The active thyroid hormone 3,5,3′ triiodothyronine (T3) is a major regulator of skeletal muscle function. The deiodinase family of enzymes controls the tissue-specific activation and inactivation of the prohormone thyroxine (T4). Here we show that type 2 deiodinase (D2) is essential for normal mouse myogenesis and muscle regeneration. Indeed, D2-mediated increases in T3 were essential for the enhanced transcription of myogenic differentiation 1 (MyoD) and for execution of the myogenic program. Conversely, the expression of T3-dependent genes was reduced and after injury regeneration markedly delayed in muscles of mice null for the gene encoding D2 (Dio2), despite normal circulating T3 concentrations. Forkhead box O3 (FoxO3) was identified as a key molecule inducing D2 expression and thereby increasing intracellular T3 production. Accordingly, FoxO3-depleted primary myoblasts also had a differentiation deficit that could be rescued by high levels of T3. In conclusion, the FoxO3/D2 pathway selectively enhances intracellular active thyroid hormone concentrations in muscle, providing a striking example of how a circulating hormone can be tissue-specifically activated to influence development locally. IntroductionThe active thyroid hormone 3,5,3′ triiodothyronine (T3) derives either directly from thyroid secretion or by the monodeiodination of the prohormone thyroxine (T4) by one of two iodothyronine selenodeiodinases. Type 1 deiodinase is expressed in the liver, kidney, and thyroid but not skeletal muscle of vertebrates, and T3 produced from T4 by this enzyme is largely released into the plasma. On the other hand, type 2 deiodinase (D2) is specifically expressed in the central nervous system, pituitary, thyroid gland, brown adipose tissue, retina, and skeletal muscle. Much of the T3 derived from D2-mediated deiodination remains within the cell (1). Thus, this deiodinase provides a mechanism by which thyroid hormone (TH) can be activated in a tissue-specific chronologically programmed fashion, such as during development, or in circumstances where there is a requirement for rapid increase in active TH in a specific tissue. The effectiveness of this mechanism has been shown in the D2-dependent feedback regulation by T4 of the thyrotropin-releasing hormone and thyroid-stimulating hormone (TSH) secretion by the hypothalamus and pituitary (2). A programmed transient increase in D2 is required for the proper increase in T3 at a critical time in the embryonic mouse brain (3) to allow normal development of the cochlea (4, 5), and a sympathetic nervous system-induced increase in D2 provides the cellular T3

The Sonic hedgehog (Shh) pathway plays a critical role in hair follicle physiology and is constitutively active in basal cell carcinomas (BCCs), the most common human malignancy. Type 3 iodothyronine deiodinase (D3), the thyroid hormone-inactivating enzyme, is frequently expressed in proliferating and neoplastic cells, but its role in this context is unknown. Here we show that Shh, through Gli2, directly induces D3 in proliferating keratinocytes and in mouse and human BCCs. We demonstrate that Gli-induced D3 reduces intracellular active thyroid hormone, thus resulting in increased cyclin D1 and keratinocyte proliferation. D3 knockdown caused a 5-fold reduction in the growth of BCC xenografts in nude mice. Shh-induced thyroid hormone degradation via D3 synergizes with the Shh-mediated reduction of the type 2 deiodinase, the thyroxine-activating enzyme, and both effects are reversed by cAMP. This previously unrecognized functional cross-talk between Shh/Gli2 and thyroid hormone in keratinocytes is a pathway by which Shh produces its proliferative effects and offers a potential therapeutic approach to BCC.basal cell carcinoma ͉ thyroxine ͉ differentiation ͉ cancer T hyroid hormone action is regulated by the activity of the deiodinases. Type 2 deiodinase (D2) activates the prohormone thyroxine (T4) by converting it to thyroid hormone (T3), whereas D3, by inactivating T3, terminates thyroid hormone action (1). All vertebrates express D2 and D3 that, in adults, contribute to plasma T3 and T4 homeostasis by their concerted actions with the hypothalamic-pituitary feedback axis. This homeostatic mechanism is possible because the Dio3 gene is transcriptionally stimulated by T3, whereas D2 is inhibited by two thyroid hormone-mediated effects, a transcriptional downregulation of Dio2 as well as protein inactivation by ubiquitination (for review, see ref.2). During development, preprogrammed changes in D2 and D3 expression are thought to regulate intracellular T3 concentrations essential to the normal development of the central nervous system, including the retina and the inner ear (3-6). However, the signals governing the changes in D2 and D3 expression during these complex processes are largely unknown.New insight into the developmental regulation of deiodinase expression has recently been obtained in the chicken growth plate, where Indian hedgehog induces WSB-1, an E3 ubiquitin ligase adaptor that inactivates D2 (7). The hedgehog pathway, acting through the Gli family of transcription factors, determines patterns of cell growth and differentiation in a wide variety of developmental settings (8-13). Given that, in general, signals regulating D2 expression affect D3 in a reciprocal fashion (2), we hypothesized that hedgehog proteins could up-regulate D3 while suppressing D2 expression. To explore this possibility, we turned to skin, a system in which Sonic hedgehog (Shh) is known to play dominant physiological as well as pathological roles (14). Both D2 and D3 are present in skin, a well recognized target of thyroid hormone...

Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome, which is characterized by cleft palate and severe defects of the skin, is an autosomal dominant disorder caused by mutations in the gene encoding transcription factor p63. Here, we report the generation of a knock-in mouse model for AEC syndrome (p63+/L514F) that recapitulates the human disorder. The AEC mutation exerts a selective dominant-negative function on wild-type p63 by affecting progenitor cell expansion during ectodermal development leading to a defective epidermal stem cell compartment. These phenotypes are associated with impairment of fibroblast growth factor (FGF) signalling resulting from reduced expression of Fgfr2 and Fgfr3, direct p63 target genes. In parallel, a defective stem cell compartment is observed in humans affected by AEC syndrome and in Fgfr2b−/− mice. Restoring Fgfr2b expression in p63+/L514F epithelial cells by treatment with FGF7 reactivates downstream mitogen-activated protein kinase signalling and cell proliferation. These findings establish a functional link between FGF signalling and p63 in the expansion of epithelial progenitor cells and provide mechanistic insights into the pathogenesis of AEC syndrome.

Thyroidectomized patients carrying Thr92Ala are at increased risk of reduced intracellular and serum T3 concentrations that are not adequately compensated for by LT4, thus providing evidence in favor of customized treatment of hypothyroidism in athyreotic patients.

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Ankyloblepharon, ectodermal defects, cleft lip/palate (AEC) syndrome is a rare autosomal dominant disorder caused by mutations in the p63 gene, essential for embryonic development of stratified epithelia. The most severe cutaneous manifestation of this disorder is the long-lasting skin fragility associated with severe skin erosions after birth. Using a knock-in mouse model for AEC syndrome, we found that skin fragility was associated with microscopic blistering between the basal and suprabasal compartments of the epidermis and reduced desmosomal contacts. Expression of desmosomal cadherins and desmoplakin was strongly reduced in AEC mutant keratinocytes and in newborn epidermis. A similar impairment in desmosome gene expression was observed in human keratinocytes isolated from AEC patients, in p63-depleted keratinocytes and in p63 null embryonic skin, indicating that p63 mutations causative of AEC syndrome have a dominant-negative effect on the wild-type p63 protein. Among the desmosomal components, desmocollin 3, desmoplakin and desmoglein 1 were the most significantly reduced by mutant p63 both at the RNA and protein levels. Chromatin immunoprecipitation experiments and transactivation assays revealed that p63 controls these genes at the transcriptional level. Consistent with reduced desmosome function, AEC mutant and p63-deficient keratinocytes had an impaired ability to withstand mechanical stress, which was alleviated by epidermal growth factor receptor inhibitors known to stabilize desmosomes. Our study reveals that p63 is a crucial regulator of a subset of desmosomal genes and that this function is impaired in AEC syndrome. Reduced mechanical strength resulting from p63 mutations can be alleviated pharmacologically by increasing desmosome adhesion with possible therapeutic implications.

SummaryPrecise control of the thyroid hormone (T3)-dependent transcriptional program is required by multiple cell systems, including muscle stem cells. Deciphering how this is achieved and how the T3 signal is controlled in stem cell niches is essentially unknown. We report that in response to proliferative stimuli such as acute skeletal muscle injury, type 3 deiodinase (D3), the thyroid hormone-inactivating enzyme, is induced in satellite cells where it reduces intracellular thyroid signaling. Satellite cell-specific genetic ablation of dio3 severely impairs skeletal muscle regeneration. This impairment is due to massive satellite cell apoptosis caused by exposure of activated satellite cells to the circulating TH. The execution of this proapoptotic program requires an intact FoxO3/MyoD axis, both genes positively regulated by intracellular TH. Thus, D3 is dynamically exploited in vivo to chronically attenuate TH signaling under basal conditions while also being available to acutely increase gene programs required for satellite cell lineage progression.

Epithelial tumor progression often involves epithelial-mesenchymal transition (EMT). We report that increased intracellular levels of thyroid hormone (TH) promote the EMT and malignant evolution of squamous cell carcinoma (SCC) cells. TH induces the EMT by transcriptionally up-regulating ZEB-1, mesenchymal genes and metalloproteases and suppresses E-cadherin expression. Accordingly, in human SCC, elevated D2 (the T3-producing enzyme) correlates with tumor grade and is associated with an increased risk of postsurgical relapse and shorter disease-free survival. These data provide the first in vivo demonstration that TH and its activating enzyme, D2, play an effective role not only in the EMT but also in the entire neoplastic cascade starting from tumor formation up to metastatic transformation, and supports the concept that TH is an EMT promoter. Our studies indicate that tumor progression relies on precise T3 availability, suggesting that pharmacological inactivation of D2 and TH signaling may suppress the metastatic proclivity of SCC.