc2–/– Nfatc4–/– mice can be attributed, in part, to the increased glucose uptake in skeletal muscle.
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Recruitment of NFAT to the resistin loci suggested that NFAT might regulate resistin gene transcription. We tested this hypothesis by examining expression of resistin mRNA using the immunosuppressant drug cyclosporine A, which inhibits calcineurin phosphatase and blocks NFAT activation. Expression of resistin mRNA in adipocytes was reduced upon cyclosporine A treatment (Fig. 12C). In addition, luciferase reporter assays indicated that the resistin promoter was regulated upon NFAT activation (Fig. 12D). Expression of constitutive nuclear NFAT or constitutive active calcineurin increased resistin promoter activity. Importantly, oligonucleotides encoding NFAT binding elements from the resistin promoter (at –700 and –2,000 bp) bound NFAT in gel mobility shift assays (Fig. 12E). Specificity of the NFAT:DNA complex was assessed by supershift analysis using antibody against NFAT and competition assays with excess amounts of unlabeled NFAT binding oligonucleotides. Together, these data demonstrate that NFAT directly regulates resistin gene transcription.
Serum of Nfatc2–/– Nfatc4–/– mice elicits increased activation in insulin signaling. Changes in the adipokine profile in Nfatc2–/– Nfatc4–/– mice, such as reduced resistin expression, might provide a cross talk mechanism for modulation of insulin signaling. We asked whether serum isolated from Nfatc2–/– Nfatc4–/– mice would confer increased activation of the insulin signaling cascade in HepG2 cells. Serum from Nfatc2–/– Nfatc4–/– mice potentiated Akt and S6K activation compared to control serum (Fig. 13A). ERK mitogen-activated kinase, however, was activated to similar extents by control serum and Nfatc2–/– Nfatc4–/– serum. Similar expression levels of Akt, S6K, ERK, and ß-actin were used as controls. These data indicate that serum from Nfatc2–/– Nfatc4–/– mice confers sustained activation of insulin signaling.
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DISCUSSION |
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Obesity has been correlated with various degrees of inflammation. Unlike the intense, acute response upon bacterial infection, obesity is associated with a sustained, low-grade inflammatory stress. We have recently demonstrated a role of NFATc2 in response to inflammatory cytokines, such as interleukin-1 (IL-1) or tumor necrosis factor alpha, in hepatocytes (59). Specifically, expression of NFATc2, but not the other three NFAT isoforms, is up-regulated by inflammatory cytokines. Thus, NFAT may be a critical contributor in inflammatory stress.
Mechanistically, induction of NFATc2 by IL-1 is mediated by transcription factor C/EBP (59). Members of the C/EBP family play a critical role in acute-phase response and in adipocyte differentiation and in obesity (40, 42, 49, 51). Thus, it is tempting to speculate that similar C/EBP-mediated regulation is responsible for NFATc2 induction upon adipocyte differentiation and in the obese state.
Induction of NFATc4, however, may be C/EBP independent. Indeed, recent studies indicate that, similar to NFATc1, induction of NFATc4 is NFAT dependent (2). It is plausible that NFATc2, which is induced at early adipocyte differentiation, may promote expression of NFATc4 to sustain NFAT activation. Hence, in a manner analogous to the induction of C/EBP by other C/EBP members (C/EBPß and C/EBP
), sustained expression of NFAT (NFATc2 and NFATc4) is required during adipocyte differentiation.
Previously, we have demonstrated that NFAT regulates PPAR2 gene transcription and thus adipocyte differentiation (58-61). PPAR is proposed as the master regulator of adipogenesis (10, 50, 54). Here, we demonstrate that ablation of NFATc2 and NFATc4 leads to reduced adiposity, supporting a positive contribution of NFAT in adipogenesis. The level of PPAR2 in Nfatc2–/– Nfatc4–/– mice, however, is similar. Given the critical role of PPAR2 in adipocyte differentiation, the remaining NFATc1 and NFATc3 may be sufficient to mediate its transcription. Indeed, we have identified a group of targets that are regulated by NFAT in a dose-dependent manner (unpublished data). Since a low level of NFAT activation is sufficient for gene transcription, we surmise that these dose-dependently regulated targets are likely essential genes. Possibly, PPAR, a master regulator in adipogenesis, may also be dose-dependently regulated by NFAT.
Role of NFAT in adipokine and insulin gene transcription. In this report, we have demonstrated that compound disruption of NFATc2 and NFATc4 alters the adipokine profile. In a manner analogous to its role in cytokine expression in immune cells, NFAT regulates resistin adipokine gene transcription. Previous studies have established that targeted disruption of resistin increases insulin sensitivity (55, 56). Hence, NFAT may contribute to glucose and insulin homeostasis, in part, by regulating resistin gene expression.
In addition to the resistin gene, NFAT may also regulate other adipokine gene transcription, such as that for adiponectin. In contrast to the case with resistin, expression of adiponectin is positively correlated with insulin sensitivity. Since ablation of NFATc2 and NFATc4 increases insulin sensitivity, it is likely that NFAT may act as a negative regulator and modulate adiponectin gene transcription. Indeed, the fact that the level of adiponectin for Nfatc2–/– Nfatc4–/– mice is similar to that for control mice, albeit with reduced adiposity in Nfatc2–/– Nfatc4–/– mice, supports this notion. Notably, recent studies demonstrated that NFATc4 and ATF3 inhibit adiponectin gene transcription (24). Future differential analysis of Nfatc2–/– Nfatc4–/– mice and control mice to identify additional adipokines is warranted.
How does NFAT contribute to both positive regulation for the resistin gene and negative regulation for the adiponectin gene? The NFAT regulatory element is composed of cytoplasmic (NFATc) and nuclear (NFATn) (reviewed in references 6, 7, and 19). NFATc was identified as calcineurin-regulated NFAT proteins (NFATc1 to NFATc4). NFATn of the IL-2 gene was identified as the AP1 (Fos plus cJun) complex (22). Optimal NFAT-mediated IL-2 gene transcription requires cooperation of NFATc and NFATn. Distinct NFATn has been identified in different NFAT targets. For example, we have demonstrated that NFAT cooperates with C/EBP to mediated PPAR gene transcription (58). It is possible that a differential NFAT partner influences activation versus repression.
Interestingly, many of the identified NFAT partners are in the bZIP group of transcription factors, such as cJun, Fos, C/EBP, ATF, and Maf. The bZIP group of transcription factors has many members within each subgroup. In addition, members of each subgroup exhibit multiple isoforms generated by alternatively splicing and/or translational initiation. For example, the C/EBP group of bZIP proteins includes C/EBP, C/EBPß, C/EBP, and C/EBP. In the C/EBP subgroup, different variants have been identified (e.g., LIP and LAP proteins) for positive or negative regulation of gene transcription. Thus, distinct bZIP members/isoforms may influence activation versus repression in NFAT-mediated gene transcription.
In response to an increase in the glucose level, pancreatic beta cells secrete insulin to maintain glucose homeostasis by promoting glucose uptake and by inhibiting glucose output from the liver. In vitro studies have demonstrated that the NFAT signaling pathway cooperates with members of the Maf transcription factor family to regulate insulin promoter activity upon acute glucose stimulation (29, 30). Our in vivo findings support the importance of the NFAT transcription factor in insulin gene regulation. Nfatc2–/– Nfatc4–/– mice exhibit reduced insulin levels at the fasting state. In addition, the Nfatc2–/– Nfatc4–/– mice are glucose and insulin hypersensitive. Insulin hypersensitivity of the Nfatc2–/– Nfatc4–/– mice may be a compensatory response due to the lack of insulin production and subsequent secretion. Alternatively, reduced insulin production/secretion is a consequence of the increased insulin sensitivity of the Nfatc2–/– Nfatc4–/– mice, which may arise from changes in adipokine signaling (such as resistin and adiponectin) and insulin receptor signaling pathway activation. Since a non-cell-autonomous mechanism is suggested for the increased insulin sensitivity in Nfatc2–/– Nfatc4–/– mice, further understanding of the cross talk between adipokines and their role in insulin activation will provide new insights on the treatment of obesity and diabetes.
Conclusions. In conclusion, we have demonstrated that NFAT contributes to insulin and glucose homeostasis. NFAT also regulates resistin adipokine gene transcription. These results expand the repertoire of NFAT function to metabolic pathogenesis and adipokine gene transcription.
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ACKNOWLEDGMENTS |
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H.Y.S. is a trainee sponsored by 5T32 GM07491. This research was supported, in part, by grants from the National Institutes of Health (C.-W.C., G.R.C., L.A.J., L.R., and P.E.S.), Howard Hughes Medical Institute (G.R.C.), American Diabetes Association (C.-W.C. and P.E.S.), and American Heart Association (C.-W.C.).
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FOOTNOTES |
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Published ahead of print on 14 August 2006.
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15 mice per group) were fed ad libitum with regular chow (10% fat content) or a high-fat diet (59% fat content). Body weight was measured once a week for 20 weeks (panel A). Food intake (panel B) was measured for 12-week-old mice, and weights of various adipose depots (panel C) and tissue organs (panel D) harvested from 24-week-old mice were also shown. Epi, epididymal fat; SC, subcutaneous fat; PR, perirenal fat; BAT, brown adipose tissue.
Cn; filled bars), on the resistin promoter (–1 to –2000) and promoterless pGL3 plasmid was determined (D). Luciferase activity was normalized to ß-galactosidase activity and presented. Resistin loci investigated by chromatin immunoprecipitation were illustrated as a hatched box, and filled triangles represent NFAT binding elements on the resistin promoter (D). Formation of NFAT-DNA complex at the –700-bp and –2,000-bp NFAT binding elements of the resistin promoter was assessed by gel mobility shift assays (E). Specificity of the NFAT:DNA complex was assessed by supershift analysis using antibody against NFAT (see asterisk for supershifted complex) and competition using excess amounts of unlabeled NFAT oligonucleotides.