Background/Purpose: Adenosine, a nucleoside released at sites of injury and hypoxia, mediates its effects via activation of G-protein-coupled receptors (A₁, A_2A, A_2B, A₃). Previously we reported that the A_2AR agonist CGS21680 inhibits osteoclast (OC) differentiation in a dose–dependent manner. Here we dissected the intracellular pathways involved in A_2AR-mediated regulation of OC differentiation.

Methods: OC differentiation was studied as M-CSF/RANKL-stimulated differentiation of murine bone marrow precursors to TRAP+/multinucleated cells in the presence/absence of CGS21680 (A_2AR agonist) and ZM241385 (A_2AR antagonist) 1µM each, and PKA activators 8-Cl-cAMP and 6-Bnz-cAMP 100nM each and the PKA inhibitor PKI 10µ/ml. cAMP EIA assay and PKA activity assay were carried out according to manufacturers’ directions. Signaling events (PKA, NFkB and MAPK) were studied by Western Blot in PKA knockdown (KO) (lentiviral shRNA for PKA) RAW264.7 cells (scrambled shRNA transfection is control for these experiments). OC marker expression was studied by RT-PCR.

Results:

CGS21680 stimulated a maximal increase in cAMP 5-10 minutes after RANKL-CGS21680 activation (35.2±3.5 fmol for CGS21680 vs. 14.4±1.9 fmol for control, p<0.001, n=6) correlating with maximal PKA activity at 15 minutes (4.96±0.2 units/ml vs. 3.07±0.2 units/ml, CGS21680 v control, p<0.001, n=4). PKA-selective cAMP analogues 8-Cl-cAMP and 6-Bnz-cAMP) inhibit OC maturation to 58±9% and 47±3% of control respectively (p<0.001, n=5) whereas a selective PKA inhibitor (PKI) increases OC differentiation (126±6% of control, p<0.001, n=5). Western Blot demonstrates that PKA activation increased over time in the presence of CGS21680, and CGS21680 inhibits NFkB nuclear translocation in control (25±1% inhibition, p<0.001, n=4) cells but not in PKA KO cells (110±2% of control, p<0.5, n=4). CGS21680 activates MAPKs (pERK1/2, p-p38 and pJNK), an effect which is blocked by ZM241385. ERK1/2 is activated by a PKA-dependent mechanism (130±6% of control, p<0.001, n=4), but p38 and pJNK activation is unaffected (112±1% and 110±2% of control, respectively, p<0.5, n=4). A_2AR activation inhibits the expression of OC differentiation markers (2±0.05 and 1.6±0.06 fold decrease for Cathepsin K and Osteopontin respectively, p<0.001, n=4) by a PKA-mediated mechanism (PKA KO cells).

Conclusion: Adenosine, acting at A_2AR, inhibits OC differentiation and regulates bone turnover via activation of PKA and inhibition of NFkB nuclear translocation. Because adenosine mediates the anti-inflammatory effects of methotrexate we speculate that the capacity of methotrexate to inhibit bone erosion in Rheumatoid Arthritis may be mediated by increases in adenosine which inhibit OC formation and function.

---

« Back to 2012 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/adenosine-a2a-receptor-stimulation-inhibits-oc-formation-by-suppressing-nfkb-translocation-to-the-nucleus-by-a-pka-erk12-mediated-mechanism/