RESUMEN
BACKGROUND: Inflammatory bowel diseases lead to progressive bowel damage and need for surgery. While the increase in prevalence of other immune-mediated diseases in IBD is well recognised, the impact of this on the natural history of IBD is unknown. AIM: To determine the impact of concomitant immune-mediated diseases on phenotypes and outcomes in IBD. METHODS: Patients with IBD enrolled in a prospective registry were queried about the presence of other immune-mediated diseases, defined as those where immune dysregulation plays a role in pathogenesis. Demographics and disease-related information were obtained. Subjects also completed measures of quality of life. Multivariable regression models compared disease phenotype and outcomes of IBD patients with and without other immune-mediated diseases. RESULTS: The cohort included 2145 IBD patients among whom 458 (21%) had another immune-mediated disease. There was no difference in CD phenotype between the two groups. UC patients were more likely to have pancolitis in the presence of another immune-mediated disease (62%) compared to those without (52%, P = 0.02). IBD patients with another immune-mediated disease had higher rates of needing anti-TNF biologics [Odds ratio (OR) 1.31, 95% CI 1.05-1.63] and surgery (OR 1.26, 95% CI 0.99-1.61). The presence of another immune-mediated disease was also associated with lower disease-specific and general physical quality of life. CONCLUSIONS: The presence of another immune-mediated disease in IBD patients was associated with higher likelihood of pancolonic involvement in UC, and a modest increase in need for IBD-related surgery and anti-TNF biological therapy. Such patients also experienced worse quality of life.
Asunto(s)
Enfermedades del Sistema Inmune/diagnóstico , Enfermedades del Sistema Inmune/epidemiología , Enfermedades Inflamatorias del Intestino/diagnóstico , Enfermedades Inflamatorias del Intestino/epidemiología , Fenotipo , Adulto , Colitis Ulcerosa/diagnóstico , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/epidemiología , Comorbilidad , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/epidemiología , Femenino , Humanos , Enfermedades del Sistema Inmune/tratamiento farmacológico , Factores Inmunológicos/farmacología , Factores Inmunológicos/uso terapéutico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Calidad de Vida , Sistema de Registros , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
BACKGROUND: Anti-tumour necrosis factor (anti-TNF) biologic associated psoriasis has been reported in inflammatory bowel disease (IBD) patients. However, little is known regarding its pathogenesis. AIM: To identify potential genetic predispositions to anti-TNF associated psoriasis in IBD patients. METHODS: This retrospective chart review included IBD patients enrolled in a prospective registry. Cases of anti-TNF associated psoriasis and idiopathic psoriasis unrelated to anti-TNF exposure were confirmed by an expert dermatologist. All patients were genotyped on the Illumina Immunochip. A weighted genetic risk score ascertaining genetic pre-disposition towards psoriasis was calculated and overall genetic pre-disposition as well as differential distribution of individual polymorphisms was compared across the three groups. RESULTS: Our study included 724 IBD patients who initiated anti-TNF therapy and did not develop psoriasis, 35 patients with anti-TNF associated psoriasis, and 38 patients with idiopathic psoriasis. Anti-TNF users who developed psoriasis had a modest but statistically significantly greater psoriasis genetic risk score than anti-TNF controls (mean 0.64 vs. 0.61, P = 0.04), and had a similar genetic risk score as those with idiopathic psoriasis (0.64 vs. 0.62, P = 0.22). Two loci associated with NOS2 and ETS1 genes achieved P < 0.05 when comparing anti-TNF associated psoriasis to anti-TNF controls. Three loci were significantly different between anti-TNF associated psoriasis and idiopathic psoriasis including a polymorphism near NOS2 encoding for inducible nitric oxide synthase that is produced by dendritic cells in skin lesions in psoriasis. CONCLUSION: Patients with anti-TNF associated psoriasis had a modestly greater genetic pre-disposition towards psoriasis but no single causative polymorphism was identified.
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Antirreumáticos/efectos adversos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Psoriasis/etiología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adolescente , Adulto , Antirreumáticos/uso terapéutico , Femenino , Genotipo , Humanos , Masculino , Fenotipo , Estudios Prospectivos , Estudios Retrospectivos , Factores de RiesgoRESUMEN
BACKGROUND: Patients hospitalised with an exacerbation of inflammatory bowel disease (IBD) often receive antibiotics in addition to intravenous steroids. However, their efficacy in this setting is unclear. AIM: To ascertain if the addition of antibiotics to intravenous steroids modifies short and long-term clinical outcomes. METHODS: Our study included IBD patients hospitalised between 2009 and 2014 who received intravenous (IV) steroids with or without adjuvant antibiotics. Outcomes of interest included length of stay (LOS), need for medical and surgical rescue therapy during the hospitalisation, and at 90 and 365 days. A meta-analysis of previously published randomised trials was additionally performed. RESULTS: A total of 354 patients were included [145 ulcerative colitis (UC); 209 Crohn's disease (CD)]. In CD, combination of IV steroids and antibiotics did not change need for in-hospital medical rescue therapy, surgery or hospitalisations at 1 year but was associated with greater LOS (6.1 vs. 4.6 days, P = 0.02). In UC, patients receiving antibiotics were less likely to require in-hospital medical rescue therapy [odds ratio (OR): 0.42, 95% confidence interval (CI): 0.19-0.93] but experienced no statistically significant differences in LOS, in-hospital surgery, re-hospitalisations or surgery by 1 year. A meta-analysis of three relevant randomised trials demonstrated no difference in clinical improvement with antibiotics over placebo (OR: 1.08, 95% CI: 0.50-2.32). CONCLUSIONS: The addition of antibiotics to intravenous steroids for treatment of IBD exacerbations was associated with a reduced need for in-hospital medical rescue therapy in ulcerative colitis without significant long-term benefit, and did not affect short- or long-term outcomes in Crohn's disease.
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Corticoesteroides/uso terapéutico , Antibacterianos/uso terapéutico , Colitis Ulcerosa/tratamiento farmacológico , Enfermedad de Crohn/tratamiento farmacológico , Corticoesteroides/administración & dosificación , Adulto , Antibacterianos/administración & dosificación , Estudios de Cohortes , Quimioterapia Combinada , Femenino , Hospitalización/estadística & datos numéricos , Humanos , Masculino , Metaanálisis como Asunto , Ensayos Clínicos Controlados Aleatorios como Asunto , Estudios RetrospectivosRESUMEN
BACKGROUND: Anti-tumour necrosis factor α (anti-TNF) agents have been implicated in drug-induced liver injury. There is minimal data on this occurrence in inflammatory bowel disease (IBD) patients. AIM: To identify the characteristics of liver enzyme elevations following anti-TNF therapy initiation in IBD. METHODS: A retrospective cohort of patients initiating anti-TNF therapy were analysed for new onset alanine transaminase (ALT) elevation (≥60 U/L). We collected data on natural history, outcomes and patient characteristics compared with controls with persistent normal liver enzymes. Likelihood of causal association was assessed using the RUCAM score. RESULTS: From 1753 patients initiating an anti-TNF (1170 infliximab, 575 adalimumab, 8 certolizumab), 102 (6%) developed new onset ALT elevation. In 54 (53%), this could be linked to an alternate aetiology. Among those with idiopathic ALT elevations, the median time to ALT elevation from anti-TNF initiation was 18 weeks and median peak ALT was 96 U/L. Six underwent liver biopsy, all demonstrating hepatitis with autoimmune features. Compared to controls, cases were on a lower dose of infliximab (5.7 vs. 6.7 mg/kg, P = 0.02) but were otherwise similar in body mass index, sex and age. On follow-up, 34 continued the anti-TNF, 14 stopped therapy and 4 initiated steroids. Most (85%) normalised their LFTs after a median of 17 weeks including 28 (82%) of those who continued anti-TNF therapy. Ten patients were transitioned to a second anti-TNF without recurrence. CONCLUSIONS: ALT elevations occurred in 6% of IBD patients initiating anti-TNF therapy. Most idiopathic elevations were mild, transient and resolved despite therapy continuation.
Asunto(s)
Terapia Biológica/métodos , Enfermedad Hepática Inducida por Sustancias y Drogas/epidemiología , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adalimumab , Adulto , Alanina Transaminasa/metabolismo , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/uso terapéutico , Terapia Biológica/efectos adversos , Certolizumab Pegol , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Humanos , Fragmentos Fab de Inmunoglobulinas/administración & dosificación , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Infliximab , Pruebas de Función Hepática , Masculino , Polietilenglicoles/administración & dosificación , Polietilenglicoles/uso terapéutico , Recurrencia , Estudios RetrospectivosRESUMEN
BACKGROUND: Patients with inflammatory bowel disease (IBD) are at higher risk for Clostridium difficile infection (CDI). Disruption of gut microbiome and interaction with the intestinal immune system are essential mechanisms for pathogenesis of both CDI and IBD. Whether genetic polymorphisms associated with susceptibility to IBD are also associated with risk of CDI is unknown. AIMS: To use a well-characterised and genotyped cohort of patients with UC to (i) identify clinical risk factors for CDI; (ii) examine if any of the IBD genetic risk loci were associated with CDI; and (iii) to compare the performance of predictive models using clinical and genetic risk factors in determining risk of CDI. METHODS: We used a prospective registry of patients from a tertiary referral hospital. Medical record review was performed to identify all ulcerative colitis (UC) patients within the registry with a history of CDI. All patients were genotyped on the Immunochip. We examined the association between the 163 risk loci for IBD and risk of CDI using a dominant genetic model. Model performance was examined using receiver operating characteristics curves. RESULTS: The study included 319 patients of whom 29 developed CDI (9%). Female gender and pancolitis were associated with increased risk, while use of anti-TNF was protective against CDI. Six genetic polymorphisms including those at TNFRSF14 [Odds ratio (OR) 6.0, P-value 0.01] were associated with increased risk while 2 loci were inversely associated. On multivariate analysis, none of the clinical parameters retained significance after adjusting for genetics. Presence of at least one high-risk locus was associated with an increase in risk for CDI (20% vs. 1%) (P = 6 × 10â»9). Compared to 11% for a clinical model, the genetic loci explained 28% of the variance in CDI risk and had a greater AUROC. CONCLUSION: Host genetics may influence susceptibility to Clostridium difficile infection in patients with ulcerative colitis.
Asunto(s)
Antibacterianos/uso terapéutico , Clostridioides difficile/aislamiento & purificación , Infecciones por Clostridium/genética , Colitis Ulcerosa/genética , Fármacos Gastrointestinales/uso terapéutico , Adulto , Infecciones por Clostridium/tratamiento farmacológico , Infecciones por Clostridium/microbiología , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/microbiología , Femenino , Técnicas de Genotipaje , Humanos , Masculino , Persona de Mediana Edad , Modelos Genéticos , Análisis Multivariante , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Factores Sexuales , Adulto JovenRESUMEN
BACKGROUND: A significant proportion of patients with IBD lose response to anti-TNF therapies. There is limited knowledge of the long-term outcomes of those who have failed two anti-TNF agents and commenced a third. AIM: To examine the safety and efficacy of third anti-TNF treatment after failure of two prior anti-TNF agents in patients with inflammatory bowel disease. METHODS: This was a retrospective study of all IBD patients [Crohn's disease (CD), ulcerative colitis (UC)] treated with a third anti-TNF agent after loss of response or intolerance to two prior anti-TNF agents at a single tertiary North American centre. Disease activity, drug therapy and Montreal phenotypes were noted at disease onset and commencement of the third anti-TNF agent. Kaplan-Meier estimates were used to calculate the probability of remaining on the third anti-TNF agent and to identify predictors of long-term clinical response. RESULTS: A total of 63 patients (64% women, 57 CD and 6 UC) were included in the analysis. The mean disease duration at initiation of third anti-TNF was 12 years. Thirty-five (55.6%) patients discontinued the third anti-TNF after a mean of 13.2 months. Probability of remaining on the third anti-TNF was 0.69, 0.55, 0.37 and 0.25 at 6, 12, 24 and 36 months respectively. Prior primary nonresponders to the first anti-TNF agent [hazard ratio (HR) 6.4, 95% CI 2.5-16.1] and persistent disease activity at 3 months after commencement of a third anti-TNF (HR 3.2, 95% CI 1.3-7.8) predicted poorer response. CONCLUSIONS: Over half of patients with inflammatory bowel disease, initiated on a third anti-TNF agent after failure of two prior anti-TNF drugs, are able to remain on the third anti-TNF at 1 year.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adolescente , Adulto , Anciano , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
The canonical Wnt/beta-catenin pathway is a highly conserved signaling cascade that is involved in development and stem cell renewal. The deregulation of this pathway is often associated with increased cell growth and neoplasia. The small GTPase Rac has been shown to influence canonical Wnt signaling by regulating beta-catenin stability through an unknown mechanism. We report that DOCK4, a guanine nucleotide exchange factor (GEF) for Rac and a member of the CDM family of unconventional GEFs, mediates Wnt-induced Rac activation in the canonical Wnt/beta-catenin pathway. DOCK4 expression regulates cellular beta-catenin levels in response to the Wnt signal, in vitro. Biochemical studies demonstrate that DOCK4 interacts with the beta-catenin degradation complex, consisting of the proteins adenomatosis polyposis coli, Axin and glycogen synthase kinase 3beta (GSK3beta). This molecular interaction enhances beta-catenin stability and Axin degradation. Furthermore, we observe that DOCK4 is phosphorylated by GSK3beta, which enhances Wnt-induced Rac activation. Using a T-cell factor reporter zebrafish we confirm that DOCK4 is required for Wnt/beta-catenin activity, in vivo. These results elucidate a novel intracellular signaling mechanism in which a Rac GEF, DOCK4 acts as a scaffold protein in the Wnt/beta-catenin pathway.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Proteína Axina , Línea Celular , Citosol/metabolismo , Proteínas Activadoras de GTPasa/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Ratones , Proteínas Represoras/metabolismo , Transducción de Señal , Pez Cebra , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The driving forces for the regulation of cell morphology are the Rho family GTPases that coordinate the assembly of the actin cytoskeleton. This dynamic feature is a result of tight coupling between the cytoskeleton and signal transduction and is facilitated by actin-binding proteins (ABPs). Mutations in the actin bundling and PDZ domain-containing protein harmonin are the causes of Usher syndrome type 1C (USH1C), a syndrome of congenital deafness and progressive blindness, as well as certain forms of non-syndromic deafness. Here, we have used the yeast two-hybrid assay to isolate molecular partners of harmonin and identified DOCK4, an unconventional guanine exchange factor for the Rho family of guanosine triphosphatases (Rho GEF GTPases), as a protein interacting with harmonin. Detailed molecular analysis revealed that a novel DOCK4 isoform (DOCK4-Ex49) is expressed in the brain, eye and inner ear tissues. We have further provided evidence that the DOCK4-Ex49 binds to nucleotide free Rac as effectively as DOCK2 and DOCK4 and it is a potent Rac activator. By immunostaining using a peptide antibody specific to DOCK4-Ex49, we showed its localization in the inner ear within the hair bundles along the stereocilia (SC). Together, our data indicate a possible Rac-DOCK4-ABP harmonin-activated signaling pathway in regulating actin cytoskeleton organization in stereocilia.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Oído Interno/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Actinas/metabolismo , Animales , Proteínas de Ciclo Celular , Línea Celular , Cilios/enzimología , Cilios/metabolismo , Proteínas del Citoesqueleto , Citoesqueleto/metabolismo , Oído Interno/enzimología , Exones , Proteínas Activadoras de GTPasa/inmunología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Ratones , Isoformas de Proteínas/metabolismo , Transducción de Señal/fisiología , Técnicas del Sistema de Dos Híbridos , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Many nuclear receptors are capable of recognizing similar DNA elements. The molecular event(s) underlying the functional specificities of these receptors (in regulating the expression of their native target genes) is a very important issue that remains poorly understood. Here we report the cloning and analysis of a novel nuclear receptor coactivator (designated NRIF3) that exhibits a distinct receptor specificity. Fluorescence microscopy shows that NRIF3 localizes to the cell nucleus. The yeast two-hybrid and/or in vitro binding assays indicated that NRIF3 specifically interacts with the thyroid hormone receptor (TR) and retinoid X receptor (RXR) in a ligand-dependent fashion but does not bind to the retinoic acid receptor, vitamin D receptor, progesterone receptor, glucocorticoid receptor, or estrogen receptor. Functional experiments showed that NRIF3 significantly potentiates TR- and RXR-mediated transactivation in vivo but has little effect on other examined nuclear receptors. Domain and mutagenesis analyses indicated that a novel C-terminal domain in NRIF3 plays an essential role in its specific interaction with liganded TR and RXR while the N-terminal LXXLL motif plays a minor role in allowing optimum interaction. Computer modeling and subsequent experimental analysis suggested that the C-terminal domain of NRIF3 directly mediates interaction with liganded receptors through an LXXIL (a variant of the canonical LXXLL) module while the other part of the NRIF3 protein may still play a role in conferring its receptor specificity. Identification of a coactivator with such a unique receptor specificity may provide new insight into the molecular mechanism(s) of receptor-mediated transcriptional activation as well as the functional specificities of nuclear receptors.
Asunto(s)
Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Compartimento Celular , Simulación por Computador , Biblioteca de Genes , Células HeLa , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Unión Proteica , Estructura Terciaria de Proteína , Receptores X Retinoide , Análisis de Secuencia de ADN , Técnicas del Sistema de Dos HíbridosRESUMEN
The mdm2 gene is positively regulated by p53 through a p53-responsive DNA element in the first intron of the mdm2 gene. mdm2 binds p53, thereby abrogating the ability of p53 to activate the mdm2 gene, and thus forming an autoregulatory loop of mdm2 gene regulation. Although the mdm2 gene is thought to act as an oncogene by blocking the activity of p53, recent studies indicate that mdm2 can act independently of p53 and block the G1 cell cycle arrest mediated by members of the retinoblastoma gene family and can activate E2F1/DP1 and the cyclin A gene promoter. In addition, factors other than p53 have recently been shown to regulate the mdm2 gene. In this article, we report that thyroid hormone (T3) receptors (T3Rs), but not the closely related members of the nuclear thyroid hormone/retinoid receptor gene family (retinoic acid receptor, vitamin D receptor, peroxisome proliferation activation receptor, or retinoid X receptor), regulate mdm2 through the same intron sequences that are modulated by p53. Chicken ovalbumin upstream promoter transcription factor I, an orphan nuclear receptor which normally acts as a transcriptional repressor, also activates mdm2 through the same intron region of the mdm2 gene. Two T3R-responsive DNA elements were identified and further mapped to sequences within each of the p53 binding sites of the mdm2 intron. A 10-amino-acid sequence in the N-terminal region of T3Ralpha that is important for transactivation and interaction with TFIIB was also found to be important for activation of the mdm2 gene response element. T3 was found to stimulate the endogenous mdm2 gene in GH4C1 cells. These cells are known to express T3Rs, and T3 is known to stimulate replication of these cells via an effect in the G1 phase of the cell cycle. Our findings, which indicate that T3Rs can regulate the mdm2 gene independently of p53, provide an explanation for certain known effects of T3 and T3Rs on cell proliferation. In addition, these findings provide further evidence for p53-independent regulation of mdm2 which could lead to the development of tumors from cells that express low levels of p53 or that express p53 mutants defective in binding to and activating the mdm2 gene.
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Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares , Proteínas Proto-Oncogénicas/genética , Receptores de Hormona Tiroidea/metabolismo , Animales , Factor de Transcripción COUP I , Línea Celular , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Intrones , Ligandos , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-mdm2 , Proto-Oncogenes , Ratas , Receptores de Calcitriol/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/metabolismo , Elementos de Respuesta , Receptores X Retinoide , Relación Estructura-Actividad , Factor de Transcripción TFIIB , Factores de Transcripción/metabolismo , Triyodotironina/farmacología , Proteína p53 Supresora de Tumor/metabolismoAsunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Análisis Mutacional de ADN/métodos , Fosfotirosina/metabolismo , Proteínas/metabolismo , Clonación Molecular/métodos , Receptores ErbB/metabolismo , Plásmidos/genética , Mutación Puntual , Conformación Proteica , Proteínas Adaptadoras de la Señalización ShcRESUMEN
Benazepril hydrochloride, a new non-sulfhydryl ACE inhibitor (ACEI) was studied in a titrated dose of 10 mg-20 mg once a day for 6 weeks in 42 mild to moderate adult hypertensive patients with sitting diastolic blood pressure (SDBP) 95-114 mm Hg. The pre-drug SDBP(mean +/- SE) of 102.5 +/- 0.8 mm Hg showed a significant reduction to 87.5 +/- 0.93 mm Hg at the end of treatment. BP was controlled (SDBP < or = 90 mm Hg) in 34 (81%) patients and a drop of at least 10 mm Hg from the pre-treatment SDBP value was noted in 34 (81%) patients. Common adverse reaction was cough in 8(19%) patients. Clinically significant changes in laboratory evaluations were not seen in any patient. Study showed that benazepril in a dose range of 10 to 20 mg per day is an effective agent for treatment of mild to moderate hypertension.
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Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Benzazepinas/uso terapéutico , Hipertensión/tratamiento farmacológico , Adulto , Presión Sanguínea/efectos de los fármacos , Femenino , Humanos , Hipertensión/fisiopatología , Masculino , Persona de Mediana EdadRESUMEN
Thyroid hormone receptor (T3R) is a member of the steroid hormone receptor gene family of nuclear hormone receptors. In most cells T3R activates gene expression only in the presence of its ligand, L-triiodothyronine (T3). However, in certain cell types (e.g., GH4C1 cells) expression of T3R leads to hormone-independent constitutive activation. This activation by unliganded T3R occurs with a variety of gene promoters and appears to be independent of the binding of T3R to specific thyroid hormone response elements (TREs). Previous studies indicate that this constitutive activation results from the titration of an inhibitor of transcription. Since the tumor suppresser p53 is capable of repressing a wide variety of gene promoters, we considered the possibility that the inhibitor is p53. Evidence to support this comes from studies indicating that expression of p53 blocks T3R-mediated constitutive activation in GH4C1 cells. In contrast with hormone-independent activation by T3R, p53 had little or no effect on T3-dependent stimulation which requires TREs. In addition, p53 mutants which oligomerize with wild-type p53 and interfere with its function also increase promoter activity. This enhancement is of similar magnitude to but is not additive with the stimulation mediated by unliganded T3R, suggesting that they target the same factor. Since p53 mutants are known to target wild-type p53 in the cell, this suggests that T3R also interacts with p53 in vivo and that endogenous levels of p53 act to suppress promoter activity. Evidence supporting both functional and physical interactions of T3R and p53 in the cell is presented. The DNA binding domain (DBD) of T3R is important in mediating constitutive activation, and the receptor DBD appears to functionally interact with the N terminus of p53 in the cell. In vitro binding studies indicate that the T3R DBD is important for interaction of T3R with p53 and that this interaction is reduced by T3. These findings are consistent with the in vivo studies indicating that p53 blocks constitutive activation but not ligand-dependent stimulation. These studies provide insight into mechanisms by which unliganded nuclear hormone receptors can modulate gene expression and may provide an explanation for the mechanism of action of the v-erbA oncoprotein, a retroviral homolog of chicken T3R alpha.
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Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Pollos , ADN/genética , ADN/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Técnicas In Vitro , Ligandos , Modelos Biológicos , Mutación , Regiones Promotoras Genéticas , Ratas , Transfección , Triyodotironina/metabolismoRESUMEN
Stimulation of the insulin receptor (IR) results in tyrosine phosphorylation of the intermediate molecules insulin receptor substrate-1 (IRS-1), IRS-2, and Shc, which then couple the IR to downstream signaling pathways by serving as binding sites for signaling molecules with SH2 domains. It has been proposed that direct binding of IRS-1, IRS-2, and Shc to an NPX-Tyr(P) motif in the juxtamembrane region of the IR is required for tyrosine phosphorylation of these molecules by the IR. In this regard, Shc and IRS-1 contain domains that are distinct from SH2 domains, referred to as the phosphotyrosine binding (PTB) or phosphotyrosine interaction (PI) domains, which bind phosphotyrosine in the context of an NPX-Tyr(P) motif. To further clarify the role of the Shc PTB/PI domain, we identified a mutation in this domain that abrogated binding of Shc to the IR in vitro. Interestingly, this mutation completely abolished Shc phosphorylation by the IR in vivo whereas mutation of the arginine in the FLVRES motif of the Shc SH2 domain did not affect Shc phosphorylation by insulin. In addition, we identified specific amino acids on the IR that are required for the IR to stimulate Shc but not IRS-1 phosphorylation in vivo. As with the PTB/PI domain Shc mutant, the ability of these mutant receptors to phosphorylate Shc correlates with the binding of the PTB/PI domain of Shc to similar sequences in vitro. These findings support a model in which binding of the PTB/PI domain of Shc directly to the NPX-Tyr(P) motif on the IR mediates Shc phosphorylation by insulin.
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Fosfoproteínas/metabolismo , Fosfotirosina/metabolismo , Receptor trkA/inmunología , Dominios Homologos src , Células 3T3 , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Clonación Molecular , Humanos , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Receptor trkA/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Transducción de SeñalRESUMEN
Shc is an Src homology 2 (SH2) domain protein thought to be an important component of the signaling pathway leading from cell surface receptors to Ras. A new phosphotyrosine interaction (PI) domain (also known as the phosphotyrosine binding (PTB) domain) has been described in the amino terminus of Shc. The Shc PI domain binding specificity is dependent on residues lying amino-terminal to the phosphotyrosine rather than carboxyl-terminal as is seen with SH2 domains. We randomly mutagenized the Shc PTB/PI domain in an effort to identify residues in the domain crucial for interaction with phosphotyrosine-containing peptides. We then screened the mutants for binding to the tyrosine-phosphorylated carboxyl-terminal tail of the epidermal growth factor (EGF) receptor. Most striking were mutations that altered a phenylalanine residue in block 4 of the domain severely impairing PI domain function. This phenylalanine residue is conserved in all but one subfamily of PI domains that have been identified to date. Reconstitution of this phenylalanine mutation into full-length Shc created a protein unable to interact with the EGF receptor in living cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Fosfopéptidos/química , Fosfotirosina/química , Proteínas/química , Dominios Homologos src , Animales , Sitios de Unión , Receptores ErbB/química , Ratones , Mutagénesis , Unión Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Relación Estructura-ActividadRESUMEN
The effects of the thyroid hormone (3,5,3'-triiodo-L-thyronine [T3]) on gene transcription are mediated by nuclear T3 receptors (T3Rs). alpha- and beta-isoform T3Rs (T3R alpha and -beta) are expressed from different genes and are members of a superfamily of ligand-dependent transcription factors that also includes the receptors for steroid hormones, vitamin D, and retinoids. Although T3 activates transcription by mediating a conformational change in the C-terminal approximately 220-amino-acid ligand-binding domain (LBD), the fundamental mechanisms of T3R-mediated transcriptional activation remain to be determined. We found that deletion of the 50-amino-acid N-terminal A/B domain of chicken T3R alpha (cT3R alpha) decreases T3-dependent stimulation of genes regulated by native thyroid hormone response elements about 10- to 20-fold. The requirement of the A/B region for transcriptional activation was mapped to amino acids 21 to 30, which contain a cluster of five basic amino acids. The A/B region of cT3R alpha is not required for T3 binding or for DNA binding of the receptor as a heterodimer with retinoid X receptor. In vitro binding studies indicate that the N-terminal region of cT3R alpha interacts efficiently with TFIIB and that this interaction requires amino acids 21 to 30 of the A/B region. In contrast, the LBD interacts poorly with TFIIB. The region of TFIIB primarily involved in the binding of cT3R alpha includes an amphipathic alpha helix contained within residues 178 to 201. Analysis using a fusion protein containing the DNA-binding domain of GAL4 and the entire A/B region of cT3R alpha suggests that this region does not contain an intrinsic activation domain. These and other studies indicate that cT3R alpha mediates at least some of its effects through TFIIB in vivo and that the N-terminal region of DNA-bound cT3R alpha acts to recruit and/or stabilize the binding of TFIIB to the transcription complex. T3 stimulation could then result from ligand-mediated changes in the LBD which may lead to the interaction of other factors with cT3R alpha, TFIIB, and/or other components involved in the initiation of transcription.
Asunto(s)
Regulación de la Expresión Génica , Receptores de Hormona Tiroidea/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Triyodotironina/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Células Cultivadas , Secuencia Conservada , Análisis Mutacional de ADN , Humanos , Ligandos , Datos de Secuencia Molecular , Unión Proteica , Receptores de Hormona Tiroidea/genética , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Factor de Transcripción TFIIBRESUMEN
Thyroid hormone (T3) receptor (T3R) regulates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by binding to and activating thyroid hormone response elements (TREs) embedded within the viral NF-kappa B and Sp1 motifs. The TREs within the NF-kappa B sites are necessary for activation by T3 in the absence of Tat, while those in the Sp1 motifs function as TREs only when Tat is expressed, suggesting that Tat and T3R interact in the cell. Transactivation of the HIV-1 LTR by T3R alpha and several receptor mutants revealed that the 50-amino-acid N-terminal A/B region of T3R alpha, known to interact with the basal transcription factor TFIIB, is critical for activation of both Tat-dependent and Tat-independent responsive sequences of the LTR. A single amino acid change in the highly conserved tau 1 region in the ligand-binding domain of T3R alpha eliminates Tat-independent but not Tat-dependent activation of the HIV-1 LTR by T3. Ro 5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(H)-one], which inhibits Tat-mediated transactivation of HIV-1, also inhibits the functional interaction between Tat and T3R alpha. Binding studies with glutathione-S-transferase fusion proteins and Western (immunoblot) analysis indicate that T3R alpha interacts with Tat through amino acids within the DNA-binding domain of T3R alpha. Mutational analysis revealed that amino acid residues in the basic and C-terminal regions of Tat are required for the binding of Tat to T3R alpha, while the N terminus of Tat is not required. These studies provide functional and physical evidence that stimulation of the HIV-1 LTR by T3 involves an interaction between T3R alpha and Tat. Our results also suggest a model in which multiple domains of T3R alpha interact with Tat and other factors to form transcriptionally important complexes.
Asunto(s)
Productos del Gen tat/metabolismo , Duplicado del Terminal Largo de VIH , VIH-1/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Antivirales/farmacología , Secuencia de Bases , Benzodiazepinonas/farmacología , Cloranfenicol O-Acetiltransferasa/genética , Productos del Gen tat/antagonistas & inhibidores , VIH-1/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Oligodesoxirribonucleótidos , Pirroles/farmacología , Receptores de Hormona Tiroidea/genética , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaAsunto(s)
Bloqueadores de los Canales de Calcio/uso terapéutico , Hipertensión/tratamiento farmacológico , Nicardipino/uso terapéutico , Adulto , Anciano , Determinación de la Presión Sanguínea , Bloqueadores de los Canales de Calcio/administración & dosificación , Femenino , Humanos , Hipertensión/fisiopatología , Masculino , Persona de Mediana Edad , Nicardipino/administración & dosificación , Resultado del TratamientoRESUMEN
SH2 domains function to bind proteins containing phosphotyrosine and are components of proteins that are important signal transducers for tyrosine kinases. We have cloned SH2 domain proteins by screening bacterial expression libraries with the tyrosine phosphorylated carboxyterminus of the epidermal growth factor (EGF) receptor. Here we report the identification of a new SH2 domain protein, Grb10. Grb10 is highly related to Grb7, an SH2 domain protein that we have previously identified. In addition to an SH2 domain, Grb7 and Grb10 have a central domain with similarity to a putative C. elegans gene likely to be involved in neuronal migration. At least three forms of Grb10 exist in fibroblasts apparently due to alternate translational start sites. Grb10 undergoes serine but not tyrosine phosphorylation after EGF treatment resulting in a shift mobility in a large fraction of Grb10 molecules. However Grb10 appears to bind poorly to EGF-Receptor and the true binding partner for the Grb10 SH2 domain is unclear. Grb10 maps to mouse chromosome 11 very close to the EGF-Receptor which is remarkably similar to Grb7 that maps near the EGF-Receptor related HER2 receptor. The finding of multiple family members with evolutionarily conserved domains indicates that these SH2 domain proteins are likely to have an important, although as of yet, unidentified function.
Asunto(s)
Proteínas/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Receptores ErbB/genética , Proteína Adaptadora GRB10 , Ratones , Datos de Secuencia Molecular , Fosforilación , Proteínas/análisis , Proteínas/química , Serina/metabolismoRESUMEN
The ligand-binding domains (LBDs) of the thyroid/retinoid receptor gene subfamily contain a series of heptad motifs important for dimeric interactions. This subfamily includes thyroid hormone receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), 9-cis RA receptors (RARs and retinoid X receptors [RXRs]), the 1,25-dihydroxyvitamin D3 receptor (VDR), and the receptors that modulate the peroxisomal beta-oxidation pathway (PPARs). These receptors bind to their DNA response elements in vitro as heterodimers with the RXRs. Unliganded receptors in vivo, in particular the T3Rs, can mediate gene silencing and ligand converts these receptors into a transcriptionally active form. The in vivo interactions of these receptors with RXR were studied by using a GAL4-RXR chimera containing the yeast GAL4 DNA-binding domain and the LBD of RXR beta. GAL4-RXR activates transcription from GAL4 response elements in the presence of 9-cis RA. Unliganded T3R, which does not bind or activate GAL4 elements, represses the activation of GAL4-RXR by 9-cis RA in HeLa cells. However, addition of T3 alone leads to transcriptional activation. These findings suggest that T3R can repress or activate transcription while tethered to the LBD of GAL4-RXR and that heterodimerization can occur in vivo without stabilization by hormone response elements. Similar ligand-dependent activation was observed in HeLa cells expressing RAR, VDR, or PPAR and in GH4C1 cells from endogenous receptors. Replacement of the last 17 amino acids of the LBD of RXRbeta with the 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein leads to a GAL4 constitutive activator that is repressed by wild-type T3R but not by a ninth heptad mutant that does not form heterodimers. This finding suggests that the ninth heptad or T3R is important for gene silencing and that the LBD of RXR does not exhibit silencing activity. This conclusion was verified with GAL4-LBD chimeras and with wild-type receptors in assays using appropriate response elements. These studies indicate that the LBD has diverse functional roles in gene regulation.