3,5-T2 Is an Alternative Ligand for the Thyroid Hormone Receptor β1

Abstract

Several liganded nuclear receptors have alternative ligands acting in a tissue-specific fashion and playing important biological roles. We present evidence that 3,5-diiodothyronine (T(2)), a naturally occurring iodothyronine that results from T(3) outer-ring deiodination, is an alternative ligand for thyroid hormone receptor β1 (TRβ1). In tilapia, 2 TRβ isoforms differing by 9 amino acids in the ligand-binding domain were cloned. Binding and transactivation studies showed that T(2) activates the human and the long tilapia TRβ1 isoform, but not the short one. A chimeric human TRβ1 (hTRβ1) that contained the 9-amino-acid insert showed no response to T(2), suggesting that the conformation of the hTRβ1 naturally allows T(2) binding and that other regions of the receptor are implicated in TR activation by T(2). Indeed, further analysis showed that the N terminus is essential for T(2)-mediated transactivation but not for that by T(3) in the long and hTRβ1, suggesting a functional interaction between the N-terminal domain and the insertion in the ligand-binding domain. To establish the functional relevance of T(2)-mediated TRβ1 binding and activation, mRNA expression and its regulation by T(2) and T(3) was evaluated for both isoforms. Our data show that long TRβ1expression is 10(6)-fold higher than that of the short isoform, and T(3) and T(2) differentially regulate the expression of these 2 TRβ1 isoforms in vivo. Taken together, our results prompted a reevaluation of the role and mechanism of action of thyroid hormone metabolites previously believed to be inactive. More generally, we propose that classical liganded receptors are only partially locked to very specific ligands and that alternative ligands may play a role in the tissue-specific action of receptors.

3,5-di-iodothyronine stimulates tilapia growth through an alternate isoform of thyroid hormone receptor β1

“…Furthermore, hepatic in vivo expression of L-TRb1 is 10 6 -fold higher than that of S-TRb1, suggesting that, at least in teleosts, the effects of THs are mainly mediated through this isoform. These observations, together with our findings that in vivo, T 2 and T 3 modulate the expression of L-TRb1 and S-TRb1 respectively, suggest a distinct signaling pathway for each TH in teleosts and prompted us to propose an extra level in the cascade of TH signaling for which T 2 is specifically made and regulated (Mendoza et al 2013).…”

“…Accordingly, studies from several laboratories have suggested that 3,5-di-iodothyronine (3,5-T 2 or T 2 ), a putative product of the outer ring deiodination pathway involved in T 3 metabolism, also possesses bioactivity (Goglia 2005). In fact, studies from our laboratory have shown that both T 3 and T 2 act directly on TH-dependent genes in teleost liver (García-G et al 2007) and that these genomic actions are mediated by different isoforms of TRb1 (Mendoza et al 2013). Indeed, results from binding and transactivating assays revealed that the effects of T 2 on genomic regulation appear to be mediated by a different isoform of TRb1 that is found only in fish and contains a 9-amino acid insert in its ligand-binding domain, and which we have denominated long TRb1 (L-TRb1).…”

“…In all cases, mRNA was quantified by real-time PCR as described previously (Mendoza et al 2013). Briefly, total RNA was extracted from juvenile tilapia liver (TRIzol, Invitrogen), and RT (M-MLV, Promega) from 2 mg of total hepatic RNA was performed using oligo(dT) primer (final volume of 25 ml).…”

“…Indeed, results from binding and transactivating assays revealed that the effects of T 2 on genomic regulation appear to be mediated by a different isoform of TRb1 that is found only in fish and contains a 9-amino acid insert in its ligand-binding domain, and which we have denominated long TRb1 (L-TRb1). By contrast, T 3 preferentially binds to and activates the short TRb1 (S-TRb1; Mendoza et al 2013). Furthermore, hepatic in vivo expression of L-TRb1 is 10 6 -fold higher than that of S-TRb1, suggesting that, at least in teleosts, the effects of THs are mainly mediated through this isoform.…”

Translating Pharmacological Findings from Hypothyroid Rodents to Euthyroid Humans: Is There a Functional Role of Endogenous 3,5-T2?

“…In particular, this link is in accordance with animal studies showing that 3,5-T2 was only a third as potent as T3 in suppressing TSH concentrations, and rather high pharmacological 3,5-T2 concentrations are required for TSH suppression (34)(35)(36). However, several animal studies in rodents and other species, for example killifish, indicated that 3,5-T2 interferes with the hypothalamus-pituitary-thyroid axis at several levels such as pituitary, thyroid, and peripheral action, thereby displaying a broad spectrum of mechanisms involved such as classical interaction with TR and also rapid effects at the cell membrane and mitochondria (10,12,14,(34)(35)(36)(37)(38)(39)(40) (for a review, see (3)). Action of 3,5-T2 via these different targets and molecular mechanisms might occur at different 3,5-T2 concentrations and depend on acute or chronic exposure (35,37,40), thus possibly explaining the unexpected bell-shaped relationship with serum TSH.…”