In Vivo Characterization of the Toxicological Properties of DPhP, One of the Main Degradation Products of Aryl Phosphate Esters.

BACKGROUND: Aryl phosphate esters (APEs) are widely used and commonly present in the environment. Health hazards associated with these compounds remain largely unknown and the effects of diphenyl phosphate (DPhP), one of their most frequent derivatives, are poorly characterized. OBJECTIVE: Our aim was to investigate whether DPhP per se may represent a more relevant marker of exposure to APEs than direct assessment of their concentration and determine its potential deleterious biological effects in chronically exposed mice. METHODS: Conventional animals (FVB mice) were acutely or chronically exposed to relevant doses of DPhP or to triphenyl phosphate (TPhP), one of its main precursors. Both molecules were measured in blood and other tissues by liquid chromatography-mass spectrometry (LC-MS). Effects of chronic DPhP exposure were addressed through liver multi-omics analysis to determine the corresponding metabolic profile. Deep statistical exploration was performed to extract correlated information, guiding further physiological analyses. RESULTS: Multi-omics analysis confirmed the existence of biological effects of DPhP, even at a very low dose of 0.1mg/mL in drinking water. Chemical structural homology and pathway mapping demonstrated a clear reduction of the fatty acid catabolic processes centered on acylcarnitine and mitochondrial ß-oxidation in mice exposed to DPhP in comparison with those treated with vehicle. An interesting finding was that in mice exposed to DPhP, mRNA, expression of genes involved in lipid catabolic processes and regulated by peroxisome proliferator-activated receptor alpha (PPARα) was lower than that in vehicle-treated mice. Immunohistochemistry analysis showed a specific down-regulation of HMGCS2, a kernel target gene of PPARα. Overall, DPhP absorption disrupted body weight-gain processes. CONCLUSIONS: Our results suggest that in mice, the effects of chronic exposure to DPhP, even at a low dose, are not negligible. Fatty acid metabolism in the liver is essential for controlling fast and feast periods, with adverse consequences on the overall physiology. Therefore, the impact of DPhP on circulating fat, cardiovascular pathologies and metabolic disease incidence deserves, in light of our results, further investigations. https://doi.org/10.1289/EHP6826.

Relevant dose of the environmental contaminant, tributyltin, promotes histomorphological changes in the thyroid gland of male rats.

Organotin compounds, such as tributyltin (TBT), are common environmental contaminants and suspected endocrine-disrupting chemicals. Tributyltin is found in antifouling paints, widely used in ships and other vessels. The present study evaluated whether a 15-day treatment with TBT at a dose of 100 ng/kg/day could induce histomorphological changes in the thyroid gland of rats. TBT promoted relevant alterations in the thyroid architecture, being the most relevant histological findings the presence of increased number of small-size follicles in the treated group. In qualitative analyses, colloid vacuolization, papillary budging structures, cystic degeneration and chronic thyroiditis, were observed. Moreover, histomorphometric analysis showed statistically significant changes in the follicular architecture of TBT-treated rats, mainly a decrease in the follicle area (colloid) and an increased epithelial height that resulted in an increased epithelial height/colloid ratio. Augmented collagen deposition was also seen in the thyroids of treated groups. In immunohistochemical (IHC) analyses, the localization of NIS protein was described and a significant increased proliferation index (evaluated by Ki67 positive cells) in the treated group was reported. As an indirect measurement of oxidative stress, mitochondrial protein SDHA was also analyzed by IHC analysis. Although the cytoplasmic expression of SDHA was observed in both groups, the staining intensity score was higher in TBT-treated group. Our results suggest that besides causing histomorphological changes, environmental relevant dose of TBT treatment can also induce oxidative alterations.

NIS expression in thyroid tumors, relation with prognosis clinicopathological and molecular features.

Thyroid cancer therapy is based on surgery followed by radioiodine treatment. The incorporation of radioiodine by cancer cells is mediated by sodium iodide symporter (NIS) (codified by the SLC5A5 gene), that is functional only when targeted to the cell membrane. We aimed to evaluate if NIS expression in thyroid primary tumors would be helpful in predicting tumor behavior, response to therapy and prognosis. NIS expression was addressed by qPCR and immunohistochemistry. In order to validate our data, we also studied SLC5A5 expression on 378 primary papillary thyroid carcinomas from The Cancer Genome Atlas (TCGA) database. In our series, SLC5A5 expression was lower in carcinomas with vascular invasion and with extrathyroidal extension and in those harboring BRAFV600E mutation. Analysis of SLC5A5 expression from TCGA database confirmed our results. Furthermore, it showed that larger tumors, with locoregional recurrences and/or distant metastases or harboring RAS, BRAF and/or TERT promoter (TERTp) mutations presented significantly less SLC5A5 expression. Regarding immunohistochemistry, 12/211 of the cases demonstrated NIS in the membrane of tumor cells, those cases showed variable outcomes concerning therapy success, prognosis and all but one were wild type for BRAF, NRAS and TERTp mutations. SLC5A5 mRNA lower expression is associated with features of aggressiveness and with key genetic alterations involving BRAF, RAS and TERTp. Mutations in these genes seem to decrease protein expression and its targeting to the cell membrane. SLC5A5 mRNA expression is more informative than NIS immunohistochemical expression regarding tumor aggressiveness and prognostic features.

Predicting thyroid nodule malignancy at several prevalence values with a combined Bethesda-molecular test.

Investigation of thyroid nodules using fine-needle aspiration cytology (FNAC) gives indeterminate results in up to 30% of samples using the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC). We present a combined Bethesda-molecular predictor of nodule malignancy to improve the accuracy of the preoperative diagnosis of thyroid nodules. To detect a molecular signature of thyroid nodule malignancy, a molecular test was performed on FNACs from 128 thyroid nodules from prospectively included patients, collected in a tertiary center. The test relied on a transcriptomic array of 20 genes selected from a previous study. An optimal set of seven genes was identified using a logistic regression model. Comparison between the combined predictor (TBSRTC + molecular) and TBSRTC alone used the area under the ROC curve (AUC). Performance of the combined predictor was calculated according to various malignancy prevalence values and benefit-to-harm ratios (B/Hr) (favoring sensitivity or specificity). In our population (36% malignancy prevalence) and with a B/Hr of 1, the combined predictor achieved 95% specificity and 76% sensitivity. The AUC was 93.5%; higher than that of TBSRTC (P = 0.004). Among indeterminate nodules (30% malignancy prevalence), sensitivity and specificity were 52.2% and 96.2%, respectively, with a B/Hr of 1, or 95.7% and 64.2% with a B/Hr of 4 (favoring sensitivity), allowing avoidance of 64% of unnecessary surgeries at the cost of only one false-positive result. In conclusion, this predictor could improve the detection of thyroid nodule malignancy, taking into account malignancy prevalence and B/Hr, and reduce the number of unnecessary thyroidectomies.

Histopathological changes induced by selective inactivation of menin on the thyroid gland in RET÷PTC3 and E7 transgenic mice. A study of 77 cases.

Multiple Endocrine Neoplasia Type 1 (MEN1) does not involve the thyroid gland, but animal studies have shown that mice with inactivation of menin could develop thyroid pathologies. The objective was to evaluate if the selective inactivation of menin in murine thyroid glands expressing RET÷PTC3 and E7 oncogenes, might induce an increased index of proliferation and a more rapid development of thyroid hyperplasia and÷or tumors. The thyroid glands of 77 mice aged 4-18 months (31 expressing the E7 oncogene and 25 the RET÷PTC3 oncogene) were analyzed for histological changes and Ki67 proliferation index. Fifty-two mice had selective inactivation of menin in the thyroid gland (16 mice with RET÷PTC3 oncogene and 19 mice with E7 oncogene). As compared to wild type, mice with inactivation of menin presented an increased Ki67 proliferation index. Mice presenting the E7 oncogene showed larger thyroid glands with a pattern of diffuse hyperplasia. Mice expressing the RET÷PTC3 oncogene presented larger thyroid glands compared to the wild type mice but smaller compared to E7 mice. The lesions in the RET÷PTC3 group were "proliferative papillary cystic changes" (60%), "cribriform" (16%), "solid" (8%) and a combination of these patterns in the rest of the thyroid glands. The inactivation of menin in the thyroid gland of young mice does not seem to change the histological pattern, but it influences the proliferation of follicular cells. Further molecular studies especially in aged mice are needed to better understand the correlation between certain oncogenes and the inactive status of menin.

Modulation of thyroidal radioiodide uptake by oncological pipeline inhibitors and Apigenin.

Targeted radioiodine therapy for thyroid cancer is based on selective stimulation of Na+/I- Symporter (NIS)-mediated radioactive iodide uptake (RAIU) in thyroid cells by thyrotropin. Patients with advanced thyroid cancer do not benefit from radioiodine therapy due to reduced or absent NIS expression. To identify inhibitors that can be readily translated into clinical care, we examined oncological pipeline inhibitors targeting Akt, MEK, PI3K, Hsp90 or BRAF in their ability to increase RAIU in thyroid cells expressing BRAFV600E or RET/PTC3 oncogene. Our data showed that (1) PI3K inhibitor GDC-0941 outperformed other inhibitors in RAIU increase mainly by decreasing iodide efflux rate to a great extent; (2) RAIU increase by all inhibitors was extensively reduced by TGF-ß, a cytokine secreted in the invasive fronts of thyroid cancers; (3) RAIU reduction by TGF-ß was mainly mediated by NIS reduction and could be reversed by Apigenin, a plant-derived flavonoid; and (4) In the presence of TGF-ß, GDC-0941 with Apigenin co-treatment had the highest RAIU level in both BRAFV600E expressing cells and RET/PTC3 expressing cells. Taken together, Apigenin may serve as a dietary supplement along with small molecule inhibitors to improve radioiodine therapeutic efficacy on invasive tumor margins thereby minimizing future metastatic events.

The targeted inactivation of TRß gene in thyroid follicular cells suggests a new mechanism of regulation of thyroid hormone production.

Thyroid epithelial cells, or thyrocytes, express functional thyroid hormone receptors but no precise role has yet been assigned to either TRα or TRß in the thyroid gland. In this study, we analyzed the impact of inactivating the TRß gene in the thyroid of mice. First, we generated a mouse line named Thyr-Cre, expressing the Cre recombinase under the control of the thyroglobulin gene promoter, which led to a complete recombination of floxed genes in thyrocytes. Thyr-Cre mice were then crossed with TRß floxed mice (TRß(flox/flox)) to obtain a thyrocyte-selective deletion of TRß. Thyr-TRß(-/-) mice were characterized by a decrease in the size and functional activity of the thyroid gland. These alterations were associated with a decrease in plasma TSH concentration. Surprisingly, Thyr-TRß(-/-) displayed elevated serum T(4) and rT(3) concentrations with no significant change in serum T(3) levels. Their intrathyroidal free T(4) and rT(3) contents were also elevated, whereas the ratio of serum T(4) to thyroid free T(4) was decreased by comparison with wild-type littermates. Also, within the thyroid, deiodinases D1 and D2 were reduced as well as the expression levels of genes encoding monocarboxylate transporters (Mct8 and Mct10). Such a decrease in intrathyroidal deiodination of T(4) and in the expression of genes encoding thyroid hormone transporters may contribute to the primary overproduction of T(4) observed in Thyr-TRß(-/-) mice. In conclusion, these data show that the control of thyroid hormone production involves not only TRß-dependent mechanisms acting at the level of hypothalamus and pituitary but also TRß-dependent mechanisms acting at the thyroid level.

Molecular characteristics of papillary thyroid carcinomas without BRAF mutation or RET/PTC rearrangement: relationship with clinico-pathological features.

About 60-70% of papillary thyroid carcinomas (PTC) present a BRAF(T1799A) gene mutation or a rearrangement of RET gene (RET/PTC). In this study, we examined whether PTC without BRAF(T1799A) mutation and without RET/PTC rearrangement named PTC-ga(-) were distinguishable from PTC-ga(+) (with one or the other gene alteration) on the basis of gene expression characteristics. We analyzed the mutational state of 116 PTC and we compared gene expression profiles of PTC-ga(+) and PTC-ga(-) from data of a 200 gene macroarray and quantitative PCR. Seventy five PTC were PTC-ga(+) and 41 were PTC-ga(-). Unsupervised analyses of macroarray data by hierarchical clustering led to a complete segregation of PTC-ga(+) and PTC-ga(-). In a series of 42 genes previously recognized as PTC 'marker' genes, 22 were found to be expressed at a comparable level in PTC-ga(-) and normal tissue. Thyroid-specific genes, TPO, TG, DIO1, and DIO2 were under-expressed in PTC-ga(+) but expressed at a normal level in PTC-ga(-). A few genes including DUOX1 and DUOX2 were selectively dys-regulated in PTC-ga(-). Tumor grade of PTC-ga(-) was lower than that of PTC-ga(+). There was a strong association between the mutational state and histiotype of PTC; 81% of PTC follicular variants were corresponded to PTC-ga(-), whereas 84% of PTC of classical form were PTC-ga(+). In conclusion, we show that PTC without BRAF(T1799A) mutation or RET/PTC rearrangement, mainly corresponding to follicular variants, maintain a thyroid differentiation expression level close to that of normal tissue and should be of better prognosis than PTC with one or the other gene alteration.

Use of ERT2-iCre-ERT2 for conditional transgenesis.

We examined the use of ERT2-iCre-ERT2 (Cre2ERT2), a tamoxifen-regulated form of Cre that has been described to have a background activity lower than that of other tamoxifen-regulated Cre constructs, for establishing performant conditional deleter mouse lines. Cre2ERT2 was inserted by homologous recombination into the Rosa26 locus. These mice were mated with R26R Cre-reporter mice. No recombination could be observed in the progenies in the absence of tamoxifen treatment. Tamoxifen treatment at E13-14 led to a high level, albeit variable, recombination in most of the tissues examined: liver, heart, kidney, brain, lung etc. Treatment of adult animals also induced recombination in these tissues, although at a lower level. Northern blot and qPCR studies suggested that these differences are not linked to significant variations of the level of expression of Cre2ERT2. Thus, Cre2ERT2 appears to be a good alternative to existing modulatable Cre systems, displaying a lack of background activity and a high-level inducibility in vivo.

Evaluation of gene expression profiles in thyroid nodule biopsy material to diagnose thyroid cancer.

CONTEXT: Detection of thyroid cancer among benign nodules on fine-needle aspiration biopsies (FNAB), which presently relies on cytological examination, is expected to be improved by new diagnostic tests set up from genomic data. OBJECTIVE: The aim of the study was to use a set of genes discriminating benign from malignant tumors, on the basis of their expression levels, to build tumor classifiers and evaluate their capacity to predict malignancy on FNAB. DESIGN: We analyzed the level of expression of 200 potentially informative genes in 56 thyroid tissue samples (benign or malignant tumors and paired normal tissue) using nylon macroarrays. Gene expression data were subjected to a weighted voting algorithm to generate tumor classifiers. The performances of the classifiers were evaluated on a series of 26 sham FNAB, i.e. FNAB carried out on thyroid nodules after surgical resection. RESULTS: A series of 19 genes with a similar expression in follicular adenomas and normal tissue and discriminating follicular adenomas+normal tissue from the following: 1) follicular thyroid carcinomas (FTCs), 2) papillary thyroid carcinomas (PTCs), or 3) both FTCs and PTCs. These were used to generate four classifiers, the FTCs, PTCs, common (FTC+PTCs), and global classifiers. In 23 of the 26 sham FNAB, the four classifiers yielded a diagnosis in agreement with the diagnosis of the pathologist used as reference; in the three other cases, the correct diagnosis was given by three of four classifiers. CONCLUSIONS: We developed a procedure of molecular diagnosis of benign vs. malignant tumors applicable to the material collected by FNAB. The molecular test complied with a preclinical validation stage; it must be now evaluated on ultrasound-guided FNAB in a large-scale prospective study.

Connexin-32 acts as a downregulator of growth of thyroid gland.

Thyroid epithelial cells communicate through gap junctions formed from connexin (Cx)32, Cx43, and Cx26. We previously reported that reexpression of Cx32 in "gap junction-deficient" FRTL-5 and FRT thyroid cell lines induces a reduction of cell proliferation rate and an activation of expression of cell differentiation. The present study aimed at determining whether Cx32 could exert similar regulatory functions in vivo. We investigated morphological and functional characteristics of thyroid gland of Cx32-deficient mice (Cx32-KO), mice overexpressing Cx32 selectively in the thyroid (Cx32-T+), and Cx32-KO mice with a thyroid-selective Cx32 complementation obtained by crossing Cx32-KO and Cx32-T+ mice. In basal conditions, Cx32-KO mice did not present any detectable thyroid alteration, whereas Cx32-T+ mice showed a thyroid hypoplasia (20% reduction) associated with a slight increase in thyroid functional activity. Under thyrotropin stimulation (following sodium perchlorate treatment), Cx32-KO mice developed a larger goiter (< or =65% increase) than wild-type littermates, whereas Cx32-T+ mice exhibited the same thyroid hyperplasia as wild-type mice. Restoration of Cx32 expression in the thyroid of Cx32-KO mice abrogated the thyroid growth increase related to Cx32 deficiency. All together, these data show that Cx32 acts as a downregulator of growth of thyroid gland; an excess of Cx32 limits growth of thyroid cells in the basal state, whereas a lack of Cx32 confers an additional growth potential to TSH-stimulated thyroid cells.

Conditional transgenesis using Dimerizable Cre (DiCre).

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. We have recently developed a conceptually new approach to regulate Cre recombinase, that we have called Dimerizable Cre or DiCre. It is based on splitting Cre into two inactive moieties and fusing them to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin associated protein), respectively. These latter can be efficiently hetero-dimerized by rapamycin, leading to the reinstatement of Cre activity. We have been able to show, using in vitro approaches, that this ligand-induced dimerization is an efficient way to regulate Cre activity, and presents a low background activity together with a high efficiency of recombination following dimerization. To test the in vivo performance of this system, we have, in the present work, knocked-in DiCre into the Rosa26 locus of mice. To evaluate the performance of the DiCre system, mice have been mated with indicator mice (Z/EG or R26R) and Cre-induced recombination was examined following activation of DiCre by rapamycin during embryonic development or after birth of progenies. No recombination could be observed in the absence of treatment of the animals, indicating a lack of background activity of DiCre in the absence of rapamycin. Postnatal rapamycin treatment (one to five daily injection, 10 mg/kg i.p) induced recombination in a number of different tissues of progenies such as liver, heart, kidney, muscle, etc. On the other hand, recombination was at a very low level following in utero treatment of DiCrexR26R mice. In conclusion, DiCre has indeed the potentiality to be used to establish conditional Cre-deleter mice. An added advantage of this system is that, contrary to other modulatable Cre systems, it offers the possibility of obtaining regulated recombination in a combinatorial manner, i.e. induce recombination at any desired time-point specifically in cells characterized by the simultaneous expression of two different promoters.

Three-dimensional organization of thyroid cells into follicle structures is a pivotal factor in the control of sodium/iodide symporter expression.

Expression of sodium/iodide symporter (NIS) by thyroid epithelial cells is primarily regulated by TSH, which acts at the level of NIS gene transcription. Knowledge of the mechanisms governing NIS expression mainly comes from studies of rat thyroid-derived cell lines forming cell monolayers. In this study we investigated the impact of the three-dimensional organization of thyroid cells into follicles on the regulation of NIS expression. We used porcine thyrocytes in primary culture that, depending on cell density and the moment TSH is added, either predominantly form a cell monolayer (CM) or reconstitute thyroid follicles (RTF). NIS expression analyzed at transcript and protein levels was remarkably high in RTF compared with CM. Cells forming RTF were NIS positive, whereas in CM, NIS was only detected in the limited number of cells forming follicle-like structures. When thyrocytes were cultured at increasing cell density to obtain a gradual shift from CM to RTF, the progressive increase in the proportion of cells enrolled in RTF was accompanied by a parallel increase in NIS expression. Other TSH-regulated genes, thyroperoxidase, Na(+),K(+)-adenosine triphosphatase alpha-subunit, and thyroglobulin, were expressed at similar levels whatever the organization of thyrocytes in culture. The transcription factor, Pax-8, was equally expressed in NIS-negative CM and NIS-positive RTF. We show that TSH highly activates NIS expression only when thyrocytes have undergone histiotypic morphogenesis. This finding suggests that TSH activation of NIS gene transcription might involve, in addition to Pax-8, a regulatory factor(s) whose synthesis and/or activity are triggered by cell-cell interaction(s) occurring in the course of folliculogenesis.

Silencing of the tumor suppressor gene SLC5A8 is associated with BRAF mutations in classical papillary thyroid carcinomas.

SLC5A8, proposed as a thyroid apical iodide transporter, was recently defined as a Na+-coupled transporter of short-chain fatty acid. To document the expression pattern of SLC5A8 in the thyroid, we analyzed the regulation of its expression in normal human thyrocytes in culture and in tissues with distinct functional activity. To determine whether SLC5A8 expression is altered in all thyroid carcinomas or only in particular subtypes, we investigated the level of its expression in a series of 50 hypofunctioning tumors. SLC5A8 expression was studied at the transcript level and compared with that of SLC26A4 or Pendrin and SLC5A5 or Na+/iodide symporter. SLC5A8 expression, unlike that of SLC5A5 and SLC26A4, was not regulated by TSH in normal human thyrocytes in culture and was not related to the functional state of thyroid tissue; toxic adenomas and adjacent resting tissues exhibited the same SLC5A8 transcript content. SLC5A8 expression was selectively down-regulated (40-fold) in papillary thyroid carcinomas of classical form (PTC-cf.). Methylation-specific PCR analyses showed that SLC5A8 was methylated in 90% of PTC-cf. and in about 20% of other papillary thyroid carcinomas. In a series of 52 PTC-cf., a low SLC5A8 expression was highly significantly associated with the presence of BRAF T1796A mutation. These data identify a relationship between the methylation-associated silencing of the tumor-suppressor gene SLC5A8 and the T1796A point mutation of the BRAF gene in the PTC-cf. subtype of thyroid carcinomas.

Evidence for transcriptional and posttranscriptional alterations of the sodium/iodide symporter expression in hypofunctioning benign and malignant thyroid tumors.

The uptake of iodide by epithelial thyroid cells requires the expression of a specific transporter, the Na(+)/I(-) symporter, NIS. Benign and malignant thyroid tumors of epithelial origin show a decrease up to a loss of iodide uptake activity. Previous studies of the human NIS (hNIS) gene expression in these tumors, based on the amplification of transcripts and/or immunohistochemical detection of the protein, have yielded divergent data; hNIS expression was found either increased or decreased. To get a new and integrated view of the alterations of hNIS expression in hypofunctioning thyroid tumors, we performed investigations of hNIS transcript and hNIS protein levels on the same tumors and paired normal tissue samples. HNIS, identified as a 75- to 80-kd species, was present in all normal tissue samples from euthyroid patients, but was undetectable, even at high membrane protein input, in all benign and malignant hypofunctioning thyroid tumors. By contrast, approximately 50% of tumors contained hNIS transcripts. This dissociation between transcript and protein levels was not found for the transcript and protein encoded by the PDS gene assayed in the same tumors. The hNIS transcript-positive tumors contained small amounts of low-molecular mass hNIS-immunoreactive species identified as nonglycosylated hNIS. Tumors containing the nonmature form of hNIS exhibited a predominant intracellular immunolabeling. In conclusion, our data show that benign and malignant hypofunctioning thyroid tumors either no longer express hNIS protein or express only a very low amount of nonglycosylated hNIS and indicate that the impairment of hNIS gene expression might result from alterations at both transcriptional and posttranscriptional levels.

The porcine sodium/iodide symporter gene exhibits an uncommon expression pattern related to the use of alternative splice sites not present in the human or murine species.

The sodium/iodide symporter (NIS) is a membrane protein mediating the active transport of iodide into the thyroid gland. NIS, expressed by human, rat, and mouse thyrocytes, is encoded by a single transcript. We identified NIS mRNA species of 3.5 and 3 kb in porcine thyrocytes. Because porcine thyrocytes in primary culture is a widely used experimental system for thyroid iodide metabolism, we further examined the origin and the function of the porcine NIS (pNIS) transcripts. We generated a porcine thyroid cDNA library from which four different clones, pNIS-D, F, J, and Delta J were isolated. pNIS-D encodes a protein of 643 amino acids highly homologous to the human, rat, and mouse NIS. pNIS-F and J differ from each other and from pNIS-D in their C-terminal part. pNIS-Delta J lacks a six-amino-acid segment within the putative transmembrane domain 10. Transiently expressed in Cos-7 cells, the four pNIS-cDNAs led to the synthesis of proteins targeted at the plasma membrane and conferred perchlorate-sensitive iodide uptake activities to Cos-7 cells, except pNIS-Delta J, which was devoid of activity. PNIS-D probably derives from the 3.5-kb transcript and pNIS-F, J, and Delta J from the 3-kb transcript. The relative abundance of pNIS-D, F, and J transcripts in porcine thyrocytes was about 60%, 35%, and 5%, respectively; the Delta J transcript was not present in detectable amount. By comparing porcine NIS genomic and cDNA sequences, splice donor and acceptor sites accounting for the generation of pNIS-F, J, and Delta J transcripts were identified. None of the combinations of alternative splice sites found in the pig was present in the human, rat or mouse NIS gene. Our data show that porcine NIS gene, contrary to the NIS gene from other species, gives rise to splice variants leading to three active and one inactive NIS proteins.

Thyroid cell proliferation in response to forced expression of gap junction proteins.

Gap junctions are known to play a role in the control of cell proliferation, and connexins (Cx) are considered to be tumor suppressors. However, the effects of Cx on cell proliferation are dependent on the Cx which is expressed and on the cell type under consideration. We previously found that restoration of cell-to-cell communication by stable transfection of two independent thyroid-derived cell lines, FRTL-5 and FRT cells, with the Cx32 gene induced a marked reduction of their proliferation rate. This study aimed i) at determining whether Cx43, which is coexpressed with Cx32 by thyroid epithelial cells, exerts the same action as Cx32 on cell proliferation and ii) at identifying alterations of the cell cycle control system that might account for the Cx32-induced proliferation slowdown in thyrocytes. In contrast with previous data on different epithelial cell types, we report that restoration of intercellular communication in FRTL-5 and FRT cells by stable expression of Cx43 did not modify their proliferation properties. Cell cycle analyses revealed that the Cx32-induced proliferation slow-down was related to a lengthening of the G1 phase. The level of expression of two regulatory proteins of the Cip/Kip cyclin-dependent kinase inhibitor family, p27kip1 and p2cip1, was increased in the two cell lines expressing Cx32. In conclusion, Cx32 and Cx43, physiologically coexpressed by thyrocytes, have a differential impact on thyroid cell proliferation in vitro. The cyclin-dependent kinase inhibitors, p27kip1 and p21cip1 might represent cell cycle effectors relaying the down-regulatory effect of Cx32 on the proliferation of thyroid epithelial cells.

Characterization and semiquantitative analyses of pendrin expressed in normal and tumoral human thyroid tissues.

The gene mutated in Pendred syndrome (PDS), the PDS gene, is expressed in the inner ear, kidney, and thyroid. It encodes a membrane protein named pendrin that is endowed with the function of anion transporter or exchanger. It has been postulated that in the thyroid pendrin could participate in the transport of iodide from the cell to the lumen of follicles. We generated antipeptide antibodies directed against the C- terminal sequence of human pendrin 1) to characterize the protein expressed in the human thyroid, and 2) to analyze its expression level in relation to the functional activity of thyroid tissue. In denaturing conditions, a single molecular species of 110-115 kDa was identified in human thyroid membrane fractions. After treatment of thyroid membranes with N-glycosidase F, pendrin had an apparent molecular mass of 85 kDa. Analyzed by ultracentrifugation on sucrose gradient in nondenaturing conditions, pendrin sedimented as a main 120- to 140-kDa component. Pendrin was assayed by semiquantitative Western blot in thyroid membrane fractions from 25 hyper- or hypofunctioning tumors and paired normal tissue samples. Pendrin was increased 2-fold in toxic adenomas, was not significantly altered in follicular adenoma, and was decreased, on the average, by 35% in papillary carcinomas compared with levels in paired normal tissue. The variations in the pendrin tissue content and PDS transcript levels, assayed by RT-PCR on duplicate samples of the same tumors, were similar. In conclusion, we show that pendrin expressed by the human thyroid gland is a mainly monomeric glycoprotein and that the level of expression of pendrin, although somewhat related, only moderately varied with the functional status of the thyroid tissue.