Inhibition of G-Protein βγ Signaling Enhances T Cell Receptor-Stimulated Interleukin 2 Transcription in CD4+ T Helper Cells

G-protein-coupled receptor (GPCR) signaling modulates the expression of cytokines that are drug targets for immune disorders. However, although GPCRs are common targets for other diseases, there are few GPCR-based pharmaceuticals for inflammation. The purpose of this study was to determine whether targeting G-protein βγ (Gβγ) complexes could provide a useful new approach for modulating interleukin 2 (IL-2) levels in CD4+ T helper cells. Gallein, a small molecule inhibitor of Gβγ, increased levels of T cell receptor (TCR)-stimulated IL-2 mRNA in primary human naïve and memory CD4+ T helper cells and in Jurkat human CD4+ leukemia T cells. Gβ1 and Gβ2 mRNA accounted for >99% of Gβ mRNA, and small interfering RNA (siRNA)-mediated silencing of Gβ1 but not Gβ2 enhanced TCR-stimulated IL-2 mRNA increases. Blocking Gβγ enhanced TCR-stimulated increases in IL-2 transcription without affecting IL-2 mRNA stability. Blocking Gβγ also enhanced TCR-stimulated increases in nuclear localization of nuclear factor of activated T cells 1 (NFAT1), NFAT transcriptional activity, and levels of intracellular Ca2+. Potentiation of IL-2 transcription required continuous Gβγ inhibition during at least two days of TCR stimulation, suggesting that induction or repression of additional signaling proteins during T cell activation and differentiation might be involved. The potentiation of TCR-stimulated IL-2 transcription that results from blocking Gβγ in CD4+ T helper cells could have applications for autoimmune diseases.


Introduction
G protein-coupled receptor (GPCR) signaling exerts multiple influences on cytokine levels with vast implications for immunodeficiency and autoimmune diseases [1]. However, although GPCRs are fairly common drug targets for neurological and cardiovascular diseases, there are fewer examples in the field of immune disorders. Of the 73 GPCRs thought to have a function in inflammation, only two so far have been successful drug targets for inflammatory disorders, yielding therapeutics for asthma (CysLT-1 receptor) and allergic rhinitis (H1 histamine receptor) [2]. Although chemokine receptors, which regulate the migration of immune cells, have

Materials and Methods Plasmids
For luciferase reporter assays, the human IL-2 promoter from −300 to 0 bp was synthesized by Life Technologies and subcloned as a HindIII cassette into pGL3 (Promega). AP1(1), NF-AT, and NFkB(2) luciferase reporter vectors, which contained transcription factor binding motifs that monitored activation of the respective transcription factors (Affymetrix/Panomics), were also subcloned into pGL3. pRL-TK Renilla (Promega) was used to normalize luciferase activities.
For live cell imaging of NFAT1 and NFAT2, GFP fusion constructs were used. For NFAT1, HA-NFAT1(1-460)-GFP (Addgene plasmid 11107) from the laboratory of Anjana Rao [24], consisting of the amino terminal regulatory domain, but lacking an intact DNA-binding domain of NFAT1, fused to the amino terminus of GFP was used (referred to as NFAT1-GFP). For NFAT2, EGFPC1-huNRATc1EE-WT (Addgene plasmid 24219) from the laboratory of Jerry Crabtree [25], consisting of the amino terminal regulatory domain, but lacking an intact DNA-binding domain of NFAT2, fused to the carboxyl terminus of GFP was used (referred to as GFP-NFAT2). Monomeric Cherry (mCherry) [26] was obtained from Roger Tsien (University of California, San Diego, CA).
For live cell imaging of intracellular Ca 2+ , a fusion of the red intensiometric Ca 2+ indicator (R-GECO1) from the plasmid, CMV-R-GECO1 (Addgene plasmid 32444) from the laboratory of Robert Campbell [27], to monomeric Cerulean (mCerulean) [28] was used. mCerulean (obtained from David Piston, Vanderbilt University, Nashville, TN) was amplified by a PCR reaction that added BamHI sites to each end and then subcloned into BamHI-digested CMV-R-GECO1, resulting in a fusion of mCerulean to the amino terminal end of R-GECO1, referred to as mCerulean-R-GECO1.

Ethics statement and study population
This study was reviewed and approved by the Geisinger Health System Internal Review Board, and all study participants signed informed consent. Peripheral blood was obtained from 30 healthy women 18 to 70 years old who did not have any autoimmune, infectious, or atopic diseases, clinical suspicion of anemia, or treatment with greater than 10 mg of prednisone within 12 hours of the blood draw.

Isolation and culture of human CD4 + T cells and Jurkat T cells
Peripheral blood mononuclear cells were isolated using Ficoll-Paque density gradient centrifugation. CD4 + T cells were isolated by depletion of non-CD4 + T cells using a CD4 + T Cell Isolation Kit II (Miltenyi Biotec). The cells were then separated into naïve and memory CD4 + T cells using a Naïve CD4 + T cell Isolation Kit (Miltenyi Biotec). Purification of the cells was confirmed by labeling samples before and after purification with fluorescently labeled antibodies to either CD4 and CD45RA (to label naïve cells) or CD4 and CD45RO (to label memory cells) and analysis using flow cytometry. 94.3% of the cells in the naïve T cell preparations were CD4 + (SE = 0.7%, ranging from 83.9% to 98.6%) and 83.8% were CD45RA + (SE = 1.4%, ranging from 68.1 to 95.9%). 95.2% of the cells in the memory T cell preparations were CD4 + (SE = 0.4%, ranging from 89.7% to 98%) and 75.0% were CD45RO + (SE = 1.8%, ranging from 55.0 to 88.6%). Cells were plated in 24-well dishes coated with 2.5 mg/mL anti-CD3 antibody (Miltenyi) in RPMI containing 10% FCS, 2.5 mg/mL anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/mL) (R&D Systems). For Type 1 T helper cell (TH1) differentiation, the media also included 20 ng/mL IL-12 and 1 mg/mL anti-IL-4 antibody (R&D Systems). For Type 2 T helper cell (TH2) differentiation, the media also included 20 ng/mL IL-4 and 2 mg/mL anti-IL-12 antibody (R&D Systems). Cells were harvested after three days.
Jurkat T cells (Clone E6-1) were obtained from ATCC and cultured in RPMI containing 10% FCS. For TCR activation, the cells were grown in wells coated with anti-CD3 (2.5 mg/mL) in the presence of soluble anti-CD28 (2.5 mg/mL).
Gallein and fluorescein (TCI America) were used at a final concentration of 15 mM.

Quantitative PCR (qPCR)
RNA was prepared using RNeasy Plus Mini Kits (Qiagen). cDNA was prepared using QuantiTect Reverse Transcription kits (Qiagen). QPCR was performed using TaqMan Gene Expression Assays (Applied Biosystems) and an Applied Biosystems qPCR machine. mRNA expression levels were determined by comparing the C t value of the mRNA of interest to that of the house-keeping gene GAPDH in the same preparation.

IL-2 ELISA
IL-2 secreted into the media by Jurkat cells was quantified using a human IL-2 Quantikine ELISA kit (R&D Systems).

Immunoblots
For Gb 1 and Gb 2 immunoblots, Gb 1 (XAB-00301-1-G) and Gb 2 (XAB-00401-1-G) antibodies from CytoSignal, LLC were used to detect expression in membranes prepared as described [30]. For determination of NFAT1 and NFAT2 expression, total cell lysates were used. NFAT1 antibody (ab2722) and NFAT2 antibody (ab2796) were obtained from Abcam Inc. 7 mg of membrane proteins or total lysates were resolved on NuPAGE 4-12% Bis-Tris gels and transferred to Invitrolon PVDF membranes (Life Technologies). The antigen-antibody complexes were detected using SuperSignal West Pico or Fempto Chemiluminescent Substrate (Pierce Biotechnology, Inc.). Chemiluminescence was imaged using a Fuji LAS-4000 Luminescent Image Analyzer. Bands in the images were quantified using ImageJ software.

Actinomycin D assay
Jurkat cells were nucleofected with Gb 1 siRNA or NT siRNA and stimulated with plate-bound anti-CD3 and soluble anti-CD28 for three days as described above and then treated with 10 mg/mL of Actinomycin D to inhibit transcription. After incubation with Actinomycin D for 0, 5, 10, 20, or 30 minutes, the cells were removed from the wells and RNA was prepared.

Luciferase Assay
Jurkat cells were nucleofected with 2 mg of a luciferase reporter plasmid and 0.1 mg of pRL-TK Renilla and then stimulated or not with plate-bound anti-CD3 (2.5 mg/mL) and soluble anti-CD28 (2.5 mg/mL) in the presence or absence of 15 mM gallein. The Dual-Luciferase Reporter Assay System (Promega) was used according to the manufacturer's instructions and data were collected using a POLARstar Optima plate reader.
Imaging and analysis of NFAT-GFP fusions in live Jurkat cells 4 × 10 6 Jurkat cells were suspended in 0.25 mL of HEPES buffered RPMI media with no serum. Plasmids expressing NFAT1-GFP (10 mg) or GFP-NFAT2 (10 mg) and mCherry (2 mg) were introduced into the cells by electroporation using 250 V and 330 mF capacitance with a BRL Cell-Porator. Cells were immediately transferred to 4 mL of bicarbonate buffered RPMI media containing 5% FCS at 37°C. After 30 min, cells were plated at a density of 3.5 × 10 5 cells per well in 250 mL on Lab-Tek II, 8 well chambered coverslips. For TCR-activating conditions, wells were pre-coated with 2.5 mg/mL anti-CD3 and soluble anti-CD28 was added to a final concentration of 2.5 mg/mL.
Cells were imaged three days after electroporation using a white light spinning disc confocal microscope comprised of an Olympus IX81 inverted microscope, UIS2 60× 1.42 N.A. objective, IX2-DSU spinning disc system, 100 watt mercury arc lamp, Hamamatsu C9100-02 electron multiplier camera, Ludl filter wheels, shutters, and xy stage, under the control of IPLab software (BD Biosciences). Excitation and emission filters for NucBlue (430/24, 470/24), GFP (470/40, 520/40), mCherry (572/35, 632/60), were obtained from Chroma. A stage incubator was used to maintain the cells at 37°C with 5% CO 2 . One hour prior to imaging, 10 mL of Nuc-Blue Live ReadyProbes Reagent (Life Technologies) was added to each well to label the nucleus. Unstimulated cells were transferred to wells that had been coated with 0.1 mg/mL poly-L-lysine for 30 min, and then imaged after 30 min. The criteria for selecting cells for imaging were visible expression of all transfected fluorescent constructs, and for TCR-stimulated cells, clear spreading on the coverslip, indicative of activation. For each condition, cells from at least five independent transfections were imaged.
All image processing was performed using ImageJ software. The background intensity, determined by averaging the intensity of a region of pixels outside the cell, was subtracted from each image. The mCherry image was thresholded to determine the pixels corresponding to the whole cell. The NucBlue image was thresholded to determine the pixels corresponding to the nucleus. The cytoplasmic pixels were determined by subtracting the nuclear pixels from the whole cell pixels. The average intensities of the nuclear and cytoplasmic pixels in the GFP image of NFAT1 or NFAT2 were determined and the nuclear to cytoplasmic intensity ratio was calculated.

Imaging and analysis of intracellular Ca 2+ in live Jurkat cells using mCerulean-R-GECO1
The mCerulean-R-GECO1 plasmid (20 ug) was introduced into Jurkat cells by electroporation and the cells were plated and stimulated with plate-bound anti-CD3 and soluble anti-CD28 as described above for the NFAT-GFP imaging experiments. Stimulated cells were imaged one or three days after electroporation using the Olympus IX81 inverted microscope. Excitation and emission filters for CFP (430/24, 470/24) and mCherry (572/35, 632/60) were obtained from Chroma. For each field imaged, a cell displaying clear spreading on the coverslip was found. As individual cells displayed different amounts of spreading, one plane of focus was not optimal for analysis of all cells in a field. Therefore, starting at the bottom of the cover slip, ten 0.75 uM z-sections were taken of each field. Cells were analyzed if they had intensity in the first z-section, indicating that they were in contact with the coverslip. Unstimulated cells were imaged three days after electroporation and were transferred to untreated wells before imaging. Since they did not spread on the untreated coverslip, a single exposure was taken for each cell and all cells were analyzed. For each condition, cells from at least three independent transfections were imaged.
All image processing was performed using ImageJ software. The background intensity, determined by averaging the intensity of a region of pixels outside the cell, was subtracted from each image. The mCerulean image was thresholded for each z-section to determine the pixels corresponding to the whole cell. For each cell, the z-section that had the largest amount of thresholded mCerulean pixels was used for analysis. The average intensities of the whole cell pixels in the mCerulean and R-GECO1 images were determined and the R-GECO1/mCerulean ratio was calculated.

Statistics
The significance of effects of siRNAs, gallein, and fluorescein on primary CD4 + T cells was determined using the Wilcoxon signed rank test (paired, non-parametric). The significance of effects on Jurkat T cells was determined using the paired T test. Values of p < 0.05 were considered significant (Ã, p < 0.05; ÃÃ, p < 0.01; ÃÃÃ, p < 0.001; ÃÃÃÃ, p < 0.0001).

Results
Gallein, a small molecule inhibitor of Gβγ signaling, enhances TCRstimulated IL-2 mRNA increases in primary human CD4 + T helper cells and Jurkat cells To determine whether Gbg plays a role in modulating TCR-stimulated IL-2 increases, we tested the effect of gallein, a small molecule inhibitor of Gbg signaling [22], in primary human CD4 + T cells grown for three days in conditions that promote either TH1 or TH2 differentiation and in the Jurkat human CD4 + T cell leukemia line, a well-established model system for studying T cell receptor signaling [31]. TH1 cells protect against intracellular organisms, but can also cause inflammation and autoimmune diseases, whereas TH2 cells protect mucosal and epithelial surfaces, but can also cause allergy and asthma [32]. The TCR was stimulated with plate-bound anti-CD3 antibodies and soluble anti-CD28 antibodies for three days. We measured IL-2 mRNA by qPCR, as levels of IL-2 are primarily regulated at the level of transcriptional induction of the IL-2 gene and stability of IL-2 mRNA [33,34]. The levels of IL-2 mRNA were greater in TH1 (Fig. 1A) than in TH2 (Fig. 1B) cells, which is characteristic of these T helper cell subsets [35] and in naïve compared to memory cells (Fig. 1, A and B), which is also consistent with previous observations [36]. Gallein significantly potentiated median TCR-stimulated IL-2 mRNA levels in each of the primary cell lineages tested by 1.6 to 1.9-fold, depending on the T cell subset (Fig. 1, A and B) and mean TCR-stimulated IL-2 mRNA levels in Jurkat cells by 2.4-fold (Fig. 1C). In contrast, fluorescein, a structurally related but inactive compound, had no effect (Fig. 1, A-C). Gallein, but not fluorescein, also increased mean TCRstimulated levels of IL-2 secreted by Jurkat cells (Fig. 1D). The gallein-dependent increase in secreted IL-2 compared to the control (1.78-fold AE 0.16) was similar to the increase in IL-2 mRNA in cells from the same samples (1.87-fold AE 0.10). M119, a Gbg inhibitor that is  [22], and which operates by the same mechanism [37], blocks the interactions of Gbg with downstream effectors, but does not interfere with GPCR-dependent Ga activation or Ga-effector interactions [21]. Our results therefore indicate that Gbg plays a role in inhibiting TCR-stimulated IL-2 increases that is downstream or independent of GPCR-Ga signaling.
siRNA-mediated silencing of Gβ 1 enhances TCR-stimulated IL-2 mRNA levels in primary human CD4 + T helper cells and Jurkat cells There are 5 Gb and 12 Gg subunit genes in the human genome and results from gene targeting approaches have demonstrated unique physiological roles for certain Gbg complexes [38]. As the first step in determining whether specific Gbg complexes are required for modulating TCR-stimulated IL-2 levels in CD4 + T helper cells, we identified the Gb and Gg subunit mRNAs that are expressed. In primary human CD4 + T cells and Jurkat T cells, only a subset of the Gb and Gg subunit mRNAs was expressed. Gb 1 and Gb 2 accounted for >99% of the total Gb subunit mRNAs in primary naïve (si NT in Fig. 2A) and memory (si NT in Fig. 2B) CD4 + T cells, and in Jurkat T cells (si NT in Fig. 2C). Gb 2 mRNA was expressed at a slightly higher level than Gb 1 mRNA in primary cells, whereas Gb 1 mRNA was expressed at a slightly higher level than Gb 2 mRNA in Jurkat cells, but these differences were not statistically significant.
In both primary T cells and Jurkat cells, Gb 1 siRNA selectively reduced the mRNA level of Gb 1 by *75-90% (Fig. 2, A-C). At the protein level in Jurkat cells, Gb 1 siRNA reduced the level of Gb 1 , but not Gb 2 to 26% of the level in the presence of NT siRNA, and Gb 2 siRNA reduced the level of Gb 2 , but not Gb 1 to 26% of the level in the presence of NT siRNA (Fig. 2D).
siRNA directed at Gb 1 potentiated TCR-stimulated IL-2 mRNA increases in Jurkat cells by 2-fold (Fig. 2E). A second Gb 1 siRNA, si b 1 (8), had similar effects as the first one on Gb 1 mRNA levels (Fig. 2F) and TCR-stimulated IL-2 mRNA levels (Fig. 2G). In contrast, siRNA directed at Gb 2 did not enhance TCR-stimulated IL-2 mRNA increases (Fig. 2E). These results indicate that Gb 1 g complexes play a role in inhibiting TCR-stimulated IL-2 increases.
As in Jurkat cells, Gb 1 siRNA enhanced TCR-stimulated increases in IL-2 mRNA in primary human naïve and memory CD4 + T cells grown for three days in conditions promoting differentiation of TH1 (Fig. 3A) and TH2 (Fig. 3B) T helper cell subsets. Again, despite the fact that TCR stimulation caused larger increases in IL-2 mRNA in TH1 cells compared to TH2 cells and in naïve T cells compared to memory T cells, each of the T cell subsets exhibited significant increases in median TCR-stimulated IL-2 mRNA levels (1.2 to 1.5-fold depending on the subset) in response to Gb 1 siRNA.
Of the 12 Gg subunits, the three most predominantly expressed isoforms at the mRNA level in both primary T cells and Jurkat cells were Gg 2 , Gg 5 , and Gg 10 (Fig. 4). Gg 8 was the next most prevalent Gg subunit in stimulated primary T cells and was expressed at the same level as Gg 2 and Gg 10 in naïve TH2 cells (Fig. 4B), but was undetectable in Jurkat cells (Fig. 4C). Also, Gg 4 was induced robustly in Jurkat cells upon stimulation (Fig. 4C), but was undetectable in unstimulated primary T cells (Fig. 4A) and only minimally detectable in stimulated primary T cells (Fig. 4B). These differences in Gg subunit mRNA expression between primary T cells and Jurkat cells may indicate differences in the composition of the Gb 1 g complexes that inhibit TCR-stimulated IL-2 mRNA increases. However, due to lack of effective antibodies and/or amount produced by the TCR-stimulated control. (D) The media was replaced with fresh media containing the same components on the second day, and IL-2 secreted between the second and third days was quantified by ELISA. Data represent the mean AE SE from 6 experiments. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. doi:10.1371/journal.pone.0116575.g001 Inhibition of Gβγ Enhances TCR-Stimulated IL-2 Transcription siRNAs, we have not determined the relative importance of particular Gg subunits for modulating TCR-stimulated IL-2 levels.
Disrupting Gβγ signaling enhances TCR-stimulated activity of a 300 bp IL-2 promoter Disrupting Gbg signaling could enhance TCR-stimulated increases in IL-2 mRNA levels by increasing IL-2 transcription and/or IL-2 mRNA stability. To determine whether inhibition of Gbg signaling increased IL-2 mRNA stability, we measured the half-life of IL-2 mRNA in Jurkat cells stimulated with plate-bound anti-CD3 antibodies and soluble anti-CD28 antibodies for three days and then treated with Actinomycin D to inhibit transcription. Gb 1 siRNA did not increase the stability of IL-2 mRNA (Fig. 5A). In the presence of Gb 1 siRNA, the t 1/2 of IL-2 mRNA (11.91 min, SE = 0.58, N = 5) was the same as in the presence of NT siRNA (11.35 min, SE = 0.56, N = 5).  To test whether blocking Gbg increased IL-2 transcription, the effect of gallein on IL-2 promoter activity was determined using a luciferase reporter plasmid containing a 300 bp region of the IL-2 promoter immediately upstream from the transcription start site, which is sufficient to confer T cell specific inducible transcription of reporter genes [39]. 72 hours of TCR stimulation resulted in a 5-fold increase in luciferase activity in the IL-2 reporter plasmid (IL2/pGL3), but not the empty vector (pGL3) (Fig. 5B). Gallein potentiated this increase by 1.47-fold (p < 0.01) (Fig. 5B). Gallein potentiated TCR-stimulated increases in IL-2 mRNA, as quantified by qPCR using portions of the same samples, by 1.56-fold (p < 0.001), indicating that increased transcription could account fully for the increased IL-2 mRNA levels.  Disrupting Gβγ signaling enhances TCR-stimulated NFAT activity The 300 bp region of the IL-2 promoter contains binding sites for multiple transcription factors, including activator protein-1 (AP-1), NFAT, and nuclear factor kappa-light chainenhancer of activated B cells (NF-kB), that play positive roles in IL-2 transcription [40] and could be targets for inhibition by Gbg (Fig. 6A). To test for regulation of these transcription factors by Gbg, we determined whether gallein affected their TCR-stimulated activity using luciferase reporter vectors that contained transcription factor binding motifs that monitored activation of the respective transcription factors (Fig. 6B). In Jurkat cells stimulated with platebound anti-CD3 antibodies and soluble anti-CD28 antibodies for three days, gallein significantly increased activity of the NFAT reporter by 1.16-fold (Fig. 6, B and C). Gallein did not increase NFAT activity in unstimulated cells (Fig. 6C). The NFAT reporter consisted of the antigen response recognition element-2 (ARRE-2) site in the human IL-2 promoter, which is a composite NFAT/AP-1 site [41]. In contrast, the AP-1 and NF-kB reporters did not exhibit significant changes in TCR-stimulated activity in response to gallein (Fig. 6B). Galleindependent increases in TCR-stimulated IL-2 mRNA in portions of the samples used for the NFAT reporter assay (1.73-fold, p < 0.01) were larger than the increases in NFAT activity. Therefore, while these results suggest that increased NFAT activity resulting from Gbg inhibition contributes to increased IL-2 transcription, this increased NFAT activity is unlikely to account entirely for the increased IL-2 transcription. Additional changes in response to Gbg inhibition are likely to be involved as well.

Blocking Gβγ does not affect NFAT expression
The three NFAT family members that are expressed in T cells are NFAT1, NFAT2, and NFAT4 [42]. NFAT2 and NFAT4 were expressed at higher mRNA levels than NFAT1, and NFAT2 mRNA exhibited the greatest fold increase upon TCR stimulation in Jurkat cells (Fig. 6D). Gallein did not affect the mRNA levels of any of these NFAT family members before or after three days of TCR stimulation (Fig. 6D).
To investigate how inhibition of Gbg signaling potentiates TCR-stimulated NFAT activity, we focused on NFAT1 and NFAT2 for two reasons. First, NFAT4 is expressed primarily in immature thymocytes and nonlymphoid tissues rather than in peripheral T cells [42]. Second, although overexpressed NFAT1, NFAT2, and NFAT4 can all activate transcription at the ARRE-2 site in the IL-2 promoter, NFAT4 binds to this site with lower affinity than NFAT1 and NFAT2 and does not appear to be part of the endogenous NFAT complex on this site, suggesting that the site is not a physiological DNA binding site for NFAT4 [43].
Gallein did not cause detectable changes in the protein expression levels of NFAT1 or NFAT2 before or after 1-3 days of TCR stimulation (Fig. 6E). NFAT1 exhibited minimal induction upon TCR stimulation, as observed previously [44], and gallein did not affect its protein expression level (Fig. 6E). Immunoblots of NFAT2 revealed three bands, which appear to correspond to isoforms A, B, and C, and which exhibited large increases in expression upon TCR stimulation, as previously demonstrated [44,45]. Gallein did not affect the expression levels of any of these isoforms (Fig. 6E).

Disrupting Gβγ signaling enhances TCR-stimulated nuclear localization of NFAT1
Because nuclear translocation of NFAT1 and NFAT2 in response to dephosphorylation by Ca 2+ /calmodulin-activated calcineurin is a major mechanism for NFAT activation [46], we investigated whether the effects of gallein on TCR-stimulated IL-2 transcription and NFAT activity were the result of increases in the nuclear localization of NFAT1 and/or NFAT2. To this end, we tested whether gallein increased the nuclear localization of fusions of GFP to NFAT1 and NFAT2 in Jurkat cells stimulated with plate-bound anti-CD3 and soluble anti-CD28 for three days.
TCR stimulation significantly increased nuclear localization of both NFAT1-GFP and GFP-NFAT2, with a greater effect on GFP-NFAT2 (Fig. 7A), consistent with the previous observation that submaximal Ca 2+ increases cause preferential nuclear localization of NFAT2 compared to NFAT1 [47]. Gallein significantly enhanced TCR-stimulated nuclear localization of NFAT1-GFP by 1.21-fold (Fig. 7A). In contrast, TCR-stimulated nuclear localization of GFP-NFAT2 exhibited a smaller increase in response to gallein that was not significant (Fig. 7A). Gallein did not affect localization of NFAT1-GFP or GFP-NFAT2 significantly in the absence of TCR stimulation. Fig. 7A shows quantitation of NFAT1-GFP and GFP-NFAT2 localization in basal and stimulated cells in the presence or absence of gallein. Fig. 7B shows representative images of NFAT1-GFP in basal and stimulated cells in the presence or absence of gallein.

Disrupting Gβγ signaling enhances TCR-stimulated increases in intracellular Ca 2+
As NFAT1 and NFAT2 translocate to the nucleus as a result of Ca 2+ /calmodulin-dependent activation of calcineurin, we investigated whether inhibiting Gbg with gallein enhanced TCR-stimulated increases in intracellular Ca 2+ . We used a calcium indicator, mCerulean-R-GECO1, consisting of a fusion of the red fluorescent Ca 2+ sensor, R-GECO1 [27], to the fluorescent protein, mCerulean [28], so that R-GECO1 fluorescence could be normalized to the expression level of the plasmid. TCR stimulation for three days resulted in a 1.7-fold increase in intracellular Ca 2+ (Fig. 7C). Inhibiting Gbg with gallein significantly potentiated intracellular Ca 2+ in TCR-stimulated cells by a factor of 1.13-fold (Fig. 7C), which may contribute to the effects on NFAT transcriptional activity (Fig. 6, B and C) and nuclear localization of NFAT1-GFP (Fig. 7, A and B). The modest size of this gallein-induced Ca 2+ increase did not result from saturation of mCerulean-R-GECO1, because in response to stimulation of Jurkat cells with 5 mM ionomycin and 2 mM CaCl 2 , the relative Ca 2+ level detected by the sensor was 1.86-fold higher than that in TCR-stimulated cells treated with gallein. Gallein did not increase intracellular Ca 2+ in the absence of TCR stimulation (Fig. 7C).

The potentiating effect of Gβγ inhibition on IL-2 transcription requires continuous Gβγ inhibition for at least two days of TCR stimulation
Ligation of the TCR and CD28 prompts CD4 + cells to secrete IL-2 rapidly, which further enhances their proliferation and survival [48]. However, the levels of IL-2 decrease as the cells Figure 6. Gallein enhances TCR-stimulated transcriptional activity of NFAT. (A) Major TCR-stimulated pathways leading to IL-2 transcription that could be inhibited by Gβγ. Interactions between the TCR and peptide-major histocompatibility complex (MHC) lead to recruitment of CD4 and its associated kinase, p56-Lck, which phosphorylates tyrosine residues in the cytoplasmic tails of the TCR subunits, leading to recruitment and phosphorylation of the tyrosine kinase, ZAP-70. CD28 costimulation provides an additional signal that is needed for complete T cell activation and regulation of IL-2 production [88]. ZAP-70 and p56-Lck then phosphorylate and activate numerous downstream target proteins, including phospholipase C-γ (PLC-γ), leading to Ras activation, Ca 2+ increases, cytoskeletal rearrangements, and ultimately, activation of transcription factors that bind to the IL-2 promoter and increase IL-2 transcription. (B-C) Gallein increases transcriptional activity of NFAT, but not AP-1 or NFκB. Jurkat cells expressing reporter plasmids for AP-1, NFAT, or NFκB, were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of gallein for three days. (B) Data from TCR-stimulated cells expressing the indicated reporter plasmids represent means AE SE from 7 experiments. ***, p < 0.001. (C) Data from cells expressing empty vector (pGL3) or the NFAT reporter plasmid represent means AE SD from triplicate determinations in a single experiment representative of 7 experiments. (D) Gallein does not affect mRNA levels of NFAT1, NFAT2, or NFAT4. Portions of the Jurkat cells used for the luciferase assays that measured activity at the NFAT ARRE-2 site were used to measure mRNA levels of NFAT1, NFAT2, and NFAT4 by qPCR. Data represent means AE SE from 7 experiments. (E) Gallein does not cause detectable changes in protein levels of NFAT1 or NFAT2. Jurkat cells were stimulated or not with plate-bound anti-CD3 and soluble anti-CD28 in the absence or presence of gallein or fluorescein. The blot of NFAT1 used lysates from cells stimulated for three days and the blot of NFAT2 used lysates from cells stimulated for two days. Similar results were obtained from cells stimulated for one, two, or three days, and in a second independent experiment. doi:10.1371/journal.pone.0116575.g006 Inhibition of Gβγ Enhances TCR-Stimulated IL-2 Transcription start to differentiate [34,39]. Accordingly, we observed an initial peak of IL-2 mRNA within 24 hours of TCR stimulation of Jurkat cells with plate-bound anti-CD3 antibodies and soluble anti-CD28 antibodies that decreased upon further stimulation (Fig. 8A). To determine whether the potentiating effect of Gbg inhibition on TCR-stimulated IL-2 transcription required three days of TCR stimulation, we measured IL-2 mRNA and activity at the 300 bp IL-2 promoter each day over a three-day period of TCR stimulation. Gallein only potentiated TCR-stimulated IL-2 mRNA levels (Fig. 8B) and IL-2 promoter activity (Fig. 8C) after 2-3 days of TCR stimulation. Consistent with these results, IL-2 secreted during the first day of TCR stimulation was not increased by gallein (Fig. 8D), in contrast to IL-2 secreted between the second and third days of TCR stimulation (Fig. 1D). Additionally, intracellular Ca 2+ in cells that had been stimulated at the TCR for one day was not increased by gallein (Fig. 8E), in contrast to cells that had been stimulated at the TCR for three days (Fig. 7C). Although by 24 hours IL-2 mRNA levels had decreased from an initial peak (Fig. 8A), activity at the IL-2 promoter increased linearly over the three days (Fig. 8C), in agreement with previous observations that the decrease in IL-2 mRNA levels after the peak reflects decreased mRNA stability [34].
The requirement of prolonged TCR stimulation for a potentiating effect of Gbg inhibition on IL-2 transcription could indicate that Gbg must be blocked continuously for the duration of TCR stimulation. Alternatively, there could be a specific interval during TCR stimulation in which blocking Gbg has an effect. To distinguish between these possibilities, we stimulated the TCR in Jurkat cells for three days and added gallein at various times, either from the beginning of TCR stimulation or after one or two days of stimulation (Fig. 9A). If gallein was added after the first day of TCR stimulation, it had minimal or no effect on IL-2 mRNA levels. This result indicates that blocking Gbg during the first day of TCR stimulation causes change(s) that are required for the increased levels of IL-2 mRNA that are observed after 2-3 days of TCR stimulation. However, treatment with gallein for just the first day was not sufficient. In cells that were stimulated at the TCR for three days, but treated with gallein for only the first day, after which it was removed, IL-2 mRNA levels were the same as in cells that had not been treated with gallein at all (Fig. 9B). These results suggest that continuous inhibition of Gbg for at least two days of TCR stimulation is required for a potentiating effect on IL-2 transcription.

Discussion
GPCR-G protein signaling modulates a vast array of cellular processes and these signaling pathways are targets for pharmaceuticals that treat cardiovascular, central nervous system, endocrine, and urogenital disorders [49], but, less frequently, immunological disorders [2]. Although Ga subunits have traditionally been thought to be responsible for much of the signaling downstream from GPCRs, the list of functions regulated by Gbg complexes continues to grow [19,20]. The results presented here indicate that Gbg complexes play a role in inhibiting TCRstimulated IL-2 transcription, suggesting that they could be useful drug targets for treating  autoimmune disorders in which modest increases in IL-2 have been shown to be beneficial, such as chronic graft-versus-host disease [8] and hepatitis C virus-induced vasculitis [9].
Blocking Gbg signaling, either with the small molecule Gbg inhibitor, gallein, or with Gb 1 siRNA led to potentiation of TCR-stimulated IL-2 transcription, indicating that Gb 1 g plays an inhibitory role. Further experiments with gallein demonstrated that Gbg inhibits TCRstimulated Ca 2+ increases, nuclear localization of NFAT1, and NFAT activity. These are direct effects of Gb 1 g complexes rather than Ga subunits, because gallein exclusively blocks Gbg complex interactions with effectors. Gallein belongs to a class of Gbg inhibitors that operate using the same reversible noncovalent mechanism [37], of which M119, the first identified member, has been the most extensively studied. The specificity of M119 for blocking Gbg and not Ga was demonstrated by inhibition of fMLP receptor-stimulated Ca 2+ increases mediated by Gbg from G i , but not M3-muscarinic receptor-stimulated Ca 2+ increases mediated by Ga q from G q [21]. Moreover, M119 does not promote dissociation of Ga i from Gbg [21].
Our observation that potentiation of IL-2 transcription required continuous Gbg inhibition during at least two days of TCR stimulation and was obtained only after IL-2 levels had decreased from an initial peak may indicate that Gbg signaling plays a role in the negative feedback mechanisms that result in the transient nature of IL-2 secretion in TCR-stimulated CD4 + T cells [50][51][52][53]. Moreover, the delayed effect of gallein, which contrasts with previously described effects that generally occurred in a matter of minutes to hours [22], may indicate that the effect of Gbg inhibition on TCR-stimulated IL-2 transcription involves induction or repression of signaling proteins during T cell activation and differentiation.
The potentiating effect of Gbg inhibition on TCR-stimulated nuclear localization of NFAT1 may result from enhancement of TCR-mediated increases in intracellular Ca 2+. There is precedent for this effect of Gbg inhibition in that pretreatment of activated primary T lymphocytes with gallein resulted in increased levels of intracellular Ca 2+ upon stimulation with CXCL11 [54]. The mechanism by which Gbg inhibition can enhance increases in intracellular Ca 2+ in T cells remains to be determined, but may involve L-type voltage-dependent Ca 2+ (Ca V 1) channels. Gbg can block activation of Ca V 1 channels [55][56][57], which are expressed in primary human T cells and Jurkat cells, and which are important for Ca 2+ -mediated NFAT translocation to the nucleus and IL-2 production [58,59]. Moreover, gallein has been demonstrated to prevent inhibition of Ca V 1 channels by Gbg [57]. Ca V 1 channels in T cells are activated by the TCR by an unknown mechanism, rather than by T cell depolarization [59]. This requirement of TCR stimulation for Ca V 1 channel activation is consistent with our observation that Gbg inhibition enhances TCR-stimulated IL-2 transcription but has no effect in the absence of TCR stimulation.
The greater magnitude of the effect of Gbg inhibition on TCR-stimulated IL-2 transcription compared to that on TCR-stimulated Ca 2+ increases, and nuclear localization and transcriptional activity of NFAT suggests that modulation of additional pathway(s) that regulate IL-2 transcription is involved. As gallein/M119 does not prevent interaction of Gbg with N-type Ca 2+ channels, inwardly rectifying K + (GIRK) channels, ERK1/2, or the adenylyl cyclase isoforms ACII, IV, and V1 [60], these effectors cannot account for the ability of gallein to enhance TCR-stimulated IL-2 transcription. In contrast, Gbg interaction with and activation of PLCb2/PLCb3, pREX guanine nucleotide exchange factor (specific for Rac), PI3Kg, and G protein-coupled receptor kinase 2 (GRK2) can be inhibited by gallein/M119 [60]. However, current evidence does not support a role for these effectors in mediating Gbg-dependent inhibition of TCR-stimulated IL-2 increases. not increased by gallein after one day of TCR stimulation. Relative Ca 2+ levels were determined using R-GECO-mCerulean as described in Materials and Methods. Data represent the means AE SE from > 330 cells for each condition.
doi:10.1371/journal.pone.0116575.g008 Figure 9. Potentiation of IL-2 mRNA increases requires continuous Gβγ inhibition during prolonged TCR stimulation. (A) Gallein had minimal or no effect on IL-2 mRNA levels when added after the first day of TCR stimulation. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies for three days. Gallein or fluorescein was added from the beginning of TCR stimulation (0 days) or at the indicated times afterwards. IL-2 mRNA levels were determined by qPCR after three days of TCR stimulation. Data represent means AE SE from 5 experiments and were normalized to the value of the untreated control. n. s., not significant; *, p < 0.05; **, p < 0.01. (B) Treatment with gallein for only the first day of a three-day TCR stimulation was not sufficient to potentiate TCR-stimulated IL-2 mRNA levels. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies for three days in the absence of gallein (none), or in the presence of gallein for the full three days or only the first day. In all cases, the media was changed after the first day and replaced with media that contained anti-CD28 and either gallein (three days), or no gallein (none and first day). Data represent the means AE SE from 4 experiments and were normalized to the untreated control. *, p < 0.05.
The above Gbg-regulated effectors appear unlikely to mediate Gbg-dependent inhibition of TCR-stimulated IL-2 increases for the following reasons. Although PLC-g plays an important role in T cell activation downstream of the TCR [61], PLC-b2 and PLC-b3 are important for chemotaxis of lymphocytes but not for TCR-mediated T cell activation [62,63]. Moreover, when the chemokine stromal cell derived factor-1a (SDF-1a) stimulates association of its receptor, CXCR4, with the TCR, TCR-stimulated IL-2 production is enhanced rather than inhibited [64]. Regarding P-Rex1, inhibition of Gbg-mediated stimulation of this exchange factor is unlikely to account for potentiation of TCR-stimulated IL-2 transcription by gallein as thymocytes from mice with Rac1/Rac2 double knockouts exhibit decreased rather than increased TCR-stimulated IL-2 production [65]. Similarly, the following results make it unlikely that inhibiting activation of PI3Kg by Gbg would lead to enhancement of TCR-stimulated IL-2 increases. A PI3Kg selective inhibitor did not affect IL-2 production in response to anti-CD3 and anti-CD28 stimulation of naïve T cells from murine lymph nodes [66], T cells from PI3Kg knockout mice exhibited decreased [67] or no change [68] in IL-2 production in response to anti-CD3 and anti-CD28 stimulation, and CD4 + T cells from PI3Kg kinase-dead knock-in mice produced decreased levels of production of IL-2 in response to anti-CD3 and anti-CD28 stimulation [69]. Finally, there is no simple scenario involving GRK2/3-GPCR regulation that can account for the similar effects of gallein and Gb 1 siRNA. Upon GPCR-G protein activation, Gbg can bind to the PH domains of GRK2/GRK3, causing translocation to the plasma membrane, and GPCR phosphorylation and desensitization [70]. Blocking interaction between Gbg and GRK2/3 with gallein might increase signaling of a GPCR that can enhance TCR-stimulated IL-2 transcription, but Gb 1 siRNA would be predicted to decrease rather than increase signaling of this GPCR. Further studies will be needed to identify the effector protein(s) that mediate Gbg-dependent inhibition of TCR-stimulated IL-2 increases.
There is precedent for transcriptional regulation by Gbg complexes, both stimulatory and inhibitory. These effects generally involved Gbg localized to the nucleus [71][72][73][74][75]. As an example of transcriptional inhibition by Gbg, Gb 1 g 2 co-localized with AP-1 complexes in the nuclei of HEK-293 cells and inhibited AP-1 activity by recruitment of histone deacetylases (HDACs) [74]. Additionally, Gb 1 and Gb 2 interacted with the glucocorticoid receptor and suppressed its transcriptional activity in the nuclei of HCT116 cells [73]. In contrast, as an example of transcription activation by Gbg, Gb 2 g 12 translocated into the nuclei of HEK293 cells upon stimulation of the angiotensin II type 1 receptor and associated with the transcription factor MEF2A, histones H2B and H4, and HDAC5, and depletion of Gb 2 decreased the activity of MEF2A [75]. In addition, Gb 1 g 2 bound to HDAC5 in total cell lysates of rat heart and HEK293A cells and inhibited its transcriptional co-repression activity, leading to activation of MEF2C [76]. Although we observed GPCR-dependent internalization of Gbg from the plasma membrane to vesicles in the cytoplasm in HEK-293 cells that exhibited partial overlap with Rab11-labeled endosomes [77], indicating potential roles for Gbg internal to the plasma membrane, the vast majority localized outside of the nucleus, although we cannot rule out the presence of minor amounts there. Subsequently, GPCR-dependent translocation of Gbg to other endomembranes including the Golgi complex and the endoplasmic reticulum, but not to the nucleus, was also reported [78].
One or more GPCRs could stimulate release of the Gbg that inhibits TCR-stimulated IL-2 transcription. The ligands for these GPCRs would have to be produced by the T cells themselves or be present in the serum, because the cells were cultured in the absence of other cells that could provide ligands in vivo, such as dendritic cells. Of the GPCRs known to inhibit production of IL-2, the A2A-adenosine receptor [12], the m opioid receptor [14], and the CB1 and CB2 cannabinoid receptors [15] appear to achieve this exclusively by increasing cAMP levels. As gallein does not prevent Gbg from interacting with adenylyl cyclase isoforms ACII, ACIV, and ACV1 [60], these receptors are unlikely to be involved, but the Edg-4/LPA2 receptor [17] and the b 2 -adrenergic receptor [13] remain as potential candidates. Alternatively, the TCR or a tyrosine kinase receptor could transactivate a GPCR [79]. Finally, the direct involvement of Gbg in regulating cytokine expression does not necessarily implicate a GPCR [80]. For instance, in the absence of GPCR stimulation, AGS family proteins can activate Gbg [81].
The potentiation of TCR-stimulated IL-2 production that results from Gbg inhibition suggests that Gbg could be a useful drug target for treating autoimmune diseases, as low dose IL-2 therapy has been shown to effectively suppress immune responses in chronic graft-versus-host disease [8] and hepatitis C virus-induced vasculitis [9]. Further experiments using in vivo models of autoimmune diseases would be required to test this hypothesis. Given the involvement of Gbg signaling in so many essential physiological processes [20], it might seem that blocking Gbg systemically would be inadvisable. However, there is precedent for therapeutic efficacy without untoward side effects in animal models of acute inflammation [22], pain [21], and heart failure [23]. Additionally, adoptive transfer of genetically modified T cells, a promising approach for treating cancer and persistent viral infections [82], could potentially be used to provide T cells with disrupted Gbg signaling.
Blocking Gbg in T cells could have the additional benefit for autoimmune diseases of preventing localization of autoreactive T cells at inflammatory sites. For instance, gallein blocks CXCL11 induced migration of activated T lymphocytes [54]. CXCR3, for which CXCL11 is a ligand, is likely to mediate the infiltration of T cells into the synovial tissue of rheumatoid arthritis patients, because this receptor is highly expressed in CD4 + T cells that accumulate in their synovial tissue [83], and accumulation of these T cells in the synovium is associated with expression of CXCR3 ligands by synovial fibroblasts [84,85]. As pharmacological blockade of lymphocyte traffic is effective for treating multiple sclerosis and Crohn's disease [86,87], inhibiting Gbg might have applications for these diseases as well.