SAT-565 The Surface Of Liganded T3 Receptor β2 For Steroid Receptor Coactivator 1 And 2 Mediates The Negative Regulation Of The Prepro-thyrotropin-releasing Hormone Gene By T3

T3 negatively regulates thyrotropin-releasing hormone (TRH) secreted from hypothalamus paraventricular nucleus (PVN). As T3 receptor (TR) _2 is known to mediate the negative regulation of prepro-TRH gene, we previously investigated the T3-dependent negative regulation of the prepro-TRH gene. We reported that T3-bound TR_2 inhibits the transcription of this gene induced by transcription factor GATA2 via tethering mechanism but not negative T3-responsive element (site4), as in the case of thyrotropin (TSH) _ gene (Vitam Horm. 106:97-127, 2018). We also confirmed that GATA2 is expressed in the TRH neuron in PVN. Interestingly, it was reported that T3-dependent repression of TSH_ gene is blunted in the mice whose steroid receptor coactivator (SRC)-1 and/or 2 were genetically ablated (Endocrinology 143(4) 1554-1557, 2002), suggesting the involvement of SRCs in the T3-dependent negative regulation. Indeed, T3-dependent inhibition of TSH_ secretion is impaired in the knock-in mice, of which TR_1 was substituted with mutant TR_1 E457A, a mutant lacking for the interaction with SRC-1 without defect with T3-binding (J. Clin. Invest. 115(9)2517-2523, 2005). Because similar TR_1 mutants have been reported (Science. 12;280(5370):1747-9, 1998), we attempted here to study their function in the context of the negative regulation of the prepro-TRH promoter in monkey kidney-derived CV-1 cells. Compared with wild-type TR_2, T3-dependent inhibition of the CAT-reporter gene, which was fused with prepro-TRH promoter (nt. -547 to nt. +84bp ), was relieved in all the mutant TR_2s including V337R, K341A, I355R, L507R, L509R, E510K and E510A (corresponding to E457A in TR_1). Unexpectedly, however, over-expression of SRC-1 and SRC-2 did not affect the T3-dependent inhibition mediated by wild-type TR_2. These results indicate that negative regulation of preproTRH gene by T3 may be mediated by unknown factor that is able to interact with the surface of T3-bound TR_2 as in the case of SRC-1 or 2. Alternatively, effect by over-expressed SRC-1 and 2 may be quickly metabolized by ubiquitin-proteasome system or exported to cytosol as previously reported. We are currently investigating the effect of SI-2, bufalin and gossypol, which were reported to degradate the SRC proteins potently (PNAS 113(18) 4970-4975, 2016 and references therein).