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Adenosine A1 receptor regulates osteoclast formation by altering TRAF6/TAK1 signaling

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Abstract

Adenosine is an endogenous nucleoside that modulates many physiological processes through four receptor subtypes (A1, A2a, A2b, A3). Previous work from our laboratory has uncovered a critical role for adenosine A1 receptor (A1 R) in osteoclastogenesis both in vivo and in vitro. Our current work focuses on understanding the details of how A1 R modulates the receptor activator of NF-κB ligand (RANKL)-induced signaling in osteoclastogenesis. Osteoclasts were generated from mouse bone marrow precursors in the presence of RANKL and macrophage-colony stimulating factor. A pharmacological antagonist of A1 R (DPCPX) inhibited RANKL-induced osteoclast differentiation, including osteoclast-specific genes (Acp5, MMP9, β 3 Integrin, α v Integrin, and CTSK) and osteoclast-specific transcription factors such as c-fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) expression in a dose-dependent manner. DPCPX also inhibited RANKL-induced activation of NF-κB and JNK/c-Jun but had little effect on other mitogen-activated protein kinases (p38 and Erk). Finally, immunoprecipitation analysis showed that blockade of A1R resulted in disruption of the association of tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β-activated kinase 1 (TAK1), a signaling event that is important for activation of NF-κB and JNK, suggesting the participation of adenosine/A1R in early signaling of RANKL. Collectively, these data demonstrated an important role of adenosine, through A1R in RANKL-induced osteoclastogenesis.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (AR56672, AR56672S1 and AR54897), the NYU-HHC Clinical and Translational Science Institute (UL1RR029893) and the Vilcek Foundation.

Disclosure

Bruce Cronstein, MD. Consultant (within the past 2 years), all <$10,000: Bristol-Myers Squibb, Novartis, CanFite Biopharmaceuticals, Cypress Laboratories, Regeneron (Westat, DSMB), Endocyte, Protalex, Allos, Inc., Savient,. Equity: CanFite Biopharmaceuticals received for membership in Scientific Advisory Board. Grants: King Pharmaceuticals, NIH, Vilcek Foundation, OSI Pharmaceuticals, URL Pharmaceuticals, Inc. Board Member: Vilcek Foundation. Intellectual Property: Patents on use of adenosine A2A receptor agonists to promote wound healing and use of A2A receptor antagonists to inhibit fibrosis. Patent on use of adenosine A1 receptor antagonists to treat osteoporosis and other diseases of bone. Patent on the use of adenosine A1 and A2B Receptor antagonists to treat fatty liver. Patent on the use of adenosine A2A receptor agonists to prevent prosthesis loosening.

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  1. New York University School of Medicine, 550 First Avenue, New York, NY, 10016, USA

    W. He & B. N. Cronstein

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  1. W. He
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Figure 1

Suppression of osteoclast formation by A2bR-selective agonist. Murine BMMs (1 × 105 cell/cm2) were cultured with M-CSF and RANKL (30 ng/ml each), with or without various concentrations of BAY 60–6,583 for 5 days in 48-well plates for TRAP staining (a). b Numbers of TRAP-positive multinuclear cells containing more than three nuclei (TRAP + MNC) were counted. c BMMs were cultured with M-CSF and RANKL (30 ng/ml each), with or without various concentrations of BAY 60–6,583 in 6-well plates for 5 days prior to RNA extraction and real-time PCR for Ctsk. β-actin served as PCR control. Relative expression was calculated relative to M-CSF only cells (fold value 1). Values are shown as means ± S.D. of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to RANKL + M-CSF cells (JPEG 96 kb)

Figure 2

Suppression of the RANKL-induced expression of Ctsk by A1R-selective antagonist, Rolofylline. BMMs were cultured with M-CSF and RANKL (30 ng/ml each), in the presence or absence of 1 μM KW3902 for 5 days. Total RNA was isolated and Ctsk mRNA levels were quantified by real-time PCR. Relative expression in mRNA levels was calculated relative to M-CSF only cells (fold value 1). Values are shown as means ± S.D. of three independent experiments. ***P < 0.001 compared to RANKL + M-CSF cells (JPEG 11 kb)

Figure 3

A1R-selective agonist N6-cyclopentyladenosine (CPA) neither directly affected Ctsk expression nor reversed the DPCPX-mediated inhibition of Ctsk expression. BMMs were cultured with M-CSF and RANKL (30 ng/ml each), with or without various concentrations of DPCPX in the presence or absence of 1 μM CPA for 5 days. Total RNA was isolated and Ctsk mRNA levels were quantified by real-time PCR. Relative expression in mRNA levels was calculated relative to M-CSF only cells (fold value 1). Values are shown as means ± S.D. of four independent experiments. **P < 0.01, ***P < 0.001 compared to RANKL + M-CSF cells (JPEG 24 kb)

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He, W., Cronstein, B.N. Adenosine A1 receptor regulates osteoclast formation by altering TRAF6/TAK1 signaling. Purinergic Signalling 8, 327–337 (2012). https://doi.org/10.1007/s11302-012-9292-9

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