Genetic basis of congenital hypothyroidism: abnormalities in the TSHbeta gene, the PIT1 gene, and the NIS gene

We have elucidated the molecular pathology of three types of congenital hypothyroidism. Thyrotropin (TSH) is the major regulator of thyroid function. In cases of isolated congenital TSH deficiency, we found that they are caused by a missense mutation in the conserved CAGYC region of the TSHbeta gene. Pit-1/GHF-1 is a pituitary specific POU-domain DNA binding factor, which transactivates the growth hormone (GH), prolactin (PRL), TSHbeta genes, and the PIT1 gene itself. In cases of combined deficiency of GH, PRL, and TSH, we found that they are caused by abnormalities in the PIT1 gene, either recessively or dominantly. Sodium dependent iodide symporter (NIS) actively transports iodide into the thyroid cells to produce thyroid hormones. In cases of iodide transport defect, we elucidated that a missense mutation in the transmembrane region of the NIS gene caused them.     

Function of galanin in the anterior pituitary of estrogen-treated Fischer 344 rats: autocrine and paracrine regulation of prolactin secretion

Estrogen is a robust stimulator of galanin synthesis and secretion in the anterior pituitary. Galanin is colocalized in lactotrophs in the estrogen-treated anterior pituitary, and its roles in lactotroph function are still being elucidated. In the present studies, we quantified the phenotypes of estrogen-treated Fischer 344 rat anterior pituitary cells expressing the galanin gene by dual in situ hybridization. The total population of galanin-positive pituitary cells increased from undetectable levels to 16% of all cells after 2 weeks of estrogen treatment. More than 90% of the galanin-positive cells coexpressed PRL messenger RNA, and one-third of the lactotrophs expressed galanin messenger RNA. We hypothesized that galanin in the anterior pituitary may contribute to the heterogeneous secretion of PRL, and that one of the functions of galanin is to regulate PRL secretion in an autocrine/paracrine manner. To test this hypothesis, we performed the reverse hemolytic plaque assay combined with in situ hybridization to measure PRL secretion and galanin gene expression within the same individual cells. PRL secretion from galanin-positive lactotrophs was significantly greater than that from galanin-negative lactotrophs. Moreover, treatment with galanin antiserum significantly attenuated PRL secretion from galanin-positive cells, and treatment with galanin significantly enhanced PRL secretion from galanin-negative lactotrophs. In conclusion, these data provide direct evidence that galanin derived from the estrogen-treated anterior pituitary stimulates PRL secretion in both autocrine and paracrine manners.     

Identification of thyroid hormone response elements in the human fatty acid synthase promoter

To investigate the regulation of the human fatty acid synthase gene by the thyroid hormone triiodothyronine, various constructs of the human fatty acid synthase promoter and the luciferase reporter gene were transfected in combination with plasmids expressing the thyroid hormone and the retinoid X receptors in HepG2 cells. The reporter gene was activated 25-fold by the thyroid hormone in the presence of the thyroid hormone receptor. When both the thyroid hormone and the retinoid X receptors were expressed in HepG2 cells, there was about a 100-fold increase in reporter gene expression. 5'-Deletion analysis disclosed two thyroid hormone response elements, TRE1 (nucleotides -870 to -650) and TRE2 (nucleotides -272 to -40), in the human fatty acid synthase promoter. The presence of thyroid hormone response elements in these two regions of the promoter was confirmed by cloning various fragments of these two regions in the minimal thymidine kinase promoter-luciferase reporter gene plasmid construct and determining reporter gene expression. The results of this cloning procedure and those of electrophoretic mobility shift assays indicated that the sequence GGGTTAcgtcCGGTCA (nucleotides -716 to -731) represents TRE1 and that the sequence GGGTCC (nucleotides -117 to -112) represents TRE2. The sequence of TRE1 is very similar to the consensus sequence of the thyroid hormone response element, whereas the sequence of TRE2 contains only a half-site of the thyroid hormone response element consensus motif because it lacks the direct repeat. The sequences on either side of TRE2 seem to influence its response to the thyroid hormone and retinoid X receptors.     

Vasoactive intestinal peptide-induced prolactin release in hypothyroid patients

VIP is an established prolactin-releasing factor. VIP gene expression at the anterior pituitary level and the central nervous system is regulated by thyroid hormones. On the other hand, primary hypothyroidism leads in many cases to amenorrhea, galactorrhea and hyperprolactinemia. In this study we assessed prolactin responses to VIP (75 micrograms iv infusion over 12 min) in a group of six hypothyroid women (mean age +/- SE, 38.8 +/- 3.3 yr; serum TSH levels, mU/L, 116.3 +/- 23.9), before treatment and after normalization of thyroid hormone levels during thyroxine (T4) replacement therapy (100-150 micrograms/day over 12-16 weeks). Furthermore, we assessed if VIP infusion had any effects on serum GH levels in these patients. In hypothyroid women, VIP infusion increased serum prolactin concentrations with peak levels being attained at 15 min (28.8 +/- 3.4 micrograms/L). The Area Under the Curve (AUC) was 1921 +/- 103 micrograms/L/2h. PRL responses to VIP were unchanged after T4 therapy, both in terms of peak levels (28.7 +/- 2.2 micrograms/L, NS) and of AUC (2079 +/- 261 micrograms/L/2h, NS). Serum GH levels were unaffected by VIP administration. In conclusion our study shows that, in hypothyroid patients, restoration of normal thyroid hormone levels by thyroxine replacement therapy does not affect lactotroph responsiveness to VIP. Therefore, our data do not support the hypothesis that VIP might contribute to the hypothyroid-induced hyperprolactinemia seen in man.