Skip to main content


Log in

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

  • Original Paper
  • Published:
Angiogenesis Aims and scope Submit manuscript


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.

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

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others


  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 425:93–98

    Article  PubMed  CAS  Google Scholar 

  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 279:45626–456633

    Article  PubMed  CAS  Google Scholar 

  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 280:9083–9087

    Article  PubMed  CAS  Google Scholar 

  4. Seuwen K, Ludwig MG, Wolf RM (2006) Receptors for protons or lipid messengers or both? J Recept Signal Transduct Res 26:599–610

    Article  PubMed  CAS  Google Scholar 

  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 285:1786–1789

    Google Scholar 

  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 19:819–821

    PubMed  CAS  Google Scholar 

  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 291:91–101

    Article  Google Scholar 

  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 14:25–34

    Article  PubMed  CAS  Google Scholar 

  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 274:2573–2584

    Article  PubMed  CAS  Google Scholar 

  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 27:1334–1347

    Article  PubMed  CAS  Google Scholar 

  11. Bergers G, Benjamin LE (2003) Tumorigenesis and the angiogenic switch. Nat Rev Cancer 3:401–410

    Article  PubMed  CAS  Google Scholar 

  12. Heath VL, Bicknell R (2009) Anticancer strategies involving the vasculature. Nat Rev Clin Oncol 6:395–404

    Article  PubMed  CAS  Google Scholar 

  13. Griffiths JR (1991) Are cancer cells acidic ? Br J Cancer 64:425–427

    Article  PubMed  CAS  Google Scholar 

  14. Gatenby RA, Gillies RJ (2004) Why do cancers have high aerobic glycolysis? Nat Rev Cancer 4:891–899

    Article  PubMed  CAS  Google Scholar 

  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 14:771–794

    Article  PubMed  CAS  Google Scholar 

  16. Gambhir SS (2002) Molecular imaging of cancer with positron emission tomography. Nat Rev Cancer 2:683–693

    Article  PubMed  CAS  Google Scholar 

  17. Vander Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324:1029–1033

    Article  PubMed  CAS  Google Scholar 

  18. Park HJ, Lyons JC, Ohtsubo T, Song CW (1999) Acidic environment causes apoptosis by increasing caspase activity. Br J Cancer 80:1892–1897

    Article  PubMed  CAS  Google Scholar 

  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 66:6699–6707

    Article  PubMed  CAS  Google Scholar 

  20. DeClerck K, Eble RC (2010) The role of hypoxia and acidosis in promoting metastasis and resistance to chemotherapy. Front Biosci 15:213–225

    Article  PubMed  CAS  Google Scholar 

  21. Brahimi-Horn MC, Pouyssegur J (2007) Hypoxia in cancer cell metabolism and pH regulation. Essays Biochem 43:165–178

    Article  PubMed  CAS  Google Scholar 

  22. Salomon Y (1979) Adenylate cyclase assay. Adv Cyclic Nucleotide Res 10:35–55

    PubMed  CAS  Google Scholar 

  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 31:102

    Article  Google Scholar 

  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 302:1055–1061

    Article  PubMed  CAS  Google Scholar 

  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 60:2178–2189

    PubMed  CAS  Google Scholar 

  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 13:259–267

    Article  PubMed  CAS  Google Scholar 

  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 94:1124–1132

    Article  PubMed  CAS  Google Scholar 

  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 114:1082–1089

    PubMed  CAS  Google Scholar 

  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 105:986–993

    Article  PubMed  CAS  Google Scholar 

  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 24:769–777

    Article  PubMed  CAS  Google Scholar 

  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 124:175–189

    Article  PubMed  CAS  Google Scholar 

  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 176:1564–1576

    Article  PubMed  Google Scholar 

  33. Tazzyman S, Lewis CE, Murdoch C (2009) Neutrophils: key mediators of tumour angiogenesis. Int J Exp Pathol 90:222–231

    Article  PubMed  CAS  Google Scholar 

  34. Murdoch C, Muthana M, Coffelt SB, Lewis CE (2008) The role of myeloid cells in the promotion of tumour angiogenesis. Nat Rev Cancer 8:618–631

    Article  PubMed  CAS  Google Scholar 

  35. Coffelt SB, Hughes R, Lewis CE (2009) Tumor-associated macrophages: effectors of angiogenesis and tumor progression. Biochim Biophys Acta 1796:11–18

    PubMed  CAS  Google Scholar 

Download references


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.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Marie-Gabrielle Ludwig.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 1605 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wyder, L., Suply, T., Ricoux, B. et al. Reduced pathological angiogenesis and tumor growth in mice lacking GPR4, a proton sensing receptor. Angiogenesis 14, 533–544 (2011).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: