Skip to main content
SpringerLink
Log in

Rat tracheal epithelial cell differentiation in vitro

  • Growth, Differentiation And Senescence
  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Summary

In vitro culture conditions enabling rat tracheal epithelial (RTE) cells to differentiate to mucociliary, mucous, or squamous phenotypes are described. Medium composition for rapid cell growth to confluence in membrane insert cultures was determined, and the effects of major modifiers of differentiation were tested. Retinoic acid (RA), collagen gel substratum, and an air-liquid interface at the level of the cell layer were required for expression of a mucociliary phenotype which most closely approximated the morphology of the tracheal epithelium in vivo. Large quantities of high molecular weight, hyaluronidase-resistant glycoconjugates, most likely mucin glycoproteins, were produced in the presence of RA when the cells were grown with or without a collagen gel and in submerged as well as in interface cultures. However, extensive ciliagenesis was dependent on the simultaneous presence of RA, collagen gel, and an air-liquid interface. When RA was omitted from the media, the cells became stratified squamous and developed a cornified apical layer in air-liquid interface cultures. This phenotype was accompanied by loss of transglutaminase (TGase) type II and keratin 18 and expression of the squamous markers TGase type I and keratin 13. The ability to modulate RTE cell phenotypes in culture will facilitate future studies investigating molecular regulation of tracheal cell proliferation, differentiation, and function.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Achtstaetter, T.; Hatzfeld, M.; Quinlan, R. A., et al. Separation of cytokeratin polypeptides by gel electrophoretic and chromatographic techniques and their identification by immunoblotting. In: Vallee, R. B., ed. Methods in enzymology, vol. 134. Orlando, FL: Academic Press; 1986:355–371.

    Google Scholar 

  • Adler, K. B.; Cheng, P.-W.; Kim, K. C. Characterization of guinea pig tracheal epithelial cells maintained in biphasic organotypic culture: cellular composition and biochemical analysis of released glycoconjugates. Am. J. Respir. Cell Mol. Biol. 2:145–154; 1990.

    PubMed  CAS  Google Scholar 

  • Adler, K. B.; Schwarz, J. E.; Whitcutt, M. J., et al. A new chamber system for maintaining differentiated guinea pig respiratory epithelial cells between air and liquid phases. Biotechniques 5:462–465; 1987.

    Google Scholar 

  • Bertolero, F.; Kaighn, E.; Gonda, M. A., et al. Mouse epidermal keratinocytes—clonal proliferation and response to hormones and growth factors in serum-free medium. Exp. Cell Res. 155:64–80; 1984.

    Article  PubMed  CAS  Google Scholar 

  • Birckbichler, P. J.; Upchurch, H. F.; Patterson, M. K., Jr., et al. A monoclonal antibody to cellular transglutaminase. Hybridoma 4:179–186; 1985.

    Article  PubMed  CAS  Google Scholar 

  • Clark, J. N.; Marchok, A. C. Characterization of mucin isolated from rat tracheal transplants. Biochem. Biophys. Acta. 588:357–367; 1979.

    PubMed  CAS  Google Scholar 

  • Cooper, D.; Schermer, A.; Sun, T.-T. Classification of human epithelia and their neoplasms using monoclonal antibodies to keratins: strategies, applications and limitations. Lab. Invest. 52:243–256; 1985.

    PubMed  CAS  Google Scholar 

  • Davies, P. J. A.; Martaugh, M. P.; Moore, W. T., Jr., et al. Retinoic acid-induced expression of tissue transglutaminase in human promyelocytic leukemia (HL-60) cells. J. Biol. Chem. 260:5166–5174; 1985.

    PubMed  CAS  Google Scholar 

  • De Luca, L. M.; Huang, F. L.; Roop, D. R. Retinoids and control of epithelial differentiation and keratin biosynthesis in hamster trachea. In: Parker, L., ed. Methods of enzymology, vol. 190. Orlando, FL: Academic Press; 1990:91–100.

    Google Scholar 

  • Denning, M. F.; Verma, A. K. Involvement of retinoic acid nuclear receptors in retinoic acid-induced tissue transglutaminase gene expression in rat tracheal 2C5 cells. Biochem. Biophys. Res. Commun. 175:344–350; 1991.

    Article  PubMed  CAS  Google Scholar 

  • Ferriola, P. C.; Walker, C.; Robertson, A. T., et al. Altered growth factor dependence and transforming growth factor gene expression in transformed rat tracheal epithelial cells. Mol. Carcinogen. 2:336–344; 1989.

    Article  CAS  Google Scholar 

  • Ferriola, P. C.; Earp, H. S.; Di Augustine, R., et al. Role of TGF alpha and its receptor in proliferation of immortalized rat tracheal epithelial cells: studies with tyrphostin and TGF alpha antisera. J. Cell. Physiol. 147:166–175; 1991.

    Article  PubMed  CAS  Google Scholar 

  • Floyd, E. E.; Jetten, A. M. Regulation of type I (epidermal) transglutaminase mRNA levels during squamous differentiation: downregulation by retinoids. Mol. Cell. Biol. 9:4846–4851; 1989.

    PubMed  CAS  Google Scholar 

  • Jetten, A. M.; Brody, A. R.; Deas, M. A., et al. Retinoic acid and substratum regulate the differentiation of rabbit tracheal epithelial cells into squamous and secretory phenotype. Lab. Invest. 56:654–664; 1987.

    PubMed  CAS  Google Scholar 

  • Jetten, A. M.; George, M. D.; Rearick, J. I. Downregulation of squamous cell-specific markers by retinoids: transglutaminase type I and cholesterol sulfotransferase. In: Parker, L., ed. Methods in enzymology, vol. 190. Orlando, FL: Academic Press; 1990:42–49.

    Google Scholar 

  • Jetten, A. M.; George, M. D.; Smits, H. L., et al. Keratin 13 expression is linked to squamous differentiation in rabbit tracheal epithelial cells and down-regulated by retinoic acid. Exp. Cell Res. 182:622–634; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Jetten, A. M.; Shirley, J. E. Characterization of transglutaminase activity in rabbit tracheal epithelial cells. Regulation by retinoids. J. Biol. Chem. 261:15097–15101; 1986.

    PubMed  CAS  Google Scholar 

  • Kim, K. C.; Rearick, J. I.; Nettesheim, P., et al. Biochemical characterization of mucous glycoproteins synthesized and secreted by hamster tracheal epithelial cells in primary culture. J. Biol. Chem. 260:4021–4027; 1985.

    PubMed  CAS  Google Scholar 

  • Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685; 1970.

    Article  PubMed  CAS  Google Scholar 

  • Lechner, J. F.; Haugen, A.; McClendon, I. A., et al. Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium. In Vitro 18:633–642; 1982.

    PubMed  CAS  Google Scholar 

  • Lee, T.-C.; Wu, R.; Brody, A. R., et al. Growth and differentiation of hamster tracheal epithelial cells in culture. Exp. Lung Res. 6:27–45; 1984.

    PubMed  CAS  Google Scholar 

  • Lichti, U.; Ben, T.; Yuspa, S. H. Retinoic acid-induced transglutaminase in mouse epidermal cells is distinct from epidermal transglutaminase. J. Biol. Chem. 260:1422–1426; 1985.

    PubMed  CAS  Google Scholar 

  • McDowell, E. M.; Keenan, K. P.; Huang, M. Effects of vitamin A-deprivation on hamster tracheal epithelium: a quantitative morphologic study. Virchows Arch. B. Cell Pathol. 45:197–219; 1984.

    Article  CAS  Google Scholar 

  • Moll, R.; Franke, W. W.; Schiller, D. L., et al. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31:11–24; 1982.

    Article  PubMed  CAS  Google Scholar 

  • Moller, P. C.; Partridge, L. R.; Cox, R. A., et al. The development of ciliated and mucus cells from basal cells in hamster tracheal epithelial cell cultures. Tissue & Cell 21:195–198; 1989.

    Article  CAS  Google Scholar 

  • Moore, W. T.; Murtaugh, M. P.; Davies, P. J. A. Retinoic acid-induced expression of tissue transglutaminase in mouse peritoneal macrophages. J. Biol. Chem. 259:12794–12802; 1984.

    PubMed  CAS  Google Scholar 

  • Nara, K.; Nakanishi, K.; Hagiwara, H., et al. Retinol-induced morphological changes of cultured bovine endothelial cells are accompanied by a marked increase in transglutaminase. J. Biol. Chem. 264:19308–19312; 1989.

    PubMed  CAS  Google Scholar 

  • Nettesheim, P.; Barrett, J. C. Tracheal epithelial cell transformation: a model system for studies on neoplastic progression. In: Goldberg, L., ed. CRC critical reviews in toxicology, vol 12. Boca Raton, FL: CRC Press; 1984:215–239.

    Google Scholar 

  • Niles, R. M.; Loewy, B. P.; Brown, K. The effect of retinoic acid on growth and proto-oncogene expression in hamster tracheal epithelial cells. Am. J. Respir. Cell Mol. Biol. 2:365–371; 1990.

    PubMed  CAS  Google Scholar 

  • Niles, R.; Kim, K. C.; Hyman, B., et al. Characterization of extended primary and secondary cultures of hamster tracheal epithelial cells. In Vitro Cell. Dev. Biol. 24:457–463; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Purkis, P. E.; Steel, J. B.; Mackenzie, I. C., et al. Antibody markers of basal cells in complex epithelia. J. Cell Sci. 97:39–50; 1990.

    PubMed  Google Scholar 

  • Randell, S. H.; Comment, C. E.; Ramaekers, F. C. S., et al. Properties of rat tracheal epithelial cells separated based on expression of cell surface alpha-galactosyl groups. Am. J. Respir. Cell Mol. Biol. 4:544–554; 1991.

    PubMed  CAS  Google Scholar 

  • Shimizu, T.; Nettesheim, P.; Mahler, J. F., et al. Cell type-specific lectin staining of the tracheobronchial epithelium of the rat: quantitative studies withGriffonia simplicifolia L isolectin B4. Histochem. Cytochem. 39:7–14; 1991.

    CAS  Google Scholar 

  • Shimizu, T.; Nettesheim, P.; Eddy, E. M., et al. Monoclonal antibody (Mab) markers for subpopulations of rat tracheal epithelial (RTE) cells. Exp. Lung Res. 18:323–342; 1992a.

    PubMed  CAS  Google Scholar 

  • Shimizu, T.; Nettesheim, P.; Ramaekers, F. C. S., et al. Expression of cell-type-specific markers during rat tracheal epithelial regeneration. Am. J. Respir. Cell Mol. Biol. 7:30–41; 1992b.

    PubMed  CAS  Google Scholar 

  • Sun, T.-T.; Eichner, R.; Nelson, W. G., et al. Keratin classes: molecular markers for different types of epithelial differentiation. J. Invest. Dermatol. 81:109s-115s; 1983.

    Article  PubMed  CAS  Google Scholar 

  • Terzaghi, M.; Nettesheim, P.; Williams, M. C. Repopulation of denuded tracheas with normal, preneoplastic and neoplastic epithelial cell populations. Cancer Res. 38:4546–4553; 1978.

    PubMed  CAS  Google Scholar 

  • Thacher, S. M.; Rice, R. H. Keratinocyte-specific transglutaminase of cultured human epidermal cells: relation to cross-linked envelope formation and terminal differentiation. Cell 40:685–695; 1985.

    Article  PubMed  CAS  Google Scholar 

  • Thomassen, D. G.; Saffiotti, U.; Kaighn, M. E. Clonal proliferation of rat tracheal epithelial cells in serum-free medium and their responses to hormones, growth factors and carcinogens. Carcinogenesis 7:2033–2039; 1986.

    Article  PubMed  CAS  Google Scholar 

  • van Scott, M. R.; Lee, N. P.; Yankaskas, J. R., et al. Effect of hormones on growth and function of cultured canine tracheal epithelial cells. Am. J. Physiol. 255:C237-C245; 1988.

    PubMed  Google Scholar 

  • Vollberg, T. M.; George, M. D.; Nervi, C., et al. Regulation of transglutaminase type I and II in normal human bronchial epithelial and lung carcinoma cells. Am. J. Respir. Cell Mol. Biol. 7:10–18; 1992.

    PubMed  CAS  Google Scholar 

  • Whitcutt, M. J.; Adler, K. B.; Wu, R. A biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells. In Vitro Cell. Dev. Biol. 24:420–428; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Wu, R.; Martin, W. R.; Robinson, C. B., et al. Expression of mucin synthesis and secretion in human tracheobronchial epithelial cells grown in culture. Am. J. Respir. Cell Mol. Biol. 3:467–478; 1990.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmacology and Toxicology, College of Veterinary Medicine, SF-00581, Helsinki, Finland

    Liisa Kaartinen

  2. Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, 27709, Research Triangle Park, North Carolina

    Paul Nettesheim & Scott H. Randell

  3. Department of Anatomy, Physiological Sciences and Radiology, North Carolina State University, College of Veterinary Medicine, 27606, Raleigh, North Carolina

    Kenneth B. Adler

Authors
  1. Liisa Kaartinen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paul Nettesheim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kenneth B. Adler
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Scott H. Randell
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kaartinen, L., Nettesheim, P., Adler, K.B. et al. Rat tracheal epithelial cell differentiation in vitro. In Vitro Cell Dev Biol - Animal 29, 481–492 (1993). https://doi.org/10.1007/BF02639383

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02639383

Key words

Search

Navigation