Cyclic AMP: a selective modulator of NF-κB action

Abstract

It has been known for several decades that cyclic AMP (cAMP), a prototypical second messenger, transducing the action of a variety of G-protein-coupled receptor ligands, has potent immunosuppressive and anti-inflammatory actions. These actions have been attributed in part to the ability of cAMP-induced signals to interfere with the function of the proinflammatory transcription factor Nuclear Factor-kappaB (NF-κB). NF-κB plays a crucial role in switching on the gene expression of a plethora of inflammatory and immune mediators, and as such is one of the master regulators of the immune response and a key target for anti-inflammatory drug design. A number of fundamental molecular mechanisms, contributing to the overall inhibitory actions of cAMP on NF-κB function, are well established. Paradoxically, recent reports indicate that cAMP, via its main effector, the protein kinase A (PKA), also promotes NF-κB activity. Indeed, cAMP actions appear to be highly cell type- and context-dependent. Importantly, several novel players in the cAMP/NF-κB connection, which selectively direct cAMP action, have been recently identified. These findings not only open up exciting new research avenues but also reveal novel opportunities for the design of more selective, NF-κB-targeting, anti-inflammatory drugs.

Fig. 1

The cAMP signaling cascade. Cyclic AMP is generated by activation of adenylyl cyclases, which convert ATP to cAMP. Physiologically, adenylyl cyclase activity is regulated by GPCRs that are either coupled to Gs or Gi proteins, which respectively stimulate and inhibit adenylyl cyclase activity. Several substances in addition allow pharmacological modulation of adenylyl cyclase activity, such as for instance cholera toxin, which stimulates adenylyl cyclase activity via activating Gs, and forskolin which directly activates adenylyl cyclases. [cAMP]_i is, furthermore, negatively regulated by phosphodiesterases which degrade cAMP to 5′AMP. At the center of the canonical cAMP signaling pathway is PKA (1). Briefly, cAMP molecules bind the PKA regulatory subunits of the PKA holoenzyme, which results in release of the two catalytic subunits that subsequently translocate to the nucleus. In the nucleus, the catalytic subunits can phosphorylate different substrates, the best known of which is the transcription factor CREB. Phosphorylated CREB induces the transcription of a plethora of genes harbouring CREB-responsive elements. Alternatively, cAMP can bind to exchange proteins directly activated by cAMP (EPACs) (2). This cascade results in the activation of Rap1

Fig. 2

The NF-κB signaling cascade. The NF-κB signaling cascade is initiated at the cell membrane and depends on the IKK complex, which, in addition to its γ regulatory subunit, contains two catalytic subunits, IKKα and IKKβ. The canonical NF-κB pathway is triggered by binding of pathogen-associated molecular patterns, cytokines or antigen to their cognate receptors. This leads to phosphorylation and consequent activation of IKKβ, which in turn phosphorylates IκB, leading to its ubiquitination and subsequent proteasomal degradation. IκB degradation exposes the nuclear localization signal of the NF-κB p65 subunit, allowing the NF-κB dimer to translocate to the nucleus, where it can switch on the transcription of its target genes, including among others cytokines, chemokines, enzymes and adhesion molecules involved in orchestrating the inflammatory response. The non-canonical pathway is initiated by extracellular stimuli involved in B-cell maturation and lymphoid organogenesis and depends on IKKα, which is activated by the NF-κB-inducing kinase (NIK). Active IKKα preferentially phosphorylates the p100 IκB family protein, which sequesters RelB in the cytoplasm. Once phosphorylated, p100 is partially degraded to p52 in the ubiquitin–proteasome pathway, allowing translocation of the p52-RelB NF-κB dimer to the nucleus. The p52-RelB NF-κB complex induces the transcription of a distinct set of NF-κB target genes, including chemokines, cytokines and other genes involved in lymphocyte function and lymphoid organogenesis

Fig. 3

Mechanisms explaining positive effects of cAMP on NF-κB activity. a cAMP activates PKA, which phosphorylates p65 at its ser 276 residue, leading to enhanced NF-κB transactivation. b At promoters, containing CREB and NF-κB responsive elements in close proximity, both transcription factors co-operatively recruit the CBP co-activator, leading to enhanced NF-κB-dependent gene expression. c In cells expressing AKIP-1, cAMP-activated PKA is targeted to NF-κB-dependent promoters, where it phosphorylates p65 at ser 276, leading to recruitment of CBP and enhanced transcriptional activation. In cells that do not express AKIP-1, PKA preferentially phosphorylates CREB, leading to competition between CREB and NF-κB for CBP and consequently reduced NF-κB-dependent gene expression (see also Fig. 4b). For more detailed information and references related to the mechanisms presented here, we refer to the text “Mechanisms of NF-κB modulation by cAMP”

Fig. 4

Mechanisms explaining negative effects of cAMP on NF-κB activity. a cAMP inhibits NF-κB activity by elevating cytoplasmic levels of IκB via: 1 inducing CREB-mediated transcription of the IκB gene, 2 blocking IKKβ activity, hence preventing IκB degradation, and 3 enhancing IκB levels by interfering with IκB ubiquitinylation and/or subsequent proteasomal degradation. b cAMP and NF-κB both depend on the limiting cofactor CBP for transcriptional activation of their respective target genes. As elevated [cAMP]_i leads to the phosphorylation of CREB, and phosphorylated CREB has a higher affinity for CBP than NF-κB, CBP will preferentially associate with active CREB, enhancing CREB-dependent transcription at the cost of NF-κB-dependent transcription. c cAMP induces the exchange of transactivating NF-κB complexes (i.e. p50–p65) for repressive complexes (i.e. p50–p50). d At certain NF-κB promoters containing CREB responsive elements (i.e. the TNF-α promoter), cAMP induces replacement of transactivating CREB-c-jun complexes at the CRE by repressive CREB-ICER complexes, leading to transcriptional inhibition. e cAMP induces the expression of c-Fos which prevents p65 homodimers from binding to their cognate responsive elements, leading to inhibited transcription of a subset of NF-κB target genes. For more detailed information and references related to the mechanisms presented here, we refer to the text “Mechanisms of NF-κB modulation by cAMP”