Defining thyrotropin-dependent and -independent steps of thyroid hormone synthesis by using thyrotropin receptor-null mice.

The thyrotropin (TSH) receptor (TSHR) is a member of the heterotrimeric G protein-coupled family of receptors whose main function is to regulate thyroid cell proliferation as well as thyroid hormone synthesis and release. In this study, we generated a TSHR knockout (TSHR-KO) mouse by homologous recombination for use as a model to study TSHR function. TSHR-KO mice presented with developmental and growth delays and were profoundly hypothyroid, with no detectable thyroid hormone and elevated TSH. Heterozygotes were apparently unaffected. Knockout mice died within 1 week of weaning unless fed a diet supplemented with thyroid powder. Mature mice were fertile on the thyroid-supplemented diet. Thyroid glands of TSHR-KO mice produced uniodinated thyroglobulin, but the ability to concentrate and organify iodide could be restored to TSHR-KO thyroids when cultured in the presence of the adenylate cyclase agonist forskolin. Consistent with this observation was the lack of detectable sodium-iodide symporter expression in TSHR-KO thyroid glands. Hence, by using the TSHR-KO mouse, we provided in vivo evidence, demonstrating that TSHR expression was required for expression of sodium-iodide symporter but was not required for thyroglobulin expression, suggesting that the thyroid hormone synthetic pathway of the mouse could be dissociated into TSHR-dependent and -independent steps.

Role of the thyroid-stimulating hormone receptor signaling in development and differentiation of the thyroid gland.

The thyroid-stimulating hormone/thyrotropin (TSH) is the most relevant hormone in the control of thyroid gland physiology in adulthood. TSH effects on the thyroid gland are mediated by the interaction with a specific TSH receptor (TSHR). We studied the role of TSHTSHR signaling on gland morphogenesis and differentiation in the mouse embryo using mouse lines deprived either of TSH (pit(dw)pit(dw)) or of a functional TSHR (tshr(hyt)tshr(hyt) and TSHR-knockout lines). The results reported here show that in the absence of either TSH or a functional TSHR, the thyroid gland develops to a normal size, whereas the expression of thyroperoxidase and the sodium/iodide symporter are reduced greatly. Conversely, no relevant changes are detected in the amounts of thyroglobulin and the thyroid-enriched transcription factors TTF-1, TTF-2, and Pax8. These data suggest that the major role of the TSH/TSHR pathway is in controlling genes involved in iodide metabolism such as sodium/iodide symporter and thyroperoxidase. Furthermore, our data indicate that in embryonic life TSH does not play an equivalent role in controlling gland growth as in the adult thyroid.

Regulation and transfer of a murine model of thyrotropin receptor antibody mediated Graves' disease.

In order to replicate a recently described murine model of Graves' disease, we immunized AKR/N (H-2k) mice i.p., every 2 weeks, with either a clone of fibroblasts expressing both the human TSH receptor (hTSHR) and murine major histocompatibility complex (MHC) class II molecules or with fibroblasts expressing the MHC class II molecules alone. Mice were bled, and their thyroid hormone levels measured, at 6, 12, and up to 18 weeks after the first immunization. Between 11-12 weeks after immunization, a significant number of mice began to die spontaneously and were found to have developed large goiters. Thirty to 40% of mice immunized with hTSHR transfected fibroblasts showed markedly increased serum T3 and T4 hormone levels by 12 weeks compared with controls, with the highest thyroid hormone levels being T3: 420 ng/dl (normal < 70) and T4: 16.5 microg/dl (normal < 5). The murine serum demonstrated the presence of antibodies to the TSHR, as evidenced by inhibition of labeled TSH binding to the hTSHR, and these sera had in vitro thyroid stimulating activity. Many of the hyperthyroid mouse exhibited weight loss and hyperactivity and, on examination, their thyroids had the histological features of thyroid hyperactivity including thyroid enlargement, thyroid cell hypertrophy, and colloid droplet formation--all consistent with Graves' disease. In contrast, a small number of mice (< 5%) developed hypothyroidism with low serum T4 levels and markedly increased TSH concentrations and evidence of thyroid hypoplasia. Both hyperthyroidism and hypothyroidism were successfully transferred to naive mice using ip cells of immunized mice. Surprisingly, hypothyroidism occurred in many recipient mice even after transfer from hyperthyroid donors. These results confirmed that immunization with naturally expressed hTSHR in mammalian cells was able to induce functional TSHR autoantibodies that either stimulated or blocked the mouse thyroid gland and induced hyperthyroidism or thyroid failure. Furthermore, both blocking and stimulating antibodies coexisted in the same mice as evidenced so clearly by the transfer of hypothyroidism from hyperthyroid mice. The addition of a Th2 adjuvant (pertussis toxin) caused approximately 50% of the animals to become hyperthyroid beginning early at 9 weeks, whereas a Th1 adjuvant (CFA) delayed the disease onset such that only 10% were hyperthyroid by 12 weeks. As with human autoimmune thyroid disease, the T cell control of this murine model may be critical and requires more extensive investigation.

A complex containing two transcription factors regulates peroxisome proliferation and the coordinate induction of beta-oxidation enzymes in Saccharomyces cerevisiae.

Expression of the POX1 gene, which encodes peroxisomal acyl coenzyme A oxidase in the yeast Saccharomyces cerevisiae, is tightly regulated and can be induced by fatty acids such as oleate. Previously we have shown that this regulation is brought about by interactions between trans-acting factor(s) and an upstream activating sequence (UAS1) in the POX1 promoter. We recently identified and isolated a transcription factor, Oaf1p, that binds to the UAS1 of POX1 and mediates its induction. A screening strategy has been developed and used to identify eight S. cerevisiae mutants, from three complementation groups, that are defective in the oleate induction of POX1. Characterization of one such mutant led to the identification of Oaf2p, a protein that is 39% identical to Oaf1p. Oaf1p and Oaf2p form a protein complex that is required for the activation of POX1 and FOX3 and for proliferation of peroxisomes. We propose a model in which these two transcription factors heterodimerize and mediate this activation process. The mutants that we have isolated, and further identification of the corresponding defective genes, provide us with an opportunity to characterize the mechanisms involved in the coordinate regulation of peroxisomal beta-oxidation enzymes.

Photoaffinity labeling of acyl-CoA oxidase with 12-azidooleoyl-CoA and 12-[(4-azidosalicyl)amino]dodecanoyl-CoA.

Synthesis of 32P-labeled CoA of high specific activity was achieved using partially purified dephospho-CoA kinase (EC 2.7.1.24) from pig liver with [gamma-32P]ATP as donor and dephospho-CoA as acceptor. A photoaffinity dodecanoic acid analog, 12-[(4-azidosalicyl)amino]dodecanoic acid was synthesized, as were its CoA derivative (ASD-CoA) and the CoA derivative of 12-azidooleic acid. The CoA derivatives were synthesized from azido fatty acid analogs by acyl-CoA synthetase. The synthesized photolabile reagents were tested as photoaffinity labels for acyl-CoA oxidase (EC 1.3.99.3) from Arthrobacter species. When a mixture of oxidase and the acyl-CoA analogs were incubated in the absence of ultraviolet light, the analogs were recognized as substrate. Acyl-CoA oxidase was incubated in the presence of acyl-CoA analogs and immediately photolyzed, which resulted in irreversible inhibition. Oleoyl-CoA and dodecanoyl-CoA protect the enzyme from photoactivated inhibition by 12-azidooleoyl-CoA and ASD-CoA, respectively. Analysis of photolyzed enzyme preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that both analogs preferentially labeled a 54,000 molecular weight protein. These results demonstrate that the photoaffinity acyl-CoA analogs have potential application as probes to identify and characterize lipid biosynthetic enzymes and to identify the active site of these proteins.