Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 Aug 1;111(2):663–672. doi: 10.1083/jcb.111.2.663

Differentiation expression during proliferative activity induced through different pathways: in situ hybridization study of thyroglobulin gene expression in thyroid epithelial cells

PMCID: PMC2116189  PMID: 2199463

Abstract

In canine thyrocytes in primary culture, our previous studies have identified three mitogenic agents and pathways: thyrotropin (TSH) acting through cyclic AMP (cAMP), EGF and its receptor tyrosine protein kinase, and the phorbol esters that stimulate protein kinase C. TSH enhances, while EGF and phorbol esters inhibit, the expression of differentiation. Given that growth and differentiation expression are often considered as mutually exclusive activities of the cells, it was conceivable that the differentiating action of TSH was restricted to noncycling (Go) cells, while the inhibition of the differentiation expression by EGF and phorbol esters only concerned proliferating cells. Therefore, the capacity to express the thyroglobulin (Tg) gene, the most prominent marker of differentiation in thyrocytes, was studied in proliferative cells (with insulin) and in quiescent cells (without insulin). Using cRNA in situ hybridization, we observed that TSH (and, to a lesser extent, insulin and insulin-like growth factor I) restored or maintained the expression of the Tg gene. Without these hormones, the Tg mRNA content became undetectable in most of the cells. EGF and 12-0-tetradecanoyl phorbol-13-acetate (TPA) inhibited the Tg mRNA accumulation induced by TSH (and/or insulin). Most of the cells (up to 90%) responded to both TSH and EGF. Nevertheless, the range of individual response was quite variable. The effects of TSH and EGF on differentiation expression were not dependent on insulin and can therefore be dissociated from their mitogenic effects. Cell cycling did not affect the induction of Tg gene. Indeed, the same cell distribution of Tg mRNA content was observed in quiescent cells stimulated by TSH alone, or in cells approximately 50% of which had performed one mitotic cycle in response to TSH + insulin. Moreover, after proliferation in "dedifferentiating" conditions (EGF + serum + insulin), thyrocytes had acquired a fusiform fibroblast-like morphology, and responded to TSH by regaining a characteristic epithelial shape and high Tg mRNA content. 32 h after the replacement of EGF by TSH, cells in mitosis presented the same distribution of the Tg mRNA content as the rest of the cell population. This implies that cell cycling (at least 27 h, as previously shown) did not affect the induction of the Tg gene which is clearly detectable after a time lag of at least 24 h. The data unequivocally show that the reexpression of differentiation and proliferative activity are separate but fully compatible processes when induced by cAMP in thyrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Full Text

The Full Text of this article is available as a PDF (2.6 MB).

Selected References

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

  1. Aouani A., Hovsépian S., Fayet G. Multihormonal regulation of thyroglobulin production by the OVNIS 6H thyroid cell line. Horm Metab Res. 1988 Feb;20(2):91–95. doi: 10.1055/s-2007-1010760. [DOI] [PubMed] [Google Scholar]
  2. Bagchi N., Brown T. R., Shivers B., Lucas S., Mack R. E. Decreased thyroidal response to thyrotropin in diabetic mice. Endocrinology. 1981 Nov;109(5):1428–1432. doi: 10.1210/endo-109-5-1428. [DOI] [PubMed] [Google Scholar]
  3. Berger C. L., de Bustros A., Roos B. A., Leong S. S., Mendelsohn G., Gesell M. S., Baylin S. B. Human medullary thyroid carcinoma in culture provides a model relating growth dynamics, endocrine cell differentiation, and tumor progression. J Clin Endocrinol Metab. 1984 Aug;59(2):338–343. doi: 10.1210/jcem-59-2-338. [DOI] [PubMed] [Google Scholar]
  4. Berger S. L., Birkenmeier C. S. Inhibition of intractable nucleases with ribonucleoside--vanadyl complexes: isolation of messenger ribonucleic acid from resting lymphocytes. Biochemistry. 1979 Nov 13;18(23):5143–5149. doi: 10.1021/bi00590a018. [DOI] [PubMed] [Google Scholar]
  5. Bergé-Lefranc J. L., Cartouzou G., Bignon C., Lissitzky S. Quantitative in situ hybridization of 3H-labeled complementary deoxyribonucleic acid (cDNA) to the messenger ribonucleic acid of thyroglobulin in human thyroid tissues. J Clin Endocrinol Metab. 1983 Sep;57(3):470–476. doi: 10.1210/jcem-57-3-470. [DOI] [PubMed] [Google Scholar]
  6. Chambard M., Mauchamp J., Chabaud O. Synthesis and apical and basolateral secretion of thyroglobulin by thyroid cell monolayers on permeable substrate: modulation by thyrotropin. J Cell Physiol. 1987 Oct;133(1):37–45. doi: 10.1002/jcp.1041330105. [DOI] [PubMed] [Google Scholar]
  7. Chebath J., Chabaud O., Mauchamp J. Modulation of thyroglobulin messenger RNA level by thyrotropin in cultured thyroid cells. Nucleic Acids Res. 1979 Jul 25;6(10):3353–3367. doi: 10.1093/nar/6.10.3353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chevalier S., Bleau G., Roberts K. D., Chapdelaine A. Proliferation and differentiation of canine prostatic epithelial cells in culture. Mol Cell Endocrinol. 1981 Nov;24(2):195–208. doi: 10.1016/0303-7207(81)90059-9. [DOI] [PubMed] [Google Scholar]
  9. Christophe D., Brocas H., Gannon F., de Martynoff G., Pays E., Vassart G. Molecular cloning of bovine thyroglobulin complementary DNA. Characterization of 2500-base-pair and 1900-base-pair fragments. Eur J Biochem. 1980 Oct;111(2):419–423. doi: 10.1111/j.1432-1033.1980.tb04956.x. [DOI] [PubMed] [Google Scholar]
  10. Contor L., Lamy F., Lecocq R., Roger P. P., Dumont J. E. Differential protein phosphorylation in induction of thyroid cell proliferation by thyrotropin, epidermal growth factor, or phorbol ester. Mol Cell Biol. 1988 Jun;8(6):2494–2503. doi: 10.1128/mcb.8.6.2494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cox K. H., DeLeon D. V., Angerer L. M., Angerer R. C. Detection of mrnas in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol. 1984 Feb;101(2):485–502. doi: 10.1016/0012-1606(84)90162-3. [DOI] [PubMed] [Google Scholar]
  12. Dumont J. E., Jauniaux J. C., Roger P. P. The cyclic AMP-mediated stimulation of cell proliferation. Trends Biochem Sci. 1989 Feb;14(2):67–71. doi: 10.1016/0968-0004(89)90046-7. [DOI] [PubMed] [Google Scholar]
  13. Dumont J. E., Willems C., Van Sande J., Nève P. Regulation of the release of thyroid hormones: role of cyclic AMP. Ann N Y Acad Sci. 1971 Dec 30;185:291–316. doi: 10.1111/j.1749-6632.1971.tb45255.x. [DOI] [PubMed] [Google Scholar]
  14. Epstein-Almog R., Orly J. Inhibition of hormone-induced steroidogenesis during cell proliferation in serum-free cultures of rat granulosa cells. Endocrinology. 1985 May;116(5):2103–2112. doi: 10.1210/endo-116-5-2103. [DOI] [PubMed] [Google Scholar]
  15. Errick J. E., Eggo M. C., Burrow G. N. Epidermal growth factor inhibits thyrotropin-mediated synthesis of tissue-specific proteins in cultured ovine thyroid cells. Mol Cell Endocrinol. 1985 Nov;43(1):51–59. doi: 10.1016/0303-7207(85)90041-3. [DOI] [PubMed] [Google Scholar]
  16. Fayet G., Hovsepian S. Demonstration of growth in porcine thyroid cell culture. Biochimie. 1979;61(8):923–930. doi: 10.1016/s0300-9084(79)80242-4. [DOI] [PubMed] [Google Scholar]
  17. Fayet G., Hovsépian S., Dickson J. G., Lissitzky S. Reorganization of porcine thyroid cells into functional follicles in a chemically defined, serum- and thyrotropin-free medium. J Cell Biol. 1982 May;93(2):479–488. doi: 10.1083/jcb.93.2.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gerard C. M., Lefort A., Libert F., Christophe D., Dumont J. E., Vassart G. Transcriptional regulation of the thyroperoxydase gene by thyrotropin and forskolin. Mol Cell Endocrinol. 1988 Dec;60(2-3):239–242. doi: 10.1016/0303-7207(88)90184-0. [DOI] [PubMed] [Google Scholar]
  19. Gray R. S., Borsey D. Q., Seth J., Herd R., Brown N. S., Clarke B. F. Prevalence of subclinical thyroid failure in insulin-dependent diabetes. J Clin Endocrinol Metab. 1980 Jun;50(6):1034–1037. doi: 10.1210/jcem-50-6-1034. [DOI] [PubMed] [Google Scholar]
  20. Griep A. E., Westphal H. Antisense Myc sequences induce differentiation of F9 cells. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6806–6810. doi: 10.1073/pnas.85.18.6806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gérard C. M., Lefort A., Christophe D., Libert F., Van Sande J., Dumont J. E., Vassart G. Control of thyroperoxidase and thyroglobulin transcription by cAMP: evidence for distinct regulatory mechanisms. Mol Endocrinol. 1989 Dec;3(12):2110–2118. doi: 10.1210/mend-3-12-2110. [DOI] [PubMed] [Google Scholar]
  22. Hansen C., Gerard C., Vassart G., Stordeur P., Christophe D. Thyroid-specific and cAMP-dependent hypersensitive regions in thyroglobulin gene chromatin. Eur J Biochem. 1988 Dec 15;178(2):387–393. doi: 10.1111/j.1432-1033.1988.tb14462.x. [DOI] [PubMed] [Google Scholar]
  23. Hayashi S., Gillam I. C., Delaney A. D., Tener G. M. Acetylation of chromosome squashes of Drosophila melanogaster decreases the background in autoradiographs from hybridization with [125I]-labeled RNA. J Histochem Cytochem. 1978 Aug;26(8):677–679. doi: 10.1177/26.8.99471. [DOI] [PubMed] [Google Scholar]
  24. Heikkila R., Schwab G., Wickstrom E., Loke S. L., Pluznik D. H., Watt R., Neckers L. M. A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G0 to G1. 1987 Jul 30-Aug 5Nature. 328(6129):445–449. doi: 10.1038/328445a0. [DOI] [PubMed] [Google Scholar]
  25. Jolin T., Morreale de Escobar G., Escobar del Rey F. Differential effects in the rat thyroidectomy, propylthiouracil and other goitrogens on plasma insulin and thyroid weight. Endocrinology. 1970 Jul;87(1):99–110. doi: 10.1210/endo-87-1-99. [DOI] [PubMed] [Google Scholar]
  26. Lamy F., Roger P., Lecocq R., Dumont J. E. Protein synthesis during induction of DNA replication in thyroid epithelial cells: evidence for late markers of distinct mitogenic pathways. J Cell Physiol. 1989 Mar;138(3):568–578. doi: 10.1002/jcp.1041380318. [DOI] [PubMed] [Google Scholar]
  27. Leffert H., Moran T., Sell S., Skelly H., Ibsen K., Mueller M., Arias I. Growth state-dependent phenotypes of adult hepatocytes in primary monolayer culture. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1834–1838. doi: 10.1073/pnas.75.4.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lissitzky S., Fayet G., Giraud A., Verrier B., Torresani J. Thyrotrophin-induced aggregation and reorganization into follicles of isolated porcine-thyroid cells. 1. Mechanism of action of thyrotrophin and metabolic properties. Eur J Biochem. 1971 Dec 22;24(1):88–99. doi: 10.1111/j.1432-1033.1971.tb19658.x. [DOI] [PubMed] [Google Scholar]
  29. Magnusson R. P., Rapoport B. Modulation of differentiated function in cultured thyroid cells: thyrotropin control of thyroid peroxidase activity. Endocrinology. 1985 Apr;116(4):1493–1500. doi: 10.1210/endo-116-4-1493. [DOI] [PubMed] [Google Scholar]
  30. Mercken L., Simons M. J., Swillens S., Massaer M., Vassart G. Primary structure of bovine thyroglobulin deduced from the sequence of its 8,431-base complementary DNA. Nature. 1985 Aug 15;316(6029):647–651. doi: 10.1038/316647a0. [DOI] [PubMed] [Google Scholar]
  31. Nakamura T., Ichihara A. Control of growth and expression of differentiated functions of mature hepatocytes in primary culture. Cell Struct Funct. 1985 Mar;10(1):1–16. doi: 10.1247/csf.10.1. [DOI] [PubMed] [Google Scholar]
  32. Nakamura T., Nakayama Y., Ichihara A. Reciprocal modulation of growth and liver functions of mature rat hepatocytes in primary culture by an extract of hepatic plasma membranes. J Biol Chem. 1984 Jul 10;259(13):8056–8058. [PubMed] [Google Scholar]
  33. Nakamura T., Nakayama Y., Teramoto H., Nawa K., Ichihara A. Loss of reciprocal modulations of growth and liver function of hepatoma cells in culture by contact with cells or cell membranes. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6398–6402. doi: 10.1073/pnas.81.20.6398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Prochownik E. V., Kukowska J., Rodgers C. c-myc antisense transcripts accelerate differentiation and inhibit G1 progression in murine erythroleukemia cells. Mol Cell Biol. 1988 Sep;8(9):3683–3695. doi: 10.1128/mcb.8.9.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Raspé E., Roger P. P., Dumont J. E. Carbamylcholine, TRH, PGF2 alpha and fluoride enhance free intracellular Ca++ and Ca++ translocation in dog thyroid cells. Biochem Biophys Res Commun. 1986 Dec 15;141(2):569–577. doi: 10.1016/s0006-291x(86)80211-x. [DOI] [PubMed] [Google Scholar]
  36. Reuse S., Roger P. P., Vassart G., Dumont J. E. Enhancement of cmyc mRNA concentration in dog thyrocytes initiating DNA synthesis in response to thyrotropin, forskolin, epidermal growth factor and phorbol myristate ester. Biochem Biophys Res Commun. 1986 Dec 30;141(3):1066–1076. doi: 10.1016/s0006-291x(86)80152-8. [DOI] [PubMed] [Google Scholar]
  37. Rodesch F. Differentiation, contact inhibition and intercellular communication in retinal pigment cells. Exp Cell Res. 1973 Jan;76(1):55–62. doi: 10.1016/0014-4827(73)90418-7. [DOI] [PubMed] [Google Scholar]
  38. Roger P. P., Dumont J. E. Factors controlling proliferation and differentiation of canine thyroid cells cultured in reduced serum conditions: effects of thyrotropin, cyclic AMP and growth factors. Mol Cell Endocrinol. 1984 Jun;36(1-2):79–93. doi: 10.1016/0303-7207(84)90087-x. [DOI] [PubMed] [Google Scholar]
  39. Roger P. P., Dumont J. E. Thyrotrophin and the differential expression of proliferation and differentiation in dog thyroid cells in primary culture. J Endocrinol. 1983 Feb;96(2):241–249. doi: 10.1677/joe.0.0960241. [DOI] [PubMed] [Google Scholar]
  40. Roger P. P., Hotimsky A., Moreau C., Dumont J. E. Stimulation by thyrotropin, cholera toxin and dibutyryl cyclic AMP of the multiplication of differentiated thyroid cells in vitro. Mol Cell Endocrinol. 1982 Apr;26(1-2):165–176. doi: 10.1016/0303-7207(82)90014-4. [DOI] [PubMed] [Google Scholar]
  41. Roger P. P., Reuse S., Servais P., Van Heuverswyn B., Dumont J. E. Stimulation of cell proliferation and inhibition of differentiation expression by tumor-promoting phorbol esters in dog thyroid cells in primary culture. Cancer Res. 1986 Feb;46(2):898–906. [PubMed] [Google Scholar]
  42. Roger P. P., Servais P., Dumont J. E. Induction of DNA synthesis in dog thyrocytes in primary culture: synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin. J Cell Physiol. 1987 Jan;130(1):58–67. doi: 10.1002/jcp.1041300110. [DOI] [PubMed] [Google Scholar]
  43. Roger P. P., Servais P., Dumont J. E. Regulation of dog thyroid epithelial cell cycle by forskolin, an adenylate cyclase activator. Exp Cell Res. 1987 Oct;172(2):282–292. doi: 10.1016/0014-4827(87)90387-9. [DOI] [PubMed] [Google Scholar]
  44. Roger P. P., Servais P., Dumont J. E. Stimulation by thyrotropin and cyclic AMP of the proliferation of quiescent canine thyroid cells cultured in a defined medium containing insulin. FEBS Lett. 1983 Jul 4;157(2):323–329. doi: 10.1016/0014-5793(83)80569-9. [DOI] [PubMed] [Google Scholar]
  45. Roger P. P., Van Heuverswyn B., Lambert C., Reuse S., Vassart G., Dumont J. E. Antagonistic effects of thyrotropin and epidermal growth factor on thyroglobulin mRNA level in cultured thyroid cells. Eur J Biochem. 1985 Oct 15;152(2):239–245. doi: 10.1111/j.1432-1033.1985.tb09189.x. [DOI] [PubMed] [Google Scholar]
  46. Santisteban P., Kohn L. D., Di Lauro R. Thyroglobulin gene expression is regulated by insulin and insulin-like growth factor I, as well as thyrotropin, in FRTL-5 thyroid cells. J Biol Chem. 1987 Mar 25;262(9):4048–4052. [PubMed] [Google Scholar]
  47. Seamon K. B., Daly J. W. Forskolin: its biological and chemical properties. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1986;20:1–150. [PubMed] [Google Scholar]
  48. Straus D. S. Effects of insulin on cellular growth and proliferation. Life Sci. 1981 Nov 23;29(21):2131–2139. doi: 10.1016/0024-3205(81)90482-3. [DOI] [PubMed] [Google Scholar]
  49. Studer H., Peter H. J., Gerber H. Natural heterogeneity of thyroid cells: the basis for understanding thyroid function and nodular goiter growth. Endocr Rev. 1989 May;10(2):125–135. doi: 10.1210/edrv-10-2-125. [DOI] [PubMed] [Google Scholar]
  50. Van Heuverswyn B., Streydio C., Brocas H., Refetoff S., Dumont J., Vassart G. Thyrotropin controls transcription of the thyroglobulin gene. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5941–5945. doi: 10.1073/pnas.81.19.5941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Westermark K., Karlsson F. A., Westermark B. Epidermal growth factor modulates thyroid growth and function in culture. Endocrinology. 1983 May;112(5):1680–1686. doi: 10.1210/endo-112-5-1680. [DOI] [PubMed] [Google Scholar]
  53. Yokoyama K., Imamoto F. Transcriptional control of the endogenous MYC protooncogene by antisense RNA. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7363–7367. doi: 10.1073/pnas.84.21.7363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. de Martynoff G., Pohl V., Mercken L., van Ommen G. J., Vassart G. Structural organization of the bovine thyroglobulin gene and of its 5'-flanking region. Eur J Biochem. 1987 May 4;164(3):591–599. doi: 10.1111/j.1432-1033.1987.tb11168.x. [DOI] [PubMed] [Google Scholar]
  55. deBustros A., Baylin S. B., Levine M. A., Nelkin B. D. Cyclic AMP and phorbol esters separately induce growth inhibition, calcitonin secretion, and calcitonin gene transcription in cultured human medullary thyroid carcinoma. J Biol Chem. 1986 Jun 15;261(17):8036–8041. [PubMed] [Google Scholar]
  56. van Roon M. A., Eier W., Charles R., Lamers W. H. The initial accumulation of carbamoylphosphate synthetase in embryonic rat hepatocytes, and the cell cycle. Differentiation. 1989 Aug;41(2):139–147. doi: 10.1111/j.1432-0436.1989.tb00741.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES