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SAT-450 Essential Role of GATA2 in the Negative Regulation of the Prepro-Thyrotropin-Releasing Hormone Gene by Liganded T3 in the Rat Paraventricular Nucleus

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...

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Autores principales: Kuroda, Go, Sasaki, Shigekazu, Matsushita, Akio, Ohba, Kenji, Sakai, Yuki, Shinkai, Shinsuke, Nakamura, Hiroko, Oki, Yutaka
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7208958/
http://dx.doi.org/10.1210/jendso/bvaa046.762
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author Kuroda, Go
Sasaki, Shigekazu
Matsushita, Akio
Ohba, Kenji
Sakai, Yuki
Shinkai, Shinsuke
Nakamura, Hiroko
Oki, Yutaka
author_facet Kuroda, Go
Sasaki, Shigekazu
Matsushita, Akio
Ohba, Kenji
Sakai, Yuki
Shinkai, Shinsuke
Nakamura, Hiroko
Oki, Yutaka
author_sort Kuroda, Go
collection PubMed
description 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.
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spelling pubmed-72089582020-05-13 SAT-450 Essential Role of GATA2 in the Negative Regulation of the Prepro-Thyrotropin-Releasing Hormone Gene by Liganded T3 in the Rat Paraventricular Nucleus Kuroda, Go Sasaki, Shigekazu Matsushita, Akio Ohba, Kenji Sakai, Yuki Shinkai, Shinsuke Nakamura, Hiroko Oki, Yutaka J Endocr Soc Thyroid 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. Oxford University Press 2020-05-08 /pmc/articles/PMC7208958/ http://dx.doi.org/10.1210/jendso/bvaa046.762 Text en © Endocrine Society 2020. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Thyroid
Kuroda, Go
Sasaki, Shigekazu
Matsushita, Akio
Ohba, Kenji
Sakai, Yuki
Shinkai, Shinsuke
Nakamura, Hiroko
Oki, Yutaka
SAT-450 Essential Role of GATA2 in the Negative Regulation of the Prepro-Thyrotropin-Releasing Hormone Gene by Liganded T3 in the Rat Paraventricular Nucleus
title SAT-450 Essential Role of GATA2 in the Negative Regulation of the Prepro-Thyrotropin-Releasing Hormone Gene by Liganded T3 in the Rat Paraventricular Nucleus
title_full SAT-450 Essential Role of GATA2 in the Negative Regulation of the Prepro-Thyrotropin-Releasing Hormone Gene by Liganded T3 in the Rat Paraventricular Nucleus
title_fullStr SAT-450 Essential Role of GATA2 in the Negative Regulation of the Prepro-Thyrotropin-Releasing Hormone Gene by Liganded T3 in the Rat Paraventricular Nucleus
title_full_unstemmed SAT-450 Essential Role of GATA2 in the Negative Regulation of the Prepro-Thyrotropin-Releasing Hormone Gene by Liganded T3 in the Rat Paraventricular Nucleus
title_short SAT-450 Essential Role of GATA2 in the Negative Regulation of the Prepro-Thyrotropin-Releasing Hormone Gene by Liganded T3 in the Rat Paraventricular Nucleus
title_sort sat-450 essential role of gata2 in the negative regulation of the prepro-thyrotropin-releasing hormone gene by liganded t3 in the rat paraventricular nucleus
topic Thyroid
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7208958/
http://dx.doi.org/10.1210/jendso/bvaa046.762
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