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Histochemistry and Cell Biology

, Volume 136, Issue 6, pp 699–708 | Cite as

Abnormal expression of Muc5b in Cftr-null mice and in mammary tumors of MMTV-ras mice

  • Hélène Valque
  • Valérie Gouyer
  • Marie-Odile Husson
  • Frédéric Gottrand
  • Jean-Luc Desseyn
Original Paper

Abstract

Gel-forming mucins are large, high molecular weight, and heavily O-glycosylated proteins that are responsible for the rheological properties of mucus gel. Among them, the mucin MUC5B has been implicated in breast cancer and cystic fibrosis. We obtained a new polyclonal serum, named CP1, which was isolated from a rabbit immunized with a mouse Muc5b peptide. The immunoprofile of Muc5b was determined on paraffin-embedded and frozen mouse tissue sections and showed a similar expression pattern in mouse to that in the human. The “nonmammary” mucin Muc5b was detected in all mammary tumors analyzed from MMTV-ras mice, suggesting that the CP1 antibody is a valuable tool for investigating the involvement of this mucin in mammary cancer. We also found that uninfected Cftr −/− mice harbored more Clara cells, which were Muc5b-positive, than did their wild-type control littermates. The number of Muc5b-positive cells increased in Cftr −/− mice infected experimentally with Pseudomonas aeruginosa, and the mice developed mucus plugs in their bronchi and bronchioles with a high frequency of Muc5b content (87%, Cohen’s kappa = 0.82; p < 0.0001). These findings suggest that mice genetically deficient in the Cftr gene are predisposed to develop mucus plugs and that MUC5B may provide a valuable target for decreasing mucus viscosity in cystic fibrosis.

Keywords

Gel-forming mucin Muc5b Breast cancer Cystic fibrosis Mucus plug 

Notes

Acknowledgments

We thank the microbiology department of the Centre de Biologie-Pathologie Pierre-Marie Degand (Lille Hospital) for access to the ABI Prism 7700; MH Gevaert and RM Siminski (Service Commun-Morphologie cellulaire, Université Lille 2) for slides; J Bara (Inserm UMR S938, Paris, France) for the 45M1 antibody; J Ryerse (Department of Pathology, Saint Louis, MO) for the anti-CCSP (R42AP) antibody; and MR Parsek and BR Borlee (Seattle, WA) for the PAO1 strain. This study was supported by the French Cystic Fibrosis Association Vaincre la Mucoviscidose (JLD, FC0203, and IC0806).

Supplementary material

418_2011_872_MOESM1_ESM.tif (1.2 mb)
Supplementary Figure 1. Location of epitopes used for anti-mouse Muc5b antibody production. A Schematic representation of the mouse mucin Muc5b. B The known CYS protein sequences of mouse-secreted gel-forming mucins were aligned to show sequence similarity. The peptide used for antibody production and shorter peptides that may also be immunogens are highlighted in gray. Until now, only one CYS domain and five CYS domains of Muc5ac have been sequenced; we named the first three (CYS#1–3) and the last two CYS#8 and #9 of Muc5ac by analogy to the nine CYS domains of the human counterparts. However, we assumed that mouse Muc2 and Muc5ac contain two and nine CYS domains, respectively, as do their human orthologues

References

  1. Ali MS, Wilson JA, Pearson JP (2002) Mixed nasal mucus as a model for sinus mucin gene expression studies. Laryngoscope 112:326–331PubMedCrossRefGoogle Scholar
  2. Arul GS, Moorghen M, Myerscough N, Alderson DA, Spicer RD, Corfield AP (2000) Mucin gene expression in Barrett’s oesophagus: an in situ hybridisation and immunohistochemical study. Gut 47:753–761PubMedCrossRefGoogle Scholar
  3. Audie JP, Janin A, Porchet N, Copin MC, Gosselin B, Aubert JP (1993) Expression of human mucin genes in respiratory, digestive, and reproductive tracts ascertained by in situ hybridization. J Histochem Cytochem 41:1479–1485PubMedCrossRefGoogle Scholar
  4. Bartlett NW, Walton RP, Edwards MR, Aniscenko J, Caramori G, Zhu J, Glanville N, Choy KJ, Jourdan P, Burnet J, Tuthill TJ, Pedrick MS, Hurle MJ, Plumpton C, Sharp NA, Bussell JN, Swallow DM, Schwarze J, Guy B, Almond JW, Jeffery PK, Lloyd CM, Papi A, Killington RA, Rowlands DJ, Blair ED, Clarke NJ, Johnston SL (2008) Mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation. Nat Med 14:199–204PubMedCrossRefGoogle Scholar
  5. Berois N, Varangot M, Sonora C, Zarantonelli L, Pressa C, Lavina R, Rodriguez JL, Delgado F, Porchet N, Aubert JP, Osinaga E (2003) Detection of bone marrow-disseminated breast cancer cells using an RT-PCR assay of MUC5B mRNA. Int J Cancer 103:550–555PubMedCrossRefGoogle Scholar
  6. Bragonzi A (2010) Murine models of acute and chronic lung infection with cystic fibrosis pathogens. Int J Med Microbiol 300:584–593PubMedCrossRefGoogle Scholar
  7. Burgel PR, Montani D, Danel C, Dusser DJ, Nadel JA (2007) A morphometric study of mucins and small airway plugging in cystic fibrosis. Thorax 62:153–161PubMedCrossRefGoogle Scholar
  8. Chen Y, Zhao YH, Kalaslavadi TB, Hamati E, Nehrke K, Le AD, Ann DK, Wu R (2004) Genome-wide search and identification of a novel gel-forming mucin MUC19/Muc19 in glandular tissues. Am J Respir Cell Mol Biol 30:155–165PubMedCrossRefGoogle Scholar
  9. Chen Y, Zhao YH, Wu R (2001) In silico cloning of mouse Muc5b gene and upregulation of its expression in mouse asthma model. Am J Respir Crit Care Med 164:1059–1066PubMedGoogle Scholar
  10. Culp DJ, Latchney LR, Fallon MA, Denny PA, Denny PC, Couwenhoven RI, Chuang S (2004) The gene encoding mouse Muc19: cDNA, genomic organization and relationship to Smgc. Physiol Genomics 19:303–318PubMedCrossRefGoogle Scholar
  11. Curran DR, Cohn L (2010) Advances in mucous cell metaplasia: a plug for mucus as a therapeutic focus in chronic airway disease. Am J Respir Cell Mol Biol 42:268–275PubMedCrossRefGoogle Scholar
  12. Davies DG, Parsek MR, Pearson JP, Iglewski BH, Costerton JW, Greenberg EP (1998) The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280:295–298PubMedCrossRefGoogle Scholar
  13. Desseyn JL (2009) Mucin CYS domains are ancient and highly conserved modules that evolved in concert. Mol Phylogenet Evol 52:284–292PubMedCrossRefGoogle Scholar
  14. Desseyn JL, Aubert JP, Van Seuningen I, Porchet N, Laine A (1997a) Genomic organization of the 3′ region of the human mucin gene MUC5B. J Biol Chem 272:16873–16883PubMedCrossRefGoogle Scholar
  15. Desseyn JL, Buisine MP, Porchet N, Aubert JP, Laine A (1998) Genomic organization of the human mucin gene MUC5B. cDNA and genomic sequences upstream of the large central exon. J Biol Chem 273:30157–30164PubMedCrossRefGoogle Scholar
  16. Desseyn JL, Gouyer V, Tetaert D (2008) Architecture of the gel-forming mucins. In: Van Seuningen I (ed) The epithelial mucins: structure/function. Roles in cancer and inflammatory diseases. Signpost, KeralaGoogle Scholar
  17. Desseyn JL, Guyonnet-Duperat V, Porchet N, Aubert JP, Laine A (1997b) Human mucin gene MUC5B, the 10.7-kb large central exon encodes various alternate subdomains resulting in a super-repeat. Structural evidence for a 11p15.5 gene family. J Biol Chem 272:3168–3178PubMedCrossRefGoogle Scholar
  18. Desseyn JL, Laine A (2003) Characterization of mouse muc6 and evidence of conservation of the gel-forming mucin gene cluster between human and mouse. Genomics 81:433–436PubMedCrossRefGoogle Scholar
  19. Desseyn JL, Rousseau K, Laine A (1999) Fifty-nine bp repeat polymorphism in the uncommon intron 36 of the human mucin gene MUC5B. Electrophoresis 20:493–496PubMedCrossRefGoogle Scholar
  20. Evans CM, Williams OW, Tuvim MJ, Nigam R, Mixides GP, Blackburn MR, DeMayo FJ, Burns AR, Smith C, Reynolds SD, Stripp BR, Dickey BF (2004) Mucin is produced by clara cells in the proximal airways of antigen-challenged mice. Am J Respir Cell Mol Biol 31:382–394PubMedCrossRefGoogle Scholar
  21. Freedman SD, Katz MH, Parker EM, Laposata M, Urman MY, Alvarez JG (1999) A membrane lipid imbalance plays a role in the phenotypic expression of cystic fibrosis in cftr(−/−) mice. Proc Natl Acad Sci USA 96:13995–14000PubMedCrossRefGoogle Scholar
  22. Gouyer V, Leir SH, Tetaert D, Liu Y, Gottrand F, Harris A, Desseyn JL (2010) The characterization of the first anti-mouse Muc6 antibody shows an increased expression of the mucin in pancreatic tissue of Cftr-knockout mice. Histochem Cell Biol 133:517–525PubMedCrossRefGoogle Scholar
  23. Guillem P, Billeret V, Buisine MP, Flejou JF, Lecomte-Houcke M, Degand P, Aubert JP, Triboulet JP, Porchet N (2000) Mucin gene expression and cell differentiation in human normal, premalignant and malignant esophagus. Int J Cancer 88:856–861PubMedCrossRefGoogle Scholar
  24. Henke MO, John G, Germann M, Lindemann H, Rubin BK (2007) MUC5AC and MUC5B mucins increase in cystic fibrosis airway secretions during pulmonary exacerbation. Am J Respir Crit Care Med 175:816–821PubMedCrossRefGoogle Scholar
  25. Henke MO, Renner A, Huber RM, Seeds MC, Rubin BK (2004) MUC5AC and MUC5B mucins are decreased in cystic fibrosis airway secretions. Am J Respir Cell Mol Biol 31:86–91PubMedCrossRefGoogle Scholar
  26. Hutton DA, Guo L, Birchall JP, Severn TL, Pearson JP (1998) MUC5B expression in middle ear mucosal glands. Biochem Soc Trans 26:S117PubMedGoogle Scholar
  27. Keates AC, Nunes DP, Afdhal NH, Troxler RF, Offner GD (1997) Molecular cloning of a major human gall bladder mucin: complete C-terminal sequence and genomic organization of MUC5B. Biochem J 324(Pt 1):295–303PubMedGoogle Scholar
  28. Kent G, Iles R, Bear CE, Huan LJ, Griesenbach U, McKerlie C, Frndova H, Ackerley C, Gosselin D, Radzioch D, O’Brodovich H, Tsui LC, Buchwald M, Tanswell AK (1997) Lung disease in mice with cystic fibrosis. J Clin Invest 100:3060–3069PubMedCrossRefGoogle Scholar
  29. Kirkham S, Sheehan JK, Knight D, Richardson PS, Thornton DJ (2002) Heterogeneity of airways mucus: variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B. Biochem J 361:537–546PubMedCrossRefGoogle Scholar
  30. Paulsen F, Langer G, Hoffmann W, Berry M (2004) Human lacrimal gland mucins. Cell Tissue Res 316:167–177PubMedCrossRefGoogle Scholar
  31. Reid CJ, Gould S, Harris A (1997) Developmental expression of mucin genes in the human respiratory tract. Am J Respir Cell Mol Biol 17:592–598PubMedGoogle Scholar
  32. Rousseau K, Wickstrom C, Whitehouse DB, Carlstedt I, Swallow DM (2003) New monoclonal antibodies to non-glycosylated domains of the secreted mucins MUC5B and MUC7. Hybrid Hybridomics 22:293–299PubMedCrossRefGoogle Scholar
  33. Roy MG, Rahmani M, Hernandez JR, Alexander SN, Ehre C, Ho SB, Evans CM (2011) Mucin production during prenatal and postnatal murine lung development. Am J Respir Cell Mol Biol 44:755–760PubMedCrossRefGoogle Scholar
  34. Sajjan U, Thanassoulis G, Cherapanov V, Lu A, Sjolin C, Steer B, Wu YJ, Rotstein OD, Kent G, McKerlie C, Forstner J, Downey GP (2001) Enhanced susceptibility to pulmonary infection with Burkholderia cepacia in Cftr(−/−) mice. Infect Immun 69:5138–5150PubMedCrossRefGoogle Scholar
  35. Seibold MA, Wise AL, Speer MC, Steele MP, Brown KK, Loyd JE, Fingerlin TE, Zhang W, Gudmundsson G, Groshong SD, Evans CM, Garantziotis S, Adler KB, Dickey BF, du Bois RM, Yang IV, Herron A, Kervitsky D, Talbert JL, Markin C, Park J, Crews AL, Slifer SH, Auerbach S, Roy MG, Lin J, Hennessy CE, Schwarz MI, Schwartz DA (2011) A common MUC5B promoter polymorphism and pulmonary fibrosis. N Engl J Med 364:1503–1512PubMedCrossRefGoogle Scholar
  36. Shen Y, Wang Y, Chen Z, Wang D, Wang X, Jin M, Bai C (2011) Role of aquaporin 5 in antigen-induced airway inflammation and mucous hyperproduction in mice. J Cell Mol Med 15:1355–1363PubMedCrossRefGoogle Scholar
  37. Sinn E, Muller W, Pattengale P, Tepler I, Wallace R, Leder P (1987) Coexpression of MMTV/v-Ha-ras and MMTV/c-myc genes in transgenic mice: synergistic action of oncogenes in vivo. Cell 49:465–475PubMedCrossRefGoogle Scholar
  38. Snouwaert JN, Brigman KK, Latour AM, Malouf NN, Boucher RC, Smithies O, Koller BH (1992) An animal model for cystic fibrosis made by gene targeting. Science 257:1083–1088PubMedCrossRefGoogle Scholar
  39. Sonora C, Mazal D, Berois N, Buisine MP, Ubillos L, Varangot M, Barrios E, Carzoglio J, Aubert JP, Osinaga E (2006) Immunohistochemical analysis of MUC5B apomucin expression in breast cancer and non-malignant breast tissues. J Histochem Cytochem 54:289–299PubMedCrossRefGoogle Scholar
  40. Tetaert D, Pierre M, Demeyer D, Husson MO, Beghin L, Galabert C, Gottrand F, Beermann C, Guery B, Desseyn JL (2007) Dietary n-3 fatty acids have suppressive effects on mucin upregulation in mice infected with Pseudomonas aeruginosa. Respir Res 8:39PubMedCrossRefGoogle Scholar
  41. Thai P, Loukoianov A, Wachi S, Wu R (2008) Regulation of airway mucin gene expression. Annu Rev Physiol 70:405–429PubMedCrossRefGoogle Scholar
  42. Thornton DJ, Howard M, Khan N, Sheehan JK (1997) Identification of two glycoforms of the MUC5B mucin in human respiratory mucus. Evidence for a cysteine-rich sequence repeated within the molecule. J Biol Chem 272:9561–9566PubMedCrossRefGoogle Scholar
  43. Thornton DJ, Rousseau K, McGuckin MA (2008) Structure and function of the polymeric mucins in airways mucus. Annu Rev Physiol 70:459–486PubMedCrossRefGoogle Scholar
  44. van Klinken BJ, Einerhand AW, Buller HA, Dekker J (1998) The oligomerization of a family of four genetically clustered human gastrointestinal mucins. Glycobiology 8:67–75PubMedCrossRefGoogle Scholar
  45. Veerman EC, van den Keijbus PA, Nazmi K, Vos W, van der Wal JE, Bloemena E, Bolscher JG, Amerongen AV (2003) Distinct localization of MUC5B glycoforms in the human salivary glands. Glycobiology 13:363–366PubMedCrossRefGoogle Scholar
  46. Verhaeghe C, Delbecque K, de Leval L, Oury C, Bours V (2007) Early inflammation in the airways of a cystic fibrosis foetus. J Cyst Fibros 6:304–308PubMedCrossRefGoogle Scholar
  47. Vincent A, Perrais M, Desseyn JL, Aubert JP, Pigny P, Van Seuningen I (2007) Epigenetic regulation (DNA methylation, histone modifications) of the 11p15 mucin genes (MUC2, MUC5AC, MUC5B, MUC6) in epithelial cancer cells. Oncogene 26:6566–6576PubMedCrossRefGoogle Scholar
  48. Wickstrom C, Davies JR, Eriksen GV, Veerman EC, Carlstedt I (1998) MUC5B is a major gel-forming, oligomeric mucin from human salivary gland, respiratory tract and endocervix: identification of glycoforms and C-terminal cleavage. Biochem J 334(Pt 3):685–693PubMedGoogle Scholar
  49. Zhu Y, Ehre C, Abdullah LH, Sheehan JK, Roy M, Evans CM, Dickey BF, Davis CW (2008) Munc13-2−/− baseline secretion defect reveals source of oligomeric mucins in mouse airways. J Physiol 586:1977–1992PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Hélène Valque
    • 1
  • Valérie Gouyer
    • 1
    • 2
  • Marie-Odile Husson
    • 1
    • 2
  • Frédéric Gottrand
    • 1
    • 2
  • Jean-Luc Desseyn
    • 1
  1. 1.Inserm U995, Faculté de Médecine, Pôle Recherche, Bât. Huriez, 5ème étageUniversity Lille 2LilleFrance
  2. 2.CHRU of LilleLilleFrance

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