Thyroid hormone action is required for normal cone opsin expression during mouse retinal development

PURPOSE. The expression of S- and M-opsins in the murine retina is altered in different transgenic mouse models with mutations in the thyroid hormone receptor (TR)-beta gene, demonstrating an important role of thyroid hormone (TH) in retinal development. METHODS. The spatial expression of S- and M-opsin was compared in congenital hypothyroidism and in two different TR mutant mouse models. One mouse model contains a ligand-binding mutation that abolishes TH binding and results in constitutive binding to nuclear corepressors. The second model contains a mutation that blocks binding of coactivators to the AF-2 domain without affecting TH binding. RESULTS. Hypothyroid newborn mice showed an increase in S- opsin expression that was completely independent of the genotype. Concerning M-opsin expression, hypothyroidism caused a significant decrease (P < 0.01) only in wild-type animals. When TR beta 1 and -beta 2 were T3-binding defective, the pattern of opsin expression was similar to TR beta ablation, showing increased S- opsin expression in the dorsal retina and no expression of M-opsin in the entire retina. In an unexpected finding, immunostaining for both opsins was detected when both subtypes of TR beta were mutated in the helix 12 AF-2 domain. CONCLUSIONS. The results show, for the first time, that the expression of S- and M-opsin is dependent on normal thyroid hormone levels during development.

Effect of pH and temperature on the global compactness, structure, and activity of cellobiohydrolase Cel7A from Trichoderma harzianum

Due to its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum (T. harzianum) has considerable potential in biomass hydrolysis application. Cellulases from Trichoderma reesei have been widely used in studies of cellulose breakdown. However, cellulases from T. harzianum are less-studied enzymes that have not been characterized biophysically and biochemically as yet. Here, we examined the effects of pH and temperature on the secondary and tertiary structures, compactness, and enzymatic activity of cellobiohydrolase Cel7A from T. harzianum (Th Cel7A) using a number of biophysical and biochemical techniques. Our results show that pH and temperature perturbations affect Th Cel7A stability by two different mechanisms. Variations in pH modify protonation of the enzyme residues, directly affecting its activity, while leading to structural destabilization only at extreme pH limits. Temperature, on the other hand, has direct influence on mobility, fold, and compactness of the enzyme, causing unfolding of Th Cel7A just above the optimum temperature limit. Finally, we demonstrated that incubation with cellobiose, the product of the reaction and a competitive inhibitor, significantly increased the thermal stability of Th Cel7A. Our studies might provide insights into understanding, at a molecular level, the interplay between structure and activity of Th Cel7A at different pH and temperature conditions.

Advanced QSAR Studies on PPAR delta Ligands Related to Metabolic Diseases

PPAR delta is a nuclear receptor that, when activated, regulates the metabolism of carbohydrates and lipids and is related to metabolic syndrome and type 2 diabetes. To understand the main interactions between ligands and PPAR delta, we have constructed 2D and 3D QSAR models and compared them with HOMO, LUMO and electrostatic potential maps of the compounds studied, as well as docking results. All QSAR models showed good statistical parameters and prediction outcomes. The QSAR models were used to predict the biological activity of an external test set, and the predicted values are in good agreement with the experimental results. Furthermore, we employed all maps to evaluate the possible interactions between the ligands and PPAR delta. These predictive QSAR models, along with the HOMO, LUMO and MEP maps, can provide insights into the structural and chemical properties that are needed in the design of new PPAR delta ligands that have improved biological activity and can be employed to treat metabolic diseases.

Ligand- and Structure-Based Drug Design Strategies and PPAR delta/alpha Selectivity

Peroxisome-proliferator-activated receptors are a class of nuclear receptors with three subtypes: a, ? and d. Their main function is regulating gene transcription related to lipid and carbohydrate metabolism. Currently, there are no peroxisome-proliferator-activated receptors d drugs being marketed. In this work, we studied a data set of 70 compounds with a and d activity. Three partial least square models were created, and molecular docking studies were performed to understand the main reasons for peroxisome-proliferator-activated receptors d selectivity. The obtained results showed that some molecular descriptors (log P, hydration energy, steric and polar properties) are related to the main interactions that can direct ligands to a particular peroxisome-proliferator-activated receptors subtype.

Signal transduction in Plasmodium-Red Blood Cells interactions and in cytoadherence

Malaria is responsible for more than 1.5 million deaths each year, especially among children (Snow et al. 2005). Despite of the severity of malaria situation and great effort to the development of new drug targets (Yuan et al. 2011) there is still a relative low investment toward antimalarial drugs. Briefly there are targets classes of antimalarial drugs currently being tested including: kinases, proteases, ion channel of GPCR, nuclear receptor, among others (Gamo et al. 2010). Here we review malaria signal transduction pathways in Red Blood Cells (RBC) as well as infected RBCs and endothelial cells interactions, namely cytoadherence. The last process is thought to play an important role in the pathogenesis of severe malaria. The molecules displayed on the surface of both infected erythrocytes (IE) and vascular endothelial cells (EC) exert themselves as important mediators in cytoadherence, in that they not only induce structural and metabolic changes on both sides, but also trigger multiple signal transduction processes, leading to alteration of gene expression, with the balance between positive and negative regulation determining endothelial pathology during a malaria infection.

Impact of Glucocorticoid Receptor Gene Polymorphisms on the Metabolic Profile of Adult Patients with the Classical Form of 21-Hydroxylase Deficiency

Background: CAH patients have an increased risk of cardiovascular disease, and it remains unknown if lifelong glucocorticoid (GC) treatment is a contributing factor. In the general population, glucocorticoid receptor gene (NR3C1) polymorphisms are associated with an adverse metabolic profile. Our aim was to analyze the association between the NR3C1 polymorphisms and the metabolic profile of CAH patients. Methodology: Sixty-eight adult patients (34SV/34SW) with a mean age of 28.4 +/- 9 years received dexamethasone (mean 0.27 +/- 0.11 mg/day) to obtain normal androgen levels. SW patients also received fludrocortisone (50 mu g/day). Metabolic syndrome (MetS) was defined by the NCEP ATPIII criteria and obesity by BMI >= 30 kg/m(2). NR3C1 alleles were genotyped, and association analyses with phenotype were carried out with Chi-square, t-test and regression analysis. Results: Obesity and MetS were observed in 23.5% and 7.3% of patients, respectively, and were not correlated with GC doses and treatment duration. BMI was positively correlated with blood pressure (BP), triglycerides (TG), LDL-c levels and HOMA-IR and inversely correlated with HDL-c levels. BclI and A3669G variants were found in 26.4% and 9.6% of alleles, respectively. Heterozygotes for the BclI polymorphism presented with higher BMI (29 kg/m(2) +/- 5.3 vs. 26 kg/m(2) +/- 5.3, respectively) and waist circumference (89 cm +/- 12.7 vs. 81 cm +/- 13, respectively) compared to wild-type subjects. Hypertension was found in 12% of patients and heterozygotes for the BclI polymorphism presented higher systolic BP than wild type subjects. Low HDL-c and high TG levels were identified in 30% and 10% of patients, respectively, and were not associated with the NR3C1 polymorphisms. A3669G carriers and non-carriers did not differ. Conclusion: In addition to GC therapy, the BclI GR variant might play an important role in obesity susceptibility in CAH patients. Genotyping of GR polymorphisms could result in the identification of a subgroup at risk patients, allowing for the establishment of personalized treatment and the avoidance of long-term adverse consequences.

Cannabinoid CB1 receptor mediates glucocorticoid effects on hormone secretion induced by volume and osmotic changes

1. The present study provides the first in vivo evidence that the cannabinoid CB1 receptor mediates the effects of dexamethasone on hormone release induced by changes in circulating volume and osmolality. Male adult rats were administered with the CB1 receptor antagonist rimonabant (10 mg/Kg, p.o.), followed or not in 1 hour by dexamethasone (1 mg/Kg, i.p.). Extracellular volume expansion (EVE, 2 mL/100 g of body weight, i.v.) was performed 2 hours after dexamethasone or vehicle treatment using either isotonic (I-EVE, 0.15 mol/L) or hypertonic (H-EVE, 0.30 mol/L) NaCl solution. Five minutes after EVE, animals were decapitated and trunk blood was collected for all plasma measurements. 2. Rimonabant potentiated oxytocin (OT) secretion induced by H-EVE and completely reversed the inhibitory effects of dexamethasone in response to the same stimulus. These data suggest that glucocorticoid modulation of OT release is mediated by the CB1 receptor. 3. Although dexamethasone did not affect vasopressin (AVP) secretion induced by H-EVE, the administration of rimonabant potentiated AVP release in response to the same stimulus, supporting the hypothesis that the CB1 receptor regulates AVP secretion independently of glucocorticoid-mediated signalling. 4. Dexamethasone alone did not affect atrial natriuretic peptide (ANP) release stimulated by I-EVE or H-EVE. However, pretreatment with rimonabant potentiated ANP secretion induced by H-EVE, suggesting a possible role for the CB1 receptor in the control of peripheral factors that modulate cardiovascular function. 5. Rimonabant also reversed the inhibitory effects of dexamethasone on H-EVE-induced corticosterone secretion, reinforcing the hypothesis that the CB1 receptor may be involved in the negative feedback exerted by glucocorticoids on the activity of the hypothalamicpituitaryadrenal axis. 6. Collectively, the results of the present study indicate that the CB1 receptor modulates neurohypophyseal hormone secretion and systemic factors, such as corticosterone and ANP, thus participating in homeostatic responses to altered extracellular volume and plasma tonicity.

Inhibition of cannabinoid CB1 receptor upregulates Slc2a4 expression via nuclear factor-kappa B and sterol regulatory element-binding protein-1 in adipocytes

Evidences have suggested that the endocannabinoid system is overactive in obesity, resulting in enhanced endocannabinoid levels in both circulation and visceral adipose tissue. The blockade of cannabinoid receptor type 1 (CB1) has been proposed for the treatment of obesity. Besides loss of body weight, CB1 antagonism improves insulin sensitivity, in which the glucose transporter type 4 (GLUT4) plays a key role. The aim of this study was to investigate the modulation of GLUT4-encoded gene (Slc2a4 gene) expression by CB1 receptor. For this, 3T3-L1 adipocytes were incubated in the presence of a highly selective CB1 receptor agonist (1 mu M arachidonyl-2'-chloroethylamide) and/or a CB1 receptor antagonist/inverse agonist (0.1, 0.5, or 1 mu M AM251, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide). After acute (2 and 4 h) and chronic (24 h) treatments, cells were harvested to evaluate: i) Slc2a4, Cnr1 (CB1 receptor-encoded gene), and Srebf1 type a (SREBP-1a type-encoded gene) mRNAs (real-time PCR); ii) GLUT4 protein (western blotting); and iii) binding activity of nuclear factor (NF)-kappa B and sterol regulatory element-binding protein (SREBP)-1 specifically in the promoter of Slc2a4 gene (electrophoretic mobility shift assay). Results revealed that both acute and chronic CB1 receptor antagonism greatly increased (similar to 2.5-fold) Slc2a4 mRNA and protein content. Additionally, CB1-induced upregulation of Slc2a4 was accompanied by decreased binding activity of NF-kappa B at 2 and 24 h, and by increased binding activity of the SREBP-1 at 24 h. In conclusion, these findings reveal that the blockade of CB1 receptor markedly increases Slc2a4/GLUT4 expression in adipocytes, a feature that involves NF-kappa B and SREBP-1 transcriptional regulation. Journal of Molecular Endocrinology (2012) 49, 97-106

Real-time PCR quantitation of glucocorticoid receptor alpha isoform

Abstract Background The expression of glucocorticoid-receptor (GR) seems to be a key mechanism in the regulation of glucocorticoid (GC) sensitivity and is potentially involved in cases of GC resistance or hypersensitivity. The aim of this study is to describe a method for quantitation of GR alpha isoform (GRα) expression using real-time PCR (qrt-PCR) with analytical capabilities to monitor patients, offering standard-curve reproducibility as well as intra- and inter-assay precision. Results Standard-curves were constructed by employing standardized Jurkat cell culture procedures, both for GRα and BCR (breakpoint cluster region), as a normalizing gene. We evaluated standard-curves using five different sets of cell culture passages, RNA extraction, reverse transcription, and qrt-PCR quantification. Intra-assay precision was evaluated using 12 replicates of each gene, for 2 patients, in a single experiment. Inter-assay precision was evaluated on 8 experiments, using duplicate tests of each gene for two patients. Standard-curves were reproducible, with CV (coefficient of variation) of less than 11%, and Pearson correlation coefficients above 0,990 for most comparisons. Intra-assay and inter-assay were 2% and 7%, respectively. Conclusion This is the first method for quantitation of GRα expression with technical characteristics that permit patient monitoring, in a fast, simple and robust way.

Gastrin-Releasing Peptide Receptor Antagonism Induces Protection from Lethal Sepsis: Involvement of Toll-like Receptor 4 Signaling

In sepsis, toll-like receptor (TLR)-4 modulates the migration of neutrophils to infectious foci, favoring bacteremia and mortality. In experimental sepsis, organ dysfunction and cytokines released by activated macrophages can be reduced by gastrin-releasing peptide (GRP) receptor (GRPR) antagonist RC-3095. Here we report a link between GRPR and TLR-4 in experimental models and in sepsis patients. RAW 264.7 culture cells were exposed to lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-alpha and RC-3095 (10 ng/mL), Male Wistar rats were subjected to cecal ligation and puncture (CLP), and RC-3095 was administered (3 mg/kg, subcutaneously); after 6 h, we removed the blood, bronchoalveolar lavage, peritoneal lavage and lung. Human patients with a clinical diagnosis of sepsis received a continuous infusion with RC-3095 (3 mg/kg, intravenous) over a period of 12 h, and plasma was collected before and after RC-3095 administration and, in a different set of patients with systemic inflammatory response syndrome (SIRS) or sepsis. GRP plasma levels were determined. RC-3095 inhibited TLR-4, extracellular-signal-related kinase (ERK)-1/2, Jun NH2-terminal kinase (JNK) and Akt and decreased activation of activator protein 1 (AP-1), nuclear factor (NF)-kappa B and interleukin (IL)-6 in macrophages stimulated by LPS. It also decreased IL-6 release from macrophages stimulated by TNF-alpha. RC-3095 treatment in CLP rats decreased lung TLR-4, reduced the migration of cells to the lung and reduced systemic cytokines and bacterial dissemination. Patients with sepsis and systemic inflammatory response syndrome have elevated plasma levels of GRP which associates with clinical outcome in the sepsis patients. These findings highlight the role of GRPR signaling in sepsis outcome and the beneficial action of GRPR antagonists in controlling the inflammatory response in sepsis through a mechanism involving at least inhibition of TLR-4 signaling. Online address: http://www.molmed.org doi: 10.2119/molmed.2012.00083

Glucocorticoid and Estrogen Receptors Are Reduced in Mitochondria of Lung Epithelial Cells in Asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell's energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GR alpha and ER beta in lung tissue. Allergic airway inflammation caused reduction in mtGR alpha, mtER beta, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GR alpha and ER beta in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease.

Characterization of a Novel Oral Glucocorticoid System and Its Possible Role in Disease

Synthetic corticosteroids are used widely for the treatment of a variety of diseases of the mouth. However, little is known as to whether the oral mucosa is able to modulate the local concentration of active corticosteroids or to produce steroids de novo. This has important clinical implications, because tissue-specific regulation of glucocorticoids is a key determinant of the clinical efficacy of these drugs. In the present study, we show that oral fibroblasts and keratinocytes expressed ACTH receptor (MC2R), glucocorticoid receptor (GR), and 11 beta-hydroxysteroid dehydrogenases (11 beta-HSDs). Unlike keratinocytes, fibroblasts lacked 11 beta-HSD2 and could not effectively deactivate exogenously administered cortisol. However, both cell types were able not only to activate cortisone into the active form cortisol, but also to synthesize cortisol de novo following stimulation with ACTH. 11 beta-HSD2, the enzyme controlling cortisol deactivation, exhibited different patterns of expression in normal (squamous epithelium and salivary glands) and diseased oral mucosa (squamous cell carcinoma and mucoepidermoid carcinoma). Blocking of endogenous cortisol catabolism in keratinocytes with the 11 beta-HSD2 inhibitor 18 beta-glycyrrhetinic acid mimicked the effect of exogenous administration of hydrocortisone and partially prevented the detrimental effects induced by pemphigus vulgaris sera. Analysis of the data demonstrates that a novel, non-adrenal glucocorticoid system is present in the oral mucosa that may play an important role in disease.

Influence of N-methyl-D-aspartate receptors on ouabain activation of nuclear factor-kappa B in the rat hippocampus

It has been shown that ouabain (OUA) can activate the Na,K-ATPase complex and mediate intracellular signaling in the central nervous system (CNS). Inflammatory stimulus increases glutamatergic transmission, especially at N-methyl-D-aspartate (NMDA) receptors, which are usually coupled to the activation of nitric oxide synthase (NOS). Nuclear factor-kappa B (NF-kappa B) activation modulates the expression of genes involved in development, plasticity, and inflammation. The present work investigated the effects of OUA on NF-kappa B binding activity in rat hippocampus and the influence of this OUA-Na,K-ATPase signaling cascade in NMDA-mediated NF-kappa B activation. The findings presented here are the first report indicating that intrahippocampal administration of OUA, in a concentration that did not alter Na,K-ATPase or NOS activity, induced an activation of NF-kappa B, leading to increases in brain-derived neurotrophic factor (Bdnf), inducible NOS (iNos), tumor necrosis factor-alpha (Tnf-alpha), and B-cell leukemia/lymphoma 2 (Bcl2) mRNA levels. This response was not linked to any significant signs of neurodegeneration as showed via Fluoro-Jade B and Nissl stain. Intrahippocampal administration of NMDA induced NF alpha B activation and increased NOS and alpha 2/3-Na,K-ATPase activities. NMDA treatment further increased OUA-induced NF-kappa B activation, which was partially blocked by MK-801, an antagonist of NMDA receptor. These results suggest that OUA-induced NF-kappa B activation is at least in part dependent on Na,K-ATPase modulatory action of NMDA receptor in hippocampus. The interaction of these signaling pathways could be associated with biological mechanisms that may underlie the basal homeostatic state linked to the inflammatory signaling cascade in the brain. (c) 2011 Wiley Periodicals, Inc.

Resistance to EGF receptor inhibitors in glioblastoma mediated by phosphorylation of the PTEN tumor suppressor at tyrosine 240

Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.

Glucocorticoid Sensitivity and Proinflammatory Cytokines Pattern in Pemphigus

Glucocorticoids (GC) represent the main treatment for pemphigus; however, some patients show GC resistance. GC sensitivity was evaluated in 19 pemphigus patients and 41 controls by the number of binding sites [B-max (fmol/mg protein)] and the affinity of GC receptor [Kd (nM)] to dexamethasone (DEX) as well as by the pattern of cytokine by DEX-mediated inhibition of concanavalin-A (Con-A)-stimulated PBMC proliferation. The Kd (15.7 +/- 2.8 vs.8.1 +/- 1.3) and Bmax (6.5 +/- 0.9 vs. 3.9 +/- 0.3) were higher in pemphigus than controls (p = 0.002). Considering the values above the 95th percentile of normal group as a cut-off (K-d > 24.9 nM and B-max > 8.1 fmol/mg protein), elevated K-d and B-max were observed in 9.8% and 2.4% of controls and 15.8% and 36.8% of patients (p = 0.02). PBMC proliferation was stimulated by Con-A and inhibited by DEX (p < 0.001) in both pemphigus and control groups. IL-6 and TNF alpha (pg/mL) basal production were higher in patients than controls. There was an increment of these cytokines after Con-A stimulation, and they were inhibited by DEX (p = 0.002) in controls and remained elevated in pemphigus (p < 0.02). Patients and controls showed no difference in basal and stimulated production of IL-8 and IL-10. There is an alteration on GC sensitivity in pemphigus patients and a higher production of proinflammatory cytokines. Therefore, in pemphigus patients, proinflammatory cytokines might be involved in the mechanism of GC resistance and/or in its maintenance.