The germinal center kinase TNIK is required for canonical NF-κB and JNK signaling in B-cells by the EBV oncoprotein LMP1 and the CD40 receptor.

The tumor necrosis factor-receptor-associated factor 2 (TRAF2)- and Nck-interacting kinase (TNIK) is a ubiquitously expressed member of the germinal center kinase family. The TNIK functions in hematopoietic cells and the role of TNIK-TRAF interaction remain largely unknown. By functional proteomics we identified TNIK as interaction partner of the latent membrane protein 1 (LMP1) signalosome in primary human B-cells infected with the Epstein-Barr tumor virus (EBV). RNAi-mediated knockdown proved a critical role for TNIK in canonical NF-κB and c-Jun N-terminal kinase (JNK) activation by the major EBV oncoprotein LMP1 and its cellular counterpart, the B-cell co-stimulatory receptor CD40. Accordingly, TNIK is mandatory for proliferation and survival of EBV-transformed B-cells. TNIK forms an activation-induced complex with the critical signaling mediators TRAF6, TAK1/TAB2, and IKKß, and mediates signalosome formation at LMP1. TNIK directly binds TRAF6, which bridges TNIK's interaction with the C-terminus of LMP1. Separate TNIK domains are involved in NF-κB and JNK signaling, the N-terminal TNIK kinase domain being essential for IKKß/NF-κB and the C-terminus for JNK activation. We therefore suggest that TNIK orchestrates the bifurcation of both pathways at the level of the TRAF6-TAK1/TAB2-IKK complex. Our data establish TNIK as a novel key player in TRAF6-dependent JNK and NF-κB signaling and a transducer of activating and transforming signals in human B-cells.

Inactivation of CD73 promotes atherogenesis in apolipoprotein E-deficient mice.

AIMS: CD73 (ecto-5'-nucleotidase) is expressed by a broad range of immune cells and attenuates inflammation in several acute disease models. This study therefore explored the role of CD73-derived adenosine in a model of chronic vascular inflammation such as atherogenesis. METHODS AND RESULTS: CD73(-/-) mice were backcrossed into the apolipoprotein E (ApoE(-/-)) background. In CD73(-/-)/ApoE(-/-) double mutants, atherosclerotic lesion formation was increased by ∼50% compared with ApoE(-/-). However, the cellular composition and extracellular matrix of the plaques did not differ. Surprisingly, we found significant activity and expression of CD73 in the plaque of ApoE(-/-) mice which increased over time. CD73 co-localized with macrophages, Tregs, and cells of mesenchymal origin. Genome-wide microarray analysis of the aorta lacking CD73 revealed upregulation of endothelin-1 (Edn1) mRNA together with changes of genes in lipid metabolism and the Wnt and nuclear factor kappa B pathways. Measurement of plasma levels verified the upregulation of Edn1 in CD73(-/-) and double mutants. Plasma triglycerides (TG) were also found to be significantly elevated in the CD73(-/-)/ApoE(-/-) mice compared with ApoE(-/-) controls. CONCLUSION: Lack of CD73 promotes atherogenesis most likely by de-inhibition of resident macrophages and T cells. Elevated Edn1 and TG levels may have contributed. This establishes CD73-derived adenosine as a direct or indirect regulator of atherogenesis.

Extracellular purine metabolism and signaling of CD73-derived adenosine in murine Treg and Teff cells.

CD73-derived adenosine acts as potent inhibitor of inflammation, and regulatory T cells (Treg) have been shown to express CD73 as a novel marker. This study explored the role of endogenously formed adenosine in modulating NF-κB activity and cytokine/chemokine release from murine Treg and effector T cells (Teff) including key enzymes/purinergic receptors of extracellular ATP catabolism. Stimulating murine splenocytes and CD4(+) T cells with anti-CD3/anti-CD28 significantly upregulated activated NF-κB in CD73(-/-) T cells (wild type: 4.36 ± 0.21; CD73(-/-): 6.58 ± 0.75; n = 4; P = 0.029). This was associated with an augmented release of proinflammatory cytokines IL-2, TNF-α, and IFN-γ. Similar changes were observed with the CD73 inhibitor APCP (50 µM) on NF-κB and IFN-γ in wild-type CD4(+) T-cells. Treatment of stimulated CD4(+) T-cells with adenosine (25 µM) potently reduced IFN-γ release which is mediated by adenosine A2a receptors (A2aR). AMP (50 µM) also reduced cytokine release which was not inhibited by APCP. In Teff, A2aR activation (CGS21680) potently inhibited the release of IL-1, IL-2, IL-3, IL-4, IL-12, IL-13, IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), CCL3, and CCL4. However, in Treg, CGS21680 did not alter cytokine/chemokine release. In summary, CD73-derived adenosine tonically inhibits active NF-κB in CD4(+) T-cells, thereby modulating the release of a broad spectrum of proinflammatory cytokines and chemokines. Downregulation of P2X7 and upregulation of CD73 in Treg after antigenic stimulation may be an important mechanism to maintain the ability of Treg to generate immunosuppressive adenosine.

Nucleoside-5'-monophosphates as prodrugs of adenosine A2A receptor agonists activated by ecto-5'-nucleotidase.

Prodrugs of adenosine A(2A) receptor agonists were developed that are activated by ecto-5'-nucleotidase (ecto-5'-NT, CD73). Because ecto-5'-NT is upregulated in inflamed tissue, the A(2A) agonists are expected to be released from their prodrug form at the sites of inflammation. 2-(Ar)alkyl-substituted AMP derivatives were synthesized and investigated. Certain 2-substituted AMP derivatives, including 2-hexylthio-AMP, 2-cyclopentylthio-AMP, 2-cyclohexylmethylthio-AMP, and 2-cyclohexylethylthio-AMP were accepted as substrates by ecto-5'-NT and readily converted to the corresponding 2-substituted adenosine derivatives. The 2-cyclohexylethylthio substitution was a good compromise between the requirements of the ecto-5'-NT and the adenosine A(2A) receptor. The corresponding AMP derivative (12g) was a similarly good substrate as AMP itself, while the resulting adenosine derivative (11g) was a relatively potent A(2A) agonist (radioligand binding to rat brain striatal membranes: K(i) = 372 nM; inhibition of anti-CD3/anti-CD28-induced IFN-gamma release in mouse CD4+ cells: EC(50) = 50 nM). Compound 11g was released from 12g by incubation with CD4+ cells isolated from wild-type mice but only to a much smaller extent by cells from ecto-5'-NT knockout mice. Compound 12g will be a new lead structure for the development of more potent and selective ecto-5'-NT-activated prodrugs of selective anti-inflammatory A(2A) receptor agonists.