Paraoxonase 2 is an ER chaperone that regulates the epithelial Na+ channel

The mammalian paraoxonases have been linked to protection against oxidative stress. However, the physiological roles of members in this family (PON1, PON2 and PON3) are still being characterized. PON2 and PON3 are expressed in the aldosterone-sensitive distal nephron of the kidney and have been shown to negatively regulate expression of the epithelial sodium channel (ENaC), a trimeric ion channel that orchestrates salt and water homeostasis. To date, the nature of this phenomenon has not been explored. Therefore, to investigate the mechanism by which PON2 regulates ENaC, we expressed PON2 along with the ENaC subunits in Fisher Rat Thyroid (FRT) cells, a system that is amenable to biochemical analyses of ENaC assembly and trafficking. We found that PON2 primarily resides in the endoplasmic reticulum (ER) in FRT cells, and its expression reduces the abundance of each ENaC subunit, reflecting enhanced subunit turnover. In contrast, no effect on the levels of mRNAs encoding the ENaC subunits was evident. Inhibition of lysosome function with chloroquine or NH4Cl did not alter the inhibitory effect of PON2 on ENaC expression. In contrast, PON2 accelerates ENaC degradation in a proteasome-dependent manner and acts prior to ENaC subunits ubiquitination. As a result of the enhanced ENaC subunits ubiquitination and degradation, both channel surface expression and ENaC-mediated Na+ transport in FRT cells were reduced by PON2. Together, our data suggest that PON2 functions as an ER chaperone to monitor ENaC biogenesis and redirect the channel for ER associated degradation.

Age-dependent effects on radiation-induced carcinogenesis in the rat thyroid

Childhood radiation exposure is a known thyroid cancer risk factor. This study evaluated the effects of age on radiation-induced thyroid carcinogenesis in rats irradiated with 8 Gy X-rays. We analyzed cell proliferation, cell death, DNA damage response, and autophagy-related markers in 4-week-old (4W) and 7-month-old (7M) rats and the incidence of thyroid tumors in 4W, 4-month-old (4M), and 7M rats 18 months after irradiation. Cell death and DNA damage response were increased in 4W rats compared to those in controls at 1 month post-irradiation. More Ki-67-positive cells were observed in 4W rats at 12 months post-irradiation. Thyroid tumors were confirmed in 61.9% (13/21), 63.6% (7/11), and 33.3% (2/6) of irradiated 4W, 4M, and 7M rats, respectively, compared to 0%, 14.3% (1/7), and 16.7% (1/6) in the respective nonirradiated controls. There were 29, 9, and 2 tumors in irradiated 4W, 4M, and 7M rats, respectively. The expression of several autophagy components was downregulated in the area surrounding radiation-induced thyroid carcinomas in 4W and 7M rats. LC3 and p62 expression levels decreased in radiation-induced follicular carcinoma in 4W rats. Radiosensitive cells causing thyroid tumors may be more prevalent in young rats, and abrogation of autophagy may be associated with radiation-induced thyroid carcinogenesis.

Trace Amine-Associated Receptor 1 Trafficking to Cilia of Thyroid Epithelial Cells

Trace amine-associated receptor 1 (rodent Taar1/human TAAR1) is a G protein-coupled receptor that is mainly recognized for its functions in neuromodulation. Previous in vitro studies suggested that Taar1 may signal from intracellular compartments. However, we have shown Taar1 to localize apically and on ciliary extensions in rodent thyrocytes, suggesting that at least in the thyroid, Taar1 may signal from the cilia at the apical plasma membrane domain of thyrocytes in situ, where it is exposed to the content of the follicle lumen containing putative Taar1 ligands. This study was designed to explore mouse Taar1 (mTaar1) trafficking, heterologously expressed in human and rat thyroid cell lines in order to establish an in vitro system in which Taar1 signaling from the cell surface can be studied in future. The results showed that chimeric mTaar1-EGFP traffics to the apical cell surface and localizes particularly to spherical structures of polarized thyroid cells, procilia, and primary cilia upon serum-starvation. Moreover, mTaar1-EGFP appears to form high molecular mass forms, possibly homodimers and tetramers, in stably expressing human thyroid cell lines. However, only monomeric mTaar1-EGFP was cell surface biotinylated in polarized human thyrocytes. In polarized rat thyrocytes, mTaar1-EGFP is retained in the endoplasmic reticulum, while cilia were reached by mTaar1-EGFP transiently co-expressed in combination with an HA-tagged construct of the related mTaar5. We conclude that Taar1 trafficking to cilia depends on their integrity. The results further suggest that an in vitro cell model was established that recapitulates Taar1 trafficking in thyrocytes in situ, in principle, and will enable studying Taar1 signaling in future, thus extending our general understanding of its potential significance for thyroid autoregulation.

Alteration of Membrane Cholesterol Content Plays a Key Role in Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Channel Activity

Altered cholesterol homeostasis in cystic fibrosis patients has been reported, although controversy remains. As a major membrane lipid component, cholesterol modulates the function of multiple ion channels by complicated mechanisms. However, whether cholesterol directly modulates cystic fibrosis transmembrane conductance regulator (CFTR) channel function remains unknown. To answer this question, we determined the effects of changing plasma membrane cholesterol levels on CFTR channel function utilizing polarized fischer rat thyroid (FRT) cells and primary human bronchial epithelial (HBE) cells. Treatment with methyl-β-cyclodextrin (MβCD) significantly reduced total cholesterol content in FRT cells, which significantly decreased forskolin (FSK)-mediated activation of both wildtype (WT-) and P67L-CFTR. This effect was also seen in HBE cells expressing WT-CFTR. Cholesterol modification by cholesterol oxidase and cholesterol esterase also distinctly affected activation of CFTR by FSK. In addition, alteration of cholesterol increased the potency of VX-770, a clinically used potentiator of CFTR, when both WT- and P67L-CFTR channels were activated at low FSK concentrations; this likely reflects the apparent shift in the sensitivity of WT-CFTR to FSK after alteration of membrane cholesterol. These results demonstrate that changes in the plasma membrane cholesterol level significantly modulate CFTR channel function and consequently may affect sensitivity to clinical therapeutics in CF patients.

Cryopreserved Rat Thyroid Autotransplantation in the Treatment of Postoperative Hypothyroidism

To verify the viability and functionality of cryopreserved thyroid autotransplantation in rats who underwent total thyroidectomy in the treatment of postoperative hypothyroidism. Thirty-two Wistar rats were randomly assigned into groups (G) with eight animals each: control (CG); simulation (SG); hypothyroidism (HTG) and transplanted (TG). At the beginning and in the 13th week of the experiment, serum levels of total T3, free T4, TSH and calcium were determined. In both the first and 14th weeks, scintigraphic examinations, 99m-Tc pertechnetate radioisotope biodistribution and histopathology were performed. In the 14th week, the expression of proliferating cell nuclear antigen (PCNA) and cellular apoptosis (caspase-3) were also evaluated. In the 13th week, the transplanted animals had normal serum levels of total T3 and free T4. TSH levels showed a tendency towards normality. In the 14th week, scintigraphic exams displayed graft isotopic uptake in all animals in the TG group. Histological examinations 13 weeks after transplantation showed the viability and functionality of thyroid follicles. PCNA revealed significant immunoreactivity of the graft (p < 0.001) when the TG was compared to the CG. There was no difference between CG and TG considering the expression of activated caspase-3. The experimental study confirmed the viability and functionality of thyroid autotransplantation implanted in skeletal muscle with evidence of cell proliferation without cellular apoptosis. This surgical strategy was effective in the treatment of postoperative hypothyroidism.

A Novel SLC5A5 Variant Reveals the Crucial Role of Kinesin Light Chain 2 in Thyroid Hormonogenesis

STRUCTURED ABSTRACT Context Iodide transport defect (ITD) (Online Mendelian Inheritance in Man #274400) is an uncommon cause of dyshormonogenic congenital hypothyroidism due to loss-of-function variants in the SLC5A5 gene, which encodes the sodium/iodide symporter (NIS), causing deficient iodide accumulation in thyroid follicular cells. Objective To determine the molecular basis of a patient´s ITD clinical phenotype. Patient The propositus was diagnosed with dyshormonogenic congenital hypothyroidism with minimal 99mTc-pertechnetate accumulation in a eutopic thyroid gland. Design Propositus SLC5A5 gene was sequenced. Functional in vitro characterization of the novel NIS variant was performed. Results Sanger sequencing revealed a novel homozygous missense p.G561E NIS variant. Mechanistically, the G561E substitution reduces iodide uptake, because targeting of G561E NIS to the plasma membrane is reduced. Biochemical analyses revealed that G561E impairs the recognition of an adjacent tryptophan-acidic motif by the kinesin-1 subunit kinesin light chain 2 (KLC2), interfering with NIS maturation beyond the endoplasmic reticulum, and reducing iodide accumulation. Structural bioinformatic analysis suggests that G561E shifts the equilibrium of the unstructured tryptophan-acidic motif towards a more structured conformation unrecognizable to KLC2. Consistently, knockdown of Klc2 causes defective NIS maturation and consequently decreases iodide accumulation in rat thyroid cells. Morpholino knockdown of klc2 reduces thyroid hormone synthesis in zebrafish larvae leading to a hypothyroid state as revealed by expression profiling of key genes related to the hypothalamic-pituitary-thyroid axis. Conclusions We report a novel NIS pathogenic variant associated with dyshormonogenic congenital hypothyroidism. Detailed molecular characterization of G561E NIS uncovered the significance of KLC2 in thyroid physiology.