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Recently the attention of the scientific community has focused on the ability of polyphenols to counteract adverse epigenetic regulation involved in the development of complex conditions such as obesity. The aim of this study was to investigate the epigenetic mechanisms underlying the anti-adiposity effect of Quercetin (3,3′,4′,5,7-pentahydroxyflavone) and of one of its derivatives, Q2 in which the OH groups have been replaced by acetyl groups. In 3 T3-L1 preadipocytes, Quercetin and Q2 treatment induce chromatin remodeling and histone modifications at the 5′ regulatory region of the two main adipogenic genes, c/EBPα and PPARγ. Chromatin immunoprecipitation assays revealed a concomitant increase of histone H3 di-methylation at Lys9, a typical mark of repressed gene promoters, and a decrease of histone H3 di-methylation at Lys 4, a mark of active transcription. At the same time, both compounds inhibited histone demethylase LSD1 recruitment to the 5′ region of c/EBPα and PPARγ genes, a necessary step for adipogenesis. The final effect is a significant reduction in c/EBPα and PPARγ gene expression and attenuated adipogenesis. Q2 supplementation in rats reduced the gain in body weight and in white adipose tissue, as well as the increase in adipocyte size determined by high fat diet. Moreover, Q2 improved dyslipidemia, glucose tolerance and decreased the hepatic lipid accumulation by activating the expression of beta-oxidation related genes. Our data suggest that Q2, as well as Quercetin, has the potential to revert the unfavorable epigenomic profiles associated with obesity onset. This opens the possibility to use these compounds in targeted prevention strategies against obesity.

Background RAS mutations are the only validated biomarkers in metastatic colorectal cancer (mCRC) for anti-epidermal growth factor receptor (EGFR) therapy. Limited clinical information is available on AXL expression, marker of epithelial to mesenchymal transition, in mCRC. Methods AXL was retrospectively assessed by immunohistochemistry in 307 patients. RAS wild-type (WT) patients (N = 136) received first-line anti-EGFR–based therapy; RAS mutant patients (N = 171) received anti-angiogenic–based regimens. Preclinical experiments were performed using human RAS WT CRC cell lines and xenograft models. AXL RNA levels were assessed in a cohort of patients with available samples at baseline and at progression to anti-EGFR treatment and in the GSE5851 dataset. Results AXL was expressed in 55/307 tumour tissues, correlating with worse survival in the overall population (AXL-positive, 23.7 months; AXL-negative, 30.8 months; HR, 1.455, P = 0.032) and in RAS WT patients (AXL-positive, 23.0 months; AXL-negative, 35.8 months; HR,1.780, P = 0.032). Progression-free survival (PFS) in the RAS WT cohort was shorter in the AXL-positive cohort (6.2 months versus 12.1 months; HR, 1.796, P = 0.013). Three-dimensional cultures obtained from a patient following anti-EGFR therapy resulted AXL-positive, showing resistance to anti-EGFR drugs and sensitivity to AXL inhibition. AXL transfection in CRC cell lines induced AXL overexpression and resistance to the EGFR blockade. At progression to cetuximab, 2/10 SW48-tumour xenograft mice showed AXL expression. Consistently, AXL RNA levels increased in 5/7 patients following anti-EGFR therapy. Moreover, in the GSE5851 dataset higher AXL RNA levels correlated with worse PFS with cetuximab in KRAS-exon2 WT chemorefractory patients. Conclusions AXL is a marker of poor prognosis in mCRC with consistent clinical and preclinical evidences of involvement in primary and acquired resistance to anti-EGFR drugs in RAS WT patients.

Thyroid hormone (TH) is a pleiotropic agent that has widespread biological functions, i.e., it controls cellular growth, tissue development and homeostasis and neoplastic transformation. Suitable TH levels are critical for the development of various types of tissues and are essential for the regulation of metabolic processes throughout life. The serum concentrations of TH affect its biological activity. Moreover, at tissue level, TH action is regulated by the expression and activity of deiodinases, i.e., the enzymes that mediate the metabolic pathways by activating and/or inactivating TH. The type I and II deiodinases (D1 and D2) initiate TH action by converting thyroxine (T4) into the active TH form (T3), whereas type III deiodinase (D3) mediates the local attenuation of TH by converting T4 and T3 into the inactive metabolites rT3 and T2, respectively. The deiodinase system is a potent mechanism of pre-receptoral control of TH action; it is often altered in such pathological conditions as cancer. D3 is widely expressed in embryonic tissues and in placenta, where it blocks excessive maternal-to-fetal transfer of TH. In contrast, during late neonatal and adult life, D3 is expressed mainly in the central nervous system and skin. Interestingly, D3 expression is re-activated in various types of human cancers. Here we review recent evidence that D3 expression plays a crucial role in human carcinogenesis, and speculate as to its complex role in the regulation of cell proliferation in several neoplastic contexts. It is conceivable that the local modulation of TH action via deiodinases is a powerful molecular tool to manipulate the intracellular TH status, thus influencing the growth and maintenance of selected hormone-dependent cancers.

Atypical lipomatous tumors (ALTs)/well-differentiated liposarcomas represent a distinctive subset of mesenchymal neoplasms featuring mature adipocytic proliferation. These tumors are characterized cytogenetically by the presence of supernumerary ring and/or long marker chromosomes that contain several copies of the chromosomal region 12q13-15, in which the HMGA2 gene is located. Deregulation of the HMGA2 gene is a common molecular alteration implicated in the development of a variety of benign tumors, such as lipomas, uterine leiomyomas, and pulmonary chondroid hamartomas. In this study, we observed HMGA2 overexpression in 7 of 12 ALT primary cell cultures examined. Subsequently, we generated an adenovirus containing the HMGA2 gene in the antisense orientation (Ad-A2as) to study the effect of HMGA2 protein suppression in ALT cells. The infection of six ALT cells, three of which were positive for HMGA2 expression, resulted in growth inhibition coupled with a significant increase in apoptosis. In addition, the growth of the ALT cells negative for HMGA2 expression was not affected by the infection with either the Ad-A2as or the control virus. On the basis of these findings, the targeting of the HMGA2 protein expression may represent a promising approach for treating the well-differentiated liposarcomas resistant to conventional therapies.

The high mobility group (HMG) proteins (HMGA1a, HMGA1b, and HMGA2) bind to DNA and interact with various transcriptional factors. Therefore, they play an important role in chromatin organization. HMGA protein expression is low in normal adult tissues, but abundant during embryonic development and in several experimental and human tumors. Blockage of HMGA expression inhibits the transformation of rat thyroid PC Cl 3 cells treated with oncogene-carrying retroviruses, thus implicating HMGA in rat thyroid transformation. To better understand the role of HMGA and to establish whether its up-regulated expression is sufficient to induce the transformed phenotype, we generated PC Cl 3 cells that overexpress the protein. We demonstrate that HMGA1b protein overexpression does not transform normal rat thyroid PC Cl 3 cells, but it deregulates their cell cycle: cells enter S-phase earlier and the G(2)-M transition is delayed. HMGA1-overexpressing cells undergo apoptosis through a pathway involving caspase-3 activation, probably consequent to the conflict between mitogenic pressure and the inability to proceed through the cell cycle. Using various HMGA1b gene mutations, we found that the third AT-hook domain and the acetylation site K60 are the protein regions required for induction of apoptosis in PC Cl 3 cells. In conclusion, although HMGA1 protein overexpression is associated with the malignant phenotype of rat and human thyroid cells, it does not transform normal thyroid cells in culture but leads them to programmed cell death.

Background: The bone synthesizing function of osteoblasts (OBs) is a highly demanding energy process that requires nutrients. However, how nutrient availability affects OBs behavior and bone mineralization remain to be fully understood. Methods: MC3T3-E1 cell line and primary OBs (OBs) cultures were treated with physiological levels of glucose (G; 5.5 mM) alone or with the addition of palmitic acid (G+PA) at different concentrations. Mitochondria morphology and activity were evaluated by fluorescence microscopy, qPCR, and oxygen consumption rate (OCR) measurement, and OBs function was assessed by mineralization assay. Results: The addition of non-lipotoxic levels of 25 μM PA to G increased mineralization in OBs. G+25 μM PA exposure reduced mitochondria size in OBs, which was associated with increased activation of dynamin-related protein 1, a mitochondrial fission protein, enhanced mitochondria OCR and ATP production, and increased expression of oxidative phosphorylation genes. Treatment with Mdivi-1, a putative inhibitor of mitochondrial fission, reduced osteogenesis and mitochondrial respiration in OBs. Conclusions: Our results revealed that OBs function was enhanced in the presence of glucose and PA at 25 μM. This was associated with increased OBs mitochondrial respiration and dynamics. These results suggest a role for nutrient availability in bone physiology and pathophysiology.

Background: The type 2 deiodinase (DIO2) converts thyroxine to 3,3′,5-triiodothyronine (T3), modulating intracellular T3. An increase in DIO2 within muscle stem cells during skeletal muscle regeneration leads to T3-dependent potentiation of differentiation. The muscle stem cell niche comprises numerous cell types, which coordinate the regeneration process. For example, muscle stem cells provide secretory signals stimulating endothelial cell-mediated vascular repair, and, in turn, endothelial cells promote muscle stem differentiation. We hypothesized that Dio2 loss in muscle stem cells directly impairs muscle stem cell-endothelial cell communication, leading to downstream disruption of endothelial cell function. Methods: We assessed the production of proangiogenic factors in differentiated C2C12 cells and in a C2C12 cell line without Dio2 (D2KO C2C12) by real-time quantitative-polymerase chain reaction and enzyme-linked immunosorbent assay. Conditioned medium (CM) was collected daily in parallel to evaluate its effects on human umbilical vein endothelial cell (HUVEC) proliferation, migration and chemotaxis, and vascular network formation. The effects of T3-treatment on vascular endothelial growth factor (Vegfa) mRNA expression in C2C12 cells and mouse muscle were assessed. Chromatin immunoprecipitation (ChIP) identified thyroid hormone receptor (TR) binding to the Vegfa gene. Using mice with a targeted disruption of Dio2 (D2KO mice), we determined endothelial cell number by immunohistochemistry/flow cytometry and evaluated related gene expression in both uninjured and injured skeletal muscle. Results: In differentiated D2KO C2C12 cells, Vegfa expression was 46% of wildtype (WT) C2C12 cells, while secreted VEGF was 45%. D2KO C2C12 CM exhibited significantly less proangiogenic effects on HUVECs. In vitro and in vivo T3 treatment of C2C12 cells and WT mice, and ChIP using antibodies against TRα, indicated that Vegfa is a direct genomic T3 target. In uninjured D2KO soleus muscle, Vegfa expression was decreased by 28% compared with WT mice, while endothelial cell numbers were decreased by 48%. Seven days after skeletal muscle injury, D2KO mice had 36% fewer endothelial cells, coinciding with an 83% decrease in Vegfa expression in fluorescence-activated cell sorting purified muscle stem cells. Conclusion: Dio2 loss in the muscle stem cell impairs muscle stem cell-endothelial cell crosstalk via changes in the T3-responsive gene Vegfa, leading to downstream impairment of endothelial cell function both in vitro and in vivo.

Thyroid hormones (THs) are key metabolic regulators coordinating short- and long-term energy needs. In skeletal muscle, THs modulate energy metabolism in pathophysiological conditions. Indeed, hypo- and hyperthyroidism are leading causes of muscle weakness and strength; however, the metabolic pathways underlying these effects are still poorly understood. Using molecular, biochemical, and isotope-tracing approaches combined with mass spectrometry and denervation experiments, we find that THs regulate glutamine metabolism and anaplerotic fluxes by up-regulating the glutamate pyruvate transaminase 2 (GPT2) gene. In humans, GPT2 autosomal recessive mutations cause a neurological syndrome characterized by intellectual disability, microcephaly, and progressive motor symptoms. Here, we demonstrate a role of the TH/GPT2 axis in skeletal muscle in which it regulates muscle weight and fiber diameter in resting and atrophic conditions and results in protection from muscle loss during atrophy. These results describe an anabolic route by which THs rewire glutamine metabolism toward the maintenance of muscle mass.

Prostate Cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in males and the fifth leading cause of death worldwide. The majority of PCas are androgen-sensitive, with a significant up-regulation of Androgen Receptor (AR) that causes a stimulatory effect on growth and progression of cancer cells. For this reason, the first-line therapy for PCa is androgen ablation, even if it ultimately fails due to the onset of hormone-refractory state, in which the malignant cells do not sense the androgen signal anymore. Besides androgens, a growing number of evidence suggests that Thyroid Hormones (THs) mediate tumor-promoting effects in a variety of human cancers, as Epithelial-to-Mesenchymal Transition (EMT), invasion and metastasis and also stimulation of angiogenesis and tumor metabolism. Moreover, epidemiological studies demonstrated an increased risk for PCa in patients with lower levels of Thyreotropin (TSH). Here, we investigated if intracellular TH metabolism affects Benign Prostatic Hyperplasia (BPH) and PCa formation and progression. We found that the intracellular TH metabolism is a crucial determinant of PCa behavior. We observed that a dynamic stage-specific expression of the THs modulating enzymes, the deiodinases, is required for the progression of BPH to PCa malignancy. By acting simultaneously on epithelial cancer cells and fibroblasts, THs exert a proliferative and pro-inflammatory effect cooperating with androgens. These findings suggest that androgens and THs may interplay and mediate a coordinate effect on human PCa formation and progression. In light of our results, future perspective could be to explore the potential benefits of THs intracellular modulators aimed to counteract PCa progression.

Thyroid hormones (THs) are key regulators of different biological processes. Their action involves genomic and non-genomic mechanisms, which together mediate the final effects of TH in target tissues. However, the proportion of the two processes and their contribution to the TH-mediated effects are still poorly understood. Skeletal muscle is a classical target tissue for TH, which regulates muscle strength and contraction, as well as energetic metabolism of myofibers. Here we address the different contribution of genomic and non-genomic action of TH in skeletal muscle cells by specifically silencing the deiodinase Dio2 or the β3-Integrin expression via CRISPR/Cas9 technology. We found that myoblast proliferation is inversely regulated by integrin signal and the D2-dependent TH activation. Similarly, inhibition of the nuclear receptor action reduced myoblast proliferation, confirming that genomic action of TH attenuates proliferative rates. Contrarily, genomic and non-genomic signals promote muscle differentiation and the regulation of the redox state. Taken together, our data reveal that integration of genomic and non-genomic signal pathways finely regulates skeletal muscle physiology. These findings not only contribute to the understanding of the mechanisms involved in TH modulation of muscle physiology but also add insight into the interplay between different mechanisms of action of TH in muscle cells.

Background: Many physiological effects of thyroid hormone (TH) are mediated by its canonical action via nuclear receptors (TH receptor α and β [TRα and TRβ]) to regulate transcription of target genes. Heterozygous dominant negative mutations in human TRα mediate resistance to thyroid hormone alpha (RTHα), characterized by features of hypothyroidism (e.g., skeletal dysplasia, neurodevelopmental retardation, constipation) in specific tissues, but near-normal circulating TH concentrations. Hitherto, 41 RTHα cases have been recorded worldwide. Methods: RTHα cases (n = 10) attending a single center underwent cutaneous assessment, recording skin lesions. Lesions excised from different RTHα patients were analyzed histologically and profiled for cellular markers of proliferation and oncogenic potential. Proliferative characteristics of dermal fibroblasts and inducible pluripotent stem cell (iPSC)-derived keratinocytes from patients and control subjects were analyzed. Results: Multiple skin tags and nevi were recorded in all cases, mainly in the head and neck area with a predilection for flexures. The affected patients had highly deleterious mutations (p.E403X, p.E403K, p.F397fs406X, p.A382PfsX7) involving TRα1 alone or mild/moderate loss-of-function mutations (p.A263V, p.L274P) common to TRα1 and TRα2 isoforms. In four patients, although lesions excised for cosmetic reasons were benign intradermal melanocytic nevi histologically, they significantly overexpressed markers of cell proliferation (K17, cyclin D1) and type 3 deiodinase. In addition, oncogenic markers typical of basal cell carcinoma (Gli-1, Gli-2, Ptch-1, n = 2 cases) and melanoma (c-kit, MAGE, CDK4, n = 1) were markedly upregulated in skin lesions. Cell cycle progression and proliferation of TRα mutation-containing dermal fibroblasts and iPSC-derived keratinocytes from patients were markedly increased. Conclusions: Our observations highlight frequent occurrence of skin tags and benign melanocytic nevi in RTHα, with cutaneous cells from patients being in a hyperproliferative state. Such excess of skin lesions, including nevi expressing oncogenic markers, indicates that dermatologic surveillance of RTHα patients, monitoring lesions for features that are suspicious for neoplastic change, is warranted.

Simple Summary Cancer cells rewire their metabolism to promote growth, survival, proliferation, and long-term maintenance. Aerobic glycolysis is a prominent trait of many cancers; contextually, glutamine addiction, enhanced glucose uptake and aerobic glycolysis sustain the metabolic needs of rapidly proliferating cancer cells. Thyroid hormone (TH) is a positive regulator of tumor progression and metastatic conversion of squamous cell carcinoma (SCC). Accordingly, overexpression of the TH activating enzyme, D2, is associated with metastatic SCC. The aim of our study was to assess the ability of TH and its activating enzyme in promoting key tracts of cancer progression such as angiogenesis, response to hypoxia and metabolic adaptation. By performing in vivo and in vitro studies, we demonstrate that TH induces VEGF-A in cancer cells and fosters aerobic glycolysis inducing pro-glycolytic mediators, thus implying that TH signal attenuation represents a therapeutic tool to contrast tumor angiogenesis and tumor progression. Abstract Cancer angiogenesis is required to support energetic demand and metabolic stress, particularly during conditions of hypoxia. Coupled to neo-vasculogenesis, cancer cells rewire metabolic programs to sustain growth, survival and long-term maintenance. Thyroid hormone (TH) signaling regulates growth and differentiation in a variety of cell types and tissues, thus modulating hyper proliferative processes such as cancer. Herein, we report that TH coordinates a global program of metabolic reprogramming and induces angiogenesis through up-regulation of the VEGF-A gene, which results in the enhanced proliferation of tumor endothelial cells. In vivo conditional depletion of the TH activating enzyme in a mouse model of cutaneous squamous cell carcinoma (SCC) reduces the concentration of TH in the tumoral cells and results in impaired VEGF-A production and attenuated angiogenesis. In addition, we found that TH induces the expression of the glycolytic genes and fosters lactate production, which are key traits of the Warburg effect. Taken together, our results reveal a TH–VEGF-A–HIF1α regulatory axis leading to enhanced angiogenesis and glycolytic flux, which may represent a target for SCC therapy.

The type 2 iodothyronine-deiodinase (D2) enzyme converts T(4) to T(3), and mice deficient in this enzyme [D2 knockout (D2KO) mice] have decreased T(3) derived from T(4) in skeletal muscle despite normal circulating T(3) levels. Because slow skeletal muscle is particularly susceptible to changes in T(3) levels, we expected D2 inactivation to result in more pronounced slow-muscle characteristics in the soleus muscle, mirroring hypothyroidism. However, ex vivo studies of D2KO soleus revealed higher rates of twitch contraction and relaxation and reduced resistance to fatigue. Immunostaining of D2KO soleus showed that these properties were associated with changes in muscle fiber type composition, including a marked increase in the number of fast, glycolytic type IIB fibers. D2KO soleus muscle fibers had a larger cross-sectional area, and this correlated with increased myonuclear accretion in myotubes formed from D2KO skeletal muscle precursor cells differentiated in vitro. Consistent with our functional findings, D2KO soleus gene expression was markedly different from that in hypothyroid wild-type (WT) mice. Comparison of gene expression between euthyroid WT and D2KO mice indicated that PGC-1α, a T(3)-dependent regulator of slow muscle fiber type, was decreased by ∼50% in D2KO soleus. Disruption of Dio2 in the C2C12 myoblast cell line led to a significant decrease in PGC-1α expression and a faster muscle phenotype upon differentiation. These results indicate that D2 loss leads to significant changes in soleus contractile function and fiber type composition that are inconsistent with local hypothyroidism and suggest that reduced levels of PCG-1α may contribute to the observed phenotypical changes.

Proliferation of pancreatic acinar cells is a critical process in the pathophysiology of pancreatic diseases, because limited or defective proliferation is associated with organ dysfunction and patient morbidity. In this context, elucidating the signalling pathways that trigger and sustain acinar proliferation is pivotal to develop therapeutic interventions promoting the regenerative process of the organ. In this study we used genetic and pharmacological approaches to manipulate both local and systemic levels of thyroid hormones to elucidate their role in acinar proliferation following caerulein-mediated acute pancreatitis in mice. In addition, molecular mechanisms mediating the effects of thyroid hormones were identified by genetic and pharmacological inactivation of selected signalling pathways.In this study we demonstrated that levels of the thyroid hormone 3,3',5-triiodo-l-thyronine (T3) transiently increased in the pancreas during acute pancreatitis. Moreover, by using genetic and pharmacological approaches to manipulate both local and systemic levels of thyroid hormones, we showed that T3 was required to promote proliferation of pancreatic acinar cells, without affecting the extent of tissue damage or inflammatory infiltration.Finally, upon genetic and pharmacological inactivation of selected signalling pathways, we demonstrated that T3 exerted its mitogenic effect on acinar cells via a tightly controlled action on different molecular effectors, including histone deacetylase, AKT, and TGFβ signalling.In conclusion, our data suggest that local availability of T3 in the pancreas is required to promote acinar cell proliferation and provide the rationale to exploit thyroid hormone signalling to enhance pancreatic regeneration. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Interest in thyroid hormone (TH) in cancer was first aroused by the demonstration, over a century ago, that breast cancer responded to thyroid extract treatment. This suggested that TH was a key regulator of tumorigenesis. However, the role of TH in the complex process of neoplastic transformation long remained obscure. Interest in the link between TH and cancer was renewed when it became clear that TH receptors and modulators are often altered in cancerous tissues and that modulation of TH might foster cancer cell expansion. Studies in two different epithelial cancers, namely the Basal Cell Carcinoma of the skin and the colon cancer have provided molecular insight the role of TH modulation at intracellular level in tumor formation, thus prompting interest in tissue-specific TH modulation as an anti-tumoral agent. Since then a large body of data has accumulated on this topic and the aim of this brief review is to try to draw together the evidence pointing to a general mechanism by which TH interferes with oncogenic pathways, thus affecting tumoral formation.

Melanoma is the most lethal form of skin cancer and its incidence is growing worldwide. In the last ten years, the therapeutic scenario of this disease has been revolutionized by the introduction of targeted therapies and immune-checkpoint inhibitors. However, in patients with many lesions and bulky tumors, in which surgery is no longer feasible, there is a need for new treatment options. Here we report, for the first time to our knowledge, a clinical case where a melanoma patient harboring the SMO p.Gln216Arg mutation has been treated with imiquimod, showing a complete and durable response. To better explain this outstanding response to the treatment, we transfected a melanoma cell line (MeWo) with the SMO p.Gln216Arg mutation in order to evaluate its role in response to the imiquimod treatment. Moreover, to better demonstrate that the antitumor activity of imiquimod was due to its role in suppressing the oncogenic SMO signaling pathway, independently of its immune modulating function, an in vivo experiment has been performed. This clinical case opens up a new scenario for the treatment of melanoma patients identifying a new potentially druggable target.

Type 2 deiodinase (D2), the principal activator of thyroid hormone (TH) signaling in target tissues, is expressed in cutaneous squamous cell carcinomas (SCCs) during late tumorigenesis, and its repression attenuates the invasiveness and metastatic spread of SCC. Although D2 plays multiple roles in cancer progression, nothing is known about the mechanisms regulating D2 in cancer. To address this issue, we investigated putative upstream regulators of D2 in keratinocyte carcinomas. We found that the expression of D2 in SCC cells is positively regulated by the NANOG transcription factor, whose expression, besides being causally linked to embryonic stemness, is associated with many human cancers. We also found that NANOG binds to the D2 promoter and enhances D2 transcription. Notably, blockage of D2 activity reduced NANOG-induced cell migration as well as the expression of key genes involved in epithelial&ndash, mesenchymal transition in SCC cells. In conclusion, our study reveals a link among endogenous endocrine regulators of cancer, thyroid hormone and its activating enzyme, and the NANOG regulator of cancer biology. These findings could provide the basis for the development of TH inhibitors as context-dependent anti-tumor agents.

The original version of this Article contained an error in the author affiliations. Silvia Varricchio, Gennaro Ilardi and Stefania Staibanow were incorrectly associated with ‘Department of Public Health, University of Naples "Federico II", Naples, Italy’ instead of the correct ‘Department of Advanced Biomedical Sciences, University of Naples “Federico II”, Naples, Italy.’ This has now been corrected in both the PDF and HTML versions of the Article.

Background: Thyroid hormones (THs) are key regulators of development, tissue differentiation, and maintenance of metabolic balance in virtually every cell of the body. Accordingly, severe alteration of TH action during fetal life leads to permanent deficits in humans. The skin is among the few adult tissues expressing the oncofetal protein type 3 deiodinase (D3), the TH inactivating enzyme. Here, we demonstrate that D3 is dynamically regulated during epidermal ontogenesis. Methods: To investigate the function of D3 in a postdevelopmental context, we used a mouse model of conditional epidermal-specific D3 depletion. Loss of D3 resulted in tissue hypoplasia and enhanced epidermal differentiation in a cell-autonomous manner. Results: Accordingly, wound healing repair and hair follicle cycle were altered in the D3-depleted epidermis. Further, in vitro ablation of D3 in primary culture of keratinocytes indicated that various markers of stratified epithelial layers were upregulated, thereby confirming the pro-differentiative action of D3 depletion and the consequent increased intracellular triiodothyronine levels. Notably, loss of D3 reduced the clearance of systemic TH in vivo, thereby demonstrating the critical requirement for epidermal D3 in the maintenance of TH homeostasis. Conclusion: In conclusion, our results show that the D3 enzyme is a key TH-signaling component in the skin, thereby providing a striking example of a physiological context for deiodinase-mediated TH metabolism, as well as a rationale for therapeutic manipulation of deiodinases in pathophysiological contexts.

Thyroid hormone action defects (THADs) have been classically considered conditions of impaired sensitivity to thyroid hormone (TH). They were originally referring to alterations in TH receptor genes (THRA and THRB), but the discovery of genetic mutations and polymorphisms causing alterations in cell membrane transport (e.g., MCT8) and metabolism (e.g., SECISBP2, DIO2) led recently to a new and broader definition of TH hyposensitivity (THH), including not only THADs but all defects that could interfere with the activity of TH. Due to the different functions and tissue-specific expression of these genes, affected patients exhibit highly variable phenotypes. Some of them are characterized by a tissue hypothyroidism or well-recognizable alterations in the thyroid function tests (TFTs), whereas others display a combination of hypo- and hyperthyroid manifestations with normal or only subtle biochemical defects. The huge effort of basic research has greatly aided the comprehension of the molecular mechanisms underlying THADs, dissecting the morphological and functional alterations on target tissues, and defining the related-changes in the biochemical profile. In this review, we describe different pictures in which a specific alteration in the TFTs (TSH, T4, and T3 levels) is caused by defects in a specific gene. Altogether these findings can help clinicians to early recognize and diagnose THH and to perform a more precise genetic screening and therapeutic intervention. On the other hand, the identification of new genetic variants will allow the generation of cell-based and animal models to give novel insight into thyroid physiology and establish new therapeutic interventions.

The deiodinase family of enzymes mediates the activation and inactivation of thyroid hormone. The role of these enzymes in the regulation of the systemic concentrations of thyroid hormone is well established and underpins the treatment of common thyroid diseases. Interest in this field has increased in the past 10 years as the deiodinases became implicated in tissue development and homeostasis, as well as in the pathogenesis of a wide range of human diseases. Three deiodinases have been identified, namely, types 1, 2 and 3 iodothyronine deiodinases, which differ in their catalytic properties and tissue distribution. Notably, the expression of these enzymes changes during the lifetime of an individual in relation to the different needs of each organ and to ageing. The systemic homeostatic role of deiodinases clearly emerges during changes in serum concentrations of thyroid hormone, as seen in patients with thyroid dysfunction. By contrast, the role of deiodinases at the tissue level allows thyroid hormone signalling to be finely tuned within a given cell in a precise time–space window without perturbing serum concentrations of thyroid hormone. This Review maps the overall functional role of the deiodinases and explores challenges and novel opportunities arising from the expanding knowledge of these ‘master’ components of the thyroid homeostatic system. This Review discusses the two main functions of the human deiodinases: the homeostatic control of plasma concentrations of thyroid hormone and the control of intracellular T3 concentrations.

Muscular dystrophy-associated mutations of TRIM32 impair ULK1-dependent autophagy response during muscle atrophy., Optimal autophagic activity is crucial to maintain muscle integrity, with either reduced or excessive levels leading to specific myopathies. LGMD2H is a muscle dystrophy caused by mutations in the ubiquitin ligase TRIM32, whose function in muscles remains not fully understood. Here, we show that TRIM32 is required for the induction of muscle autophagy in atrophic conditions using both in vitro and in vivo mouse models. Trim32 inhibition results in a defective autophagy response to muscle atrophy, associated with increased ROS and MuRF1 levels. The proautophagic function of TRIM32 relies on its ability to bind the autophagy proteins AMBRA1 and ULK1 and stimulate ULK1 activity via unanchored K63-linked polyubiquitin. LGMD2H-causative mutations impair TRIM32’s ability to bind ULK1 and induce autophagy. Collectively, our study revealed a role for TRIM32 in the regulation of muscle autophagy in response to atrophic stimuli, uncovering a previously unidentified mechanism by which ubiquitin ligases activate autophagy regulators.

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., SCOPUS: ar.j, info:eu-repo/semantics/published

Thyroid hormone (TH) is a key metabolic regulator that acts by coordinating short- and long-term energy needs. Accordingly, significant metabolic changes are observed depending on thyroid status. Although it is established that hyperthyroidism augments basal energy consumption, thus resulting in an enhanced metabolic state, the net effects on cellular respiration and generation of reactive oxygen species (ROS) remain unclear. To elucidate the effects of augmented TH signal in muscle cells, we generated a doxycycline-inducible cell line in which the expression of the TH-activating enzyme, type 2 deiodinase (D2), is reversibly turned on by the "Tet-ON" system. Interestingly, increased intracellular TH caused a net shift from oxidative phosphorylation to glycolysis and a consequent increase in the extracellular acidification rate. As a result, mitochondrial ROS production, and both the basal and doxorubicin-induced production of cellular ROS were reduced. Importantly, the expression of a set of antioxidant genes was up-regulated, and, among them, the mitochondrial scavenger Sod2 was specifically induced at transcriptional level by D2-mediated TH activation. Finally, we observed that attenuation of oxidative stress and increased levels of SOD2 are key elements of the differentiating cascade triggered by TH and D2, thereby establishing that D2 is essential in coordinating metabolic reprogramming of myocytes during myogenic differentiation. In conclusion, our findings indicate that TH plays a key role in oxidative stress dynamics by regulating ROS generation. Our novel finding that TH and its intracellular metabolism act as mitochondrial detoxifying agents sheds new light on metabolic processes relevant to muscle physiology.

Thyroid hormone is a pleiotropic factor that controls many cellular processes in multiple cell types such as cancer stem cells (CSC). Thyroid hormone concentrations in the blood are stable, but the action of the deiodinases (D2–D3) provides cell-specific regulation of thyroid hormone activity. Deregulation of deiodinase function and thyroid hormone status has been implicated in tumorigenesis. Therefore, we investigated the role of thyroid hormone metabolism and signaling in colorectal CSCs (CR-CSC), where deiodinases control cell division and chemosensitivity. We found that increased intracellular thyroid hormone concentration through D3 depletion induced cell differentiation and sharply mitigated tumor formation. Upregulated BMP4 expression and concomitantly attenuated Wnt signaling accompanied these effects. Furthermore, we demonstrate that BMP4 is a direct thyroid hormone target and is involved in a positive autoregulatory feedback loop that modulates thyroid hormone signaling. Collectively, our findings highlight a cell-autonomous metabolic mechanism by which CR-CSCs exploit thyroid hormone signaling to facilitate their self-renewal potential and suggest that drug-induced cell differentiation may represent a promising therapy for preventing CSC expansion and tumor progression. Cancer Res; 76(5); 1237–44. ©2015 AACR.

Liver diseases affect millions of people worldwide. In most of the cases, severe hepatic dysfunction and liver cancer stem from mild and common clinical signs including hepatic steatosis, insulin resistance, liver inflammation, and oxidative stress, all together referred to as Nonalcoholic Fatty Liver Disease (NAFLD). Nutraceuticals endowed with antioxidant activity have been shown to reduce NAFLD risk factors and exert hepatoprotective effects. Here, we test the protective effect exerted on liver by the antioxidant Taurisolo, a nutraceutical formulation produced by grape pomace and enriched in Resveratrol and Polyphenols. We analyze the effect of Taurisolo on liver cells by profiling the metabolome of in vitro cultured hepatic HuH7 cells and of C57BL-6J mice fed a High Fat Diet and treated with the nutraceutical. Both in vitro and in vivo, we provide evidence that Taurisolo reduces risk factor markers associated with NAFLD. Taurisolo stimulates glucose uptake and reduces hepatic cholesterol and serum triglycerides. Furthermore, we give new insights into the mechanism of action of Taurisolo. The nutraceutical increases mitochondrial activity and promotes respiration and ATP production, fostering catabolic reactions like fatty acid &beta, oxidation and amino acid catabolism. On the contrary, Taurisolo reduces anabolic reactions like biosynthesis of cholesterol, bile acids, and plasma membrane lipids.

Thyroid hormones (THs) are key determinants of cellular metabolism and regulate a variety of pathways that are involved in the metabolism of carbohydrates, lipids and proteins in several target tissues. Notably, hyperthyroidism induces a hyper-metabolic state characterized by increased resting energy expenditure, reduced cholesterol levels, increased lipolysis and gluconeogenesis followed by weight loss, whereas hypothyroidism induces a hypo-metabolic state characterized by reduced energy expenditure, increased cholesterol levels, reduced lipolysis and gluconeogenesis followed by weight gain. Thyroid hormone is also a key regulator of mitochondria respiration and biogenesis. Besides mirroring systemic TH concentrations, the intracellular availability of TH is potently regulated in target cells by a mechanism of activation/inactivation catalyzed by three seleno-proteins: type 1 and type 2 iodothyronine deiodinase (D1 and D2) that convert the biologically inactive precursor thyroxine T4 into T3, and type 3 iodothyronine deiodinase (D3) that inactivates TH action. Thus, the pleiotropic effects of TH can fluctuate among tissues and strictly depend on the cell-autonomous action of the deiodinases. Here we review the mechanisms of TH action that mediate metabolic regulation. This review traces the critical impact of peripheral regulation of TH by the deiodinases on the pathways that regulate energy metabolism and the balance among energy intake, expenditure and storage in specific target tissues.

The three iodothyronine deiodinases catalyze the metabolic pathway that removes one iodine residue from the T4 molecule, thus producing either the active T3 or the inactive metabolite rT3. Hence, deiodination is a potent mechanism by which to modulate thyroid hormone (TH) action at cellular level, thereby allowing cells to customize their own T3 availability, both spatially and temporally, irrespective of TH serum concentrations. Sonic hedgehog (Hh) regulates patterning and growth of a remarkable variety of tissues throughout embryogenesis. Its constitutive activation is associated with cancer development.Recent evidences from two independent systems implicate the Hh signaling pathway in regulation of TH action via modulation of deiodinase expression. Interestingly, many critical developmental events, for example, amphibian metamorphosis, are tightly regulated by the TH and Hh signaling pathways. This review provides an overview of recent data referring to the intricate regulation of deiodinase activity and intracellular TH action by the Hh pathway.This functional cross-talk provides a paradigm for interaction between two key signaling pathways critical during development and neoplastic transformation. This interaction may be relevant in other tissues and situations in which the two signaling pathways participate. Deciphering these mechanisms constitutes an exciting field for future research.

Thyroid hormone activation is catalyzed by two deiodinases, D1 and D2. Whereas D1 is a stable plasma membrane protein, D2 is resident in the endoplasmic reticulum (ER) and has a 20-min half-life due to selective ubiquitination and proteasomal degradation. Here we have shown that stable retention explains D2 residency in the ER, a mechanism that is nevertheless over-ridden by fusion to the long-lived plasma membrane protein, sodium-iodine symporter. Fusion to D2, but not D1, dramatically shortened sodium-iodine symporter half-life through a mechanism dependent on an 18-amino acid D2-specific instability loop. Similarly, the D2-specific loop-mediated protein destabilization was also observed after D2, but not D1, was fused to the stable ER resident protein SEC62. This indicates that the instability loop in D2, but not its subcellular localization, is the key determinant of D2 susceptibility to ubiquitination and rapid turnover rate. Our data also show that the 6 N-terminal amino acids, but not the 12 C-terminal ones, are the ones required for D2 recognition by WSB-1.

Summary Precise 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., Graphical Abstract, Highlights • D3 is induced in proliferating satellite cells thereby reducing thyroid signaling • D3 depletion causes massive cell apoptosis in vitro and in vivo • Apoptosis requires FoxO3, a TH target gene • Satellite cells customize TH signature and adapt it to their functional needs, Deiodinases inactivate thyroid hormone (TH) signaling, allowing a precise control of TH action at the cellular level. Dentice et al. find that type 3 deiodinase acts as a survival factor during skeletal muscle repair, and attenuation of TH signaling is required to prevent muscle stem cell apoptosis and promote lineage progression in vivo.

Type 3 deiodinase (D3), the physiologic inactivator of thyroid hormones, is induced during tissue injury and regeneration. This has led to the hypotheses that D3 impacts injury tolerance by reducing local T3 signaling and contributes to the fall in serum triiodothyronine (T3) observed in up to 75% of sick patients (termed the low T3 syndrome). Here we show that a novel mutant mouse with hepatocyte-specific D3 deficiency has normal local responses to toxin-induced hepatonecrosis, including normal degrees of tissue necrosis and intact regeneration, but accelerated systemic recovery from illness-induced hypothyroxinemia and hypotriiodothyroninemia, demonstrating that peripheral D3 expression is a key modulator of the low T3 syndrome.

International audience; Ubiquitination is a critical posttranslational regulator of protein stability and/or subcellular localization. Here we show that ubiquitination can also regulate proteins by transiently inactivating enzymatic function through conformational change in a dimeric enzyme, which can be reversed upon de-ubiquitination. Our model system is the thyroid hormone activating type 2 deiodinase (D2), an endoplasmic reticulum resident type 1 integral membrane enzyme. D2 exists as a homodimer maintained by interacting surfaces at its transmembrane and globular cytosolic domains. The D2 dimer associates with the Hedgehog-inducible ubiquitin ligase WSB-1, the ubiquitin conjugase UBC-7, and VDU-1, a D2-specific deubiquitinase. Upon binding of T4, its natural substrate, D2 is ubiquitinated, which inactivates the enzyme by interfering with D2's globular interacting surfaces that are critical for dimerization and catalytic activity. This state of transient inactivity and change in dimer conformation persists until de-ubiquitination. The continuous association of D2 with this regulatory protein complex supports rapid cycles of deiodination, conjugation to ubiquitin, and enzyme reactivation by de-ubiquitination, allowing tight control of thyroid hormone action.

WSB-1 is a SOCS-box-containing WD-40 protein of unknown function that is induced by Hedgehog signalling in embryonic structures during chicken development. Here we show that WSB-1 is part of an E3 ubiquitin ligase for the thyroid-hormone-activating type 2 iodothyronine deiodinase (D2). The WD-40 propeller of WSB-1 recognizes an 18-amino-acid loop in D2 that confers metabolic instability, whereas the SOCS-box domain mediates its interaction with a ubiquitinating catalytic core complex, modelled as Elongin BC-Cul5-Rbx1 (ECS(WSB-1)). In the developing tibial growth plate, Hedgehog-stimulated D2 ubiquitination via ECS(WSB-1) induces parathyroid hormone-related peptide (PTHrP), thereby regulating chondrocyte differentiation. Thus, ECS(WSB-1) mediates a mechanism by which 'systemic' thyroid hormone can effect local control of the Hedgehog-PTHrP negative feedback loop and thus skeletogenesis.

Thyroid transcription factor gene 1 (TTF-1) is a homeobox-containing gene involved in thyroid organogenesis. During early thyroid development, the homeobox gene Nkx-2.5 is expressed in thyroid precursor cells coincident with the appearance of TTF-1. The aim of this study was to investigate the molecular mechanisms underlying thyroid-specific gene expression. We show that the Nkx-2.5 C terminus interacts with the TTF-1 homeodomain and, moreover, that the expression of a dominant-negative Nkx-2.5 isoform (N188K) in thyroid cells reduces TTF-1-driven transcription by titrating TTF-1 away from its target DNA. This process reduced the expression of several thyroid-specific genes, including pendrin and thyroglobulin. Similarly, down-regulation of TTF-1 by RNA interference reduced the expression of both genes, whose promoters are sensitive to and directly associate with TTF-1 in the chromatin context. In conclusion, we demonstrate that pendrin and thyroglobulin are downstream targets in vivo of TTF-1, whose action is a prime factor in controlling thyroid differentiation in vivo.

The type 2 iodothyronine selenodeiodinase (D2) is an endoplasmic reticulum (ER)-resident selenoprotein that activates T4 to T3, playing a critical role in thyroid homeostasis. D2 has an approximately 45-min half-life due to selective ubiquitin-mediated ER-associated degradation (ERAD), a process of particular interest because it is accelerated by exposure to D2 substrates, T4 or rT3. The present in vitro binding studies indicate that glutathione-S-transferase (GST)-human D2 fusion proteins specifically associate with a mammalian homolog of the ubiquitin conjugase UBC7 (MmUBC7), with localization to amino acids 169-234 of D2. Coexpression of D2 with an inactive D2 mutant or a truncated version containing amino acids 169-234 stabilizes D2 half-life, supporting the importance of the carboxyl region of D2 for ERAD. Mammalian UBC6 (MmUBC6) does not directly associate with D2 but can associate with a complex containing UBC7 and D2. At the same time, functional studies in human embryonic kidney-293 cells indicate that D2 activity half-life and protein levels are stabilized only when inactive mutants of both UBC6 and UBC7 are overexpressed with D2, suggesting that redundancy may exist at the level of the E2 for both basal and substrate-accelerated D2 ERAD. In conclusion, D2 ERAD in human cells proceeds via an association between UBC7 and the carboxyl region of D2, a unique mechanism for the control of thyroid hormone activation.