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Adenosine Inhibition of Lipopolysaccharide-Induced Interleukin-6 Secretion by the Osteoblastic Cell Line MG-63

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Abstract

Adenosine is known to inhibit inflammatory responses in many cell systems via a family of purine receptors termed “P1.” The P1 family consists of the adenosine receptors (ADORA) of subtypes A1, A2a, A2b, and A3. In order to assess whether adenosine has anti-inflammatory actions in osteoblastic cells, we investigated its effects on lipopolysaccharide (LPS)-induced interleukin 6 (IL-6) release in an in vitro inflammatory functional response model. We showed that the osteoblastic cell line MG-63 expresses ADORA1, A2a, and A2b but not A3. Treatment of MG-63 cells with adenosine and pharmacological ADORA agonist 5′-N-ethylcarboxamidoadenosine or 2–[4-(2-p-carboxyethyl)phenylamino]-5′-N-ethylcarboxamidoadenosine (CGS21680) inhibits LPS-induced IL-6 release. This inhibition was protein kinase A (PKA)-dependent and mimicked by treatment with the adenylate cyclase activator forskolin. Treatment of MG-63 with the ADORA2a-specific antagonist ZM241385 partially reversed the inhibitory effects of ADORA stimulation on LPS-induced IL-6 release. Overall, these data suggest that ADORA2a is involved in the regulation of LPS-induced IL-6 release, thus illustrating a regulatory role for adenosine receptors in the control of inflammation and potentially osteoclastogenesis and bone resorption.

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References

  1. Drury AN, Szent-Györgyi A (1929) The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. J Physiol 68:213–237

    PubMed  CAS  Google Scholar 

  2. Fredholm BB, Chen J-F, Masino SA, Vaugeois J-M (2005) Actions of adenosine at its receptors in the CNS: insights from knockouts and drugs. Annu Rev Pharmacol Toxicol 45:385–412

    Article  PubMed  CAS  Google Scholar 

  3. Fredholm BB, Ijzerman AP, Jacobson KA, Klotz K-N, Linden J (2001) International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev 53:527–552

    PubMed  CAS  Google Scholar 

  4. Linden J (2005) Adenosine in tissue protection and tissue regeneration. Mol Pharmacol 67:1385–1387

    Article  PubMed  CAS  Google Scholar 

  5. Schulte G, Fredholm BB (2003) Signalling from adenosine receptors to mitogen-activated protein kinases. Cell Signal 15:813

    Article  PubMed  CAS  Google Scholar 

  6. Burnstock G (2002) Potential therapeutic targets in the rapidly expanding field of purinergic signalling. Clin Med J R Coll Physicians 2:45–53

    CAS  Google Scholar 

  7. Lappas CM, Sullivan GW, Linden J (2005) Adenosine A2a agonists in development for the treatment of inflammation. Expert Opin Investig Drugs 14:797–806

    Article  PubMed  CAS  Google Scholar 

  8. Kowaluk EA (1998) Adenosine modulation: a novel approach to analgesia and inflammation. Expert Opin Investig Drugs 7:535–543

    Article  PubMed  CAS  Google Scholar 

  9. Hayashida M, Fukuda K-i, Fukunaga A (2005) Clinical application of adenosine and ATP for pain control. J Anesthesia 19:225

    Article  Google Scholar 

  10. Londos C, Cooper DMF, Wolff J (1980) Subclasses of external adenosine receptors. Proc Natl Acad Sci USA 77:2551–2554

    Article  PubMed  CAS  Google Scholar 

  11. Hasko G, Deitch EA, Szabo C, Nemeth ZH, Vizi ES (2002) Adenosine: a potential mediator of immunosuppression in multiple organ failure. Curr Opin Pharmacol 2:440

    Article  PubMed  CAS  Google Scholar 

  12. Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50:413–492

    PubMed  CAS  Google Scholar 

  13. Shimegi S (1996) ATP and adenosine act as a mitogen for osteoblast-like cells (MC3T3-E1). Calcif Tissue Int 58:109–113

    PubMed  CAS  Google Scholar 

  14. Burnstock G (1990) Overview. Purinergic mechanisms. Ann N Y Acad Sci 603:1b–17

    Article  Google Scholar 

  15. Olah M, Stiles G (1995) Adenosine receptor subtypes: characterization and therapeutic regulation. Annu Rev Pharmacol Toxicol 35:581–606

    Article  PubMed  CAS  Google Scholar 

  16. Pintor J, Miras-Portugal M-T, Fredholm BB (2000) Research on purines and their receptors comes of age. Trends Pharmacol Sci 21:453–456

    Article  PubMed  CAS  Google Scholar 

  17. Bruns RF, Lu GH, Pugsley TA (1986) Characterization of the A2 adenosine receptor labeled by [3H]NECA in rat striatal membranes. Mol Pharmacol 29:331–346

    PubMed  CAS  Google Scholar 

  18. Dixon A, Gubitz A, Sirinathsinghji D, Richardson P, Freeman T (1996) Tissue distribution of adenosine receptor mRNAs in the rat. Br J Pharmacol 118:1461–1468

    PubMed  CAS  Google Scholar 

  19. Kadowaki M, Takeda M, Tokita K, Hanaoka K, Tomoi M (2000) Molecular identification and pharmacological characterization of adenosine receptors in the guinea-pig colon. Br J Pharmacol 129:871–876

    Article  PubMed  CAS  Google Scholar 

  20. Koolpe M, Pearson D, Benton HP (1999) Expression of both P1 and P2 purine receptor genes by human articular chondrocytes and profile of ligand-mediated prostaglandin E2 release. Arthritis Rheum 42:258–267

    Article  PubMed  CAS  Google Scholar 

  21. Linden J (1994) Cloned adenosine A3 receptors: pharmacological properties, species differences and receptor functions. Trends Pharmacol Sci 15:298–306

    Article  PubMed  CAS  Google Scholar 

  22. Tesch AM, MacDonald MH, Kollias-Baker C, Benton HP (2002) Chondrocytes respond to adenosine via A2 receptors and activity is potentiated by an adenosine deaminase inhibitor and a phosphodiesterase inhibitor. Osteoarthritis Cartilage 10:34–43

    Article  PubMed  CAS  Google Scholar 

  23. Tesch AM, MacDonald MH, Kollias-Baker C, Benton HP (2002) Effects of an adenosine kinase inhibitor and an adenosine deaminase inhibitor on accumulation of extracellular adenosine by equine articular chondrocytes. Am J Vet Res 63:1512–1519

    Article  PubMed  CAS  Google Scholar 

  24. Tesch AM, MacDonald MH, Kollias-Baker C, Benton HP (2004) Endogenously produced adenosine regulates articular cartilage matrix homeostasis: enzymatic depletion of adenosine stimulates matrix degradation. Osteoarthritis Cartilage 12:349

    Article  PubMed  CAS  Google Scholar 

  25. Fiebich BL, Akundi RS, Biber K, Hamke M, Schmidt C, Butcher RD, Calker DV, Willmroth F (2005) IL-6 expression induced by adenosine A2b receptor stimulation in U373 MG cells depends on p38 mitogen activated kinase and protein kinase C. Neurochem Int 46:510–512

    Article  CAS  Google Scholar 

  26. Fiebich BL, Biber K, Gyufko K, Berger M, Bauer J, van Calker D (1996) Adenosine A2b Receptors mediate an increase in interleukin (IL)-6 mRNA and IL-6 protein synthesis in human astroglioma cells. J Neurochem 66:1426–1431

    Article  PubMed  CAS  Google Scholar 

  27. Meng F, Xie G, Chalmers D, Morgan C, Watson S, Akil H (1994) Cloning and expression of the A2a adenosine receptor from guinea pig brain. Neurochem Res 19:613–621

    Article  PubMed  CAS  Google Scholar 

  28. Furlong TJ, Pierce KD, Selbie LA, Shine J (1992) Molecular characterization of a human brain adenosine A2 receptor. Mol Brain Res 15:62–66

    Article  PubMed  CAS  Google Scholar 

  29. Linden J, Thai T, Figler H, Jin X, Robeva AS (1999) Characterization of human A2b adenosine receptors: radioligand binding, Western blotting, and coupling to Gq in human embryonic kidney 293 cells and HMC-1 mast cells. Mol Pharmacol 56:705–713

    PubMed  CAS  Google Scholar 

  30. Genetos D, Geist D, Liu D, Donahue H, Duncan R (2005) Fluid shear-induced ATP secretion mediates prostaglandin release in MC3T3-E1 osteoblasts. J Bone Miner Res 20:41–49

    Article  PubMed  CAS  Google Scholar 

  31. Li J, Liu D, Ke HZ, Duncan RL, Turner CH (2005) The P2X7 nucleotide receptor mediates skeletal mechanotransduction. J Biol Chem 280:42952–42959

    Article  PubMed  CAS  Google Scholar 

  32. You J, Jacobs CR, Steinberg TH, Donahue HJ (2002) P2Y purinoceptors are responsible for oscillatory fluid flow-induced intracellular calcium mobilization in osteoblastic cells. J Biol Chem 277:48724–48729

    Article  PubMed  CAS  Google Scholar 

  33. Billiau A, Edy VG, Heremans H, Van Damme J, Desmyter J, Georgiades JA, De Somer P (1977) Human interferon: mass production in a newly established cell line, MG-63. Antimicrob Agents Chemother 12:11–15

    PubMed  CAS  Google Scholar 

  34. Angeli A, Dovio A, Sartori ML, Masera RG, Ceoloni B, Prolo P, Racca S, Chiappelli F (2002) Interactions between glucocorticoids and cytokines in the bone microenvironment. Ann N Y Acad Sci 966:97–107

    Article  PubMed  CAS  Google Scholar 

  35. Dovio A, Masera RG, Sartori ML, Racca S, Angeli A (2001) Autocrine up-regulation of glucocorticoid receptors by interleukin-6 in human osteoblast-like cells. Calcif Tissue Int 69:299–304

    Article  Google Scholar 

  36. Suda T, Udagawa N, Nakamura I, Miyaura C, Takahashi N (1995) Modulation of osteoclast differentiation by local factors. Bone 17:87S–91S

    Article  PubMed  CAS  Google Scholar 

  37. Wong P, Quinn J, Sims N, Nieuwenhuijze A, Campbell I, Wicks I (2006) Interleukin-6 modulates production of T lymphocyte-derived cytokines in antigen-induced arthritis and drives inflammation-induced osteoclastogenesis. Arthritis Rheum 54:158–168

    Article  PubMed  CAS  Google Scholar 

  38. Ohsaki Y, Takahashi S, Scarcez T, Demulder A, Nishihara T, Williams R, Roodman GD (1992) Evidence for an autocrine/paracrine role for interleukin-6 in bone resorption by giant cells from giant cell tumors of bone. Endocrinology 131:2229–2234

    Article  PubMed  CAS  Google Scholar 

  39. Reddy S, Takahashi S, Dallas M, Williams R, Neckers L, Roodman GD (1994) Interleukin-6 antisense deoxyoligonucleotides inhibit bone resorption by giant cells from human giant cell tumors of bone. J Bone Miner Res 9:753–757

    Article  PubMed  CAS  Google Scholar 

  40. Roodman GD, Kurihara N, Ohsaki Y, Kukita A, Hosking D, Demulder A, Smith JF, Singer FR (1992) Interleukin 6. A potential autocrine/paracrine factor in Paget’s disease of bone. J Clin Invest 89:46–52

    Article  PubMed  CAS  Google Scholar 

  41. Kurihara N, Bertolini D, Suda T, Akiyama Y, Roodman GD (1990) IL-6 stimulates osteoclast-like multinucleated cell formation in long term human marrow cultures by inducing IL-1 release. J Immunol 144:4226–4230

    PubMed  CAS  Google Scholar 

  42. Manolagas SC (1998) The role of IL-6 type cytokines and their receptors in bone. Ann N Y Acad Sci 840:194–204

    Article  PubMed  CAS  Google Scholar 

  43. Jilka RL, Weinstein RS, Bellido T, Parfitt AM, Manolagas SC (1998) Osteoblast programmed cell death (apoptosis): modulation by growth factors and cytokines. J Bone Miner Res 13:793–802

    Article  PubMed  CAS  Google Scholar 

  44. Weitzmann MN, Pacifici R (2006) Estrogen deficiency and bone loss: an inflammatory tale. J Clin Invest 116:1186–1194

    Article  PubMed  CAS  Google Scholar 

  45. Stein B, Yang MX (1995) Repression of the interleukin-6 promoter by estrogen receptor is mediated by NF-kappa B and C/EBP beta. Mol Cell Biol 15:4971–4979

    PubMed  CAS  Google Scholar 

  46. Baumhauer J, O’Keefe R, Schon L, Pinzur M (2006) Cytokine-induced osteoclastic bone resorption in charcot arthropathy: an immunohistochemical study. Foot Ankle Int 27:797–800

    PubMed  Google Scholar 

  47. Sezer O (2005) Myeloma bone disease. Hematology 10:19–24

    Article  PubMed  CAS  Google Scholar 

  48. Iwamoto J, Takeda T, Ichimura S (2002) Increased bone resorption with decreased activity and increased recruitment of osteoblasts in osteogenesis imperfecta type I. J Bone Miner Metab 20:174–179

    Article  PubMed  Google Scholar 

  49. Tanaka Y, Nakayamada S, Okada Y (2005) Osteoblasts and osteoclasts in bone remodeling and inflammation. Curr Drug Targets Inflam Allergy 4:325

    Article  CAS  Google Scholar 

  50. Evans BAJ, Elford C, Pexa A, Francis K, Hughes AC, Deussen A, Ham J (2006) Human osteoblast precursors produce extracellular adenosine, which modulates their secretion of IL-6 and osteoprotegerin. J Bone Miner Res 21:228–236

    Article  PubMed  CAS  Google Scholar 

  51. Wang X, Seed B (2003) A PCR primer bank for quantitative gene expression analysis. Nucl Acids Res 31:e154

    Article  PubMed  CAS  Google Scholar 

  52. Schlotzer-Schrehardt U, Zenkel M, Decking U, Haubs D, Kruse FE, Junemann A, Coca-Prados M, Naumann GOH (2005) Selective upregulation of the A3 adenosine receptor in eyes with pseudoexfoliation syndrome and glaucoma. Invest Ophthalmol Vis Sci 46:2023–2034

    Article  PubMed  Google Scholar 

  53. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354

    Article  PubMed  CAS  Google Scholar 

  54. Bost KL, Ramp WK, Nicholson NC, Bento JL, Marriott I, Hudson MC (1999) Staphylococcus aureus infection of mouse or human osteoblasts induces high levels of interleukin-6 and interleukin-12 production. J Infect Dis 180:1912–1920

    Article  PubMed  CAS  Google Scholar 

  55. Palmer TM, Gettys TW, Stiles GL (1995) Differential interaction with and regulation of multiple G-proteins by the rat A3 adenosine receptor. J Biol Chem 270:16895–16902

    Article  PubMed  CAS  Google Scholar 

  56. Robinson AJ, Dickenson JM (2001) Regulation of p42/p44 MAPK and p38 MAPK by the adenosine A1 receptor in DDT1MF-2 cells. Eur J Pharmacol 413:151

    Article  PubMed  CAS  Google Scholar 

  57. Peck W, Carpenter J, Shuster R (1976) Adenosine-mediated stimulation of bone cell adenylate cyclase activity. Endocrinology 99:901–909

    PubMed  CAS  Google Scholar 

  58. Takahashi N, Tetsuka T, Uranishi H, Okamoto T (2002) Inhibition of the NF-kB transcriptional activity by protein kinase A. Eur J Biochem 269:4559–4565

    Article  PubMed  CAS  Google Scholar 

  59. Vassiliou E, Jing H, Ganea D (2003) Prostaglandin E2 inhibits TNF production in murine bone marrow-derived dendritic cells. Cell Immunol 223:120–132

    Article  PubMed  CAS  Google Scholar 

  60. Song Z, Chen T, Deaciuc IV, Uriarte S, Hill D, Barve S, McClain CJ (2004) Modulation of endotoxin stimulated interleukin-6 production in monocytes and Kupffer cells by S-adenosylmethionine (SAMe). Cytokine 28:214–223

    Article  PubMed  CAS  Google Scholar 

  61. Phelps PT, Anthes JC, Correll CC (2006) Characterization of adenosine receptors in the human bladder carcinoma T24 cell line. Eur J Pharmacol 536:28–37

    Article  PubMed  CAS  Google Scholar 

  62. Olah ME (1997) Identification of A2a adenosine receptor domains involved in selective coupling to Gs. Analysis of chimeric A1/A2a adenosine receptors. J Biol Chem 272:337–344

    PubMed  CAS  Google Scholar 

  63. Pierce KD, Furlong TJ, Selbie LA, Shine J (1992) Molecular cloning and expression of an adenosine A2b receptor from human brain. Biochem Biophys Res Commun 187:86

    Article  PubMed  CAS  Google Scholar 

  64. Schulte G, Fredholm BB (2003) The Gs-coupled adenosine A2b receptor recruits divergent pathways to regulate ERK1/2 and p38. Exp Cell Res 290:168–176

    Article  PubMed  CAS  Google Scholar 

  65. Eliasson L, Ma X, Renstrom E, Barg S, Berggren P-O, Galvanovskis J, Gromada J, Jing X, Lundquist I, Salehi A, Sewing S, Rorsman P (2003) SUR1 regulates PKA-independent cAMP-induced granule priming in mouse pancreatic B-cells. J Gen Physiol 121:181–197

    Article  PubMed  CAS  Google Scholar 

  66. Fujimoto K, Shibasaki T, Yokoi N, Kashima Y, Matsumoto M, Sasaki T, Tajima N, Iwanaga T, Seino S (2002) Piccolo, a Ca2+ sensor in pancreatic beta-cells. Involvement of cAMP-GEFII.Rim2.piccolo complex in cAMP-dependent exocytosis. J Biol Chem 277:50497–50502

    Article  PubMed  CAS  Google Scholar 

  67. Kang G, Joseph JW, Chepurny OG, Monaco M, Wheeler MB, Bos JL, Schwede F, Genieser H-G, Holz GG (2003) Epac-selective cAMP analog 8-pCPT-2’-O-Me-cAMP as a stimulus for Ca2+-induced Ca2+ release and exocytosis in pancreatic beta-cells. J Biol Chem 278:8279–8285

    Article  PubMed  CAS  Google Scholar 

  68. Kashima Y, Miki T, Shibasaki T, Ozaki N, Miyazaki M, Yano H, Seino S (2001) Critical role of cAMP-GEFII.Rim2 complex in incretin-potentiated insulin secretion. J Biol Chem 276:46046–46053

    Article  PubMed  CAS  Google Scholar 

  69. Ozaki N, Shibasaki T, Kashima Y, Miki T, Takahashi K, Ueno H, Sunaga Y, Yano H, Matsuura Y, Iwanaga T, Takai Y, Seino S (2000) cAMP-GEFII is a direct target of cAMP in regulated exocytosis. Nat Cell Biol 2:805–811

    Article  PubMed  CAS  Google Scholar 

  70. Kaneko M, Takahashi T (2004) Presynaptic mechanism underlying cAMP-dependent synaptic potentiation. J Neurosci 24:5202–5208

    Article  PubMed  CAS  Google Scholar 

  71. Sakaba T, Neher E (2003) Direct modulation of synaptic vesicle priming by GABAB receptor activation at a glutamatergic synapse. Nature 424:775–778

    Article  PubMed  CAS  Google Scholar 

  72. Levy O, Coughlin M, Cronstein BN, Roy RM, Desai A, Wessels MR (2006) The adenosine system selectively inhibits TLR-mediated TNF-alpha production in the human newborn. J Immunol 177:1956–1966

    PubMed  CAS  Google Scholar 

  73. Martin L, Pingle SC, Hallam DM, Rybak LP, Ramkumar V (2006) Activation of the adenosine A3 receptor in RAW 264.7 cells inhibits lipopolysaccharide-stimulated tumor necrosis factor-alpha release by reducing calcium-dependent activation of nuclear factor-kappaB and extracellular signal-regulated kinase 1/2. J Pharmacol Exp Ther 316:71–78

    Article  PubMed  CAS  Google Scholar 

  74. Leibovich SJ, Chen JF, Pinhal-Enfield G, Belem PC, Elson G, Rosania A, Ramanathan M, Montesinos C, Jacobson M, Schwarzschild MA, Fink JS, Cronstein B (2002) Synergistic up-regulation of vascular endothelial growth factor expression in murine macrophages by adenosine A2a receptor agonists and endotoxin. Am J Pathol 160:2231–2244

    PubMed  CAS  Google Scholar 

  75. Ramanathan M, Pinhal-Enfield G, Hao I, Leibovich SJ (2007) Synergistic up-regulation of vascular endothelial growth factor (VEGF) expression in macrophages by adenosine A2a receptor agonists and endotoxin involves transcriptional regulation via the hypoxia response element in the VEGF promoter. Mol Biol Cell 18:14–23

    Article  PubMed  CAS  Google Scholar 

  76. Baharav E, Bar-Yehuda S, Madi L, Silberman D, Rath-Wolfson L, Halpren M, Ochaion A, Weinberger A, Fishman P (2005) Antiinflammatory effect of A3 adenosine receptor agonists in murine autoimmune arthritis models. J Rheumatol 32:469–476

    PubMed  CAS  Google Scholar 

  77. Bar-Yehuda S, Madi L, Silberman D, Gery S, Shkapenuk M, Fishman P (2005) CF101, an agonist to the A3 adenosine receptor, enhances the chemotherapeutic effect of 5-fluorouracil in a colon carcinoma murine model. Neoplasia 7:85–90

    Article  PubMed  CAS  Google Scholar 

  78. Victor-Vega C, Desai A, Montesinos M, Cronstein B (2002) Adenosine A2a receptor agonists promote more rapid wound healing than recombinant human platelet–derived growth factor (becaplermin gel). Inflammation 26:19–24

    Article  PubMed  CAS  Google Scholar 

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  1. Joseph M. Russell

    Present address: Department of Medical Microbiology and Immunology, School of Medicine, University of California, One Shields Avenue, Davis, CA, 95616-8645, USA

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  1. Department of Veterinary Medicine, Anatomy, Physiology, and Cell Biology, University of California, Davis, CA, 95616, USA

    Joseph M. Russell, Graham S. Stephenson, Clare E. Yellowley & Hilary P. Benton

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Russell, J.M., Stephenson, G.S., Yellowley, C.E. et al. Adenosine Inhibition of Lipopolysaccharide-Induced Interleukin-6 Secretion by the Osteoblastic Cell Line MG-63. Calcif Tissue Int 81, 316–326 (2007). https://doi.org/10.1007/s00223-007-9060-y

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