Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Mammary gland development is delayed in mice deficient for aminopeptidase N


Development of the mammary gland requires the coordinated action of proteolytic enzymes during two phases of remodelling. Firstly, new ducts and side-branches thereof need to be established during pregnancy to generate an extensive ductal tree allowing the secretion and transport of milk. A second wave of remodelling occurs during mammary involution after weaning. We have analysed the role of the cell surface protease aminopeptidase N (Anpep, APN, CD13) during these processes using Anpep deficient and Anpep over-expressing mice. We find that APN deficiency significantly delays mammary gland morphogenesis during gestation. The defect is characterised by a reduction in alveolar buds and duct branching at mid-pregnancy. Conversely over-expression of Anpep leads to accelerated ductal development. This indicates that Anpep plays a critical role in the proteolytic remodelling of mammary tissue during adult mammary development.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4


APN, Anpep:

Aminopeptidase N


Bacterial artificial chromosome

ES cells:

Embryonic stem cells


Horse radish peroxidase


Matrix metallo-proteinase


Polymerase chain reaction


  1. Alexander CM, Selvarajan S, Mudgett J, Werb Z (2001) Stromelysin-1 regulates adipogenesis during mammary gland involution. J Cell Biol 152(4):693–703

  2. Atherton AJ, Monaghan P, Warburton MJ, Gusterson BA (1992) Immunocytochemical localization of the ectoenzyme aminopeptidase N in the human breast. J Histochem Cytochem 40(5):705–710

  3. Atherton AJ, Anbazhagan R, Monaghan P, Bartek J, Gusterson BA (1994a) Immunolocalisation of cell surface peptidases in the developing human breast. Differentiation 56(1–2):101–106

  4. Atherton AJ, O’Hare MJ, Buluwela L, Titley J, Monaghan P, Paterson HF, Warburton MJ, Gusterson BA (1994b) Ectoenzyme regulation by phenotypically distinct fibroblast sub-populations isolated from the human mammary gland. J Cell Sci 107(Pt 10):2931–2939

  5. Benaud C, Dickson RB, Thompson EW (1998) Roles of the matrix metalloproteinases in mammary gland development and cancer. Breast Cancer Res Treat 50(2):97–116

  6. Bhagwat SV, Lahdenranta J, Giordano R, Arap W, Pasqualini R, Shapiro LH (2001) CD13/APN is activated by angiogenic signals and is essential for capillary tube formation. Blood 97(3):652–659

  7. Clarkson RW, Wayland MT, Lee J, Freeman T, Watson CJ (2004) Gene expression profiling of mammary gland development reveals putative roles for death receptors and immune mediators in post-lactational regression. Breast Cancer Res 6(2):R92–R109

  8. Crawford HC, Matrisian LM (1996) Mechanisms controlling the transcription of matrix metalloproteinase genes in normal and neoplastic cells. Enzyme Protein 49(1–3):20–37

  9. Delmas B, Gelfi J, Kut E, Sjostrom H, Noren O, Laude H (1994) Determinants essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain of aminopeptidase-N that is distinct from the enzymatic site. J Virol 68(8):5216–5224

  10. Farrelly N, Lee YJ, Oliver J, Dive C, Streuli CH (1999) Extracellular matrix regulates apoptosis in mammary epithelium through a control on insulin signaling. J Cell Biol 144(6):1337–1348

  11. Friedl P, Gilmour D (2009) Collective cell migration in morphogenesis, regeneration and cancer. Nat Rev Mol Cell Biol 10(7):445

  12. Fu X, Kassim SY, Parks WC, Heinecke JW (2001) Hypochlorous acid oxygenates the cysteine switch domain of pro-matrilysin (MMP-7). A mechanism for matrix metalloproteinase activation and atherosclerotic plaque rupture by myeloperoxidase. J Biol Chem 276(44):41279–41287

  13. Gilmore AP, Metcalfe AD, Romer LH, Streuli CH (2000) Integrin-mediated survival signals regulate the apoptotic function of Bax through its conformation and subcellular localization. J Cell Biol 149(2):431–446

  14. Guzman-Rojas L, Rangel R, Salameh A, Edwards JK, Dondossola E, Kim Y-G, Saghatelian A, Giordano RJ, Kolonin MG, Staquicini FI, Koivunen E, Sidman RL, Arap W, Pasqualini R (2012) Cooperative effects of aminopeptidase N (CD13) expressed by nonmalignant and cancer cells within the tumor microenvironment. Proc Nat Acad Sci 109(5):1637–1642. doi:10.1073/pnas.1120790109

  15. Jerry DJ, Kuperwasser C, Downing SR, Pinkas J, He C, Dickinson E, Marconi S, Naber SP (1998) Delayed involution of the mammary epithelium in BALB/c-p53null mice. Oncogene 17(18):2305–2312

  16. Kolb AF, Siddell SG (1997) Genomic targeting of a bicistronic DNA fragment by Cre-mediated site-specific recombination. Gene 203(2):209–216

  17. Lassnig C, Sanchez CM, Egerbacher M, Walter I, Majer S, Kolbe T, Pallares P, Enjuanes L, Muller M (2005) Development of a transgenic mouse model susceptible to human coronavirus 229E. Proc Natl Acad Sci U S A 102(23):8275–8280

  18. Li M, Hu J, Heermeier K, Hennighausen L, Furth PA (1996) Apoptosis and remodeling of mammary gland tissue during involution proceeds through p53-independent pathways. Cell Growth Differ 7(1):13–20

  19. Lilla J, Stickens D, Werb Z (2002) Metalloproteases and adipogenesis: a weighty subject. Am J Pathol 160(5):1551–1554

  20. Lund LR, Romer J, Thomasset N, Solberg H, Pyke C, Bissell MJ, Dano K, Werb Z (1996) Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways. Development 122(1):181–193

  21. Lund LR, Romer J, Bugge TH, Nielsen BS, Frandsen TL, Degen JL, Stephens RW, Dano K (1999) Functional overlap between two classes of matrix-degrading proteases in wound healing. EMBO J 18(17):4645–4656

  22. Master SR, Hartman JL, D’Cruz CM, Moody SE, Keiper EA, Ha SI, Cox JD, Belka GK, Chodosh LA (2002) Functional microarray analysis of mammary organogenesis reveals a developmental role in adaptive thermogenesis. Mol Endocrinol 16(6):1185–1203

  23. Page-McCaw A, Ewald AJ, Werb Z (2007) Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol 8(3):221–233

  24. Rangel R, Sun Y, Guzman-Rojas L, Ozawa MG, Sun J, Giordano RJ, Van Pelt CS, Tinkey PT, Behringer RR, Sidman RL, Arap W, Pasqualini R (2007a) Impaired angiogenesis in aminopeptidase N-null mice. PNAS 104(11):4588–4593. doi:10.1073/pnas.0611653104

  25. Rangel R, Sun Y, Guzman-Rojas L, Ozawa MG, Sun J, Giordano RJ, Van Pelt CS, Tinkey PT, Behringer RR, Sidman RL, Arap W, Pasqualini R (2007b) Impaired angiogenesis in aminopeptidase N-null mice. Proc Natl Acad Sci U S A 104(11):4588–4593

  26. Rossiter H, Barresi C, Ghannadan M, Gruber F, Mildner M, Fadinger D, Tschachler E (2007) Inactivation of VEGF in mammary gland epithelium severely compromises mammary gland development and function. FASEB J 21(14):3994–4004. doi:10.1096/fj.07-8720com

  27. Schedin P, Strange R, Mitrenga T, Wolfe P, Kaeck M (2000) Fibronectin fragments induce MMP activity in mouse mammary epithelial cells: evidence for a role in mammary tissue remodeling. J Cell Sci 113(Pt 5):795–806

  28. Sorrell DA, Szymanowska M, Boutinaud M, Robinson C, Clarkson RW, Stein T, Flint DJ, Kolb AF (2005) Regulation of genes encoding proteolytic enzymes during mammary gland development. J Dairy Res 72(4):433–441

  29. Stein T, Morris JS, Davies CR, Weber-Hall SJ, Duffy MA, Heath VJ, Bell AK, Ferrier RK, Sandilands GP, Gusterson BA (2004) Involution of the mouse mammary gland is associated with an immune cascade and an acute-phase response, involving LBP, CD14 and STAT3. Breast Cancer Res 6(2):R75–R91

  30. Sternlicht MD, Lochter A, Sympson CJ, Huey B, Rougier JP, Gray JW, Pinkel D, Bissell MJ, Werb Z (1999) The stromal proteinase MMP3/stromelysin-1 promotes mammary carcinogenesis. Cell 98(2):137–146

  31. Tonner E, Barber MC, Allan GJ, Beattie J, Webster J, Whitelaw CBA, Flint DJ (2002) Insulin-like growth factor binding protein-5 (IGFBP-5) induces premature cell death in the mammary glands of transgenic mice. Development 129(19):4547–4557

  32. Winnicka B, O’Conor C, Schacke W, Vernier K, Grant CL, Fenteany FH, Pereira FE, Liang B, Kaur A, Zhao R, Montrose DC, Rosenberg DW, Aguila HL, Shapiro LH (2010) CD13 is dispensable for normal hematopoiesis and myeloid cell functions in the mouse. J Leukoc Biol 88(2):347–359. doi:10.1189/jlb.0210065

  33. Wiseman BS, Sternlicht MD, Lund LR, Alexander CM, Mott J, Bissell MJ, Soloway P, Itohara S, Werb Z (2003) Site-specific inductive and inhibitory activities of MMP-2 and MMP-3 orchestrate mammary gland branching morphogenesis. J Cell Biol 162(6):1123–1133

  34. Witty JP, Wright JH, Matrisian LM (1995) Matrix metalloproteinases are expressed during ductal and alveolar mammary morphogenesis, and misregulation of stromelysin-1 in transgenic mice induces unscheduled alveolar development. Mol Biol Cell 6(10):1287–1303

  35. Yeager CL, Ashmun RA, Williams RK, Cardellichio CB, Shapiro LH, Look AT, Holmes KV (1992) Human aminopeptidase N is a receptor for human coronavirus 229E. Nature 357(6377):420–422

Download references


The work was supported by the EU framework VI project grant (QOL-2000-00874) and the Hannah Development Fund.

Author information

Correspondence to Andreas F. Kolb.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kolb, A.F., Sorrell, D., Lassnig, C. et al. Mammary gland development is delayed in mice deficient for aminopeptidase N. Transgenic Res 22, 425–434 (2013). https://doi.org/10.1007/s11248-012-9654-7

Download citation


  • Mammary gland
  • Protease
  • Gene locus
  • Homologous recombination
  • Development