Abstract: SUN 212

The Role of GATA2 in the Negative Regulation of the Prepro- Thyrotropin-Releasing Hormone Gene By Liganded T3 Receptor β 2

Presenter: Go Kuroda


Abstract


T3 inhibits thyrotropin-releasing hormone (TRH) secreted from hypothalamus paraventricular nucleus (PVN). Although T3 receptor (TR) β2 is known to mediate the negative regulation of prepro-TRH gene, its mechanism is still unknown. view more

T3 inhibits thyrotropin-releasing hormone (TRH) secreted from hypothalamus paraventricular nucleus (PVN). Although T3 receptor (TR) β2 is known to mediate the negative regulation of prepro-TRH gene, its mechanism is still unknown. We previously studied the T3-dependent negative regulation of the thyrotropin β subunit (TSHβ) gene, and reported that (1) inhibition of this gene is not the mirror image of the positive regulation by T3 because unliganded TR is not transcriptional activator, (2) it does not require the putative negative T3-responsive element (nTRE), which is previously reported immediately downstream to transcription start site, and (3) it is mediated via the tethering of TRbeta 2 by a transcription factor, GATA2, a critical activator for the TSHβ gene in thyrotroph. Interestingly, it was reported that the transcription factor, Sim1, a determinant of PVN differentiation in hypothalamus, induces the expressions of TRβ2 and GATA2 in cultured neuron cells. This led us to analyze the function of GATA2 and TRβ2 in the transcriptional regulation of prepro-TRH promoter encompassing between nt. -547 and nt. +84, which is known to be sufficient for its expression in PVN. Using CAT reporter gene with CV1 cells, which has been studied both positive and negative regulations by T3, we found that the promoter is activated by GATA2 approximately 6 fold but not by mutant GATA2, of which DNA-binding affinity was impaired. The deletion and mutation analyses identified a functional GATA-responsive element between nt. -357 and nt. -352bp. Although it has been reported that signaling from melanocortin 4 receptor may stimulate TRH expression via protein kinase A (PKA) pathway, stimulation by a PKA activator, forskolin, was modest (2 fold). When TRβ2 was co-expressed, T3 (10 nM) reduced the GATA2-dependent promoter activity to approximately 40%. Similar results were observed in C-cell derived CA-77 cells, which express endogenous TRH. Unliganded TRβ2 alone failed to stimulate this promoter, suggesting again that the negative regulation of this gene is not the mirror image of the positive regulation. Although it has been proposed that the inhibition by T3-bound TRβ2 might be mediated by putative negative TRE (site4), it was maintained after the mutation of this DNA sequence. These results suggest that, as in the case of the TSHβ gene, the preproTRH gene is transactivated by GATA2 and that T3 may negatively regulate this gene by TRβ2 tethered with GATA2 but not by reported nTRE.

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