Background/Purpose: Orphan receptors comprise a large number of evolutionarily conserved molecules that have unknown ligands but may have potent biologic effects. Among these receptors are the nuclear receptors (NR) which are important regulators of gene expression; subgroup A of NR (NR4A) includes 3 members, Nurr77 (NR4A1), Nurr-1 (NR4A2) and Nor 1 (NR4A3), important regulators of many physiological and pathological conditions. NR4A2 is involved in the regulation of bone homeostasis by transactivation of the osteoprotegerin promoter in an osteoblast cell line and is also thought to reduce NFkB signaling. Because stimulation of A2AR, which markedly reduces bone resorption by osteoclasts, increases expression of NR4A2 in cells of myeloid origin (monocytes/macrophages) we determined whether NR4A2 might also play a role in regulating bone resorption as well.

Methods: Immunohistochemistry of articular bone was used to determine NR4A2 expression in bone. Osteoclast precursors were isolated from primary bone marrow cells and, along with the murine macrophage cell line RAW 264.7 cells, were induced to differentiate into osteoclasts following treatment with M-CSF and RANKL (30ng/ml). NR4A2 expression was measured in vitro by RT-PCR and Western Blot.

Results: NR4A2 is expressed in articular bone (osteoclasts, osteoblasts and osteocytes) but not articular chondrocytes. During osteoclast differentiation induced by RANKL treatment of primary osteoclast precursors there is a marked reduction of NR4A2 mRNA expression (75% of control, p<0.01). Similarly, RANKL induction of osteoclast differentiation by RAW 264.7 cells is also associated with diminished NR4A2 mRNA expression (Day 3, 99.8% reduction, p<0.01). Moreover immunofluorescence and western blotting data show cytosolic and nuclear NR4A2 negatively correlates with expression of cathepsin-K, consistent with reduced NR4A2 expression during osteoclast differentiation (nuclear NR4A2 is decreased by 27% and 49% at days 3 and 6 of osteoclast differentiation, respectively). Interestingly treatment of osteoclast precursors with CGS21680 (1 uM), a selective A2AR agonist, reverses this expression pattern, significantly increasing NR4A2 mRNA expression in both primary macrophages (2 fold increase vs control; p<0.05) and RAW 264.7 cells (51 fold increase vs control; p<0.01).

Conclusion: Reduced expression of NR4A2 is characteristic of osteoclast differentiation and stimulation of A2AR receptors on osteoclast precursors reverses both the reduction in NR4A2 expression and osteoclast differentiation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a2a-adenosine-receptor-a2ar-stimulation-modulates-nr4a2-orphan-receptor-expression-during-osteoclast-differentiation/