RESUMEN
Introduction: Lrba is a cytoplasmic protein involved in vesicular trafficking. Lrba-deficient (Lrba-/-) mice exhibit substantially higher levels of IgA in both serum and feces than wild-type (WT) mice. Transforming growth factor ß1 (TGFß1) and its receptors (TGFßR I and II) is essential for differentiating IgA+ B cells. Furthermore, increased IgA production suggests a potential connection between Lrba and the TGFßR signaling pathway in IgA production. However, the specific function of Lrba in B cell biology remains unknown. Aim: Given the increased IgA levels in Lrba-/- mice, the goal in this work was to explore the lymph organs where the switch to IgA occurs, and if TGFßR function is affected. Methods: Non-immunized Lrba-/- mice were compared with Lrba+/+ mice. IgA levels in the serum and feces, as well as during peripheral B cell development, were determined. IgA+ B cells and plasma cells were assessed in the small intestine and secondary lymphoid organs, such as the spleen, mesenteric lymph nodes, and Peyer's patches. The TGFßR signaling pathway was evaluated by determining the expression of TGFßR on B cells. Additionally, SMAD2 phosphorylation was measured under basal conditions and in response to recombinant TGFß. Finally, confocal microscopy was performed to investigate a possible interaction between Lrba and TGFßR in B cells. Results: Lrba-/- mice exhibited significantly higher levels of circulating IgA, IgA+ B, and plasma cells than in peripheral lymphoid organs those in WT mice. TGFßR expression on the membrane of B cells was similar in both Lrba-/- and Lrba+/+ mice. However, intracellular TGFßR expression was reduced in Lrba-/- mice. SMAD2 phosphorylation showed increased levels under basal conditions; stimulation with recombinant TGFß elicited a poorer response than in that in Lrba+/+ B cells. Finally, we found that Lrba colocalizes with TGFßR in B cells. Conclusion: Lrba is essential in controlling TGFßR signaling, subsequently regulating SMAD2 phosphorylation on B cells. This mechanism may explain the increased differentiation of IgA+ B cells and production of IgA-producing plasma cells.
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Linfocitos B , Diferenciación Celular , Inmunoglobulina A , Transducción de Señal , Animales , Ratones , Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular/inmunología , Inmunoglobulina A/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Ganglios Linfáticos Agregados/inmunología , Ganglios Linfáticos Agregados/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteína Smad2/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Maternal malnutrition can alter developmental biology, programming health and disease in offspring. The increase in sugar consumption during the peripubertal period, a worldwide concern, also affects health through adulthood. Studies have shown that maternal exposure to a low protein diet (LPD) is associated with an increase in prostate disease with aging. However, the combined effects of maternal LPD and early postnatal sugar consumption on offspring prostate disorders were not investigated. The effects on aging were evaluated using a maternal gestational model with lactational LPD (6% protein) and sugar consumption (10%) from postnatal day (PND) 21-90, associating the consequences on ventral prostate (VP) rats morphophysiology on PND540. An increase was shown in mast cells and in the VP of the CTR + SUG and Gestational and Lactational Low Protein (GLLP) groups. In GLLP + SUG, a significant increase was shown in TGF-ß1 expression in both the systemic and intra-prostatic forms, and SMAD2/3p had increased. The study identified maternal LPD and sugar consumption as risk factors for prostatic homeostasis in senility, activating the TGFß1-SMAD2/3 pathway, a signaling pathway with potential markers for prostatic disorders.
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Desnutrición , Fenómenos Fisiologicos Nutricionales Maternos , Efectos Tardíos de la Exposición Prenatal , Próstata , Enfermedades de la Próstata , Animales , Masculino , Femenino , Embarazo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Enfermedades de la Próstata/patología , Enfermedades de la Próstata/etiología , Enfermedades de la Próstata/metabolismo , Desnutrición/complicaciones , Próstata/metabolismo , Próstata/patología , Ratas , Inflamación/patología , Inflamación/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Dieta con Restricción de Proteínas/efectos adversos , Proteína Smad2/metabolismo , Ratas Wistar , Proteína smad3/metabolismo , Proteína smad3/genética , Transducción de Señal , Animales Recién Nacidos , Mastocitos/metabolismoRESUMEN
Octreotide (OCT) is used to inhibit hormone secretion and growth in somatotroph tumors, although a significant percentage of patients are resistant. It has also been tested in nonfunctioning (NF) tumors but with poor results, with these outcomes having been associated with SSTR2 levels and impaired signaling. We investigated whether OCT inhibitory effects can be improved by TGF-ß1 in functioning and nonfunctioning somatotroph tumor cells. OCT effects on hormone secretion and proliferation were analyzed in the presence of TGF-ß1 in WT and SSTR2-overexpressing secreting GH3 and silent somatotroph tumor cells. The mechanism underlying these effects was assessed by studying SSTR and TGFßR signaling pathways mediators. In addition, we analyzed the effects of OCT/TGF-ß1 treatment on tumor growth and cell proliferation in vivo. The inhibitory effects of OCT on GH- and PRL-secretion and proliferation were improved in the presence of TGF-ß1, as well as by SSTR2 overexpression. The OCT/TGF-ß1 treatment induced downregulation of pERK1/2 and pAkt, upregulation of pSmad3, and inhibition of cyclin D1. In vivo experiments showed that OCT in the presence of TGF-ß1 blocked tumor volume growth, decreased cell proliferation, and increased tumor necrosis. These results indicate that SSTR2 levels and the stimulation of TGF-ß1/TGFßR/Smad2/3 pathway are important for strengthening the antiproliferative and antisecretory effects of OCT.
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Antineoplásicos Hormonales/farmacología , Proliferación Celular/efectos de los fármacos , Octreótido/farmacología , Neoplasias Hipofisarias/tratamiento farmacológico , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Somatotrofos/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , Animales , Línea Celular , Femenino , Humanos , Ratones Desnudos , Fosforilación , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/metabolismo , Neoplasias Hipofisarias/patología , Ratas , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Transducción de Señal , Somatotrofos/metabolismo , Somatotrofos/patología , Carga Tumoral/efectos de los fármacosRESUMEN
Poor prognosis associated with the dysregulated expression of activin A in a number of malignancies has been related to with numerous aspects of tumorigenesis, including angiogenesis. The present study investigated the prognostic significance of activin A immunoexpression in blood vessels and cancer cells in a number of oral squamous cell carcinoma (OSCC) cases and applied in vitro strategies to determine the impact of activin A on angiogenesis. In a cohort of 95 patients with OSCC, immunoexpression of activin A in both blood vessels and tumor cells was quantified and the association with clinicopathological parameters and survival was analyzed. Effects of activin A on the tube formation, proliferation and migration of human umbilical vein endothelial cells (HUVECs) were evaluated in gainoffunction (treatment with recombinant activin A) or lossoffunction [treatment with activin Aantagonist follistatin or by stable transfection with short hairpin RNA (shRNA) targeting activin A] conditions. Conditioned medium from an OSCC cell line with shRNAmediated depletion of activin A was also tested. The profile of pro and antiangiogenic factors regulated by activin A was assessed with a human angiogenesis quantitative PCR (qPCR) array. Vascular endothelial growth factor A (VEGFA) and its major isoforms were evaluated by reverse transcriptionqPCR and ELISA. Activin A expression in blood vessels demonstrated an independent prognostic value in the multivariate analysis with a hazard ratio of 2.47 [95% confidence interval (CI), 1.304.71; P=0.006) for diseasespecific survival and 2.09 (95% CI, 1.074.08l: P=0.03) for diseasefree survival. Activin A significantly increased tubular formation of HUVECs concomitantly with an increase in proliferation. This effect was validated by reduced proliferation and tubular formation of HUVECs following inhibition of activin A by follistatin or shRNA, as well as by treatment of HUVECs with conditioned medium from activin Adepleted OSCC cells. Activin Aknockdown increased the migration of HUVECs. In addition, activin A stimulated the phosphorylation of SMAD2/3 and the expression and production of total VEGFA, significantly enhancing the expression of its proangiogenic isoform 121. The present findings suggest that activin A is a predictor of the prognosis of patients with OSCC, and provide evidence that activin A, in an autocrine and paracrine manner, may contribute to OSCC angiogenesis through differential expression of the isoform 121 of VEGFA.
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Activinas/metabolismo , Neoplasias de la Boca/patología , Neovascularización Patológica/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Activinas/análisis , Activinas/antagonistas & inhibidores , Activinas/genética , Adulto , Anciano , Anciano de 80 o más Años , Comunicación Autocrina/efectos de los fármacos , Comunicación Autocrina/genética , Movimiento Celular , Proliferación Celular , Femenino , Folistatina/farmacología , Folistatina/uso terapéutico , Técnicas de Silenciamiento del Gen , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Persona de Mediana Edad , Mucosa Bucal/patología , Neoplasias de la Boca/irrigación sanguínea , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/mortalidad , Comunicación Paracrina/efectos de los fármacos , Comunicación Paracrina/genética , Fosforilación/efectos de los fármacos , Fosforilación/genética , Pronóstico , Isoformas de Proteínas/metabolismo , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/irrigación sanguínea , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/mortalidadRESUMEN
PURPOSE: Epithelial to mesenchymal transition (EMT) plays an important role in acquired resistance to gefitinib in lung cancer. This study aimed to explore the underlying mechanism of gefitinib-induced EMT in lung adenocarcinoma cells harboring EGFR mutation. METHODS: CXC chemokine receptor 4 (CXCR4) expression was determined through qRT-PCR, Western blot and flow cytometry assays in lung cancer cell line (PC9) bearing mutated EGFR. Functional role of CXCR4 was inhibited applying siRNAs as well as the specific antagonist AMD3100. The expression of EMT markers was determined, and the migration of PC9 cells was measured with transwell assay. RESULTS: We found that gefitinib promoted the migratory capacity of PC9 cells in vitro, which correlated with EMT occurrence through upregulation of CXCR4. Blocking CXCR4 significantly suppressed gefitinib-induced enhancement of migration and EMT. Moreover, we determined that the upregulation of CXCR4 by gefitinib was dependent on TGF-ß1/Smad2 signaling activity. CONCLUSIONS: Our study suggested a potential mechanism by which gefitinib induced EMT in cells harboring EGFR mutation through a pathway involving TGF-ß1 and CXCR4. Thus, the combination of CXCR4 antagonist and TGFßR inhibitors might provide an alternative strategy to overcome progression of lung cancer after gefitinib treatment.
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Adenocarcinoma del Pulmón/patología , Antineoplásicos/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Receptores ErbB/genética , Gefitinib/farmacología , Neoplasias Pulmonares/patología , Receptores CXCR4/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Adenocarcinoma del Pulmón/genética , Bencilaminas/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL12/metabolismo , Ciclamas/farmacología , Humanos , Neoplasias Pulmonares/genética , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , ARN Interferente Pequeño/farmacología , Receptores CXCR4/antagonistas & inhibidores , Proteína Smad2/metabolismo , Regulación hacia ArribaRESUMEN
Megalin/LRP2 is a receptor that plays important roles in the physiology of several organs, such as kidney, lung, intestine, and gallbladder and also in the physiology of the nervous system. Megalin expression is reduced in diseases associated with fibrosis, including diabetic nephropathy, hepatic fibrosis and cholelithiasis, as well as in some breast and prostate cancers. One of the hallmarks of these conditions is the presence of the cytokine transforming growth factor beta (TGF-ß). Although TGF-ß has been implicated in the reduction of megalin levels, the molecular mechanism underlying this regulation is not well understood. Here, we show that treatment of two epithelial cell lines (from kidney and gallbladder) with TGF-ß1 is associated with decreased megalin mRNA and protein levels, and that these effects are reversed by inhibiting the TGF-ß1 type I receptor (TGF-ßRI). Based on in silico analyses, the two SMAD-binding elements (SBEs) in the megalin promoter are located at positions -57 and -605. Site-directed mutagenesis of the SBEs and chromatin immunoprecipitation (ChIP) experiments revealed that SMAD2/3 transcription factors interact with SBEs. Both the presence of SMAD2/3 and intact SBEs were associated with repression of the megalin promoter, in the absence as well in the presence of TGF-ß1. Also, reduced megalin expression and promoter activation triggered by high concentration of albumin are dependent on the expression of SMAD2/3. Interestingly, the histone deacetylase inhibitor Trichostatin A (TSA), which induces megalin expression, reduced the effects of TGF-ß1 on megalin mRNA levels. These data show the significance of TGF-ß and the SMAD2/3 signalling pathway in the regulation of megalin and explain the decreased megalin levels observed under conditions in which TGF-ß is upregulated, including fibrosis-associated diseases and cancer.
Asunto(s)
Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Secuencia de Bases , Sitios de Unión , Biomarcadores , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Smad2/genética , Proteína smad3/genética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The transforming growth factor-beta (TGF-ß) pathway is involved in the regulation of cell growth and differentiation. In normal cells or in the early stages of cancer, this pathway can control proliferation stimuli by inducing cell cycle arrest or apoptosis (through the MAP-kinase protein p38MAPK), while in late stages it seems to act as a tumor promoter. This feature is known as the TGF-ß dual role in cancer and it is not completely explained. This seems to arise through the accumulation of mutations in cancer development that affect the normal function of these pathways. In this work we propose a Boolean model of the crosstalk between the TGF-ß, p38 MAPK and cell cycle checkpoint pathways which qualitatively describes this dual behavior. The model shows that for the wild type case, TGF-ß acts as tumor supressor by inducing cell cycle arrest or apoptosis, as expected. However, the loss of function (LoF) of its two signaling proteins: SMAD2 and SMAD3 has immortalization effects due to the activation of the PI3K/AKT pathway that contributes to inhibit apoptosis. In silico mutations of the model elements were compared with cell phenotypes in experiments presenting agreement. In addition, we performed a series of double gene perturbations (that simulate random deleterious mutations) to determine the main regulators of the network. The results suggest that SMAD2/3 and p38MAPK are key players in processing the network input. In addition, when the LoF of SMAD2/3 is combined with the LoF of p38MAPK and p53, cell cycle arrest is completely abrogated. In conclusion, the model allows to visualize, through in silico mutations, the dual role of TGF-ß: for the wild-type case TGF-ß is able to block proliferation, however deleterious mutations can impair cell cycle arrest promoting cellular proliferation.
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Sistema de Señalización de MAP Quinasas , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Apoptosis , Ciclo Celular , Humanos , Modelos Biológicos , Neoplasias/patología , Proteína Smad2/metabolismo , Proteína smad3/metabolismoRESUMEN
Many metabolic syndromes lead to energetic disturbs which ends to an intense catabolic state. The branched-chain amino acid leucine shows very positive effects on muscle protein metabolism. However, it is still not clear how leucine acts improving the protein turnover. This study aimed to evaluate in vitro the effects of leucine supplementation in minimising the signalling pathway of protein degradation when mTOR was inhibited. Our studies were conducted in murine C2C12 myotubes exposed to 2mM leucine or 2mM isoleucine in control situation and compared to the inhibition of mTOR by rapamycin. Then, the expression of proteins related to protein synthesis and degradation signalling pathway was obtained by Western Blot. At this concentration, the leucine was sufficient to maintain the expression of proteins evaluated as in control situation. However, when the cells were exposed to rapamycin (80nM), leucine inhibited the expression of SMAD and FoxO3a, showing that leucine was able to modulate the degradation pathway when protein synthesis is compromised. Furthermore, leucine had no effect in modifying the expression of subunits of ubiquitin-proteasome system, showing that leucine had no direct effect in ubiquitin-proteasome system, but acted leading to the phosphorylation of SMAD and FoxO3, which inhibited the activity of transcriptional of these proteins. No similar results were observed in cells exposed to isoleucine under the same experimental protocol, likely showing that leucine has specific action over another branched-chain amino acids. In conclusion, the present study shows that leucine can modulate degradation pathways even under inhibition of mTOR by rapamycin.
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Leucina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteolisis , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Animales , Línea Celular , Suplementos Dietéticos , Proteína Forkhead Box O3/metabolismo , Ratones , Fibras Musculares Esqueléticas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Proteína Smad2/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
Growth differentiation factor 11 (GDF11), a member of the transforming growth factor-ß family, has been shown to act as a negative regulator in cardiac hypertrophy. Ca2+ signaling modulates cardiomyocyte growth; however, the role of Ca2+-dependent mechanisms in mediating the effects of GDF11 remains elusive. Here, we found that GDF11 induced intracellular Ca2+ increases in neonatal rat cardiomyocytes and that this response was blocked by chelating the intracellular Ca2+ with BAPTA-AM or by pretreatment with inhibitors of the inositol 1,4,5-trisphosphate (IP3) pathway. Moreover, GDF11 increased the phosphorylation levels and luciferase activity of Smad2/3 in a concentration-dependent manner, and the inhibition of IP3-dependent Ca2+ release abolished GDF11-induced Smad2/3 activity. To assess whether GDF11 exerted antihypertrophic effects by modulating Ca2+ signaling, cardiomyocytes were exposed to hypertrophic agents (100 nM testosterone or 50 µM phenylephrine) for 24 h. Both treatments increased cardiomyocyte size and [³H]-leucine incorporation, and these responses were significantly blunted by pretreatment with GDF11 over 24 h. Moreover, downregulation of Smad2 and Smad3 with siRNA was accompanied by inhibition of the antihypertrophic effects of GDF11. These results suggest that GDF11 modulates Ca2+ signaling and the Smad2/3 pathway to prevent cardiomyocyte hypertrophy.
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Señalización del Calcio , Cardiomegalia/metabolismo , Factores de Diferenciación de Crecimiento/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Factores de Diferenciación de Crecimiento/genética , Miocitos Cardíacos/efectos de los fármacos , Fenilefrina/farmacología , Ratas , Ratas Sprague-Dawley , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismo , Testosterona/farmacologíaRESUMEN
INTRODUCTION: The present study aimed to elucidate the potential antifibrotic effects of pinocembrin (PIN), a flavanone found abundantly in honey and propolis, by studying its effect on different oxidative stress, inflammatory and fibrosis markers in an experimental model of CCl4-induced liver fibrosis. MATERIAL AND METHODS: PIN (20 mg/kg) was given orally 3 times/week for 6 consecutive weeks alternating with CCl4 (0.5 mL/kg, 1:1 mixture with corn oil, i. p.) twice weekly. Different hepatotoxicity indices, oxidative stress, inflammatory and liver fibrosis markers were assessed. RESULTS: PIN significantly restored liver transaminases and total cholesterol to normal levels. Also, PIN ameliorated oxidative stress injury evoked by CCl4 as evidenced by inhibition of reduced glutathione depletion and lipid peroxidation as well as elevation of antioxidant enzyme superoxide dismutase (SOD). Further, PIN upregulated the nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2), thereby inducing the expression and activity of the cytoprotective enzyme hemeoxygenase-1 (HO-1). Moreover, PIN alleviated pro-inflammatory cytokines such as TNF-α via inhibiting nuclear factor-κB (NF-κB) activation. As markers of fibrosis, collagen and α-SMA expression increased markedly in the CCl4 group and PIN prevented these alterations. In addition, PIN down-regulated TGFß1 and p-Smad2/3, thereby inhibiting TGFß1/Smad signaling pathway. CONCLUSION: These results suggest that PIN possess potent antifibrotic effects that can be explained on its antioxidant properties. It ameliorates oxidative stress and inflammation during induction of fibrogenesis via its ability to augment celular antioxidant defenses, activating Nrf2-mediated HO-1 expression and modulating NF-κB and TGF-ß1/Smad signaling pathway.
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Antiinflamatorios/farmacología , Antioxidantes/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Flavanonas/farmacología , Mediadores de Inflamación/metabolismo , Cirrosis Hepática Experimental/prevención & control , Hígado/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Biomarcadores/metabolismo , Tetracloruro de Carbono , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Hemo Oxigenasa (Desciclizante)/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/metabolismo , Cirrosis Hepática Experimental/patología , Masculino , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Fosforilación , Ratas Wistar , Transducción de Señal/efectos de los fármacosRESUMEN
The transforming growth factor type-beta (TGF-ß) induces skeletal muscle atrophy characterised by a decrease in the fibre's diameter and levels of myosin heavy chain (MHC), also as an increase of MuRF-1 expression. In addition, TGF-ß induces muscle atrophy by a mechanism dependent on reactive oxygen species (ROS). TGF-ß signals by activating both canonical Smad-dependent, and non-canonical signalling pathways such as ERK1/2, JNK1/2, and p38 MAPKs. However, the participation of canonical and non-canonical signalling pathways in the TGF-ß atrophic effect on skeletal muscle is unknown. We evaluate the impact of Smad and MAPK signalling pathways on the TGF-ß-induced atrophic effect in C2C12 myotubes. The results indicate that TGF-ß activates Smad2/3, ERK1/2 and JNK1/2, but not p38 in myotubes. The pharmacological inhibition of Smad3, ERK1/2 and JNK1/2 activation completely abolished the atrophic effect of TGF-ß. Finally, the inhibition of these canonical and non-canonical pathways did not decrease the ROS increment, while the inhibition of ROS production entirely abolished the phosphorylation of Smad3, ERK1/2 and JNK1/2. These results suggest that TGF-ß requires Smad3, ERK1/2 and JNK1/2 activation to produce skeletal muscle atrophy. Moreover, the induction of ROS by TGF-ß is an upstream event to canonical and non-canonical pathways.
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Atrofia Muscular/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Fosforilación , Proteína Smad2/metabolismo , Proteína smad3/metabolismoRESUMEN
Adult mesenchymal stromal cell-based interventions have shown promising results in a broad range of diseases. However, their use has faced limited effectiveness owing to the low survival rates and susceptibility to environmental stress on transplantation. We describe the cellular and molecular characteristics of multilineage-differentiating stress-enduring (Muse) cells derived from adipose tissue (AT), a subpopulation of pluripotent stem cells isolated from human lipoaspirates. Muse-AT cells were efficiently obtained using a simple, fast, and affordable procedure, avoiding cell sorting and genetic manipulation methods. Muse-AT cells isolated under severe cellular stress, expressed pluripotency stem cell markers and spontaneously differentiated into the three germ lineages. Muse-AT cells grown as spheroids have a limited proliferation rate, a diameter of â¼15 µm, and ultrastructural organization similar to that of embryonic stem cells. Muse-AT cells evidenced high stage-specific embryonic antigen-3 (SSEA-3) expression (â¼60% of cells) after 7-10 days growing in suspension and did not form teratomas when injected into immunodeficient mice. SSEA-3+ -Muse-AT cells expressed CD105, CD29, CD73, human leukocyte antigen (HLA) class I, CD44, and CD90 and low levels of HLA class II, CD45, and CD34. Using lipopolysaccharide-stimulated macrophages and antigen-challenged T-cell assays, we have shown that Muse-AT cells have anti-inflammatory activities downregulating the secretion of proinflammatory cytokines, such as interferon-γ and tumor necrosis factor-α. Muse-AT cells spontaneously gained transforming growth factor-ß1 expression that, in a phosphorylated SMAD2-dependent manner, might prove pivotal in their observed immunoregulatory activity through decreased expression of T-box transcription factor in T cells. Collectively, the present study has demonstrated the feasibility and efficiency of obtaining Muse-AT cells that can potentially be harnessed as immunoregulators to treat immune-related disorders. Stem Cells Translational Medicine 2017;6:161-173.
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Tejido Adiposo/patología , Carcinogénesis/patología , Inmunomodulación , Células Madre Pluripotentes/citología , Factor de Crecimiento Transformador beta1/farmacología , Animales , Biomarcadores/metabolismo , Carcinogénesis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Estratos Germinativos/citología , Humanos , Inmunomodulación/efectos de los fármacos , Cariotipo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación/efectos de los fármacos , Células Madre Pluripotentes/trasplante , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismo , Bazo/citología , Estrés Fisiológico , Teratoma/patologíaRESUMEN
The temporal and spatial patterns of Smad and Yes-associated protein 1 (YAP1) expression were investigated in skeletal muscle (gastrocnemius muscle and extensor digitorum longus) at different growth stages (2 days old, 2 and 6 months old) in Hu sheep. Smads were differentially expressed in sheep skeletal muscle, with high expression in the gastrocnemius muscle and lower expression in the extensor digitorum longus. Expression of Smad2, Smad3, and Smad4 at the 2-day-old stage was significantly higher than at other stages (P < 0.05). The expression of Smad7 in 2-day-old sheep was lower than in 6-month-old sheep, with the lowest levels at 2 months. Smad expression was higher in males than in females at the 2-day-old stage, and expression in 2- and 6-month-old males was lower than that in 2-day-old females. Smad3 expression was higher in the 2-day- and 2-month-old males than in the females. There was a positive correlation (P < 0.01) between YAP1 and Smad2 expression in gastrocnemius muscle at the 2-month-old stage. YAP1 and Smad4/7 expression were positively correlated (P < 0.01) in extensor digitorum longus at the 2-day-old stage. YAP1 expression was negatively correlated with Smad7 in the extensor digitorum longus at 6 months. A significant difference between Smad2 and Smad3 (P < 0.01) expression in muscle was observed, consistent with Smad3 and Smad4 expression, indicating that these inhibit transforming growth factor-ß signaling in the same way. There was a positive correlation (P < 0.01) between YAP1 and MSTN expression, suggesting that YAP1 participates in muscle growth in sheep.
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Proteínas Adaptadoras Transductoras de Señales/genética , Envejecimiento/genética , Músculo Esquelético/metabolismo , Proteína Smad2/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Músculo Esquelético/crecimiento & desarrollo , Miostatina/genética , Miostatina/metabolismo , Ovinos , Oveja Doméstica , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Proteína smad7/genética , Proteína smad7/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
UNLABELLED: Introduction and Aim. TGF-ß signalling is involved in pathogenesis and progress of hepatocellular carcinoma (HCC). This bioinformatics study consequently aims to determine the underlying molecular mechanism of TGF- ß activation in HCC cells. MATERIAL AND METHODS: Dataset GSE10393 was downloaded from Gene Expression Omnibus, including 2 Huh-7 (HCC cell line) samples treated by TGF- ß (100 pmol/L, 48 h) and 2 untreated samples. Differentially expressed genes (DEGs) were screened using Limma package (false discovery rate < 0.05 and |log2 fold change| > 1.5), and then enrichment analyses of function, pathway, and disease were performed. In addition, protein-protein interaction (PPI) network was constructed based on the PPI data from multiple databases including INACT, MINT, BioGRID, UniProt, BIND, BindingDB, and SPIKE databases. Transcription factor (TF)-DEG pairs (Bonferroni adjusted p-value < 0.01) from ChEA database and DEG-DEG pairs were used to construct TF-DEG regulatory network. Furthermore, TF-pathway-DEG complex network was constructed by integrating DEG-DEG pairs, TF-DEG pairs, and DEG-pathway pairs. RESULTS: Totally, 209 DEGs and 30 TFs were identified. The DEGs were significantly enriched in adhesion-related functions. PPI network indicted hub genes such as CUL4B and NEDD4. According to the TF-DEG regulatory network, the two hub genes were targeted by SMAD2, SMAD3, and HNF4A. Besides, the 11 pathways in TF-pathway-DEG network were mainly enriched by UGT1A family and CYP3A7, which were predicted to be regulated by SMAD2, SMAD3, SOX2, TP63, and HNF4A. CONCLUSIONS: TGF- ß might influence biological processes of HCC cells via SMAD2/SMAD3-NEDD4, HNF4A-CUL4B/NEDD4, SOX2/TP63/HNF4A-CYP3A7, and SMAD2/SMAD3/SOX2/TP63/HNF4A-UGT1As regulatory pathways.
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Carcinoma Hepatocelular/genética , Proteínas Cullin/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Glucuronosiltransferasa/genética , Neoplasias Hepáticas/genética , Factor de Crecimiento Transformador beta/metabolismo , Ubiquitina-Proteína Ligasas/genética , Carcinoma Hepatocelular/metabolismo , Adhesión Celular/genética , Línea Celular Tumoral , Proteínas Cullin/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Bases de Datos Genéticas , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Perfilación de la Expresión Génica , Glucuronosiltransferasa/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Ubiquitina-Proteína Ligasas Nedd4 , Mapas de Interacción de Proteínas , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
PURPOSE: One of the hypotheses regarding the genesis of epithelial ovarian cancer involves the action of androgens on the proliferation of epithelial ovarian cells, as well as inclusion cysts. The purpose of the present study was to evaluate whether DHT causes changes in the TGF-ß1 pathway that might modify the anti-proliferative effect of the latter. METHODS: The levels of TGF-ß1 protein, of its receptors (TGFBR1 and TGFBR2), of Smad2/3 (canonical signaling pathway protein) and of p21 (cell cycle protein) were assessed in ovarian tissues, epithelial ovarian cancer cell lines (A2780) and control cell lines (HOSE) through the use of immunohistochemistry and immunocytochemistry. Additionally, cell lines were treated with 100 nmol/L DHT, 10 ng/mL of TGF-ß1 and DHT + TGF-ß1 during 72 h in the presence and absence of a siRNA against androgen receptor. After treatment, TGFBR1 and TGFBR2 levels were detected through Western blotting and p21 was assessed through immunocytochemistry. RESULTS: Epithelial ovarian cancer tissues showed a decrease in TGF-ß1 I receptor (p < 0.05) and a change in Smad2/3 protein levels. Additionally, after treatment of cell lines with DHT, protein levels of TGF-ß1 receptors (TGFBR1-TGFBR2) showed a decrease (p < 0.05) that might cause a potential disorder in TGF-ß1 response, represented by the significant decrease in p21 protein levels in the presence of DHT (p < 0.001). CONCLUSIONS: Overall, our results indicate a defect in the canonical TGF-ß signaling pathway in epithelial ovarian cancer caused by androgen action, thus suggesting eventual changes in such tissue proliferation rates.
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Dihidrotestosterona/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Ováricas/metabolismo , Ovario/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Andrógenos/farmacología , Apoptosis , Western Blotting , Carcinoma Epitelial de Ovario , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Humanos , Técnicas para Inmunoenzimas , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Ovario/citología , Ovario/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Smad2/genética , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta1/genética , Células Tumorales CultivadasRESUMEN
BACKGROUND: Pure apocynin, which can be traditionally isolated and purified from several plant species such as Picrorhiza kurroa Royle ex Benth (Scrophulariaceae), acts as an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity inhibiting its production of reactive oxygen species (ROS). Transforming growth factor type beta 1 (TGF-ß1) is a growth factor that produces inhibition of myogenesis, diminution of regeneration and induction of atrophy in skeletal muscle. The typical signalling that is activated by TGF-ß involves the Smad pathway. PURPOSE: To evaluate the effect of TGF-ß and the effect of apocynin on TGF-ß1 expression in skeletal muscle cells. STUDY DESIGN: Controlled laboratory study. In vitro assays were performed with C2C12 cells incubated with TGF-ß1 in presence or absence of apocynin (NOX inhibitor), SB525334 (TGF-ß-receptor I inhibitor), or chelerythrine (PKC inhibitor). METHODS: TGF-ß1 and atrogin-1 expression was evaluated by RT-qPCR and/or ELISA; Smad3 phosphorylation by western blot; Smad4 nuclear translocation by indirect immunofluorescence; and ROS levels by DCF probe fluorescent measurements. RESULTS: We show that myoblasts respond to TGF-ß1 by increasing its own gene expression in a time- and dose-dependent fashion which was abolished by SB525334 and siRNA for Smad2/3. TGF-ß1 also induced ROS. Remarkably, apocynin inhibited the TGF-ß1 induced ROS as well as the autoinduction of TGF-ß1 gene expression. We also show that TGF-ß-induced ROS production and TGF-ß1 expression require PKC activity as indicated by the inhibition using chelerythrine. CONCLUSION: These results strongly suggest that TGF-ß induces its own expression through a TGF-ß-receptor/Smad-dependent mechanism and apocynin is able to inhibit this process, suggesting that requires NOX-induced ROS in skeletal muscle cells.
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Acetofenonas/farmacología , Fibras Musculares Esqueléticas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Animales , Benzofenantridinas/farmacología , Línea Celular , Imidazoles/farmacología , Ratones , Fibras Musculares Esqueléticas/metabolismo , Proteína Quinasa C/metabolismo , Quinoxalinas/farmacología , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: Halofuginone (HF) is a low-molecular-weight alkaloid that has been demonstrated to interfere with Metalloproteinase-2 (MMP-2) and Tumor Growth Factor-ß (TGF-ß) function and, to present antiangiogenic, antiproliferative and proapoptotic properties in several solid tumor models. Based on the fact that high levels of Vascular Endothelial Growth Factor (VEGF) and increased angiogenesis have been described in acute myeloid leukemia and associated with disease progression, we studied the in vivo effects of HF using an Acute Promyelocytic Leukemia (APL) mouse model. METHODS: NOD/SCID mice were transplanted with leukemic cells from hCG-PML/RARA transgenic mice (TM) and treated with HF 150 µg/kg/day for 21 days. The leukemic infiltration and the percentage of VEGF+ cells were evaluated by morphology and flow cytometry. The effect of HF on the gene expression of several pro- and antiangiogenic factors, phosphorylation of SMAD2 and VEGF secretion was assessed in vitro using NB4 and HUVEC cells. RESULTS: HF treatment resulted in hematological remission with decreased accumulation of immature cell and lower amounts of VEGF in BM of leukemic mice. In vitro, HF modulated gene expression of several pro- and antiangiogenic factors, reduced VEGF secretion and phosphorylation of SMAD2, blocking TGF-ß-signaling. CONCLUSION: Taken together, our results demonstrate that HF inhibits SMAD2 signaling and reduces leukemia growth and angiogenesis.
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Leucemia Promielocítica Aguda/metabolismo , Piperidinas/metabolismo , Quinazolinonas/metabolismo , Proteína Smad2/genética , Animales , Modelos Animales de Enfermedad , Humanos , Inmunofenotipificación , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Neovascularización Patológica , Fosforilación , Proteína Smad2/metabolismo , Células Tumorales CultivadasRESUMEN
Considering that interleukin 10 (IL10), transforming growth factor beta1 (TGFB1), and interferon gamma (IFNG) are involved in the susceptibility of BeWo trophoblast cells to Toxoplasma gondii infection, the aim of the present study was to investigate the effector mechanisms triggered by these cytokines in the control of T. gondii in BeWo cells. For this purpose, infected/uninfected BeWo cells were treated with IL10, TGFB1 (50 ng/ml), and IFNG (20 or 100 ng/ml) in order to verify the phosphorylation of signal transducers and activators of transcription 1 (STAT1), STAT3, and Smad2, parasite intracellular proliferation, as well as the Th1/Th2/IL17A cytokine production. The treatment of BeWo cells with IL10 and TGFB1 favored T. gondii proliferation, and these findings were associated with STAT3 and Smad2 phosphorylation, respectively (P < 0.05). Also, these cytokine treatments were able to down-modulate TNF alpha (TNFA) and IL6 production (P < 0.05). Low concentration of IFNG was unable to control T. gondii infection but was able to trigger STAT1 phosphorylation and up-regulate IL6 and IL17A production; whereas a high concentration of IFNG was unable to activate STAT1 but down-modulated IL6 and TNFA and increased T. gondii proliferation (P < 0.05). IL10, TGFB1, and IFNG regulate a differential T. gondii proliferation in BeWo cells because they distinctly trigger intracellular signaling pathways and cytokine production, especially IL6 and TNFA. Our data open new windows to understand the mechanisms triggered by IL10, TGFB1, and IFNG at the maternal-fetal interface in the presence of T. gondii, contributing to recognizing the importance of these effector mechanisms involved in the vertical transmission of this parasite.
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Citocinas/metabolismo , Interferón gamma/farmacología , Interleucina-10/farmacología , Transducción de Señal/efectos de los fármacos , Toxoplasmosis/prevención & control , Factor de Crecimiento Transformador beta1/farmacología , Trofoblastos/efectos de los fármacos , Trofoblastos/parasitología , Línea Celular Tumoral , Coriocarcinoma/patología , Susceptibilidad a Enfermedades , Femenino , Humanos , Técnicas In Vitro , Interleucina-16/metabolismo , Fosforilación , Embarazo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/fisiología , Proteína Smad2/metabolismo , Toxoplasma/aislamiento & purificación , Toxoplasmosis/metabolismo , Toxoplasmosis/patología , Trofoblastos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Neoplasias Uterinas/patologíaRESUMEN
We examined the effects of co-culturing CD4+ CD25+ Treg cells with sirolimus or cyclosporin A on Treg cell proliferation and differentiation and on transforming growth factor-ß (TGF-ß) and Foxp3 expression. CD4+ CD25+ Treg cells were harvested from mononuclear cells of spleens of C57BL/6 mice using immunomagnetic beads and divided into control, sirolimus, and cyclosporine groups. Following a 96-h co-culture, Treg cells were assayed by flow cytometry. FoxP3 and TGF-ß mRNA levels and secretion were assayed by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Smad protein of the TGF-ß signaling pathway was assayed by western blot and its effect on CD4+ CD25+ FoxP3+ Treg cell proliferation was determined. Sirolimus-promoted differentiation and proliferation was examined using a TGF-ß neutralizing antibody. Sirolimus-treated CD4+ T cell TGF-ß secretion increased 2.5X over control levels (P < 0.01), but that of the cyclosporine group decreased marginally (P > 0.05). The CD4+ cell proportion decreased significantly (41.25 vs 69.22%, P < 0.01) and slightly (65.21 vs 69.22, P > 0.05) in the cyclosporine and sirolimus groups, respectively. T cell Foxp3 mRNA expression was significantly higher in the sirolimus-treated than in the cyclosporine (53.7 vs 40.2%, P < 0.05) and control groups (P < 0.01), but was significantly lower in the cyclosporine group than in controls (23.6 vs 40.2%, P < 0.01). Overall, sirolimus promoted CD4+ CD25+ Treg cell proliferation and growth in vitro, whereas cyclosporin A inhibited proliferation. Sirolimus might promote CD4+ CD25+ FoxP3+ regulatory T cell proliferation by inducing TGF-ß secretion in vivo.
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Inmunosupresores/farmacología , Sirolimus/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Biomarcadores , Proliferación Celular/efectos de los fármacos , Ciclosporina/farmacología , Factores de Transcripción Forkhead/metabolismo , Masculino , Ratones , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunologíaRESUMEN
Seizures have been shown to upregulate the expression of numerous extracellular matrix molecules. Tenascin C (TNC) is an extracellular matrix protein involved in several physiological roles and in pathological conditions. Though TNC upregulation has been described after excitotoxins injection, to date there is no research work on the signal transduction pathway(s) participating in TNC protein overproduction. The aim of this study was to evaluate the role of TGF-ß signaling pathway on TNC upregulation. In this study, we used male rats, which were injected with saline or pilocarpine to induce status epilepticus (SE) and killed 24h, 3 and 7 days after pilocarpine administration. For evaluating biochemical changes, we measured protein content of TNC, TGF-ß1 and phospho-Smad2/3 for localization of TNC in coronal brain hippocampus at 24h, 3 and 7 days after pilocarpine-caused SE. We found a significant increase of TNC protein content in hippocampal homogenates after 1, 3, and 7 days of pilocarpine-caused SE, together with an enhancement of TNC immunoreactivity in several hippocampal layers and the dentate gyrus field where more dramatic changes occurred. We also observed a significant enhancement of protein content of both the TGF-ß1 and the critical downstream transduction effector phospho-Smad2/3 throughout the chronic exposure. Interestingly, animals injected with SB-431542, a TGF-ß-type I receptor inhibitor, decreased TNC content in cytosolic fraction and diminished phospho-Smad2/3 content in both cytoplasmic and nuclear fraction compared with pilocarpine vehicle-injected. These findings suggest the participation of TGF-ß signaling pathway on upregulation of TNC which in turn support the idea that misregulation of this signaling pathway produces changes that may contribute to disease.