Centre for Molecular Medicine Norway, Nordic EMBL Partnership and Biotechnology Centre, University of Oslo, Oslo, Norway
Professor, Centre Director Kjetil Taskén, Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo, PO Box 1137 Blindern, N-0318 Oslo, Norway. E-mail: firstname.lastname@example.org
Professor, Centre Director Kjetil Taskén, Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo, PO Box 1137 Blindern, N-0318 Oslo, Norway. E-mail: email@example.com
Cyclic AMP is the intracellular second messenger for a variety of immunoregulatory inflammatory mediators such as prostaglandin E2, adenosine and histamine that signal to effector T cells from monocytes, macrophages and regulatory T cells. Protein kinase A (PKA) type I localizes to lipid rafts in effector T cells during T cell activation and directly modulates proximal signal events including phosphorylation of C-terminal Src kinase (Csk), which initiates a negative signal pathway that fine-tunes the T cell activation process. The PKA-Csk immunoregulatory pathway is scaffolded by the A kinase anchoring protein ezrin, the Csk binding protein phosphoprotein associated with glycosphingolipid-enriched membrane microdomains and the linker protein ezrin/radixin/moesin binding protein of 50 kDa. This pathway is hyperactivated in chronic infections with an inflammatory component such as HIV, other immunodeficiencies and around solid tumours as a consequence of local inflammation leading to inhibition of anti-tumour immunity.
ezrin/radixin/moesin (ERM) binding protein of 50 kDa
phosphoprotein associated with glycosphingolipid-enriched membrane microdomains
protein kinase A
T cell receptor
The common and versatile second messenger cAMP controls numerous cellular processes and is known to be a potent inhibitor of T cell functions such as antigen-induced proliferation and cytokine production (Skalhegg et al., 1992; Aandahl et al., 2002). The cAMP-protein kinase A (PKA) signalling pathway in T cells is initiated by binding of prostaglandin E2 (PGE2) and other extracellular ligands such as catecholamines, serotonin, adenosine and histamine to GPCR, leading to activation of AC and the subsequent generation of cAMP from ATP (Kammer, 1988; Hanoune and Defer, 2001; Loza and Penn, 2010; Mosenden and Tasken, 2011). Endogenous cAMP levels in effector T cells are also in part controlled by regulatory T cells as detailed later.
Human regulatory T cells comprise 5–10% of the peripheral CD4+ T cell population (Ng et al., 2001), are functionally characterized by their ability to suppress effector T cells, have an essential role in maintaining immunological tolerance (Sakaguchi et al., 2006) and are implicated in various clinical conditions such as autoimmune disease, cancer and chronic viral infections. Regulatory T cells can be subdivided into naturally occurring regulatory T cells that are derived from the thymus and adaptive or peripherally induced regulatory T cells that develop from naïve T cells in the periphery during immune activation (Bluestone and Abbas, 2003). The role of cAMP in induction of regulatory T cells and mediation of regulatory T cell suppression is getting increasing attention [reviewed in Yaqub and Tasken (2008)]. Naturally occurring regulatory T cells have been shown to have high cAMP levels which least partly can be explained by the forkhead box P3 (FOXP3)-dependent down-regulation of PDE3B (Gavin et al., 2007) and/or up-regulation of AC 9 by down-regulation of miR-142-3p (Huang et al., 2009), and are shown to mediate their suppressive function by transferring cAMP to effector T cells through gap junctions (Bopp et al., 2007). Furthermore, the ectoenzymes CD39 and CD73 expressed on naturally occurring regulatory T cells may metabolize ATP from dying cells to generate adenosine which acts via the adenosine A2a receptor on activated effector T cells, thereby suppressing their function by intracellular production of cAMP (Deaglio et al., 2007). In addition, continuous antigen exposure to T cells leads to generation of adaptive regulatory T cells (Aandahl et al., 2004). Adaptive regulatory T cells express COX-2, leading to secretion of PGE2. PGE2 stimulates FOXP3 expression in regulatory T cells in addition to inhibiting effector T cell function through activation of the cAMP-PKA-C-terminal Src kinase (Csk) signalling pathway (Figure 1B) (Mahic et al., 2006).
Although other effectors of cAMP such as the guanine exchange factor Epac and cyclic nucleotide-gated ion channels have been identified and extensively characterized in other cell types (Kaupp and Seifert, 2002; Gloerich and Bos, 2010), there is little evidence for expression in T cells, although two reports find Epac in Jurkat or primary T cells (Fuld et al., 2005; Gerlo et al., 2006). Consequently, the functionally important effector system in effector T cells appears to be PKA. Ligand-induced changes in cAMP concentration vary in duration, amplitude and localization in the cell. Spatiotemporal organization of cAMP microdomains is accomplished through coupled control of cAMP production by ACs and degradation by cAMP PDEs, a subset of the superfamily of more than 10 classes of cyclic nucleotide PDEs that degrade cAMP and cGMP (Baillie et al., 2005; Willoughby and Cooper, 2007). Targeting of PKA by A kinase anchoring proteins (AKAPs) and integration of a wide repertoire of proteins involved in signal transduction into complex signal networks further increase the specificity required for the precise regulation of numerous cellular and physiologic processes. The cAMP-PKA pathway is strongly involved in the regulation and modulation of immune responses and is the most potent and acute inhibitor of activation of lymphocytes.
Cyclic AMP and PKA regulate immune function at multiple levels. Possible targets for PKA phosphorylation that can modulate an immune response are the transcription factors cAMP response element binding protein, nuclear factor of activated T cells and NF-κB, and upstream targets like HePTP, Ras, Raf, MAP/ERK kinase (MEK), MAPK, phospholipase Cγ1/2 (PLCγ1/2), PLCβ, Csk and RhoA [reviewed in Skalhegg et al. (1994); Torgersen et al. (2008); Mosenden and Tasken (2011)]. However, based on studies with selective agonists, activation of PKA type I (RIα2C2; RI, type I regulatory subunit) has been shown to be necessary and sufficient for mediating inhibition of T cell receptor (TCR)-induced T cell activation by cAMP (Skalhegg et al., 1994). Similarly, PKA type I negatively regulates activation of B cells through the B cell antigen receptor (Levy et al., 1996) and NK cell cytotoxicity elicited through specific NK cell receptors (Torgersen et al., 1997). Although PKA can modulate TCR signalling at multiple levels [reviewed in Torgersen et al. (2002); Mosenden and Tasken (2011)], the observed inhibitory effects of cAMP on TCR-induced ζ chain phosphorylation point to an important role for Csk, which is the most upstream PKA target reported so far. PKA phosphorylates S364 in Csk, and thereby induces a two- to fourfold increase in phosphotransferase activity of Csk in lipid rafts of T cells (Vang et al., 2001).
So far, two different mechanisms are reported to regulate Csk activity. PKA, through phosphorylation of S364, increases Csk kinase activity two- to fourfold leading to reduced Lck activity and ζ chain phosphorylation (Torgersen et al., 2001; Vang et al., 2001; Vang et al., 2003). In addition, Csk binding protein (Cbp)/phosphoprotein associated with glycosphingolipid-enriched membrane microdomain (PAG) recruits Csk to the site of action in lipid rafts (Brdicka et al., 2000; Kawabuchi et al., 2000), and the interaction between Csk-Src homology 2 and Cbp/PAG through phosphorylated Y314 increases Csk activity (Takeuchi et al., 2000). Thus, PKA phosphorylation of Csk and Csk interaction with Cbp/PAG may act together in turning on Csk activity, providing a powerful mechanism for terminating activation through antigen receptors dependent on Src kinase signalling.
PKA is targeted to the TCR signalling machinery by interaction with the AKAP ezrin
Ezrin is a 78-kDa protein belonging to the ezrin/radixin/moesin (ERM) family of proteins that plays structural and regulatory roles in the assembly and stabilization of specialized plasma membrane domains by linking microfilaments to the membrane. ERM proteins have a highly homologous N-terminal band four-point-one ERM (FERM) domain that binds directly to a number of transmembrane proteins including CD44 and CD43 (Tsukita et al., 1994; Yonemura et al., 1998) in addition to indirect binding to other membrane proteins via scaffold proteins like ERM binding protein of 50 kDa (EBP50). The actin cytoskeleton interaction is via the C-terminus (Algrain et al., 1993). Most of ezrin's interactions are dependent on conformational activation of the molecule. In the dormant state, binding sites for interaction partners are masked due to an intramolecular interaction between the FERM domain and the C-terminus. Upon phosphorylation of a C-terminal threonine by protein kinase C or Rho kinase, the intramolecular bond is released and other interactions can occur (Bretscher et al., 2002). Mapping studies of ezrin reveal that the PKA RIα binding sequence is located in the α-helical region between the FERM domain and the C-terminus (Ruppelt et al., 2007). Interestingly, ezrin, EBP50 and PAG come together in a complex to scaffold the PKA-Csk inhibitory pathway in T cells (Bretscher et al., 2002).
Functional evidence that PKA type I regulation of T cell responses is dependent on AKAP anchoring by ezrin comes from the observations that disruption of PKA type I binding to ezrin using and anchoring disruptor peptides Ht31 and RI anchoring disruptor (RIAD) or the peptides from the RI specifier region in ezrin displaces PKA type I from lipid rafts and releases the T cells from cAMP-mediated inhibition of proliferation and IL-2 production (Carlson et al., 2006; Ruppelt et al., 2007; Jarnaess et al., 2008). Furthermore, small interfering RNA (siRNA)-mediated knockdown of ezrin abrogated cAMP regulation of IL-2 secretion (Ruppelt et al., 2007), whereas reconstitution with an siRNA-resistant wild-type ezrin, but not an ezrin variant where the PKA binding domain had been mutated, re-established the cAMP-regulation of IL-2 secretion (Ruppelt et al., 2007; Jarnaess et al., 2008). In addition, disruption of the ezrin-EBP50-PAG scaffold at the level of the ezrin-EBP50 interaction also disrupts cAMP-regulation of IL-2 secretion (Figure 1D) (Stokka et al., 2010).
Type I PKA is displaced from AKAPs by the soluble, high-affinity binder RIAD made by combined bioinformatic design and peptide array substitutions starting with a consensus PKA type I binding sequence derived from dual-specificity AKAPs (Carlson et al., 2006). Recently, we disrupted type I PKA anchoring in peripheral T cells by expressing a soluble ezrin fragment with the RIAD sequence inserted in place of the endogenous A-kinase binding domain under the lck distal promoter in mice (Mosenden et al., 2011). The RIAD-transgenic mice showed reduced sensitivity to PGE2- and cAMP-mediated inhibition of effector T cell function, enhanced T cell responsiveness assessed as IL-2 secretion and resistance to murine AIDS, a disease model induced by infection with a mixture of attenuated murine leukaemia viruses (LP-BM5) (Mosenden et al., 2011). Hyperactivation of the cAMP-type I PKA pathway is involved in the T cell dysfunction of HIV infection, and our findings underscores the cAMP-type I PKA pathway in T cells as a putative target for therapeutic intervention in immunodeficiency diseases. Furthermore, our unpublished data suggest that effector T cells protected from Treg suppression through cAMP also show significantly improved anti-tumour immune responses to solid tumours compared to mice with normal type I PKA anchoring (Cornez I, Taskén K, unpubl. data).
Immunological hyperactivation induces a chronic inflammatory state with cAMP-mediated hyporesponsiveness of CD8+ and CD4+ T lymphocytes in HIV-infected patients
HIV causes a chronic infection leading to severe dysfunction of the immune system (Figure 1A) with a markedly increased incidence of opportunistic infections and certain forms of malignancies, ultimately leading to death (Douek et al., 2003). Immune hyperactivation by chronic antigen infusion of non-viable microbial antigens itself leads to functional immunodeficiency in animal models (Hazenberg et al., 2003). Correspondingly, the extent of CD38 expression, as an indicator for hyperimmune activation, has long been recognized as a far better prognostic parameter than HIV RNA levels or CD4 counts (Giorgi et al., 2002; Rodriguez et al., 2006), and assessment of CD38 expression in T cell subsets may further improve prognostic value of CD38 as a surrogate parameter for future CD4 loss and disease progression (Holm et al., 2008). Furthermore, in parallel with CD4 loss and immunodeficiency, the ability to react with new antigens and to boost memory responses becomes seriously impaired. However, signs of hyporesponsiveness can be detected long before clinical immunodeficiency. Moreover, this appears to be true both for HIV-specific cellular immunity and for non-HIV antigens. As to HIV-specific responses, it is known that a large number of HIV-specific CD4+ T cell clones get preferentially infected and rapidly lost in primary infection (Douek et al., 2003). We and others have characterized suppressive HIV-specific regulatory T cells, and further characterized the clinical values of reversing the hyporesponsiveness due to overexpression of programmed cell death-1 on effector T cells, particularly HIV-specific clones (Aandahl et al., 2004; Holm et al., 2008; Torheim et al., 2009; Pettersen et al., 2010).
Impaired responses to common T cell-dependent vaccines such as tetanus toxoid in HIV-infected patients have been reported previously (Kvale et al., 2002). Low vaccine efficacy may be linked to hyperimmune activation, a notion reviewed by Dalgleish (Cadogan and Dalgleish, 2008), and where one important mechanism contributing to poor vaccine responses is dysregulated intracellular signalling in T cells. Elevated intracellular cAMP can indirectly reduce or block T cell activation that is expected after interaction between major histocompatibility complex, TCR and specific antigenic peptide (Mahic et al., 2006). HIV patients have higher levels of PKA-mediated inhibition of TCR signalling and T cell activation than uninfected, healthy controls (Aandahl et al., 1998; 1999). We hypothesized that inhibition of PKA, through decreased production of PGE2 by COX inhibitors, could reverse this process and restore T cell responsiveness to vaccine antigens. When treating HIV-infected patients with COX-2 inhibitors, we could indeed find increased proliferative responses (Johansson et al., 2004) and decreased levels of CD38 (Kvale et al., 2006) in patients on highly active antiretroviral therapy and even more pronounced reduction in CD38 as well as tetanus vaccine boosting effects in treatment-naive patients (Pettersen et al., 2011).
Another important source of PGE2 is monocytes expressing COX-2 (Treves et al., 1982). The latter fits very well with the current concept of increased microbial translocation in chronic HIV which results in high levels of circulating lipopolysaccharides (LPS) (Brenchley et al., 2006), potent inducers of COX-2 enzyme in monocytes. The LPS levels also relate to more rapid disease progression in individual patients.
PGE2-/COX-2-mediated modulation of anti-tumour immune responses in colorectal cancer
COX-2 levels are elevated in as many as 85% of human colorectal cancers (CRCs) and approximately 50% of colorectal adenomas leading to a chronic inflammation around the cancer (Eberhart et al., 1994). Studies have shown that COX inhibition by non-steroidal anti-inflammatory drugs or aspirin reduces the risk of CRC and may be beneficial in large population groups at risk (Kraus and Arber, 2011). Selective COX-2 inhibitors are also associated with a decline in the incidence of CRC and reduced mortality rate, although COX-2 inhibitors have been associated with serious cardiovascular events in this context (Psaty and Potter, 2006). PGE2 has been shown to be an important mediator of COX-2 associated effects, and PGE2 levels are elevated in CRC biopsies compared with normal mucosa and even in patient blood samples (Yaqub et al., 2008). Homozygous deletion of the gene for the PGE2 receptor prostaglandin E receptor 2 (EP2) reduced the number and size of colorectal polyps in a polyposis mouse model (Fujino et al., 2011). Beside an anti-angiogenic effect (Fujino et al., 2011), COX inhibition promotes apoptosis and alters tumour growth (Sheng et al., 2001) and stimulates cell migration and invasion by up-regulation of matrix metalloproteinases (Gupta and DuBois, 2001). PGE2 and COX-2 overexpression also correlates with CRC risk and metastasis of CRC (Tsujii et al., 1997), making this pathway relevant also in follow-up after treatment of the primary cancer. Furthermore, our observations show that the PGE2 produced also inhibits anti-tumour immunity in primary CRC through the EP2/EP4 prostanoid receptor – cAMP – PKA – Csk pathway in effector T cells that inhibit T cell activation (Figure 1A and B) (Yaqub and Tasken, 2008; Yaqub et al., 2008; Oberprieler et al., 2010). In addition, our prospective studies of disease recurrence in patients with metastatic CRC that underwent liver resection of metastases showed strong correlation between the degree of Treg and PGE2-mediated suppression of anti-tumour activity and disease recurrence, further substantiating the significance of COX-2 and PGE2-mediated regulation of anti-tumour immune activity (K.W. Brudvik et al., submitted).
Other human tumours also express high levels of COX-2 (Eberhart et al., 1994; Brosens et al., 2005; Steffensen et al., 2007). Tumour-derived PGE2 contributes to cancer progression among others by promoting the conversion of adaptive Treg cells into a regulatory phenotype (Baratelli et al., 2005; Sharma et al., 2005). Treg cells induced in the periphery have been shown to accumulate at tumour sites, where they may impair the development of effective anti-tumour immune responses (Liu et al., 2007). Patients with several forms of cancer have been shown to have increased numbers of circulating and tumour-associated Treg cells compared with healthy controls. In patients with gastrointestinal malignancies, the percentage of Treg cells in peripheral blood correlates inversely with disease prognosis (de Souza and Bonorino, 2009). Moreover, depletion of Treg cells in animal models enhances anti-tumour responses (Sakaguchi et al., 2001; Tanaka et al., 2002).
Therapeutic targeting of the cAMP/PGE2 pathway in disease
The activities of both type I PKA and PDE4 seem to be important for regulation of TCR-induced signalling and T cell function. Type I PKA is activated by external stimuli such as PGE2 produced by regulatory T cells (Mahic et al., 2006), monocytes (Bryn et al., 2006) and adrenergic agonists as well as by an intrinsic mechanism upon TCR stimulation, both of which induces cAMP production in lipid rafts and inhibits proximal T cell signalling (Abrahamsen et al., 2004). However, CD28 co-stimulation leads to recruitment of a PDE4/β-arrestin complex that releases cAMP inhibition allowing a full T cell response to occur (Bjorgo et al., 2010). Finally, ezrin is the essential link between cytoskeletal F-actin and a lipid raft-associated multiprotein complex including EBP50, Cbp/PAG, Csk and PKA type I that negatively regulates immune responses upon T cell activation.
As high levels of endogenous cAMP may be responsible for the anergic phenotype of Treg cells, as well as being central to their suppressive action, uncoupling of the cAMP-type I PKA-Csk pathway in responder T cells would potentially render these cells resistant to Treg cell-mediated suppression. Interestingly, pharmacological intervention that seeks to normalize the elevated cAMP levels in effector T cells has been shown to attenuate Treg cell-mediated suppression of IL-2 secretion (Bopp et al., 2007). The ectoenzymes CD39 and CD73 together generate adenosine on the Treg cell surface. Loss of CD39 and inhibition of CD73 enzymatic activity as well as use of an adenosine receptor antagonist abrogated Treg cell-mediated suppressive function in the presence of 5′AMP in vitro and in vivo (Kobie et al., 2006; Deaglio et al., 2007; Mandapathil and Whiteside, 2011). Thus, perturbed cAMP signalling in responder T cells would potentially render them resistant to Treg cell-mediated suppression and facilitate anti-tumour immune responses. Our published data from characterization of transgenic mice perturbed in the cAMP-type I PKA-Csk pathway by T cell-specific expression of RIAD show that this intervention protects from disease (Mosenden et al., 2011). Moreover, our work with characterizing PKA type I selective cAMP antagonists shows that such compounds also may effectively block cAMP-mediated immune suppression both in patent samples ex vivo and in animal disease models (Aandahl et al., 1998; 1999; Aukrust et al., 1999; Rahmouni et al., 2001; Holm et al., 2003; Nayjib et al., 2008; Yaqub et al., 2008). In contrast, preclinical studies demonstrate that cAMP-elevating agents like vasoactive intestinal peptide or PDE inhibitors can alleviate autoimmune diseases via the increase of cAMP in self-reactive effector T cells (Delgado et al., 2002; Aricha et al., 2006). Selective inhibitors of PDE4 have been proposed as novel anti-inflammatory agents for T cell-mediated diseases in that rolipram has been documented to suppress TNF- and IFN-γ production in animal models of rheumatoid arthritis and has been suggested to have chondroprotective effects (Essayan, 2001). Clinical trials with the PDE4 inhibitor piclamilast documented significant symptom relief in patients with autoimmune diseases such as rheumatoid arthritis, along with decreased levels of IL-6 and C-reactive protein (Souness et al., 2000). Furthermore, inhibition of PDEs can alleviate autoimmune diseases via the increase of cAMP in self-reactive effector T cells (Aricha et al., 2006). Lastly, PDE7 is also up-regulated post-activation, and PDE7 or PDE4/7 inhibitors may also have application in inflammatory diseases (Giembycz, 2008). In conclusion, the cAMP-type I PKA-Csk pathway emerges as a putative target for therapeutic perturbation in chronic inflammation in cancer as well as in chronic viral disease, whereas pacing the same pathway may be beneficial in autoimmune diseases.
Our work is supported by grants from the Research Council of Norway and The Norwegian Cancer Society. KWB is a fellow of The Norwegian Cancer Society.