Juan Pablo Nicola

The Na+/I− symporter mediates active iodide uptake in the intestine

Absorption of dietary iodide, presumably in the small intestine, is the first step in iodide (I(-)) utilization. From the bloodstream, I(-) is actively taken up via the Na(+)/I(-) symporter (NIS) in the thyroid for thyroid hormone biosynthesis and in such other tissues as lactating breast, which supplies I(-) to the newborn in the milk. The molecular basis for intestinal I(-) absorption is unknown. We sought to determine whether I(-) is actively accumulated by enterocytes and, if so, whether this process is mediated by NIS and regulated by I(-) itself. NIS expression was localized exclusively at the apical surface of rat and mouse enterocytes. In vivo intestine-to-blood transport of pertechnetate, a NIS substrate, was sensitive to the NIS inhibitor perchlorate. Brush border membrane vesicles accumulated I(-) in a sodium-dependent, perchlorate-sensitive manner with kinetic parameters similar to those of thyroid cells. NIS was expressed in intestinal epithelial cell line 6, and I(-) uptake in these cells was also kinetically similar to that in thyrocytes. I(-) downregulated NIS protein expression and its own NIS-mediated transport both in vitro and in vivo. We conclude that NIS is functionally expressed on the apical surface of enterocytes, where it mediates active I(-) accumulation. Therefore, NIS is a significant and possibly central component of the I(-) absorption system in the small intestine, a system of key importance for thyroid hormone biosynthesis and thus systemic intermediary metabolism.

The Acute Inhibitory Effect of Iodide Excess on Sodium/Iodide Symporter Expression and Activity Involves the PI3K/Akt Signaling Pathway

Iodide (I(-)) is an irreplaceable constituent of thyroid hormones and an important regulator of thyroid function, because high concentrations of I(-) down-regulate sodium/iodide symporter (NIS) expression and function. In thyrocytes, activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) cascade also inhibits NIS expression and function. Because I(-) excess and PI3K/Akt signaling pathway induce similar inhibitory effects on NIS expression, we aimed to study whether the PI3K/Akt cascade mediates the acute and rapid inhibitory effect of I(-) excess on NIS expression/activity. Here, we reported that the treatment of PCCl3 cells with I(-) excess increased Akt phosphorylation under normal or TSH/insulin-starving conditions. I(-) stimulated Akt phosphorylation in a PI3K-dependent manner, because the use of PI3K inhibitors (wortmannin or 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) abrogated the induction of I(-) effect. Moreover, I(-) inhibitory effect on NIS expression and function were abolished when the cells were previously treated with specific inhibitors of PI3K or Akt (Akt1/2 kinase inhibitor). Importantly, we also found that the effect of I(-) on NIS expression involved the generation of reactive oxygen species (ROS). Using the fluorogenic probes dihydroethidium and mitochondrial superoxide indicator (MitoSOX Red), we observed that I(-) excess increased ROS production in thyrocytes and determined that mitochondria were the source of anion superoxide. Furthermore, the ROS scavengers N-acetyl cysteine and 2-phenyl-1,2-benzisoselenazol-3-(2H)-one blocked the effect of I(-) on Akt phosphorylation. Overall, our data demonstrated the involvement of the PI3K/Akt signaling pathway as a novel mediator of the I(-)-induced thyroid autoregulation, linking the role of thyroid oxidative state to the Wolff-Chaikoff effect.

Functional Toll-Like Receptor 4 Conferring Lipopolysaccharide Responsiveness Is Expressed in Thyroid Cells

Lipopolysaccharide (LPS), a glycolipid found in the cell wall of Gram-negative bacteria, exerts pleiotropic biological effects in different cell types. LPS is mainly recognized by the Toll-like receptor (TLR) 4/MD2/Cluster of differentiation 14 complex (CD14). We previously demonstrated that LPS produced a direct action on thyroid cells, including up-regulation of thyroglobulin gene expression. This work aimed to study further the effect of LPS on thyroid function and to elucidate the mechanism by which LPS is recognized by the thyroid cell. We could detect the transcript and protein expression of TLR4, MD2, and CD14 in thyroid cells, and that these proteins are localized at the plasma membrane. The sodium iodide symporter (NIS) is the transporter involved in the iodide uptake, the first step in thyroid hormonogenesis. We demonstrated that LPS increases the TSH-induced iodide uptake and NIS protein expression. The LPS agonist lipid A reproduced LPS effect, whereas the LPS antagonist, polymyxin B, abrogated it. By the use of anti-TLR4 blocking antibodies and the transient expression of TLR4 dominant-negative forms, we evidenced the involvement of TLR4 in the LPS action. The enrichment of TLR4 expressing Fisher rat thyroid cell line-5 (FRTL-5) cells confirmed that TLR4 confers LPS responsiveness to thyroid cells. In conclusion, we revealed for the first time that all the components of the LPS receptor complex are expressed in thyroid cells. Evidence that the effects of LPS on rodent thyroid function involve TLR4-induced signaling was obtained. The fact that thyroid cells are able to recognize and respond to LPS supports a role of the endotoxin as a potential modifier of thyroid function.

Nuclear Factor (NF)-κB-dependent Thyroid Hormone Receptor β1 Expression Controls Dendritic Cell Function via Akt Signaling

Despite considerable progress in our understanding of the interplay between immune and endocrine systems, the role of thyroid hormones and their receptors in the control of adaptive immunity is still uncertain. Here, we investigated the role of thyroid hormone receptor (TR) β1 signaling in modulating dendritic cell (DC) physiology and the intracellular mechanisms underlying these immunoregulatory effects. Exposure of DCs to triiodothyronine (T3) resulted in a rapid and sustained increase in Akt phosphorylation independently of phosphatidylinositol 3-kinase activation, which was essential for supporting T3-induced DC maturation and interleukin (IL)-12 production. This effect was dependent on intact TRβ1 signaling as small interfering RNA-mediated silencing of TRβ1 expression prevented T3-induced DC maturation and IL-12 secretion as well as Akt activation and IκB-ϵ degradation. In turn, T3 up-regulated TRβ1 expression through mechanisms involving NF-κB, suggesting an autocrine regulatory loop to control hormone-dependent TRβ1 signaling. These findings were confirmed by chromatin immunoprecipitation analysis, which disclosed a new functional NF-κB consensus site in the promoter region of the TRB1 gene. Thus, a T3-induced NF-κB-dependent mechanism controls TRβ1 expression, which in turn signals DCs to promote maturation and function via an Akt-dependent but PI3K-independent pathway. These results underscore a novel unrecognized target that regulates DC maturation and function with critical implications in immunopathology at the cross-roads of the immune-endocrine circuits.

The KCNQ1-KCNE2 K+ channel is required for adequate thyroid I− uptake

The KCNQ1 α subunit and the KCNE2 β subunit form a potassium channel in thyroid epithelial cells. Genetic disruption of KCNQ1‐KCNE2 causes hypothyroidism in mice, resulting in cardiac hypertrophy, dwarfism, alopecia, and prenatal mortality. Here, we investigated the mechanistic requirement for KCNQ1‐KCNE2 in thyroid hormone biosynthesis, utilizing whole‐animal dynamic positron emission tomography. The KCNQ1‐specific antagonist (—)‐[3R,4S]‐chromanol 293B (C293B) significantly impaired thyroid cell I− uptake, which is mediated by the Na+/I− symporter (NIS), in vivo (dSUV/dt: vehicle, 0.028±0.004 min−1; 10 mg/kg C293B, 0.009±0.006 min‐1) and in vitro (EC50: 99±10 μM C293B). Na+‐dependent nicotinate uptake by SMCT, however, was unaffected. Kcne2 deletion did not alter the balance of free vs. thyroglobulin‐bound I− in the thyroid (distinguished using ClO4−, a competitive inhibitor of NIS), indicating that KCNQ1‐KCNE2 is not required for Duox/TPO‐mediated I− organification. However, Kcne2 deletion doubled the rate of free I− efflux from the thyroid following ClO4− injection, a NIS‐independent process. Thus, KCNQ1‐KCNE2 is necessary for adequate thyroid cell I− uptake, the most likely explanation being that it is prerequisite for adequate NIS activity.—Purtell, K., Paroder‐Belenitsky, M., Reyna‐Neyra, A., Nicola, J. P., Koba, W., Fine, E., Carrasco, N., Abbott, G. W. The KCNQ1‐KCNE2 K+ channel is required for adequate thyroid I− uptake. FASEB J. 26, 3252–3259 (2012). www.fasebj.org

Mechanism of anion selectivity and stoichiometry of the Na+/I- symporter (NIS)

I- uptake in the thyroid, the first step in thyroid hormone biosynthesis, is mediated by the Na+/I- symporter (NIS) with an electrogenic 2Na+ : 1I- stoichiometry. We have obtained mechanistic information on NIS by characterizing the congenital I- transport defect-causing NIS mutant G93R. This mutant is targeted to the plasma membrane but is inactive. Substitutions at position 93 show that the longer the side chain of the neutral residue at this position, the higher the Km for the anion substrates. Unlike WT NIS, which mediates symport of Na+ and the environmental pollutant perchlorate electroneutrally, G93T/N/Q/E/D NIS, strikingly, do it electrogenically with a 2∶1 stoichiometry. Furthermore, G93E/Q NIS discriminate between anion substrates, a discovery with potential clinical relevance. A 3D homology model of NIS based on the structure of the bacterial Na+/galactose transporter identifies G93 as a critical player in the mechanism of the transporter: the changes from an outwardly to an inwardly open conformation during the transport cycle use G93 as a pivot.

NF-κB p65 Subunit Mediates Lipopolysaccharide-Induced Na+/I- Symporter Gene Expression by Involving Functional Interaction with the Paired Domain Transcription Factor Pax8

The Gram-negative bacterial endotoxin lipopolysaccharide (LPS) elicits a variety of biological responses. Na(+)/I(-) symporter (NIS)-mediated iodide uptake is the main rate-limiting step in thyroid hormonogenesis. We have recently reported that LPS stimulates TSH-induced iodide uptake. Here, we further analyzed the molecular mechanism involved in the LPS-induced NIS expression in Fisher rat thyroid cell line 5 (FRTL-5) thyroid cells. We observed an increase in TSH-induced NIS mRNA expression in a dose-dependent manner upon LPS treatment. LPS enhanced the TSH-stimulated NIS promoter activity denoting the NIS-upstream enhancer region (NUE) as responsible for the stimulatory effects. We characterized a novel putative conserved kappaB site for the transcription factor nuclear factor-kappaB (NF-kappaB) within the NUE region. NUE contains two binding sites for the transcription factor paired box 8 (Pax8), main regulator of NIS transcription. A physical interaction was observed between the NF-kappaB p65 subunit and paired box 8 (Pax8), which appears to be responsible for the synergic effect displayed by these transcription factors on NIS gene transcription. Moreover, functional blockage of NF-kappaB signaling and site-directed mutagenesis of the kappaB cis-acting element abrogated LPS stimulation. Silencing expression of p65 confirmed its participation as an effector of LPS-induced NIS stimulation. Furthermore, chromatin immunoprecipitation corroborated that NIS is a novel target gene for p65 transactivation in response to LPS. Moreover, we were able to corroborate the LPS-stimulatory effect on thyroid cells in vivo in LPS-treated rats, supporting that thyrocytes are capable of responding to systemic infections. In conclusion, our results reveal a new mechanism involving p65 in the LPS-induced NIS expression, denoting a novel aspect in thyroid cell differentiation.

Dietary iodide controls its own absorption through post-transcriptional regulation of the intestinal Na+/I− symporter

•  Expression of the Na+/I− symporter (NIS) at the apical surface of the epithelium of the small intestine is key to I− absorption, the first step in I− metabolism. •  Intracellular I− at high concentrations in enterocytes decreases its own NIS‐mediated uptake by a newly discovered mechanism, downregulating NIS expression at the plasma membrane, increasing NIS protein degradation and decreasing NIS mRNA levels by reducing NIS mRNA stability, involving the NIS 3′‐untranslated region. •  In conclusion, we have uncovered that I− regulates intestinal NIS expression, and thus its own intestinal absorption, by a complex array of post‐transcriptional mechanisms.

The iodide-transport-defect-causing mutation R124H: a δ-amino group at position 124 is critical for maturation and trafficking of the Na+/I- symporter.

Summary Na+/I− symporter (NIS)-mediated active accumulation of I− in thyrocytes is a key step in the biosynthesis of the iodine-containing thyroid hormones T3 and T4. Several NIS mutants have been identified as a cause of congenital I− transport defect (ITD), and their investigation has yielded valuable mechanistic information on NIS. Here we report novel findings derived from the thorough characterization of the ITD-causing mutation R124H, located in the second intracellular loop (IL-2). R124H NIS is incompletely glycosylated and colocalizes with endoplasmic reticulum (ER)-resident protein markers. As a result, R124H NIS is not targeted to the plasma membrane and therefore does not mediate any I− transport in transfected COS-7 cells. Strikingly, however, the mutant is intrinsically active, as revealed by its ability to mediate I− transport in membrane vesicles. Of all the amino acid substitutions we carried out at position 124 (K, D, E, A, W, N and Q), only Gln restored targeting of NIS to the plasma membrane and NIS activity, suggesting a key structural role for the &dgr;-amino group of R124 in the transporters maturation and cell surface targeting. Using our NIS homology model based on the structure of the Vibrio parahaemolyticus Na+/galactose symporter, we propose an interaction between the &dgr;-amino group of either R or Q124 and the thiol group of C440, located in IL-6. We conclude that the interaction between IL-2 and IL-6 is critical for the local folding required for NIS maturation and plasma membrane trafficking.

Asn441 plays a key role in folding and function of the Na+/I− symporter (NIS)

The Na+/I– symporter (NIS) is a plasma membrane glycoprotein that mediates active I– transport in the thyroid, the first step in the biosynthesis of the iodine‐containing thyroid hormones T3 and T4. Several NIS mutants have been identified as a cause of congenital I– transport defect (ITD), and their investigation has yielded valuable mechanistic information on NIS. Here we report a thorough characterization of the ITD‐causing NIS mutation in which the sixth intracellular loop residues 439‐443 are missing. This mutant protein was intracellularly retained, incompletely glycosylated, and intrinsically inactive. Engineering 5 Ala at positions 439‐443 partially recovered cell surface targeting and activity (~15%). Strikingly, NIS with the sequence 439‐AANAA‐443, in which Asn was restored at position 441, was targeted to the plasma membrane and exhibited ~95% the transport activity of WT NIS. Based on our NIS homology model, we propose that the side chain of N441, a residue conserved throughout most of the SLC5 family, interacts with the main chain amino group of G444, capping the α‐helix of transmembrane segment XII and thus stabilizing the structure of the molecule. Our data provide insight into a critical interhelical interaction required for NIS folding and activity.—Li, W., Nicola, J. P., Amzel, L. M., Carrasco, N., Asn441 plays a key role in folding and function of the Na+/I– symporter (NIS). FASEB J. 27, 3229–3238 (2013). www.fasebj.org