RESUMEN
BACKGROUND: Propylthiouracil (PTU) and methimazole (MMI) are drugs that are widely used to treat Graves' disease. Although both exert an antithyroid effect primarily by blocking thyroid peroxidase activity, their molecular structure and other actions are different. We hypothesized that PTU and MMI may have differential effects on thyroid-specific gene expression and function. METHODS: The effects of PTU and MMI on thyroid-specific gene expression and function were examined in rat thyroid FRTL-5 cells using DNA microarray, reverse transcriptase (RT)-polymerase chain reaction (PCR), real-time PCR, Western blot, immunohistochemistry, and radioiodine uptake studies. RESULTS: DNA microarray analysis showed a marked increase in sodium/iodide symporter (NIS) gene expression after PTU treatment, whereas MMI had no effect. RT-PCR and real-time PCR analysis revealed that PTU-induced NIS mRNA levels were comparable to those elicited by thyroid-stimulating hormone (TSH). PTU increased 5'-1880-bp and 5'-1052-bp activity of the rat NIS promoter. While PTU treatment also increased NIS protein levels, the size of the induced protein was smaller than that induced by TSH, and the protein localized predominantly in the cytoplasm rather than the plasma membrane. Accumulation of (125)I in FRTL-5 cells was increased by PTU stimulation, but this effect was weaker than that produced by TSH. CONCLUSIONS: We found that PTU induces NIS expression and iodide uptake in rat thyroid FRTL-5 cells in the absence of TSH. Although PTU and MMI share similar antithyroid activity, their effects on other thyroid functions appear to be quite different, which could affect their therapeutic effectiveness.
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Propiltiouracilo/farmacología , Simportadores/biosíntesis , Glándula Tiroides/metabolismo , Tirotropina/farmacología , Animales , Enfermedad de Graves/metabolismo , Yoduros/metabolismo , Metimazol/farmacología , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Simportadores/genética , Glándula Tiroides/efectos de los fármacosRESUMEN
CONTEXT: A functional thyroid-stimulating autoantibodies (TSAb) assay using a thyroid-stimulating hormone receptor chimera (Mc4) appears to be clinically more useful than the commonly used assay, a binding assay that measures all the antibodies binding to the thyroid-stimulating hormone receptor without functional discrimination, in diagnosing patient with Graves' disease (GD). OBJECTIVE: The objective of the study was to investigate whether an Mc4 assay can predict relapse/remission of hyperthyroidism after antithyroid drug (ATD) treatment in patients with GD. DESIGN: An Mc4 assay was used to prospectively track TSAb activity in GD patients treated with ATD over a 5-yr period. SETTING AND PATIENTS: GD patients from the Chieti University participated in this study. INTERVENTIONS: Interventions included the assessment of patients' sera using the Mc4 assay, the Mc4-derivative assay (Thyretain), and a human monoclonal thyroid-stimulating hormone receptor antibody, M22 assay. MAIN OUTCOME MEASURES: The Mc4 assay, a sensitive index of remission and recurrence, was used in this study. RESULTS: The TSAb levels significantly decreased only in the remitting group as evidenced by Mc4 assay values at the end of ATD (0.96 ± 1.47, 10.9 ± 26.6. and 24.7 ± 37.5 arbitrary units for the remitting, relapsing, and unsuspended therapy groups, respectively). Additional prognostic help was obtained by thyroid volume measurements at the end of treatment. Although not statistically significant, the Mc4 assay has a trend toward improved positive predictive value (95.4 vs. 84.2 or 87.5%), specificity (96.4 vs. 86.4 and 90.9%), and accuracy (87.3 vs. 83.3 and 80.9%) comparing the Mc4, Thyretain, and M22 assays, respectively. Thyretain has a trend toward improved negative predictive value (82.6 vs. 81.8 and 76.9%) and sensitivity (80 vs. 77.8 and 70%) comparing Thyretain, Mc4, and M22 assays, respectively. CONCLUSION: The Mc4 assay is a clinically useful index of remission and relapse in patients with GD. Larger studies are required to confirm these findings.
Asunto(s)
Enfermedad de Graves/diagnóstico , Inmunoglobulinas Estimulantes de la Tiroides/análisis , Receptores de HL/análisis , Receptores de Tirotropina/análisis , Proteínas Recombinantes de Fusión , Adulto , Animales , Antitiroideos/uso terapéutico , Autoanticuerpos/análisis , Autoanticuerpos/sangre , Células CHO , Ensayos Clínicos como Asunto/métodos , Cricetinae , Cricetulus , Femenino , Estudios de Seguimiento , Enfermedad de Graves/sangre , Enfermedad de Graves/tratamiento farmacológico , Enfermedad de Graves/inmunología , Células HEK293 , Humanos , Inmunoglobulinas Estimulantes de la Tiroides/sangre , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Receptores de HL/química , Receptores de HL/fisiología , Receptores de Tirotropina/química , Receptores de Tirotropina/fisiología , Proteínas Recombinantes de Fusión/farmacología , Recurrencia , Inducción de Remisión , Pruebas de Función de la Tiroides/métodos , Adulto JovenRESUMEN
CONTEXT: Stimulating thyrotropin receptor (TSHr) autoantibodies (TSAb) are the cause of hyperthyroidism in Graves' disease. In a patient's serum, TSAb can coexist with antagonist TSHr autoantibodies that block TSAb stimulatory activity (TSBAb); both can vary in amount and time. OBJECTIVE: The objective of the study was to create a functional assay that detects only TSAb, thus having an increased accuracy for diagnosing Graves' disease. DESIGN: A TSHr chimera (Mc4) that retains an agonist-sensitive TSAb epitope but replaces a TSBAb epitope was stably transfected in cells to establish the Mc4 assay. SETTING: The study was conducted at the Chieti University (Outpatient Endocrine Clinic) and the University of Pisa (the Department of Endocrinology). PATIENTS: The assay was validated using sera from 170 individuals with Graves' disease, Hashimoto's thyroiditis, and nonautoimmune hyperthyroidism and normal subjects from Chieti University. A second blinded study evaluated sera from 175 patients with autoimmune thyroid disease (mainly Graves' disease) from the University of Pisa. INTERVENTIONS: Interventions included the assessment of patients' sera using human wild-type TSHr (WT-TSHr), Mc4 chimera, and binding (TRAb) assays. MAIN OUTCOME MEASURES: The Mc4 assay has the best accuracy for diagnosing Graves' disease. RESULTS: The Mc4 assay has a better diagnostic accuracy than WT-TSHr and second-generation TRAb assays. Indeed, the sensitivity of the WT-TSHr, TRAb, and Mc4 assays was 97.3, 86.5, and 100%, respectively, whereas the specificity was 93.1, 97, and 98.5%, respectively. CONCLUSION: The Mc4 assay is a functional assay with improved sensitivity and specificity for the detection of TSAb and is clinically useful in diagnosing Graves' disease.
Asunto(s)
Enfermedad de Graves/diagnóstico , Inmunoglobulinas Estimulantes de la Tiroides/análisis , Receptores de HL/análisis , Receptores de Tirotropina/análisis , Proteínas Recombinantes de Fusión , Adulto , Anciano , Animales , Autoanticuerpos/análisis , Autoanticuerpos/sangre , Células CHO , Estudios de Casos y Controles , Células Cultivadas , Cricetinae , Femenino , Enfermedad de Graves/sangre , Enfermedad de Graves/inmunología , Células HEK293 , Humanos , Inmunoglobulinas Estimulantes de la Tiroides/sangre , Masculino , Persona de Mediana Edad , Visón , Receptores de HL/química , Receptores de HL/fisiología , Receptores de Tirotropina/química , Receptores de Tirotropina/fisiología , Proteínas Recombinantes de Fusión/análisis , Pruebas de Función de la Tiroides/métodosRESUMEN
OBJECTIVE: Wnt5a is a secreted glycoprotein highly present in atherosclerotic lesions. Uptake of oxidized-low density lipoprotein (ox-LDL) by monocytes/macrophages plays a critical role in atherosclerosis. The objective of this study was to determine if Wnt5a mRNA expression correlates with the severity of atherosclerotic lesions, and if, ox-LDL can induce Wnt5a mRNA in macrophages. METHODS: Wnt5a mRNA in tissue sections from carotid arteries of patients undergoing endarterectomy was quantified via RT-PCR and correlated with plaque severity. Human monocyte-derived macrophages and differentiated THP-1 cells, a human monocytic cell line, were treated with ox-LDL or native-LDL. Subsequently, Wnt5a transcripts were quantified by RT-PCR. RESULTS: Regions of the arteries with more severe plaques had detectable and significant levels of Wnt5a mRNA, while regions of the arteries containing less vulnerable plaques had low or non-detectable Wnt5a. Ox-LDL, but not native-LDL, induced Wnt5a mRNA in both human monocyte-derived macrophages and differentiated THP-1 cells. CONCLUSION: Our results demonstrate that the expression of Wnt5a correlates with the severity of atherosclerotic lesions, and that ox-LDL induces Wnt5a mRNA expression in human macrophages. These findings are consistent with the hypothesis that Wnt5a plays a critical role in atherosclerosis progression and that a source of Wnt5a is ox-LDL stimulated macrophages.
RESUMEN
BACKGROUND: One form of sepsis, or endotoxic shock, is a hyperactivated systemic response caused by excessive expression of proinflammatory mediators, which results from Gram-negative bacterial lipopolysaccharide-stimulated Toll-like receptor-4 signaling. This lipopolysaccharide signaling is known to consist of a MyD88-dependent nuclear factor-κB-mediated pathway that results in production of proinflammatory mediators (tumor necrosis factor-α, interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, inducible nitric oxide synthase, cyclooxygenase-2) and a MyD88-independent interferon regulatory factor-mediated pathway that regulates production of Type 1 interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1). In prior studies, phenylmethimazole markedly decreased virally induced Toll-like receptor-3 expression and signaling and significantly suppressed murine colitis in an experimental model wherein lipopolysaccharide is known to play an important role. OBJECTIVE: In this study, we probed the hypothesis that phenylmethimazole inhibits lipopolysaccharide-mediated Toll-like receptor-4 signaling and is efficacious in attenuating inflammatory changes and improving survival in an in vivo murine model of endotoxic shock. DESIGN: Experimental animal model. SETTING: University laboratory. SUBJECTS: Male C57BL/6J mice weighing 18-22 g. INTERVENTIONS: Phenylmethimazole (1 mg/kg) was administered intraperitoneally to mice before a lethal lipopolysaccharide challenge (25 mg/kg). RAW264.7 mouse macrophage cells were pretreated with phenylmethimazole followed by lipopolysaccharide stimulation. MEASUREMENTS AND MAIN RESULTS: : Macroscopic observations revealed that phenylmethimazole was significantly protective in controlling clinical manifestations of endotoxic shock and death under conditions wherein flunixin of meglumine and prednisolone were marginally effective. A combination of enzyme-linked immunosorbent assay, Northern blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blot analyses showed that phenylmethimazole attenuated lipopolysaccharide-induced increases in production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-6, interferon-γ), endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), inducible nitric oxide synthase and cyclooxygenase-2, interferon regulatory factor-1, interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1), and signal transducer and activator of transcription-1 phosphorylation in multiple tissues in mice. Consistent with these observations, electrophoretic mobility shift assay demonstrated that phenylmethimazole inhibited in vitro lipopolysaccharide-induced nuclear factor-κB and interferon regulatory factor-1 activation in RAW 264.7 mouse macrophages. CONCLUSIONS: Collectively, these results provide direct evidence that phenylmethimazole diminishes lipopolysaccharide-induced MyD88-dependent as well as MyD88-independent signaling pathways and is protective in an experimental model of endotoxic shock.
Asunto(s)
Citocinas/biosíntesis , Citocinas/efectos de los fármacos , Metimazol/análogos & derivados , Choque Séptico/inmunología , Choque Séptico/prevención & control , Tionas/uso terapéutico , Animales , Modelos Animales de Enfermedad , Inflamación/inmunología , Masculino , Metimazol/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Choque Séptico/metabolismoRESUMEN
Visceral adipocytes and associated macrophages produce and release excessive amounts of biologically active inflammatory cytokines via the portal and systemic vascular system, which induce insulin resistance in insulin target tissues such as fat, liver, and muscle. Free fatty acids (FFAs) absorbed via the portal system or released from adipocytes also induce insulin resistance. In this report, we show that phenylmethimazole (C10) blocks basal IL6 and leptin production as well as basal Socs-3 expression in fully differentiated 3T3L1 cells (3T3L1 adipocytes) without affecting insulin-stimulated AKT signaling. In addition, C10 inhibits palmitate-induced IL6 and iNos up-regulation in both 3T3L1 adipocytes and RAW 264.7 macrophages, LPS-induced NF-κB and IFN-ß activation in 3T3L1 cells, and LPS-induced iNos, Ifn-ß, Il1ß, Cxcl10, and Il6 expression in RAW 264.7 macrophages. C10 also blocks palmitate-induced Socs-3 up-regulation and insulin receptor substrate-1 (IRS-1) serine 307 phosphorylation in 3T3L1 adipocytes. Additionally, we show for the first time that although palmitate increases IRS-1 serine 307 phosphorylation in 3T3L1 adipocytes, AKT serine 473 phosphorylation is enhanced, not reduced, by palmitate. These results suggest that through inhibition of FFA-mediated signaling in adipocytes and associated macrophages, as well as possibly other insulin target cells/tissues (i.e. non-immune cells), C10 might be efficacious to prevent or reverse cytokine-induced insulin resistance seen in obesity-related insulin resistance and type 2 diabetes mellitus.
Asunto(s)
Adipocitos/efectos de los fármacos , Mediadores de Inflamación/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Metimazol/análogos & derivados , Palmitatos/metabolismo , Tionas/farmacología , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Quimiocina CXCL10/antagonistas & inhibidores , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/análisis , Resistencia a la Insulina , Interferón beta/antagonistas & inhibidores , Interleucina-1beta/antagonistas & inhibidores , Interleucina-6/antagonistas & inhibidores , Leptina/antagonistas & inhibidores , Lipopolisacáridos/antagonistas & inhibidores , Macrófagos/metabolismo , Metimazol/farmacología , Ratones , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Palmitatos/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/análisis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/antagonistas & inhibidores , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Ulcerative colitis is an autoimmune-inflammatory disease characterized by abnormally increased expression of Toll-like receptor-4 (TLR4) in colonic epithelial cells, increased production of pro-inflammatory cytokines (e.g., TNF-alpha, IL-1beta, IL-6, IL-12), chemokines (e.g., IP-10), and endothelial cell adhesion molecules (e.g., VCAM-1), plus enhanced leukocyte infiltration into colonic interstitium. Previously, we have shown that phenyl methimazole (C10) markedly decreases virally-induced TLR-3 expression and signaling and potently inhibits both TNF-alpha-induced VCAM-1 expression and the resultant leukocyte-endothelial cell adhesion. In this study we probed the hypothesis that C10 is efficacious in a TLR-4- and VCAM-1-associated murine model [the dextran sulfate sodium (DSS) model] of human colitis. C10 was administered intraperitoneally coincident with or after DSS treatment was initiated. Macroscopic colon observations revealed that C10 significantly reversed DSS-induced shortening of the colon (P<0.05) and reduced the presence of blood in the colon. Histological analyses of colonic tissues revealed that C10 distinctly attenuated both DSS-induced edema as well as leukocyte infiltration in the colonic mucosa and resulted in pronounced protection against DSS-induced crypt damage (P<0.001). Northern blot analyses and immunohistochemistry of colonic tissue revealed that C10 markedly diminished DSS-induced expression of pertinent inflammatory mediators: TNF-alpha, IL-1beta, IL-6, IL-12, IP-10, TLR-4 and VCAM-1. Most importantly, C10 significantly improved survival and protected mice against DSS-induced colitic-death: 75% by comparison to 12.5% with identical treatment with DMSO-control (log rank test: P=0.005). These results provide direct evidence that C10 suppresses DSS-induced colitis by inhibiting expression of key inflammatory mediators and leukocyte infiltration, and is a potentially attractive therapeutic for colitis.
Asunto(s)
Colitis Ulcerosa/prevención & control , Metimazol/análogos & derivados , Tionas/uso terapéutico , Receptor Toll-Like 4/antagonistas & inhibidores , Molécula 1 de Adhesión Celular Vascular/inmunología , Animales , Northern Blotting , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Citocinas/biosíntesis , Citocinas/inmunología , Sulfato de Dextran , Modelos Animales de Enfermedad , Inmunohistoquímica , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Masculino , Metimazol/farmacología , Metimazol/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Tionas/farmacología , Receptor Toll-Like 4/biosíntesis , Molécula 1 de Adhesión Celular Vascular/biosíntesisRESUMEN
Although it is well known that an excess of iodide suppresses thyroid function and blood flow in vivo, the underlying molecular mechanisms are not fully known. The functional effect of iodide occurs at multiple steps, which include inhibition of sodium/iodide symporter (NIS) expression, transient block of organification, and inhibition of hormonal release. The vascular effect likely involves suppression of the vascular endothelial growth factor (VEGF) gene. In this report, we show that excess iodide coordinately suppresses the expression of the NIS and VEGF genes in FRTL-5 thyroid cells. We also demonstrate that the mechanism of iodide suppression of NIS gene expression is transcriptional, which is synergized by the addition of thyroglobulin. Based on the findings of reporter gene assays and electrophoretic gel mobility shift analysis, we also report two novel DNA binding proteins that responded specifically to iodide and modulated NIS promoter activity. The results suggest that excess iodide affects thyroid vascular function in addition to iodide uptake. This study provides additional insights into the mechanism of action of excess iodide on thyroid function.
Asunto(s)
Yoduros/farmacología , Simportadores/genética , Glándula Tiroides/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Yoduros/metabolismo , Ratas , Simportadores/antagonistas & inhibidores , Tiroglobulina/metabolismo , Tiroglobulina/farmacología , Glándula Tiroides/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Thyroglobulin (Tg), a major product of the thyroid gland, serves as a macromolecular precursor of thyroid hormone biosynthesis. In addition, Tg stored in the thyroid follicles is a potent regulator of thyroid-specific gene expression. In conjunction with thyroid stimulating hormone (TSH) and iodide, Tg regulates thyroid follicle function, which is the minimal functional unit of the thyroid gland. In the present study, we show that Tg stimulates growth of FRTL-5 thyroid cells in the absence of TSH, insulin and serum. Unlike TSH, Tg did not increase cellular cyclic AMP (cAMP) levels; rather, the TSH signal counteracted Tg-induced cell growth. A specific inhibitor of A-kinase, H-89, did not modulate the effect of Tg. Tg increased kinase activity of Akt to the same level as TSH, insulin and 5% serum, while LY294002 abolished Tg-induced growth. Interestingly, low Tg concentrations maximized growth-promotion activity and induction of the apical iodide transporter (PDS; SLC26A4), whereas high Tg concentrations suppressed both cell growth and the expression of thyroid-specific genes. These results suggest that a low levels of Tg in the follicular lumen might stimulates cell growth and iodide transport to accelerate the iodide organification process; however, elevated Tg levels in the follicle might then shut down all of these functions.
Asunto(s)
Proliferación Celular , AMP Cíclico/metabolismo , Tiroglobulina/metabolismo , Glándula Tiroides/fisiología , Tirotropina/metabolismo , Animales , Línea Celular , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Expresión Génica/efectos de los fármacos , Ratas , Timidina/metabolismo , Tiroglobulina/farmacología , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Tirotropina/farmacologíaRESUMEN
Wnt5a is a noncanonical member of the Wnt family of signaling molecules that has been linked to various physiological and pathological processes including cell differentiation, cell migration, cell growth, vascular remodeling, cancer and chronic inflammation. To understand the role of Wnt5a in these processes, it is necessary to determine the function and expression level of Wnt5a. In this study we developed a sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA) for detecting Wnt5a. We found that a rabbit anti-human Wnt5a is a suitable capture antibody for establishing a sandwich ELISA. We used two systems to detect Wnt5a: (1) goat anti-mouse Wnt5a and horseradish peroxidase (HRP) conjugated F(ab')(2) donkey anti-goat IgG as detection and enzyme-linked antibodies respectively, or (2) biotinylated goat anti-mouse Wnt5a and HRP-streptavidin as detection antibody and enzyme-linked avidin respectively. A sandwich ELISA using either of these systems failed to detect recombinant mouse (rm)-Wnt5a diluted in Hank's balanced salt solution supplemented with Ca(2+) and Mg(2+) and 1% bovine serum albumin (HBBS+, 1% BSA). Addition of polyethylene glycol (PEG) to the HBBS+, buffer during the binding stage of rm-Wnt5a, afforded the detection of rm-Wnt5a. The use of PEG during both the binding of rm-Wnt5a and detection antibody stages of the assay yielded the maximum signal for rm-Wnt5a. The relationship between the ELISA signal and concentration of Wnt5a was linear with an R(2) of 0.9934. In summary, we have developed a specific and sensitive sandwich ELISA that detects rm-Wnt5a.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas Wnt/metabolismo , Animales , Reacciones Antígeno-Anticuerpo/inmunología , Equidae , Cabras , Humanos , Ratones , Unión Proteica , Conejos , Sensibilidad y Especificidad , Proteína Wnt-5aRESUMEN
Increased expression of major histocompatibility complex (MHC) class-I genes and aberrant expression of MHC class-II genes in thyroid epithelial cells (TECs) are associated with autoimmune thyroid diseases. Previous studies have shown that methimazole (MMI) reduces MHC class-I expression and inhibits interferon-gamma (IFN-gamma or IFNG as listed in the MGI Database)-induced expression of the MHC class-II genes in TECs. The action of MMI on the MHC class-I genes is transcriptional, but its mechanism has not been investigated previously. In the present study, we show that in Fisher rat thyroid cell line 5 cells, the ability of MMI and its novel derivative phenylmethimazole (C10) to decrease MHC class-I promoter activity is similar to TSH/cAMP suppression of MHC class-I and TSH receptor genes, and involves a 39 bp silencer containing a cAMP response element (CRE)-like site. Furthermore, we show that C10 decreases MHC class-I gene expression to a greater extent than MMI and at 10- to 50-fold lower concentrations. C10 also reduces the IFN-gamma-induced increase in the expression of MHC class-I and MHC class-II genes more effectively than MMI. Finally, we show that in comparison to MMI, C10 is a better inhibitor of specific protein-DNA complexes that are formed with a CRE-like element on the MHC class-II promoter. These data support the conclusion that the immunosuppressive mechanism by which MMI and C10 inhibit MHC gene expression mimics 'normal' hormonal suppression by TSH/cAMP.
Asunto(s)
Antitiroideos/farmacología , Células Epiteliales/metabolismo , Expresión Génica/efectos de los fármacos , Genes MHC Clase II , Genes MHC Clase I , Metimazol/análogos & derivados , Metimazol/farmacología , Tionas/farmacología , Glándula Tiroides/metabolismo , Animales , Antitiroideos/administración & dosificación , Línea Celular , AMP Cíclico/metabolismo , ADN , Silenciador del Gen , Interferón gamma/farmacología , Metimazol/administración & dosificación , Regiones Promotoras Genéticas/efectos de los fármacos , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes , Elementos de Respuesta , Tionas/administración & dosificación , Sitio de Iniciación de la TranscripciónRESUMEN
PURPOSE: To evaluate whether (a) Wnt5a expression in pancreatic cancer and malignant melanoma cells might be associated with constitutive levels of Toll-like receptor 3 (TLR3) and/or TLR3 signaling; (b) phenylmethimazole (C10), a novel TLR signaling inhibitor, could decrease constitutive Wnt5a and TLR3 levels together with cell growth and migration; and (c) the efficacy of C10 as a potential inhibitor of pancreatic cancer and malignant melanoma cell growth in vivo. EXPERIMENTAL DESIGN: We used a variety of molecular biology techniques including but not limited to PCR, Western blotting, and ELISA to evaluate the presence of constitutively activated TLR3/Wnt5a expression and signaling. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based technology and scratch assays were used to evaluate inhibition of cell growth and migration, respectively. TLR3 regulation of cell growth was confirmed using small interfering RNA technology. Nude and severe combined immunodeficient mice were implanted with human pancreatic cancer and/or melanoma cells and the effects of C10 on tumor growth were evaluated. RESULTS: We show that constitutive TLR3 expression is associated with constitutive Wnt5a in human pancreatic cancer and malignant melanoma cell lines, that C10 can decrease constitutive TLR3/Wnt5a expression and signaling, suggesting that they are interrelated signal systems, and that C10 inhibits growth and migration in both of these cancer cell lines. We also report that C10 is effective at inhibiting human pancreatic cancer and malignant melanoma tumor growth in vivo in nude or severe combined immunodeficient mice and associate this with inhibition of signal transducers and activators of transcription 3 activation. CONCLUSIONS: C10 may have potential therapeutic applicability in pancreatic cancer and malignant melanoma.
Asunto(s)
Antitiroideos/farmacología , Melanoma/metabolismo , Metimazol/análogos & derivados , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Neoplasias Cutáneas/metabolismo , Tionas/farmacología , Receptor Toll-Like 3/metabolismo , Proteínas Wnt/metabolismo , Animales , Línea Celular Tumoral , Quimiocina CXCL10/antagonistas & inhibidores , Quimiocina CXCL10/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Interferón beta/antagonistas & inhibidores , Interferón beta/metabolismo , Interleucina-6/antagonistas & inhibidores , Interleucina-6/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/patología , Metimazol/farmacología , Ratones , Ratones Desnudos , Ratones SCID , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , ARN Interferente Pequeño/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Receptor Toll-Like 3/antagonistas & inhibidores , Receptor Toll-Like 3/genética , Proteínas Wnt/antagonistas & inhibidores , Proteína Wnt-5aRESUMEN
Atherosclerosis is an inflammatory disease involving the accumulation of macrophages in the intima. Wnt5a is a noncanonical member of the Wnt family of secreted glycoproteins. Recently, human macrophages have been shown to express Wnt5a upon stimulation with bacterial pathogens in vitro and in granulomatous lesions in the lung of Mycobacterium tuberculosis-infected patients. Wnt5a expression has also been liked to Toll-like receptor-4 (TLR-4), an innate immune receptor implicated in atherosclerosis. These observations, along with the fact that Wnt5a is involved in cell migration and proliferation, led us to postulate that Wnt5a plays a role in atherosclerosis. To investigate this hypothesis, we characterized Wnt5a expression in murine and human atherosclerotic lesions. Tissue sections derived from the aortic sinus to the aortic arch of apolipoprotein E-deficient mice and sections derived from the carotid arteries of patients undergoing endarterectomy were subjected to immunohistochemical analysis. All samples were found to be positive for Wnt5a with predominant staining in the areas of macrophage accumulation within the intima. In parallel, we probed for the presence of TLR-4 and found coincident TLR-4 and Wnt5a expression. For both the Wnt5a and TLR-4 staining, consecutive tissue sections treated with an isotype- and species-matched Ig served as a negative control and exhibited little, if any, reactivity. Quantitative RT-PCR revealed that Wnt5a mRNA expression in RAW264.7 murine macrophages can be induced by stimulation with LPS, a known ligand for TLR-4. Combined, these findings demonstrate for the first time Wnt5a expression in human and murine atherosclerotic lesions and suggest that cross talk between TLR-4 and Wnt5a is operative in atherosclerosis.
Asunto(s)
Enfermedades de la Aorta/metabolismo , Aterosclerosis/metabolismo , Enfermedades de las Arterias Carótidas/metabolismo , Macrófagos/química , Proteínas Proto-Oncogénicas/análisis , Proteínas Wnt/análisis , Animales , Enfermedades de la Aorta/patología , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerosis/patología , Enfermedades de las Arterias Carótidas/patología , Línea Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunohistoquímica , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/metabolismo , Receptor Toll-Like 4/análisis , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt-5aRESUMEN
The increased expression of VCAM-1 on endothelial segments within plaque regions could be used as a target to deliver polymeric drug carriers selectively to sites of atherosclerosis. We probed the hypothesis that polymeric particles conjugated with a ligand for VCAM-1 exhibit selective and avid adhesion to sites of atherosclerosis. Particles made from polystyrene or the biodegradable polymer poly(sebacic acid)-block-polyethylene glycol (PSA-PEG) were conjugated with an antibody to VCAM-1 (alpha-VCAM-1) or IgG (negative control). The particles were injected into the jugular vein of ApoE(-/-) (a murine model of atherosclerosis) or wild type mice and their adhesion to the aorta determined. alpha-VCAM-1 particles exhibited significantly greater adhesion to ApoE(-/-) mouse aorta [32 +/- 5 (mean +/- SEM) particles/mm(2) for polystyrene particles and 31 +/- 7 particles/mm(2) for PSA-PEG particles] compared to the level of adhesion to wild type mouse aorta (18 +/- 1 particles/mm(2) for polystyrene particles and 6 +/- 1 particles/mm(2) for PSA-PEG particles). Within ApoE(-/-) mice, the alpha-VCAM-1 particles exhibited significantly greater adhesion to the aorta (32 +/- 5 particles/mm(2) for polystyrene particles and 31 +/- 7 particles/mm(2) for PSA-PEG particles) compared to the adhesion of IgG particles (1 +/- 1 particles/mm(2) for polystyrene particles and 2 +/- 1 particles/mm(2) for PSA-PEG particles). Detailed analysis of the adhesion revealed that alpha-VCAM-1 particles exhibited focal adhesion to plaque regions, in particular the periphery of the plaques, within the ApoE(-/-) mouse aorta. Combined the data demonstrate that polymeric particles conjugated with a ligand to VCAM-1 exhibit selective, avid and focal adhesion to sites of atherosclerosis providing strong evidence that VCAM-1 ligand bearing polymeric particles could be used for targeting drugs selectively to atherosclerotic tissue.
Asunto(s)
Anhídridos/metabolismo , Aterosclerosis/metabolismo , Adhesiones Focales/metabolismo , Polietilenglicoles/metabolismo , Poliestirenos/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Análisis de Varianza , Animales , Anticuerpos Monoclonales/metabolismo , Aorta/metabolismo , Portadores de Fármacos/metabolismo , Ligandos , Ratones , Especificidad por SustratoRESUMEN
We previously showed that immunization of mice with murine fibroblasts transfected with the thyrotropin receptor (TSHR) and a murine major histocompatibility complex (MHC) class II molecule induces immune thyroid disease with the humoral and histological features of human Graves' disease in about 20% of mice. In this model, based on the proliferative response of T cells from hyperthyroid mice to a panel of overlapping TSHR peptides, we now demonstrate that TSHR 121-140 peptide contains an immunodominant T cell epitope. Supporting this conclusion, spleen cells from mice immunized with TSHR 121-140 peptide showed a strong proliferative response to fibroblasts transfected with the TSHR and a murine I-A(k) molecule, but not either alone. Also, intranasal administration of 100 mug of TSHR 121-140 peptide led to suppressed proliferative response of lymph node cells to the peptide. Interestingly, however, administration of this peptide enhanced, rather than suppressed, the frequency and severity of Graves' disease induced by the immunization of the fibroblasts transfected with the TSHR and a murine I-A(k) molecule. Spleen cells from hyperthyroid mice that were pretreated with intranasal peptide tended to produce lesser amounts of IL-4, IL-10 and IFN-gamma than those from normothyroid control mice. Although precise mechanisms of this enhancement remain to be determined, the results suggest that attempts to treat Graves' disease by intranasal administration of an immunodominant TSHR T cell epitope may aggravate, not prevent, the disease.
Asunto(s)
Epítopos de Linfocito T/inmunología , Enfermedad de Graves/inmunología , Enfermedad de Graves/prevención & control , Inmunoterapia/métodos , Receptores de Tirotropina/inmunología , Administración Intranasal , Animales , Proliferación Celular , Citocinas/metabolismo , Epítopos de Linfocito T/administración & dosificación , Femenino , Fibroblastos/inmunología , Antígenos de Histocompatibilidad Clase II/administración & dosificación , Antígenos de Histocompatibilidad Clase II/inmunología , Ratones , Péptidos/administración & dosificación , Péptidos/inmunología , Receptores de Tirotropina/administración & dosificación , Linfocitos T/inmunología , TransfecciónRESUMEN
Quercetin is the most consumed flavonoid present in fruits and vegetables. There has been increased interest in the possible health benefits of quercetin and other flavonoids. Because it is reported that these compounds have some antithyroid properties, we were interested whether, and by what mechanism, quercetin might regulate thyroid cell growth and function. In this report we show that quercetin inhibits thyroid cell growth in association with inhibition of insulin-modulated phosphatidylinositol 3-kinase-Akt kinase activity. Furthermore, quercetin decreases TSH-modulated RNA levels of the thyroid-restricted gene sodium/iodide symporter (NIS). We associated down-regulation of NIS RNA levels with inhibition of iodide uptake at comparable quercetin concentrations and could show that the inhibitory effect of quercetin on NIS RNA levels and iodide uptake is reproduced by inhibitors of the phospholipase-A(2)/lipoxygenase pathway. The specific inhibitor of protein kinase A, H89, only partially inhibited TSH-increased NIS expression and did not reproduce the quercetin effect. The quercetin studies thus reveal that the phospholipase-A(2)/lipoxygenase pathway appears to play an important role in TSH regulation of NIS gene expression, whereas quercetin inhibition of growth appears to involve an effect on insulin/IGF-I-Akt signaling. The data raise the possibility that quercetin may be a novel disruptor of thyroid function, which has potential effects on, or use in, the therapy of thyroid diseases.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Quercetina/farmacología , Glándula Tiroides/efectos de los fármacos , Animales , Antitiroideos/farmacología , Células Cultivadas , Evaluación Preclínica de Medicamentos , Disruptores Endocrinos/farmacología , Yodo/metabolismo , Fosfolipasas A2/fisiología , Ratas , Transducción de Señal/efectos de los fármacos , Simportadores/genética , Simportadores/metabolismo , Simportadores/fisiología , Glándula Tiroides/metabolismo , Glándula Tiroides/fisiología , Tirotropina/farmacologíaRESUMEN
TGF-beta-like activities of proteins unrelated to the cytokine could mimic its actions in fibrosis and cell proliferation. Thyroglobulin (Tg) has been identified as having a TGF-beta receptor (TGFbetaR)-binding activity and is deposited in the glomerulus in certain immune-complex diseases. The aim of the present study is to determine whether Tg can reproduce the transcriptional activity of TGF-beta1 in the mouse glomerular mesangial cell (MC), and to examine whether such activity is manifested through TGFbetaR. Real-time RT-PCR was employed to examine the effects of TGF-beta1 and bovine Tg on the expression of three genes (TGF-beta1, plasminogen activator inhibitor 1 [PAI-1], and Pax-8) regulated by TGF-beta1 in other cell types. In addition, a pentacosapeptide TGF-beta1 antagonist, beta(1)(25) (41-65) was employed to determine whether the transcriptional activity of Tg was mediated through the TGF-beta binding site on the TGFbetaR. A 6h exposure to TGF-beta1 resulted in increased TGF-beta1 and PAI-1 transcript, and a decrease in Pax-8. Similarly, a 6h exposure to Tg resulted in increases of about 5-fold in TGF-beta1 and PAI-1 mRNA and a decrease of 53% in Pax-8. In comparison with other proteins, Tg had the greatest positive effect on TGF-beta1 transcript levels. beta(1)(25) (41-65) significantly reduced the TGF-beta1-, but not the Tg-induced changes in TGF-beta1, PAI-1 and Pax-8 transcript levels. We conclude from these studies that Tg possesses a TGF-beta-mimetic transcriptional activity in the MC that is not mediated by its binding to TGFbetaR. These results suggest that Tg and other proteins could initiate glomerular injury by reproducing the actions of TGF-beta1 in the mesangial cell.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Células Mesangiales/efectos de los fármacos , Tiroglobulina/farmacología , Factor de Crecimiento Transformador beta/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Células Mesangiales/metabolismo , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box/genética , Fragmentos de Péptidos/farmacología , Serpina E2 , Serpinas/genética , Tiroglobulina/fisiología , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
High basal levels of TLR3 and Wnt5a RNA are present in papillary thyroid carcinoma (PTC) cell lines consistent with their overexpression and colocalization in PTC cells in vivo. This is not the case in thyrocytes from normal tissue and in follicular carcinoma (FC) or anaplastic carcinoma (AC) cells or tissues. The basally expressed TLR3 are functional in PTC cells as evidenced by the ability of double-strand RNA (polyinosine-polycytidylic acid) to significantly increase the activity of transfected NF-kappaB and IFN-beta luciferase reporter genes and the levels of two end products of TLR3 signaling, IFN-beta and CXCL10. Phenylmethimazole (C10), a drug that decreases TLR3 expression and signaling in FRTL-5 thyrocytes, decreases TLR3 levels and signaling in PTC cells in a concentration-dependent manner. C10 also decreased Wnt5a RNA levels coordinate with decreases in TLR3. E-cadherin RNA levels, whose suppression may be associated with high Wnt5a, increased with C10 treatment. C10 simultaneously decreased PTC proliferation and cell migration but had no effect on the growth and migration of FC, AC, or FRTL-5 cells. C10 decreases high basal phosphorylation of Tyr705 and Ser727 on Stat3 in PTC cells and inhibits IL-6-induced Stat3 phosphorylation. IL-6-induced Stat3 phosphorylation is important both in up-regulating Wnt5a levels and in cell growth. In sum, high Wnt5a levels in PTC cells may be related to high TLR3 levels and signaling; and the ability of phenylmethimazole (C10) to decrease growth and migration of PTC cells may be related to its suppressive effect on TLR3 and Wnt5a signaling, particularly Stat3 activation.
Asunto(s)
Carcinoma Papilar/genética , Metimazol/análogos & derivados , Metimazol/farmacología , Proteínas Proto-Oncogénicas/fisiología , Neoplasias de la Tiroides/genética , Receptor Toll-Like 3/fisiología , Proteínas Wnt/fisiología , Carcinoma Papilar/patología , División Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Humanos , Proteínas Proto-Oncogénicas/genética , Neoplasias de la Tiroides/patología , Receptor Toll-Like 3/genética , Proteínas Wnt/genética , Proteína Wnt-5aRESUMEN
We previously reported that hormones important for the normal growth and function of FRTL-5 rat thyroid cells, TSH, or its cAMP signal plus insulin or IGF-I, could transcriptionally suppress constitutive and gamma-interferon (IFN)-increased synthesis of the 90K protein (also known as Mac-2BP). Here we cloned the 5'-flanking region of the rat 90K gene and identified a minimal promoter containing an interferon response element and a consensus E-box or upstream stimulator factor (USF) binding site, which are highly conserved in both the human and murine genes. We show that suppression of constitutive and gamma-IFN-increased 90K gene expression by TSH/cAMP plus insulin/IGF-I depends on the ability of the hormones to decrease the binding of USF to the E-box, located upstream of the interferon response element. This site is required for the constitutive expression of the 90K gene. Transfection with USF1 and USF2 cDNAs increases constitutive promoter activity, attenuates the ability of TSH/cAMP plus insulin/IGF-I to decrease constitutive or gamma-IFN-increased 90K gene expression but does not abrogate the ability of gamma-IFN itself to increase 90K gene expression.
Asunto(s)
Región de Flanqueo 5'/genética , Proteínas/genética , Factores Estimuladores hacia 5'/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Proteínas Portadoras , Línea Celular , AMP Cíclico/farmacología , Ensayo de Cambio de Movilidad Electroforética , Proteínas de la Matriz Extracelular , Regulación de la Expresión Génica/efectos de los fármacos , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Interferón gamma/farmacología , Luciferasas/genética , Luciferasas/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Unión Proteica , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Elementos de Respuesta/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Tirotropina/farmacología , Transfección , Factores Estimuladores hacia 5'/genéticaRESUMEN
Wnt binding to cell surface receptors can activate a 'canonical' pathway that increases cellular beta-catenin or a 'noncanonical' Ca(++) pathway which can increase protein kinase C (PKC) activity. Although components of both Wnt/beta-catenin-signaling pathways exist in thyrocytes, their biological role is largely unknown. In evaluating the biological role of Wnt signaling in differentiated FRTL-5 thyroid cells, we showed that TSH increased canonical Wnt-1 but, surprisingly, decreased the active form of beta-catenin. Transient overexpression of Wnt-1 or beta-catenin in FRTL-5 cells increased active beta-catenin (ABC), decreased thyroperoxidase (TPO) mRNA, and suppressed TPO-promoter activity. The target of beta-catenin suppressive action was a consensus T cell factor/lymphoid enhancing factor (TCF/LEF)-binding site 5'-A/T A/T CAAAG-3', -137 to -129 bp on the rat TPO promoter. beta-Catenin overexpression significantly increased complex formation between beta-catenin/TCF-1 and an oligonucleotide containing the TCF/LEF sequence, suggesting that the beta-catenin/TCF-1 complex acts as a transcriptional repressor of the TPO gene. Stable over-expression of Wnt-1 in FRTL-5 cells significantly increased the growth rate without increasing beta-catenin levels. Increased growth was blunted by a PKC inhibitor, staurosporin. Wnt-1 overexpression increased serine phosphorylation, without affecting tyrosine phosphorylation, of signal transducers and activators of transcription 3 (STAT3) protein. In addition, these final results suggest that TSH-induced increase in Wnt-1 levels in thyrocytes contributes to enhanced cellular growth via a PKC pathway that increases STAT3 serine phosphorylation and activation, whereas TSH-induced decrease in activation of beta-catenin simultaneously relieves transcriptional suppression of TPO. We hypothesize that Wnt signaling contributes to the ability of TSH to simultaneously increase cell growth and functional, thyroid-specific, gene expression.