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Abstract
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 Gβγ. 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.
Highlights
The cellular effects of the second messenger cAMP are often dependent on the context of concentration changes
Inhibiting the A2A adenosine receptor (A2AR) in primary human CD4+ T helper cells and Jurkat cells enhances T cell receptor (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 [45], and CD28, which provides an additional signal that is needed for complete T cell activation and regulation of IL-2 production [46]
The results described above demonstrate an inhibitory role of Gs-coupled receptor (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 vasoactive intestinal peptide (VIP) receptors [16, 17]
Summary
The cellular effects of the second messenger cAMP are often dependent on the context of concentration changes. 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 [9] 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. 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. 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
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