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.
Highlights
Negative feedback regulation in the hypothalamus-pituitary-thyroid (H-P-T) axis is the central mechanism for the homeostasis of thyroid function [1]
We found that the major activator for the TSHβ gene is GATA2 while Pit-1 protects the GATA2 function from suppression by the sequence downstream to GATA-REs [27]
Because these cells express endogenous GATA2 [30] or GATA4 [14], we speculated that, if a functional GATA-RE exists in the human D2 promoter, it will be within this region
Summary
Negative feedback regulation in the hypothalamus-pituitary-thyroid (H-P-T) axis is the central mechanism for the homeostasis of thyroid function [1]. Based on the prediction of several GATA-responsive elements (GATA-REs) in the D2 genes by the computer searches, Dentice et al [15] compared the activation of the D2 promoters by GATA4 with that of Nkx-2.5, another transcription factor in cardiomyocytes [14]. Their reporter assay with Hela cells suggested that activation by GATA4 may be much weaker than that by Nkx-2.5, further study should be necessary because the protein level of GATA4 was not verified in their report
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