Skip to main content
SpringerLink
Log in

Adenosine A2A receptor (A2AR) is a fine-tune regulator of the collagen1:collagen3 balance

  • Original Article
  • Published:
Purinergic Signalling Aims and scope Submit manuscript

Abstract

Adenosine is a potent endogenous anti-inflammatory and immunosuppressive metabolite that is a potent modulator of tissue repair. However, the adenosine A2A receptor (A2AR)-mediated promotion of collagen synthesis is detrimental in settings such as scarring and scleroderma. The signaling cascade from A2AR stimulation to increased collagen production is complex and obscure, not least because cAMP and its downstream molecules PKA and Epac1 have been reported to inhibit collagen production. We therefore examined A2AR-stimulated signaling for collagen production by normal human dermal fibroblasts (NHDF). Collagen1 (Col1) and collagen3 (Col3) content after A2AR activation by CGS21680 was studied by western blotting. Contribution of PKA and Epac was analyzed by the PKA inhibitor PKI and by knockdowns of the PKA-Cα, -Cβ, -Cγ, Epac1, and Epac2. CGS21680 stimulates Col1 expression at significantly lower concentrations than those required to stimulate Col3 expression. A2AR stimulates Col1 expression by a PKA-dependent mechanism since PKA inhibition or PKA-Cα and -Cβ knockdown prevents A2AR-mediated Col1 increase. In contrast, A2AR represses Col3 via PKA but stimulates both Col1 and Col3 via an Epac2-dependent mechanism. A2AR stimulation with CGS21680 at 0.1 μM increased Col3 expression only upon PKA blockade. A2AR activation downstream signaling for Col1 and Col3 expression proceeds via two distinct pathways with varying sensitivity to cAMP activation; more highly cAMP-sensitive PKA activation stimulates Col1 expression, and less cAMP-sensitive Epac activation promotes both Col1 and Col3 expression. These observations may explain the dramatic change in Col1:Col3 ratio in hypertrophic and immature scars, where adenosine is present in higher concentrations than in normal skin.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

Col1:

Collagen type I

Col3:

Collagen type III

A2AR:

Adenosine A2 receptor

NHDF:

Normal human dermal fibroblasts

References

  1. Williams JC (1980) Adenine nucleotide degradation by the obligate intracellular bacterium Rickettsia typhi. Infect Immun 28(1):74–81

    PubMed  CAS  PubMed Central  Google Scholar 

  2. Hasko G, Cronstein BN (2004) Adenosine: an endogenous regulator of innate immunity. Trends Immunol 25(1):33–39

    Article  PubMed  CAS  Google Scholar 

  3. Ohta A, Sitkovsky M (2001) Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage. Nature 414(6866):916–920

    Article  PubMed  CAS  Google Scholar 

  4. Stadelmann WK, Digenis AG, Tobin GR (1998) Physiology and healing dynamics of chronic cutaneous wounds. Am J Surg 176(2A Suppl):26S–38S

    Article  PubMed  CAS  Google Scholar 

  5. Victor-Vega C, Desai A, Montesinos MC, Cronstein BN (2002) Adenosine A2A receptor agonists promote more rapid wound healing than recombinant human platelet-derived growth factor (Becaplermin gel). Inflammation 26(1):19–24

    Article  PubMed  CAS  Google Scholar 

  6. Montesinos MC, Desai A, Chen JF, Yee H, Schwarzschild MA, Fink JS, Cronstein BN (2002) Adenosine promotes wound healing and mediates angiogenesis in response to tissue injury via occupancy of A(2A) receptors. Am J Pathol 160(6):2009–2018

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  7. Montesinos MC, Gadangi P, Longaker M, Sung J, Levine J, Nilsen D, Reibman J, Li M, Jiang CK, Hirschhorn R, Recht PA, Ostad E, Levin RI, Cronstein BN (1997) Wound healing is accelerated by agonists of adenosine A2 (G alpha s-linked) receptors. J Exp Med 186(9):1615–1620

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  8. Katebi M, Fernandez P, Chan ES, Cronstein BN (2008) Adenosine A2A receptor blockade or deletion diminishes fibrocyte accumulation in the skin in a murine model of scleroderma, bleomycin-induced fibrosis. Inflammation 31(5):299–303

    Article  PubMed  CAS  Google Scholar 

  9. Lazzerini PE, Natale M, Gianchecchi E, Capecchi PL, Montilli C, Zimbone S, Castrichini M, Balistreri E, Ricci G, Selvi E, Garcia-Gonzalez E, Galeazzi M, Laghi-Pasini F (2012) Adenosine A2A receptor activation stimulates collagen production in sclerodermic dermal fibroblasts either directly and through a cross-talk with the cannabinoid system. J Mol Med (Berl) 90(3):331–342

    Article  CAS  Google Scholar 

  10. Perez-Aso M, Chiriboga L, Cronstein BN (2012) Pharmacological blockade of adenosine A2A receptors diminishes scarring. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology 26(10):4254–4263. doi:10.1096/fj.12-209627

    Article  CAS  Google Scholar 

  11. Chan ES, Montesinos MC, Fernandez P, Desai A, Delano DL, Yee H, Reiss AB, Pillinger MH, Chen JF, Schwarzschild MA, Friedman SL, Cronstein BN (2006) Adenosine A(2A) receptors play a role in the pathogenesis of hepatic cirrhosis. Br J Pharmacol 148(8):1144–1155. doi:10.1038/sj.bjp.0706812

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  12. Chan ES, Fernandez P, Merchant AA, Montesinos MC, Trzaska S, Desai A, Tung CF, Khoa DN, Pillinger MH, Reiss AB, Tomic-Canic M, Chen JF, Schwarzschild MA, Cronstein BN (2006) Adenosine A2A receptors in diffuse dermal fibrosis: pathogenic role in human dermal fibroblasts and in a murine model of scleroderma. Arthritis and Rheumatism 54(8):2632–2642. doi:10.1002/art.21974

    Article  PubMed  CAS  Google Scholar 

  13. Kaul A, Chandra M, Misra MK (2006) Adenosine deaminase in ischemia reperfusion injury in patients with myocardial infarction. Journal of Enzyme Inhibition and Medicinal Chemistry 21(5):543–546. doi:10.1080/14756360600774520

    Article  PubMed  CAS  Google Scholar 

  14. Chan ES, Liu H, Fernandez P, Luna A, Perez-Aso M, Bujor AM, Trojanowska M, Cronstein BN (2013) Adenosine A2A receptors promote collagen production by a Fli1- and CTGF-mediated mechanism. Arthritis Res Ther 15(3):R58

    Article  PubMed  CAS  Google Scholar 

  15. Che J, Chan ES, Cronstein BN (2007) Adenosine A2A receptor occupancy stimulates collagen expression by hepatic stellate cells via pathways involving protein kinase A, Src, and extracellular signal-regulated kinases 1/2 signaling cascade or p38 mitogen-activated protein kinase signaling pathway. Mol Pharmacol 72(6):1626–1636. doi:10.1124/mol.107.038760

    Article  PubMed  CAS  Google Scholar 

  16. Lazarova T, Brewin KA, Stoeber K, Robinson CR (2004) Characterization of peptides corresponding to the seven transmembrane domains of human adenosine A2a receptor. Biochemistry 43(40):12945–12954

    Article  PubMed  CAS  Google Scholar 

  17. Thevenin D, Roberts MF, Lazarova T, Robinson CR (2005) Identifying interactions between transmembrane helices from the adenosine A2A receptor. Biochemistry 44(49):16239–16245

    Article  PubMed  CAS  Google Scholar 

  18. Ohta A, Gorelik E, Prasad SJ, Ronchese F, Lukashev D, Wong MK, Huang X, Caldwell S, Liu K, Smith P, Chen JF, Jackson EK, Apasov S, Abrams S, Sitkovsky M (2006) A2A adenosine receptor protects tumors from antitumor T cells. Proc Natl Acad Sci USA 103(35):13132–13137

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  19. Bingham TC, Fisher EA, Parathath S, Reiss AB, Chan ES, Cronstein BN (2010) A2A adenosine receptor stimulation decreases foam cell formation by enhancing ABCA1-dependent cholesterol efflux. J Leukoc Biol 87(4):683–690

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  20. Kreckler LM, Gizewski E, Wan TC, Auchampach JA (2009) Adenosine suppresses lipopolysaccharide-induced tumor necrosis factor-alpha production by murine macrophages through a protein kinase A- and exchange protein activated by cAMP-independent signaling pathway. J Pharmacol Exp Ther 331(3):1051–1061

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  21. Seidel MG, Klinger M, Freissmuth M, Holler C (1999) Activation of mitogen-activated protein kinase by the A(2A)-adenosine receptor via a rap1-dependent and via a p21(ras)-dependent pathway. J Biol Chem 274(36):25833–25841

    Article  PubMed  CAS  Google Scholar 

  22. Fredholm BB, Arslan G, Halldner L, Kull B, Schulte G, Wasserman W (2000) Structure and function of adenosine receptors and their genes. Naunyn Schmiedebergs Arch Pharmacol 362(4–5):364–374

    Article  PubMed  CAS  Google Scholar 

  23. Schulte G, Fredholm BB (2003) Signalling from adenosine receptors to mitogen-activated protein kinases. Cell Signal 15(9):813–827

    Article  PubMed  CAS  Google Scholar 

  24. Insel PA, Murray F, Yokoyama U, Romano S, Yun H, Brown L, Snead A, Lu D, Aroonsakool N (2012) cAMP and Epac in the regulation of tissue fibrosis. Br J Pharmacol 166(2):447–456

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  25. Yokoyama U, Patel HH, Lai NC, Aroonsakool N, Roth DM, Insel PA (2008) The cyclic AMP effector Epac integrates pro- and anti-fibrotic signals. Proc Natl Acad Sci USA 105(17):6386–6391

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  26. Ji Z, Mei FC, Miller AL, Thompson EB, Cheng X (2008) Protein kinase A (PKA) isoform RIIbeta mediates the synergistic killing effect of cAMP and glucocorticoid in acute lymphoblastic leukemia cells. J Biol Chem 283(32):21920–21925

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  27. Jimenez SA, Hitraya E, Varga J (1996) Pathogenesis of scleroderma. Collagen Rheum Dis Clin North Am 22(4):647–674

    Article  CAS  Google Scholar 

  28. Fredholm BB, Ijzerman AP, Jacobson KA, Klotz KN, Linden J (2001) International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev 53(4):527–552

    PubMed  CAS  Google Scholar 

  29. Fernandez P, Trzaska S, Wilder T, Chiriboga L, Blackburn MR, Cronstein BN, Chan ES (2008) Pharmacological blockade of A2A receptors prevents dermal fibrosis in a model of elevated tissue adenosine. Am J Pathol 172(6):1675–1682. doi:10.2353/ajpath.2008.070952

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  30. Eltzschig HK, Carmeliet P (2011) Hypoxia and inflammation. N Engl J Med 364(7):656–665. doi:10.1056/NEJMra0910283

    Article  PubMed  CAS  Google Scholar 

  31. Eckle T, Kohler D, Lehmann R, El Kasmi K, Eltzschig HK (2008) Hypoxia-inducible factor-1 is central to cardioprotection: a new paradigm for ischemic preconditioning. Circulation 118(2):166–175. doi:10.1161/CIRCULATIONAHA.107.758516

    Article  PubMed  CAS  Google Scholar 

  32. Eckle T, Krahn T, Grenz A, Kohler D, Mittelbronn M, Ledent C, Jacobson MA, Osswald H, Thompson LF, Unertl K, Eltzschig HK (2007) Cardioprotection by ecto-5'-nucleotidase (CD73) and A2B adenosine receptors. Circulation 115(12):1581–1590. doi:10.1161/CIRCULATIONAHA.106.669697

    Article  PubMed  CAS  Google Scholar 

  33. Synnestvedt K, Furuta GT, Comerford KM, Louis N, Karhausen J, Eltzschig HK, Hansen KR, Thompson LF, Colgan SP (2002) Ecto-5'-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. J Clin Invest 110(7):993–1002. doi:10.1172/JCI15337

    PubMed  CAS  PubMed Central  Google Scholar 

  34. Higgins DF, Kimura K, Bernhardt WM, Shrimanker N, Akai Y, Hohenstein B, Saito Y, Johnson RS, Kretzler M, Cohen CD, Eckardt KU, Iwano M, Haase VH (2007) Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition. J Clin Invest 117(12):3810–3820. doi:10.1172/JCI30487

    PubMed  CAS  PubMed Central  Google Scholar 

  35. Kong T, Westerman KA, Faigle M, Eltzschig HK, Colgan SP (2006) HIF-dependent induction of adenosine A2B receptor in hypoxia. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology 20(13):2242–2250. doi:10.1096/fj.06-6419com

    Article  CAS  Google Scholar 

  36. Grenz A, Bauerle JD, Dalton JH, Ridyard D, Badulak A, Tak E, McNamee EN, Clambey E, Moldovan R, Reyes G, Klawitter J, Ambler K, Magee K, Christians U, Brodsky KS, Ravid K, Choi DS, Wen J, Lukashev D, Blackburn MR, Osswald H, Coe IR, Nurnberg B, Haase VH, Xia Y, Sitkovsky M, Eltzschig HK (2012) Equilibrative nucleoside transporter 1 (ENT1) regulates postischemic blood flow during acute kidney injury in mice. J Clin Invest 122(2):693–710. doi:10.1172/JCI60214

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  37. Koeppen M, Eckle T, Eltzschig HK (2011) Interplay of hypoxia and A2B adenosine receptors in tissue protection. Adv Pharmacol 61:145–186. doi:10.1016/B978-0-12-385526-8.00006-0

    Article  PubMed  CAS  Google Scholar 

  38. Clambey ET, McNamee EN, Westrich JA, Glover LE, Campbell EL, Jedlicka P, de Zoeten EF, Cambier JC, Stenmark KR, Colgan SP, Eltzschig HK (2012) Hypoxia-inducible factor-1 alpha-dependent induction of FoxP3 drives regulatory T-cell abundance and function during inflammatory hypoxia of the mucosa. Proc Natl Acad Sci U S A 109(41):E2784–E2793. doi:10.1073/pnas.1202366109

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  39. Milne GR, Palmer TM (2011) Anti-inflammatory and immunosuppressive effects of the A2A adenosine receptor. ScientificWorldJournal 11:320–339

    Article  PubMed  CAS  Google Scholar 

  40. Ohta A, Madasu M, Kini R, Subramanian M, Goel N, Sitkovsky M (2009) A2A adenosine receptor may allow expansion of T cells lacking effector functions in extracellular adenosine-rich microenvironments. J Immunol 183(9):5487–5493

    Article  PubMed  CAS  Google Scholar 

  41. Yano N, Suzuki D, Endoh M, Zhao TC, Padbury JF, Tseng YT (2007) A novel phosphoinositide 3-kinase-dependent pathway for angiotensin II/AT-1 receptor-mediated induction of collagen synthesis in MES-13 mesangial cells. J Biol Chem 282(26):18819–18830

    Article  PubMed  CAS  Google Scholar 

  42. Pidoux G, Tasken K (2010) Specificity and spatial dynamics of protein kinase A signaling organized by A-kinase-anchoring proteins. J Mol Endocrinol 44(5):271–284

    Article  PubMed  CAS  Google Scholar 

  43. Skalhegg BS, Tasken K (2000) Specificity in the cAMP/PKA signaling pathway. Differential expression, regulation, and subcellular localization of subunits of PKA. Front Biosci 5(5):D678–D693

    Article  PubMed  CAS  Google Scholar 

  44. Insel PA, Bourne HR, Coffino P, Tomkins GM (1975) Cyclic AMP-dependent protein kinase: pivotal role in regulation of enzyme induction and growth. Science 190(4217):896–898

    Article  PubMed  CAS  Google Scholar 

  45. Breckler M, Berthouze M, Laurent AC, Crozatier B, Morel E, Lezoualc'h F (2011) Rap-linked cAMP signaling Epac proteins: compartmentation, functioning and disease implications. Cell Signal 23(8):1257–1266

    Article  PubMed  CAS  Google Scholar 

  46. Dao KK, Teigen K, Kopperud R, Hodneland E, Schwede F, Christensen AE, Martinez A, Doskeland SO (2006) Epac1 and cAMP-dependent protein kinase holoenzyme have similar cAMP affinity, but their cAMP domains have distinct structural features and cyclic nucleotide recognition. J Biol Chem 281(30):21500–21511

    Article  PubMed  CAS  Google Scholar 

  47. Butkowski RJ (1982) Estimation of the size of collagenous proteins by electrophoresis and gel chromatography. In: Cunningham LW, Frederickson DW (eds) Methods in enzymology, vol 82. Academic Press, New York, pp 410–423

    Google Scholar 

  48. Rentz TJ, Poobalarahi F, Bornstein P, Sage EH, Bradshaw AD (2007) SPARC regulates processing of procollagen I and collagen fibrillogenesis in dermal fibroblasts. J Biol Chem 282(30):22062–22071

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the National Institutes of Health (AR56672 and AR54897), NYU-HHC (Health and Hospitals Corporation) Clinical and Translational Science Institute (UL1RR029893), NYU Cancer Institute Center Support Grant, and NIH/National Cancer Institute (5 P30CA16087-31).

Conflicts of interest

AM and BNC have filed a patent on use of adenosine A2AR agonists to prevent prosthesis loosening (pending). MP-A does not have any disclosures. BNC holds patents numbers 5,932,558; 6,020,321; 6,555,545; 7,795,427; adenosine A1R and A2BR antagonists to treat fatty liver (pending); and adenosine A2AR agonists to prevent prosthesis loosening (pending). BNC is a consultant for Bristol-Myers Squibb, Novartis, CanFite Biopharmaceuticals, Cypress Laboratories, Regeneron (Westat, DSMB), Endocyte, Protalex, Allos, Inc., Savient, Gismo Therapeutics, Antares Pharmaceutical, Medivector, King Pharmaceutical, Celizome, Tap Pharmaceuticals, Prometheus Laboratories, Sepracor, Amgen, Combinatorx, Kyowa Hakka, Hoffman-LaRoche, and Avidimer Therapeutics. BNC has stock in CanFite Biopharmaceuticals.

Author information

Authors and Affiliations

  1. Division of Translational Medicine, Department of Medicine, New York University School of Medicine, 550 First Avenue, MSB 255, New York, NY, 10016, USA

    Miguel Perez-Aso, Aránzazu Mediero & Bruce N. Cronstein

Authors
  1. Miguel Perez-Aso
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aránzazu Mediero
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bruce N. Cronstein
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Miguel Perez-Aso.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplemental Figure 1

TGF-β1 promotes collagen type I and collagen type III increase after 24 h. NHDF cells were incubated with TGF-β1 at 0.01 ng/ml during 24 h (PDF 21 kb)

Supplemental Figure 2

PMA reduces Col1 and Col3 at nanomolar and micromolar concentrations. a NHDF cells were incubated with increasing concentrations of PMA during 24 h. Statistics was performed by ANOVA followed by Newman–Keuls post-test: Col1 **P < 0.01 and *P < 0.05 or Col3 ### P < 0.001, ## P < 0.01, and # P < 0.05 vs. non-stimulated control. Data represent means ± SEM of three independent experiments (PDF 74 kb)

Supplemental Figure 3

Validation of collagen1 and 3 antibodies. a Specificity of collagen antibodies; 1 μg of purified collagen1 and 3 were incubated with anti-Col1 and anti-Col3 antibodies. b Standard curves of purified collagen1 and collagen3 show a higher affinity for the anti-Col3 antibody than for the anti-Col1. Data represent means ± SEM. c Increasing loading of two different sets of protein extracts from NHDF were incubated with collagen1 and collagen3 antibodies (PPTX 410 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Perez-Aso, M., Mediero, A. & Cronstein, B.N. Adenosine A2A receptor (A2AR) is a fine-tune regulator of the collagen1:collagen3 balance. Purinergic Signalling 9, 573–583 (2013). https://doi.org/10.1007/s11302-013-9368-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11302-013-9368-1

Keywords

Search

Navigation