Partial thyrocyte-specific Gαs deficiency leads to rapid-onset hypothyroidism, hyperplasia, and papillary thyroid carcinoma-like lesions in mice.

Thyroid function is controlled by thyroid-stimulating hormone (TSH), which binds to its G protein-coupled receptor [thyroid-stimulating hormone receptor (TSHR)] on thyrocytes. TSHR can potentially couple to all G protein families, but it mainly activates the Gs- and Gq/11-mediated signaling cascades. To date, there is a knowledge gap concerning the role of the individual G protein cascades in thyroid pathophysiology. Here, we demonstrate that the thyrocyte-specific deletion of Gs-protein α subunit (Gαs) in adult mice [tamoxifen-inducible Gs protein α subunit deficient (iTGαsKO) mice] rapidly impairs thyrocyte function and leads to hypothyroidism. Consequently, iTGαsKO mice show reduced food intake and activity. However, body weight and the amount of white adipose tissue were decreased only in male iTGαsKO mice. Unexpectedly, hyperplastic follicles and papillary thyroid cancer-like tumor lesions with increased proliferation and slightly increased phospho-ERK1/2 staining were found in iTGαsKO mice at an older age. These tumors developed from nonrecombined thyrocytes still expressing Gαs in the presence of highly elevated serum TSH. In summary, we report that partial thyrocyte-specific Gαs deletion leads to hypothyroidism but also to tumor development in thyrocytes with remaining Gαs expression. Thus, these mice are a novel model to elucidate the pathophysiological consequences of hypothyroidism and TSHR/Gs/cAMP-mediated tumorigenesis.-Patyra, K., Jaeschke, H., Löf, C., Jännäri, M., Ruohonen, S. T., Undeutsch, H., Khalil, M., Kero, A., Poutanen, M., Toppari, J., Chen, M., Weinstein, L. S., Paschke, R., Kero, J. Partial thyrocyte-specific Gαs deficiency leads to rapid-onset hypothyroidism, hyperplasia, and papillary thyroid carcinoma-like lesions in mice.

A mouse model with tamoxifen-inducible thyrocyte-specific cre recombinase activity.

We have created a mouse model expressing tamoxifen-inducible Cre recombinase (CreER(T2) ) under the control of the thyroglobulin (Tg) gene promoter to be able to study the role of defined genetic modifications in the regulation of thyroid function. We chose the thyroglobulin promoter, as it is expressed specifically in the thyroid. In order to obtain reliable expression under the control of the Tg promoter, we used a P1 artificial chromosome (PAC) containing a large piece of the Tg promoter. A tamoxifen inducible CreER(T2) construct was selected to avoid the possible consequences of the gene deletion for the development of the thyroid gland, and to study the role of gene deletion in the adult thyroid. Transgenic lines (TgCreER(T2) ) carrying this construct were generated and analyzed by crossing the TgCreER(T2) mice with the ROSA26LacZ reporter strain. The activity and specificity of the Cre recombinase was tested by staining for ß-galactosidase activity and by immunohistochemistry using an anti-Cre-antibody. In the TgCreER(T2) xROSA26LacZ reporter line, Cre-mediated recombination occurred specifically in the thyrocytes only after tamoxifen administration, and no significant staining was observed in controls. The recombination efficiency was nearly complete, since almost all thyrocytes showed X-gal staining. We could also induce the recombination in utero by giving tamoxifen to the pregnant female. In addition, mice expressing TgCreER(T2) had no obvious histological changes, hormonal alterations, or different response to growth stimuli as compared to controls. These results demonstrate that the TgCreER(T2) mouse line is a powerful tool to study temporally controlled deletion of floxed genes in the thyroid.

Hyperthyroidism and Papillary Thyroid Carcinoma in Thyrotropin Receptor D633H Mutant Mice.

BACKGROUND: Constitutively active thyrotropin receptor (TSHR) mutations are the most common etiology of non-autoimmune hyperthyroidism (NAH). Thus far, the functionality of these mutations has been tested in vitro, but the in vivo models are lacking. METHODS: To understand the pathophysiology of NAH, the patient-derived constitutively active TSHR D633H mutation was introduced into the murine Tshr by homologous recombination. RESULTS: In this model, both subclinical and overt hyperthyroidism was observed, depending on the age, sex, and genotype. Homozygous mice presented hyperthyroidism at two months of age, while heterozygous animals showed only suppressed thyrotropin. Interestingly, at six months of age, thyroid hormone concentrations in all mutant mice were analogous to wild-type mice, and they showed colloid goiter with flattened thyrocytes. Strikingly, at one year of age, nearly all homozygous mice presented large papillary thyroid carcinomas. Mechanistically, this papillary thyroid carcinoma phenotype was associated with an overactive thyroid and strongly increased stainings of proliferation-, pERK-, and NKX2-1 markers, but no mutations in the "hot-spot" areas of common oncogenes (Braf, Nras, and Kras) were found. CONCLUSIONS: This is the first study to reveal the dynamic age-, sex-, and genotype-dependent development of NAH. Furthermore, the study shows that a constitutively active TSHR can trigger a malignant transformation of thyrocytes.

Detection of Novel Gene Variants Associated with Congenital Hypothyroidism in a Finnish Patient Cohort.

BACKGROUND: Congenital hypothyroidism (CH) is defined as the lack of thyroid hormones at birth. Mutations in at least 15 different genes have been associated with this disease. While up to 20% of CH cases are hereditary, the majority of cases are sporadic with unknown etiology. Apart from a monogenic pattern of inheritance, multigenic mechanisms have been suggested to play a role in CH. The genetics of CH has not been studied in Finland so far. Therefore, multigenic sequencing of CH candidate genes was performed in a Finnish patient cohort with both familial and sporadic CH. METHODS: A targeted next-generation sequencing (NGS) panel, covering all exons of the major CH genes, was applied for 15 patients with sporadic and 11 index cases with familial CH. RESULTS: Among the familial cases, six pathogenic mutations were found in the TPO, PAX8, and TSHR genes. Furthermore, pathogenic NKX2.1 and TG mutations were identified from sporadic cases, together with likely pathogenic variants in the TG, NKX2.5, SLC26A4, and DUOX2 genes. All identified novel pathogenic mutations were confirmed by Sanger-sequencing and characterized in silico and/or in vitro. CONCLUSION: In summary, the CH panel provides an efficient, cost-effective, and multigenic screening tool for both known and novel CH gene mutations. Hence, it may be a useful method to identify accurately the genetic etiology for dyshormogenic, familial, or syndromic forms of CH.