Xin Cheng1,2, Chengcheng Yin3, Yongqiang Deng2, Zubing Li4. 1. The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, 430079, China. 2. Department of Stomatology, Shenzhen University General Hospital, Shenzhen University Clinical Medical Academy, 1098 Xueyuan Road, Nanshan District, Shenzhen, 518055, Guangdong Province, China. 3. School and Hospital of Stomatology, China Medical University, Shenyang, 110002, China. 4. The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, 430079, China. lizubing@whu.edu.cn.
Abstract
BACKGROUND: Adenosine is a purine nucleoside involved in regulating bone homeostasis through binding to A1, A2A, A2B, and A3 adenosine receptors (A1R, A2AR, A2BR, and A3R, respectively). However, the underlying mechanisms by which adenosine and receptor subtypes regulate osteoclast differentiation remain uncertain. This study aims to assess the role of exogenous adenosine and receptor subtypes in receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation and explore the underlying molecular mechanisms. METHODS AND RESULTS: The nanofibrous mats incorporated with adenosine exhibited robust ability to facilitate rat critical-size calvarial defect healing with decreased number of osteoclasts. Moreover, exogenous adenosine substantially enhanced the expression of A2AR and suppressed tartrate-resistant acid phosphatase-positive osteoclast formation and expression of osteoclast-related genes Ctsk, NFATc1, MMP9, and ACP5. This enhancement and suppression could be reversed by adding an A2AR antagonist, ZM241385, in RAW264.7 cells. Finally, RNA sequencing showed that the expression of Fos-related antigen 2 (Fra2) was distinctly downregulated through stimulation of adenosine in RAW264.7 cells treated with RANKL. This downregulation was reversed by ZM241385 according to real-time PCR, Western blot, and immunofluorescence analyses. CONCLUSIONS: These findings demonstrated that exogenous adenosine binding to A2AR attenuated osteoclast differentiation via the inhibition of activating protein-1 (AP-1, including Fra2 subunit) pathway both in vitro and in vivo.
BACKGROUND: Adenosine is a purine nucleoside involved in regulating bone homeostasis through binding to A1, A2A, A2B, and A3 adenosine receptors (A1R, A2AR, A2BR, and A3R, respectively). However, the underlying mechanisms by which adenosine and receptor subtypes regulate osteoclast differentiation remain uncertain. This study aims to assess the role of exogenous adenosine and receptor subtypes in receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation and explore the underlying molecular mechanisms. METHODS AND RESULTS: The nanofibrous mats incorporated with adenosine exhibited robust ability to facilitate rat critical-size calvarial defect healing with decreased number of osteoclasts. Moreover, exogenous adenosine substantially enhanced the expression of A2AR and suppressed tartrate-resistant acid phosphatase-positive osteoclast formation and expression of osteoclast-related genes Ctsk, NFATc1, MMP9, and ACP5. This enhancement and suppression could be reversed by adding an A2AR antagonist, ZM241385, in RAW264.7 cells. Finally, RNA sequencing showed that the expression of Fos-related antigen 2 (Fra2) was distinctly downregulated through stimulation of adenosine in RAW264.7 cells treated with RANKL. This downregulation was reversed by ZM241385 according to real-time PCR, Western blot, and immunofluorescence analyses. CONCLUSIONS: These findings demonstrated that exogenous adenosine binding to A2AR attenuated osteoclast differentiation via the inhibition of activating protein-1 (AP-1, including Fra2 subunit) pathway both in vitro and in vivo.
Authors: Bertil B Fredholm; Adriaan P IJzerman; Kenneth A Jacobson; Joel Linden; Christa E Müller Journal: Pharmacol Rev Date: 2011-02-08 Impact factor: 25.468