Pflügers Archiv - European Journal of Physiology

, Volume 463, Issue 2, pp 391–398 | Cite as

Altered expression of tight junction proteins in mammary epithelium after discontinued suckling in mice

  • Alexander G. Markov
  • Natalia M. Kruglova
  • Yulia A. Fomina
  • Michael Fromm
  • Salah AmashehEmail author
Transport Physiology


Milk production is modulated by the paracellular barrier function of tight junction (TJ) proteins located in the mammary epithelium. The aim of our study was the molecular analysis of TJs in native lactating murine mammary gland epithelium as this process may strongly challenge epithelial barrier properties and regulation. Mammary gland tissue specimens from lactating control mice and animals after a 20-h interruption of suckling were prepared; histological analyses were performed by light and electron microscopy; and expression of TJ proteins was detected by PCR, Western blotting, immunofluorescent staining, and confocal laser scanning microscopy. Discontinuation of suckling resulted in a substantial accumulation of milk in mammary glands, an increase of alveolar size, and a flattening of epithelial cells without effects on inflammatory indicators. In control tissues, PCR and Western blots showed signals for occludin, and claudin-1, -2, -3, -4, -5, -7, -8, -15, and -16. After a 20-h accumulation of milk, expression of two sealing TJ proteins, claudin-1 and -3, was markedly increased, whereas two TJ proteins involved in cation transport, claudin-2 and -16, were reduced. Real-time PCR validated increased transcripts of claudin-1 and claudin-3. During extension of mammary glands in the process of lactation, claudin-1 and -3 are markedly induced and claudin-2 and -16 are decreased. Volume and composition of milk might be strongly dependent on this counter-regulation of sealing claudins with permeability-mediating claudins, indicating a physiological process of a tightening of TJs against a back-leak of solutes and ions from the alveolar lumen.


Mammary gland Lactation Claudins Occluding 



Tight junction



The study has been supported by the Deutsche Forschungsgemeinschaft (DFG FOR 721), the Sonnenfeld-Stiftung Berlin, the Partnership Program FU Berlin–University St. Petersburg, and by Saint Petersburg University Research Grant No.

Conflict of interest

There is no conflict of interest.


  1. 1.
    Alekseev NP, Markov AG, Tolkunov YA (1992) Transepithelial potential difference in the goat mammary gland and its change during hand milking, and administration of oxytocin and catecholamines. J Dairy Res 59:469–478PubMedCrossRefGoogle Scholar
  2. 2.
    Alexandre MD, Lu Q, Chen YH (2005) Overexpression of claudin-7 decreases the paracellular Cl conductance and increases the paracellular Na+ conductance in LLC-PK1 cells. J Cell Sci 118:2683–2693PubMedCrossRefGoogle Scholar
  3. 3.
    Amasheh S, Dullat S, Fromm M, Schulzke JD, Buhr HJ, Kroesen AJ (2009) Inflamed pouch mucosa possesses altered tight junctions indicating recurrence of inflammatory bowel disease. Int J Colorectal Dis 24:1149–1156PubMedCrossRefGoogle Scholar
  4. 4.
    Amasheh S, Fromm M, Günzel D (2011) Claudins of intestine and nephron—a correlation of molecular tight junction structure and barrier function. Acta Physiol (Oxf) 201:133–140CrossRefGoogle Scholar
  5. 5.
    Amasheh S, Meiri N, Gitter AH, Schöneberg T, Mankertz J, Schulzke JD, Fromm M (2002) Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells. J Cell Sci 115:4969–4976PubMedCrossRefGoogle Scholar
  6. 6.
    Amasheh S, Milatz S, Krug SM, Bergs M, Amasheh M, Schulzke JD, Fromm M (2009) Na+ absorption defends from paracellular back-leakage by claudin-8 upregulation. Biochem Biophys Res Commun 378:45–50PubMedCrossRefGoogle Scholar
  7. 7.
    Amasheh S, Schmidt T, Mahn M, Florian P, Mankertz J, Tavalali S, Gitter AH, Schulzke JD, Fromm M (2005) Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells. Cell Tissue Res 321:89–96PubMedCrossRefGoogle Scholar
  8. 8.
    Barmeyer C, Amasheh S, Tavalali S, Mankertz J, Zeitz M, Fromm M, Schulzke JD (2004) IL-1beta and TNFalpha regulate sodium absorption in rat distal colon. Biochem Biophys Res Commun 317:500–507PubMedCrossRefGoogle Scholar
  9. 9.
    Blackman B, Russell T, Nordeen SK, Medina D, Neville MC (2005) Claudin 7 expression and localization in the normal murine mammary gland and murine mammary tumors. Breast Cancer Res 7:R248–R255PubMedCrossRefGoogle Scholar
  10. 10.
    Blanchard AA, Watson PH, Shiu RP, Leygue E, Nistor A, Wong P, Myal Y (2006) Differential expression of claudin 1, 3, and 4 during normal mammary gland development in the mouse. DNA Cell Biol 25:79–86PubMedCrossRefGoogle Scholar
  11. 11.
    Cattan V, Bernard G, Rousseau A, Bouhout S, Chabaud S, Auger FA, Bolduc S (2011) Mechanical stimuli-induced urothelial differentiation in a human tissue-engineered tubular genitourinary graft. Eur Urol [Epub ahead of print]Google Scholar
  12. 12.
    Ernens I, Clegg R, Schneider YJ, Larondelle Y (2007) Ability of cultured mammary epithelial cells in a bicameral system to secrete milk fat. J Dairy Sci 90:677–681PubMedCrossRefGoogle Scholar
  13. 13.
    Florian P, Amasheh S, Lessidrensky M, Todt I, Bloedow A, Ernst A, Fromm M, Gitter AH (2003) Claudins in the tight junctions of stria vascularis marginal cells. Biochem Biophys Res Commun 304:5–10PubMedCrossRefGoogle Scholar
  14. 14.
    Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S (1998) Claudin-1 and −2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141:1539–1550PubMedCrossRefGoogle Scholar
  15. 15.
    Furuse M, Hata M, Furuse K, Yoshida Y, Haratake A, Sugitani Y, Noda T, Kubo A, Tsukita S (2002) Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice. J Cell Biol 156:1099–1111PubMedCrossRefGoogle Scholar
  16. 16.
    Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S (1993) Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol 123:1777–1788PubMedCrossRefGoogle Scholar
  17. 17.
    Günzel D, Amasheh S, Pfaffenbach S, Richter JF, Kausalya PJ, Hunziker W, Fromm M (2009) Claudin-16 affects transcellular Cl- secretion in MDCK cells. J Physiol 587:3777–3793PubMedCrossRefGoogle Scholar
  18. 18.
    Higuchi T, Tadokoro Y, Honda K, Negoro H (1986) Detailed analysis of blood oxytocin levels during suckling and parturition in the rat. J Endocrinol 110:251–256PubMedCrossRefGoogle Scholar
  19. 19.
    Hoevel T, Macek R, Mundigl O, Swisshelm K, Kubbies M (2002) Expression and targeting of the tight junction protein CLDN1 in CLDN1-negative human breast tumor cells. J Cell Physiol 191:60–68PubMedCrossRefGoogle Scholar
  20. 20.
    Hsu CC, Tsai WC, Chen CP, Lu YM, Wang JS (2010) Effects of negative pressures on epithelial tight junctions and migration in wound healing. Am J Physiol Cell Physiol 299:C528–C534PubMedCrossRefGoogle Scholar
  21. 21.
    Ikari A, Okude C, Sawada H, Sasaki Y, Yamazaki Y, Sugatani J, Degawa M, Miwa M (2008) Activation of a polyvalent cation-sensing receptor decreases magnesium transport via claudin-16. Biochim Biophys Acta 1778:283–290PubMedCrossRefGoogle Scholar
  22. 22.
    Ikenouchi J, Furuse M, Furuse K, Sasaki H, Tsukita S, Tsukita S (2005) Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells. J Cell Biol 171:939–945PubMedCrossRefGoogle Scholar
  23. 23.
    Jakab C, Halász J, Szász AM, Kiss A, Schaff Z, Rusvai M, Gálfi P, Kulka J (2008) Expression of claudin-1, -2, -3, -4, -5 and −7 proteins in benign and malignant canine mammary gland epithelial tumours. J Comp Pathol 139:238–245PubMedCrossRefGoogle Scholar
  24. 24.
    Kausalya PJ, Amasheh S, Günzel D, Wurps H, Müller D, Fromm M, Hunziker W (2006) Disease-associated mutations affect intracellular traffic and paracellular Mg2+ transport function of claudin-16. J Clin Invest 116:878–891PubMedCrossRefGoogle Scholar
  25. 25.
    Kiuchi-Saishin Y, Gotoh S, Furuse M, Takasuga A, Tano Y, Tsukita S (2002) Differential expression patterns of claudins, tight junction membrane proteins, in mouse nephron segments. J Am Soc Nephrol 13:875–886PubMedGoogle Scholar
  26. 26.
    Krug SM, Amasheh S, Richter JF, Milatz S, Günzel D, Westphal JK, Huber O, Schulzke JD, Fromm M (2009) Tricellulin forms a barrier to macromolecules in tricellular tight junctions without affecting ion permeability. Mol Biol Cell 20:3713–3724PubMedCrossRefGoogle Scholar
  27. 27.
    Lincoln DW, Hill A, Wakerley JB (1973) The milk ejection reflex on the rat: an intermittent function not abolished by surgical levels of anaesthesia. J Endocrinol 57:459–476PubMedCrossRefGoogle Scholar
  28. 28.
    Markov AG (1991) Study of durations and periodicity of nursing in lactating mice. Sechenov Physiol J USSR 77:142–148Google Scholar
  29. 29.
    Markov AG, Veshnyakova A, Fromm M, Amasheh M, Amasheh S (2010) Segmental expression of claudin proteins correlates with tight junction barrier properties in rat intestine. J Comp Physiol B 180:591–598PubMedCrossRefGoogle Scholar
  30. 30.
    Milatz S, Krug SM, Rosenthal R, Günzel D, Müller D, Schulzke JD, Amasheh S, Fromm M (2010) Claudin-3 acts as a sealing component of the tight junction for ions of either charge and uncharged solutes. Biochim Biophys Acta 1798:2048–2057PubMedCrossRefGoogle Scholar
  31. 31.
    Neville MC (2005) Calcium secretion into milk. J Mammary Gland Biol Neoplasia 10:119–128PubMedCrossRefGoogle Scholar
  32. 32.
    Nguyen DA, Neville MC (1998) Tight junction regulation in the mammary gland. J Mammary Gland Biol Neoplasia 3:233–246PubMedCrossRefGoogle Scholar
  33. 33.
    Nguyen DA, Parlow AF, Neville MC (2001) Hormonal regulation of tight junction closure in the mouse mammary epithelium during the transition from pregnancy to lactation. J Endocrinol 170:347–356PubMedCrossRefGoogle Scholar
  34. 34.
    Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, Hashimoto N, Furuse M, Tsukita S (2003) Size-selective loosening of the blood–brain barrier in claudin-5-deficient mice. J Cell Biol 161:653–660PubMedCrossRefGoogle Scholar
  35. 35.
    Reiter B, Kraft R, Günzel D, Zeissig S, Schulzke JD, Fromm M, Harteneck C (2006) TRPV4-mediated regulation of epithelial permeability. FASEB J 20:1802–1812PubMedCrossRefGoogle Scholar
  36. 36.
    Rosenthal R, Milatz S, Krug SM, Oelrich B, Schulzke JD, Amasheh S, Günzel D, Fromm M (2010) Claudin-2, a component of the tight junction, forms a paracellular water channel. J Cell Sci 123:1913–1921PubMedCrossRefGoogle Scholar
  37. 37.
    Simon DB, Lu Y, Choate KA, Velazquez H, Al Sabban E, Praga M, Casari G, Bettinelli A, Colussi G, Rodriguez-Soriano J, McCredie D, Milford D, Sanjad S, Lifton RP (1999) Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption. Science 285:103–106PubMedCrossRefGoogle Scholar
  38. 38.
    Steed E, Rodrigues NT, Balda MS, Matter K (2009) Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family. BMC Cell Biol 10:95PubMedCrossRefGoogle Scholar
  39. 39.
    Tolkunov YA, Markov AG (1997) Integrity of the secretory epithelium of the lactating mouse mammary gland during extended periods after suckling. J Dairy Res 64:39–45PubMedCrossRefGoogle Scholar
  40. 40.
    Troeger H, Loddenkemper C, Schneider T, Schreier E, Epple HJ, Zeitz M, Fromm M, Schulzke JD (2009) Structural and functional changes of the duodenum in human norovirus infection. Gut 58:1070–1077PubMedCrossRefGoogle Scholar
  41. 41.
    Tsukita S, Furuse M, Itoh M (2001) Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol 2:285–293PubMedCrossRefGoogle Scholar
  42. 42.
    Turner MD, Rennison ME, Handel SE, Wilde CJ, Burgoyne RD (1992) Proteins are secreted by both constitutive and regulated secretory pathways in lactating mouse mammary epithelial cells. J Cell Biol 117:269–278PubMedCrossRefGoogle Scholar
  43. 43.
    Van Itallie CM, Fanning AS, Anderson JM (2003) Reversal of charge selectivity in cation or anion-selective epithelial lines by expression of different claudins. Am J Physiol Renal Physiol 285:F1078–F1084PubMedGoogle Scholar
  44. 44.
    Van Itallie CM, Rahner C, Anderson JM (2001) Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability. J Clin Invest 107:1319–1327PubMedCrossRefGoogle Scholar
  45. 45.
    Wolburg H, Wolburg-Buchholz K, Kraus J, Rascher-Eggstein G, Liebner S, Hamm S, Duffner F, Grote EH, Risau W, Engelhardt B (2003) Localization of claudin-3 in tight junctions of the blood–brain barrier is selectively lost during experimental autoimmune encephalomyelitis and human glioblastoma multiforme. Acta Neuropathol 105:586–592PubMedGoogle Scholar
  46. 46.
    Yu AS, Enck AH, Lencer WI, Schneeberger EE (2003) Claudin-8 expression in MDCK cells augments the paracellular barrier to cation permeation. J Biol Chem 278:17350–17359PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Alexander G. Markov
    • 1
  • Natalia M. Kruglova
    • 1
  • Yulia A. Fomina
    • 1
  • Michael Fromm
    • 2
  • Salah Amasheh
    • 2
    Email author
  1. 1.Biological and Soil FacultySt. Petersburg UniversitySt. PetersburgRussia
  2. 2.Institute of Clinical PhysiologyCharité Campus Benjamin FranklinBerlinGermany

Personalised recommendations