Br J Pharmacol. 2013 Jul;169(6):1372-88.
Activation of adenosine A2A receptor reduces osteoclast formation via PKA- and ERK1/2-mediated suppression of NFκB nuclear translocation.
Mediero A, Perez-Aso M, Cronstein BN.
Source
Department of Medicine, Division of Translational Medicine, NYU School of Medicine, New York,
NY 10016, USA.
Abstract
BACKGROUND AND PURPOSE:
We previously reported that adenosine, acting at adenosine A2A receptors (A2A R), inhibits osteoclast (OC) differentiation in vitro (A2A R activation OC formation reduces by half) and in vivo. For a better understanding how adenosine A2A R stimulation regulates OC differentiation, we dissected the signalling pathways involved in A2A R signalling.
EXPERIMENTAL APPROACH:
OC differentiation was studied as TRAP+ multinucleated cells following M-CSF/RANKL stimulation of either primary murine bone marrow cells or the murine macrophage line, RAW264.7, in presence/absence of the A2A R agonist CGS21680, the A2A R antagonist ZM241385, PKA activators (8-Cl-cAMP 100 nM, 6-Bnz-cAMP) and the PKA inhibitor (PKI). cAMP was quantitated by EIA and PKA activity assays were carried out. Signalling events were studied in PKA knockdown (lentiviral shRNA for PKA) RAW264.7 cells (scrambled shRNA as control). OC marker expression was studied by RT-PCR.
KEY RESULTS:
A2A R stimulation increased cAMP and PKA activity which and were reversed by addition of ZM241385. The direct PKA stimuli 8-Cl-cAMP and 6-Bnz-cAMP inhibited OC maturation whereas PKI increased OC differentiation. A2A R stimulation inhibited p50/p105 NFκB nuclear translocation in control but not in PKA KO cells. A2A R stimulation activated ERK1/2 by a PKA-dependent mechanism, an effect reversed by ZM241385, but not p38 and JNK activation. A2A R stimulation inhibited OC expression of differentiation markers by a PKA-mechanism.
CONCLUSIONS AND IMPLICATIONS:
A2A R activation inhibits OC differentiation and regulates bone turnover via PKA-dependent inhibition of NFκB nuclear translocation, suggesting a mechanism by which adenosine could target bone destruction in inflammatory diseases like Rheumatoid Arthritis.
© 2013 The British Pharmacological Society.
PMID:23647065[PubMed – in process]

FASEB J. 2013 Nov 8. [Epub ahead of print]
Adenosine 2A receptor promotes collagen production by human fibroblasts via pathways involving cyclic AMP and AKT but independent of Smad2/3.
Perez-Aso M, Fernandez P, Mediero A, Chan ES, Cronstein BN.
Source
*Department of Medicine, New York University School of Medicine, New York, NewYork, USA; and.
Abstract
Activation of adenosine A2A receptor (A2AR) promotes fibrosis and collagen synthesis. However, the underlying mechanism is still unclear, not least because cAMP, its principal effector, has been found to inhibit TGFβ1-induced collagen synthesis. Here, we show that in primary normal human dermal fibroblasts, A2AR stimulation with CGS21680 elicits a modest cAMP increase (150±12% of control; EC50 54.8 nM), which stimulates collagen1 (Col1) and collagen3 (Col3), but maximal cAMP resulting from direct activation of adenylyl cyclase by forskolin (15,689±7038% of control; EC50 360.7 nM) inhibits Col1 and increases Col3. Similar to Col1 expression, fibroblast proliferation increased following physiological cAMP increases by CGS21680 but was inhibited by cAMP increases beyond the physiological range by forskolin. The A2AR-mediated increase of Col1 and Col3 was mediated by AKT, while Col3, but not Col1, expression was dependent on p38 and repressed by ERK. TGFβ1 induced phosphorylation of Smad2/3 and increased Col3 expression, which was prevented by Smad3 depletion. In contrast, CGS21680 did not activate Smad2/3, and Smad2/3 knockdown did not prevent CGS21680-induced Col1 or Col3 increases. Our results indicate that cAMP is a concentration-dependent switch for collagen production via noncanonical, AKT-dependent, Smad2/3-independent signaling. These observations explain the paradoxical effects of cAMP on collagen expression.-Perez-Aso, M., Fernandez, P., Mediero, A., Chan, E. S., and Cronstein, B. N.
Adenosine 2A receptor promotes collagen production by human fibroblasts via pathways involving cyclic AMP and AKT but independent of Smad2/3.
PMID:24200882[PubMed – as supplied by publisher]

Rheumatol Int. 2008 Dec;29(2):167-71.
Cyclic adenosine 5′-monophosphate in synovial fluid of rheumatoid arthritis and osteoarthritis patients.
Morovic-Vergles J, Culo MI, Gamulin S, Culo F.
Source
Division of Clinical Immunology and Rheumatology, Department of Internal Medicine, Dubrava University Hospital, and Department of Pathophysiology, Medical School University of Zagreb, Av. G. Suska 6, Zagreb, 10040, Croatia. jmorovic@kbd.hr
Abstract
The relationship between synovial fluid (SF) cAMP level and IL-18 and PGE2 SF levels in rheumatoid arthritis (RA) and osteoarthritis (OA) patients, and between SF cAMP level and disease as well as inflammatory activity in RA were investigated in 17 RA and 19 OA patients. Erythrocyte sedimentation rate (ESR), serum (S) C-reactive protein (CRP) level and SF IL-18 level were higher in RA than in OA patients. SF PGE2 level was similar in both groups. SF cAMP level was higher in OA than in RA patients. In RA patients, SF cAMP level showed negative correlation with Disease Activity Score including a 28-joint count and S CRP, ESR and SF IL-18 level.
The results suggest that cAMP promotes anti-inflammatory response in RA and OA patients, which is higher in the latter.
Promotion of anti-inflammatory response by cAMP elevating agents might be useful in the treatment of RA.
PMID:18695981[PubMed – indexed for MEDLINE]

Biochem Pharmacol. 2008 Aug 15;76(4):482-94. Epub 2008 Jun 17.
The A3 adenosine receptor agonist CF502 inhibits the PI3K, PKB/Akt and NF-kappaB signaling pathway in synoviocytes from rheumatoid arthritis patients and in adjuvant-induced arthritis rats.
Ochaion A, Bar-Yehuda S, Cohen S, Amital H, Jacobson KA, Joshi BV, Gao ZG, Barer F, Patoka R, Del Valle L, Perez-Liz G, Fishman P.
Can-Fite BioPharma Ltd., Kiryat-Matalon, Petah-Tikva 49170, Israel.
The A(3) adenosine receptor (A(3)AR) is over-expressed in inflammatory cells and was defined as a target to combat inflammation. Synthetic agonists to this receptor, such as IB-MECA and Cl-IB-MECA, exert an anti-inflammatory effect in experimental animal models of adjuvant- and collagen-induced arthritis. In this study we present a novel A(3)AR agonist, CF502, with high affinity and selectivity at the human A(3)AR. CF502 induced a dose dependent inhibitory effect on the proliferation of fibroblast-like synoviocytes (FLS) via de-regulation of the nuclear factor-kappa B (NF-kappaB) signaling pathway. Furthermore, CF502 markedly suppressed the clinical and pathological manifestations of adjuvant-induced arthritis (AIA) in a rat experimental model when given orally at a low dose (100 microg/kg). As is typical of other G-protein coupled receptors, the A(3)AR expression level was down-regulated shortly after treatment with agonist CF502 in paw and in peripheral blood mononuclear cells (PBMCs) derived from treated AIA animals. Subsequently, a decrease in the expression levels of protein kinase B/Akt (PKB/Akt), IkappaB kinase (IKK), I kappa B (IkappaB), NF-kappaB and tumor necrosis factor-alpha (TNF-alpha) took place. In addition, the expression levels of glycogen synthase kinase-3 beta (GSK-3beta), beta-catenin, and poly(ADP-ribose)polymerase (PARP), known to control the level and activity of NF-kappaB, were down-regulated upon treatment with CF502. Taken together, CF502 inhibits FLS growth and the inflammatory manifestations of arthritis, supporting the development of A(3)AR agonists for the treatment of rheumatoid arthritis.
PMID: 18602896 [PubMed – indexed for MEDLINE]

Arthritis Res Ther. 2006;8(1):R33. Epub 2006 Jan 13.
The PI3K-NF-kappaB signal transduction pathway is involved in mediating the anti-inflammatory effect of IB-MECA in adjuvant-induced arthritis.
Fishman P, Bar-Yehuda S, Madi L, Rath-Wolfson L, Ochaion A, Cohen S, Baharav E.
Can-Fite BioPharma Ltd, Kiryat-Matalon, Petah-Tikva, Israel. pnina@canfite.co.il
The anti-inflammatory effect of adenosine was previously found to be mediated via activation of the A3 adenosine receptor (A3AR).
The aim of the present study was to decipher the molecular mechanism involved with the inhibitory effect of IB-MECA, an A3AR agonist, on adjuvant-induced arthritis. The adjuvant-induced arthritis rats responded to IB-MECA treatment with a decrease in the clinical score and the pathological score of the disease. The response to IB-MECA was neutralized by the antagonist MRS 1220, confirming that the efficacy of the synthetic agonist was A3AR mediated. The A3AR protein expression level was highly expressed in the synovia, in the peripheral blood mononuclear cells and in the drain lymph node (DLN) tissues of adjuvant-induced arthritis rats in comparison with naïve animals. Downregulation of A3AR expression was noted upon treatment with IB-MECA. Analysis of synovia and DLN protein extracts revealed a decreased expression level of PI3K, PKB/Akt, IKK, NF-kappaB and tumor necrosis factor alpha, known to affect survival and apoptosis of inflammatory cells, whereas the caspase-3 level was upregulated.Taken together, high A3AR expression is found in the synovia, in the immune cells in the DLN and in peripheral blood mononuclear cells. IB-MECA, an orally bioavailable molecule, activates the A3AR, inducing receptor downregulation and the initiation of a molecular mechanism that involves de-regulation of the PI3K-NF-kappaB signaling pathway. As a result, a potent anti-inflammatory effect manifested in the improvement of the disease clinical score and pathological score occurs. The finding that the A3AR expression level in the peripheral blood mononuclear cells and in the DLN reflects the receptor status in the remote inflammatory site suggests use of the A3AR as a follow-up biomarker.
PMID: 16507132 [PubMed – indexed for MEDLINE]

Expert Opin Investig Drugs. 2007 Oct;16(10):1601-13.
The anti-inflammatory effect of A3 adenosine receptor agonists: a novel targeted therapy for rheumatoid arthritis.
Bar-Yehuda S, Silverman MH, Kerns WD, Ochaion A, Cohen S, Fishman P.
Can-Fite BioPharma, 10 Bareket Street, PO Box 7537, Petach-Tikva 49170, Israel.
Targeting the A(3) adenosine receptor (A(3)AR) to combat inflammation is a new concept based on two findings. First, A(3)AR is highly expressed in inflammatory cells, whereas low expression is found in normal tissues. This receptor was also found to be overexpressed in peripheral blood mononuclear cells, reflecting receptor status in the remote inflammatory process. Second, A(3)AR activation with a specific agonist induces de-regulation of the NF-kappaB signaling pathway in inflammatory cells, as well as initiation of immunomodulatory effects. The A(3)AR agonist CF-101 (known generically as IB-MECA) induces anti-inflammatory effects in experimental animal models of collagen- and adjuvant-induced arthritis. Combined therapy with CF-101 and methotrexate in adjuvant-induced arthritis rats yielded an additive anti-inflammatory effect. Methotrexate induced upregulation of A(3)AR, rendering the inflammatory cells more susceptible to CF-101. In Phase I and in Phase IIa human studies, CF-101 was safe, well tolerated and showed strong evidence of an anti-inflammatory effect in rheumatoid arthritis patients. In peripheral blood mononuclear cells withdrawn from the patients at base line, a statistically significant correlation between A(3)AR expression level and response to the drug was noted. It is suggested that A(3)AR may serve as a biologic marker to predict patient response to the drug. Taken together, this information suggests that A(3)AR agonists may be a new family of orally bioavailable drugs to be developed as potent inhibitors of autoimmune-inflammatory diseases.
PMID: 17922624 [PubMed – indexed for MEDLINE]