| Thyroid hormone is a master regulator of cellular metabolism and growth. In adults, thyroid hormone signaling regulates energy expenditure throughout the whole life. In children, thyroid hormone also mediates the proliferation and differentiation of growing tissues. Many actions of thyroid hormone are initiated by binding of the active form of the hormone T3 to nuclear receptors in target cells.T4 is the predominant iodothyronine secreted by the thyroid gland under normal conditions. As a consequence, T4 has to be converted by so-called outer ring deiodination(ORD) to T3. T4 and T3 also can be inactivated by inner ring deiodination. Iodothyronine deiodinases are involved in these reactions. In human and mammal D1 and D2 are main activators of thyroid hormone by converting T4 to T3,whereas D3 is the only inactivators catalyzing the degradation of T4 to reverse T3(rT3) and of T3 to 3,3’-T2.There are two dio3 genes in zebrafish due to gene duplication, one is called dio3 a while the other named dio3 b. Our goals were to determine whether or not both dio3 gene(s) are functional and, if so, whether they have similar or different roles during development. Our present study shows that both zebrafish dio3 paralogs are syntenic to human DIO3 and that in vitro studies each encodes a functional selenoenzyme that potently inactivates both T4 and T3. While the enzyme kinetics of both zebrafish Dio3 proteins are similar, their expression patterns and gene structures are markedly divergent and we report here that dio3 b is the first D3 gene from any species to possess a large intron separating its open frame from its selenocysteine insertion sequence(SECIS) element, a critical 3’UTR sequence required for selenoprotein synthesis, selenocysteine insertion at UGA codons, and deiodinase catalytic activity. Maximal sensitivity enzyme assays show that zebrafish D3 a could catalyze rT3 by outer ring deiodination, this is different with all the other D3 s.These results indicates that the role of D3 a and D3 b is different in development.Further studies of zebrafish D1 showed minimal catalysis of either T4 or T3. This suggests that, in the zebrafish, Dio2 is the primary activator of T4 and the Dio3 enzymes are the major inactivators of both T4 and T3.In humans, the placenta has the highest D3 activity of any normal tissue so we hypothesized that D3 plays an important role in development. Morpholino mediated knockdown of D3 b severely perturbs embryonic development and dramatically reduces embryo size at 72 hpf. There is no obvious abnormal after D3 a knock down.Thyroid hormone antagonist significantly ameliorated the growth failure indicating that this phenotype caused by D3 b is specific and dependent on the thyroid hormone receptor signaling.Exposure to high dose of T3 induced the upregulation of D3 activity and the mRNA expression of both dio3 genes.Thyroid hormone plays an important role in amphibian metamorphosis.We found that the genes involved in thyroid hormone induced apoptosis and tp53 were upregulated after D3 b knockdown. Combined injection of p53 morpholinos and D3 b morpholinos eliminated the phenotype of apoptosis and microsomia.This indicates that the increase of thyroid hormone receptor signaling in embryonic structures that normally express dio3 b result in the upregulation of apoptosis signalling to cause the microsomia.The mammalian fetus can not avoid the effect of the maternal thyroid hormone.So it is very complicated to study the role of D3 during embryo development using mammalian model.Out study using zebrafish,which is external fertilized, directly shows that embryonic D3 deficiency alone is sufficient cause microsomia.Intrauterine growth retardation in humans is a global pediatric health problem that is incompletely understood and increasingly link in risks of adult disease. our data raise the intriguing possibility that even transient abnormalities in embryonic deiodination could cause intrauterine growth retardation and provide the new insight into the clinical diagnosis and treatment. |
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