sFRP2 gene a activates -catenin signaling The thyroid hormone receptor TR (cid:1) 1 regulates intestinal development and homeostasis by controlling epithelial proliferation in the crypts. This involves positive control of the Wnt/ (cid:2) -cateninpathway.Tofurtherinvestigatetheeffectofthyroidhor- mone-TR (cid:1) 1 signaling on the intestinal epithelium proliferating compartment, we performed a comparative transcription profile analysis on laser microdissected crypt cells recovered from wild type animals with normal or perturbed hormonal status, as well as from TR knock-out mice. Statistical analysis and an in silico approach allowed us to identify 179 differentially regulated genes and to group them into organized functional net-works. We focused on the “cell cycle/cell proliferation” network and, in particular, on the Frizzled-related protein sFRP2, whose expression was greatly increased in response to thyroid hormones. In vitro and in vivo analyses showed that the expression of sFRP2 is directly regulated by TR (cid:1) 1 and that it activates (cid:2) -catenin signaling via Frizzled receptors. Indeed, sFRP2 stabilizes (cid:2) -catenin, activates its target genes, and enhances cell proliferation. In conclusion, these new data, in conjunction with our previous results,

Objectives. - The tissue renin-angiotensin system (tRAS) plays a key role in the maintenance of cellular homeostasis but is also implicated in atherosclerosis. Thyroid hormone (TH) contributes, via genomic effects, to control of tRAS gene expression in the arterial wall and vascular smooth muscle cells (VSMCs). We investigated the specific functions of TH receptors-alpha and -beta (TR alpha and TR beta) on tRAS gene expression in the aorta and VSMCs, and the potential protective effect of TR alpha against atherosclerosis. Material and methods. - Using aorta and cultured aortic VSMCs from TR alpha and TR beta deficient mice, tRAS gene expression was analyzed by determining mRNA levels on real-time PCR. Gene regulation under cholesterol loading mimicking atherosclerosis conditions was also examined in VSMCs in vitro. Results. - TR alpha deletion significantly increased expression of angiotensinogen (AGT) and angiotensin II receptor type 1 subtype a (AT(1)R(a)) at transcriptional level in aorta, a tissue with high TR alpha expression level. TR alpha activity thus seems to be required for maintenance of physiological levels of AGTand AT(1)R(a) expression in the arterial wall. In addition, during cholesterol loading, TR alpha deletion significantly increased cholesterol content in VSMCs, with a weaker decrease in AGTexpression. Conclusion. - TR alpha seems to have an inhibitory impact on AGTand AT I R a expression, and loss of TR alpha function in TR alpha 0/0 mice increases tRAS expression in the aortic wall. More importantly, TR alpha deletion significantly increases VSMC cholesterol content. Our results are consistent with a protective role of TR alpha against atherosclerosis. (C) 2018 Elsevier Masson SAS. All rights reserved.

Rebecca Bahn – Mayo Medical School, Rochester, MN, USA Paul Banga – King’s College London School of Medicine, London, UK Luigi Bartalena – University of Insubria, Varese, Italy Bernadette Biondi – University of Naples Federico II, Naples, Italy Anita Boelen – Academic Medical Center, Amsterdam, Netherlands Steen Bonnema – Odense University Hospital, Odense, Denmark Georg Brabant – University of Lübeck, Lübeck, Germany Monica Dentice – University of Naples, Naples, Italy Creswell Eastman – The University of Sydney, Westmead, NSW, Australia Grigoris Effraimidis – Rigshospitalet, Copenhagen, Denmark Murat Erdogan – Ibni-i-Sina Hastanesi, Ankara, Turkey Valentin Fadeyev – Federal Endocrinological Scientific Centre, Moscow, Russia Ulla Feldt-Rasmussen – Copenhagen Univ. Hosp., Rigshospitalet, Copenhagen, Denmark Dagmar Führer-Sakel – University Hospital Essen, Essen, Germany George Kahaly – Gutenberg University Medical Center, Mainz, Germany Gerd Krause – Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany Rui Maciel – Universidade Federal de São Paulo, São Paulo, Brazil Ana Luiza Maia – Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil Ralf Paschke – University of Calgary, Calgary, AB, Canada

Thyroid hormone (TH) regulates gene transcription by binding to TH receptors (TRs). TRs regulate the genes of lipid metabolism and the renin-angiotensin system (RAS). We examined the effect of TRα deletion in ApoE–/– mice (DKO mice) on the following: (i) the expression of genes controlling cholesterol metabolism and tissue (t)RAS in the liver and aorta and (ii) the expression of these genes and the regulation of cholesterol content in cultured vascular smooth muscle cells (VSMCs). TRα deletion in ApoE–/– mice led to the repression of genes involved in the synthesis and influx of cholesterol in the liver. However, TRα deletion in the arterial wall suppressed the expression of genes involved in the esterification and excretion of cholesterol and enhanced the expression of angiotensinogen (AGT). The VSMCs of the ApoE–/– and DKO mice increased their cholesterol content during cholesterol loading, but failed to increase the expression of ATP-binding cassette transporter A1 (ABCA1). T3 addition partially corrected these abnormalities in the cells of the ApoE–/– mice but not those of the DKO mice. In conclusion, TRα deletion in ApoE–/– mice slightly increases the expression of tRAS in the aorta and aggravates the dysregulation of cholesterol content in the VSMCs.

Our previous work demonstrated a key function of the thyroid hormone nuclear receptor TRα1, a T3-modulated transcription factor, in controlling intestinal development and homeostasis via the Wnt and Notch pathways. Importantly, increased expression of TRα1 in the intestinal epithelium in a mutated Apc genetic background (vil-TRα1/Apc+/1638N mice) accelerated tumorigenesis and contributed to a more aggressive tumor phenotype compared to that of the Apc mutants alone. Therefore, the aim of this study was to determine the relevance of this synergistic effect in human colorectal cancers and to gain insights into the mechanisms involved. We analyzed cohorts of patients by in silico and experimental approaches and observed increased TRα1 expression and a significant correlation between TRα1 levels and Wnt activity. TRα1 loss-of-function and gain-of-function in Caco2 cell lines not only confirmed that TRα1 levels control Wnt activity but also demonstrated the role of TRα1 in regulating cell proliferation and migration. Finally, upon investigation of the molecular mechanisms responsible for the Wnt-TRα1 association, we described the repression by TRα1 of several Wnt inhibitors, including Frzb, Sox17 and Wif1. In conclusion, our results underline an important functional interplay between the thyroid hormone nuclear receptor TRα1 and the canonical Wnt pathway in intestinal cancer initiation and progression. More importantly, we show for the first time that the expression of TRα1 is induced in human colorectal cancers.

Most living organisms show circadian rhythms in physiology and behavior. These oscillations are generated by endogenous circadian clocks, present in virtually all cells where they control key biological processes. To study peripheral clocks in vivo, we developed an original model, the Rev-Luc mouse to follow noninvasively and longitudinally Rev-Luc oscillations in peripheral clocks using in vivo bioluminescence imaging. We found in vitro and in vivo a robust diurnal rhythm of Rev-Luc, mainly in liver, intestine, kidney and adipose tissues. We further confirmed in vivo that Rev-Luc peripheral tissues are food-entrainable oscillators, not affected by age or sex. These data strongly support the relevance of the Rev-Luc model for circadian studies, especially to investigate in vivo the establishment and the entrainment of the rhythm throughout ontogenesis. We then showed that Rev-Luc expression develops dynamically and gradually, both in amplitude and in phase, during fetal and postnatal development. We also demonstrate for the first time that the immature peripheral circadian system of offspring in utero is mainly entrained by maternal cues from feeding regimen. The prenatal entrainment will also differentially determine the Rev-Luc expression in pups before weaning underlining the importance of the maternal chrononutrition on the circadian system entrainment of the offspring.

Thyroid hormone receptors (TRs) were cloned based on their homology with the retroviral oncogene v-ERBA. In Vertebrates two genes, THRA and THRB, encode respectively many isotypes and isoforms of receptors TRα and TRβ, resulting from alternative splicing and/or internal transcription start sites. We present here a wide overview of this diversity and of their mechanisms of action as transcription regulators, as well as alternative actions through cytoplasmic signaling.

Thyroid hormone receptors (TRs) were cloned based on their homology with the retroviral oncogene v-ERBA. In Vertebrates two genes, THRA and THRB, encode respectively many isotypes and isoforms of receptors TR alpha and TR beta, resulting from alternative splicing and/or internal transcription start sites. We present here a wide overview of this diversity and of their mechanisms of action as transcription regulators, as well as alternative actions through cytoplasmic signaling.

The obesity epidemic is a significant global health issue. Improved understanding of the mechanisms that regulate appetite and body weight will provide the rationale for the design of anti-obesity therapies. Thyroid hormones play a key role in metabolic homeostasis through their interaction with thyroid hormone receptors (TRs), which function as ligand-inducible transcription factors. The TR-beta isoform (TRβ) is expressed in the ventromedial hypothalamus (VMH), a brain area important for control of energy homeostasis. Here, we report that selective knockdown of TRβ in the VMH of adult mice results in severe obesity due to hyperphagia and reduced energy expenditure. The observed increase in body weight is of a similar magnitude to murine models of the most extreme forms of monogenic obesity. These data identify TRβ in the VMH as a major physiological regulator of food intake and energy homeostasis.

UHRF2 has been implicated as a novel regulator for both DNA methylation (5mC) and hydroxymethylation (5hmC), but its physiological function and role in DNA methylation/hydroxymethylation are unknown. Here we show that in mice, UHRF2 is more abundantly expressed in the brain and a few other tissues. Uhrf2 knock-out mice are viable and fertile and exhibit no gross defect. Although there is no significant change of DNA methylation, the Uhrf2 null mice exhibit a reduction of 5hmC in the brain, including the cortex and hippocampus. Furthermore, the Uhrf2 null mice exhibit a partial impairment in spatial memory acquisition and retention. Consistent with the phenotype, gene expression profiling uncovers a role for UHRF2 in regulating neuron-related gene expression. Finally, we provide evidence that UHRF2 binds 5hmC in cells but does not appear to affect the TET1 enzymatic activity. Together, our study supports UHRF2 as a bona fide 5hmC reader and further demonstrates a role for 5hmC in neuronal function.

Current literature makes a distinction between two pathways for thyroid hormone signaling: genomic and nongenomic. However, this classification is a source of confusion. We propose a clarification in the nomenclature that may help to avoid unproductive controversies and favor progress in this field of research. Four types of thyroid hormone signaling are defined, and the experimental criteria for classification are discussed.

Significance Thyroid hormone (T3) controls both developmental and physiological processes. Its nuclear receptors (TR) are transcription factors. Methyl dioxygenase ten-eleven translocation protein 3 (TET3) is characterized here as a TR coregulator. It stabilizes and promotes TR chromatin association in a dioxygenase-independent manner, thus increasing the sensitivity of the cell to T3. Mutations in TR cause the resistance to thyroid hormone syndrome (RTH) symptom, the severity of which varies with the particular mutation. Only some mutated TR can be stabilized by TET3. The availability of TET3 is therefore a parameter modulating TR activity, and its differential interaction with mutated TR might explain different severity of RTH. Furthermore, TET3 is likely to function as a general coregulator for nuclear receptors, as it enhances chromatin association of additional members of this superfamily.

Maria Alevizaki – Athens University, Athens, Greece Ana Aranda – Universidad Autónoma de Madrid, Madrid, Spain Rebecca Bahn – Mayo Medical School, Rochester, Minn., USA Paul Banga – King’s College London School of Medicine, London, UK Luigi Bartalena – University of Insubria, Varese, Italy Bernadette Biondi – University of Naples Federico II, Naples, Italy Anita Boelen – Academic Medical Center, Amsterdam, Netherlands Georg Brabant – University of Lübeck, Lübeck, Germany Henning Dralle – Martin Luther University, Halle/Saale, Germany Creswell J. Eastman – Th e University of Sydney, Westmead, N.S.W., Australia Murat Erdogan – Ibni-i-Sina Hastanesi, Ankara, Turkey Valentin Fadeyev – Federal Endocrinological Scientifi c Centre, Moscow, Russia Ulla Feldt-Rasmussen – Copenhagen Univ. Hosp., Rigshospitalet, Copenhagen, Denmark Laszlo Hegedus – Odense University Hospital, Odense, Denmark George J. Kahaly – Gutenberg University Medical Center, Mainz, Germany Rui Maciel – Universidade Federal de São Paulo, São Paulo, Brazil Ana Luiza Maia – Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil Jens Mittag – University of Lübeck, Lübeck, Germany Ralf Paschke – Universität Leipzig, Leipzig, Germany Robin P. Peeters – Erasmus MC, Rotterdam, Netherlands

MicroRNA-135a (miR-135a) down-modulates parameters of cancer progression and its expression is decreased in metastatic breast cancers (as compared to non-metastatic tumors) as well as in prostate tumors relative to normal tissue. These expression and activity patterns are opposite to those of the Estrogen-Related Receptor α (ERRα), an orphan member of the nuclear receptor family. Indeed high expression of ERRα correlates with poor prognosis in breast and prostate cancers, and the receptor promotes various traits of cancer aggressiveness including cell invasion. Here we show that miR-135a down-regulates the expression of ERRα through specific sequences of its 3'UTR. As a consequence miR-135a also reduces the expression of downstream targets of ERRα. miR-135a also decreases cell invasive potential in an ERRα-dependent manner. Our results suggest that the decreased expression of miR-135a in metastatic tumors leads to elevated ERRα expression, resulting in increased cell invasion capacities.

Resistance to thyroid hormone (RTH) is a rare genetic disease caused by reduced tissue sensitivity to thyroid hormone. The hallmark of RTH is elevated serum levels of thyroid hormone with unsuppressed thyrotropin (TSH). However, the most common form of RTH results from minor defects in the ligand-binding domain or hinge domain of the TRβ gene, resulting in impaired T3-induced transcriptional activity, often showing mild presentation. Early diagnosis can be challenging. The objective of the current study was to characterize this specific group of RTH patients. This was a retrospective study. Patients diagnosed as RTH with TRβ mutations were enrolled in a single institute between 2004 and 2014. A total of 14 patients were diagnosed as RTH with mutation in THβ gene. The median age at diagnosis was 22.5 (IQR: 13.25–32.75). Goiter was the most common clinical finding. TSH was significantly elevated after TRH injection (median peak was 21.83 μIU/l, IQR: 13.59–31.48), 9.2-fold compared to the basal level. We found 10 mutations in TRβ gene, all located in the last four exons, and including one novel mutation, H271D. In vitro study found that H271D mutation reduced TR affinity to T3. Four patients with intact thyroid were diagnosed after 16 years old, defined as late manifestation. Compared to those diagnosed before 10 years old, patients with late manifestation presented with normal growth and mental development. Interestingly, three of them carried R438H mutation. We identified a novel p.H271D mutation in TRβ associated with RTH. Endocrinologists should be alert that RTH is frequently found in euthyroid patients with mild symptoms and often leads to misleading diagnosis as well as inappropriate treatment.

Hypothyroidism is associated with an increased occurrence of atherosclerosis, suggesting some protective role for thyroid hormones (THs). Hypercholesterolemia is one of the major risk factor to develop this disease. Here, we show that the well-known TH cholesterol lowering effect was dependent on TH nuclear receptor (TR)β liver activity. But most importantly, TRα was also shown to contribute of slowing down atherosclerosis progression via an independent mechanism. Introduction of TRα(0/0) deletion in the ApoE(-/-) background accelerated the appearance of plaques. Earlier cholesterol accumulation was detected in aorta macrophages, likely due to impaired cholesterol efflux. The IL-1β inflammatory cytokine was elevated in serum and macrophages in correlation with an activation of the AKT/nuclear factor κB pathway in these cells. Inhibition of AKT prevented inflammation and restored normal cholesterol efflux. Similar low-grade inflammation was identified in TRα(0/0) male mice. Thus, the mere absence of TRα is associated with elevated levels of cytokines likely responsible for cholesterol accumulation and atherosclerosis. This TRα protective activity should be relevant for other inflammatory pathologies.

A three-component probe harnesses the extraordinary properties of a solid-state fluorophore for the detection of living cells exhibiting a particular peptidase activity. The off-on mode by which the probe operates, the bright fluorescence of the resulting precipitate, and the rapid response allow an exceptional signal-to-background ratio during microscopic imaging. A tertiary carbamate link between the spacer and phenolic fluorophore is at the heart of the probe's long-term stability. The degree of chlorination of the probe determines its response time and thus its suitability for live-cell analysis. Our probe also allows highly resolved localization of peptidase activity during gel analysis or on agar. In comparison, probes releasing soluble fluorophores demonstrate complete diffusion of the fluorescent signal. These results demonstrate the probe's potential for diverse biomedical applications, including high-fidelity flow cytometry and sensitive colony assays.

The protein of retroviral origin ENS-1/ERNI plays a major role during neural plate development in chick embryos by controlling the activity of the epigenetic regulator HP1γ, but its function in the earlier developmental stages is still unknown. ENS-1/ERNI promoter activity is down-regulated upon differentiation but the resulting protein expression has never been examined. In this study, we present the results obtained with custom-made antibodies to gain further insights into ENS-1 protein expression in Chicken embryonic stem cells (CES) and during their differentiation. First, we show that ENS-1 controls the activity of HP1γ in CES and we examined the context of its interaction with HP1γ. By combining immunofluorescence and western blot analysis we show that ENS-1 is localized in the cytoplasm and in the nucleus, in agreement with its role on gene's promoter activity. During differentiation, ENS-1 decreases in the cytoplasm but not in the nucleus. More precisely, three distinct forms of the ENS-1 protein co-exist in the nucleus and are differently regulated during differentiation, revealing a new level of control of the protein ENS-1. In silico analysis of the Ens-1 gene copies and the sequence of their corresponding proteins indicate that this pattern is compatible with at least three potential regulation mechanisms, each accounting only partially. The results obtained with the anti-ENS-1 antibodies presented here reveal that the regulation of ENS-1 expression in CES is more complex than expected, providing new tracks to explore the integration of ENS-1 in CES cells regulatory networks.

Resistance to thyroid hormone (RTH) was first described in 1967 (1), and the first mutations in the THRB gene were identified in 1989 (2, 3), only 3 years after the cloning of the THR genes (4, 5). The cardinal features of this syndrome of reduced sensitivity to thyroid hormone are elevated serum levels of free thyroid hormone with nonsuppressed TSH, often with goiter and no clear symptoms and signs of thyrotoxicosis (6). In fact, signs of decreased and increased thyroid hormone action in different tissues may coexist. During the First International Workshop on Resistance to Thyroid Hormone in Cambridge, United Kingdom in 1993, a consensus statement was issued to establish a unified nomenclature of THRB gene mutations in RTH (7), as defined above. In the ensuing years more than 3000 cases have been identified, 80% of which harbored mutations in the THRB gene. More recently, two syndromes with reduced cellular access of the biologically active thyroid hormone, T3, were identified. These are caused by defects of thyroid hormone cell membrane transport (8, 9) and a defect reducing the intracellular metabolism generating T3 from T4 (10). To accommodate these new findings, it was proposed to broaden the definition of hormone resistance. Thus, the Fifth International Workshop on Resistance to Thyroid Hormone, which took place in Lyon, France, in 2005, saw the introduction of the term “reduced sensitivity to thyroid hormone (RSTH) to encompass all defects that can interfere with the biological activity of a chemically intact thyroid hormone secreted in normal or excessive amounts.” Following the 10th International Workshop on Resistance to Thyroid Hormone and Action that took place in Quebec City, Canada, in 2012, a number of investigators took on the task to develop a nomenclature for inherited forms of impaired sensitivity to thyroid hormone (Table 1). The term “impaired” was to substitute for “reduced” because nascent data indicate that syndromes of increased sensitivity may also exist. We are cognizant that no nomenclature can fit perfectly all aspects of the described syndromes because variability exists. Several aspects were taken into consideration: the already existing nomenclature, new findings, and anticipated putative discoveries. For example, in over 2000 publications “RTH” is used to define a phenotype of congenitally increased free T4 with nonsuppressed TSH, irrespective of the presence or absence of a THRB gene mutation (see non-TR-RTH). In view of the identification of THRA gene mutations that present a distinct phenotype (11, 12), we propose using the term “RTH α”, and in new publications to use “RTH β” when a THRB gene mutation is present in association with the RTH phenotype. This allows the naming of new gene defects in individuals with the RTH phenotype. The use of the abbreviation “THR” as a synonym for RTH is discouraged, not only because the hormone is not resistant, but also because this abbreviation is used to denote other circumstances. Indeed, a Medline search using THR yielded over 20 000 references, only a few related to resistance to thyroid hormone. Table 1. Inheritable Forms of Impaired Sensitivity to Thyroid Hormone