Essential Role of GATA2 in the Negative Regulation of Type 2 Deiodinase Gene by Liganded Thyroid Hormone Receptor β2 in Thyrotroph

Abstract

The inhibition of thyrotropin (thyroid stimulating hormone; TSH) by thyroid hormone (T3) and its receptor (TR) is the central mechanism of the hypothalamus-pituitary-thyroid axis. Two transcription factors, GATA2 and Pit-1, determine thyrotroph differentiation and maintain the expression of the β subunit of TSH (TSHβ). We previously reported that T3-dependent repression of the TSHβ gene is mediated by GATA2 but not by the reported negative T3-responsive element (nTRE). In thyrotrophs, T3 also represses mRNA of the type-2 deiodinase (D2) gene, where no nTRE has been identified. Here, the human D2 promoter fused to the CAT or modified Renilla luciferase gene was co-transfected with Pit-1 and/or GATA2 expression plasmids into cell lines including CV1 and thyrotroph-derived TαT1. GATA2 but not Pit-1 activated the D2 promoter. Two GATA responsive elements (GATA-REs) were identified close to cAMP responsive element. The protein kinase A activator, forskolin, synergistically enhanced GATA2-dependent activity. Gel-shift and chromatin immunoprecipitation assays with TαT1 cells indicated that GATA2 binds to these GATA-REs. T3 repressed the GATA2-induced activity of the D2 promoter in the presence of the pituitary-specific TR, TRβ2. The inhibition by T3-bound TRβ2 was dominant over the synergism between GATA2 and forskolin. The D2 promoter is also stimulated by GATA4, the major GATA in cardiomyocytes, and this activity was repressed by T3 in the presence of TRα1. These data indicate that the GATA-induced activity of the D2 promoter is suppressed by T3-bound TRs via a tethering mechanism, as in the case of the TSHβ gene.

Liganded T3 receptor β2 inhibits the positive feedback autoregulation of the gene for GATA2, a transcription factor critical for thyrotropin production

“…Interestingly, however, the D2 gene harbors the two highly conserved GATA-REs [47]. As expected, our previous work revealed that liganded TRβ2 suppresses the D2 promoter by a tethering mechanism via GATA2 on these GATA-REs [47]. In the GATA2 gene, the critical function of +9.5 kb GATA-RE ( Fig 1A) was evidenced based on hematopoietic stem/progenitor cell depletion observed in the mice harboring the deletion of this GATA-RE [45].…”

“…Although T3 has been known to repress type-2 deiodinase (D2) gene/activity in several organs and cultured cells including thyrotroph [67], no DNA sequence similar to the nTRE reported in the TSHβ gene has been identified in this gene to date [68,69]. Interestingly, however, the D2 gene harbors the two highly conserved GATA-REs [47]. As expected, our previous work revealed that liganded TRβ2 suppresses the D2 promoter by a tethering mechanism via GATA2 on these GATA-REs [47].…”

“…These PCR products were digested by EcoRI and Bgl-II. The human D2 promoter in hD2-CAT [47] was replaced by these PCR products after digestion with restriction enzymes (EcoRI and Bgl-II), generating 1S-CAT, (-3.9)1S-CAT and (+9.5)1S-CAT, respectively ( Fig 1B). In these constructs, the pUC-derived AP-1-like sequence was deleted because it might also mediate artifactual T3-dependent inhibition [ [13,46] and references therein].…”

New Insights into Vertebrate Thyroid Hormone Receptor Evolution

“…This means that THs induce their repression instead of their activation. This is the case of genes of the TH pathway such as the thyrotropin-releasing hormone (TRH) gene or the deiodinase 2 [49,50]. If Pm542 and Pm8742 are somehow specialized in the negative TREs, they may account for the drop of global TH levels observed in lamprey metamorphosis.…”

Novel aspects of T3 actions on GH and TSH synthesis and secretion: physiological implications

“…However, recent studies have shown that the negative regulation of human TSHB gene exerted by T 3 is independent of nTRE (Matsushita et al 2007, Matsunaga et al 2015.…”