Adenosine Regulates Bone Metabolism Via A₁, A_2A and A_2B Receptors in Bone Marrow Cells From Normal and Patients with Multiple Myeloma

Multiple myeloma is characterized by osteolytic bone lesions, wherein coupled bone remodeling is disrupted with increased osteoclast activation and decreased osteoblast differentiation. We have previously demonstrated that adenosine, acting via A_2A receptors, diminishes human and murine osteoclast formation and others have reported that adenosine, acting at A_2Breceptors, promotes osteoblast differentiation in murine osteoblast precursors and cell lines. In this study, we examined the effect of adenosine on osteoblast and osteoclast differentiation derived from multiple myeloma (MM) patients.

Human bone marrow was collected from multiple myeloma patients. Bone marrow stromal cells (BMSCs) and bone marrow derived mononuclear (BMMs) cells were isolated and osteoblasts and osteoclastswere cultured, respectively. Adenosine A₁ receptor agonist CHA and antagonist Rolofylline, A_2A receptor agonist CGS and antagonist ZM, and A_2b receptor agonist BAY and antagonist MRS 1754, A₃receptor agonist IB-MECA and antagonist MRS 1191; and dipyridamole, a nucleoside transport inhibitor, were added to the culture media. Alkaline phosphatase (ALP) activity assay was used to quantitate the osteoblast differentiation. In vitro osteoblast calcification was determined by alizarin red staining. TRAP+ staining was used to examine the osteoclast differentiation and bone resorption assay was used to study the osteoclast activity.

We found that A₁R blockade by rolofyllineand A_2aR ligation by CGS21680 inhibited differentiation of both normal and MM BMMs into TRAP+ multinucleated cells (IC₅₀= 1nM for A₁R, IC₅₀= 10μM for A_2AR;p<0. 001, n=3 for both). The inhibition of osteoclast differentiation by Rolofylline was also seen in bone resorption assay (Pit formation assay). The A_2A receptor antagonist completely reversed the effects of CGS21680 on osteoclast differentiation. Moreover, enhanced adenosine accumulation in the presence of dipyridamole (0. 5μM) and A_2BR activation promoted the differentiation of BMSCs from myeloma patients into osteoblasts shown byArlizarin red staining and ALP activity assay (by 1. 8 ± 0. 41 and 1. 57 ± 0. 26 fold, respectively, p<0. 05, compared with osteogenic media only, n=3 for both).

These results indicate that adenosine A₂ receptors may be useful targets for the treatment and prevention of MM-induced bone disease.

No relevant conflicts of interest to declare.