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G ATA2 mediates the negative regulation of the prepro-thyrotropin-releasing hormone gene by liganded T3 receptor ?2 in the rat hypothalamic paraventricular nucleus.

ABSTRACT: Thyroid hormone (T3) inhibits thyrotropin-releasing hormone (TRH) synthesis in the hypothalamic paraventricular nucleus (PVN). Although the T3 receptor (TR) ?2 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 ? subunit (TSH?) gene suggest that there is a tethering mechanism, whereby liganded TR?2 interferes with the function of the transcription factor, GATA2, a critical activator of the TSH? gene. Interestingly, the transcription factors Sim1 and Arnt2, the determinants of PVN differentiation in the hypothalamus, are reported to induce expression of TR?2 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 TR?2 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 TR?2 interferes with this activation via a tethering mechanism as in the case of the TSH? gene.

SUBMITTER: Kuroda G

PROVIDER: S-EPMC7671546 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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G ATA2 mediates the negative regulation of the prepro-thyrotropin-releasing hormone gene by liganded T3 receptor β2 in the rat hypothalamic paraventricular nucleus.

Kuroda Go G Sasaki Shigekazu S Matsushita Akio A Ohba Kenji K Sakai Yuki Y Shinkai Shinsuke S Nakamura Hiroko Misawa HM Yamagishi Satoru S Sato Kohji K Hirahara Naoko N Oki Yutaka Y Ito Masahiko M Suzuki Tetsuro T Suda Takafumi T

PloS one 20201117 11

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, GATA ...[more]

PMID: 33201916

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Project description: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.

Dataset Information

G ATA2 mediates the negative regulation of the prepro-thyrotropin-releasing hormone gene by liganded T3 receptor ?2 in the rat hypothalamic paraventricular nucleus.

Publications

G ATA2 mediates the negative regulation of the prepro-thyrotropin-releasing hormone gene by liganded T3 receptor β2 in the rat hypothalamic paraventricular nucleus.

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