title: Rolofylline, an adenosine A1 receptor antagonist, inhibits osteoclast differentiation as an inverse agonist.
abstract: OBJECTIVE
  Adenosine may be generated by hydrolysis of extracellular nucleotides by ectonucleotidases, including ectonucleoside triphosphate diphosphohydrolase 1 (CD39), ecto-5'-nucleotidase (CD73), nucleotide pyrophosphatase phosphodiesterase 1 (NPP-1) and tissue non-specific alkaline phosphatase (TNAP). Previous work from our laboratory has uncovered a critical role for adenosine A1 receptors (A1 R) in osteoclastogenesis; blockade or deletion of these receptors diminishes osteoclast differentiation. Interestingly, selective A1 R agonists neither affect basal osteoclastogenesis nor do they reverse A1 R antagonist-mediated inhibition of osteoclastogenesis. In this study, we determined whether ectonucleotidase-mediated adenosine production was required for A1 R antagonist-mediated inhibition, and, when we saw no effect, determined whether A1 R was constitutively activated and the antagonist was acting as an inverse agonist to diminish osteoclast differentiation.
  
  
  METHODS
  Osteoclast formation derived from wild-type, CD39 knockout (KO), CD73 KO, NPP-1 KO and TNAP KO mice was examined by tartrate-resistant acid phosphatase staining of receptor activator of NF-κB ligand-macrophage colony-stimulating factor-stimulated osteoclasts and osteoclast gene expression (Ctsk, Acp5, MMP-9 and NFATc1). Intracellular cAMP concentration was determined by elisa.
  
  
  RESULTS
  Rolofylline inhibited osteoclast formation in a dose-dependent manner (IC50 = 20-70 nM) in mice lacking all four of these phosphatases, although baseline osteoclast formation was significantly less in precursors from CD73 KO mice. Rolofylline (1 μM) stimulates cAMP production in bone marrow macrophages by 10.23 ± 0.89-fold.
  
  
  CONCLUSIONS
  Based on these findings, we hypothesize that the A1 R is constitutively activated in osteoclast precursors, thereby diminishing basal AC activity, and that A1 R antagonists act as inverse agonists to release A1 R-mediated inhibition of basal AC activity and permit osteoclast differentiation. The constitutive activity of A1 R promotes osteoclast formation and down-regulation of this activity blocks osteoclast formation.
authors: New York University School of Medicine, New York, NY, USA.
authors: Wenjie
authors: W
authors: He
authors: Tuere
authors: T
authors: Wilder
authors: Bruce N
authors: BN
authors: Cronstein
mesh_terms: D015720:5'-Nucleotidase / Q000172:deficiency / Q000235:genetics; D058916:Adenosine A1 Receptor Antagonists / Q000494:pharmacology*; D000469:Alkaline Phosphatase / Q000172:deficiency / Q000235:genetics; D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D001081:Apyrase / Q000172:deficiency / Q000235:genetics; D001854:Bone Marrow Cells; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D005260:Female; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects*; D010727:Phosphoric Diester Hydrolases / Q000172:deficiency / Q000235:genetics; D011755:Pyrophosphatases / Q000172:deficiency / Q000235:genetics; D014970:Xanthines / Q000494:pharmacology*
chemical_list: D058916:Adenosine A1 Receptor Antagonists; D015703:Antigens, CD; D014970:Xanthines; C073525:rolofylline; D000242:Cyclic AMP; C510878:ALPL protein, mouse; D000469:Alkaline Phosphatase; D015720:5'-Nucleotidase; D010727:Phosphoric Diester Hydrolases; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; D011755:Pyrophosphatases; D001081:Apyrase; C067869:CD39 antigen
keywords: adenosine A1 receptor; inverse agonist; osteoclastogenesis
references: Br J Pharmacol. 1996 Apr;117(8):1645-52
references: 8732272
references: Arthritis Rheum. 2010 Feb;62(2):534-41
references: 20112380
references: FASEB J. 2001 May;15(7):1139-48
references: 11344082
references: J Biol Chem. 2003 Apr 11;278(15):13468-79
references: 12560324
references: J Biochem. 1978 May;83(5):1471-83
references: 26678
references: Calcif Tissue Int. 2007 Oct;81(4):316-26
references: 17705048
references: J Biol Chem. 2012 May 4;287(19):15718-27
references: 22403399
references: Purinergic Signal. 2007 Mar;3(1-2):171-80
references: 18404431
references: Crit Rev Eukaryot Gene Expr. 2003;13(2-4):243-53
references: 14696971
references: Mol Pharmacol. 1998 May;53(5):886-93
references: 9584215
references: Cell Death Dis. 2011 Jun 02;2:e165
references: 21633388
references: Bone. 2007 Oct;41(4):549-61
references: 17669706
references: Histochem Cell Biol. 1997 Mar;107(3):183-91
references: 9105889
references: J Histochem Cytochem. 2002 Mar;50(3):333-40
references: 11850436
references: Handb Exp Pharmacol. 2009;(193):25-58
references: 19639278
references: Biochemistry. 1999 Oct 12;38(41):13473-9
references: 10521254
references: Front Biosci (Elite Ed). 2011 Jun 01;3:888-95
references: 21622100
references: Am J Pathol. 2012 Feb;180(2):775-86
references: 22138579
references: PLoS One. 2012;7(5):e37100
references: 22666342
references: J Med Chem. 1997 Jun 6;40(12):1773-8
references: 9191953
references: Clin Orthop Relat Res. 1978 Sep;(135):218-25
references: 709934
references: J Pharmacol Exp Ther. 2007 Feb;320(2):637-45
references: 17077318
references: Br J Pharmacol. 2010 Aug;160(7):1577-9
references: 20649561
references: Mol Pharmacol. 1992 Nov;42(5):831-7
references: 1331765
references: Neuroscience. 2007 Dec 19;150(4):863-79
references: 18031938
references: Bone. 2010 Nov;47(5):926-37
references: 20696287
references: J Cell Physiol. 2012 Jun;227(6):2622-31
references: 21882189
references: Bone. 2003 Jan;32(1):1-7
references: 12584029
references: Purinergic Signal. 2012 Jun;8(2):327-37
references: 22311477
references: J Exp Med. 2004 Dec 6;200(11):1395-405
references: 15583013
references: J Biol Chem. 2005 Apr 29;280(17):16909-15
references: 15722352
references: Curr Pharm Des. 2002;8(26):2333-44
references: 12369948
references: J Immunol. 2007 Jun 15;178(12):8127-37
references: 17548651
references: Science. 2006 Dec 15;314(5806):1792-5
references: 17170310
references: Proc Natl Acad Sci U S A. 2005 Dec 20;102(51):18706-11
references: 16352729
references: Pharmacol Ther. 2005 Jul;107(1):1-30
references: 15963349
references: J Physiol. 1998 Sep 1;511 ( Pt 2):495-500
references: 9706025
references: Br J Pharmacol. 2010 Aug;160(7):1573-6
references: 20649560
references: Sci Transl Med. 2012 May 23;4(135):135ra65
references: 22623741
references: Arterioscler Thromb Vasc Biol. 2005 Apr;25(4):686-91
references: 15625282
references: J Immunol. 2011 Oct 15;187(8):4256-67
references: 21918191
references: Crit Rev Biochem Mol Biol. 2000;35(6):393-432
references: 11202013
references: Hepatology. 2010 May;51(5):1702-11
references: 20146261
references: J Bone Miner Res. 2000 Oct;15(10):1879-88
references: 11028439
references: FASEB J. 2011 Apr;25(4):1264-74
references: 21233486
references: J Bone Miner Res. 2006 Feb;21(2):228-36
references: 16418778
references: Bone. 2011 Apr 1;48(4):938-44
references: 21193069
references: J Exp Med. 1998 Oct 19;188(8):1433-43
references: 9782120