Selective inhibition of KCa3.1 channels mediates adenosine regulation of the motility of human T cells.

Adenosine, a purine nucleoside, is present at high concentrations in tumors, where it contributes to the failure of immune cells to eliminate cancer cells. The mechanisms responsible for the immunosuppressive properties of adenosine are not fully understood. We tested the hypothesis that adenosine's immunosuppressive functions in human T lymphocytes are in part mediated via modulation of ion channels. The activity of T lymphocytes relies on ion channels. KCa3.1 and Kv1.3 channels control cytokine release and, together with TRPM7, regulate T cell motility. Adenosine selectively inhibited KCa3.1, but not Kv1.3 and TRPM7, in activated human T cells. This effect of adenosine was mainly mediated by A2A receptors, as KCa3.1 inhibition was reversed by SCH58261 (selective A2A receptor antagonist), but not by MRS1754 (A2B receptor antagonist), and it was mimicked by the A2A receptor agonist CGS21680. Furthermore, it was mediated by the cAMP/protein kinase A isoform (PKAI) signaling pathway, as adenylyl-cyclase and PKAI inhibition prevented adenosine effect on KCa3.1. The functional implication of the effect of adenosine on KCa3.1 was determined by measuring T cell motility on ICAM-1 surfaces. Adenosine and CGS21680 inhibited T cell migration. Comparable effects were obtained by KCa3.1 blockade with TRAM-34. Furthermore, the effect of adenosine on cell migration was abolished by pre-exposure to TRAM-34. Additionally, adenosine suppresses IL-2 secretion via KCa3.1 inhibition. Our data indicate that adenosine inhibits KCa3.1 in human T cells via A2A receptor and PKAI, thereby resulting in decreased T cell motility and cytokine release. This mechanism is likely to contribute to decreased immune surveillance in solid tumors.

Modulation of Kv1.3 channels by protein kinase A I in T lymphocytes is mediated by the disc large 1-tyrosine kinase Lck complex.

The cAMP/PKA signaling system constitutes an inhibitory pathway in T cells and, although its biochemistry has been thoroughly investigated, its possible effects on ion channels are still not fully understood. K(V)1.3 channels play an important role in T-cell activation, and their inhibition suppresses T-cell function. It has been reported that PKA modulates K(V)1.3 activity. Two PKA isoforms are expressed in human T cells: PKAI and PKAII. PKAI has been shown to inhibit T-cell activation via suppression of the tyrosine kinase Lck. The aim of this study was to determine the PKA isoform modulating K(V)1.3 and the signaling pathway underneath. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), a nonselective activator of PKA, inhibited K(V)1.3 currents both in primary human T and in Jurkat cells. This inhibition was prevented by the PKA blocker PKI(6-22). Selective knockdown of PKAI, but not PKAII, with siRNAs abolished the response to 8-BrcAMP. Additional studies were performed to determine the signaling pathway mediating PKAI effect on K(V)1.3. Overexpression of a constitutively active mutant of Lck reduced the response of K(V)1.3 to 8-Br-cAMP. Moreover, knockdown of the scaffolding protein disc large 1 (Dlg1), which binds K(V)1.3 to Lck, abolished PKA modulation of K(V)1.3 channels. Immunohistochemistry studies showed that PKAI, but not PKAII, colocalizes with K(V)1.3 and Dlg1 indicating a close proximity between these proteins. These results indicate that PKAI selectively regulates K(V)1.3 channels in human T lymphocytes. This effect is mediated by Lck and Dlg1. We thus propose that the K(V)1.3/Dlg1/Lck complex is part of the membrane pathway that cAMP utilizes to regulate T-cell function.

PGE2 inhibits natural killer and gamma delta T cell cytotoxicity triggered by NKR and TCR through a cAMP-mediated PKA type I-dependent signaling.

Natural killer (NK) and unconventional gammadelta T cells, by their ability to sense ligands induced by oncogenic stress on cell surface and to kill tumor cells without a need for clonal expansion, show a great therapeutic interest. They use numerous activating and inhibitory receptors which can function with some independence to trigger or inhibit destruction of target cells. Previous reports demonstrated that PGE(2) is able to suppress the destruction of some tumor cell lines by NK and gammadelta T cells but it remained uncertain if PGE(2) interferes with the different activating receptors governing the cytolytic responses of NK and gammadelta T cells. In this report, using the model of specific redirected lysis of the mouse FcgammaR(+) cell line P815, we clearly demonstrate that the major NK receptors (NKR): NKG2D, CD16 and natural cytotoxicity receptors (NCR: NKp30, NKp44, NKp46) and gammadelta T cell receptors TCR Vgamma9Vdelta2, NKG2D and CD16 are all inhibited by PGE(2). As is the case with gammadelta T cells, we show that PGE(2) binds on E-prostanoid 2 (EP2) and EP4 receptors on NK cells. Finally, we delineate that the signaling of the blockade by PGE(2) is mediated through a cAMP-dependent activation of PKA type I which inhibits early signaling protein of cytotoxic cells. In the discussion, we focused on how these data should impact particular approaches in the treatment of cancer.

Regulation of arginase-1 expression in macrophages by a protein kinase A type I and histone deacetylase dependent pathway.

The aim of the current study was to investigate the cAMP-dependent regulation of arginase-1 (ARG1) expression in RAW-macrophages. Basal ARG1 mRNA expression was low and increased upon incubation with the cAMP analogue Br-cAMP. We used selective agonists of protein kinase A type I (PKAI), type II (PKAII) and exchange protein directly activated by cAMP (EPAC) to determine the pathway responsible for ARG1 expression. Activation of PKAI led to a significant up-regulation of ARG1 mRNA expression and arginase enzyme activity. In contrast, neither activation of PKAII nor activation of EPAC affected ARG1 expression. In addition, it has been shown that histone deacetylase (HDAC) activity plays a critical role in cAMP-dependent transcriptional regulation. Incubation with Br-cAMP and the HDAC inhibitor trichostatin A (TSA) led to a concentration-dependent suppression of ARG1 expression. These data indicate that cAMP-dependent activation of ARG1 expression is mediated by PKAI and requires histone deacetylation.