Advertisement

Molecular mechanisms in uterine epithelium during trophoblast binding: the role of small GTPase RhoA in human uterine Ishikawa cells

  • Carola Heneweer
  • Martina Schmidt
  • Hans-Werner Denker
  • Michael Thie
Open Access
Research

Abstract

Background

Embryo implantation requires that uterine epithelium develops competence to bind trophoblast to its apical (free) poles. This essential element of uterine receptivity seems to depend on a destabilisation of the apico-basal polarity of endometrial epithelium. Accordingly, a reorganisation of the actin cytoskeleton regulated by the small GTPase RhoA plays an important role in human uterine epithelial RL95-2 cells for binding of human trophoblastoid JAR cells. We now obtained new insight into trophoblast binding using human uterine epithelial Ishikawa cells.

Methods

Polarity of Ishikawa cells was investigated by electron microscopy, apical adhesiveness was tested by adhesion assay. Analyses of subcellular distribution of filamentous actin (F-actin) and RhoA in apical and basal cell poles were performed by confocal laser scanning microscopy (CLSM) with and without binding of JAR spheroids as well as with and without inhibition of small Rho GTPases by Clostridium difficile toxin A (toxin A). In the latter case, subcellular distribution of RhoA was additionally investigated by Western blotting.

Results

Ishikawa cells express apical adhesiveness for JAR spheroids and moderate apico-basal polarity. Without contact to JAR spheroids, significantly higher signalling intensities of F-actin and RhoA were found at the basal as compared to the apical poles in Ishikawa cells. RhoA was equally distributed between the membrane fraction and the cytosol fraction. Levels of F-actin and RhoA signals became equalised in the apical and basal regions upon contact to JAR spheroids. After inhibition of Rho GTPases, Ishikawa cells remained adhesive for JAR spheroids, the gradient of fluorescence signals of F-actin and RhoA was maintained while the amount of RhoA was reduced in the cytosolic fraction with a comparable increase in the membrane fraction.

Conclusion

Ishikawa cells respond to JAR contact as well as to treatment with toxin A with rearrangement of F-actin and small GTPase RhoA but seem to be able to modify signalling pathways in a way not elucidated so far in endometrial cells. This ability may be linked to the degree of polar organisation observed in Ishikawa cells indicating an essential role of cell phenotype modification in apical adhesiveness of uterine epithelium for trophoblast in vivo.

Notes

Acknowledgements

We thank K.-D. Schulz (Marburg, Germany) for providing the Ishikawa cell line, as well as H. G. Adelmann (Loughborough, UK) for constructive criticisms in advanced digital image processing and for kind provision of his Gauss bandpass and homomorphic filter plugins, and J. Huesing (Essen, Germany) for help with statistical analysis. The skilful technical assistance of K. Baden, B. Gobs, B. Maranca and D. Schuenke is gratefully acknowledged.

References

  1. 1.
    Carson DD, Bagchi I, Dey SK, Enders AC, Fazleabas AT, Lessey BA, Yoshinaga K: Embryo implantation. Dev Biol. 2000, 223: 217-237. 10.1006/dbio.2000.9767.CrossRefPubMedGoogle Scholar
  2. 2.
    Denker HW: Endometrial receptivity: cell biological aspects of an unusual epithelium. A review. Anat Anz. 1994, 176: 53-60.CrossRefGoogle Scholar
  3. 3.
    Denker HW, Thie M: The regulatory function of the uterine epithelium for trophoblast attachment: experimental approaches. Ital J Anat Embryol. 2001, 106 (2Suppl 2): 291-306.PubMedGoogle Scholar
  4. 4.
    Thie M, Denker HW: In vitro studies on endometrial adhesiveness for trophoblast: cellular dynamics in uterine epithelial cells. Cells Tissues Organs. 2002, 172: 237-252. 10.1159/000066963.CrossRefPubMedGoogle Scholar
  5. 5.
    Albers A, Thie M, Hohn HP, Denker HW: Differential expression and localization of integrins and CD44 in the membrane domains of human uterine epithelial cells during the menstrual cycle. Acta Anat. 1995, 153: 12-19.CrossRefGoogle Scholar
  6. 6.
    Carson DD: The glycobiology of implantation. Front Biosci. 2002, 7: d1535-1544.CrossRefGoogle Scholar
  7. 7.
    Kim JJ, Fazleabas AT: Uterine receptivity and implantation: the regulation and action of insulin-like growth factor binding protein-1 (IGFBP-1), HOXA10 and forkhead transcription factor-1 (FOXO-1) in the baboon endometrium. Reprod Biol Endocrinol. 2004, 2: 34-10.1186/1477-7827-2-34.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Lessey BA, Castelbaum AJ: Integrins and implantation in the human. Rev Endocr Metab Disord. 2002, 3: 107-117. 10.1023/A:1015450727580.CrossRefPubMedGoogle Scholar
  9. 9.
    Thie M, Denker HW: Endometrial receptivity for trophoblast attachment: Model studies using cell lines. Microscopy of Reproduction and Development. A Dynamic Approach. Edited by: Motta PM. 1997, Rome: Antonio Delfino Editore S.r.l., 241-249.Google Scholar
  10. 10.
    Thie M, Rospel R, Dettmann W, Benoit M, Ludwig M, Gaub HE, Denker HW: Interactions between trophoblast and uterine epithelium: monitoring of adhesive forces. Hum Reprod. 1998, 13: 3211-3219. 10.1093/humrep/13.11.3211.CrossRefPubMedGoogle Scholar
  11. 11.
    Bentin-Ley U, Horn T, Sjogren A, Sorensen S, Falck Larsen J, Hamberger L: Ultrastructure of human blastocyst-endometrial interactions in vitro. J Reprod Fertil. 2000, 120: 337-350. 10.1530/reprod/120.2.337.CrossRefPubMedGoogle Scholar
  12. 12.
    Murphy CR: The plasma membrane transformation of uterine epithelial cells during pregnancy. J Reprod Fertil Suppl. 2000, 55: 23-28.PubMedGoogle Scholar
  13. 13.
    Nikas G: Endometrial receptivity: changes in cell-surface morphology. Semin Reprod Med. 2000, 18: 229-235. 10.1055/s-2000-12561.CrossRefPubMedGoogle Scholar
  14. 14.
    Tinel H, Denker HW, Thie M: Calcium influx in human uterine epithelial RL95-2 cells triggers adhesiveness for trophoblast-like cells. Model studies on signalling events during embryo implantation. Mol Hum Reprod. 2000, 6: 1119-1130. 10.1093/molehr/6.12.1119.CrossRefPubMedGoogle Scholar
  15. 15.
    Kimber SJ, Spanswick C: Blastocyst implantation: the adhesion cascade. Semin Cell Dev Biol. 2000, 11: 77-92. 10.1006/scdb.2000.0154.CrossRefPubMedGoogle Scholar
  16. 16.
    Paria BC, Lim H, Das SK, Reese J, Dey SK: Molecular signaling in uterine receptivity for implantation. Semin Cell Dev Biol. 2000, 11: 67-76. 10.1006/scdb.2000.0153.CrossRefPubMedGoogle Scholar
  17. 17.
    Denker HW: Epithel-Epithel-Interaktionen bei der Embryo-Implantation: Ansaetze zur Loesung eines zellbiologischen Paradoxons. Anat Anz. 1986, 160 (Suppl Verh Anat Ges 80): 93-114.Google Scholar
  18. 18.
    Denker HW: Trophoblast – endometrial interactions at embryo implantation: A cell biological paradox. Trophoblast Invasion and Endometrial Receptivity. Novel Aspects of the Cell Biology of Embryo Implantation. Trophoblast Research. Edited by: Denker HW, Aplin JD. 1990, New York: Plenum Medical Book, 4: 3-29.CrossRefGoogle Scholar
  19. 19.
    Denker HW: Implantation: a cell biological paradox. J Exp Zool. 1993, 266: 541-558.CrossRefGoogle Scholar
  20. 20.
    John NJ, Linke M, Denker HW: Quantitation of human choriocarcinoma spheroid attachment to uterine epithelial cell monolayers. In Vitro Cell Dev Biol Anim. 1993, 29A: 461-468.CrossRefGoogle Scholar
  21. 21.
    Thie M, Harrach-Ruprecht B, Sauer H, Fuchs P, Albers A, Denker HW: Cell adhesion to the apical pole of epithelium: a function of cell polarity. Eur J Cell Biol. 1995, 66: 180-91.PubMedGoogle Scholar
  22. 22.
    Thie M, Fuchs P, Butz S, Sieckmann F, Hoschuetzky H, Kemler R, Denker HW: Adhesiveness of the apical surface of uterine epithelial cells: the role of junctional complex integrity. Eur J Cell Biol. 1996, 70: 221-232.PubMedGoogle Scholar
  23. 23.
    Thie M, Herter P, Pommerenke H, Durr F, Sieckmann F, Nebe B, Rychly J, Denker HW: Adhesiveness of the free surface of a human endometrial monolayer for trophoblast as related to actin cytoskeleton. Mol Hum Reprod. 1997, 3: 275-283. 10.1093/molehr/3.4.275.CrossRefPubMedGoogle Scholar
  24. 24.
    Heneweer C, Kruse LH, Kindhauser F, Schmidt M, Jakobs KH, Denker HW, Thie M: Adhesiveness of human uterine epithelial RL95-2 cells to trophoblast: rho protein regulation. Mol Hum Reprod. 2002, 8: 1014-1022. 10.1093/molehr/8.11.1014.CrossRefPubMedGoogle Scholar
  25. 25.
    Heneweer C, Adelmann HG, Kruse LH, Denker HW, Thie M: Human uterine epithelial RL95-2 cells reorganize their cytoplasmic architecture with respect to Rho protein and F-actin in response to trophoblast binding. Cells Tissues Organs. 2003, 175: 1-8. 10.1159/000073432.CrossRefPubMedGoogle Scholar
  26. 26.
    Nishida M, Kasahara K, Kaneko M, Iwasaki H, Hayashi K: Establishment of a new human endometrial adenocarcinoma cell line, Ishikawa cells, containing estrogen and progesterone receptors. Nippon Sanka Fujinka Gakkai Zasshi. 1985, 37: 1103-11.PubMedGoogle Scholar
  27. 27.
    Lessey BA, Castelbaum AJ: Integrins and implantation in the human. Rev Endocr Metab Disord. 2002, 3: 107-117. 10.1023/A:1015450727580.CrossRefPubMedGoogle Scholar
  28. 28.
    Li Q, Wang J, Armant DR, Bagchi MK, Bagchi IC: Calcitonin down-regulates E-cadherin expression in rodent uterine epithelium during implantation. J Biol Chem. 2002, 277: 46447-46455. 10.1074/jbc.M203555200.CrossRefPubMedGoogle Scholar
  29. 29.
    Savaris R, Chies JA: Copper ions dynamically regulate beta3 integrin subunit expression in Ishikawa cells. Contraception. 2003, 67: 247-249. 10.1016/S0010-7824(02)00511-5.CrossRefPubMedGoogle Scholar
  30. 30.
    Widra EA, Weeraratna A, Stepp MA, Stillman RJ, Patierno SR: Modulation of implantation-associated integrin expression but not uteroglobin by steroid hormones in an endometrial cell line. Mol Hum Reprod. 1997, 3: 563-568. 10.1093/molehr/3.7.563.CrossRefPubMedGoogle Scholar
  31. 31.
    Emons G, Schroder B, Ortmann O, Westphalen S, Schulz KD, Schally AV: High affinity binding and direct antiproliferative effects of luteinizing hormone-releasing hormone analogs in human endometrial cancer cell lines. J Clin Endocrinol Metab. 1993, 77: 1458-1464. 10.1210/jc.77.6.1458.CrossRefPubMedGoogle Scholar
  32. 32.
    Irmer G, Burger C, Ortmann O, Schulz KD, Emons G: Expression of luteinizing hormone releasing hormone and its mRNA in human endometrial cancer cell lines. J Clin Endocrinol Metab. 1994, 79: 916-919. 10.1210/jc.79.3.916.CrossRefPubMedGoogle Scholar
  33. 33.
    Pattillo RA, Ruckert A, Hussa R, Bernstein R, Delfs E: The JAR cell line – continuous human multihormone production and controls [abstract]. In Vitro. 1971, 6: 398-399.Google Scholar
  34. 34.
    Chaves-Olarte E, Low P, Freer E, Norlin T, Weidmann M, von Eichel-Streiber C, Thelestam M: A novel cytotoxin from Clostridium difficile serogroup F is a functional hybrid between two other large clostridial cytotoxins. J Biol Chem. 1999, 274: 11046-11052. 10.1074/jbc.274.16.11046.CrossRefPubMedGoogle Scholar
  35. 35.
    Adelmann HG: A frequency-domain Gaussian filter module for quantitative and reproducible high-pass, low-pass and band-pass filtering of images. Am Lab. 1997, 29: 27-33.Google Scholar
  36. 36.
    Adelmann HG: Butterworth equations for homomorphic filtering of images. Comput Biol Med. 1998, 28: 169-10.1016/S0010-4825(98)00004-3.CrossRefPubMedGoogle Scholar
  37. 37.
    Cross RHM: A reliable epoxy resin mixture and its application in routine biological transmission electron microscopy. Micron Microsc Acta. 1989, 20: 1-7. 10.1016/0739-6260(89)90002-6.CrossRefGoogle Scholar
  38. 38.
    Bishop AL, Hall A: Rho GTPases and their effector proteins. Biochem J. 2000, 348: 241-255. 10.1042/0264-6021:3480241.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Schmidt A, Hall A: Guanine nucleotide exchange factors for Rho GTPases: turning on the switch. Genes Dev. 2002, 16: 1587-1609. 10.1101/gad.1003302.CrossRefPubMedGoogle Scholar
  40. 40.
    Zajchowski LD, Robbins SM: Lipid rafts and little caves: Compartmentalized signalling in membrane microdomains. Eur J Biochem. 2002, 269: 737-752. 10.1046/j.0014-2956.2001.02715.x.CrossRefPubMedGoogle Scholar
  41. 41.
    Etienne-Manneville S, Hall A: Rho GTPases in cell biology. Nature. 2002, 420: 629-635. 10.1038/nature01148.CrossRefPubMedGoogle Scholar
  42. 42.
    Reuther GW, Der CJ: The Ras branch of small GTPases: Ras family members don't fall far from the tree. Curr Opin Cell Biol. 2000, 12: 157-165. 10.1016/S0955-0674(99)00071-X.CrossRefPubMedGoogle Scholar
  43. 43.
    Quilliam LA, Rebhun JF, Castro AF: A growing family of guanine nucleotide exchange factors is responsible for activation of Ras-family GTPases. Prog Nucleic Acid Res Mol Biol. 2002, 71: 391-444.CrossRefGoogle Scholar
  44. 44.
    Maillet M, Robert SJ, Cacquevel M, Gastineau M, Vivien D, Bertoglio J, Zugaza JL, Fischmeister R, Lezoualc'h F: Crosstalk between Rap1 and Rac regulates secretion of sAPPalpha. Nat Cell Biol. 2003, 5: 633-639. 10.1038/ncb1007.CrossRefPubMedGoogle Scholar
  45. 45.
    Kyo S, Nakamura M, Kiyono T, Maida Y, Kanaya T, Tanaka M, Yatabe N, Inoue M: Successful immortalization of endometrial glandular cells with normal structural and functional characteristics. Am J Pathol. 2003, 163: 2259-2269.CrossRefGoogle Scholar
  46. 46.
    Boyer B, Valles AM, Edme N: Induction and regulation of epithelial-mesenchymal transitions. Biochem Pharmacol. 2000, 60: 1091-1099. 10.1016/S0006-2952(00)00427-5.CrossRefPubMedGoogle Scholar
  47. 47.
    Hay ED: Epithelial-mesenchymal transitions. Semin Dev Biol. 1990, 1: 347-356.Google Scholar
  48. 48.
    Hay ED: An overview of epithelio-mesenchymal transformation. Acta Anat. 1995, 154: 8-20.CrossRefGoogle Scholar
  49. 49.
    Savagner P: Leaving the neighborhood: molecular mechanisms involved during epithelial-mesenchymal transition. Bioessays. 2001, 23: 912-923. 10.1002/bies.1132.CrossRefPubMedGoogle Scholar
  50. 50.
    Thiery JP, Chopin D: Epithelial cell plasticity in development and tumor progression. Cancer Metastasis Rev. 1999, 18: 31-42. 10.1023/A:1006256219004.CrossRefPubMedGoogle Scholar

Copyright information

© Heneweer et al; licensee BioMed Central Ltd. 2005

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Authors and Affiliations

  • Carola Heneweer
    • 1
  • Martina Schmidt
    • 2
  • Hans-Werner Denker
    • 1
  • Michael Thie
    • 1
    • 3
  1. 1.Institute of AnatomyUniversity HospitalEssenGermany
  2. 2.Institute of PharmacologyUniversity HospitalEssenGermany
  3. 3.Stiftung caesarBonnGermany

Personalised recommendations