Stimulation of cell proliferation and inhibition of differentiation expression by tumor-promoting phorbol esters in dog thyroid cells in primary culture.

12-O-Tetradecanoylphorbol-13-acetate (TPA) and 4 beta-phorbol 12, 13-dibutyrate (PDBU) are potent tumor promoters and share several biological activities of epidermal growth factor (EGF). We have shown previously that EGF stimulates DNA synthesis and proliferation and inhibits TSH-induced markers of differentiation in dog thyroid follicle-derived primary cultures. Using this system, we have examined the biological action of TPA and PDBU in reference to that of EGF. Low concentrations (1.6-16 nM) and to a lesser extent higher concentrations (greater than 1.6 microM) of TPA and PDBU stimulated cell proliferation in a 1% serum, hormone-supplemented medium and triggered the DNA synthesis revealed by autoradiography in cells which were quiescent before stimulation in serum-free conditions. EGF, TSH, and dibutyryl cyclic adenosine 3':5'-monophosphate separately also induce DNA synthesis, but they produce little if any effects additive to those of TPA. In fact, TPA appeared to inhibit the mitogenic effects of EGF. Moreover like EGF, phorbol esters strongly inhibited in 2 days the morphological effects of TSH and basal and TSH-stimulated iodide transport capacity and thyroglobulin messenger RNA accumulation, two markers of thyroid differentiation. TPA also inhibited the expression of differentiation stimulated by dibutyryl cyclic adenosine 3':5'-monophosphate indicating a post-cyclic adenosine 3':5'-monophosphate site of action. TPA and EGF shared long-term morphological effects such as the induction of an elongated fusiform shape, but not acute effects. The thyroid cells progressively and spontaneously escaped both the mitogenic and differentiation-inhibiting effects of TPA and PDBU, while, as shown previously, these parameters are stably modified by continuous culture with EGF. This suggests specific desensitization processes to phorbol esters. As evidence is accumulating that phorbol esters act at least partly by stimulating the calcium-activated, phospholipid-dependent protein kinase C, our results shed light on the possible key role of this kinase in carcinogenesis and in the normal control of proliferation and expression of differentiation in the thyroid gland. Additionally they suggest that complex interactions occur between the mechanisms of action of EGF and of phorbol esters in the thyroid cell.

Antagonistic effects of thyrotropin and epidermal growth factor on thyroglobulin mRNA level in cultured thyroid cells.

Both thyrotropin (TSH) and epidermal growth factor (EGF) are potent mitogenic agents when added to dog thyroid cells in primary culture [Roger, P. P. and Dumont, J. E. (1984) Mol. Cell. Endocrinol. 36, 79-93]. The concomitant effect of these agents on the differentiation state of the cells was appreciated using cell morphology, iodide trapping, thyroglobulin synthesis and cytoplasmic thyroglobulin mRNA content as markers. Together with previous results [Mol. Cell. Endocrinol. 36, 79-93 (1984)] it is shown that cells cultured in the continuous presence of TSH maintain all the parameters at a near normal level. In the absence of TSH, thyroglobulin mRNA decreased to very low, though still detectable levels. Addition of TSH restored subnormal mRNA levels. Culture of cells in the presence of EGF for 4-6 days affected profoundly their morphology, abolished iodide trapping and decreased thyroglobulin synthesis and cytoplasmic mRNA content to undetectable levels. Addition of TSH to cells previously exposed to EGF reversed the growth factor effect on all four indexes. The redifferentiating effect of TSH was well observed within 3-4 days and was mimicked by the adenylate cyclase activators, forskolin and cholera toxin. When administered simultaneously, TSH and EGF achieved an intermediate situation, EGF antagonizing partially the effect of TSH on the expression of thyroglobulin gene. Another growth factor, fibroblast growth factor, while promoting thyroid cell proliferation also, did not interfere at all with TSH effects on cytoplasmic thyroglobulin mRNA content. Our results make the dog thyroid cell in primary culture an appropriate model to study the mechanisms involved in gene regulation by cyclic AMP and growth factors.

Transcriptional control of thyroglobulin gene expression by cyclic AMP.

Transcription of thyroglobulin (Tg) gene is under the positive control of thyrotropin (TSH). The mechanism of this control has been further investigated. Rats were treated with triiodothyronine (T3) to decrease their endogenous TSH production. Following the intravenous injection of bovine TSH, a 3-fold stimulation of Tg gene transcription could be detected in isolated nuclei as early as 1 h after treatment. The TSH effect was also observed in tissue fragments incubated in vitro under conditions where a concomitant stimulation of cAMP accumulation was detected. Forskolin, a universal activator of adenylate cyclase, was able to mimic TSH action on Tg gene transcription. We conclude that TSH controls transcription of Tg gene directly via its known interaction with receptors on thyrocytes and that cAMP is a physiological mediator of this effect.

Thyrotropin controls transcription of the thyroglobulin gene.

The availability of rat thyroglobulin cDNA clones was exploited to study the regulation of thyroglobulin gene transcription by thyrotropin (TSH). Groups of rats were subjected to treatments leading to reduction or increase in the rat serum TSH (rTSH) levels. Thyroid gland nuclei were isolated, incubated in vitro in the presence of 32P-labeled uridine triphosphate, and thyroglobulin transcripts were quantitated by hybridization to immobilized rat thyroglobulin cDNA clones. Transcription of the thyroglobulin gene was found to be very active in thyroid nuclei from control animals. It represented about 10% of total RNA polymerase II activity. Chronic hyperstimulation of the thyroid glands with endogenous rTSH was achieved in rats treated with the goitrogen propylthiouracil. No significant increase of thyroglobulin gene transcription could be measured in thyroid nuclei from these animals. On the contrary, a dramatic decrease in thyroglobulin gene transcription was observed in those animals in which endogenous rTSH levels had been suppressed by hypophysectomy or by the administration of triiodothyronine. Injection of exogenous bovine TSH in such animals readily restored transcriptional activity of the gene. Our results identify transcription as an important regulatory step involved in TSH action. They suggest that normal TSH levels induce close to maximal expression of the thyroglobulin gene but that continuous presence of TSH is required in order to maintain the gene in an activated state.

[Structure and expression of thyroglobulin gene]. / Structure et expression du gène de la thyroglobuline.

Thyroglobulin is composed of two 300000 dalton polypeptide chains, translated from an 8000 base mRNA. Preparation of a full length cDNA and its cloning in E. coli have lead to the demonstration that the polypeptides of thyroglobulin protomers were identical. Used as molecular probes, the cloned cDNA allowed the isolation of a fragment of thyroglobulin gene. Electron microscopic studies have demonstrated that this gene contains more than 90% intronic material separating small size exons (less than 200 bp). Sequencing of bovine thyroglobulin structural gene is in progress. Preliminary results show evidence for the existence of repetitive segments. Availability of cloned DNA complementary to bovine and human thyroglobulin mRNA allows the study of genetic defects of thyroglobulin gene expression in the human and in various animal models.

The effect of 131I for diagnostic purposes on serum thyroglobulin (hTg) levels in subjects with thyroidal disorders.

The concentration of serum thyroglobulin was measured in sera of subjects with various thyroid disorders, before and after the administration of tracer doses of 131I. The mean serum human thyroglobulin (hTg) concentration before administration of the isotope was 35.2 ng/ml +/- 7.8 (SE) in 13 subjects and 36.3 ng/ml +/- 7.9 (SE) 24 h after the administration of 131I. The data indicate that no significant release of thyroglobulin occurs 24 h after the administration of tracer doses of 131I. In 2 of the 3 subjects, however, in whom samples were obtained at 4 and 8 h after diagnostic administration of 131I, a modest rise in serum Tg levels was observed. Determinations of serum thyroglobulin levels within 24 h after the administration of a tracer dose of 131I are nevertheless valid providing one allows sufficient time to elapse for tracer decay.

Thyroglobulin and thyroid hormone release after intravenous administration of bovine thyrotropin in man.

To elucidate the mechanism of thyroglobulin (Tg) release in man, the effects of an iv injection of a submaximal dose of bovine TSH (bTSH) on the serum levels of Tg were compared with the effects on serum T3 and T4. After the administration of bTSH, short term kinetics (0-4 h) were studied in eight subjects receiving 0.5 IU bTSH and seven subjects receiving 1 IU bTSH. Serum Tg did not significantly increase in either of the short term studies. By contrast, serum T3 increased significantly and linearly after the administration of 0.5 and 1 IU bTSH; serum T4 also rose but only after 1 IU bTSH. Long term kinetics (0-120 h) were studied in seven additional subjects after the iv administration of 1 IU bTSH; serum bTSH was no longer detectable after 8 h. Maximum serum concentrations of T3 were obtained at about 4 h, maximum serum concentrations of T4 were obtained between 4-8 h. Serum Tg levels increased linearly with time during the first 24 h. Maximum serum Tg levels correlated well with basal serum Tg values (r = 0.97; P < 0.001). The maximal increment in Tg correlated inversely with the maximal increment in T3 (r = 0.71; P < 0.05). The half-life of Tg was estimated to be approximately 4 days by measuring the disappearance rate of Tg after its peak level was attained.