Human pregnane X receptor is expressed in breast carcinomas, potential heterodimers formation between hPXR and RXR-alpha.

BACKGROUND The human pregnane X receptor (hPXR) is an orphan nuclear receptor that induces transcription of response elements present in steroid-inducible cytochrome P-450 gene promoters. This activation requires the participation of retinoid X receptors (RXRs), needed partners of hPXR to form heterodimers. We have investigated the expression of hPXR and RXRs in normal, premalignant, and malignant breast tissues, in order to determine whether their expression profile in localized infiltrative breast cancer is associated with an increased risk of recurrent disease. METHODS Breast samples from 99 patients including benign breast diseases, in situ and infiltrative carcinomas were processed for immunohistochemistry and Western-blot analysis. RESULTS Cancer cells from patients that developed recurrent disease showed a high cytoplasmic location of both hPXR isoforms. Only the infiltrative carcinomas that relapsed before 48 months showed nuclear location of hPXR isoform 2. This location was associated with the nuclear immunoexpression of RXR-alpha. CONCLUSION Breast cancer cells can express both variants 1 and 2 of hPXR. Infiltrative carcinomas that recurred showed a nuclear location of both hPXR and RXR-alpha; therefore, the overexpression and the subcellular location changes of hPXR could be considered as a potential new prognostic indicator.

Análisis del gen adra2a receptor alfa 2a adrenergico en pacientes con trastorno de hiperactividad y déficit de atención

El trastorno de hiperactividad y déficit de atención (THDA), es definido clínicamente como una alteración en el comportamiento, caracterizada por inatención, hiperactividad e impulsividad. Estos aspectos son clasificados en tres subtipos, que son: Inatento, hiperactivo impulsivo y mixto. Clínicamente se describe un espectro amplio que incluye desordenes académicos, trastornos de aprendizaje, déficit cognitivo, trastornos de conducta, personalidad antisocial, pobres relaciones interpersonales y aumento de la ansiedad, que pueden continuar hasta la adultez. A nivel global se ha estimado una prevalencia entre el 1% y el 22%, con amplias variaciones, dadas por la edad, procedencia y características sociales. En Colombia, se han realizado estudios en Bogotá y Antioquia, que han permitido establecer una prevalencia del 5% y 15%, respectivamente. La causa específica no ha sido totalmente esclarecida, sin embargo se ha calculado una heredabilidad cercana al 80% en algunas poblaciones, demostrando el papel fundamental de la genética en la etiología de la enfermedad. Los factores genéticos involucrados se relacionan con cambios neuroquímicos de los sistemas dopaminérgicos, serotoninérgicos y noradrenérgicos, particularmente en los sistemas frontales subcorticales, corteza cerebral prefrontal, en las regiones ventral, medial, dorsolateral y la porción anterior del cíngulo. Basados en los datos de estudios previos que sugieren una herencia poligénica multifactorial, se han realizado esfuerzos continuos en la búsqueda de genes candidatos, a través de diferentes estrategias. Particularmente los receptores Alfa 2 adrenérgicos, se encuentran en la corteza cerebral, cumpliendo funciones de asociación, memoria y es el sitio de acción de fármacos utilizados comúnmente en el tratamiento de este trastorno, siendo esta la principal evidencia de la asociación de este receptor con el desarrollo del THDA. Hasta la fecha se han descrito más de 80 polimorfismos en el gen (ADRA2A), algunos de los cuales se han asociado con la entidad. Sin embargo, los resultados son controversiales y varían según la metodología diagnóstica empleada y la población estudiada, antecedentes y comorbilidades. Este trabajo pretende establecer si las variaciones en la secuencia codificante del gen ADRA2A, podrían relacionarse con el fenotipo del Trastorno de Hiperactividad y el Déficit de Atención.

Fluorescence imaging reveals the nuclear behavior of peroxisome proliferator-activated receptor/retinoid X receptor heterodimers in the absence and presence of ligand.

In a global approach combining fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET), we address the behavior in living cells of the peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors involved in lipid and glucose metabolism, inflammation control, and wound healing. We first demonstrate that unlike several other nuclear receptors, PPARs do not form speckles upon ligand activation. The subnuclear structures that may be observed under some experimental conditions result from overexpression of the protein and our immunolabeling experiments suggest that these structures are subjected to degradation by the proteasome. Interestingly and in contrast to a general assumption, PPARs readily heterodimerize with retinoid X receptor (RXR) in the absence of ligand in living cells. PPAR diffusion coefficients indicate that all the receptors are engaged in complexes of very high molecular masses and/or interact with relatively immobile nuclear components. PPARs are not immobilized by ligand binding. However, they exhibit a ligand-induced reduction of mobility, probably due to enhanced interactions with cofactors and/or chromatin. Our study draws attention to the limitations and pitfalls of fluorescent chimera imaging and demonstrates the usefulness of the combination of FCS, FRAP, and FRET to assess the behavior of nuclear receptors and their mode of action in living cells.

Functional interactions of peroxisome proliferator-activated receptor, retinoid-X receptor, and Sp1 in the transcriptional regulation of the acyl-coenzyme-A oxidase promoter.

Peroxisome proliferator-activated receptor (PPARs) are members of the nuclear receptor superfamily. For transcriptional activation of their target genes, PPARs heterodimerize with the retinoid-X receptor (RXR). The convergence of the PPAR and RXR signaling pathways has been shown to have an important function in lipid metabolism. The promoter of the gene encoding the acyl-coenzyme-A oxidase (ACO), the rate-limiting enzyme in peroxisomal beta-oxidation of fatty acids, is a target site of PPAR action. In this study, we examined the role and the contribution of both cis-and trans-acting factors in the transcriptional regulation of this gene using transient transfections in insect cells. We identified several functional cis-acting elements present in the promoter of the ACO gene and established that PPAR-dependent as well as PPAR-independent mechanisms can activate the ACO promoter in these cells. We show that the PPAR/RXR heterodimer exerts its effect through two response elements within the ACO promoter, in synergy with the transcription factor Sp1 via five Sp1-binding sites. Furthermore, this functional interaction also occurs when Sp1 is co-expressed with PPAR or RXR alone, indicating that activation can occur independently of PPAR/RXR heterodimers.

Signaling cross-talk between peroxisome proliferator-activated receptor/retinoid X receptor and estrogen receptor through estrogen response elements.

Peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs) are nuclear hormone receptors that are activated by fatty acids and 9-cis-retinoic acid, respectively. PPARs and RXRs form heterodimers that activate transcription by binding to PPAR response elements (PPREs) in the promoter of target genes. The PPREs described thus far consist of a direct tandem repeat of the AGGTCA core element with one intervening nucleotide. We show here that the vitellogenin A2 estrogen response element (ERE) can also function as a PPRE and is bound by a PPAR/RXR heterodimer. Although this heterodimer can bind to several other ERE-related palindromic response elements containing AGGTCA half-sites, only the ERE is able to confer transactivation of test reporter plasmids, when the ERE is placed either close to or at a distance from the transcription initiation site. Examination of natural ERE-containing promoters, including the pS2, very-low-density apolipoprotein II and vitellogenin A2 genes, revealed considerable differences in the binding of PPAR/RXR heterodimers to these EREs. In their natural promoter context, these EREs did not allow transcriptional activation by PPARs/RXRs. Analysis of this lack of stimulation of the vitellogenin A2 promoter demonstrated that PPARs/RXRs bind to the ERE but cannot transactivate due to a nonpermissive promoter structure. As a consequence, PPARs/RXRs inhibit transactivation by the estrogen receptor through competition for ERE binding. This is the first example of signaling cross-talk between PPAR/RXR and estrogen receptor.

Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers.

The nuclear hormone receptors called PPARs (peroxisome proliferator-activated receptors alpha, beta, and gamma) regulate the peroxisomal beta-oxidation of fatty acids by induction of the acyl-CoA oxidase gene that encodes the rate-limiting enzyme of the pathway. Gel retardation and cotransfection assays revealed that PPAR alpha heterodimerizes with retinoid X receptor beta (RXR beta; RXR is the receptor for 9-cis-retinoic acid) and that the two receptors cooperate for the activation of the acyl-CoA oxidase gene promoter. The strongest stimulation of this promoter was obtained when both receptors were exposed simultaneously to their cognate activators. Furthermore, we show that natural fatty acids, and especially polyunsaturated fatty acids, activate PPARs as potently as does the hypolipidemic drug Wy 14,643, the most effective activator known so far. Moreover, we discovered that the synthetic arachidonic acid analogue 5,8,11,14-eicosatetraynoic acid is 100 times more effective than Wy 14,643 in the activation of PPAR alpha. In conclusion, our data demonstrate a convergence of the PPAR and RXR signaling pathways in the regulation of the peroxisomal beta-oxidation of fatty acids by fatty acids and retinoids.

Associação entre os polimorfismos HaeIII e MspI do gene para o receptor alfa de estrogênio e densidade mamográfica em mulheres após a menopausa

OBJETIVO: Avaliar a presença dos polimorfismos HaeIII e MspI do gene para o receptor de estrogênio alfa, bem como fatores clínicos e suas possíveis associações com a densidade mamográfica em mulheres após a menopausa. MÉTODOS: Foram avaliadas 115 mulheres após a menopausa, não usuárias de terapia hormonal e sem lesão mamária clínica ou mamograficamente identificada. A densidade mamográfica foi determinada por três observadores independentes, tomando-se como base a classificação dos padrões mamográficos do ACR-BIRADS®, 2003 (duas avaliações subjetivas e uma computadorizada - Adobe Photoshop® 7.0). Amostras de raspado bucal foram obtidas para extração de DNA e em seguida foi realizada uma PCR-RFLP (Polymerase Chain Reation - Restriction Fragment Length Polymorphism) para análise de polimorfismos presentes no íntron 1 e éxon 1 do gene do REalfa (HaeIII e MspI). RESULTADOS: O polimorfismo HaeIII foi encontrado em 43 (37,4%) das 115 mulheres, ao passo que o MspI estava presente em 96 (83,5%) das mesmas. Houve alto grau de concordância entre os três observadores na determinação da densidade mamográfica. Trinta e quatro (29,6%) mulheres tinham mamas densas, e 81 (70,4%), mamas lipossubstituídas. CONCLUSÃO: Não houve associação entre o polimorfismo HaeIII do gene para o receptor de estrogênio alfa e densidade mamográfica (Fisher = 0,712). Houve associação próxima à significância estatística entre o polimorfismo MspI e densidade (Fisher = 0,098). Idade, paridade e índice de massa corporal mostraram-se associados com densidade mamográfica.

Ligand-dependent activation of transcription in vitro by retinoic acid receptor α/retinoid X receptor α heterodimers that mimics transactivation by retinoids in vivo

All-trans and 9-cis retinoic acids (RA) signals are transduced by retinoic acid receptor/retinoid X receptor (RAR/RXR) heterodimers that act as functional units controlling the transcription of RA-responsive genes. With the aim of elucidating the underlying molecular mechanisms, we have developed an in vitro transcription system using a chromatin template made up of a minimal promoter and a direct repeat with 5-spacing-based RA response element. RARα and RXRα were expressed in and purified from baculovirus-infected Sf9 cells, and transcription was carried out by using naked DNA or chromatin templates. Transcription from naked templates was not affected by the presence of RA and/or RAR/RXR heterodimers. In contrast, very little transcription occurred from chromatin templates in the absence of RA or RAR/RXR heterodimers whereas their addition resulted in a dosage-dependent stimulation of transcription that never exceeded that occurring on naked DNA templates. Most importantly, the addition of synthetic agonistic or antagonistic retinoids to the chromatin transcription system mimicked their stimulatory or inhibitory action in vivo, and activation by a RXR-specific retinoid was subordinated to the binding of an agonist ligand to the RAR partner. Moreover, the addition of the p300 coactivator generated a synergistic enhancement of transcription. Thus, the dissection of this transcription system ultimately should lead to the elucidation of the molecular mechanisms by which RAR/RXR heterodimers control transcription in a ligand-dependent manner.

Influence of a fat overload on lipogenic regulators in metabolic syndrome patients

Several epidemiological studies have related an increase of lipids in the postprandial state to an individual risk for the development of CVD, possibly due to the increased plasma levels of TAG and fatty acids (FA) through enzymes of FA metabolism. The interaction between nutrition and the human genome determines gene expression and metabolic response. The aim of the present study was to evaluate the influence of a fat overload on the gene mRNA levels of lipogenic regulators in peripheral blood mononuclear cells (PBMC) from patients with the metabolic syndrome. The study included twenty-one patients with criteria for the metabolic syndrome who underwent a fat overload. Measurements were made before and after the fat overload of anthropometric and biochemical variables and also the gene mRNA levels of lipogenic factors. The main results were that the fat overload led to an increased mRNA levels of sterol regulatory element binding protein-1 (SREBP1), retinoid X receptor α (RXRα) and liver X receptor α (LXRα) in PBMC, and this increase was associated with the FA synthase (FASN) mRNA levels. We also found that TAG levels correlated with FASN mRNA levels. In addition, there was a positive correlation of SREBP1 with RXRα and of LXRα with the plasma lipoperoxide concentration. The fat overload led to an increase in regulators of lipogenesis in PBMC from patients with the metabolic syndrome.

Orphan nuclear receptor NGFI-B forms dimers with nonclassical interface

The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptor's subfamily 4A (Nr4a). NGFI-B was shown to be capable of binding both as a monomer to an extended half-site containing a single AAAGGTCA motif and also as a homodimer to a widely separated everted repeat, as opposed to a large number of nuclear receptors that recognize and bind specific DNA sequences predominantly as homo- and/or heterodimers. To unveil the structural organization of NGFI-B in solution, we determined the quaternary structure of the NGFI-B LBD by a combination of ab initio procedures from small-angle X-ray scattering (SAXS) data and hydrogen-deuterium exchange followed by mass spectrometry. Here we report that the protein forms dimers in solution with a radius of gyration of 2.9 nm and maximum dimension of 9.0 nm. We also show that the NGFI-B LBD dimer is V-shaped, with the opening angle significantly larger than that of classical dimer's exemplified by estrogen receptor (ER) or retinoid X receptor (RXR). Surprisingly, NGFI-B dimers formation does not occur via the classical nuclear receptor dimerization interface exemplified by ER and RXR, but instead, involves an extended surface area composed of the loop between helices 3 and 4 and C-terminal fraction of the helix 3. Remarkably, the NGFI-B dimer interface is similar to the dimerization interface earlier revealed for glucocorticoid nuclear receptor (GR), which might be relevant to the recognition of cognate DNA response elements by NGFI-B and to antagonism of NGFI-B-dependent transcription exercised by GR in cells. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society.

Imatinib atenua a fibrose miocárdica em associação com a inibição da atividade do PDGFRα

FUNDAMENTO: O Imatinib é um inibidor do receptor tirosina-quinase que foi confirmada como exercendo um efeito inibidor sobre a atividade do receptor do PDGF, fator de crescimento plaquetário (PDGFRα e PDGFRβ). OBJETIVO: Investigar o efeito protetor do Imatinib na fibrose miocárdica em acetato de deoxicorticosterona (DOCA)/ratos com hipertensão induzida por sal. MÉTODOS: Sessenta ratos Sprague-Dawley machos, uninefrectomizados foram distribuídos em três grupos: ratos controles (grupo CON): grupo deoxicorticosterona (grupo DOCA); grupo deoxicorticosterona e Imatinib (grupo DOCA IMA). A Pressão Arterial Sistólica (PAS) foi medida quinzenalmente. Foi estudada a porção apical do ventrículo esquerdo. Foram empregados: coloração vermelho sirius, coloração de hematoxilina-eosina, imuno-histoquímica e ensaio de western blot. RESULTADOS: A PAS nos grupos DOCA e IMA+DOCA foi maior que no grupo CON nos dias 14 e 28. Os animais do grupo DOCA apresentaram fibrose intersticial e perivascular grave no dia 28, e as expressões de PI, PIII, tenascina-C e fibronectina foram significativamente maiores que nos grupos DOCA+IMA e CON. Quando comparados com o grupo CON, os grupos DOCA e DOCA+IMA apresentaram resposta inflamatória de tecido miocárdico e infiltração de monócitos/macrófagos de diferentes graus. As expressões proteicas do PDGF-A, PDGF-C e PDGFRα foram significativamente maiores nos grupos DOCA e DOCA+IMA que no grupo CON, mas a expressão proteica do p-PDGFRα no grupo DOCA+IMA foi menor que no DOCA. CONCLUSÃO: O Imatinib pode exercer efeitos inibitórios sobre a fibrose miocárdica em ratos com hipertensão induzida por DOCA/sal, os quais podem ser atribuídos à inibição da atividade do PDGFR-α.

Hypermethylated 14-3-3-sigma and ESR1 gene promoters in serum as candidate biomarkers for the diagnosis and treatment efficacy of breast cancer metastasis

Background: Numerous hypermethylated genes have been reported in breast cancer, and the silencing of these genes plays an important role in carcinogenesis, tumor progression and diagnosis. These hypermethylated promoters are very rarely found in normal breast. It has been suggested that aberrant hypermethylation may be useful as a biomarker, with implications for breast cancer etiology, diagnosis, and management. The relationship between primary neoplasm and metastasis remains largely unknown. There has been no comprehensive comparative study on the clinical usefulness of tumor-associated methylated DNA biomarkers in primary breast carcinoma and metastatic breast carcinoma. The objective of the present study was to investigate the association between clinical extension of breast cancer and methylation status of Estrogen Receptor1 (ESR1) and Stratifin (14-3-3-σ) gene promoters in disease-free and metastatic breast cancer patients. Methods: We studied two cohorts of patients: 77 patients treated for breast cancer with no signs of disease, and 34 patients with metastatic breast cancer. DNA was obtained from serum samples, and promoter methylation status was determined by using DNA bisulfite modification and quantitative methylation-specific PCR. Results: Serum levels of methylated gene promoter 14-3-3-σ significantly differed between Control and Metastatic Breast Cancer groups (P < 0.001), and between Disease-Free and Metastatic Breast Cancer groups (P < 0.001). The ratio of the 14-3-3-σ level before the first chemotherapy cycle to the level just before administration of the second chemotherapy cycle was defined as the Biomarker Response Ratio [BRR]. We calculated BRR values for the "continuous decline" and "rise-and-fall" groups. Subsequent ROC analysis showed a sensitivity of 75% (95% CI: 47.6 - 86.7) and a specificity of 66.7% (95% CI: 41.0 - 86.7) to discriminate between the groups for a cut-off level of BRR = 2.39. The area under the ROC curve (Z = 0.804 ± 0.074) indicates that this test is a good approach to post-treatment prognosis. Conclusions: The relationship of 14-3-3-σ with breast cancer metastasis and progression found in this study suggests a possible application of 14-3-3-σ as a biomarker to screen for metastasis and to follow up patients treated for metastatic breast cancer, monitoring their disease status and treatment response.

A gut feeling of the PXR, PPAR and NF-kappaB connection.

Bowel diseases reveal the complex interplay of sensing and signalling pathways in maintaining healthy homeostasis of the intestine. Recent studies of the xenobiotic nuclear receptor, pregnane X receptor and the inflammatory mediator nuclear transcription factor kappaB (NF-kappaB) reveal a functional link between xenobiotic neutralization and inflammation and explain how certain xenobiotics can affect the immune response. Furthermore, another nuclear receptor, peroxisome proliferator-activated receptor gamma (PPAR gamma) has been shown to produce beneficial effects in experimental inflammatory bowel diseases by repression of NF-kappaB thereby reducing inflammation, whilst its close relative PPAR beta/delta appears at a central position in signalling pathways involved in the progression of colon cancer. Recently accumulated knowledge on the action of these nuclear receptors and NF-kappaB in intestinal homeostasis may provide the rationale for the development of innovative treatment strategies with selective receptor modulators.

Cells derived from the coelomic epithelium contribute to multiple gastrointestinal tissues in mouse embryos.

Gut mesodermal tissues originate from the splanchnopleural mesenchyme. However, the embryonic gastrointestinal coelomic epithelium gives rise to mesenchymal cells, whose significance and fate are little known. Our aim was to investigate the contribution of coelomic epithelium-derived cells to the intestinal development. We have used the transgenic mouse model mWt1/IRES/GFP-Cre (Wt1(cre)) crossed with the Rosa26R-EYFP reporter mouse. In the gastrointestinal duct Wt1, the Wilms' tumor suppressor gene, is specific and dynamically expressed in the coelomic epithelium. In the embryos obtained from the crossbreeding, the Wt1-expressing cell lineage produces the yellow fluorescent protein (YFP) allowing for colocalization with differentiation markers through confocal microscopy and flow cytometry. Wt1(cre-YFP) cells were very abundant throughout the intestine during midgestation, declining in neonates. Wt1(cre-YFP) cells were also transiently observed within the mucosa, being apparently released into the intestinal lumen. YFP was detected in cells contributing to intestinal vascularization (endothelium, pericytes and smooth muscle), visceral musculature (circular, longitudinal and submucosal) as well as in Cajal and Cajal-like interstitial cells. Wt1(cre-YFP) mesenchymal cells expressed FGF9, a critical growth factor for intestinal development, as well as PDGFRα, mainly within developing villi. Thus, a cell population derived from the coelomic epithelium incorporates to the gut mesenchyme and contribute to a variety of intestinal tissues, probably playing also a signaling role. Our results support the origin of interstitial cells of Cajal and visceral circular muscle from a common progenitor expressing anoctamin-1 and SMCα-actin. Coelomic-derived cells contribute to the differentiation of at least a part of the interstitial cells of Cajal.

Regulation of human class I alcohol dehydrogenases by bile acids

Class I alcohol dehydrogenases (ADH1s) are the rate-limiting enzymes for ethanol and vitamin A (retinol) metabolism in the liver . Because previous studies have shown that human ADH1 enzymes may participate in bile acid metabolism, we investigated whether the bile acid-activated nuclear receptor farnesoid X receptor (FXR) regulates ADH1 genes. In human hepatocytes, both the endogenous FXR ligand chenodeoxycholic acid and synthetic FXR-specific agonist GW4064 increased ADH1 mRNA, protein, and activity. Moreover, overexpression of a constitutively active form of FXR induced ADH1A and ADH1B expression, whereas silencing of FXR abolished the effects of FXR agonists on ADH1 expression and activity. Transient transfection studies and electrophoretic mobility shift assays revealed functional FXR response elements in the ADH1A and ADH1B proximal promoters, thus indicating that both genes are direct targets of FXR. These findings provide the first evidence for direct connection of bile acid signaling and alcohol metabolism.