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Resistance to thyroid hormone beta (RTHβ) caused by germline mutations in genes encoding thyroid hormone receptor beta (TRβ) is a rare disorder. Little information is available regarding the clinical experience of this syndrome in Japan. We retrospectively reviewed the records of 34 patients with RTHβ (21 adult females and 13 adult males) with positive TRβ mutations identified at our division between 2000 and 2020. Of the 24 patients with available clinical history, 10 (41.7%) received inappropriate treatments such as antithyroid drugs, thyroidectomy, or radioactive iodine. Diagnostic delay and inappropriate management of RTHβ are still present in Japan. Every patient except one demonstrated thyroid hormone profiles indicative of syndrome of inappropriate secretion of thyrotropin (SITSH), characterized by a hormonal profile of hyperthyroxinemia with a non-suppressed TSH concentration. Since the most common forms of hyperthyroidism including Graves' disease feature elevated thyroid hormone levels with suppressed TSH concentrations, early diagnosis of SITSH is critical for preventing inappropriate management. One patient positive for anti-thyroglobulin antibody (Tg-Ab) and anti-thyroperoxidase antibody (TPO-Ab) showed remarkably elevated TSH (>200 μIU/mL) despite thyroid hormone concentrations within the reference ranges. At least one thyroid autoantibody (Tg-Ab, TPO-Ab, or thyrotropin receptor antibodies) was identified in 37.9% (11/29) of the patients tested. One patient developed overt Graves' disease nine years after ‍RTHβ diagnosis. These findings suggest that RTHβ is frequently comorbid with additional autoimmune thyroid disorders. ‍Further research is required to identify the most appropriate treatments for RTHβ patients who develop a second thyroid ‍disorder.

The transcription factor GATA2 regulates gene expression in several cells and tissues, including hematopoietic tissues and the central nervous system. Recent studies revealed that loss-of-function mutations in GATA2 are associated with hematological disorders. Our earlier in vitro studies showed that GATA2 plays an essential role in the hypothalamus–pituitary–thyroid axis (HPT axis) by regulating the genes encoding prepro-thyrotropin-releasing hormone (preproTRH) and thyroid-stimulating hormone β (TSHβ). However, the effect of GATA2 mutants on the transcriptional activity of their promoters remains unelucidated. In this study, we created five human GATA2 mutations (R308P, T354M, R396Q, R398W, and S447R) that were reported to be associated with hematological disorders and analyzed their functional properties, including transactivation potential and DNA-binding capacity toward the preproTRH and the TSHβ promoters. Three mutations (T354M, R396Q, and R398W) within the C-terminal zinc-finger domain reduced the basal GATA2 transcriptional activity on both the preproTRH and the TSHβ promoters with a significant loss of DNA binding affinity. Interestingly, only the R398W mutation reduced the GATA2 protein expression. Subsequent analysis demonstrated that the R398W mutation possibly facilitated the GATA2 degradation process. R308P and S447R mutants exhibited decreased transcriptional activity under protein kinase C compared to the wild-type protein. In conclusion, we demonstrated that naturally occurring GATA2 mutations impair the HPT axis through differential functional mechanisms in vitro.

Thyroid hormone (T3) inhibits thyrotropin-releasing hormone (TRH) synthesis in the hypothalamic paraventricular nucleus (PVN). Although the T3 receptor (TR) β2 is known to mediate the negative regulation of the prepro-TRH gene, its molecular mechanism remains unknown. Our previous studies on the T3-dependent negative regulation of the thyrotropin β subunit (TSHβ) gene suggest that there is a tethering mechanism, whereby liganded TRβ2 interferes with the function of the transcription factor, GATA2, a critical activator of the TSHβ gene. Interestingly, the transcription factors Sim1 and Arnt2, the determinants of PVN differentiation in the hypothalamus, are reported to induce expression of TRβ2 and GATA2 in cultured neuronal cells. Here, we confirmed the expression of the GATA2 protein in the TRH neuron of the rat PVN using immunohistochemistry with an anti-GATA2 antibody. According to an experimental study from transgenic mice, a region of the rat prepro-TRH promoter from nt. -547 to nt. +84 was able to mediate its expression in the PVN. We constructed a chloramphenicol acetyltransferase (CAT) reporter gene containing this promoter sequence (rTRH(547)-CAT) and showed that GATA2 activated the promoter in monkey kidney-derived CV1 cells. Deletion and mutation analyses identified a functional GATA-responsive element (GATA-RE) between nt. -357 and nt. -352. When TRβ2 was co-expressed, T3 reduced GATA2-dependent promoter activity to approximately 30%. Unexpectedly, T3-dependent negative regulation was maintained after mutation of the reported negative T3-responsive element, site 4. T3 also inhibited the GATA2-dependent transcription enhanced by cAMP agonist, 8-bromo-cAMP. A rat thyroid medullary carcinoma cell line, CA77, is known to express the preproTRH mRNA. Using a chromatin immunoprecipitation assay with this cell line where GATA2 expression plasmid was transfected, we observed the recognition of the GATA-RE by GATA2. We also confirmed GATA2 binding using gel shift assay with the probe for the GATA-RE. In CA77 cells, the activity of rTRH(547)-CAT was potentiated by overexpression of GATA2, and it was inhibited in a T3-dependent manner. These results suggest that GATA2 transactivates the rat prepro-TRH gene and that liganded TRβ2 interferes with this activation via a tethering mechanism as in the case of the TSHβ gene.

T3 inhibits thyrotropin-releasing hormone (TRH) synthesis in hypothalamic paraventricular nucleus (PVN). Although T3 receptor (TR) β2 is known to mediate the negative regulation of prepro-TRH gene, its molecular mechanism remains unknown. Our previous studies on the T3-dependent negative regulation of the thyrotropin β subunit (TSHβ) gene indicate the tethering mechanism, where T3-bound TRβ2 interferes with the function of the transcription factor GATA2, which is essential for TSHβ expression. Interestingly, the transcription factor Sim1, a determinant of PVN differentiation in hypothalamus, is reported to induce the expressions of TRβ2 and GATA2. Indeed, our immunohistochemistry revealed the expression of GATA2 in the TRH neuron of the rat PVN. According to the experimental report with transgenic mice, the DNA sequence from nt. -547 to nt. +84 is sufficient for the expression of the prepro-TRH gene in PVN. Using the CAT reporter gene harboring this region, we found that this promoter is activated by GATA2 approximately 6-fold in CV1 cells. The deletion and mutation analyses identified a functional GATA-responsive element (GATA-RE) between nt. -357 and nt. -352. When TRβ2 was co-expressed, T3 reduced GATA2-dependent promoter activity to approximately 30%. T3-dependent repression was maintained after the mutation of the putative negative T3 responsive element (site4). Although the melanocortin 4 receptor signaling is known to stimulate the prepro-TRH promoter via protein kinase A pathway in the PVN, inhibition by T3 was dominant over the 8-bromo-cAMP-induced activation. We observed the in vivo recognition of GATA-RE by GATA2 using chromatin immunoprecipitation assay with CA77 cells, which express endogenous TRH. The electrophoretic mobility shift assay also demonstrated that GATA2 bound to oligonucleotide containing the GATA-RE. These results suggest that, as in the case of the TSHβ gene, GATA2 transactivates the prepro-TRH gene and that T3-bound TRβ2 interferes with its function, resulting in the negative regulation of this gene.

The serum concentration of thyrotropin (thyroid stimulating hormone, TSH) is drastically reduced by small increase in the levels of thyroid hormones (T3 and its prohormone, T4); however, the mechanism underlying this relationship is unknown. TSH consists of the chorionic gonadotropin α (CGA) and the β chain (TSHβ). The expression of both peptides is induced by the transcription factor GATA2, a determinant of the thyrotroph and gonadotroph differentiation in the pituitary. We previously reported that the liganded T3 receptor (TR) inhibits transactivation activity of GATA2 via a tethering mechanism and proposed that this mechanism, but not binding of TR with a negative T3-responsive element, is the basis for the T3-dependent inhibition of the TSHβ and CGA genes. Multiple GATA-responsive elements (GATA-REs) also exist within the GATA2 gene itself and mediate the positive feedback autoregulation of this gene. To elucidate the effect of T3 on this non-linear regulation, we fused the GATA-REs at -3.9 kb or +9.5 kb of the GATA2 gene with the chloramphenicol acetyltransferase reporter gene harbored in its 1S-promoter. These constructs were co-transfected with the expression plasmids for GATA2 and the pituitary specific TR, TRβ2, into kidney-derived CV1 cells. We found that liganded TRβ2 represses the GATA2-induced transactivation of these reporter genes. Multi-dimensional input function theory revealed that liganded TRβ2 functions as a classical transcriptional repressor. Then, we investigated the effect of T3 on the endogenous expression of GATA2 protein and mRNA in the gonadotroph-derived LβT2 cells. In this cell line, T3 reduced GATA2 protein independently of the ubiquitin proteasome system. GATA2 mRNA was drastically suppressed by T3, the concentration of which corresponds to moderate hypothyroidism and euthyroidism. These results suggest that liganded TRβ2 inhibits the positive feedback autoregulation of the GATA2 gene; moreover this mechanism plays an important role in the potent reduction of TSH production by T3.

Thyroid hormone (T3) activates (positive regulation) or represses (negative regulation) target genes at the transcriptional level. The molecular mechanism of the former has been elucidated in detail; however, the mechanism for negative regulation has not been established. The best example of the gene that is negatively regulated by T3 is the thyrotropin (thyroid-stimulating hormone) β subunit (TSHβ) gene. Analogous to the T3-responsive element (TRE) in positive regulation, a negative TRE (nTRE) has been postulated in the TSHβ gene. However, TSHβ promoter analysis, performed in the presence of transcription factors Pit1 and GATA2, which are determinants of thyrotroph differentiation in the pituitary, revealed that the nTRE is dispensable for inhibition by T3. We propose a tethering model in which the T3 receptor is tethered to GATA2 via protein-protein interaction and inhibits GATA2-dependent transactivation of the TSHβ gene in a T3-dependent manner.