Regulation of pituitary thyrotropin gene expression during Xenopus metamorphosis: negative feedback is functional throughout metamorphosis

Several hypotheses have been proposed to explain the increase and sustained expression of pituitary thyrotropin (TSH) in the presence of elevated plasma thyroid hormone (TH) concentrations at metamorphic climax in amphibians. It has been proposed that the negative feedback of TH on TSH is inoperative until metamorphic climax, and that it is established at this time by the upregulation of pituitary deiodinase type II (DII); DII converts thyroxine (T(4)) to 3,5,3'-triiodothyronine (T(3)). However, earlier investigators, using indirect measures of TSH, reported that TH negative feedback on TSH was functional in premetamorphic tadpoles. In an effort to understand pituitary TSH regulation during amphibian metamorphosis, we analyzed multiple pituitary genes known or hypothesized to be involved in TSH regulation in tadpoles of Xenopus laevis. Tadpole pituitary explant cultures were used to examine direct negative feedback on TSH mRNA expression. Negative feedback is operative in the early prometamorphic tadpole pituitary and both T(3) and T(4) can downregulate TSH mRNA expression throughout metamorphosis. The expression of both DII and TH receptor betaA mRNAs increased during development and peaked at climax; however, these increases coincided with similar increases in deiodinase type III, which inactivates TH. Moreover, corticotropin-releasing factor (CRF) receptors, CRF binding protein and thyrotropin-releasing hormone receptor type 2 mRNA expression also peaked at climax. Our data suggest that the regulation of TSH is more complex than the timing of DII expression, and likely involves a balance between stimulation of TSH synthesis and secretion by neuropeptides (e.g. CRF) of hypothalamic or pituitary origin, increased pituitary sensitivity to neuropeptides through upregulation of their receptors, and intrapituitary TH levels.

Hormonal control of the transcription factor Pax8 and its role in the regulation of thyroglobulin gene expression in thyroid cells

The transcription factor Pax8 plays an important role in the expression of the differentiated phenotype of thyroid follicular cells. It has recently been shown that Pax8 is necessary for thyroglobulin (Tg) gene expression in the fully differentiated rat thyroid cell line PC. We have used the PC model system to investigate the role of Pax8 as a mediator of TSH regulation of Tg gene expression. We have demonstrated that Pax8 expression, as well as Tg expression, is severely reduced in cells grown in the absence of hormones and serum. The re-addition of TSH or forskolin to the culture medium is able to restore to wild-type levels the expression of both Pax8 and Tg. We have determined that the action of TSH/forskolin on Pax8 is at the transcriptional level. However, the re-expression of Pax8 can be observed several hours before that of Tg, suggesting that either another factor is needed or that Pax8 itself must be post-translationally modified by a newly synthesized protein to become active. To distinguish between these two possibilities we have stably transfected into PC cells an exogenous Pax8 that is expressed independently of TSH. Our results indicate that in these cells the Tg promoter is still dependent on TSH despite the constitutive presence of Pax8. Furthermore, we also show that in this condition Tg gene transcription requires de novo protein synthesis. In conclusion, TSH regulates the expression of Pax8 at a transcriptional level and also regulates the activity of Pax8 by controlling the expression of one or more as yet unknown factors.

Effect of neonatal hyperthyroidism upon the regulation of TSH secretion in rats

The neo-T4 syndrome was induced in rats by administration of 30 μg T4/in 5 doses starting on the first day of life. In the first experiment (A), neo-T4 and saline-control rats were divided into two populations, one of which was thyroidectomized on day 25. All rats then received 5 μg T4/100 g b.w. on days 42, 43 and 44, and were sacrificed on day 45. In the second experiment (B), neo-T4 and saline-control rats were thyroidectomized at 25 days of age and were sacrificed 10, 20 and 40 days after the operation. In both experiments, pituitary and plasma TSH and pituitary GH were determined. T4 administration has the same effect on plasma and pituitary TSH regulation in neo-T4 and control rats, thyroidectomized or not. The increase in pituitary GH produced by T4 is smaller in the thyroidectomized neoT4 animals. The comparison between T4-induced increases of pituitary TSH in thyroidectomized neo-T4 and saline control-rats, and the corresponding decreases in non-thyroidectomized animals suggests an alteration in TSH synthesis which has previously been compared with that found in rats with lesions of the hypothalamus. However, the changes in pituitary and plasma TSH levels in neo-T4 rats at different intervals after thyroidectomy do not coincide with those described for rats with hypothalamic lesions. The possible perturbation in pituitary TSH synthesis proposed for neo-T4 rats accords with the lack of response to TRH found in adult animals treated with thyroxine, an effect which remains even when plasma T4 levels decrease.