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

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.

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.

    Article  PubMed  CAS  Google Scholar 

  2. 2.

    Denker HW: Endometrial receptivity: cell biological aspects of an unusual epithelium. A review. Anat Anz. 1994, 176: 53-60.

    Article  CAS  Google 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.

    PubMed  CAS  Google 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.

    Article  PubMed  Google 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.

    Article  CAS  PubMed  Google Scholar 

  6. 6.

    Carson DD: The glycobiology of implantation. Front Biosci. 2002, 7: d1535-1544.

    Article  PubMed  Google 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.

    Article  PubMed  PubMed Central  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google Scholar 

  12. 12.

    Murphy CR: The plasma membrane transformation of uterine epithelial cells during pregnancy. J Reprod Fertil Suppl. 2000, 55: 23-28.

    PubMed  CAS  Google Scholar 

  13. 13.

    Nikas G: Endometrial receptivity: changes in cell-surface morphology. Semin Reprod Med. 2000, 18: 229-235. 10.1055/s-2000-12561.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Google Scholar 

  19. 19.

    Denker HW: Implantation: a cell biological paradox. J Exp Zool. 1993, 266: 541-558.

    Article  CAS  PubMed  Google 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.

    Article  CAS  PubMed  Google 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.

    PubMed  CAS  Google 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.

    PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  Google Scholar 

  38. 38.

    Bishop AL, Hall A: Rho GTPases and their effector proteins. Biochem J. 2000, 348: 241-255. 10.1042/0264-6021:3480241.

    Article  PubMed  PubMed Central  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google Scholar 

  41. 41.

    Etienne-Manneville S, Hall A: Rho GTPases in cell biology. Nature. 2002, 420: 629-635. 10.1038/nature01148.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  CAS  PubMed  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  CAS  PubMed  PubMed Central  Google 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.

    Article  PubMed  CAS  Google 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.

    Article  CAS  PubMed  Google Scholar 

  49. 49.

    Savagner P: Leaving the neighborhood: molecular mechanisms involved during epithelial-mesenchymal transition. Bioessays. 2001, 23: 912-923. 10.1002/bies.1132.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google Scholar 

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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.

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Correspondence to Michael Thie.

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The author(s) declare that they have no competing interests.

Authors' contributions

CH conceived of the study and carried out the confocal laser scanning microscopical and electron microscopical investigations. MS provided expertise in small Rho GTPases and conducted biochemical experiments. HWD participated in the design of the study and helped to draft the manuscript. MT conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

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Heneweer, C., Schmidt, M., Denker, H. et al. Molecular mechanisms in uterine epithelium during trophoblast binding: the role of small GTPase RhoA in human uterine Ishikawa cells . J Exp Clin Assist Reprod 2, 4 (2005). https://doi.org/10.1186/1743-1050-2-4

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