Inhibition of G-protein ßγ 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 ßγ (Gßγ) 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 Gßγ, 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. Gß1 and Gß2 mRNA accounted for >99% of Gß mRNA, and small interfering RNA (siRNA)-mediated silencing of Gß1 but not Gß2 enhanced TCR-stimulated IL-2 mRNA increases. Blocking Gßγ enhanced TCR-stimulated increases in IL-2 transcription without affecting IL-2 mRNA stability. Blocking Gßγ 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 Gßγ 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 Gßγ in CD4+ T helper cells could have applications for autoimmune diseases.

Inhibition of G-Protein ßγ Signaling Decreases Levels of Messenger RNAs Encoding Proinflammatory Cytokines in T Cell Receptor-Stimulated CD4(+) T Helper Cells.

BACKGROUND: Inhibition of G-protein ßγ (Gßγ) signaling was found previously to enhance T cell receptor (TCR)-stimulated increases in interleukin 2 (IL-2) mRNA in CD4(+) T helper cells, suggesting that Gßγ might be a useful drug target for treating autoimmune diseases, as low dose IL-2 therapy can suppress autoimmune responses. Because IL-2 may counteract autoimmunity in part by shifting CD4(+) T helper cells away from the Type 1 T helper cell (TH1) and TH17 subtypes towards the TH2 subtype, the purpose of this study was to determine if blocking Gßγ signaling affected the balance of TH1, TH17, and TH2 cytokine mRNAs produced by CD4(+) T helper cells. METHODS: Gallein, a small molecule inhibitor of Gßγ, and siRNA-mediated silencing of the G-protein ß1 subunit (Gß1) were used to test the effect of blocking Gßγ on mRNA levels of cytokines in primary human TCR-stimulated CD4(+) T helper cells. RESULTS: Gallein and Gß1 siRNA decreased interferon-γ (IFN-γ) and IL-17A mRNA levels in TCR-stimulated CD4(+) T cells grown under TH1-promoting conditions. Inhibiting Gßγ also decreased mRNA levels of STAT4, which plays a positive role in TH1 differentiation and IL-17A production. Moreover, mRNA levels of the STAT4-regulated TH1-associated proteins, IL-18 receptor ß chain (IL-18Rß), mitogen-activated protein kinase kinase kinase 8 (MAP3K8), lymphocyte activation gene 3 (LAG-3), natural killer cell group 7 sequence (NKG7), and oncostatin M (OSM) were also decreased upon Gßγ inhibition. Gallein also increased IL-4, IL-5, IL-9, and IL-13 mRNA levels in TCR-stimulated memory CD4(+) T cells grown in TH2-promoting conditions. CONCLUSIONS: Inhibiting Gßγ to produce these shifts in cytokine mRNA production might be beneficial for patients with autoimmune diseases such as rheumatoid arthritis (RA), Crohn's disease (CD), psoriasis, multiple sclerosis (MS), and Hashimoto's thyroiditis (HT), in which both IFN-γ and IL-17A are elevated.

Inhibition of Gαs/cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4(+) T Helper Cells.

BACKGROUND: 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. As the effect of cAMP is likely to depend on its cellular context, the current study investigated whether the mechanism of activation of Gαs and adenylyl cyclase influences their effect on T cell receptor (TCR)-stimulated interleukin-2 (IL-2) mRNA levels. METHODS: The effect of blocking Gs-coupled receptor (GsPCR)-mediated Gs activation on TCR-stimulated IL-2 mRNA levels in CD4(+) T cells was compared with that of knocking down Gαs expression or inhibiting adenylyl cyclase activity. The effect of knocking down Gαs expression on TCR-stimulated cAMP accumulation was compared with that of blocking GsPCR signaling. RESULTS: ZM-241385, an antagonist to the Gs-coupled A2A adenosine receptor (A2AR), enhanced TCR-stimulated IL-2 mRNA levels in primary human CD4(+) T helper cells and in Jurkat T cells. A dominant negative Gαs construct, GαsDN3, also enhanced TCR-stimulated IL-2 mRNA levels. Similar to GsPCR antagonists, GαsDN3 blocked GsPCR-dependent activation of both Gαs and Gßγ. In contrast, Gαs siRNA and 2',5'-dideoxyadenosine (ddA), an adenylyl cyclase inhibitor, decreased TCR-stimulated IL-2 mRNA levels. Gαs siRNA, but not GαsDN3, decreased TCR-stimulated cAMP synthesis. Potentiation of IL-2 mRNA levels by ZM-241385 required at least two days of TCR stimulation, and addition of ddA after three days of TCR stimulation enhanced IL-2 mRNA levels. CONCLUSIONS: GsPCRs play an inhibitory role in the regulation of TCR-stimulated IL-2 mRNA levels whereas Gαs and cAMP can play a stimulatory one. Additionally, TCR-dependent activation of Gαs does not appear to involve GsPCRs. These results suggest that the context of Gαs/cAMP activation and the stage of T cell activation and differentiation determine the effect on TCR-stimulated IL-2 mRNA levels.