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OBJECTIVE AND DESIGN: This study investigated the opposite mechanisms by which IL-1ß and TGF-ß1 modulated the inflammatory and migratory phenotypes in cultured human intimal vascular smooth muscle cells vSMCs. MATERIALS AND TREATMENT: Primary human vSMCs, obtained from twelve hypertensive patients who underwent carotid endarterectomy, were incubated for 24 hours with either 40 pM TGF-ß1, or 1 nmol/L IL-1ß, or their combination in presence or absence of anti-TGF-ß neutralizing antibody. METHODS: The expression levels of matrix metalloproteases and their inhibitors, and the elastolytic enzyme cathepsin S (CTSS) and its inhibitor cystatin C were evaluated with RT-PCR. CTSS activity was measured by fluorometry. RESULTS: TGF-ß1 reversed IL-1ß-induced expression of iNOS, CXCL6, IL1R1, MMP12, and CTSS, while upregulated TIMP2 expression. Furthermore, anti-TGF-ß neutralizing antibody abrogated TGF-ß effects. Combination with IL-1ß and TGF-ß1 induced the expression of IL1α, IL1ß, IL1R1, and CTSS, but suppressed CST3 expression. CTSS expression in the combination treatment was higher than that of cells treated with anti-TGF-ß antibodies alone. Moreover, IL-1ß-induced CTSS enzymatic activity was reduced when human vSMCs were co-treated with TGF-ß, whereas this reduction was abrogated by anti-TGF-ß neutralizing antibody. CONCLUSION: TGF-ß1 abrogated IL-1ß-induced expression of inflammatory genes and elastolytic activity in cultured human vSMCs. Thus, TGF-ß1 can play a crucial role in impairing IL-1ß-induced vascular inflammation and damage involved in the etiology of cardiovascular diseases.
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Músculo Liso Vascular , Factor de Crecimiento Transformador beta1 , Catepsinas/genética , Células Cultivadas , Humanos , Interleucina-1beta , Factor de Crecimiento Transformador betaRESUMEN
AIMS: We conducted a systematic review assessing the reporting quality of studies validating models based on machine learning (ML) for clinical diagnosis, with a specific focus on the reporting of information concerning the participants on which the diagnostic task was evaluated on. METHOD: Medline Core Clinical Journals were searched for studies published between July 2015 and July 2018. Two reviewers independently screened the retrieved articles, a third reviewer resolved any discrepancies. An extraction list was developed from the Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis guideline. Two reviewers independently extracted the data from the eligible articles. Third and fourth reviewers checked, verified the extracted data as well as resolved any discrepancies between the reviewers. RESULTS: The search results yielded 161 papers, of which 28 conformed to the eligibility criteria. Detail of data source was reported in 24 of the 28 papers. For all of the papers, the set of patients on which the ML-based diagnostic system was evaluated was partitioned from a larger dataset, and the method for deriving such set was always reported. Information on the diagnostic/non-diagnostic classification was reported well (23/28). The least reported items were the use of reporting guideline (0/28), distribution of disease severity (8/28 patient flow diagram (10/28) and distribution of alternative diagnosis (10/28). A large proportion of studies (23/28) had a delay between the conduct of the reference standard and ML tests, while one study did not and four studies were unclear. For 15 studies, it was unclear whether the evaluation group corresponded to the setting in which the ML test will be applied to. CONCLUSION: All studies in this review failed to use reporting guidelines, and a large proportion of them lacked adequate detail on participants, making it difficult to replicate, assess and interpret study findings. PROSPERO REGISTRATION NUMBER: CRD42018099167.
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Simulación por Computador , Diagnóstico por Computador , Aprendizaje Automático , Proyectos de Investigación/normas , Diagnóstico por Computador/métodos , Diagnóstico por Computador/normas , HumanosRESUMEN
PURPOSE: Leptomeningeal carcinomatosis (MC) is commonly associated with HER2-positive breast cancer (HER2-BC), with a poor prognosis and no standardised treatment. We conducted a phase I dose-escalation study of intrathecal (IT) administration of trastuzumab in HER2-BC patients with MC to determine the maximum tolerated dose (MTD), which was based on both the achievement of a trastuzumab intra-cerebrospinal fluid concentration close to a conventional therapeutic plasma concentration (30 mg/L) and/or dose-limiting toxicity (DLT). METHODS: The protocol planned IT administration of trastuzumab (30 mg, 60 mg, 100 mg or 150 mg dose levels) once a week, over the course of at least 4 weeks. Sixteen patients with MC from HER2-BC received IT trastuzumab. Intra-cerebrospinal fluid samples were obtained before each injection for pharmacokinetics. RESULTS: We did not observe DLT of IT trastuzumab. Eleven patients had no toxicity attributed to IT trastuzumab. For 60 mg or higher dose levels, minor toxicities attributed to IT trastuzumab included headache (2 patients), nausea (2 patients), vomiting (1 patient), cervical pain (1 patient) and peripheral neuropathy (1 patient). Two patients experienced immediate toxicity including headache or vomiting. The mean residual intra-cerebrospinal fluid concentration of trastuzumab was 27.9 mg/L for the 150 mg dose level. Three patients achieved a clinical response, seven patients had stable disease and four patients had progressive disease. CONCLUSIONS: The MTD and recommended phase II weekly dose of IT trastuzumab in patients with HER2-BC and MC is 150 mg. A phase II trial using this dose regimen in MC from HER2-BC is ongoing. REGISTRATION IDENTIFICATION: ClinicalTrials.gov Identifier: NCT01373710 (https://clinicaltrials.gov/ct2/show/NCT01373710?term=trastuzumab+intrathecal&rank=1).
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Carcinomatosis Meníngea/tratamiento farmacológico , Trastuzumab/administración & dosificación , Adulto , Anciano , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Estudios de Factibilidad , Femenino , Humanos , Inyecciones Espinales , Dosis Máxima Tolerada , Carcinomatosis Meníngea/metabolismo , Carcinomatosis Meníngea/secundario , Persona de Mediana Edad , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Trastuzumab/efectos adversos , Trastuzumab/líquido cefalorraquídeo , Trastuzumab/farmacocinética , Adulto JovenRESUMEN
PURPOSE: The aim of this study was to describe pharmacogenomics-based inclusion criteria (enrichment) and the main characteristics of clinical trials involving oncology-targeted therapies. METHODS: Clinical trials of oncology-targeted therapies approved after 2005 with pharmacogenomic testing required or recommended in their label were retrieved from a mapping of the ClinicalTrials.gov database. RESULTS: We examined information for 12 drugs and 858 trials. Overall, 434 trials (51%) were enriched on the biomarker first mentioned in the label and 145 (17%) were enriched on another biomarker, whereas 270 trials (31%) included all patients. The median proportion of trials corresponding to both the drug's indication and drug's target was 35%. Of the 361 trials that tested drugs in another disease than the first one in the label, 219 (61%) were without enrichment and 87 (24%) were actually enriched but on another biomarker than the first one in the label. CONCLUSION: Several drugs have been tested in trials enriched on many different biomarkers. Nonetheless, most targeted therapies have been developed only using biomarker-positive patients; therefore, exclusion of biomarker-negative patients from treatment relies on only preclinical data and on biological understanding of the disease and target.Genet Med 18 8, 796-805.
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Biomarcadores de Tumor/genética , Terapia Molecular Dirigida/métodos , Neoplasias/tratamiento farmacológico , Variantes Farmacogenómicas , Antineoplásicos/uso terapéutico , Ensayos Clínicos como Asunto , Etiquetado de Medicamentos , Humanos , Neoplasias/genética , Estados Unidos , United States Food and Drug AdministrationRESUMEN
UNLABELLED: The involvement of the renin-angiotensin-aldosterone system (RAAS) and cortisol in increased cardiovascular risk is well known. If numerous relationships between RAAS and corticosteroids have been described, their interactions within the arterial wall, especially during the transdifferentiation of vascular smooth muscle cells (VSMCs) and the atheroma formation, are not established. Here, we clarified the relationships between mRNA levels of corticosteroid and angiotensin system components using cortisol, fludrocortisone, and angiotensin II treatments of cultured VSMCs maintained in a contractile phenotype or induced to a lipid storing phenotype. We then determined the quantitative relationships between the mRNA content of these components measured with reverse transcription polymerase chain reaction (RT-PCR), in the atheroma plaque and nearby macroscopically intact tissue (MIT) from 27 human carotid endarterectomy samples. In both VSMC phenotypes, cortisol markedly increased both angiotensinogen (AGT) and AT1-receptor (AT1R) mRNA levels. These effects of cortisol were mediated via glucocorticoid receptor-α (GRα) without any illicit activation of the mineralocorticoid receptor (MR). Angiotensin II increased GRα, 11ßHSD1, CYP11B1, as well as CYP11B2 mRNAs and decreased AT1R in contractile VSMC; only GRα and CYP11B2 were increased in lipid storing VSMCs, while MR and AGT mRNAs decreased. In endarterectomy specimens, positive correlations between mRNA levels of AGT and aldosterone synthase or 11ßHSD1 in MIT and of AT1R and MR in atheroma were detected. The arterial tissue angiotensin system is a target for local glucocorticoids and arterial glucocorticoids for angiotensin II. Both systems appear activated in lipid storing VSMCs and strongly correlated in vivo, and their mutual amplification may contribute to the development of atheroma. KEY MESSAGE: Cortisol increases angiotensin II signaling in VSMCs via GRα. Angiotensin II stimulates cortisol signaling through increased GRα and 11ß-HSD1. Corticoid and angiotensin receptors are strongly correlated in the arterial wall. These correlations are maintained at different stages of atheroma development. An auto-amplification loop between angiotensin and cortisol signaling favors atherogenesis.
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Corticoesteroides/metabolismo , Angiotensinas/metabolismo , Arterias Carótidas/patología , Miocitos del Músculo Liso/citología , Placa Aterosclerótica/patología , Anciano , Angiotensina II/metabolismo , Diferenciación Celular , Transdiferenciación Celular , Fludrocortisona/química , Humanos , Hidrocortisona/metabolismo , Lípidos/química , Contracción Muscular , Músculo Liso Vascular/citología , Fenotipo , Placa Aterosclerótica/metabolismo , Receptores de Mineralocorticoides/metabolismoRESUMEN
TGF-ß is protective in atherosclerosis but deleterious in metastatic cancers. Our aim was to determine whether TGF-ß transcriptional regulation is tissue-specific in early atherosclerosis. The computational methods included 5 steps: (i) from microarray data of human atherosclerotic carotid tissue, to identify the 10 best co-expressed genes with TGFB1 (TGFB1 gene cluster), (ii) to choose the 11 proximal promoters, (iii) to predict the TFBS shared by the promoters, (iv) to identify the common TFs co-expressed with the TGFB1 gene cluster, and (v) to compare the common TFs in the early lesions to those identified in advanced atherosclerotic lesions and in various cancers. Our results show that EGR1, SP1 and KLF6 could be responsible for TGFB1 basal expression, KLF6 appearing specific to atherosclerotic lesions. Among the TFs co-expressed with the gene cluster, transcriptional activators (SLC2A4RG, MAZ) and repressors (ZBTB7A, PATZ1, ZNF263) could be involved in the fine-tuning of TGFB1 expression in atherosclerosis.
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Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/metabolismo , Regulación de la Expresión Génica , Miocitos del Músculo Liso/metabolismo , Factor de Crecimiento Transformador beta1/genética , Sitios de Unión , Células Cultivadas , Simulación por Computador , Proteína 1 de la Respuesta de Crecimiento Precoz/fisiología , Expresión Génica , Humanos , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/fisiología , Modelos Genéticos , Familia de Multigenes , Músculo Liso Vascular/patología , Especificidad de Órganos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/fisiología , Factor de Transcripción Sp1/fisiología , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
High cortisol and aldosterone levels increase cardiovascular risk, but the respective roles of each hormone within the arterial wall remain controversial. We tested the hypothesis that cortisol production within the arterial wall may contribute to atherosclerotic remodeling and act through illicit activation of the mineralocorticoid receptor (MR). Gene expression studies of the corticoid system components and marker genes of the atherosclerotic process in human carotid atheroma plaque and nearby macroscopically intact tissue (MIT) were considered together with clinical data and compared with pharmacological stimulations of human vascular smooth muscle cells (VSMCs) in contractile or lipid-storing phenotypes. The components of corticoid production and action were present and active within the human carotid wall and VSMCs. Atheroma plaque and lipid-storing VSMCs expressed 11ß-hydroxysteroid deshydrogenase-1 (11ß-HSD1) at two- to tenfold higher levels than MIT or contractile VSMCs. The 11ß-HSD1 expression was stimulated by cortisol and cortisone, especially in lipid-storing VSMCs. MR mRNA level was lower in atheroma and lipid-storing VSMCs and downregulated via MR by fludrocortisone and cortisol. Cortisol upregulated collagen1 and MCP-1 mRNAs via the glucocorticoid receptor (GRα), in both VSMC phenotypes, whereas fludrocortisone stimulated the collagen1 expression only in lipid-storing VSMCs. The GRα mRNA level in MIT was higher in patients with previous stroke and correlated positively with the collagen1 mRNA but negatively with diastolic blood pressure. Local cortisol production by 11ß-HSD1, and its action via high parietal GRα could be relevant from the first step of atherosclerotic remodeling and auto-amplify with transdifferentiation of VSMCs during atheroma progression.
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Arterias/metabolismo , Hidrocortisona/metabolismo , Placa Aterosclerótica/metabolismo , Accidente Cerebrovascular/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cortisona/genética , Cortisona/metabolismo , Fludrocortisona/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Hidrocortisona/genética , Lípidos/genética , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Placa Aterosclerótica/genética , ARN Mensajero/genética , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Accidente Cerebrovascular/genéticaRESUMEN
Nitric oxide (NO) is believed to play a key role in adverse microvascular changes during sepsis. A deficit in NO has been evoked as a potential mechanism of microcirculatory deterioration in the early phase of septic shock. The aim of this study was to evaluate simultaneously and continuously both hepatic microcirculation and local NO production during early experimental sepsis. Wistar male rats were divided into two groups: a sepsis group undergoing cecal ligation and puncture (CLP) peritonitis and a control group undergoing sham surgery. Hepatic microcirculation was continuously monitored using a laser Doppler probe and local nitric oxide (NO) production by means of a specific electrode. Constitutive and inducible NO synthase production was assessed 2h after surgery, at onset of shock, and at 2 and 3h after shock. In control animals, hepatic microcirculatory perfusion and NO production remained stable throughout the experiment. In septic animals, whereas a fall in microcirculatory perfusion was noted as early as 2h after CLP, NO concentration remained stable and further increased after the onset of shock. At this time, inducible NO synthase was the only isoform significantly elevated. In this non-resuscitated experimental model of sepsis, an absolute liver deficit of NO could not explain the early adverse changes in the local microvascular system.
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Circulación Hepática , Hígado/irrigación sanguínea , Hígado/metabolismo , Microcirculación , Óxido Nítrico/deficiencia , Choque Séptico/metabolismo , Choque Séptico/fisiopatología , Animales , Velocidad del Flujo Sanguíneo , Ciego/microbiología , Ciego/cirugía , Modelos Animales de Enfermedad , Flujometría por Láser-Doppler , Ligadura , Masculino , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Punciones , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Choque Séptico/genética , Choque Séptico/microbiología , Factores de TiempoRESUMEN
BACKGROUND: Perilipin1, a lipid droplet associated protein has an important role in the regulation of lipolysis and lipid storage in adipocytes. Perilipin1 is also expressed in foam cells of atheroma plaques and could therefore play a role in the accumulation of lipids in arterial wall and in the development of atherosclerosis. The aim of the study was to investigate this possible role of perilipin1 in atherogenesis. METHODS: Mice deficient in perilipin1 (Plin1-/-) were crossed with Ldlr-/- mice. Ldlr-/- and Plin1-/- Ldlr-/- mice received an atherogenic diet during 10 or 20 weeks. Blood pressure and plasma lipids concentrations were measured. Aortas were collected at the end of the atherogenic diet periods for quantification of atheroma lesions (en face method), histological and immunohistological studies RESULTS: Ldlr-/- and Plin1-/- Ldlr-/- mice had comparable blood pressure and plasma lipids levels. Plin1-/- Ldlr-/- mice had a lower body weight and decreased adiposity. The atherosclerotic lesion area in Plin1-/-Ldlr-/- mice was moderately increased after 10 weeks of atherogenic diet (ns) and significantly higher after 20 weeks (p < 0.01). Histology of atheroma plaques was comparable with no sign of increased inflammation in Plin1-/- Ldlr-/- mice. CONCLUSION: Perilipin1 ablation in mice results in increased atherosclerosis independently of modifications of risk factors such as raised blood pressure or plasma lipids levels. These data strongly support an atheroprotective role for perilipin1.
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Aterosclerosis/fisiopatología , Proteínas Portadoras/fisiología , Fosfoproteínas/fisiología , Adiposidad , Animales , Aorta/inmunología , Aorta/metabolismo , Aorta/patología , Aortitis/etiología , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Peso Corporal , Proteínas Portadoras/genética , Cruzamientos Genéticos , Dieta Aterogénica/efectos adversos , Hiperlipidemias/sangre , Hiperlipidemias/etiología , Hipertensión/etiología , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Perilipina-1 , Fosfoproteínas/genética , Placa Aterosclerótica/etiología , Placa Aterosclerótica/patología , ARN Mensajero/metabolismo , Receptores de LDL/genética , Receptores de LDL/fisiología , Índice de Severidad de la EnfermedadRESUMEN
To understand the role of TGF-ß signaling in cardiovascular development, we generated mice with conditional deletion of the TGF-ß type II receptor (TßRII) gene (Tgfbr2) in cells expressing the smooth muscle cell-specific protein SM22α. The SM22α promoter was active in tissues involved in cardiovascular development: vascular smooth muscle cells (VSMCs), epicardium and myocardium. All SM22-Cre(+/-)/Tgfbr2 (flox/flox) embryos died during the last third of gestation. About half the mutant embryos exhibited heart defects (ventricular myocardium hypoplasia and septal defects). All mutant embryos displayed profound vascular abnormalities in the descending thoracic aorta (irregular outline and thickness, occasional aneurysms and elastic fiber disarray). Restriction of these defects to the descending thoracic aorta occurred despite similar levels of Tgfbr2 invalidation in the other portions of the aorta, the ductus arteriosus and the pulmonary trunk. Immunocytochemistry identified impairment of VSMC differentiation in the coronary vessels and the descending thoracic aorta as crucial for the defects. Ventricular myocardial hypoplasia, when present, was associated to impaired α-SMA differentiation of the epicardium-derived coronary VSMCs. Tgfbr2 deletion in the VSMCs of the descending thoracic aorta diminished the number of α-SMA-positive VSMC progenitors in the media at E11.5 and drastically decreased tropoelastin (from E11.5) and fibulin-5 (from E.12.5) synthesis and/or deposition. Defective elastogenesis observed in all mutant embryos and the resulting dilatation and probable rupture of the descending thoracic aorta might explain the late embryonic lethality. To conclude, during mouse development, TGF-ß plays an irreplaceable role on the differentiation of the VSMCs in the coronary vessels and the descending thoracic aorta.
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Aorta Torácica/anomalías , Cardiopatías Congénitas/genética , Miocitos del Músculo Liso/metabolismo , Pericardio/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/genética , Animales , Aorta Torácica/metabolismo , Aorta Torácica/patología , Diferenciación Celular , Tejido Elástico/patología , Elastina/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Cardiopatías Congénitas/metabolismo , Cardiopatías Congénitas/patología , Masculino , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas Musculares/genética , Miocitos del Músculo Liso/patología , Pericardio/patología , Embarazo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: TGF-beta shifts from inhibition to stimulation of vascular smooth muscle cell (vSMC) growth when cell density increases. How proliferation and apoptosis contribute to this shift is still unknown. METHODS: In sparse and confluent V8 vSMC treated or not with TGF-beta(1) (1 ng/ml) for 3 days, cell number, mitotic activity, cell-cycle-regulatory protein levels, caspase-3 and phosphoinositide 3-kinase (PI3-K) activities were studied. RESULTS: In TGF-beta(1)-treated cells, (i) the growth curve rose constantly compared to controls, reaching post-confluent densities; (ii) mitotic activity, which was constant at all cell densities, was lower than in sparse but higher than in contact-inhibited control cells, and (iii) apoptosis occurred at sparse densities only. The mechanism of proliferation control by TGF-beta(1) was very unconventional in V8 vSMCs: (i) p15(INK4b) and cyclin D levels were similar in cells treated or not with TGF-beta(1), and (ii) p27(Kip1) levels remained very low even at high densities while cyclin E levels were not markedly decreased. TGF-beta(1)-induced apoptosis in sparse cultures and its reversal in dense cultures were inversely correlated to PI3-K activation. CONCLUSIONS: TGF-beta(1) slowed sparse V8 vSMC growth by inhibiting proliferation and inducing apoptosis. TGF-beta(1)-treated confluent vSMCs escaped contact inhibition and kept growing through unconventional regulation of p27(Kip1), cyclin E and suppression of apoptosis.
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Apoptosis , Proliferación Celular , Inhibición de Contacto , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Caspasa 3/metabolismo , Línea Celular Transformada , Ciclina D , Ciclina E/metabolismo , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclinas/metabolismo , Replicación del ADN , Masculino , Mitosis , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Ratas , Ratas Wistar , Proteína de Retinoblastoma/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
Members of the TGF-beta/BMP family of growth factors induce odontoblast differentiation and reparative dentin synthesis, and their use has been proposed to stimulate pulp healing during dental therapeutics in human. However, factors that modulate TGF-beta and/or BMP signalling during odontoblast differentiation and physiology remain largely unknown. To identify them, we compared expression profiles of TGF-beta/BMP-related genes in pulp fibroblast- and odontoblast-like cells cultured from human dental pulp explants using cDNA gene arrays. We evidenced that the gene encoding ecotropic viral integration site-1 (EVI1), a transcription factor that inhibits TGF-beta/BMP signalling, was under-expressed in odontoblast-like cells. This result was verified by real-time PCR and, at the protein level, by immunohistochemistry. In vivo, real-time PCR analysis revealed that EVI1 was expressed in the dental pulp, at a level similar to brain, but lower than in lung, kidney or trachea. The protein was localized in dental pulp samples in pulp core and subodontoblast cells. Staining intensity progressively decreased from the radicular to the coronal pulp where EVI1 staining was almost undetectable in odontoblasts. Our data suggest that fine regulation of the EVI1 level in the human dental pulp might be important in the TGF-beta/BMP-induced modulation of dental pulp cell kinetics and/or odontoblast differentiation.
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Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas de Unión al ADN/análisis , Pulpa Dental/metabolismo , Factores de Transcripción/análisis , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Adolescente , Proteínas Morfogenéticas Óseas/genética , Diferenciación Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , Pulpa Dental/citología , Regulación hacia Abajo , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Proteína del Locus del Complejo MDS1 y EV11 , Odontoblastos/citología , Odontoblastos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Proto-Oncogenes/genética , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The orphan nuclear receptor, steroidogenic factor 1 (SF-1), plays a major role in adrenal and gonadal development, as well as in sexual differentiation. It has been demonstrated that the expression of a number of genes regulated by SF-1 is inhibited by the transforming growth factor, (TGF-beta). To date, however, the influence of TGF-beta on the expression of SF-1 gene has not been reported. A Northern blot analysis with the use of a radiolabeled cDNA probe, and immunodetection with antibodies directed against SF-1, demonstrated that the Sf-1 transcript and the SF-1 protein levels were lowered by TGF-beta in Y-1 adrenocortical cells, both in untreated and adenylyl cyclase activator, forskolin-treated cells. An examination of the Sf-1 transcript stability in the presence of actinomycin D revealed no influence of TGF-beta on the rate of Sf-1 mRNA decay. Inhibition of Sf-1 expression by TGF-beta was abolished by cycloheximide, suggesting that the growth factor inhibitory effect requires ongoing protein synthesis. We conclude that in Y-1 cells TGF-beta inhibits the expression of SF-1 gene at a transcriptional level, and we postulate that the inhibitory effect of TGF-beta on steroid hormone synthesis in the adrenal cortex could be due to an attenuated transcription of Sf-1.
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Corteza Suprarrenal/metabolismo , Proteínas de Homeodominio/genética , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta/fisiología , Neoplasias de la Corteza Suprarrenal , Animales , Northern Blotting , Línea Celular Tumoral , Colforsina/farmacología , Cicloheximida/farmacología , Dactinomicina/farmacología , Proteínas de Homeodominio/biosíntesis , Ratones , ARN Mensajero/biosíntesis , Receptores Citoplasmáticos y Nucleares/biosíntesis , Factor Esteroidogénico 1 , Factores de Tiempo , Factores de Transcripción/biosíntesis , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The objective of our study was to investigate the effect of stimulation of the cAMP-dependent pathway on the expression of an orphan nuclear receptor, SF-1/Ad4BP in mouse adrenal tumour, Y-1 cells in culture. We evaluated the temporal pattern of the effects of corticotropin (ACTH) and the adenylyl cyclase activator forskolin on the level of SF-1 mRNA, and compared the time course of induction of SF-1 with that of CYP11A1. Forskolin, corticotropin and 8-Br-cAMP significantly elevated the level of the SF-1 transcript, after 1.5 h of incubation, with a concomitant increase of SF-1 protein level, observed after 6 h. The CYP11A1 transcript increased gradually over the incubation period, and reached the maximal level after 12 to 24 h. The steady-state level of the SF-1 transcript was unaffected by forskolin when the cells were incubated with actinomycin D, indicating that stimulation of the cAMP pathway results in enhanced transcription of the gene. The effect of forskolin was augmented by cycloheximide, suggesting that an inhibitory protein, whose synthesis was inhibited by cycloheximide, could be involved in negative regulation of SF-1 expression. It is concluded that SF-1 expression is positively regulated by the cAMP pathway at the transcriptional level, and can represent the primary event in cAMP-mediated induction of steroid hormone synthesis in Y-1 cells.
Asunto(s)
AMP Cíclico/metabolismo , Transducción de Señal , Factor Esteroidogénico 1/genética , Factor Esteroidogénico 1/metabolismo , Hormona Adrenocorticotrópica/farmacología , Animales , Northern Blotting , Western Blotting , Proteína de Unión a CREB/genética , Línea Celular Tumoral , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Colforsina/farmacología , Cicloheximida/farmacología , Dactinomicina/farmacología , Histona Acetiltransferasas/genética , Ratones , Coactivador 1 de Receptor Nuclear , Fosfoproteínas/genética , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genéticaRESUMEN
The roles of transforming growth factor-beta (TGFbeta) in heart or skeletal muscle development and physiology are still the subject of controversies. Our aim was to block, in transgenic mice, the TGFbeta signalling pathway by a dominant negative mutant of the TGFbeta type II receptor fused to the enhanced green fluorescent protein (TbetaRII-KR-EGFP) under the control of a 7.1 kbp mouse beta-myosin heavy chain (betaMHC) promoter to investigate the roles of TGFbeta in the heart and slow skeletal muscles. First, we generated two transgenic lines overexpressing EGFP under the control of the 7.1 kbp betaMHC promoter. In embryos, EGFP was detectable as early as 7.5 days post coitum. In embryos, newborns and adults, EGFP was expressed mainly in the cardiac ventricles and in slow skeletal muscles. EGFP expression was intense in the bladder but weak in the intestines. In contrast to the endogenous betaMHC promoter, the activity of the 7.1 kbp betaMHC promoter in the transgene was not repressed after birth and remained high in adult transgenic mice. We obtained two founders with the transgene comprising the TbetaRII-KR-EGFP sequence under the control of the 7.1 kbp betaMHC promoter. These founders were generated at a very low frequency and expressed barely detectable levels of TbetaRII-KR-EGFP mRNA. Our failure to obtain transgenic lines overexpressing the dominant negative receptor suggests that the blocking of the TGFbeta signalling pathway in the heart and slow skeletal muscles could be embryonically lethal. To conclude, the 7.1 kbp betaMHC promoter directs high levels of transgene expression in the cardiac ventricles and in slow skeletal muscles of the mouse. Analysis of the consequences of the blocking of the TGFbeta signalling pathway in the heart will require the use of tissue specific means of conditional gene invalidation.
Asunto(s)
Genes Letales , Regiones Promotoras Genéticas , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Miosinas Ventriculares/genética , Animales , Secuencia de Bases , Northern Blotting , Cartilla de ADN , Efecto Fundador , Proteínas Fluorescentes Verdes/genética , Ratones , Ratones Transgénicos , Músculo Esquelético/metabolismo , Mutación , Miocardio/metabolismo , Reacción en Cadena de la Polimerasa , Receptores de Factores de Crecimiento Transformadores beta/genéticaRESUMEN
In DM (differentiation medium), Sol 8 myoblasts spontaneously form myotubes and express the betaMHC (beta-myosin heavy chain), their main marker of terminal differentiation. This marker is detectable at 24 h, and increases up to 72 h. Our aim was to define temporal effects of TGFbeta (transforming growth factor beta) on betaMHC expression in Sol 8 cells. TGFbeta1 (1 ng/ml) added at time zero to DM decreased MyoD expression and completely inhibited betaMHC expression in Sol 8 cells. This inhibition of betaMHC expression was progressively lost when TGFbeta1 was added from 8 to 34 h. After 34 h, the cells were irreversibly differentiated, and TGFbeta1 did not inhibit betaMHC accumulation any longer. Two independent approaches showed that a TGFbeta autocrine regulatory loop retarded and partially impaired Sol 8 cell terminal differentiation. First, permanent immunoneutralization of the active TGFbetas released by the cells into DM increased betaMHC levels at 72 h compared with controls. Secondly, a dominant-negative mutant of the TGFbeta type II receptor was overexpressed in Sol 8 cells under the control of the betaMHC promoter. Both the dominant-negative receptor and the betaMHC gene were expressed after 24 h in DM. The delayed blocking of the TGFbeta signalling pathway by the dominant-negative receptor was as effective as permanent immunoneutralization to promote betaMHC expression. To conclude, TGFbeta inhibits Sol 8 cell terminal differentiation within a narrow time interval (24-34 h) that coincides with the onset of betaMHC expression.
Asunto(s)
Comunicación Autocrina/fisiología , Diferenciación Celular/fisiología , Mioblastos/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/fisiología , Ratones , Ratones Endogámicos C3H , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Cadenas Pesadas de Miosina/biosíntesis , ARN Mensajero/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta1RESUMEN
In Lyon hypertensive (LH) rats, a model of low-renin genetic hypertension, we investigated adrenal sensitivity to angiotensin II in terms of angiotensin II receptor (AT1 and AT2 receptors) regulation, morphological changes, and aldosterone and corticosterone secretion. Twelve-week-old LH rats, compared with normotensive LN and LL rats, were either untreated or treated for 4 weeks with AT1 receptor antagonist irbesartan (50 mg/kg/d), angiotensin-converting enzyme inhibitor perindopril (3 mg/kg/d), or perindopril (3 mg/kg/d) plus angiotensin II infusion (200 ng/kg/min). At 16 weeks, untreated LH rats had high systolic blood pressure (P<0.05), low aldosterone (P<0.05), and increased corticosterone (P<0.05) plasma levels. AT1-receptor binding density in the zona glomerulosa was similar in the three strains. In LH rats, angiotensin II infusion increased the relative adrenal weight from 10.5+/-0.3 to 16.7+/-0.7 mg/100g (P<0.05), whereas this change was very modest in normotensive rats. Zona glomerulosa enlarged and plasma aldosterone increased after angiotensin II infusion in the 3 strains, but more markedly in LH versus normotensive rats (2.4- versus 1.3- and 1.6-fold, respectively; 20- versus 10-fold in normotensive rats, P<0.05). Surprisingly, after angiotensin II infusion, despite the absence of angiotensin II receptors in the three strains, the zona fasciculata-reticularis enlarged 1.5-fold and plasma corticosterone increased 1.7-fold only in LH rats (P<0.05), suggesting an indirect control of this compartment by angiotensin II. The hypertrophy and hypersecretory activity of both zona glomerulosa and zona fasciculata-reticularis in LH rats in response to angiotensin II point to the adrenal cortex as a pivotal tissue in the pathophysiology of hypertension in LH rats.
Asunto(s)
Corteza Suprarrenal/metabolismo , Corteza Suprarrenal/patología , Angiotensina II/farmacología , Hipertensión/metabolismo , Hipertensión/patología , Corteza Suprarrenal/efectos de los fármacos , Glándulas Suprarrenales/efectos de los fármacos , Glándulas Suprarrenales/patología , Aldosterona/sangre , Animales , Presión Sanguínea/efectos de los fármacos , Corticosterona/sangre , Hipertensión/genética , Masculino , Tamaño de los Órganos , Ratas , Ratas Mutantes , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/metabolismoRESUMEN
The human corticotropin-releasing factor receptor type 1 (hCRF-R1) functional transcript is mainly expressed in the anterior pituitary corticotrophs, a tissue usually not available for clinical investigation. Splice variants translated into defective isoforms of the receptor have been described in few peripheral tissues. The aim of this work was to determine whether peripheral white blood cells from healthy individuals, an accessible tissue for clinical investigation, were suitable for the analysis of the hCRF-R1 transcript and gene. We report that: (i) specific amplification of the hCRF-R1 transcript from peripheral white blood cells mRNAs is feasible; (ii) this transcript is similar to the functional transcript; (iii) the draft sequence of chromosome 17 and unrelated sequences allow direct sequencing of all 14 exons of the gene, adjacent splice sites and related branch points. In conclusion, these approaches would be suitable for studies in patients having isolated secondary glucocorticoids deficiency to implicate the hCRH-R1 in the etiology of the disease.