T3 rapidly modulates TSH beta mRNA stability and translational rate in the pituitary of hypothyroid rats

Whereas it is well known that T3 inhibits TSH beta gene transcription, its effects on TSH beta mRNA stability and translation have been poorly investigated. This study examined these possibilities, by evaluating the TSH beta transcripts poly(A) tail length, translational rate and binding to cytoskeleton, in pituitaries of thyroidectomized and sham-operated rats treated with T3 or saline, and killed 30 min thereafter. The hypothyroidism induced an increase of TSH beta transcript poly(A) tail, as well as of its content in ribosomes and attachment to cytoskeleton. The hypothyroid rats acutely treated with T3 exhibited a reduction of TSH beta mRNA poly(A) tail length and recruitment to ribosomes, indicating that this treatment decreased the stability and translation rate of TSH beta mRNA. Nevertheless, acute T3 administration to sham-operated rats provoked an increase of TSH beta transcripts binding to ribosomes. These data add new insight to an important role of T3 in rapidly regulating TSH gene expression at posttranscriptional level. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

The Thyroid Hormone Receptor (TR) beta-Selective Agonist GC-1 Inhibits Proliferation But Induces Differentiation and TR beta mRNA Expression in Mouse and Rat Osteoblast-Like Cells

Previous studies showed anabolic effects of GC-1, a triiodothyronine (T3) analogue that is selective for both binding and activation functions of thyroid hormone receptor (TR) beta 1 over TR alpha 1, on bone tissue in vivo. The aim of this study was to investigate the responsiveness of rat (ROS17/2.8) and mouse (MC3T3-E1) osteoblast-like cells to GC-1. As expected, T3 inhibited cellular proliferation and stimulated mRNA expression of osteocalcin or alkaline phosphatase in both cell lineages. Whereas equimolar doses of T3 and GC-1 equally affected these parameters in ROS17/2.8 cells, the effects of GC-1 were more modest compared to those of T3 in MC3T3-E1 cells. Interestingly, we showed that there is higher expression of TR alpha 1 than TR beta 1 mRNA in rat (similar to 20-90%) and mouse (similar to 90-98%) cell lineages and that this difference is even higher in mouse cells, which highlights the importance of TR alpha 1 to bone physiology and may partially explain the modest effects of GC-1 in comparison with T3 in MC3T3-E1 cells. Nevertheless, we showed that TR beta 1 mRNA expression increases (similar to 2.8- to 4.3-fold) as osteoblastic cells undergo maturation, suggesting a key role of TR beta 1 in mediating T3 effects in the bone forming cells, especially in mature osteoblasts. It is noteworthy that T3 and GC-1 induced TR beta 1 mRNA expression to a similar extent in both cell lineages (similar to 2- to 4-fold), indicating that both ligands may modulate the responsiveness of osteoblasts to T3. Taken together, these data show that TR beta selective T3 analogues have the potential to directly induce the differentiation and activity of osteoblasts.

Environmental signals: Synthetic humic substances act as xeno-estrogen and affect the thyroid system of Xenopus laevis

According to outdated paradigms humic substances (HS) are considered to be refractory or inert that do not directly interact with aquatic organisms. However, they are taken up and induce biotransformation activities and may act as hormone-like substances. In the present study, we tested whether HS can interfere with endocrine regulation in the amphibian Xenopus laevis. In order to exclude contamination with phyto-hormones, which may occur in environmental isolates, the artificial HS 1500 was applied. The in vivo results showed that HS 1500 causes significant estrogenic effects on X. laevis during its larval development and results of semi-quantitative RT-PCR revealed a marked increase of the estrogenic biomarker estrogen receptor mRNA (ER-mRNA). Furthermore, preliminary RT-PCR results showed that the thyroid-stimulating hormone (TSH beta-mRNA) is enhanced after exposure to HS1500, indicating a weak adverse effect on T3/T4 availability. Hence, HS may have estrogenic and anti-thyroidal effects on aquatic animals, and therefore may influence the structure of aquatic communities and they may be considered environmental signaling chemicals. (c) 2005 Elsevier Ltd. All rights reserved.

GABAA receptor beta subunit mRNA expression in the human alcoholic brain

A competitive RT-PCR assay was used to quantify the expression of the GABA(A) receptor beta(1), beta(2) and beta(3) isoform mRNA transcripts in the superior frontal cortex and motor cortex of 21 control and 22 alcoholic cases. A single set of primers was designed that permitted amplification of all three transcripts and the internal standard simultaneously; differentiation of the individual transcripts was achieved by restriction enzyme digestion. Construction of a standard curve, using the internal standard and a concentration range of beta(2) cRNA-enabled quantitation of mRNA expression levels. No significant difference in mRNA expression was found between the control and alcoholic case groups in either the superior frontal or motor cortex for the beta(2) or beta(3) isoforms. A significant interaction was found between isoform and area, although, the two case groups did not partition on this measure. The interaction was due to a significant difference between superior frontal and motor cortex for the beta(3) isoform; this regional comparison was not significant for beta(2) mRNA. Age at death and post-mortem delay (PMD) had no significant effect on beta mRNA expression in either case group in either region. A beta(1) signal could not be detected in the RT-PCR assay. (C) 2004 Elsevier Ltd. All rights reserved.

Changes in oestrogen receptor-alpha and -beta during progression to acquired resistance to tamoxifen and fulvestrant (Faslodex, ICI 182,780) in MCF7 human breast cancer cells

Cell culture models of antioestrogen resistance often involve applying selective pressures of oestrogen deprivation simultaneously with addition of tamoxifen or fulvestrant (Faslodex, ICI 182,780) which makes it difficult to distinguish events in development of antioestrogen resistance from those in loss of response to oestrogen or other components. We describe here time courses of loss of antioestrogen response using either oestrogen-maintained or oestrogen-deprived MCF7 cells in which the only alteration to the culture medium was addition of 10(-6) M tamoxifen or 10(-7) M fulvestrant. In both oestrogen-maintained and oestrogen-deprived models, loss of growth response to tamoxifen was not associated with loss of response to fulvestrant. However, loss of growth response to fulvestrant was associated in both models with concomitant loss of growth response to tamoxifen. Measurement of oestrogen receptor alpha (ER alpha) and oestrogen receptor beta (ER beta) mRNA by real-time RT-PCR together with ER alpha and ER beta protein by Western immunoblotting revealed substantial changes to ER alpha levels but very little alteration to ER beta levels following development of antioestrogen resistance. In oestrogen-maintained cells, tamoxifen resistance was associated with raised levels of ERa mRNA/protein. However by contrast, in oestrogen-deprived MCF7 cells, where oestrogen deprivation alone had already resulted in increased levels of ERa mRNA/protein, long-term tamoxifen exposure now reduced ER alpha levels. Whilst long-term exposure to fulvestrant reduced ERa. mRNA/protein levels in the oestrogen-maintained cells to a level barely detectable by Western immunoblotting and non-functional in inducing gene expression (ERE-LUC reporter or pS2), in oestrogen-deprived cells the reduction was much less substantial and these cells retained an oestrogen-induction of both the ERE-LUC reporter gene and the endogenous pS2 gene which could still be inhibited by antioestrogen. This demonstrates that whilst ER alpha can be abrogated by fulvestrant and increased by tamoxifen in some circumstances, this does not always hold true and mechanisms other than alteration to ER must be involved in the development of antioestrogen resistant growth. (c) 2006 Elsevier Ltd. All rights reserved.

Low level laser therapy (LLLT) decreases pulmonary microvascular leakage, neutrophil influx and IL-1 beta levels in airway and lung from rat subjected to LPS-induced inflammation

Background and Objective. Low level laser therapy (LLLT) is a known anti-inflammatory therapy. Herein we studied the effect of LLLT on lung permeability and the IL-1 beta level in LPS-induced pulmonary inflammation. Study Design/Methodology. Rats were divided into 12 groups (n = 7 for each group). Lung permeability was measured by quantifying extravasated albumin concentration in lung homogenate, inflammatory cells influx was determined by myeloperoxidase activity, IL-1P in BAL was determined by ELISA and IL-1P mRNA expression in trachea was evaluated by RT-PCR. The rats were irradiated on the skin over the upper bronchus at the site of tracheotomy after LPS. Results. LLLT attenuated lung permeability. In addition, there was reduced neutrophil influx, myeloperoxidase activity and both IL-1 beta in BAL and IL-1 beta mRNA expression in trachea obtained from animals subjected to LPS-induced inflammation. Conclusion. LLLT reduced the lung permeability by a mechanism in which the IL-1 beta seems to have an important role.

Modulation of thyroid hormone receptors, TR alpha and TR beta, by using different doses of triiodothyronine (T3) at different times

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Stimulation-specific contribution of p38 and JNK to IFN-beta gene expression in human macrophages

Induction of interferon-beta (IFN-beta) gene expression is a tightly regulated process, and a plethora of studies identified the signal transduction pathway TANK-binding kinase-1 (TBK-1)/IFN regulatory factor-3 (IRF-3) as essential to the induction of IFN-beta gene expression. Data regarding the role of p38 and JNK are rare, however. We investigated the contribution of these kinases to IFN-beta expression in human macrophages treated with poly(I:C), lipopolysaccharide (LPS), Sendai virus, or vesicular stomatitis virus (VSV). We found that all the stimuli induced IFN-beta mRNA, albeit to a different extent. Whereas LPS and VSV induced the phosphorylation of p38 and JNK, neither poly(I:C) nor Sendai virus led to the detection of phosphospecific signals. When inhibiting p38, a VSV-triggered IFN-beta mRNA response was inhibited, whereas inhibiting JNK suppressed an LPS-triggered response, but only when macrophages were primed with IFN-gamma. Neither poly(I:C)-induced nor Sendai virus-induced IFN-beta mRNA expression was affected when p38 and JNK were inhibited. Collectively, the data show that the contribution of p38 and JNK to the expression of IFN-beta occurs in a stimulation-specific manner in human macrophages.

Cloning, expression, and distribution of a Ca(2+)-activated K+ channel beta-subunit from human brain.

We have cloned and expressed a Ca(2+)-activated K+ channel beta-subunit from human brain. The open reading frame encodes a 191-amino acid protein possessing significant homology to a previously described subunit cloned from bovine muscle. The gene for this subunit is located on chromosome 5 at band q34 (hslo-beta). There is no evidence for alternative RNA splicing of this gene product. hslo-beta mRNA is abundantly expressed in smooth muscle, but expression levels are low in most other tissues, including brain. Brain subregions in which beta-subunit mRNA expression is relatively high are the hippocampus and corpus callosum. The coexpression of hslo-beta mRNA together with hslo-alpha subunits in either Xenopus oocytes or stably transfected HEK 293 cells give rise to Ca(2+)-activated potassium currents with a much increased calcium and/or voltage sensitivity. These data indicate that the beta-subunit shows a tissue distribution different to that of the alpha-subunit, and in many tissues there may be no association of alpha-subunits with beta-subunits. These beta-subunits can play a functional role in the regulation of neuronal excitability by tuning the Ca2+ and/or the voltage dependence of alpha-subunits.

Human immunodeficiency virus type 1 infection alters chemokine beta peptide expression in human monocytes: implications for recruitment of leukocytes into brain and lymph nodes.

Two chemokine (chemoattractant cytokines) beta peptides, macrophage inflammatory proteins 1 alpha and 1 beta (MIP-1 alpha and MIP-1 beta), were induced in human monocyte cultures following infection with the human immunodeficiency virus type 1 (HIV-1). Induction depended on productive viral infection: not only did the kinetics of MIP-1 peptide induction closely follow those of viral replication, but monocyte cultures inoculated with heat-inactivated virus or infected in the presence of AZT failed to produce these chemokine beta peptides. In addition, HIV infection markedly altered the pattern of beta chemokine expression elicited by tumor necrosis factor (TNF), itself a potent proinflammatory cytokine upregulated during the development of AIDS. Reverse transcription (RT)-PCR and RT-in situ PCR studies on brain tissue from patients with AIDS dementia demonstrated elevated MIP-1 alpha and MIP-1 beta mRNA expression relative to comparable samples from HIV-1-infected patients without dementia. Cells expressing chemokines in HIV-1-infected brains were identified morphologically as microglia and astrocytes. As MIP-1 alpha and MIP-1 beta are potent chemoattractants for both monocytes and specific subpopulations of lymphocytes, this dysregulation of beta chemokine expression may influence the trafficking of leukocytes during HIV infection. These data, taken together, suggest a mechanism by which HIV-1-infected monocytes might recruit uninfected T cells and monocytes to sites of active viral replication or inflammation, notably the brain and lymph nodes.

Thyroid-stimulating hormone (TSH)-directed induction of the CREM gene in the thyroid gland participates in the long-term desensitization of the TSH receptor.

Thyroid gland function is regulated by the hypothalamic-pituitary axis via the secretion of TSH, according to environmental, developmental, and circadian stimuli. TSH modulates both the secretion of thyroid hormone and gland trophism through interaction with a specific guanine nucleotide-binding protein-coupled receptor (TSH receptor; TSH-R), which elicits the activation of the cAMP-dependent signaling pathway. After TSH stimulation, the levels of TSH-R RNA are known to decrease dramatically within a few hours. This phenomenon ultimately leads to homologous long-term desensitization of the TSH-R. Here we show that TSH drives the induction of the inducible cAMP early repressor (ICER) isoform of the cAMP response element (CRE) modulator gene both in rat thyroid gland and in the differentiated thyroid cell line FRTL-5. The kinetics of ICER protein induction mirrors the down-regulation of TSH-R mRNA. ICER binds to a CRE-like sequence in the TSH-R promoter and represses its expression. Thus, ICER induction by TSH in the thyroid gland represents a paradigm of the molecular mechanism by which pituitary hormones elicit homologous long-term desensitization.

Role of glycogen synthase kinase 3 beta as a negative regulator of dorsoventral axis formation in Xenopus embryos.

The dorsoventral axis is established early in Xenopus development and may involve signaling by Wnts, a family of Wnt1-protooncogene-related proteins. The protein kinase shaggy functions in the wingless/Wnt signaling pathway, which operates during Drosophila development. To assess the role of a closely related kinase, glycogen synthase kinase 3 beta (GSK-3 beta), in vertebrate embryogenesis, we cloned a cDNA encoding a Xenopus homolog of GSK-3 beta (XGSK-3 beta). XGSK-3 beta-specific transcripts were detected by Northern analysis in Xenopus eggs and early embryos. Microinjection of the mRNA encoding a catalytically inactive form of rat GSK-3 beta into a ventrovegetal blastomere of eight-cell embryos caused ectopic formation of a secondary body axis containing a complete set of dorsal and anterior structures. Furthermore, in isolated ectodermal explants, the mutant GSK-3 beta mRNA activated the expression of neural tissue markers. Wild-type XGSK-3 beta mRNA suppressed the dorsalizing effects of both the mutated GSK-3 beta and Xenopus dishevelled, a proposed upstream signaling component of the same pathway. These results strongly suggest that XGSK-3 beta functions to inhibit dorsoventral axis formation in the embryo and provide evidence for conservation of the Wnt signaling pathway in Drosophila and vertebrates.

A novel mutation (Q40P) in PAX8 associated with congenital hypothyroidism and thyroid hypoplasia: Evidence for phenotypic variability in mother and child

Congenital hypothyroidism associated with thyroid hypoplasia can be caused by several genetic defects, including mutations in the TSH beta -subunit, the TSH receptor, the G(A)alpha -subunit, and the transcription factor PAX8. Four girls with sporadic congenital hypothyroidism and hypoplastic thyroid glands were analyzed for mutations in PAX8 and TTF2 (FKHL15). Mutations in the coding region of the TSH beta -subunit gene, the TSH receptor gene, and exons 8 and 9 of G(mu)alpha had been excluded previously. Serum TSH concentrations were 150 mU/liter or more, TG levels were within normal limits, and thyroid autoantibodies were absent. Technetium scintigraphies did not reveal the presence of thyroid tissue, but ultrasonography documented hypoplastic, normally located glands.One patient was found to harbor a heterozygous transversion 119A -->C in exon 3 of PAX8 replacing a conserved glutamine by proline in the paired box domain (Q40P). Analysis of her family members revealed that her mother, who has a thyroid gland of normal size and mild, adult-onset autoimmune hypothyroidism, is also heterozygous for this mutation. Functional analyses of the PAX8 Q40P mutation showed impaired binding to a PAX8 response element and absent transactivation of a thyroid peroxidase promoter luciferase reporter gene.These findings confirm the important role of PAX8 in the development of the thyroid, but they indicate that PAX8 gene mutations may have a variable penetrance or expressivity. The absence of mutations in the coding sequences of the analyzed genes in the three other patients supports the concept that the pathogenesis of congenital hypothyroidism associated with thyroid hypoplasia is diverse.

Exercício e restrição alimentar aumentam o RNAm de proteínas do trânsito de Ca2+ miocárdico em ratos

FUNDAMENTO: Treinamento físico (TF) aumenta a sensibilidade dos hormônios tireoidianos (HT) e a expressão gênica de estruturas moleculares envolvidas no movimento intracelular de cálcio do miocárdio, enquanto a restrição alimentar (RIA) promove efeitos contrários ao TF. OBJETIVO: Avaliar os efeitos da associação TF e RIA sobre os níveis plasmáticos dos HT e a produção de mRNA dos receptores HT e estruturas moleculares do movimento de cálcio do miocárdio de ratos. MÉTODOS: Utilizaram-se ratos Wistar Kyoto divididos em: controle (C, n = 7), RIA (R50, n = 7), exercício físico (EX, n = 7) e exercício físico + RIA (EX50, n = 7). A RIA foi de 50% e o TF foi natação (1 hora/dia, cinco sessões/semana, 12 semanas consecutivas). Avaliaram-se as concentrações séricas de triiodotironina (T3), tiroxina (T4) e hormônio tireotrófico (TSH). O mRNA da bomba de cálcio do retículo sarcoplasmático (SERCA2a), fosfolamban (PLB), trocador Na+/Ca+2 (NCX), canal lento de cálcio (canal-L), rianodina (RYR), calsequestrina (CQS) e receptor de HT (TRα1 e TRβ1) do miocárdio foram avaliados por reação em cadeia da polimerase (PCR) em tempo real. RESULTADOS: RIA reduziu o T4, TSH e mRNA do TRα1 e aumentou a expressão da PLB, NCX e canal-L. TF aumentou a expressão do TRβ1, canal-L e NCX. A associação TF e RIA reduziu T4 e TSH e aumentou o mRNA do TRβ1, SERCA2a, NCX, PLB e correlação do TRβ1 com a CQS e NCX. CONCLUSÃO: Associação TF e RIA aumentou o mRNA das estruturas moleculares cálcio transiente, porém o eixo HT-receptor não parece participar da transcrição gênica dessas estruturas.