Partial Resistance to Thyroid Hormone-Induced Tachycardia and Cardiac Hypertrophy in Mice Lacking Thyroid Hormone Receptor ß.

Background: Thyroid hormones regulate cardiac functions mainly through direct actions in the heart and by binding to the thyroid hormone receptor (TR) isoforms α1 and ß. While the role of the most abundantly expressed isoform, TRα1, is widely studied and well characterized, the role of TRß in regulating heart functions is still poorly understood, primarily due to the accompanying elevation of circulating thyroid hormone in TRß knockout mice (TRß-KO). However, their hyperthyroidism is ameliorated at thermoneutrality, which allows studying the role of TRß without this confounding factor. Methods: Here, we noninvasively monitored heart rate in TRß-KO mice over several days using radiotelemetry at different housing temperatures (22°C and 30°C) and upon 3,3',5-triiodothyronine (T3) administration in comparison to wild-type animals. Results: TRß-KO mice displayed normal average heart rate at both 22°C and 30°C with only minor changes in heart rate frequency distribution, which was confirmed by independent electrocardiogram recordings in freely-moving conscious mice. Parasympathetic nerve activity was, however, impaired in TRß-KO mice at 22°C, and only partly rescued at 30°C. As expected, oral treatment with pharmacological doses of T3 at 30°C led to tachycardia in wild-types, accompanied by broader heart rate frequency distribution and increased heart weight. The TRß-KO mice, in contrast, showed blunted tachycardia, as well as resistance to changes in heart rate frequency distribution and heart weight. At the molecular level, these observations were paralleled by a blunted cardiac mRNA induction of several important genes, including the pacemaker channels Hcn2 and Hcn4, as well as Kcna7. Conclusions: The phenotyping of TRß-KO mice conducted at thermoneutrality allows novel insights on the role of TRß in cardiac functions in the absence of the usual confounding hyperthyroidism. Even though TRß is expressed at lower levels than TRα1 in the heart, our findings demonstrate an important role for this isoform in the cardiac response to thyroid hormones.

A Comparison of Miller Straight Blade and Macintosh Blade Laryngoscopes for Intubation in Morbidly Obese Patients.

The primary objective of this study was to demonstrate whether the Miller blade laryngoscope could provide better visualization of the vocal cords in morbidly obese patients than the Macintosh blade laryngoscope. The secondary objective was to identify the patient-measured factors associated with better visualization of the vocal cords when using the Miller vs. Macintosh blade, as well as whether the application of external pressure might improve the visibility of the glottis during intubation. A prospective, observational study encompassing 110 patients with a BMI > 40 undergoing elective bariatric surgery and intubation procedure was performed. The evaluation of the vocal cords was performed according to the Cormack-Lehane scale and POGO scale in the same patient during intubation, performed with a Miller and a Macintosh blade laryngoscope, in a random matter. The following parameters were assessed: body weight, height, BMI, neck circumference, thyromental distance, sternomental distance, mouth opening, and Mallampati scale and their impact on visualization of the vocal cords using the Miller blade without the application of external pressure. The Miller blade provides an improved view of the glottis compared to the Macintosh blade measured with both the Cormac-Lehane scale (45 (40.91%) without external pressure application on the larynx, and 18 (16.36%) with external pressure application on the larynx) and the POGO scale (45 (40.91%) without external pressure application on the larynx, and 19 (17.27%) with external pressure application on the larynx). The application of laryngeal pressure improved the view of the glottis. Among the measured features, a significant improvement in the visibility of the glottis could be found in patients with a BMI over 44.244 kg/m2 and a neck circumference over 46 cm. To conclude, the usage of the Miller blade improves the visibility of the glottis compared to the Macintosh blade in morbidly obese patients. The recommendation to use the Miller blade in this group of patients requires further investigation, taking into account the effectiveness of the intubation. Trial Registration: NCT05494463.

Maternal thyroid hormone receptor ß activation in mice sparks brown fat thermogenesis in the offspring.

It is well established that maternal thyroid hormones play an important role for the developing fetus; however, the consequences of maternal hyperthyroidism for the offspring remain poorly understood. Here we show in mice that maternal 3,3',5-triiodothyronine (T3) treatment during pregnancy leads to improved glucose tolerance in the adult male offspring and hyperactivity of brown adipose tissue (BAT) thermogenesis in both sexes starting early after birth. The activated BAT provides advantages upon cold exposure, reducing the strain on other thermogenic organs like muscle. This maternal BAT programming requires intact maternal thyroid hormone receptor ß (TRß) signaling, as offspring of mothers lacking this receptor display the opposite phenotype. On the molecular level, we identify distinct T3 induced alterations in maternal serum metabolites, including choline, a key metabolite for healthy pregnancy. Taken together, our results connect maternal TRß activation to the fetal programming of a thermoregulatory phenotype in the offspring.

Resistance to thyroid hormone induced tachycardia in RTHα syndrome.

Mutations in thyroid hormone receptor α1 (TRα1) cause Resistance to Thyroid Hormone α (RTHα), a disorder characterized by hypothyroidism in TRα1-expressing tissues including the heart. Surprisingly, we report that treatment of RTHα patients with thyroxine to overcome tissue hormone resistance does not elevate their heart rate. Cardiac telemetry in male, TRα1 mutant, mice indicates that such persistent bradycardia is caused by an intrinsic cardiac defect and not due to altered autonomic control. Transcriptomic analyses show preserved, thyroid hormone (T3)-dependent upregulation of pacemaker channels (Hcn2, Hcn4), but irreversibly reduced expression of several ion channel genes controlling heart rate. Exposure of TRα1 mutant male mice to higher maternal T3 concentrations in utero, restores altered expression and DNA methylation of ion channels, including Ryr2. Our findings indicate that target genes other than Hcn2 and Hcn4 mediate T3-induced tachycardia and suggest that treatment of RTHα patients with thyroxine in high dosage without concomitant tachycardia, is possible.

Single-cell RNA-based phenotyping reveals a pivotal role of thyroid hormone receptor alpha for hypothalamic development.

Thyroid hormone and its receptor TRα1 play an important role in brain development. Several animal models have been used to investigate this function, including mice heterozygous for the TRα1R384C mutation, which confers receptor-mediated hypothyroidism. These mice display abnormalities in several autonomic functions, which was partially attributed to a developmental defect in hypothalamic parvalbumin neurons. However, whether other cell types in the hypothalamus are similarly affected remains unknown. Here, we used single-nucleus RNA sequencing to obtain an unbiased view on the importance of TRα1 for hypothalamic development and cellular diversity. Our data show that defective TRα1 signaling has surprisingly little effect on the development of hypothalamic neuronal populations, but it heavily affects hypothalamic oligodendrocytes. Using selective reactivation of the mutant TRα1 during specific developmental periods, we find that early postnatal thyroid hormone action seems to be crucial for proper hypothalamic oligodendrocyte maturation. Taken together, our findings underline the well-known importance of postnatal thyroid health for brain development and provide an unbiased roadmap for the identification of cellular targets of TRα1 action in mouse hypothalamic development.

In situ laboratory for plastic degradation in the Red Sea.

Degradation and fragmentation of plastics in the environment are still poorly understood. This is partly caused by the lack of long-term studies and methods that determine weathering duration. We here present a novel study object that preserves information on plastic age: microplastic (MP) resin pellets from the wreck of the SS Hamada, a ship that foundered twenty-nine years ago at the coast of Wadi el Gemal national park, Egypt. Its sinking date enabled us to precisely determine how long MP rested in the wreck and a nearby beach, on which part of the load was washed off. Pellets from both sampling sites were analyzed by microscopy, X-ray tomography, spectroscopy, calorimetry, gel permeation chromatography, and rheology. Most pellets were made of low-density polyethylene, but a minor proportion also consisted of high-density polyethylene. MP from inside the wreck showed no signs of degradation compared to pristine reference samples. Contrary, beached plastics exhibited changes on all structural levels, which sometimes caused fragmentation. These findings provide further evidence that plastic degradation under saltwater conditions is comparatively slow, whereas UV radiation and high temperatures on beaches are major drivers of that process. Future long-term studies should focus on underlying mechanisms and timescales of plastic degradation.

Miscibility and Phase Separation in PMMA/SAN Blends Investigated by Nanoscale AFM-IR.

The miscibility and phase separation of poly(methyl methacrylate) (PMMA) and styrene-acrylonitrile (SAN) have already been investigated using various methods. However, these methods have limitations that often result in inconsistent characterization. Consequently, the reasons for the dependence of miscibility on composition as well as on processing temperature have not yet been proved. The phase separation of PMMA/SAN blends was therefore investigated for the first time using a novel technique, nanoscale AFM-IR. It couples nanoscale atomic force microscopy (AFM) with infrared (IR) spectroscopy. Therefore, the phase morphology can be chemically identified and precisely classified within the nm-regime. The PMMA/SAN blends, on the other hand, were analyzed of their changes in morphology under different thermal treatments. It was possible to visualize and define the phase separation, as well as dependence of the miscibility on the mixing ratio. In the miscible domain, no two individual phases could be detected down to the nanometer range. It was shown that with increasing temperature, the morphology changes and two different phases are formed, where the phase boundaries can be sharply defined. The onset of these changes could be identified at temperatures of about 100 °C.

Maternal Thyroid Hormone Programs Cardiovascular Functions in the Offspring.

Background: Maternal thyroid hormone (TH) plays an essential role for fetal development, especially for the cardiovascular system and its central control. However, the precise consequences of altered TH action during the different periods in pregnancy remain poorly understood. Methods: To address this question, we used mice heterozygous for a mutant thyroid hormone receptor α1 (TRα1) and wild-type controls that were born to wild-type mothers treated with 3,3',5-triiodothyronine (T3) during the first or the second half of pregnancy. We then phenotyped the offspring animals as adults by in vivo measurements and postmortem tissue analyses. Results: Maternal T3 treatment in either half of the pregnancy did not affect postnatal growth development. Serum thyroxine and hypophyseal thyrotropin subunit beta or deiodinase type II expression was also not affected in any group, only TRα1 mutant males exhibited a reduction in serum T3 levels after the treatment. Likewise, hepatic deiodinase type I was not altered, but serum selenium levels were reduced by the maternal treatment in wild-type offspring of both genders. Most interestingly, a significant increase in heart weight was found in adult wild-types born to mothers that received T3 during the first or second half of pregnancy, while TRα1 mutant males were protected from this effect. Moreover, we detected a significant increase in heart rate selectively in male mice that were exposed to elevated maternal T3 in the second half of the pregnancy. Conclusion: Taken together, our findings demonstrate that maternal TH is of particular relevance during the second half of pregnancy for establishing cardiac properties, with specific effects depending on TRα1 or gender. The data advocate routinely monitoring TH levels during pregnancy to avoid adverse cardiac effects in the offspring.

Maternal Brown Fat Thermogenesis Programs Glucose Tolerance in the Male Offspring.

Environmental temperature is a driving factor in evolution, and it is commonly assumed that metabolic adaptations to cold climates are the result of transgenerational selection. Here, we show in mice that even minor changes in maternal thermogenesis alter the metabolic phenotype already in the next generation. Male offspring of mothers genetically lacking brown adipose tissue (BAT) thermogenesis display increased lean mass and improved glucose tolerance as adults, while females are unaffected. The phenotype is replicated in offspring of mothers kept at thermoneutrality; conversely, mothers with higher gestational BAT thermogenesis produce male offspring with reduced lean mass and impaired glucose tolerance. Running-wheel exercise reverses the offspring's metabolic impairments, pointing to the muscle as target of these fetal programming effects. Our data demonstrate that gestational BAT activation negatively affects metabolic health of the male offspring; however, these unfavorable fetal programming effects may be negated by active lifestyle.

Dopamine receptor D1- and D2-agonists do not spark brown adipose tissue thermogenesis in mice.

Brown adipose tissue (BAT) thermogenesis is considered a potential target for treatment of obesity and diabetes. In vitro data suggest dopamine receptor signaling as a promising approach; however, the biological relevance of dopamine receptors in the direct activation of BAT thermogenesis in vivo remains unclear. We investigated BAT thermogenesis in vivo in mice using peripheral administration of D1-agonist SKF38393 or D2-agonist Sumanirole, infrared thermography, and in-depth molecular analyses of potential target tissues; and ex vivo in BAT explants to identify direct effects on key thermogenic markers. Acute in vivo treatment with the D1- or D2-agonist caused a short spike or brief decrease in BAT temperature, respectively. However, repeated daily administration did not induce lasting effects on BAT thermogenesis. Likewise, neither agonist directly affected Ucp1 or Dio2 mRNA expression in BAT explants. Taken together, the investigated agonists do not seem to exert lasting and physiologically relevant effects on BAT thermogenesis after peripheral administration, demonstrating that D1- and D2-receptors in iBAT are unlikely to constitute targets for obesity treatment via BAT activation.

Central Hypothyroidism Impairs Heart Rate Stability and Prevents Thyroid Hormone-Induced Cardiac Hypertrophy and Pyrexia.

Background: Tachycardia, cardiac hypertrophy, and elevated body temperature are major signs of systemic hyperthyroidism, which are considered to reflect the excessive thyroid hormone (TH) action in the respective peripheral tissues. However, recent observations indicate that the central actions of TH also contribute substantially to cardiovascular regulation and thermogenesis. Methods: In this study, we dissect the individual contributions of peripheral TH action versus the central effects in body temperature regulation and cardiovascular functions by taking advantage of mice lacking the TH transporters monocarboxylate transporter 8 (MCT8) and organic anion transporting polypeptide 1C1 (OATP1C1) (M/O double knock-out [dko]), which exhibit elevated serum triiodothyronine (T3) levels while their brain is in a profoundly hypothyroid state. We compared these animals with wild-type (WT) mice that were treated orally with T3 to achieve similarly elevated serum T3 levels, but are centrally hyperthyroid. For the studies, we used radiotelemetry, infrared thermography, gene expression profiling, Western blot analyses, and enzyme linked immunosorbent assays (ELISA) assays. Results: Our analyses revealed mild hyperthermia and cardiac hypertrophy in T3-treated WT mice but not in M/O dko animals, suggesting that central actions of TH are required for these hyperthyroid phenotypes. Although the average heart rate was unaffected in either model, the M/O dko exhibited an altered heart rate frequency distribution with tachycardic bursts in active periods and bradycardic episodes during resting time, demonstrating that the stabilization of heart rate by the autonomic nervous system can be impaired in centrally hypothyroid animals. Conclusions: Our studies unravel distinct phenotypical traits of hyperthyroidism that depend on an intact central nervous system, and provide valuable insight into the cardiovascular pathology of the Allan-Herndon-Dudley syndrome, a condition caused by the lack of MCT8 in humans.

Thyroid-Hormone-Induced Browning of White Adipose Tissue Does Not Contribute to Thermogenesis and Glucose Consumption.

Regulation of body temperature critically depends on thyroid hormone (TH). Recent studies revealed that TH induces browning of white adipose tissue, possibly contributing to the observed hyperthermia in hyperthyroid patients and potentially providing metabolic benefits. Here, we show that browning by TH requires TH-receptor ß and occurs independently of the sympathetic nervous system. The beige fat, however, lacks sufficient adrenergic stimulation and is not metabolically activated despite high levels of uncoupling protein 1 (UCP1). Studies at different environmental temperatures reveal that TH instead causes hyperthermia by actions in skeletal muscle combined with a central body temperature set-point elevation. Consequently, the metabolic and thermogenic effects of systemic hyperthyroidism were maintained in UCP1 knockout mice, demonstrating that neither beige nor brown fat contributes to the TH-induced hyperthermia and elevated glucose consumption, and underlining that the mere presence of UCP1 is insufficient to draw conclusions on the therapeutic potential of browning agents.

Aortic effects of thyroid hormone in male mice.

It is well established that thyroid hormones are required for cardiovascular functions; however, the molecular mechanisms remain incompletely understood, especially the individual contributions of genomic and non-genomic signalling pathways. In this study, we dissected how thyroid hormones modulate aortic contractility. To test the immediate effects of thyroid hormones on vasocontractility, we used a wire myograph to record the contractile response of dissected mouse aortas to the adrenergic agonist phenylephrine in the presence of different doses of T3 (3,3',5-triiodothyronine). Interestingly, we observed reduced vasoconstriction under low and high T3 concentrations, indicating an inversed U-shaped curve with maximal constrictive capacity at euthyroid conditions. We then tested for possible genomic actions of thyroid hormones on vasocontractility by treating mice for 4 days with 1 mg/L thyroxine in drinking water. The study revealed that in contrast to the non-genomic actions the aortas of these animals were hyperresponsive to the contractile stimulus, an effect not observed in endogenously hyperthyroid TRß knockout mice. To identify targets of genomic thyroid hormone action, we analysed aortic gene expression by microarray, revealing several altered genes including the well-known thyroid hormone target gene hairless. Taken together, the findings demonstrate that thyroid hormones regulate aortic tone through genomic and non-genomic actions, although genomic actions seem to prevail in vivo. Moreover, we identified several novel thyroid hormone target genes that could provide a better understanding of the molecular changes occurring in the hyperthyroid aorta.

Dopamine directly increases mitochondrial mass and thermogenesis in brown adipocytes.

Brown adipose tissue (BAT) is key to energy homeostasis. By virtue of its thermogenic potential, it may dissipate excessive energy, regulate body weight and increase insulin sensitivity. Catecholamines are critically involved in the regulation of BAT thermogenesis, yet research has focussed on the effects of noradrenaline and adrenaline. Some evidence suggests a role of dopamine (DA) in BAT thermogenesis, but the cellular mechanisms involved have not been addressed. We employed our extensively characterised murine brown adipocyte cells. D1-like and D2-like receptors were detectable at the protein level. Stimulation with DA caused an increase in cAMP concentrations. Oxygen consumption rates (OCR), mitochondrial membrane potential (Δψm) and uncoupling protein 1 (UCP1) levels increased after 24 h of treatment with either DA or a D1-like specific receptor agonist. A D1-like receptor antagonist abolished the DA-mediated effect on OCR, Δψm and UCP1. DA induced the release of fatty acids, which did not additionally alter DA-mediated increases of OCR. Mitochondrial mass (as determined by (i) CCCP- and oligomycin-mediated effects on OCR and (ii) immunoblot analysis of mitochondrial proteins) also increased within 24 h. This was accompanied by an increase in peroxisome proliferator-activated receptor gamma co-activator 1 alpha protein levels. Also, DA caused an increase in p38 MAPK phosphorylation and pharmacological inhibition of p38 MAPK abolished the DA-mediated effect on Δψm In summary, our study is the first to reveal direct D1-like receptor and p38 MAPK-mediated increases of thermogenesis and mitochondrial mass in brown adipocytes. These results expand our understanding of catecholaminergic effects on BAT thermogenesis.

Phosphatidylinositide 3-kinase (PI3K) and PI3K-related kinase (PIKK) activity contributes to radioresistance in thyroid carcinomas.

Anaplastic (ATC) and certain follicular thyroid-carcinomas (FTCs) are radioresistant. The Phosphatidylinositide 3-kinase (PI3K) pathway is commonly hyperactivated in thyroid-carcinomas. PI3K can modify the PI3K-related kinases (PIKKs) in response to radiation: How PIKKs interact with PI3K and contribute to radioresistance in thyroid-carcinomas is unknown. Further uncertainties exist in how these interactions function under the radioresistant hypoxic microenvironment. Under normoxia/anoxia, ATC (8505c) and FTC (FTC-133) cells were irradiated, with PI3K-inhibition (via GDC-0941 and PTEN-reconstitution into PTEN-null FTC-133s) and effects on PIKK-activation, DNA-damage, clonogenic-survival and cell cycle, assessed. FTC-xenografts were treated with 5 × 2 Gy, ± 50 mg/kg GDC-0941 (twice-daily; orally) for 14 days and PIKK-activation and tumour-growth assessed. PIKK-expression was additionally assessed in 12 human papillary thyroid-carcinomas, 13 FTCs and 12 ATCs. GDC-0941 inhibited radiation-induced activation of Ataxia-telangiectasia mutated (ATM), ATM-and Rad3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Inhibition of ATM and DNA-PKcs was PI3K-dependent, since activation was reduced in PTEN-reconstituted FTC-133s. Inhibition of PIKK-activation was greater under anoxia: Consequently, whilst DNA-damage was increased and prolonged under both normoxia and anoxia, PI3K-inhibition only reduced clonogenic-survival under anoxia. GDC-0941 abrogated radiation-induced cell cycle arrest, an effect most likely linked to the marked inhibition of ATR-activation. Importantly, GDC-0941 inhibited radiation-induced PIKK-activation in FTC-xenografts leading to a significant increase in time taken for tumours to triple in size: 26.5 ± 5 days (radiation-alone) versus 31.5 ± 5 days (dual-treatment). PIKKs were highly expressed across human thyroid-carcinoma classifications, with ATM scoring consistently lower. Interestingly, some loss of ATM and DNA-PKcs was observed. These data provide new insight into the mechanisms of hypoxia-associated radioresistance in thyroid-carcinoma.

GDC-0941 inhibits metastatic characteristics of thyroid carcinomas by targeting both the phosphoinositide-3 kinase (PI3K) and hypoxia-inducible factor-1α (HIF-1α) pathways.

CONTEXT: Phosphoinositide 3-kinase (PI3K) regulates the transcription factor hypoxia-inducible factor-1 (HIF-1) in thyroid carcinoma cells. Both pathways are associated with aggressive phenotype in thyroid carcinomas. OBJECTIVE: Our objective was to assess the effects of the clinical PI3K inhibitor GDC-0941 and genetic inhibition of PI3K and HIF on metastatic behavior of thyroid carcinoma cells in vitro and in vivo. DESIGN: Vascular endothelial growth factor ELISA, HIF activity assays, proliferation studies, and scratch-wound migration and cell spreading assays were performed under various O(2) tensions [normoxia, hypoxia (1 and 0.1% O(2)), and anoxia] with or without GDC-0941 in a panel of four thyroid carcinoma cell lines (BcPAP, WRO, FTC133, and 8505c). Genetic inhibition was achieved by overexpressing phosphatase and tensin homolog (PTEN) into PTEN-null cells and by using a dominant-negative variant of HIF-1α (dnHIF). In vivo, human enhanced green fluorescence protein-expressing follicular thyroid carcinomas (FTC) were treated with GDC-0941 (orally). Spontaneous lung metastasis was confirmed by viewing enhanced green fluorescence protein-positive colonies cultured from lung tissue. RESULTS: GDC-0941 inhibited hypoxia/anoxia-induced HIF-1α and HIF-2α expression and HIF activity in thyroid carcinoma cells. Basal (three of four cell lines) and/or hypoxia-induced (four of four) secreted vascular endothelial growth factor was inhibited by GDC-0941, whereas selective HIF targeting predominantly affected hypoxia/anoxia-mediated secretion (P < 0.05-0.0001). Antiproliferative effects of GDC-0941 were more pronounced in PTEN mutant compared with PTEN-restored cells (P < 0.05). Hypoxia increased migration in papillary cells and cell spreading/migration in FTC cells (P < 0.01). GDC-0941 reduced spreading and migration in all O(2) conditions, whereas dnHIF had an impact only on hypoxia-induced migration (P < 0.001). In vivo, GDC-0941 reduced expression of HIF-1α, phospho-AKT, GLUT-1, and lactate dehydrogenase A in FTC xenografts. DnHIF expression and GDC-0941 reduced FTC tumor growth and metastatic lung colonization (P < 0.05). CONCLUSIONS: PI3K plays a prominent role in the metastatic behavior of thyroid carcinoma cells irrespective of O(2) tension and appears upstream of HIF activation. GDC-0941 significantly inhibited the metastatic phenotype, supporting the clinical development of PI3K inhibition in thyroid carcinomas.

Hypoxia-inducible factor in thyroid carcinoma.

Intratumoural hypoxia (low oxygen tension) is associated with aggressive disease and poor prognosis. Hypoxia-inducible factor-1 is a transcription factor activated by hypoxia that regulates the expression of genes that promote tumour cell survival, progression, metastasis, and resistance to chemo/radiotherapy. In addition to hypoxia, HIF-1 can be activated by growth factor-signalling pathways such as the mitogen-activated protein kinases- (MAPK-) and phosphatidylinositol-3-OH kinases- (PI3K-) signalling cascades. Mutations in these pathways are common in thyroid carcinoma and lead to enhanced HIF-1 expression and activity. Here, we summarise current data that highlights the potential role of both hypoxia and MAPK/PI3K-induced HIF-1 signalling in thyroid carcinoma progression, metastatic characteristics, and the potential role of HIF-1 in thyroid carcinoma response to radiotherapy. Direct or indirect targeting of HIF-1 using an MAPK or PI3K inhibitor in combination with radiotherapy may be a new potential therapeutic target to improve the therapeutic response of thyroid carcinoma to radiotherapy and reduce metastatic burden.

Wnt signaling in estrogen-induced lactotroph proliferation.

Prolactinomas are the most common type of functioning pituitary adenoma in humans, but the control of lactotroph proliferation remains unclear. Here, using microarray analysis, we show that estrogen treatment increased expression of Wnt4 mRNA in adult Fischer rat pituitary tissue. Dual immunofluorescence analysis revealed that Wnt4 expression was not confined to lactotrophs, but that it was expressed in all anterior pituitary cell types. Estradiol induced proliferation in the somatolactotroph GH3 cell line, in parallel with Wnt4 mRNA and protein induction. A reporter gene assay for TCF- and LEF-dependent transcription revealed that there was no activation of the canonical Wnt pathway in GH3 cells upon stimulation with Wnt-conditioned culture medium or coexpression of constitutively active mutant ß-catenin. Expression of ß-catenin in both GH3 cells and normal rat anterior pituitary cells was restricted to the cell membrane and was unaltered by treatment with estradiol, with no nuclear ß-catenin being detected under any of the conditions tested. We show for the first time that Wnt4 affects non-canonical signaling in the pituitary by inhibiting Ca(2+) oscillations in GH3 cells, although the downstream effects are as yet unknown. In summary, Wnt4 is expressed in the adult pituitary gland, and its expression is increased by estrogen exposure, suggesting that its involvement in adult tissue plasticity is likely to involve ß-catenin-independent signaling pathways.

Wnt/beta-catenin signaling mediates antineoplastic effects of imatinib mesylate (gleevec) in anaplastic thyroid cancer.

CONTEXT: Dysregulation of Wnt signaling is a key step in neoplastic thyrocyte proliferation. However, it is unclear whether the selective tyrosine kinase (TK) inhibitor, imatinib mesylate, is linked to the Wnt/beta-catenin cascade and is able to modulate the pathway. OBJECTIVE: Conflicting data are reported on the therapeutic effects of imatinib in anaplastic thyroid carcinomas (ATCs), but the molecular mechanism of action is unclear. Here, we further delineated the antitumor effects and the potential efficacy of imatinib in dedifferentiated thyroid carcinomas. RESULTS: Tissue microarray of histologically proven ATCs (n = 12) demonstrated that six of 12 tumors expressed at least one of the imatinib-sensitive TKs. Similarily, imatinib-sensitive TKs were detected in seven of 10 thyroid cancer cell lines derived from metastatic papillary, follicular, and ATCs. Coimmunoprecipitation in ARO cells demonstrated a direct link between c-abl and beta-catenin. Imatinib (10 microM for 48 h) drastically reduced beta-catenin expression and redistributed it from the nucleus to the cell membrane. It stabilized adherens junctions by increasing beta-catenin/E-cadherin binding and reduced the invasive potential of thyroid cancer. Furthermore, imatinib (10 microM for 48 h) attenuated T cell factor/lymphoid enhancer factor activity, reduced cyclin D1 levels and dose-dependently suppressed thyrocyte proliferation by half without affecting apoptosis. CONCLUSION: Our data provide a molecular mechanism for the antitumor activity of imatinib that may help to develop it as a therapeutic option in a subset of ATC patients.