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Intracellular and extracellular sites of iodination in dispersed hog thyroid cells.

ABSTRACT: Iodination and hormone synthesis has been studied in isolated hog thyroid cells in suspension. We characterized three iodination processes by use of pharmacological agents. (1) Intracellular iodination dependent on active iodide transport, which was inhibited by NaClO4 or ouabain, but not by catalase. This iodination was linear for 6h with no apparent Km for iodide of 1.5 muM, was stimulated by thyrotropin or N6O2'-dibutyryladenosine 3':5'-cyclic monophosphate, yielded mostly iodinated thyroglobulin and was efficient for tetraiodothyronine synthesis. (2) Extracellular iodination, which was sensitive to catalase, but not to NaClO4 or ouabain. This iodination plateaued after 2h and the apparent Km was 16.5 muM. This process was insensitive to thyrotropin and dibutyryl cyclic AMP. The major products were iodoprotein other then thyroglobulin and iodolipid and the yield of tetraiodothyronine was low. (3) Intracellular iodination from passively diffused iodide, which was not sensitive to inhibitors. Other characteristics of passive intracellular iodination were intermediate between active intracellular iodination and extracellular iodination. The fact that the three processes are inhibited by similar concentrations of methimazole, and their apparent Km values, when corrected for the concentrating effect of iodide trapping, are all of the same order as the Km of purified thyroid peroxidases, suggest that although their locations are different, the enzymic systems involved are identical. These results show that, besides an extracellular site of iodination, dispersed thyroid cells process an intracellular site of iodination with biochemical characteristics of physiological relevance.

SUBMITTER: Rousset B

PROVIDER: S-EPMC1162403 | biostudies-other | 1980 Dec

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:The thyroid gland has a special relationship with oxidative stress. While generation of oxidative substances is part of normal iodide metabolism during thyroid hormone synthesis, the gland must also defend itself against excessive oxidation in order to maintain normal function. Antioxidant and detoxification enzymes aid thyroid cells to maintain homeostasis by ameliorating oxidative insults, including during exposure to excess iodide, but the factors that coordinate their expression with the cellular redox status are not known. The antioxidant response system comprising the ubiquitously expressed NFE2-related transcription factor 2 (Nrf2) and its redox-sensitive cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (Keap1) defends tissues against oxidative stress, thereby protecting against pathologies that relate to DNA, protein, and/or lipid oxidative damage. Thus, it was hypothesized that Nrf2 should also have important roles in maintaining thyroid homeostasis. METHODS:Ubiquitous and thyroid-specific male C57BL6J Nrf2 knockout (Nrf2-KO) mice were studied. Plasma and thyroids were harvested for evaluation of thyroid function tests by radioimmunoassays and of gene and protein expression by real-time polymerase chain reaction and immunoblotting, respectively. Nrf2-KO and Keap1-KO clones of the PCCL3 rat thyroid follicular cell line were generated using CRISPR/Cas9 technology and were used for gene and protein expression studies. Software-predicted Nrf2 binding sites on the thyroglobulin enhancer were validated by site-directed in vitro mutagenesis and chromatin immunoprecipitation. RESULTS:The study shows that Nrf2 mediates antioxidant transcriptional responses in thyroid cells and protects the thyroid from oxidation induced by iodide overload. Surprisingly, it was also found that Nrf2 has a dramatic impact on both the basal abundance and the thyrotropin-inducible intrathyroidal abundance of thyroglobulin (Tg), the precursor protein of thyroid hormones. This effect is mediated by cell-autonomous regulation of Tg gene expression by Nrf2 via its direct binding to two evolutionarily conserved antioxidant response elements in an upstream enhancer. Yet, despite upregulating Tg levels, Nrf2 limits Tg iodination both under basal conditions and in response to excess iodide. CONCLUSIONS:Nrf2 exerts pleiotropic roles in the thyroid gland to couple cell stress defense mechanisms to iodide metabolism and the thyroid hormone synthesis machinery, both under basal conditions and in response to excess iodide.

Project description:Background: The production of thyroid hormones [triiodothyronine (T3), thyroxine (T4)] depends on the organization of the thyroid in follicles, which are lined by a monolayer of thyrocytes with strict apicobasal polarity. This polarization supports vectorial transport of thyroglobulin (Tg) for storage into, and recapture from, the colloid. It also allows selective addressing of channels, transporters, ion pumps, and enzymes to their appropriate basolateral [Na+/I- symporter (NIS), SLC26A7, and Na+/K+-ATPase] or apical membrane domain (anoctamin, SLC26A4, DUOX2, DUOXA2, and thyroperoxidase). How these actors of T3/T4 synthesis reach their final destination remains poorly understood. The PI 3-kinase isoform Vps34/PIK3C3 is now recognized as a main component in the general control of vesicular trafficking and of cell homeostasis through the regulation of endosomal trafficking and autophagy. We recently reported that conditional Vps34 inactivation in proximal tubular cells in the kidney prevents normal addressing of apical membrane proteins and causes abortive macroautophagy. Methods: Vps34 was inactivated using a Pax8-driven Cre recombinase system. The impact of Vps34 inactivation in thyrocytes was analyzed by histological, immunolocalization, and messenger RNA expression profiling. Thyroid hormone synthesis was assayed by 125I injection and plasma analysis. Results: Vps34 conditional knockout (Vps34cKO) mice were born at the expected Mendelian ratio and showed normal growth until postnatal day 14 (P14), then stopped growing and died at ?1 month of age. We therefore analyzed thyroid Vps34cKO at P14. We found that loss of Vps34 in thyrocytes causes (i) disorganization of thyroid parenchyma, with abnormal thyrocyte and follicular shape and reduced PAS+ colloidal spaces; (ii) severe noncompensated hypothyroidism with extremely low T4 levels (0.75?±?0.62??g/dL) and huge thyrotropin plasma levels (19,300?±?10,500?mU/L); (iii) impaired 125I organification at comparable uptake and frequent occurrence of follicles with luminal Tg but nondetectable T4-bearing Tg; (iv) intense signal in thyrocytes for the lysosomal membrane marker, LAMP-1, as well as Tg and the autophagy marker, p62, indicating defective lysosomal proteolysis; and (v) presence of macrophages in the colloidal space. Conclusions: We conclude that Vps34 is crucial for thyroid hormonogenesis, at least by controlling epithelial organization, Tg iodination as well as proteolytic T3/T4 excision in lysosomes.

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Intracellular and extracellular sites of iodination in dispersed hog thyroid cells.

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