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. Endwrinol. 36,[79][80][81][82][83][84][85][86][87][88][89][90][91][92][93]. Thc 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 markcrs. Together with previous results [Mal. Cd/. Endacrinol. 36, 79-93 (1984)j it is shown that cclls cultured in the continuous presence of TSH maintain all the parameters at a near normal level. In the absencc of TSH, thyroglobulin mRNA decreased to very low, though stil! detectable levels. Addition of TSH restored subnormal mRNA levels. Culture of cells in the presence or E G F 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 E G F reversed the growth factor effect on all four indexes. The rediffercntiating effect of TSH was well observed within 3-4 days and was mimicked by the adcnylate cyclase activators, forskolin and cholera toxin. When administered simultancously, TSH and EGF achieved an intermediate situation, EGP 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.

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.

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.

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.