, Volume 14, Issue 4, pp 533–544 | Cite as

Reduced pathological angiogenesis and tumor growth in mice lacking GPR4, a proton sensing receptor

  • Lorenza Wyder
  • Thomas Suply
  • Bérangère Ricoux
  • Eric Billy
  • Christian Schnell
  • Birgit U. Baumgarten
  • Sauveur Michel Maira
  • Claudia Koelbing
  • Mireille Ferretti
  • Bernd Kinzel
  • Matthias Müller
  • Klaus Seuwen
  • Marie-Gabrielle Ludwig
Original Paper


The G protein-coupled receptor GPR4 is activated by acidic pH and recent evidence indicates that it is expressed in endothelial cells. In agreement with these reports, we observe a high correlation of GPR4 mRNA expression with endothelial marker genes, and we confirm expression and acidic pH dependent function of GPR4 in primary human vascular endothelial cells. GPR4-deficient mice were generated; these are viable and fertile and show no gross abnormalities. However, these animals show a significantly reduced angiogenic response to VEGF (vascular endothelial growth factor), but not to bFGF (basic fibroblast growth factor), in a growth factor implant model. Accordingly, in two different orthotopic models, tumor growth is strongly reduced in mice lacking GPR4. Histological analysis of tumors indicates reduced tumor cell proliferation as well as altered vessel morphology, length and density. Moreover, GPR4 deficiency results in reduced VEGFR2 (VEGF Receptor 2) levels in endothelial cells, accounting, at least in part, for the observed phenotype. Our data suggest that endothelial cells sense local tissue acidosis via GPR4 and that this signal is required to generate a full angiogenic response to VEGF.


Angiogenesis Acidosis Hypoxia Endothelium Tumor 



We would like to thank Melanie Muller, Juliane Vauxlaire, Corinne Manlius, Marina Maurer, Agnes Feige, Barbara Wilmering-Wetter, Marianne Lemaister, Thierry Doll, Imke Renz-Albrecht and Caterina Safina for excellent technical help, John Monahan for help with Affymetrix analysis, Julie Boisclair for help in characterizing the GPR4 deficient mice, Andreas Theuer for writing the program for Ki67 quantification and Jeanette Wood, Georg Martiny-Baron and Francesco Hofmann for support and critical discussions.

Conflict of interest

Thomas Suply, Bérangère Ricoux, Eric Billy, Christian Schnell, Birgit U Baumgarten, Sauveur Michel Maira, Claudia Koelbing, Mireille Ferretti, Bernd Kinzel, Matthias Müller, Klaus Seuwen and Marie-Gabrielle Ludwig are employees of Novartis AG, Switzerland. Lorenza Wyder is a former employee of Novartis AG.

Supplementary material

10456_2011_9238_MOESM1_ESM.doc (1.6 mb)
Supplementary material 1 (DOC 1605 kb)


  1. 1.
    Ludwig MG, Vanek M, Guerini D, Gasser JA, Jones CE, Junker U, Hofstetter H, Wolf RM, Seuwen K (2003) Proton-sensing G-protein-coupled receptors. Nature 425:93–98PubMedCrossRefGoogle Scholar
  2. 2.
    Wang JQ, Kon J, Mogi C, Tobo M, Damirin A, Sato K, Komachi M, Malchinkhuu E, Murata N, Kimura T, Kuwabara A, Wakamatsu K, Koizumi H, Uede T, Tsujimoto G, Kurose H, Sato T, Harada A, Misawa N, Tomura H, Okajima F (2004) TDAG8 is a proton-sensing and psychosine-sensitive G-protein-coupled receptor. J Biol Chem 279:45626–456633PubMedCrossRefGoogle Scholar
  3. 3.
    Ishii S, Kihara Y, Shimizu T (2005) Identification of T cell death-associated gene 8 (TDAG8) as a novel acid sensing G-protein-coupled receptor. J Biol Chem 280:9083–9087PubMedCrossRefGoogle Scholar
  4. 4.
    Seuwen K, Ludwig MG, Wolf RM (2006) Receptors for protons or lipid messengers or both? J Recept Signal Transduct Res 26:599–610PubMedCrossRefGoogle Scholar
  5. 5.
    Lum H, Qiao J, Walter RJ, Huang F, Subbaiah PV, Kim KS, Holian O (2003) Inflammatory stress increases receptor for lysophosphatidylcholine in human microvascular endothelial cells. Am J Physiol Heart Circ Physiol 285:1786–1789Google Scholar
  6. 6.
    Kim KS, Ren J, Jiang Y, Ebrahem Q, Tipps R, Cristina K, Xiao YJ, Qiao J, Taylor KL, Lum H, Anand-Apte B, Xu Y (2005) GPR4 plays a critical role in endothelial cell function and mediates the effects of sphingosylphosphorylcholine. FASEB J 19:819–821PubMedGoogle Scholar
  7. 7.
    Qiao J, Huang F, Naikawadi RP, Kim KS, Said T, Lum H (2006) Lysophosphatidylcholine impairs endothelial barrier function through the G protein-coupled receptor GPR4. Am J Physiol Lung Cell Mol Physiol 291:91–101CrossRefGoogle Scholar
  8. 8.
    Huang F, Mehta D, Predescu S, Kim KS, Lum H (2007) A novel lysophospholipid- and pH-sensitive receptor, GPR4, in brain endothelial cells regulates monocyte transmigration. Endothelium 14:25–34PubMedCrossRefGoogle Scholar
  9. 9.
    Zou Y, Kim CH, Chung JH, Kim JY, Chung SW, Kim MK, Im DS, Lee J, Yu BP, Chung HY (2007) Upregulation of endothelial adhesion molecules by lysophosphatidylcholine. Involvement of G protein-coupled receptor GPR4. FEBS J 274:2573–2584PubMedCrossRefGoogle Scholar
  10. 10.
    Yang LV, Radu CG, Roy M, Lee S, McLaughlin J, Teitell MA, Iruela-Arispe ML, Witte ON (2007) Vascular abnormalities in mice deficient for the G protein-coupled receptor GPR4 that functions as a pH sensor. Mol Cell Biol 27:1334–1347PubMedCrossRefGoogle Scholar
  11. 11.
    Bergers G, Benjamin LE (2003) Tumorigenesis and the angiogenic switch. Nat Rev Cancer 3:401–410PubMedCrossRefGoogle Scholar
  12. 12.
    Heath VL, Bicknell R (2009) Anticancer strategies involving the vasculature. Nat Rev Clin Oncol 6:395–404PubMedCrossRefGoogle Scholar
  13. 13.
    Griffiths JR (1991) Are cancer cells acidic ? Br J Cancer 64:425–427PubMedCrossRefGoogle Scholar
  14. 14.
    Gatenby RA, Gillies RJ (2004) Why do cancers have high aerobic glycolysis? Nat Rev Cancer 4:891–899PubMedCrossRefGoogle Scholar
  15. 15.
    Chiche J, Brahimi-Horn MC, Pouyssegur J (2010) Tumor hypoxia induces a metabolic shift causing acidosis: a common feature in cancer. J Cell Mol Med 14:771–794PubMedCrossRefGoogle Scholar
  16. 16.
    Gambhir SS (2002) Molecular imaging of cancer with positron emission tomography. Nat Rev Cancer 2:683–693PubMedCrossRefGoogle Scholar
  17. 17.
    Vander Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324:1029–1033PubMedCrossRefGoogle Scholar
  18. 18.
    Park HJ, Lyons JC, Ohtsubo T, Song CW (1999) Acidic environment causes apoptosis by increasing caspase activity. Br J Cancer 80:1892–1897PubMedCrossRefGoogle Scholar
  19. 19.
    Rofstad EK, Mathiesen B, Kindem K, Galappathi K (2006) Acidic extracellular pH promotes experimental metastasis of human melanoma cells in athymic nude mice. Cancer Res 66:6699–6707PubMedCrossRefGoogle Scholar
  20. 20.
    DeClerck K, Eble RC (2010) The role of hypoxia and acidosis in promoting metastasis and resistance to chemotherapy. Front Biosci 15:213–225PubMedCrossRefGoogle Scholar
  21. 21.
    Brahimi-Horn MC, Pouyssegur J (2007) Hypoxia in cancer cell metabolism and pH regulation. Essays Biochem 43:165–178PubMedCrossRefGoogle Scholar
  22. 22.
    Salomon Y (1979) Adenylate cyclase assay. Adv Cyclic Nucleotide Res 10:35–55PubMedGoogle Scholar
  23. 23.
    Hüsken D, Asselbergs F, Kinzel B, Natt F, Weiler J, Martin P, Häner R, Hall J (2003) mRNA fusion constructs serve in a general cell-based assay to profile oligonucleotide activity. Nucleic Acids Res 31:102CrossRefGoogle Scholar
  24. 24.
    Wood J, Bonjean K, Ruetz S, Bellahcène A, Devy L, Foidart JM, Castronovo V, Green JR (2002) Novel antiangiogenic effects of the bisphosphonate compound zoledronic acid. J Pharmacol Exp Ther 302:1055–1061PubMedCrossRefGoogle Scholar
  25. 25.
    Wood JM, Bold G, Buchdunger E, Cozens R, Ferrari S, Frei J, Hofmann F, Mestan J, Mett H, O’Reilly T, Persohn E, Rosel J, Schnell C, Stover D, Theuer A, Towbin H, Wenger F, Woods-Cook K, Menrad A, Siemeister G, Schirner M, Thierauch KH, Schneider MR, Drevs J, Martiny-Baron G, Totzke F (2000) PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res 60:2178–2189PubMedGoogle Scholar
  26. 26.
    Martiny-Baron G, Holzer P, Billy E, Schnell C, Brueggen J, Ferretti M, Schmiedeberg N, Wood JM, Furet P, Imbach P (2010) The small molecule specific EphB4 kinase inhibitor NVP-BHG712 inhibits VEGF driven angiogenesis. Angiogenesis 13:259–267PubMedCrossRefGoogle Scholar
  27. 27.
    Ehrbar M, Djonov VG, Schnell C, Tschanz SA, Martiny-Baron G, Schenk U, Wood J, Burri PH, Hubbell JA, Zisch AH (2004) Cell-demanded liberation of VEGF121 from fibrin implants induces local and controlled blood vessel growth. Circ Res 94:1124–1132PubMedCrossRefGoogle Scholar
  28. 28.
    Chae SS, Paik JH, Furneaux H, Hla T (2004) Requirement for sphingosine 1-phosphate receptor-1 in tumor angiogenesis demonstrated by in vivo RNA interference. J Clin Invest 114:1082–1089PubMedGoogle Scholar
  29. 29.
    Fiedler W, Serve H, Döhner H, Schwittay M, Ottmann OG, O’Farrell AM, Bello CL, Allred R, Manning WC, Cherrington JM, Louie SG, Hong W, Brega NM, Massimini G, Scigalla P, Berdel WE, Hossfeld DK (2005) A phase 1 study of SU11248 in the treatment of patients with refractory or resistant acute myeloid leukemia (AML) or not amenable to conventional therapy for the disease. Blood 105:986–993PubMedCrossRefGoogle Scholar
  30. 30.
    Wedam SB, Low JA, Yang SX, Chow CK, Choyke P, Danforth D, Hewitt SM, Berman A, Steinberg SM, Liewehr DJ, Plehn J, Doshi A, Thomasson D, McCarthy N, Koeppen H, Sherman M, Zujewski J, Camphausen K, Chen H, Swain SM (2006) Antiangiogenic and antitumor effects of bevacizumab in patients with inflammatory and locally advanced breast cancer. J Clin Oncol 24:769–777PubMedCrossRefGoogle Scholar
  31. 31.
    Grunewald M, Avraham I, Dor Y, Bachar-Lustig E, Itin A, Jung S, Chimenti S, Landsman L, Abramovitch R, Keshet E (2006) VEGF-induced adult neovascularization: recruitment, retention, and role of accessory cells. Cell 124:175–189PubMedCrossRefGoogle Scholar
  32. 32.
    Coffelt SB, Lewis CE, Naldini L, Brown JM, Ferrara N, De Palma M (2010) Elusive identities and overlapping phenotypes of proangiogenic myeloid cells in tumors. Am J Pathol 176:1564–1576PubMedCrossRefGoogle Scholar
  33. 33.
    Tazzyman S, Lewis CE, Murdoch C (2009) Neutrophils: key mediators of tumour angiogenesis. Int J Exp Pathol 90:222–231PubMedCrossRefGoogle Scholar
  34. 34.
    Murdoch C, Muthana M, Coffelt SB, Lewis CE (2008) The role of myeloid cells in the promotion of tumour angiogenesis. Nat Rev Cancer 8:618–631PubMedCrossRefGoogle Scholar
  35. 35.
    Coffelt SB, Hughes R, Lewis CE (2009) Tumor-associated macrophages: effectors of angiogenesis and tumor progression. Biochim Biophys Acta 1796:11–18PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Lorenza Wyder
    • 1
    • 2
  • Thomas Suply
    • 1
  • Bérangère Ricoux
    • 1
  • Eric Billy
    • 1
  • Christian Schnell
    • 1
  • Birgit U. Baumgarten
    • 1
  • Sauveur Michel Maira
    • 1
  • Claudia Koelbing
    • 1
  • Mireille Ferretti
    • 1
  • Bernd Kinzel
    • 1
  • Matthias Müller
    • 1
  • Klaus Seuwen
    • 1
  • Marie-Gabrielle Ludwig
    • 1
  1. 1.Novartis Institutes for Biomedical ResearchBaselSwitzerland
  2. 2.Actelion Pharmaceuticals LtdAllschwilSwitzerland

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