The anti-inflammatory target A3 adenosine receptor is over-expressed in rheumatoid arthritis, psoriasis and Crohn’s disease

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Abstract

The Gi protein associated A3 adenosine receptor (A3AR) was recently defined as a novel anti-inflammatory target. The aim of this study was to look at A3AR expression levels in peripheral blood mononuclear cells (PBMCs) of patients with autoimmune inflammatory diseases and to explore transcription factors involved receptor expression.

Over-expression of A3AR was found in PBMCs derived from patients with rheumatoid arthritis (RA), psoriasis and Crohn’s disease compared with PBMCs from healthy subjects. Bioinformatics analysis demonstrated the presence of DNA binding sites for nuclear factor-κB (NF-κB) and cyclic AMP-responsive element binding protein (CREB) in the A3AR gene promoter. Up-regulation of NF-κB and CREB was found in the PBMCs from patients with RA, psoriasis and Crohn’s disease. The PI3K-PKB/Akt signaling pathway, known to regulate both the NF-κB and CREB, was also up-regulated in the patients’ PBMCs.

Taken together, NF-κB and CREB are involved with the over-expression of A3AR in patients with autoimmune inflammatory diseases. The receptor may be considered as a specific target to combat inflammation.

Introduction

Effective anti-inflammatory drugs to treat autoimmune diseases today are mainly monoclonal antibodies against tumor necrosis factor-α (TNF-α) or other inflammatory cytokines. Due to the critical role of these cytokines in regulating normal immune and inflammatory responses, these drugs induce adverse effects and efforts are still directed towards more specific targets which are expressed solely by pathological but not normal cells [1], [2], [3], [4], [5].

Recently the A3 adenosine receptor (A3AR) was identified as a new target to combat RA due to its unique characteristics. The A3AR belongs to the Gi protein associated family of adenosine receptors which includes also the A1, A2A and A2B sub-members. The A1 and the A3 adenosine receptors are negatively associated with cAMP whereas activation of the A2A and A2B stimulates adenylyl cyclase and cAMP formation [6].

The A3AR was found to be over-expressed in cells from synovial and paw tissues of rats with adjuvant induced arthritis (AIA). High receptor expression was also found in PBMCs derived from the AIA rats, reflecting receptor status in the remote inflammatory sites [7], [8], [9].

Based on these findings, synthetic highly selective agonists to the A3AR were introduced for the treatment of AIA. CF101, chemically known as 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-9-yl]-N-methyl-β-d-ribofura-nuronamide (IB-MECA) induced marked amelioration of the clinical and pathological manifestations of AIA. Mechanistically, CF101 decreased the expression levels of PI3K, PKB/Akt, IKK and IκB resulting in down-regulation of NF-κB, inhibition of TNF-α and apoptosis of inflammatory cells. In addition, a direct anti-proliferative effect of CF101 towards auto-reactive T cells was observed [7], [8], [9], [10], [11].

Examination of A3AR expression levels in the PBMCs of RA patients revealed receptor up-regulation in both early diagnosed patients which were not yet under treatment and in patients chronically treated with methotrexate (MTX) [9], [12].

These data prompted the initiation of a pre-clinical and clinical programs utilizing CF101 as an anti-inflammatory drug candidate. In a Phase I study in healthy subjects, CF101 was found to be safe and well tolerated with a linear pharmacokinetic activity [13]. In a Phase IIa study conducted in patients with RA, CF101 administered twice daily for 12 weeks resulted in an improvement of disease signs and symptoms and appeared to be safe and well tolerated. Analysis of A3AR expression levels at base line showed statistically significant direct correlation with patient responses to CF101, suggesting A3AR utilization as a biomarker to predict patients’ response to the drug prior to treatment initiation [14].

The aim of the present study was to explore whether the A3AR target is over-expressed in additional autoimmune inflammatory diseases. We thus looked at receptor expression levels in PBMCs derived from patients with RA, psoriasis and Crohn’s disease. We further studied the molecular mechanism involved with receptor over-expression and performed bioinformatics analysis to look at the transcription factors in the A3AR promoter gene regulating receptor expression and functionality.

Section snippets

Reagents

Rabbit polyclonal antibodies against rat A3AR (developed against amino acids 151–230 with the internal region of the A3AR, Santa Cruz Biotechnology, CA, USA) and the signaling proteins TNF-α (rat anti TNF-α, R&D systems, MN, USA), PI3K (rabbit polyclonal antibody raised against amino acids 189–390 mapping near the N-terminus of PI3K p110α unit, Santa Cruz Biotechnology, CA, USA), phosphorylated PKB/Akt (PKB/Akt phosphorylated at pSer473, Sigma MI, USA), IκB (rabbit polyclonal antibody against a

A3AR is highly expressed in PBMCs derived from patients with autoimmune inflammatory diseases

Based on former studies which showed that A3AR over-expression in the inflammatory tissues is reflected in the PBMCs, we looked at the A3AR expression levels in PBMCs from patients with RA, psoriasis and Crohn’s disease (n = 25 from each disease) and from healthy subjects (n = 50). Table 1 depicts patients’ characteristics.

Over-expression of the A3AR protein was noted in the PBMCs of the three diseases: RA – 1.8 ± 0.18-, psoriasis – 3.9 ± 0.62-, Crohn’s disease – 3.26 ± 0.57-fold higher than the level in

Discussion

In this study we first show that the A3AR is over-expressed in PBMCs derived from RA, psoriasis and Crohn’s disease patients in comparison to low receptor expression in PBMCs from healthy subjects.

These data raised the question whether A3AR up-regulation is a manifestation of the inflammatory condition or does it play a role in mediating disease pathogenesis. Earlier studies showed that A3AR is up-regulated under hypoxic conditions such as cancer or inflammation. Receptor up-regulation was

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