Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Sep 2;114(6):1149–1158. doi: 10.1083/jcb.114.6.1149

Apical and basolateral transferrin receptors in polarized BeWo cells recycle through separate endosomes

PMCID: PMC2289140  PMID: 1910050

Abstract

Contrary to most other epithelia, trophoblasts in the human placenta, which form the physical barrier between the fetal and the maternal blood circulation, express high numbers of transferrin receptors on their apical cell surface. This study describes the establishment of a polarized trophoblast-like cell line BeWo, which exhibit a high expression of transferrin receptors on the apex of the cells. Cultured on permeable filter supports, BeWo cells formed a polarized monolayer with microvilli on their apical cell surface. Across the monolayer a transepithelial resistance developed of approximately 600 omega.cm2 within 4 d. Depletion of Ca2+ from the medium decreased the resistance to background levels, showing its dependence on the integrity of tight junctions. Within the same period of time the secretion of proteins became polarized. In addition, the compositions of integral membrane proteins at the apical and basolateral plasma membrane domains were distinct as determined by domain-selective iodination. Similar to placental trophoblasts, binding of 125I-labeled transferrin to BeWo monolayers revealed that the transferrin receptor was expressed at both plasma membrane domains. Apical and basolateral transferrin receptors were found in a 1:2 surface ratio and exhibited identical dissociation constants and molecular weights. After uptake, transferrin recycled predominantly to the domain of administration, indicating separate recycling pathways from the apical and basolateral domain. This was confirmed by using diaminobenzidine cytochemistry, a technique by which colocalization of endocytosed 125I-labeled and HRP-conjugated transferrin can be monitored. No mixing of the two types of ligands was observed, when both ligands were simultaneously internalized for 10 or 60 min from opposite domains, demonstrating that BeWo cells possess separate populations of apical and basolateral early endosomes. In conclusion, the trophoblast-like BeWo cell line can serve as a unique model to compare the apical and basolateral endocytic pathways of a single ligand, transferrin, in polarized epithelial cells.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ajioka R. S., Kaplan J. Characterization of endocytic compartments using the horseradish peroxidase-diaminobenzidine density shift technique. J Cell Biol. 1987 Jan;104(1):77–85. doi: 10.1083/jcb.104.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson G. J., Mackerras A., Powell L. W., Halliday J. W. Improved purification of the human placental transferrin receptor and a novel immunoradiometric assay for receptor protein. Biochim Biophys Acta. 1986 Nov 19;884(2):225–233. doi: 10.1016/0304-4165(86)90167-4. [DOI] [PubMed] [Google Scholar]
  3. Bomsel M., Prydz K., Parton R. G., Gruenberg J., Simons K. Endocytosis in filter-grown Madin-Darby canine kidney cells. J Cell Biol. 1989 Dec;109(6 Pt 2):3243–3258. doi: 10.1083/jcb.109.6.3243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  5. Carson D. D., Tang J. P., Julian J., Glasser S. R. Vectorial secretion of proteoglycans by polarized rat uterine epithelial cells. J Cell Biol. 1988 Dec;107(6 Pt 1):2425–2435. doi: 10.1083/jcb.107.6.2425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ciechanover A., Schwartz A. L., Dautry-Varsat A., Lodish H. F. Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents. J Biol Chem. 1983 Aug 25;258(16):9681–9689. [PubMed] [Google Scholar]
  7. Contractor S. F., Eaton B. M. Role of transferrin in iron transport between maternal and fetal circulations of a perfused lobule of human placenta. Cell Biochem Funct. 1986 Jan;4(1):69–74. doi: 10.1002/cbf.290040111. [DOI] [PubMed] [Google Scholar]
  8. Courtoy P. J., Quintart J., Baudhuin P. Shift of equilibrium density induced by 3,3'-diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles. J Cell Biol. 1984 Mar;98(3):870–876. doi: 10.1083/jcb.98.3.870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dautry-Varsat A., Ciechanover A., Lodish H. F. pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2258–2262. doi: 10.1073/pnas.80.8.2258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Douglas G. C., King B. F. Uptake and processing of 125I-labelled transferrin and 59Fe-labelled transferrin by isolated human trophoblast cells. Placenta. 1990 Jan-Feb;11(1):41–57. doi: 10.1016/s0143-4004(05)80442-4. [DOI] [PubMed] [Google Scholar]
  11. Enns C. A., Shindelman J. E., Tonik S. E., Sussman H. H. Radioimmunochemical measurement of the transferrin receptor in human trophoblast and reticulocyte membranes with a specific anti-receptor antibody. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4222–4225. doi: 10.1073/pnas.78.7.4222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Enns C. A., Sussman H. H. Similarities between the transferrin receptor proteins on human reticulocytes and human placentae. J Biol Chem. 1981 Dec 25;256(24):12620–12623. [PubMed] [Google Scholar]
  13. Fuller S. D., Simons K. Transferrin receptor polarity and recycling accuracy in "tight" and "leaky" strains of Madin-Darby canine kidney cells. J Cell Biol. 1986 Nov;103(5):1767–1779. doi: 10.1083/jcb.103.5.1767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fuller S., von Bonsdorff C. H., Simons K. Vesicular stomatitis virus infects and matures only through the basolateral surface of the polarized epithelial cell line, MDCK. Cell. 1984 Aug;38(1):65–77. doi: 10.1016/0092-8674(84)90527-0. [DOI] [PubMed] [Google Scholar]
  15. GITLIN D., KUMATE J., URRUSTI J., MORALES C. THE SELECTIVITY OF THE HUMAN PLACENTA IN THE TRANSFER OF PLASMA PROTEINS FROM MOTHER TO FETUS. J Clin Invest. 1964 Oct;43:1938–1951. doi: 10.1172/JCI105068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Glasser S. R., Julian J., Decker G. L., Tang J. P., Carson D. D. Development of morphological and functional polarity in primary cultures of immature rat uterine epithelial cells. J Cell Biol. 1988 Dec;107(6 Pt 1):2409–2423. doi: 10.1083/jcb.107.6.2409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Godefroy O., Huet C., Blair L. A., Sahuquillo-Merino C., Louvard D. Differentiation of a clone isolated from the HT29 cell line: polarized distribution of histocompatibility antigens (HLA) and of transferrin receptors. Biol Cell. 1988;63(1):41–55. [PubMed] [Google Scholar]
  18. Godefroy O., Huet C., Ibarra C., Dautry-Varsat A., Louvard D. Establishment of polarized endocytosis in differentiable intestinal HT29-18 subclones. New Biol. 1990 Oct;2(10):875–886. [PubMed] [Google Scholar]
  19. Gumbiner B. Structure, biochemistry, and assembly of epithelial tight junctions. Am J Physiol. 1987 Dec;253(6 Pt 1):C749–C758. doi: 10.1152/ajpcell.1987.253.6.C749. [DOI] [PubMed] [Google Scholar]
  20. Hubbard A. L., Cohn Z. A. The enzymatic iodination of the red cell membrane. J Cell Biol. 1972 Nov;55(2):390–405. doi: 10.1083/jcb.55.2.390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hubbard A. L. Endocytosis. Curr Opin Cell Biol. 1989 Aug;1(4):675–683. doi: 10.1016/0955-0674(89)90033-1. [DOI] [PubMed] [Google Scholar]
  22. Hughson E. J., Cutler D. F., Hopkins C. R. Basolateral secretion of kappa light chain in the polarised epithelial cell line, Caco-2. J Cell Sci. 1989 Oct;94(Pt 2):327–332. doi: 10.1242/jcs.94.2.327. [DOI] [PubMed] [Google Scholar]
  23. Hughson E. J., Hopkins C. R. Endocytic pathways in polarized Caco-2 cells: identification of an endosomal compartment accessible from both apical and basolateral surfaces. J Cell Biol. 1990 Feb;110(2):337–348. doi: 10.1083/jcb.110.2.337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Klausner R. D., Van Renswoude J., Ashwell G., Kempf C., Schechter A. N., Dean A., Bridges K. R. Receptor-mediated endocytosis of transferrin in K562 cells. J Biol Chem. 1983 Apr 25;258(8):4715–4724. [PubMed] [Google Scholar]
  25. Kliman H. J., Nestler J. E., Sermasi E., Sanger J. M., Strauss J. F., 3rd Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology. 1986 Apr;118(4):1567–1582. doi: 10.1210/endo-118-4-1567. [DOI] [PubMed] [Google Scholar]
  26. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  27. McClelland A., Kühn L. C., Ruddle F. H. The human transferrin receptor gene: genomic organization, and the complete primary structure of the receptor deduced from a cDNA sequence. Cell. 1984 Dec;39(2 Pt 1):267–274. doi: 10.1016/0092-8674(84)90004-7. [DOI] [PubMed] [Google Scholar]
  28. Mostov K. E., Simister N. E. Transcytosis. Cell. 1985 Dec;43(2 Pt 1):389–390. doi: 10.1016/0092-8674(85)90166-7. [DOI] [PubMed] [Google Scholar]
  29. Nelson D. M., Meister R. K., Ortman-Nabi J., Sparks S., Stevens V. C. Differentiation and secretory activities of cultured human placental cytotrophoblast. Placenta. 1986 Jan-Feb;7(1):1–16. doi: 10.1016/s0143-4004(86)80012-1. [DOI] [PubMed] [Google Scholar]
  30. Oliver C. Endocytic pathways at the lateral and basal cell surfaces of exocrine acinar cells. J Cell Biol. 1982 Oct;95(1):154–161. doi: 10.1083/jcb.95.1.154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Parton R. G., Prydz K., Bomsel M., Simons K., Griffiths G. Meeting of the apical and basolateral endocytic pathways of the Madin-Darby canine kidney cell in late endosomes. J Cell Biol. 1989 Dec;109(6 Pt 2):3259–3272. doi: 10.1083/jcb.109.6.3259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pattillo R. A., Gey G. O. The establishment of a cell line of human hormone-synthesizing trophoblastic cells in vitro. Cancer Res. 1968 Jul;28(7):1231–1236. [PubMed] [Google Scholar]
  33. Podbilewicz B., Mellman I. ATP and cytosol requirements for transferrin recycling in intact and disrupted MDCK cells. EMBO J. 1990 Nov;9(11):3477–3487. doi: 10.1002/j.1460-2075.1990.tb07556.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rabin M., McClelland A., Kühn L., Ruddle F. H. Regional localization of the human transferrin receptor gene to 3q26.2----qter. Am J Hum Genet. 1985 Nov;37(6):1112–1116. [PMC free article] [PubMed] [Google Scholar]
  35. Rindler M. J., Traber M. G. A specific sorting signal is not required for the polarized secretion of newly synthesized proteins from cultured intestinal epithelial cells. J Cell Biol. 1988 Aug;107(2):471–479. doi: 10.1083/jcb.107.2.471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rodman J. S., Mercer R. W., Stahl P. D. Endocytosis and transcytosis. Curr Opin Cell Biol. 1990 Aug;2(4):664–672. doi: 10.1016/0955-0674(90)90108-q. [DOI] [PubMed] [Google Scholar]
  37. Rodriguez-Boulan E., Nelson W. J. Morphogenesis of the polarized epithelial cell phenotype. Science. 1989 Aug 18;245(4919):718–725. doi: 10.1126/science.2672330. [DOI] [PubMed] [Google Scholar]
  38. Sargiacomo M., Lisanti M., Graeve L., Le Bivic A., Rodriguez-Boulan E. Integral and peripheral protein composition of the apical and basolateral membrane domains in MDCK cells. J Membr Biol. 1989 Mar;107(3):277–286. doi: 10.1007/BF01871942. [DOI] [PubMed] [Google Scholar]
  39. Seligman P. A., Schleicher R. B., Allen R. H. Isolation and characterization of the transferrin receptor from human placenta. J Biol Chem. 1979 Oct 25;254(20):9943–9946. [PubMed] [Google Scholar]
  40. Simons K., Fuller S. D. Cell surface polarity in epithelia. Annu Rev Cell Biol. 1985;1:243–288. doi: 10.1146/annurev.cb.01.110185.001331. [DOI] [PubMed] [Google Scholar]
  41. Simons K., Wandinger-Ness A. Polarized sorting in epithelia. Cell. 1990 Jul 27;62(2):207–210. doi: 10.1016/0092-8674(90)90357-k. [DOI] [PubMed] [Google Scholar]
  42. Snider M. D., Rogers O. C. Intracellular movement of cell surface receptors after endocytosis: resialylation of asialo-transferrin receptor in human erythroleukemia cells. J Cell Biol. 1985 Mar;100(3):826–834. doi: 10.1083/jcb.100.3.826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sporn L. A., Marder V. J., Wagner D. D. Differing polarity of the constitutive and regulated secretory pathways for von Willebrand factor in endothelial cells. J Cell Biol. 1989 Apr;108(4):1283–1289. doi: 10.1083/jcb.108.4.1283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stoorvogel W., Geuze H. J., Griffith J. M., Schwartz A. L., Strous G. J. Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells. J Cell Biol. 1989 Jun;108(6):2137–2148. doi: 10.1083/jcb.108.6.2137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stoorvogel W., Geuze H. J., Griffith J. M., Strous G. J. The pathways of endocytosed transferrin and secretory protein are connected in the trans-Golgi reticulum. J Cell Biol. 1988 Jun;106(6):1821–1829. doi: 10.1083/jcb.106.6.1821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stoorvogel W., Schwartz A. L., Strous G. J., Fallon R. J. A pool of intracellular phosphorylated asialoglycoprotein receptors which is not involved in endocytosis. J Biol Chem. 1991 Mar 25;266(9):5438–5444. [PubMed] [Google Scholar]
  47. Stoorvogel W., Strous G. J., Geuze H. J., Oorschot V., Schwartz A. L. Late endosomes derive from early endosomes by maturation. Cell. 1991 May 3;65(3):417–427. doi: 10.1016/0092-8674(91)90459-c. [DOI] [PubMed] [Google Scholar]
  48. Theil E. C. Regulation of ferritin and transferrin receptor mRNAs. J Biol Chem. 1990 Mar 25;265(9):4771–4774. [PubMed] [Google Scholar]
  49. Truman P., Ford H. C. The brush border of the human term placenta. Biochim Biophys Acta. 1984 Jun 25;779(2):139–160. doi: 10.1016/0304-4157(84)90006-6. [DOI] [PubMed] [Google Scholar]
  50. Unemori E. N., Bouhana K. S., Werb Z. Vectorial secretion of extracellular matrix proteins, matrix-degrading proteinases, and tissue inhibitor of metalloproteinases by endothelial cells. J Biol Chem. 1990 Jan 5;265(1):445–451. [PubMed] [Google Scholar]
  51. Vanderpuye O. A., Kelley L. K., Morrison M. M., Smith C. H. The apical and basal plasma membranes of the human placental syncytiotrophoblast contain different erythrocyte membrane protein isoforms. Evidence for placental forms of band 3 and spectrin. Biochim Biophys Acta. 1988 Aug 18;943(2):277–287. doi: 10.1016/0005-2736(88)90559-7. [DOI] [PubMed] [Google Scholar]
  52. Vanderpuye O. A., Kelley L. K., Smith C. H. Transferrin receptors in the basal plasma membrane of the human placental syncytiotrophoblast. Placenta. 1986 Sep-Oct;7(5):391–403. doi: 10.1016/s0143-4004(86)80027-3. [DOI] [PubMed] [Google Scholar]
  53. Vanderpuye O. A., Smith C. H. Proteins of the apical and basal plasma membranes of the human placental syncytiotrophoblast: immunochemical and electrophoretic studies. Placenta. 1987 Nov-Dec;8(6):591–608. doi: 10.1016/0143-4004(87)90030-0. [DOI] [PubMed] [Google Scholar]
  54. Wice B., Menton D., Geuze H., Schwartz A. L. Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation in vitro. Exp Cell Res. 1990 Feb;186(2):306–316. doi: 10.1016/0014-4827(90)90310-7. [DOI] [PubMed] [Google Scholar]
  55. van Deurs B., Hansen S. H., Petersen O. W., Melby E. L., Sandvig K. Endocytosis, intracellular transport and transcytosis of the toxic protein ricin by a polarized epithelium. Eur J Cell Biol. 1990 Feb;51(1):96–109. [PubMed] [Google Scholar]
  56. van Dijk J. P. Regulatory aspects of placental iron transfer--a comparative study. Placenta. 1988 Mar-Apr;9(2):215–226. doi: 10.1016/0143-4004(88)90018-5. [DOI] [PubMed] [Google Scholar]
  57. van de Rijn M., Geurts van Kessel A. H., Kroezen V., van Agthoven A. J., Verstijnen K., Terhorst C., Hilgers J. Localization of a gene controlling the expression of the human transferrin receptor to the region q12 leads to qter of chromosome 3. Cytogenet Cell Genet. 1983;36(3):525–531. doi: 10.1159/000131967. [DOI] [PubMed] [Google Scholar]
  58. van der Ende A., du Maine A., Schwartz A. L., Strous G. J. Effect of ATP depletion and temperature on the transferrin-mediated uptake and release of iron by BeWo choriocarcinoma cells. Biochem J. 1989 May 1;259(3):685–692. doi: 10.1042/bj2590685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. van der Ende A., du Maine A., Schwartz A. L., Strous G. J. Modulation of transferrin-receptor activity and recycling after induced differentiation of BeWo choriocarcinoma cells. Biochem J. 1990 Sep 1;270(2):451–457. doi: 10.1042/bj2700451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. van der Ende A., du Maine A., Simmons C. F., Schwartz A. L., Strous G. J. Iron metabolism in BeWo chorion carcinoma cells. Transferrin-mediated uptake and release of iron. J Biol Chem. 1987 Jun 25;262(18):8910–8916. [PubMed] [Google Scholar]
  61. van der Heul C., Kroos M. J., van Eijk H. G. Binding sites of iron transferrin on rat reticulocytes. Inhibition by specific antibodies. Biochim Biophys Acta. 1978 Aug 17;511(3):430–441. doi: 10.1016/0005-2736(78)90279-1. [DOI] [PubMed] [Google Scholar]
  62. von Bonsdorff C. H., Fuller S. D., Simons K. Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters. EMBO J. 1985 Nov;4(11):2781–2792. doi: 10.1002/j.1460-2075.1985.tb04004.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES