TR Alpha 2 Exerts Dominant Negative Effects on Hypothalamic Trh Transcription In Vivo

Mammalian thyroid hormone receptors (TRs) have multiple isoforms, including the bona fide receptors that bind T3 (TRa1, TRb1 and TRb2) and a non-hormone-binding variant, TRa2. Intriguingly, TRa2 is strongly expressed in the brain, where its mRNA levels exceed those of functional TRs. Ablation of TRa2 in mice results in over-expression of TRa1, and a complex phenotype with low levels of free T3 and T4, without elevated TSH levels, suggesting an alteration in the negative feedback at the hypothalamic-pituitary level. As the hypothesis of a potential TRH response defect has never been tested, we explored the functional role of TRa2 in negative feedback on transcription of hypothalamic thyrotropin, Trh. The in vivo transcriptional effects of TRa2 on hypothalamic Trh were analysed using an in vivo reporter gene approach. Effects on Trh-luc expression were examined to that of two, T3 positively regulated genes used as controls. Applying in vivo gene transfer showed that TRa2 over-expression in the mouse hypothalamus abrogates T3-dependent repression of Trh and T3 activation of positively regulated promoters, blocking their physiological regulation. Surprisingly, loss of function studies carried out by introducing a shTRa2 construct in the hypothalamus also blocked physiological T3 dependent regulation. Thus, modulating hypothalamic TRa2 expression by either gain or loss of function abrogated T3 dependent regulation of Trh transcription, producing constant transcriptional levels insensitive to feedback. This loss of physiological regulation was reflected at the level of the endogenous Trh gene, were gain or loss of function held mRNA levels constant. These result