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.
Similar content being viewed by others
References
Gruber HE et al (1986) Osteoblast and osteoclast cell number and cell activity in postmenopausal osteoporosis. Miner Electrolyte Metab 12(4):246–254
Lozo P et al (2004) Bone histology in postmenopausal osteoporosis—variations in cellular activity. Acta Med Croatica 58(1):5–11
Reddy SV (2004) Etiology of Paget's disease and osteoclast abnormalities. J Cell Biochem 93(4):688–696
Neale SD et al (2000) Osteoclast differentiation from circulating mononuclear precursors in Paget’s disease is hypersensitive to 1,25-dihydroxyvitamin D(3) and RANKL. Bone 27(3):409–416
Roato I et al (2005) Mechanisms of spontaneous osteoclastogenesis in cancer with bone involvement. FASEB J 19(2):228–230
Roato I et al (2008) Osteoclasts are active in bone forming metastases of prostate cancer patients. PLoS One 3(11):e3627
Tjoa ST et al (2008) Formation of osteoclast-like cells from peripheral blood of periodontitis patients occurs without supplementation of macrophage colony-stimulating factor. J Clin Periodontol 35(7):568–575
Sakellari D, Menti S, Konstantinidis A (2008) Free soluble receptor activator of nuclear factor-kappab ligand in gingival crevicular fluid correlates with distinct pathogens in periodontitis patients. J Clin Periodontol 35(11):938–943
Hirayama T et al (2002) Osteoclast formation and activity in the pathogenesis of osteoporosis in rheumatoid arthritis. Rheumatology (Oxford) 41(11):1232–1239
Gravallese EM (2002) Bone destruction in arthritis. Ann Rheum Dis 61(Suppl 2):ii84–ii86
Faccio R et al (2003) Dynamic changes in the osteoclast cytoskeleton in response to growth factors and cell attachment are controlled by beta3 integrin. J Cell Biol 162(3):499–509
Faccio R et al (2003) c-Fms and the alphavbeta3 integrin collaborate during osteoclast differentiation. J Clin Invest 111(5):749–758
McHugh KP et al (2000) Mice lacking beta3 integrins are osteosclerotic because of dysfunctional osteoclasts. J Clin Invest 105(4):433–440
Nakamura I, Gailit J, Sasaki T (1996) Osteoclast integrin alphaVbeta3 is present in the clear zone and contributes to cellular polarization. Cell Tissue Res 286(3):507–515
Grigoriadis AE et al (1994) c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science 266(5184):443–448
David JP et al (2001) Carbonic anhydrase II is an AP-1 target gene in osteoclasts. J Cell Physiol 188(1):89–97
Matsuo K et al (2004) Nuclear factor of activated T-cells (NFAT) rescues osteoclastogenesis in precursors lacking c-Fos. J Biol Chem 279(25):26475–26480
Cao X et al (1993) Cloning of the promoter for the avian integrin beta 3 subunit gene and its regulation by 1,25-dihydroxyvitamin D3. J Biol Chem 268(36):27371–27380
Reddy SV et al (1995) Characterization of the mouse tartrate-resistant acid phosphatase (TRAP) gene promoter. J Bone Miner Res 10(4):601–606
Takayanagi H et al (2002) Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell 3(6):889–901
Ikeda F et al (2004) Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation. J Clin Invest 114(4):475–484
David JP et al (2002) JNK1 modulates osteoclastogenesis through both c-Jun phosphorylation-dependent and -independent mechanisms. J Cell Sci 115(Pt 22):4317–4325
Mizukami J et al (2002) Receptor activator of NF-kappaB ligand (RANKL) activates TAK1 mitogen-activated protein kinase kinase kinase through a signaling complex containing RANK, TAB2, and TRAF6. Mol Cell Biol 22(4):992–1000
Lee SW et al (2002) TAK1-dependent activation of AP-1 and c-Jun N-terminal kinase by receptor activator of NF-kappaB. J Biochem Mol Biol 35(4):371–376
Huang H et al (2006) Osteoclast differentiation requires TAK1 and MKK6 for NFATc1 induction and NF-kappaB transactivation by RANKL. Cell Death Differ 13(11):1879–1891
Besse A et al (2007) TAK1-dependent signaling requires functional interaction with TAB2/TAB3. J Biol Chem 282(6):3918–3928
Sorrentino A et al (2008) The type I TGF-beta receptor engages TRAF6 to activate TAK1 in a receptor kinase-independent manner. Nat Cell Biol 10(10):1199–1207
Adhikari A, Xu M, Chen ZJ (2007) Ubiquitin-mediated activation of TAK1 and IKK. Oncogene 26(22):3214–3226
Kara FM et al (2010) Adenosine A(1) receptors regulate bone resorption in mice: adenosine A(1) receptor blockade or deletion increases bone density and prevents ovariectomy-induced bone loss in adenosine A(1) receptor-knockout mice. Arthritis Rheum 62(2):534–541
Kara FM et al (2010) Adenosine A1 receptors (A1Rs) play a critical role in osteoclast formation and function. FASEB J 24(7):2325–2333
Lomaga MA et al (1999) TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev 13(8):1015–1024
Kim N et al (2005) Osteoclast differentiation independent of the TRANCE-RANK-TRAF6 axis. J Exp Med 202(5):589–595
Naito A et al (1999) Severe osteopetrosis, defective interleukin-1 signalling and lymph node organogenesis in TRAF6-deficient mice. Genes Cells 4(6):353–362
Mediero A et al (2011) Adenosine A(2A) receptor ligation inhibits osteoclast formation. Am J Pathol 180:775–786
Yamashita T et al (2007) NF-kappaB p50 and p52 regulate receptor activator of NF-kappaB ligand (RANKL) and tumor necrosis factor-induced osteoclast precursor differentiation by activating c-Fos and NFATc1. J Biol Chem 282(25):18245–18253
Iotsova V et al (1997) Osteopetrosis in mice lacking NF-kappaB1 and NF-kappaB2. Nat Med 3(11):1285–1289
Franzoso G et al (1997) Requirement for NF-kappaB in osteoclast and B-cell development. Genes Dev 11(24):3482–3496
Brust TB, Cayabyab FS, MacVicar BA (2007) C-Jun N-terminal kinase regulates adenosine A1 receptor-mediated synaptic depression in the rat hippocampus. Neuropharmacology 53(8):906–917
Liu AM, Wong YH (2004) G16-mediated activation of nuclear factor kappaB by the adenosine A1 receptor involves c-Src, protein kinase C, and ERK signaling. J Biol Chem 279(51):53196–53204
Ikeda F et al (2008) JNK/c-Jun signaling mediates an anti-apoptotic effect of RANKL in osteoclasts. J Bone Miner Res 23(6):907–914
Takaesu G et al (2001) Interleukin-1 (IL-1) receptor-associated kinase leads to activation of TAK1 by inducing TAB2 translocation in the IL-1 signaling pathway. Mol Cell Biol 21(7):2475–2484
Morlon A, Munnich A, Smahi A (2005) TAB2, TRAF6 and TAK1 are involved in NF-kappaB activation induced by the TNF-receptor, Edar and its adaptator Edaradd. Hum Mol Genet 14(23):3751–3757
Ninomiya-Tsuji J et al (1999) The kinase TAK1 can activate the NIK-I kappaB as well as the MAP kinase cascade in the IL-1 signalling pathway. Nature 398(6724):252–256
Verzijl D, Ijzerman AP (2011) Functional selectivity of adenosine receptor ligands. Purinergic Signal 7(2):171–192
Merrill JT et al (1997) Adenosine A1 receptor promotion of multinucleated giant cell formation by human monocytes: a mechanism for methotrexate-induced nodulosis in rheumatoid arthritis. Arthritis Rheum 40:1308–1315
Merrill JT et al (1997) Inhibition of methotrexate-induced rheumatoid nodulosis by colchicine: evidence from an in vitro model and regression in 7 of 14 patients. J Clin Rheumatol 3(6):328–333
Merrill JT et al (1997) Adenosine A1 receptor promotion of multinucleated giant cell formation by human monocytes: a mechanism for methotrexate-induced nodulosis in rheumatoid arthritis. Arthritis Rheum 40(7):1308–1315
Shryock JC, Ozeck MJ, Belardinelli L (1998) Inverse agonists and neutral antagonists of recombinant human A1 adenosine receptors stably expressed in Chinese hamster ovary cells. Mol Pharmacol 53(5):886–893
Teramachi J et al (2011) Adenosine abolishes MTX-induced suppression of osteoclastogenesis and inflammatory bone destruction in adjuvant-induced arthritis. Lab Invest 91(5):719–731
Gharibi B et al (2011) Contrasting effects of A1 and A2b adenosine receptors on adipogenesis. Int J Obes doi:10.1038/ijo.2011.129
Russell JM et al (2007) Adenosine inhibition of lipopolysaccharide-induced interleukin-6 secretion by the osteoblastic cell line MG-63. Calcif Tissue Int 81(4):316–326
Evans BAJ et al (2006) Human osteoblast precursors produce extracellular adenosine, which modulates their secretion of IL-6 and osteoprotegerin. J Bone Miner Res 21(2):228–236
Chan ES, Cronstein BN (2010) Methotrexate—how does it really work? Nat Rev Rheumatol 6(3):175–178
Montesinos C et al (2000) Reversal of the antiinflammatory effects of methotrexate by the nonselective adenosine receptor antagonists theophylline and caffeine. Evidence that the antiinflammatory effects of methotrexate are mediated via multiple adenosine receptors in rat adjuvant arthritis. Arthritis Rheum 43(3):656–663
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.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
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)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11302-012-9292-9