L-thyroxine directly affects expression of thyroid hormone-sensitive genes: regulatory effect of RXR β

Abstract

l-thyroxine (T4) has been considered mainly a prohormone, the hormonal action of which is related to its conversion to 3,5,3′-triiodothyronine (T3) in peripheral tissues. In this study we investigated in transient transfection assays whether T4 might directly affect the expression of thyroid hormone (TH) sensitive genes. The reporter construct ME-TRE-TK-CAT or TSH-TRE-TK-CAT containing the nucleotide sequence of the TH response element (TRE) of either malic enzyme (ME) or TSH β genes, was transfected with either TH receptor (TR) α alone or in combination with retinoid X receptor (RXR) β into NIH3T3 cells. Addition of 100 nM T4 to the culture medium in the presence of TR α increased the basal level of ME-TRE-TK-CAT expression by 4.5-fold. T4 action was due to a direct interaction with TR α and not to its conversion to T3, since T4 effect persisted in the presence of 5′-deiodinase inhibitors (propylthiouracil, iopanoic acid) effectively preventing T3 generation, as assessed by the absence of T3 by HPLC in the cellular extracts of transfected cells. In a dose-response study half-maximal stimulation by T4 was achieved at a concentration of 100 nM, whereas 50% of maximal induction was produced by 1 nM T3 and 6 nM triiodothyroacetic acid (TRIAC). Coexpression of RXR β greatly enhanced the transcriptional activity of the ME-TRE-TK-CAT gene when either T3, T4 or TRIAC was added to the culture medium of NIH3T3 cells, but established a hormonal hierarchy in the reporter activation different than that observed in the presence of TR α alone (TRIAC>T3≥T4, instead of T3>TRIAC>T4). T4 at a concentration of 100 nM could activate the TH/TR-dependent down-regulation mediated by the negative TSH-TRE, although at a lower level than that obtained with similar concentrations of T3 (35 and 55% inhibition, respectively). Our results demonstrate that, in addition to the action mediated through its monodeiodination to T3, T4 exerts a direct effect on genes that are either positively or negatively regulated by TH. Moreover, RXR β, forming heterodimers with TRs, appeared to exert a central role in modulating the sensitivity of TH-responsive genes to different iodothyronines.