Summary
The control of growth and differentiation of tracheal epithelial cells is poorly understood. Retinoic acid seems to be essential for the growth and secretory cell differentiation of hamster tracheal epithelial (HTE) cells in culture. In this study, we tested the hypothesis that one way by which retinoic acid (RA) stimulates growth is by decreasing transforming growth factor beta (TGFβ) expression or activity or both. HTE cells were very sensitive to TGFβ-induced growth inhibition. TGFβ1 was more potent than TGFβ2 with 50% inhibition of growth achieved at a concentration less than 0.1 ng/ml. A single TGFβ1 transcript of 2.4 kb was expressed in HTE cells, and the amount increased by fourfold as cell proliferation decreased and differentiation increased. No TGFβ2 mRNA could be detected in proliferating undifferentiated HTE cells, but two distinct mRNAs (5.1 and 3.5 kb) were observed to be induced in a transient fashion in RA-treated cells which correlated with the onset of differentiation. The amount of biologically active TGFβ in conditioned media from HTE cells at different stages of growth and differentiation in primary culture was determined by the mink lung epithelial cell growth inhibition assay and the use of neutralizing antibodies. These assays indicated a large increase in the total amount of TGFβ at the time the cells slowed their growth and started to differentiate. The activity was due primarily to TGFβ1. Interestingly, cells treated with RA had a major component of “preactivated” (non-latent) TGFβ1 compared to control cells. Addition of TGFβ1 neutralizing antibodies directly to HTE cultures delayed the onset of both growth arrest and differentiation. These results do not support the hypothesis that RA stimulates HTE cell growth by decreasing TGFβ; rather they suggest that endogenously produced TGFβ may play a role in the initiation of growth arrest which precedes differentiation.
Similar content being viewed by others
References
Bascom, C. C.; Wolfshol, J. R.; Coffey, R. J., et al. Complex regulation of transforming growth factorβ1,β2, andβ3 mRNA expression in mouse fibroblasts and keratinocytes by transforming growth factorsβ1 andβ2. Mol. Cell. Biol. 9:5508–5515; 1989.
Bradford, M. M. A rapid and sensitive assay for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72:248–254; 1976.
Cathala, G.; Savouret, J. F.; Mendez, B., et al. A method for isolation of intact translationally active ribonucleic acid. DNA 2:329–335; 1983.
Christensen, T. G.; Haddad, C. E.; Breuer, R., et al. Quantitative assessment of the effect of retinoic acid on cell shape and secretory granule formation in primary cultures of hamster tracheal epithelial cells. J. Cell Biol. 107:349a; 1990.
Church, G.; Gillbert, W. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81:1991–1995; 1984.
Danielpour, D.; Dart, L. L.; Flanders, K. C., et al. Immunodetection and quantitation of the two forms of transforming growth factor-beta (TGF-β1 and TGF-β2) secreted by cells in culture. J. Cell. Physiol. 138:79–86; 1989.
Danielpour, D.; Kim, K. Y.; Winokur, T. S., et al. Differential regulation of the expression of transforming growth factor-βs 1 and 2 by retinoic acid, epidermal growth factor, and dexamethasone in NRK-49F and A549 cells. J. Cell. Physiol. 148:235–244; 1991.
Derynck, R.; Jarrett, J. A.; Chen, E. Y., et al. Human transforming growth factor-beta cDNA sequence and expression in tumor cell lines. Nature 316:701–705; 1985.
Derynck, R.; Lindquist, P. B.; Lee, A., et al. A new type of transforming growth factor-β, TGF-β3. EMBO J. 7:3737–3743; 1988.
Glick, A. B.; Flanders, K. C.; Danielpour, D., et al. Retinoic acid induces transforming growth factor-β2 in cultured keratinocytes and mouse epidermis. Cell Regulation 1:87–97; 1989.
Hafez, M. M.; Infante, D.; Winawer, S., et al. Transforming growth factorβ1 acts as an autocrine-negative growth regulator in colon enterocytic differentiation but not in goblet cell maturation. Cell Growth & Differentiation 1:617–626; 1990.
Hanks, S. K.; Amour, R.; Baldwin, J. H., et al. Amino acid sequence of the BSC-1 cell growth inhibitor (polyergin) deduced from the nucleotide sequence of the cDNA. Proc. Natl. Acad. Sci. USA 85:79–82; 1988.
Jetten, A. M.; Shirley, J. E.; Stoner, G. Regulation of proliferation and differentiation of respiratory tract epithelial cells by TGFβ. Exp. Cell Res. 167:539–549; 1986.
Knabbe, C.; Lippman, M. E.; Wakefield, L. M., et al. Evidence that transforming growth factor-β is a hormonally regulated negative growth factor in human breast cancer cells. Cell 48:417–428; 1987.
Lancillotti, F.; Darwiche, N.; Celli, G., et al. Retinoid status and the control of keratin expression and adhesion during the histogenesis of squamous metaplasia of tracheal epithelium. Cancer Res. 52:6144–6152; 1992.
Masui, T.; Wakefield, L. M.; Lechner, J. F., et al. Typeβ transforming growth factor is the primary differentiation-inducing serum factor for normal human bronchial epithelial cells. Proc. Natl. Acad. Sci. USA 83:2438–2442; 1986.
Niles, R. M.; Kim, K. C.; Hyman, B., et al. Characterization of extended primary and secondary cultures of hamster tracheal epithelial cells. In Vitro 24:457–462; 1988.
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.
Pelton, R. W.; Johnson, M. D.; Perkett, E. A., et al. Expression of transforming growth factor-β1,β2, and -β3 mRNA and protein in the murine lung. Am. J. Respir. Cell Mol. Biol. 5:522–530; 1991.
Qian, S. W.; Kondaiah, P.; Roberts, A. B., et al. cDNA cloning by PCR of rat transforming growth factorβ1. Nucleic Acids Res. 18: 3059–3064; 1990.
Seyedin, S. M.; Segarini, P. R.; Rosen, D. M., et al. Cartilage-inducing factor-B is a unique protein structurally and functionally related to transforming growth factor-beta. J. Biol. Chem. 262:1946–1949; 1987.
Sing, G. K.; Ruscetti, F. W.; Beckwith, M., et al. Growth inhibition of a human lymphoma cell line: induction of a transforming growth factor-β-mediated autocrine negative loop by phorbol myristate acetate. Cell Growth & Differentiation 1:549–557; 1990.
Sporn, M. B.; Roberts, A. B. TGF-β: problems and prospects. Cell Regulation 1:875–882; 1990.
Twardzik, D. R.; Ranchalis, J. E.; McPherson, J. M., et al. Inhibition and promotion of differentiated-like phenotype of a human lung carcinoma in athymic mice by natural and recombinant forms of transforming growth factor-β. J. Natl. Cancer Res. 81:1182–1185; 1989.
Wasano, K.; Kim, K. C.; Niles, R. M., et al. Membrane differentiation markers of airway epithelial secretory cells. J. Histochem. Cytochem. 36:167–178; 1988.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Niles, R.M., Thompson, N.L. & Fenton, F. Expression of TGF-β during in vitro differentiation of hamster tracheal epithelial cells. In Vitro Cell Dev Biol - Animal 30, 256–262 (1994). https://doi.org/10.1007/BF02632048
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02632048