Pendrin: the thyrocyte apical membrane iodide transporter?

In the thyroid, the transport of iodide from the extracellular space to the follicular lumen requires two steps: the transport in the cell at the basal side and in the lumen at the apical side. The first step is mediated by the Na(+)/I(-) symporter (NIS). In most reviews and textbooks, the second step is presented as mediated by pendrin. In this review, we analyze this assumption. There are several arguments supporting the concept that indeed pendrin plays an important role in thyroid physiology. However, biochemical, clinical and histological data on the thyroid of a patient with Pendred syndrome do not suggest an essential role in iodide transport, which is corroborated by the lack of a thyroid phenotype in pendrin knockout mice. Experiments in vivo and in vitro on polarized and unpolarized cells show that iodide is transported transport of iodide at the apex of the thyroid cell. Moreover, ectopic expression of pendrin in transfected non-thyroid cells is capable of mediating iodide efflux. It is concluded that pendrin may participate in the iodide efflux into thyroid lumen but not as the unique transporter. Moreover, another role of pendrin in mediating Cl(-)/HCO(3)(-) exchange and controlling luminal pH is suggested.

Functional model of rat thyroid follicles cultured in Matrigel.

BACKGROUND: Long-term maintenance of functional activity of thyroid cells is an essential requirement for basic in vitro studies on the physiology and pathology of the thyroid. An important prerequisite of thyrocytes' functional activity in vivo and in vitro is their follicle organization. AIM: This study aimed at developing a method of cultivation of functionally active rat thyroid follicles in Matrigel under three-dimensional conditions. METHODS: Undamaged rat thyroid follicles were isolated by enzymatic digestion with collagenase/dispase, then embedded into Matrigel, and cultivated for 2 weeks. Thyroglobulin, thyroxine and zonula occludens-1 (ZO-1) localization were revealed by immunofluorescence analysis. Iodide organification was tested by protein-bound 125I (PBI) measurement. RESULTS: Integrity of the follicles was preserved during the whole period of cultivation and was confirmed by 3D reconstruction of ZO-1 localization. Thyroglobulin was detected in the thyrocyte cytoplasm, as well as in the intrafollicular lumen. Thyroxine was observed predominantly at the apical side of thyrocytes. Also, generated cultures were characterized by a high level of iodide organification: PB125I represented 39% of the total radioactivity in the Matrigel drop embedding the follicles; at the same time, methimazole almost totally inhibited this process (0.2% of total radioactivity). CONCLUSION: The method of rat thyrocyte cultivation in Matrigel, as described here allows to maintain the structural integrity and the functional activity of thyroid follicles in vitro and could be used for wide ranges of basic and applied researches in thyroidology.

Role of caveolin-1 in thyroid phenotype, cell homeostasis, and hormone synthesis: in vivo study of caveolin-1 knockout mice.

In human thyroid, caveolin-1 is localized at the apex of thyrocytes, but its role there remains unknown. Using immunohistochemistry, (127)I imaging, transmission electron microscopy, immunogold electron microscopy, and quantification of H(2)O(2), we found that in caveolin-1 knockout mice thyroid cell homeostasis was disrupted, with evidence of oxidative stress, cell damage, and apoptosis. An even more striking phenotype was the absence of thyroglobulin and iodine in one-half of the follicular lumina and their presence in the cytosol, suggesting that the iodide organification and binding to thyroglobulin were intracellular rather than at the apical membrane/extracellular colloid interface. The latter abnormality may be secondary to the observed mislocalization of the thyroid hormone synthesis machinery (dual oxidases, thyroperoxidase) in the cytosol. Nevertheless, the overall uptake of radioiodide, its organification, and secretion as thyroid hormones were comparable to those of wild-type mice, suggesting adequate compensation by the normal TSH retrocontrol. Accordingly, the levels of free thyroxine and TSH were normal. Only the levels of free triiodothyronine showed a slight decrease in caveolin-1 knockout mice. However, when TSH levels were increased through low-iodine chow and sodium perchlorate, the induced goiter was more prominent in caveolin-1 knockout mice. We conclude that caveolin-1 plays a role in proper thyroid hormone synthesis as well as in cell number homeostasis. Our study demonstrates for the first time a physiological function of caveolin-1 in the thyroid gland. Because the expression and subcellular localization of caveolin-1 were similar between normal human and murine thyroids, our findings in caveolin-1 knockout mice may have direct relevance to the human counterpart.

Expression, Localization, and Regulation of the Sodium Bicarbonate Cotransporter NBCe1 in the Thyroid.

BACKGROUND: The intrafollicular space of thyroid follicles is the storage compartment for thyroid hormones. Its pH has been established at around 7.6 at least after thyrotropin (TSH) stimulation. This alkaline intrafollicular pH is thought to be critical for iodide coupling to thyroglobulin and internalization of iodinated thyroglobulin. At least in mice, this alkalinization requires the expression of pendrin (Slc26a4) within the apical membrane, and a lack of pendrin results in acidic follicular lumen pH. Yet, the mechanism importing HCO3- into the cytoplasm is unknown. This study investigated whether the rather ubiquitous sodium bicarbonate cotransporter NBCe1 (SLC4A4) might play this role. It also examined which variant was expressed and where it was localized in both rat and human thyroid tissue. Lastly, the dependence of its expression on TSH was studied. METHODS: Reverse transcription polymerase chain reaction, immunofluorescence, and Western blotting were used to test whether TSH stimulated NBCe1 protein expression in vivo. Subcellular localization of NBCe1 was performed using immunofluorescence in both rat and human thyroid. Cultured thyroid cells were also used to attempt to define how TSH affects NBCe1 expression. RESULTS: Only transcripts of the NBCe1-B variant were detected in both rat and human thyroid. Of interest, NBCe1-C was not detected in human tissues, not even in the brain. On immunofluorescence microscopy, the immunostaining of NBCe1 mainly appeared in the basolateral membrane upon stimulation with TSH. This TSH induction of basolateral membrane expression of NBCe1 protein was confirmed in vivo in rat thyroid and in vitro on human thyroid slices. CONCLUSIONS: This study demonstrates the expression of the sodium bicarbonate cotransporter NBCe1-B in rat and human thyroid. Additionally, the data suggest that TSH blocks the degradation of NBCe1 protein by trafficking it to the basolateral membrane. Hence, TSH increases NBCe1 half-life without increasing its synthesis.

Thyrotropin stimulates the generation of inositol 1,4,5-trisphosphate in human thyroid cells.

CONTEXT: Dual activation by TSH of the phospholipase C and cAMP cascades has been reported in human thyroid cells. In contrast, Singh et al. reported convincing data in FRTL-5 thyrocytes arguing against such an effect in this model. Their data in FRTL-5 cells indicated no increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to TSH. Therefore, the authors questioned results previously obtained on human cells by cruder methodology. OBJECTIVE: We investigated the formation of inositol phosphates by HPLC techniques in human thyroid slices to separate the inositol phosphate isomers. RESULTS: Ins(1,4,5)P3, inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate were increased after TSH stimulation. The effect of TSH in human thyroid cells was reproduced by recombinant TSH and prevented by antibodies blocking the TSH receptor. Thyroid-stimulating antibodies at concentrations eliciting a cAMP response equivalent to TSH failed to stimulate inositol phosphate generation. CONCLUSIONS: TSH, but not thyroid-stimulating antibodies, activates both cAMP and the phospholipase C cascade in human thyroid as now demonstrated by an increase in Ins(1,4,5)P3 and its inositol phosphate metabolites. Therefore, this effect cannot be extrapolated to the FRTL-5 cell line. The apparent discrepancy may be due to a difference between species (human vs. rat) or to the loss of the fresh tissue properties in a cell line. The dual effect of TSH in human cells, through cAMP on secretion of thyroid hormones and through the diacylglycerol, Ins(1,4,5)P3 Ca2+ pathway on thyroid hormone synthesis, implies the possible separation of these effects in thyroid disease.

Thyroid-specific inactivation of KIF3A alters the TSH signaling pathway and leads to hypothyroidism.

Kinesins, including the kinesin 2/KIF3 molecular motor, play an important role in intracellular traffic and can deliver vesicles to distal axon terminals, to cilia, to nonpolarized cell surfaces or to epithelial cell basolateral membranes, thus taking part in the establishment of cellular polarity. We report here the consequences of kinesin 2 motor inactivation in the thyroid of 3-week-old Kif3a(Δ)(/flox) Pax8(Cre/)(+) mutant mice. Our results indicate first that 3-week-old Pax8(Cre/)(+) mice used in these experiments present minor thyroid functional defects resulting in a slight increase in circulating bioactive TSH and intracellular cAMP levels, sufficient to maintain blood thyroxine levels in the normal range. Second, Kif3a inactivation in thyrocytes markedly amplified the phenotype observed in Pax8(Cre/)(+) mice, resulting in altered TSH signaling upstream of the second messenger cAMP and mild hypothyroidism. Finally, our results in mouse embryonic fibroblasts indicate that Kif3a inactivation in the absence of any Pax8 gene alteration leads to altered G protein-coupled receptor plasma membrane expression, as shown for the ß2 adrenergic receptor, and we suggest that a similar mechanism may explain the altered TSH signaling and mild hypothyroidism detected in Kif3a(Δ)(/flox) Pax8(Cre/)(+) mutant mice.

Acrylamide does not induce tumorigenesis or major defects in mice in vivo.

Chronic administration of acrylamide has been shown to induce thyroid tumors in rat. In vitro acrylamide also causes DNA damage, as demonstrated by the comet assay, in various types of cells including human thyroid cells and lymphocytes, as well as rat thyroid cell lines. In this work, mice were administered acrylamide in their drinking water in doses comparable with those used in rats, i.e., around 3-4 mg/kg per day for mice treated 2, 6, and 8 months. Some of the mice were also treated with thyroxine (T(4)) to depress the activity of the thyroid. Others were treated with methimazole that inhibits thyroid hormone synthesis and consequently secretion and thus induces TSH secretion and thyroid activation. These moderate treatments were shown to have their known effect on the thyroid (e.g. thyroid hormone and thyrotropin serum levels, thyroid gland morphology...). Besides, T(4) induced an important polydipsia and degenerative hypertrophy of adrenal medulla. Acrylamide exerted various discrete effects and at high doses caused peripheral neuropathy, as demonstrated by hind-leg paralysis. However, it did not induce thyroid tumorigenesis. These results show that the thyroid tumorigenic effects of acrylamide are not observed in another rodent species, the mouse, and suggest the necessity of an epidemiological study in human to conclude on a public health policy.