Cloning and characterization of a novel isoform of iodotyrosine dehalogenase 1 (DEHAL1) DEHAL1C from human thyroid: comparisons with DEHAL1 and DEHAL1B.

The human iodotyrosine dehalogenase 1 (DEHAL1) gene is composed of six exons. Two isoforms (DEHAL1 and DEHAL1B) have been published in GenBank, both of which have a nitroreductase domain and arise from differential splicing in exon 5. We recently showed that the DEHAL1 isoform is a transmembrane protein that efficiently catalyzes the NADPH-dependent deiodination of mono (L-MIT) and diiodotyrosine (L-DIT) in human embryonic kidney-293 (HEK293) cells. In the present study, we establish the existence of a new transcript, DEHAL1C, in the human thyroid with a terminal exon that lacks in the DEHAL1 transcript. This exon is the complete exon 5, which is spliced in the DEHAL1B mRNA variant. These two variants encode proteins with differing C-terminal domains. Using quantitative reverse transcription polymerase chain reaction, we found that the expression of the mRNA of DEHAL1C and DEHAL1B was lower than that of DEHAL1 mRNA in the thyroid. We also observed that human DEHAL1B and DEHAL1C proteins are rapidly degraded in stably transfected HEK293 cells, unlike the DEHAL1 protein, and that exposure to the proteasome inhibitor MG132 resulted in accumulation of these proteins that was markedly time- and concentration-dependent. These findings show that the cytoplasmic tail could play a role in the stability of the protein.

Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity.

Duox2 (and probably Duox1) is a glycoflavoprotein involved in thyroid hormone biosynthesis, as the thyroid H2O2 generator functionally associated with Tpo (thyroperoxidase). So far, because of the impairment of maturation and of the targeting process, transfecting DUOX into nonthyroid cell lines has not led to the expression of a functional H2O2-generating system at the plasma membrane. For the first time, we investigated the H2O2-generating activity in the particulate fractions from DUOX2- and DUOX1-transfected HEK293 and Chinese hamster ovary cells. The particulate fractions of these cells stably or transiently transfected with human or porcine DUOX cDNA demonstrate a functional NADPH/Ca2+-dependent H2O2-generating activity. The immature Duox proteins had less activity than pig thyrocyte particulate fractions, and their activity depended on their primary structures. Human Duox2 seemed to be more active than human Duox1 but only half as active as its porcine counterpart. TPO co-transfection produced a slight increase in the enzymatic activity, whereas p22(phox), the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H2O2 formation, it was shown that mature thyroid NADPH oxidase does not release O2*- but H2O2. Using a spin-trapping technique combined with electron paramagnetic resonance spectroscopy, we confirmed this result but also demonstrated that the partially glycosylated form of Duox2, located in the endoplasmic reticulum, generates superoxide in a calcium-dependent manner. These results suggest that post-translational modifications during the maturation process of Duox2 could be implicated in the mechanism of H2O2 formation by favoring intramolecular superoxide dismutation.

CCAAT/enhancer-binding protein-homologous protein expression and transcriptional activity are regulated by 3',5'-cyclic adenosine monophosphate in thyroid cells.

The cAMP pathway activates p38-MAPKs in the FRTL-5 rat thyroid cell line, contributing to the increased expression of the Na+/I- symporter (NIS) mRNA. This study investigates the cAMP-dependent expression and transcriptional activity of the p38-MAPK substrate CCAAT/enhancer-binding protein-homologous protein (CHOP). CHOP is expressed in the rat thyroid gland and in confluent PCCL3 and FRTL-5 cells. In FRTL-5 cells, TSH withdrawal induced a rapid down-regulation of CHOP that could be prevented by forskolin (Fk). Moreover, TSH and Fk were able to reinduce CHOP expression. The use of pharmacological inhibitors indicated that cAMP-induced CHOP expression was dependent on protein kinase A (PKA), mammalian target of rapamycin pathway, and reactive oxygen species. Transfection of a CHOP trans- reporting system revealed strong stimulation of the transcriptional activity of CHOP by Fk, by chlorophenylthio-cAMP, and by the catalytic subunit of PKA. CHOP transcriptional activity was significantly reduced by the p38-MAPK inhibitor SB203580, by transfection of a dominant-negative variant of p38alpha-MAPK, or by mutation of two serine residues in CHOP targeted by p38-MAPKs. Finally, cAMP-induced NIS mRNA expression was higher in FRTL-5 cells stably transfected with CHOP cDNA than in control cells. Likewise, the activity of the NIS promoter was higher in cells overexpressing CHOP than in control cells. These findings suggest that the stimulation of CHOP expression and transcriptional activity by the cAMP pathway may contribute to the regulation of genes involved in thyroid cell differentiation.

Thyroid hormone regulates the expression of NeuroD/BHF1 during the development of rat cerebellum.

During the postnatal development of the rat cerebellum, there is an extensive proliferation of granular neurones in the external layer, followed by their migration and differentiation in the internal layer. These processes are impaired by neonatal hypothyroidism and can be restored by thyroid hormone therapy. They are also abolished in transgenic mice in which the neuroD gene is not expressed. This gene encodes a basic helix-loop-helix (bHLH) transcription factor (NeuroD), which induces the differentiation of neuronal precursors. We studied the expression of neuroD/BHF1-A mRNA during the postnatal development of euthyroid and hypothyroid rats, and compared it with that of neurotrophin-3 (NT-3), a marker of granular neurone differentiation. In euthyroid animals, the neuroD/BHF1-A mRNA increases 6-fold between days 4 and 15 after birth, and then decreases to 50% of this level in the adult. NT-3 mRNA expression followed a similar pattern, although it was increased only 3-fold. Hypothyroidism reduced both mRNA levels by 35-45%, depending on the postnatal stage. In hypothyroid pups, the injection of triiodothyronine (T3) restored normal levels of both mRNAs within 6 h. In 15-day old hypothyroid rats, the amount of NeuroD protein was reduced by about 35%. It increased about 2-fold 24 h after T3 injection. In conclusion, our results indicate that thyroid hormones (TH) regulate the expression of NeuroD during the "critical period" of cerebellum development. This regulation may constitute an early event in the control of differentiation of the cerebellar granular neurones by TH.