The cellular effects of the second messenger cAMP are often dependent on the context of concentration changes. For instance, cAMP stimulates proliferation in certain cell types whereas it inhibits proliferation in others [1, 2], which can be determined by the expression of other signaling components [3, 4]. A-kinase anchoring proteins (AKAPs)  and cAMP-degrading phosphodiesterases (PDEs)  can determine the physiological effects of cAMP by regulating the spatial and temporal organization of cAMP pathway components. Differential effects of cAMP can result from the selective involvement of protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac) [7, 8], which in turn can be determined by the intensity and localization of upstream signals . Given this complexity of cAMP regulation and effects, it is not surprising that the role of cAMP in regulating T cell activation and function has been controversial. cAMP is generally known as an immunosuppressant, but it is also required for generating optimal immune responses.
On one hand, studies utilizing agonists and antagonists of GsPCRs, cAMP analogs, and cholera toxin have demonstrated an inhibitory role of cAMP on T cells. For instance, numerous studies of GsPCRs such as the A2AR[10, 11, 12, 13], PGE2 receptors [14, 15], and vasoactive intestinal peptide (VIP) receptors [16, 17] have demonstrated inhibition of TCR-stimulated production of IL-2, a growth factor for effector and regulatory T cells that has been used to augment immune responses to treat cancer  and persistent viral infections , and, at lower doses, to suppress immune responses in chronic graft-versus-host disease  and hepatitis C virus-induced vasculitis . Moreover, treatment of cultures of human T lymphocytes and monocytes with forskolin to activate adenylyl cyclase, cAMP phosphodiesterase (PDE) inhibitors, or a cell-permeable cAMP analog inhibited phytohemagglutinin (PHA)-stimulated IL-2 production . Additionally, treatment of proliferating T lymphocytes with cAMP analogs inhibited cell replication  and led to phosphorylation and activation of Csk, the most proximal PKA substrate . Overexpression of Csk resulted in decreased levels of IL-2 in Jurkat T cells activated by anti-CD3 antibodies and phorbol 12-myristate 13-acetate (PMA) . Furthermore, treatment of Jurkat cells with cholera toxin, which constitutively activates Gαs, inhibited TCR-stimulated increases in inositol trisphosphate (IP3) and Ca2+ .
On the other hand, there is precedent for cAMP playing a positive role in T cell function. Mice that lacked Gαs had reduced cAMP levels, decreased Ca2+ influx, and impaired TH1 and TH17 differentiation , and T cells from mice that lacked the AC7 isoform of adenylyl cyclase were defective in T cell help and memory function . Moreover, the EP2 and EP4 receptors for PGE2 facilitated TH17 expansion by means of the cAMP/PKA pathway , and Gαs activation by cholera toxin induced TH17 cells and protected against inhalation anthrax . Additionally, treatment of mouse spleen cell cultures with low concentrations of dibutyryl cAMP increased humoral immune responses and enhanced PMA/ionomycin-stimulated lymphoproliferation, whereas incubation of the cells with ddA decreased both of these responses in parallel with decreasing basal levels of cAMP . Furthermore, transient adhesion-dependent cAMP increases were stimulatory to TCR signaling, although sustained increases in response to forskolin were inhibitory . The amplitude and duration of cAMP increases may also determine the effect on TCR-stimulated IL-2 synthesis. For instance, antigen stimulation of a murine T cell line produced a transient rise in cAMP that correlated with T cell proliferation and IL-2 production . Moreover, a study in Jurkat T cells suggested that sustained increases in cAMP were required to inhibit PHA-stimulated IL-2 production whereas smaller and transient cAMP increases were not sufficient for inhibition and sometimes even caused increases in IL-2 .
Prior studies suggest that the context in which cAMP levels are increased can determine the effect on T cell function. For instance, VIP receptors can inhibit production of IL-2 in T cells stimulated by the TCR or ConA, but not by PMA and the Ca2+ ionophore, A23187 [16, 17]. Similarly, although forskolin and a cAMP analog inhibited IL-2 production by T cells stimulated by PMA/A23187, the EC50 was about 10-fold higher than that in T cells activated by PHA . Additionally, dibutyryl cAMP augmented synergistic stimulation of DNA synthesis in guinea pig lymphocytes by diacylglycerol and low concentrations of A23187 while having an inhibitory effect in the presence of higher concentrations of the ionophore .
Given the complexities of the effects of cAMP on immune function in general and IL-2 production in particular, cAMP levels and effects are likely to be controlled by multiple inputs that are integrated according to the cellular context. As a step towards elucidating how the nature of the upstream activation of Gαs and adenylyl cyclase might influence their effect on TCR-stimulated IL-2 mRNA levels, the purpose of the current study was to compare the effects of blocking GsPCR-mediated Gs activation versus inhibiting cAMP synthesis at the level of Gαs or adenylyl cyclase. We found that the former resulted in increased TCR-stimulated IL-2 mRNA levels in contrast to the latter, which caused decreases. Moreover, cAMP increases stimulated by the TCR were inhibited by Gαs siRNA, but not by a dominant negative Gαs construct, GαsDN3, consistent with the conclusion that the TCR stimulates cAMP synthesis via Gαs, but not a GsPCR. Taken together, these results suggest that the source and context of activated Gαs and cAMP determine whether they increase or decrease levels of TCR-stimulated IL-2 mRNA.
GαsDN3 was produced as described , where it was referred to as αs(α3β5/G226A/A366S). GαsDN3-CFP was produced by subcloning an EcoRI fragment from GαsDN3 containing the α3β5, G226A, and A366S mutations in place of the corresponding fragment in Gαs-CFP . Gαs-YFP was produced as described for Gαs-CFP using enhanced YFP containing a substitution of Met for Gln-69 instead of enhanced CFP. All Gαs subunit constructs contain mutations that encode the EE epitope as described . YFP-N-β1 and YFP-C-γ7 were produced as described . The human HA-tagged β2AR cDNA was kindly provided by Brian Kobilka (Stanford University, Stanford, CA). β2AR-GFP was produced as described . mRFP-Mem was produced as described . For luciferase reporter assays, a 1 kB sequence encoding the human IL-2 promoter from -950 to +48 bp from Panomics/Affymetrix was subcloned into pGL3 (Promega). pRL-TK Renilla (Promega) was used to normalize luciferase activities. Subcloning and mutagenesis procedures were verified by restriction enzyme analysis and DNA sequencing.
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 20 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 hour of the blood draw.
Isolation and culture of human CD4+ T cells and Jurkat T cells
Peripheral blood mononuclear cells (PBMCs) 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. 93.5% of the cells in the naïve T cell preparations were CD4+ (SE = 0.8%, ranging from 83.9% to 98.2%) and 84.3% were CD45RA+ (SE = 1.6%, ranging from 68.1% to 94.2%). 94.8% of the cells in the memory T cell preparations were CD4+ (SE = 0.4%, ranging from 89.7% to 97.4%) and 74.1% were CD45RO+ (SE = 2.2%, ranging from 55.0% to 87.3%). Cells were plated at a density of 2–9 × 106 cells/ml (depending on yield) in 24-well dishes coated with 2.5 µg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 µg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems). For TH1 differentiation, the media also included 20 ng/ml IL-12 and 1 µg/ml anti-IL-4 antibody (R&D Systems). For TH2 differentiation, the media also included 20 ng/ml IL-4 and 2 µg/ml anti-IL-12 antibody (R&D Systems). Cells were harvested after 3 days.
Jurkat T cells (Clone E6-1) were obtained from ATCC and cultured in RPMI containing 10% fetal bovine serum. For TCR activation, the cells were grown in wells coated with anti-CD3 (2.5 µg/ml) in the presence of soluble anti-CD28 (2.5 µg/ml).
ZM-241385, ddA, siRNA, and plasmid treatments
10 µM ZM-241385 and 150 µM ddA were added when the T cells were placed in activating/differentiating media.
siRNAs were produced by Dharmacon. The sequence of Gαs siRNA, CGAUGUGACUGCCAUCAUC, was from . The non-targeting (NT) siRNA used was ON-TARGETplus Non-targeting Pool (Dharmacon, D-001810-10-20). 4 × 106 Jurkat cells were nucleofected with 10 µM siRNA in 100 µl of Cell Line Nucleofector Kit V using Program X-005. After two days, the cells were nucleofected again with siRNA in the same manner and then stimulated or not with plate-bound anti-CD3 and soluble anti-CD28 for 3 days.
4 × 106 Jurkat cells were nucleofected with 3.5 µg of αsDN3 or empty vector (pcDNAI/Amp) and then stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days.
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 Ct value of the mRNA of interest to that of the house-keeping gene GAPDH in the same preparation.
Imaging of fluorescent fusion proteins
HEK-293 cells (ATCC, CRL-1573) were plated at a density of 105 cells per well on Lab-Tek II, 4 well chambered coverslips and transiently transfected using 0.25 µl of LipofectAMINE 2000 Reagent. Cells were imaged 2 days after transfection at 63 × using a Zeiss Axiovert 200 fluorescence microscope under the control of IPLab software as described . Using the motorized x-y-z stage, time course images of cells located at 5–6 positions in the well were collected simultaneously as described . Images for each color channel and DIC were collected at each position in the well every 60 seconds. Following the second time point, cells were stimulated with 10 µM isoproterenol (final concentration) and images were collected for 30 minutes. For each experimental condition, cells were imaged from plates transfected on 3 different days.
Time course images were analyzed using IPLab software. Changes in the plasma membrane intensity of labeled proteins were measured in cells co-expressing a membrane marker (mRFP-Mem) that was used to segment membrane pixels and correct for intensity changes due to changes in cell shape as described . Briefly, a segment of pixels covering a length of the plasma membrane was identified using the image of the membrane marker. The average intensities of these pixels in the background- and bleach-corrected images of the labeled protein and membrane marker were determined. The membrane marker intensity values were normalized to a starting value of one and the labeled protein intensity values were divided by the normalized membrane marker values. The corrected labeled protein intensities were normalized to a starting value of one and averaged with values from multiple cells.
Using Jurkat cell membranes prepared as described , a polyclonal antibody directed at Gαs residues 28–42 , prepared in the laboratory of Henry Bourne (University of California, San Francisco), was used to detect expression of Gαs, and Gβ1 (XAB-00301-1-G) and Gβ2 (XAB-00401-1-G) antibodies from CytoSignal, LLC were used to detect expression of Gβ1 and Gβ2, respectively. Membrane proteins 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 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. For quantification of Gαs, both the long and short forms of Gαs  were measured together.
Actinomycin D assay
Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 for three days in the presence or absence of 150 µM ddA and then treated with 10 µg/mL of Actinomycin D to inhibit transcription. After incubation with Actinomycin D for 0, 10, 20, 30, or 60 minutes, the cells were removed from the wells, RNA was prepared, and IL-2 mRNA levels were determined by qPCR.
Jurkat cells were nucleofected with 2 µg of a luciferase reporter plasmid and 0.1 µg of pRL-TK Renilla and then stimulated or not with plate-bound anti-CD3 (2.5 µg/mL) and soluble anti-CD28 (2.5 µg/mL) in the presence or absence of 150 µM ddA. 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.
cAMP accumulation assay
4 × 106 Jurkat cells were nucleofected with 3.5 µg αsDN3 or pcDNAI/Amp and then labeled with 40 µCi of [3H]-adenine for 24 hours, or nucleofected twice with siRNAs as described above and then labeled with 40 µCi of [3H]-adenine for 24 hours before the assay. On the day of the assay, the cells were pelleted, washed once, and then resuspended in HEPES-buffered RPMI without bicarbonate with 10% fetal bovine serum, and 1 x 106 cells in 0.5 mL were plated per well in triplicate in 24-well plates. For TCR activation, the wells were pre-coated with 2.5 µg/ml anti-CD3 and 2.5 µg/ml soluble anti-CD28 was added to the media. For stimulation of the A2AR, 300 µM CGS-21680 was added to the media. The media also contained 1 mM 1-methyl-3-isobutylxanthine, a phosphodiesterase inhibitor. Cells were incubated for 40 minutes at 37oC. Reactions were terminated by adding an equal volume of TCA stop buffer (10% TCA, 2 mM ATP, and 2 mM cAMP). Nucleotides were separated on ion exchange columns . cAMP accumulation was determined as 1000 X [3H]cAMP/([3H]ATP + [3H]cAMP). Relative cAMP levels in stimulated cells were expressed as the ratio of the value in stimulated cells to the basal value.
The significance of effects of 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 (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
Inhibiting the A2AR in primary human CD4+ T helper cells and Jurkat cells enhances TCR-stimulated IL-2 mRNA increases
As prior reports suggested that the effect of cAMP increases on TCR-stimulated IL-2 synthesis might depend on the nature and context of these increases[16, 17, 22, 32, 33], we directly compared the effects of inhibiting different upstream activators of cAMP synthesis in CD4+ T helper cells co-stimulated by antibodies to CD3, which associates with the TCR and links it to downstream signaling molecules , and CD28, which provides an additional signal that is needed for complete T cell activation and regulation of IL-2 production . The cells were stimulated for three days, an interval during which primary CD4+ T cells proliferate and differentiate into polarized phenotypes[47, 48, 49].
First, we studied the effect of antagonizing the A2AR, which is known to have anti-inflammatory effects mediated by Gαs  and can decrease TCR-stimulated IL-2 . ATP released from necrotic and apoptotic cells, regulatory T cells, and effector T cells is converted to adenosine by extracellular ectonucleotidases, or cell surface ectonucleotidases in the case of regulatory T cells, resulting in suppression of T cell function by autocrine or paracrine signaling loops . We tested the effect of ZM-241385 , an antagonist to the A2AR, on TCR-stimulated IL-2 mRNA increases in primary human CD4+ T cells grown 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  (Fig. 1). 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 [54, 55], and because our own comparisons of qPCR-determined IL-2 mRNA levels and secreted IL-2  and those of others  demonstrated a strong correlation between mRNA and protein levels. There was more IL-2 mRNA in TH1 cells than in TH2 cells and in naïve compared to memory cells, as previously reported , but ZM-241385 significantly enhanced mean TCR-stimulated IL-2 mRNA levels in each of the primary cell lineages tested, by 1.9 to 3.5-fold, depending on the T cell subset (Fig. 1A), and by 1.8-fold in Jurkat cells (Fig. 1B).
A dominant negative Gαs construct, GαsDN3, which blocks signaling from Gs-coupled receptors, enhances TCR-stimulated IL-2 mRNA increases
To determine whether the results of antagonizing the A2AR with ZM-241385 could be generalized to other Gs-coupled receptors under our TCR-activating conditions, we tested the effect of a dominant negative Gαs construct, Gαs(α3β5/G226A/A366S), referred to here as GαsDN3, which exhibits increased receptor affinity and blocks stimulation of cAMP synthesis by GsPCRs [35, 58]. GαsDN3 potentiated the TCR-stimulated increase in IL-2 mRNA by 1.31-fold (Fig. 2). The increased effectiveness of ZM-241385 compared to GαsDN3 is most likely due to the less than 100% efficiency of plasmid expression in nucleofected Jurkat cells.
GαsDN3 blocks signaling of both Gαs and Gβγ
We observed previously that GαsDN3 inhibited stimulation of cAMP production by GsPCRs , a Gαs-mediated function, but we didn’t determine whether Gβγ function was also inhibited. This was relevant because we determined previously that inhibition of Gβγ by either Gβ1 siRNA, which inhibits both Gβ1γ and Gα signaling downstream from G protein-coupled receptors (GPCRs), and by gallein, which specifically blocks Gβγ-effector interactions downstream of GPCR-G protein interactions , potentiated rather than inhibited TCR-stimulated increases in IL-2 transcription in CD4+ T helper cells . Thus, the enhancing effects of both ZM-241385 and GαsDN3 on TCR-stimulated IL-2 mRNA levels might be the result of inhibiting both Gβγ and Gαs together, or Gβγ alone, rather than Gαs.
GαsDN3 contains three sets of mutations in Gαs, substitutions of Gαi2 homologs for Gαs residues in the α3β5 loop, G226A, and A366S . Previously, we demonstrated that the α3β5 loop mutations increased the apparent affinity of Gαs for the β-adrenergic receptor (βAR)  using an assay that measures a Gαs-dependent increase in the affinity of the βAR for the agonist isoproterenol that occurs in the absence of bound guanine nucleotide [60, 61]. The G226A mutation prevents an activating conformational change in Gαs [62, 63], and A366S elevates basal GDP release, causing Gαs to be constitutively activated and to spend more time in the empty state .
We hypothesized that the combined effects of the α3β5 loop mutations, G226A, and A366S in GαsDN3 might prevent activation of both Gαs and Gβγ derived from Gs by causing the formation of a stable receptor-Gs complex that does not dissociate upon agonist binding. To investigate this possibility, we imaged the basal and agonist-stimulated localization patterns of the Gs subunits and the β2AR in the presence and absence of GαsDN3 in live HEK-293 cells expressing fluorescent fusion proteins. We focused on the β2AR because we determined previously that activation of the β2AR resulted in internalization from the plasma membrane of both Gαs and Gβγ, as well as the β2AR itself, allowing us to use internalization as a readout for signaling of each of these components . Previous studies showed that the A2AR does not internalize upon prolonged agonist stimulation . Gαs and GαsDN3 were visualized using fusion proteins in which CFP or YFP was inserted into an internal loop of Gαs . Gβ1 and Gγ7 were imaged exclusively in the form of Gβ1γ7 complexes using the strategy of bimolecular fluorescence complementation [65, 66], which involves the production of a fluorescent signal by two nonfluorescent fragments of YFP or CFP when they are brought together by interactions between proteins fused to each fragment. When expressed together, fusion proteins consisting of an amino-terminal YFP fragment (residues 1–158) fused to Gβ1, YFP-N-Gβ1, and a carboxy-terminal YFP fragment (residues 159–238) fused to Gγ7, YFP-C-Gγ7, produce a fluorescent signal in the plasma membrane that is not obtained with either subunit alone . The β2AR was visualized using a fusion of GFP to the carboxyl terminus of the β2AR .
We tested for effects of GαsDN3 on basal localization and agonist-dependent internalization of the β2AR in HEK-293 cells co-expressing β2AR-GFP, GαsDN3-CFP, and unlabeled Gβ1γ7. In cells co-expressing β2AR-GFP, Gαs-CFP, and unlabeled Gβ1γ7, both β2AR-GFP (Fig. 3A, open circles, Fig. 4A) and Gαs-CFP (Fig. 3A, open squares, Fig. 4A) internalized upon stimulation of the cells with the β-adrenergic agonist, isoproterenol. As reported previously , the β2AR and the Gs subunits internalized with different kinetics (Fig. 3A) and did not co-localize during internalization (Fig. 4A). Expression of GαsDN3-CFP did not affect the average intensity of the β2AR-GFP signal or the degree to which it associated with the plasma membrane. However, upon stimulation with isoproterenol, neither β2AR-GFP (Fig. 3A, filled circles, Fig. 4B) nor GαsDN3-CFP (Fig. 3A, filled squares, Fig. 4B) internalized.
In the presence of GαsDN3-CFP, internalization of both the Gαs and Gβγ subunits of Gs was also blocked. In cells co-expressing Gαs-YFP, GαsDN3-CFP, and unlabeled β2AR and Gβ1γ7, neither Gαs-YFP (Fig. 3B, filled circles, Fig. 4D), nor GαsDN3-CFP (Fig. 3B, filled squares, Fig. 4D) internalized upon stimulation, in contrast to the internalization responses of both Gαs-YFP (Fig. 3B, open circles, Fig. 4C) and Gαs-CFP (Fig. 3B, open squares, Fig. 4C) that occurred upon stimulation of cells expressing these constructs. Similarly, in cells co-expressing YFP-N-Gβ1, YFP-C-Gγ7, GαsDN3-CFP, and unlabeled β2AR, neither YFP-N-Gβ1/YFP-C-Gγ7 (Fig. 3C, filled circles, Fig. 4F), nor GαsDN3-CFP (Fig. 3C, filled squares, Fig. 4F) internalized upon stimulation, in contrast to the internalization responses of both YFP-N-Gβ1/YFP-C-Gγ7 (Fig. 3C, open circles, Fig. 4E) and Gαs-CFP (Fig. 3C, open squares, Fig. 4E) that occurred upon stimulation of cells expressing these constructs. These results suggest that, similar to the effect of ZM-241385 on A2AR signaling, GαsDN3 blocks GsPCR-stimulated Gαs and Gβγ signaling, consistent with the formation of a stable GPCR-Gs complex that does not dissociate upon binding of agonist.
Gαs siRNA and ddA, an adenylyl cyclase inhibitor, decrease TCR-stimulated IL-2 mRNA levels
The results described above demonstrate an inhibitory role of GsPCR/Gs signaling on TCR-stimulated IL-2 mRNA production, in agreement with numerous previous studies of GsPCRs such as the A2AR[10, 11, 12, 13], PGE2 receptors [14, 15], and VIP receptors [16, 17]. If the observed potentiating effects of ZM-241385 and GαsDN3 on TCR-stimulated IL-2 mRNA production were simply the result of blocking adenylyl cyclase stimulation by activated Gαs, then Gαs siRNA and ddA, an adenylyl cyclase inhibitor, would be expected to have similar potentiating effects.
Expression of Gαs siRNA in Jurkat cells decreased Gαs mRNA to 30% (Fig. 5A) and Gαs protein to 26% (Fig. 5B) of the levels in cells expressing NT siRNA. Gβ1 and Gβ2 mRNA account for >99% of Gβ mRNA in Jurkat cells  and Gαs siRNA caused slight decreases in Gβ1 and Gβ2 protein expression, but these decreases were not statistically significant (Fig. 5B). Larger and significant decreases in Gβ1 and Gβ2 protein expression enhanced or had no effect, respectively, on TCR-stimulated IL-2 mRNA levels . Surprisingly, in contrast to the potentiating effects of ZM-241385 and GαsDN3 on TCR-stimulated IL-2 mRNA levels, Gαs siRNA decreased TCR-stimulated IL-2 mRNA to 39% of the value obtained with NT siRNA (Fig. 5C), and ddA decreased TCR-stimulated IL-2 mRNA to 41% of the control value (Fig. 5D).
Inhibiting adenylyl cyclase decreases TCR-stimulated activity of the IL-2 promoter
Inhibiting adenylyl cyclase activity could decrease TCR-stimulated increases in IL-2 mRNA levels by decreasing IL-2 transcription and/or IL-2 mRNA stability. To determine whether inhibition of adenylyl cyclase decreased 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. ddA did not decrease the stability of IL-2 mRNA (Fig. 6A). The t1/2 of IL-2 mRNA from cells treated with ddA (27.30 min, SE = 1.95, N = 4) was the same as that from untreated cells (25.16, SE = 1.87, N = 4).
To test whether inhibiting adenylyl cyclase activity decreased IL-2 transcription, the effect of ddA on IL-2 promoter activity was determined using a luciferase reporter plasmid containing a 1 kB sequence encoding the human IL-2 promoter from -950 to +48 bp. Three days of TCR stimulation increased luciferase activity in the IL-2 reporter plasmid (IL2/pGL3), but not the empty vector (pGL3) (Fig. 6B). ddA reduced the stimulated value of IL2/pGL3 to 55% of the control value (Fig. 6, B and C).
Gαs siRNA, but not GαsDN3, decreases TCR-stimulated cAMP
The above results demonstrated an important difference between the effects of ZM-241385 and GαsDN3, on the one hand, and Gαs siRNA and ddA, on the other. Namely, the former enhanced TCR-stimulated IL-2 mRNA levels whereas the latter had the opposite effect. Additionally, ZM-241385 and GαsDN3 inhibited signaling of both the Gαs and Gβγ subunits of Gs, whereas the latter specifically inhibited Gαs/cAMP signaling. As both GsPCRs and the TCR [32, 67, 68] can stimulate cAMP increases, these results raised the possibility that the source and context of Gs activation can determine whether TCR-stimulated IL-2 production is enhanced or inhibited. As a first step in investigating this possibility, we tested whether TCR-mediated stimulation of cAMP production is mediated by GsPCRs.
Consistent with previous reports of cAMP elevation in response to TCR activation[32, 67, 68], TCR stimulation increased cAMP accumulation in Jurkat cells (Fig. 7, A and B). Gαs siRNA decreased the TCR-stimulated cAMP increase, indicating that this increase is mediated by activated Gαs (Fig. 7A), in agreement with a previous report showing TCR-stimulated increases of cAMP in lipid rafts, TCR-stimulated recruitment of Gαs to the lipid rafts, and inhibition of TCR-stimulated cAMP increases by inhibitory Gαs antibodies . However, GαsDN3 did not inhibit the TCR-stimulated cAMP increase (Fig. 7B), although it did inhibit A2AR-stimulated cAMP increases (Fig. 7C). These results suggest that the TCR stimulates the Gαs/cAMP pathway via a mechanism that does not involve a GsPCR, which is consistent with a previous study showing that maximal cAMP increases in response to the TCR and to PGE2 were additive . These two apparently independent mechanisms of stimulating the Gαs/cAMP pathway in T cells could produce differences in the kinetics, amplitude, and/or localization of cAMP increases, which would have implications for the resulting effect on TCR-stimulated IL-2 increases.
Evidence for an inhibitory effect of cAMP on TCR-stimulated IL-2 mRNA levels after at least 2 days of TCR stimulation
As GsPCRs such as the A2AR function to terminate TCR responses , we hypothesized that the duration of TCR-stimulation might influence whether cAMP had an enhancing or inhibiting effect on TCR-stimulated levels of IL-2 mRNA. Ligation of the TCR and CD28 prompts CD4+ T cells to secrete IL-2 rapidly, which further enhances their proliferation and survival . However, the levels of IL-2 decrease as the cells start to differentiate [55, 70]. 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). The enhancing effect of the A2AR antagonist, ZM-241385, was only observed after this initial peak, occurring after at least two days of TCR stimulation (Fig. 8A). This result may be explained in part by our observation that during the course of a 3-day stimulation of the TCR, expression of A2AR mRNA increased ~4-fold (data not shown), consistent with a previous report that the A2AR exhibited increased NFAT-dependent expression upon TCR engagement and that CGS-21680-stimulated cAMP levels were higher in cells that had been stimulated previously with anti-CD3 antibodies . Thus, TCR-stimulated increases in GsPCR activity may function as a built-in negative feedback mechanism.
The delayed potentiating effect of ZM-241385 on TCR-stimulated IL-2 mRNA levels might indicate merely that A2AR expression was initially limiting. Alternatively, cAMP inhibition might only have an enhancing effect if it occurred after prolonged TCR stimulation. To distinguish between these possibilities, we stimulated the TCR in Jurkat cells for three days and added ddA one hour before harvesting them (Fig. 8B). In contrast to the inhibitory effect of ddA when added from the initiation of TCR stimulation (Fig. 5D), when ddA was added one hour before the cells were harvested and IL-2 mRNA levels were determined, TCR-stimulated IL-2 mRNA levels were enhanced, consistent with an inhibitory effect of cAMP at this stage of TCR-stimulation (Fig. 8B). These results suggest that TCR-stimulated changes in the T cell (see Discussion) influence whether cAMP plays an enhancing or inhibitory role in regulation of IL-2 mRNA levels.
The effect of cAMP on IL-2 production in T cells has generally been thought to be inhibitory[10, 11, 12, 13, 14, 15, 16, 17, 22, 24], although there is also some evidence to the contrary [32, 33]. The results presented here demonstrate that the effect of inhibiting cAMP increases on IL-2 mRNA levels in TCR-stimulated CD4+ T cells depends on the means by which this is accomplished. These results support both an inhibitory role for GsPCRs and a stimulatory one for Gαs and cAMP in the regulation of TCR-stimulated IL-2 mRNA levels (Fig. 9). The source of the activated Gαs that plays a positive role has not been identified yet, but the TCR is one possibility, as it appears to stimulate cAMP synthesis via a non-canonical mechanism that involves activation of Gαs, but not GsPCRs, and as discussed below, the cAMP increases stimulated by the TCR compared to GsPCRs are likely to be more modest and transient, characteristics associated with an enhancing effect on TCR-stimulated IL-2 [32, 33].
Based on prior results [16, 17, 22] and those presented here, the context of Gαs/cAMP signaling appears to be an important determinant of its effect on IL-2 production by activated T cells. The presence or absence of uninhibited Gβγ signaling is one important contextual difference between TCR-stimulated T cells in which GsPCRs versus Gαs or adenylyl cyclase are blocked. Whereas knocking down Gαs expression and inhibiting adenylyl cyclase activity each decreased levels of TCR-stimulated IL-2 mRNA, potentiation of these mRNA levels was obtained when both Gαs and Gβγ signaling were blocked, which is important in light of our previous observation that inhibition of Gβγ alone with the small molecule inhibitor, gallein, enhanced TCR-stimulated IL-2 transcription . This could indicate that inhibition of both the Gαs and Gβγ components of Gs is necessary to obtain a stimulatory effect, and that simultaneous Gβγ signaling determines the effect of Gαs/cAMP signaling on TCR-stimulated IL-2 mRNA levels in a manner analogous to B-Raf, which is a cell type-specific molecular switch that determines whether cAMP has a stimulatory or inhibitory effect on MAPK activity in central nervous system parenchymal cells . Alternatively, inhibiting the Gβγ component of Gs alone may be sufficient to potentiate TCR-stimulated IL-2 transcription, even without a decrease in Gαs activity and cAMP levels. As gallein blocks the interactions of Gβγ with downstream effectors rather than exclusively inhibiting Gβγ derived from a particular G protein heterotrimer such as Gs [59, 71], it is currently not possible to distinguish between these two possibilities.
One potential mechanism by which simultaneous Gβγ activation could influence the effect of Gαs/cAMP signaling on TCR-stimulated IL-2 transcription is via inhibition of TCR-stimulated increases in intracellular Ca2+ levels (Fig. 9). We determined previously that inhibiting Gβγ led to increased levels of intracellular Ca2+ in TCR-stimulated CD4+ T cells . The mechanism for this effect of Gβγ inhibition remains to be determined, but may involve L-type voltage-dependent Ca2+ (CaV1) channels, which are expressed in primary human T cells and Jurkat cells, are activated by the TCR by an unknown mechanism, rather than by T cell depolarization , and are important for Ca2+-mediated NFAT translocation to the nucleus and IL-2 production [72, 73] (Fig. 9). Gβγ can block activation of CaV1 channels [74, 75, 76] and gallein can prevent this effect of Gβγ . TCR-stimulated signaling involves increases in intracellular Ca2+ in response to IP3 generated by activated PLC-γ, resulting in activation of a variety of downstream pathways including translocation of NFAT to the nucleus and activation of IL-2 transcription  (Fig. 9). Whereas GsPCR stimulated Gs might simultaneously increase cAMP via Gαs and decrease Ca2+ via Gβγ (Fig. 9), activation of Gαs/cAMP signaling alone might potentiate TCR-stimulated Ca2+ increases, as has been demonstrated for transient adhesion-dependent cAMP increases .
Most of our experiments involved antagonizing or blocking GsPCR signaling, knocking down Gαs expression, or inhibiting adenylyl cyclase activity from the initial onset of a 3-day interval of TCR stimulation. The observed negative effects of blocking Gαs/cAMP signaling are consistent with the decreased T cell functioning seen in knockout animals for Gαs  and for the AC7 isoform of adenylyl cyclase , in which cAMP signaling is blocked before the initiation of TCR stimulation. In contrast, our data showing that potentiation of IL-2 mRNA levels by ZM-241385 required at least two days of TCR stimulation and that addition of ddA after three days of TCR stimulation enhanced IL-2 mRNA levels (Fig. 8) suggest that the inhibitory effects of cAMP on IL-2 transcription occur only after the initiation of TCR stimulation. Of note, the potentiating effect of Gβγ inhibition on IL-2 transcription required continuous Gβγ inhibition for at least two days of TCR stimulation , implicating a delayed effect of both the Gαs and Gβγ components of GsPCRs relative to initiation of TCR stimulation. Previous reports suggest a possible mechanism for differential effects of cAMP on TCR-stimulated IL-2 depending on the duration of TCR stimulation. TCR-stimulation initially leads to phosphorylation of CRE-binding protein (CREB), which then recruits p300 and CREB-binding protein (CBP) and binds to the IL-2 promoter to activate transcription [68, 78, 79, 80] (Activating Step 1 in Fig. 9). Later it is replaced by cAMP response element (CRE) modulator (CREM), which exerts an inhibitory effect on IL-2 transcription that occurs after the initial increase in TCR-stimulated IL-2 levels  (Inhibitory Step 2 in Fig. 9). cAMP also inhibits IL-2 transcription via inducible cAMP early represser (ICER), a cAMP inducible CREM family member that can form NFAT/ICER complexes on several NFAT/AP-1 composite sites in the IL-2 promoter leading to repression of transcription  (Inhibitory Step 2 in Fig. 9). ICER mRNA  and protein  were not detected until after three hours of exposure of human medullary thymocytes to forskolin treatment. Furthermore, a study of the kinetics of inhibition of IL-2 transcription by forskolin demonstrated a delay in inhibition of IL-2 mRNA accumulation that correlated with a delay in inhibition of NF-κB activity . Therefore, our results, taken together with these previous reports, are consistent with a stimulatory role for cAMP during the early stages of T cell activation that would be blocked by Gαs siRNA and ddA.
Based on the ability of TCR stimulation to elevate cAMP by a mechanism that is inhibited by Gαs siRNA, but not by GαsDN3, Gαs activation by the TCR appears to involve a non-canonical mechanism that does not involve a GsPCR. There is precedent for non-GPCR-dependent G protein activation and such a mechanism may operate in T cells . For instance, the TCR signals to integrins , integrins can activate Gαs, leading to translocation of phosphorylated CREB to the nucleus , and transient adhesion-dependent cAMP increases are stimulatory to TCR signaling . Additionally, Ric-8B  and Cysteine String Protein (CSP)  can catalyze nucleotide exchange on free Gαs-GDP.
Our results showing that TCR-stimulated cAMP increases do not appear to involve GsPCRs, which inhibit TCR-stimulated IL-2 production, raise the possibility that TCR-stimulated cAMP plays a positive role in IL-2 transcription. One relevant characteristic that distinguishes the cAMP responses stimulated by the TCR versus GsPCRs is the differences in amplitude of the cAMP increases. Reported increases in cAMP in response to TCR stimulation were ~2-fold  or 4–6-fold  rather than the ~13-fold increase induced by PGE2 . The levels of cAMP that we measured after 40 minutes of stimulation of either the TCR (Fig. 7, A and B) or the A2AR (Fig. 7C) were similar. However, increases in the expression of A2AR mRNA in response to TCR stimulation (unpublished)  suggest that levels of A2AR-stimulated cAMP are likely to be greater after several days of TCR stimulation. In contrast, expression of the TCR on the cell surface, as determined by flow cytometry, was ~4-fold lower after a 3-day TCR stimulation than in unstimulated cells (data not shown), similar to a previous report in which co-stimulation of naïve T cells with antigen and anti-CD28 for 10 hours resulted in down-regulation of ~90% of the TCRs . Taken together, these results suggest that by three days of TCR stimulation cAMP increases in response to A2AR stimulation would be greater than those due to TCR stimulation.
Another variable that might determine the directionality of the effect of Gαs/cAMP signaling on TCR-stimulated IL-2 mRNA levels is the kinetics of the cAMP response. Recently it has become possible to monitor cAMP increases in real time in single cells using a FRET-based cAMP sensor, AKAR2, which detects conformational changes induced by PKA phosphorylation . Use of this probe in T cells showed that adhesion-dependent cAMP increases peaked in less than 2 minutes and returned to baseline within 10 minutes . Whereas these transient cAMP increases were stimulatory to TCR signaling, sustained increases in response to forskolin were inhibitory . Anti-CD3-stimulated cAMP increases with similar transient kinetics have been reported in T cell populations. Stimulation of Jurkat cells with anti-CD3 produced a cAMP increase that peaked at 1 minute , and antibody-mediated cross-linking of anti-CD3 antibodies on primary human T lymphocytes produced peak cAMP levels within 2 minutes . In contrast, the kinetics of GsPCR-stimulated cAMP increases appears to be somewhat slower. For instance, PGE2-stimulated cAMP increases peaked at 5 minutes . Taken together with previous reports demonstrating a positive correlation between small and transient cAMP increases and IL-2 production [32, 33], these observations are consistent with a positive effect on TCR-stimulated IL-2 production of modest and transient cAMP increases in response to a non-canonical Gαs activator such as the TCR itself in contrast to the negative effect of GsPCR stimulation (Fig. 9).
Inhibition of GsPCR signaling in TCR-stimulated CD4+ T helper cells enhanced TCR-stimulated increases in IL-2 mRNA, but knocking down Gαs expression, or inhibiting adenylyl cyclase activity had the opposite effect. As inhibiting GsPCRs blocks both the Gαs and Gβγ components of Gs, and inhibiting Gβγ alone enhances TCR-stimulated increases in IL-2 mRNA, the presence of simultaneously activated Gβγ may determine the effect of activating the Gαs/cAMP pathway. Additionally, the TCR appears to stimulate cAMP synthesis via a non-canonical mechanism that involves activation of Gαs, but not GsPCRs. As prior reports showed that TCR-stimulated cAMP increases are smaller and more transient than those induced by GsPCRs, and modest and transient cAMP increases have been associated with enhancement of T cell function, the TCR may be a source of Gαs/cAMP signaling that plays a positive role in IL-2 transcription. Finally, as potentiation of IL-2 mRNA levels by upon A2AR antagonism required at least two days of TCR stimulation, and inhibition of adenylyl cyclase after three days of TCR stimulation enhanced IL-2 mRNA levels, the stage of T cell activation and differentiation appears to determine the effect of Gαs/cAMP signaling on TCR-stimulated IL-2 mRNA levels.
The authors declare that they have no competing interests.