Abstract
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.
Highlights
G protein-coupled receptor (GPCR) signaling exerts multiple influences on cytokine levels with vast implications for immunodeficiency and autoimmune diseases [1]
To determine whether Gbg plays a role in modulating TCR-stimulated interleukin 2 (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]
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 consistent with previous observations [36]
Summary
G protein-coupled receptor (GPCR) signaling exerts multiple influences on cytokine levels with vast implications for immunodeficiency and autoimmune diseases [1]. 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]. Chemokine receptors, which regulate the migration of immune cells, have. While targeting GPCR signaling to regulate cytokine levels may well prove to be a useful therapeutic approach, targeting signaling distal to the GPCRs may be advantageous, as multiple GPCRs can influence cytokine levels
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