RESUMO
The absence of TSH-stimulated radioiodide uptake in differentiated thyroid cancer is associated with a high recurrence rate and reduced survival. We studied regulation of the sodium/iodide symporter gene in human papillary thyroid cancer cell lines (BHP) and primary human thyroid cells. BHP cells expressed very low levels of sodium/iodide symporter mRNA and did not concentrate iodide, but iodide uptake was restored to levels seen in FRTL-5 rat thyroid cells by stable transfection of a sodium/iodide symporter cDNA. Sodium/iodide symporter gene expression, therefore, was necessary and sufficient for iodide uptake in BHP cells. We cloned the human sodium/iodide symporter gene 5'-flanking region and analyzed progressive 5'-deletions in transient transfections. We identified a region, -596 to -268, essential to confer full promoter activity in primary normal human thyroid cells. Sodium/iodide symporter promoter activity in four BHP cell lines, however, was markedly reduced, consistent with down-regulation of the endogenous sodium/iodide symporter gene. Nuclear extracts from BHP 2-7 cells had reduced or absent binding to regions of the sodium/iodide symporter promoter shown to be critical for expression, compared with nuclear extracts from FRTL-5 cells. Competition studies indicated that these nuclear proteins were not known thyroid transcription factors. Modifications of the sodium/iodide symporter promoter with demethylation or histone acetylation did not increase sodium/iodide symporter expression, and no deletions of the critical regulatory region were identified in the endogenous gene in BHP cells. Regulation of the sodium/iodide symporter 5'-flanking region in transient transfection paralleled endogenous sodium/iodide symporter expression. Reduced expression of potential novel nuclear factor(s) in these cell lines may contribute to reduced sodium/iodide symporter expression resulting in absence of iodide uptake in some papillary thyroid cancers.
Assuntos
Carcinoma Papilar/genética , Proteínas de Transporte/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/genética , Regiões Promotoras Genéticas/fisiologia , Simportadores , Glândula Tireoide/fisiologia , Neoplasias da Glândula Tireoide/genética , Sequência de Bases/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Genoma , Humanos , Iodetos/metabolismo , Iodetos/farmacocinética , Mutação , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados , Fragmentos de Peptídeos/genética , RNA Mensageiro/metabolismo , Valores de Referência , Glândula Tireoide/citologia , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Fator Nuclear 1 de Tireoide , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma) induce apoptosis and exert antiproliferative effects on several carcinoma cell lines. The present study investigates the expression of PPARgamma and the possibility that agonists for PPARgamma also inhibit the growth of human thyroid carcinoma cells. We examined this hypothesis using six cell lines, designated BHP thyroid carcinoma cells, which originated from patients with papillary thyroid carcinoma. RT-PCR analysis revealed that the thyroid carcinoma cell lines BHP2-7, 7-13, 10-3, and 18-21 express PPARgamma. More PPARgamma was expressed in carcinoma than in adjacent normal thyroid tissue in three of six samples of human papillary carcinoma of the thyroid. PPARgamma-positive thyroid carcinoma cells were treated with agonists of PPARgamma, troglitazone, BRL 49653, and 15-deoxy-12,14-prostaglandin J2. Troglitazone (10 micromol/L), BRL 49653 (10 micromol/L), and 15-deoxy-12,14-prostaglandin J2 (1 microg/mL) decreased [(3)H]thymidine incorporation and reduced cell number, respectively, in BHP carcinoma cell lines that expressed PPARgamma. Under low serum conditions, ligands for PPARgamma induced condensation of the nucleus and fragmentation of chromatin into nucleosome ladders. These findings indicate that the death of thyroid carcinoma cells is a form of apoptosis. To investigate the molecular mechanism of the apoptosis, we assessed expression of the apoptosis-regulatory genes bcl-2, bax, and c-myc. Troglitazone significantly increased the expression of c-myc messenger RNA but had no effect on the expression of bcl-2 and bax in thyroid carcinoma cells. These results suggest that, at least in part, the induction of apoptosis in human papillary thyroid carcinoma cells may be due to an increase of c-myc. Troglitazone (500 mg/kg.day) significantly inhibited tumor growth and prevented distant metastasis of BHP18-21 tumors in nude mice in vivo. Taken together, these results suggest that PPARgamma agonist inhibit cell growth of some types of human thyroid cancer.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Papilar/tratamento farmacológico , Cromanos/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Tiazóis/farmacologia , Tiazolidinedionas , Neoplasias da Glândula Tireoide/tratamento farmacológico , Fatores de Transcrição/agonistas , Animais , Northern Blotting , Carcinoma Papilar/patologia , Divisão Celular/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/análise , Receptores Citoplasmáticos e Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rosiglitazona , Timidina/metabolismo , Neoplasias da Glândula Tireoide/patologia , Fatores de Transcrição/genética , Troglitazona , Células Tumorais CultivadasRESUMO
Gene therapy with thyroglobulin (TG) promoter and a prodrug/suicide gene combination may prove useful as a treatment for thyroid carcinoma. However, most poorly differentiated and anaplastic thyroid carcinomas have lost the ability to express the TG gene expression accompanied by loss of transcription factors [thyroid transcription factor-1 (TTF-1), TTF-2, or Pax-8] interacting with the TG promoter. In anticipation of developing transcriptionally targeted gene therapy of TG-nonproducing thyroid carcinomas, we investigated the effect of TTF-1 gene transfer on TG promoter activity and the cytotoxic effect obtained by the TG promoter-driven HSV-TK gene along with ganciclovir in thyroid carcinoma and nonthyroidal cells. Using a chimeric construct containing the 5'-flanking region of the rat TG gene between -826 and +39 bp and the luciferase gene, TG promoter activity was detected in a normal rat thyroid cell line (FRTL-5), but not in a dedifferentiated line of thyroid cells (FRT) expressing Pax-8 but not TTF-1, TTF-2, or TG [TTF-1(-)/TTF-2(-)/Pax-8(+)/TG(-)], or in a human papillary thyroid carcinoma cell line [BHP15-3; TTF-1(-)/TTF-2(-)/Pax-8(-)/TG(-)], a human pulmonary cell line [H441; TTF-1(+)/TTF-2(-)/Pax-8(-)/TG(-)], or a dog kidney epithelial cell line [MDCK; TTF-1(-)/TTF-2(-)/Pax-8(+)/TG(-)]. Cotransfection of the TTF-1 expression vector stimulated TG promoter activity in FRT and BHP15-3 dedifferentiated thyroid cells, but not in H441 pulmonary cells. Only weak activation was observed in MDCK kidney cells. We then constructed recombinant adenovirus vectors, AdTTF-1 and ADTGTK: AdTTF-1 contained cytomegalovirus promoter and rat TTF-1 cDNA; AdTGTK carried the TG promoter-driven HSV-TK gene. Infection with AdTGTK and combined with GCV treatment induced a cytotoxic effect in FRTL-5 cells but not in dedifferentiated thyroid or nonthyroid cells. Cotransduction of AdTTF-1 and AdTGTK permitted 90% cytotoxicity for BHP15-3 and >95% cytotoxicity for FRT, as well as for BHP7-13 and BHP18-21v thyroid cancer cell lines [both/TTF1(-)/TTF-2(-)/Pax-8(+)/TG(-)]. In contrast, little cytotoxicity was seen for H441 and MDCK cell lines even with 300 microg/ml of ganciclovir. These results suggest that cotransduction of a TG promoter-controlled suicide gene and the TTF-1 gene by adenoviral vectors confers transcriptionally targeted gene-mediated cytotoxicity in poorly differentiated thyroid carcinoma cells unable to express the TG gene.
Assuntos
Carcinoma Papilar/genética , Terapia Genética/métodos , Proteínas Nucleares/genética , Tireoglobulina/genética , Neoplasias da Glândula Tireoide/genética , Fatores de Transcrição/genética , Ativação Transcricional , Adenoviridae/genética , Animais , Carcinoma Papilar/tratamento farmacológico , Carcinoma Papilar/terapia , Ganciclovir/farmacocinética , Ganciclovir/farmacologia , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos/genética , Células HeLa , Humanos , Regiões Promotoras Genéticas , Ratos , Simplexvirus/enzimologia , Simplexvirus/genética , Timidina Quinase/genética , Timidina Quinase/metabolismo , Tireoglobulina/biossíntese , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/terapia , Fator Nuclear 1 de Tireoide , Transdução Genética , Células Tumorais CultivadasRESUMO
The sodium iodide symporter (NIS) is a novel autoantigen in autoimmune thyroid disease (ATD). A recent study has described the development of a bioassay for human (h) NIS antibody detection, but this will not detect antibodies that bind the symporter without modulating its activity. Therefore, the establishment of a binding assay is of importance to determine the overall prevalence of hNIS antibodies in ATD patients. An in vitro transcription and translation system was used to produce [35S]-labeled hNIS. The radiolabeled ligand reacted specifically in immunoprecipitation experiments with rabbit antiserum raised against a peptide fragment of hNIS. Subsequently, the reactivity of control and ATD sera to translated [35S]hNIS was determined using RIAs. A significant difference in the frequency of hNIS antibody-positive sera was found when patients with either Graves' disease (GD) or autoimmune hypothyroidism (AH) were compared with normal controls (P = 0.01 and P = 0.03, respectively). Of 49 GD and 29 AH sera tested, 11 (22%) and 7 (24%), respectively, were found to contain hNIS antibodies. Differences were also significant when the antibody-binding indices of the control group of sera were compared with those of both the GD and the AH patient sera (P < 0.0001 and P = 0.001, respectively). In contrast, sera from 10 patients with Addison's disease and 10 patients with vitiligo (without associated ATD) were all negative for antibody reactivity to the symporter. No differences were detected when the antibody binding indices of either the Addison's disease or the vitiligo sera were compared with those of the normal sera group (P = 0.9 and P = 0.6, respectively). Eight of the 11 (73%) GD and 3 of the 7 (43%) AH sera, which were positive for hNIS antibodies in the immunoprecipitation assay, were also found to inhibit iodide uptake in hNIS-transfected CHO-K1 cells, suggesting the existence of antibodies in some serum samples that bind to the symporter without modulating its function. Overall, a significant correlation was found between the iodide uptake inhibition and the binding assays for hNIS antibody detection (r = 0.49, P < 0.0001). In summary, we have developed a specific and quantitative assay for the detection of hNIS binding antibodies in sera of patients with ATD. This system offers the advantage of studying antibody reactivity against conformational epitopes and will be useful in understanding the role of NIS autoreactivity in the pathogenesis of ATD.
Assuntos
Autoanticorpos/sangue , Proteínas de Transporte/imunologia , Doença de Graves/imunologia , Proteínas de Membrana/imunologia , Simportadores , Tireoidite Autoimune/imunologia , Doença de Addison/sangue , Doença de Addison/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células CHO , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cricetinae , Feminino , Doença de Graves/sangue , Humanos , Hipotireoidismo/sangue , Hipotireoidismo/imunologia , Iodetos/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Conformação Proteica , Coelhos , Radioimunoensaio , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Tireoidite Autoimune/sangue , TransfecçãoRESUMO
Deficiencies in cellular cyclic AMP (cAMP) and nitric oxide (NO) production are thought to be involved in the pathogenesis of diabetic neuropathy. We used a human neuroblastoma cell line, SH-SY5Y, to investigate the effect of cilostazol, a specific cAMP phosphodiesterase inhibitor, on NO production and Na+, K+-ATPase activity. SH-SY5Y cells were cultured under 5 or 50 mM glucose for 5-6 days, the cells were then exposed to cilostazol or other chemicals and nitrite, cAMP and Na+, K+-ATPase activity were measured. In cells grown in 50 mM glucose, cilostazol was observed to increase significantly both NO production and cellular cAMP accumulation in a time- and dose-dependent manner. Cilostazol also significantly recovered reduced levels of protein kinase A activity (PKA) in 50 mM glucose. Furthermore, a PKA inhibitor, H-89 significantly suppressed the increase in NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production by activating PKA. Cilostazol did not affect either sorbitol or myo-inositol concentrations. Dexamethasone, which is known to induce inducible NO synthase, had no effect on NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production catalyzed by neuronal constitutive NO synthase (ncNOS) in SH-SY5Y cells. L-arginine, which is an NO agonist enhanced Na+, K+-ATPase activity in cells grown in 50 mM glucose, NG-nitro-L-arginine methyl ester (L-NAME), which is an NOS inhibitor inhibited basal Na+, K+-ATPase activity in 5 mM glucose and suppressed the increased enzyme activity induced by cilostazol in 50 mM glucose. The above results confirmed our previous observation that NO regulates Na+, K+-ATPase activity in SH-SY5Y cells and suggest that cilostazol increases Na+, K+-ATPase activity, at least in part, by stimulating NO production. The present results also suggest that cilostazol has a beneficial effect on diabetic neuropathy by improving Na+, K+-ATPase activity via directly increasing cAMP and NO production in nerves.
Assuntos
Óxido Nítrico/biossíntese , Inibidores de Fosfodiesterase/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Sulfonamidas , Tetrazóis/farmacologia , Cilostazol , AMP Cíclico/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dexametasona/farmacologia , Humanos , Inositol/análise , Isoquinolinas/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Sorbitol/análise , Células Tumorais CultivadasRESUMO
Tumor necrosis factor-alpha (TNF-alpha) is known to modulate the expression of thyroid-specific genes, such as thyroglobulin (TG), contributing to the pathogenesis of autoimmune thyroid disease. In the present study, we show that TNF-alpha suppresses DNA-binding activity of thyroid transcription factors, Pax-8 and thyroid transcription factor-1 (TTF-1), which is, in part, involved in TNF-alpha-induced decrease in TG gene expression. Transfected into rat thyroid FRTL-5 cells, the activity of reporter plasmid containing the rat TG promoter ligated to a luciferase gene was significantly suppressed in the presence of TNF-alpha. In gel mobility shift analyses, protein-DNA complexes formed by TTF-1 and Pax-8 were reduced when the nuclear extracts prepared from TNF-alpha-treated FRTL-5 cells were used. The suppressive effect of TNF-alpha on TTF-1-DNA complex formation is, in part, caused by suppression of TTF-1 gene transcription by TNF-alpha. Expressions of TTF-1 messenger RNA and protein, which were assessed by Northern blot and Western blot analyses, respectively, were decreased by TNF-alpha treatment of FRTL-5 cells. In contrast, TNF-alpha did not affect the expression of Pax-8 messenger RNA. Treatment of FRTL-5 cells with TNF-alpha caused a decrease in Pax-8 protein in nuclear extracts and accumulation of the protein in the cytoplasm, as assessed by Western blot analyses. Mutation of the TTF-1/Pax-8-binding site lost the TNF-alpha-induced decrease in TG promoter activity in a transfection experiment. These results indicate that TNF-alpha suppresses the activity of TTF-1 and Pax-8 by different mechanisms, which, in part, seem to be involved in TNF-alpha-induced decrease in TG gene expression.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Glândula Tireoide/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Animais , Sequência de Bases/genética , Sítios de Ligação/fisiologia , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quimera/genética , Citoplasma/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Mutação/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , Ratos , Tireoglobulina/genética , Glândula Tireoide/citologia , Fator Nuclear 1 de Tireoide , Transativadores/antagonistas & inibidores , Transativadores/genética , Transativadores/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) are cytokines that can individually or additively suppress thyroid cell function and the expression of thyroid-specific genes, such as thyroglobulin (TG) and thyroperoxidase (TPO). Thyroid transcription factor-2 (TTF-2) is a DNA-binding protein that modulates the expression of TG and TPO genes. In the present study, we examine the effects of TNF-alpha and IFN-gamma on TTF-2 gene expression, as well as the DNA-binding activity of TTF-2. FRTL-5 cells were maintained in 5H medium containing 0.2% calf serum for 7 days, then incubated with TNF-alpha, IFN-gamma, or TNF-alpha plus IFN-gamma. Total RNA was isolated and Northern blotted. TNF-alpha (50 ng/ml) only slightly suppressed (61+/-2% compared with control), whereas IFN-gamma (100 U/ml) modestly decreased TTF-2 messenger RNA (mRNA) levels (34+/-4%). TNF-alpha and IFN-gamma simultaneously caused a marked decrease in TTF-2 mRNA levels (13+/-2%). The suppressive effects of TNF-alpha and IFN-gamma on TTF-2 mRNA levels were concentration dependent and maximal at 50 ng/ml TNF-alpha with 100 U/ml IFN-gamma. The suppressive effect was also time dependent, reaching a maximum 12 h after exposure. Moreover, the suppressive effects of TNF-alpha and IFN-gamma upon rat TG and TTF-2 mRNA levels were similar. To test whether TNF-alpha and IFN-gamma alter TTF-2-binding to DNA, we performed electrophoretic mobility shift assays using a TTF-2-binding element in the rat TG gene as a probe. Formation of the TTF-2/DNA complex was decreased by TNF-alpha and/or IFN-gamma. Our results demonstrate that TNF-alpha and IFN-gamma additively reduce the gene expression and DNA-binding of TTF-2. These data suggest that TTF-2 is involved in the TNF-alpha and IFN-gamma-induced suppression of thyroid-specific gene expression.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Proteínas Repressoras/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Fatores de Transcrição Forkhead , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , Ratos , Proteínas Repressoras/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Full-length of steroid receptor coactivator-1 (F-SRC-1) has been shown to interact with thyroid hormone receptors (TRs) in a ligand-dependent manner and to stimulate receptor-dependent transcription. To identify functional domains of F-SRC-1, several internal deletion mutants of F-SRC-1 were constructed. Although in vitro pull down assay with TR showed interaction of all of these mutants with TR, lack of mid legion (amino acids 398-1172) lost enhancing activity of TR-mediated transcription in a transient transfection assay. However, F-SRC-1 mutant lacking CBP-interacting domain still preserved enhancing activity. Surprisingly, F-SRC-1 mutants also increased basal level of viral promoter activity depending upon their deleted region. Yeast activation function assay revealed that these F-SRC-1 mutants had intrinsic activation function when bound to DNA. Analyses of small fragments of F-SRC-1 identified three separable activation domains. In vitro binding assay showed that TBP and TFIIB bound to C-terminal half of F-SRC-1. These results suggest that F-SRC-1 can function via both CBP-dependent and independent manners using various sets of activation domains and that direct interactions between F-SRC-1 and TBP or TFIIB may not be important for CBP-independent transcription.
Assuntos
Proteínas Nucleares/fisiologia , Receptores dos Hormônios Tireóideos/metabolismo , Transativadores/fisiologia , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Vírus do Sarcoma Aviário/genética , Proteína de Ligação a CREB , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação Viral da Expressão Gênica , Histona Acetiltransferases , Ligantes , Proteínas Nucleares/metabolismo , Coativador 1 de Receptor Nuclear , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta/genética , Deleção de Sequência , Proteína de Ligação a TATA-Box , Transativadores/metabolismo , Fator de Transcrição TFIIB , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transfecção , Leveduras/genética , Leveduras/metabolismoRESUMO
EMO syndrome, a rare complication of Graves' disease, exhibits exophthalmos, pretibial myxedema, and osteoarthropathy. The presence of functional thyrotropin receptors (TSHR) in adipocytes and osteoblasts, both of which we have recently observed, may be related to these extrathyroidal manifestations of Graves' disease. In addition, the expression of TSHR in the heart has recently been reported. We describe here a patient with Graves' disease exhibiting EMO syndrome with a papillary fibroelastoma in the left ventricle. Pathological examinations showed that the fibroelastoma contained Alcian blue-stained mucinous materials that were also observed in the subcutaneous tissue of pretibial myxedema.
Assuntos
Fibroma/complicações , Doença de Graves/complicações , Neoplasias Cardíacas/complicações , Dermatoses da Perna/complicações , Mixedema/complicações , Osteoartropatia Hipertrófica Secundária/etiologia , Fibroma/patologia , Neoplasias Cardíacas/patologia , Ventrículos do Coração , Humanos , Dermatoses da Perna/patologia , Masculino , Pessoa de Meia-Idade , SíndromeRESUMO
We studied the effect of extracellular adenosine on iodide (I-) transport in FRTL-5 thyroid cells. I- accumulation increases after a 48 h exposure to adenosine in a concentration-dependent manner, reaching a maximum of 7.9-fold basal levels at 72 h after the addition of 300 microM adenosine. Neither I- efflux nor intracellular cyclic adenosine monophosphate accumulation is affected by the exposure to adenosine. The stimulation of I- transport by adenosine is partly as a result of an increase in Na+/I- symporter (NIS) mRNA and protein levels. Northern blot analysis revealed that adenosine increases NIS mRNA levels at 24 h, reaching a maximum at 36 h. Western blot analysis demonstrated that adenosine increases NIS protein levels at 36 h, reaching a maximum at 72 h, in parallel with the kinetics of adenosine-induced I- transport. Adenosine increased the promoter activity of a full-length NIS promoter-luciferase chimera, suggesting that the effect of adenosine on NIS mRNA levels is transcriptional. The stimulatory effect of adenosine on NIS mRNA levels, is mimicked by N6-(L-2-phenylisopropyl) adenosine (PIA), an A1 adenosine receptor agonist, and inhibited by 1,3-dipropyl-8-cyclopentylxanthine, an A1 adenosine receptor antagonist, suggesting that the effect is mediated via the A1 adenosine receptor stimulation in FRTL-5 cells. Incubating cells with islet-activating protein inhibited the adenosine-induced NIS mRNA levels. In sum, extracellular adenosine increases NIS gene expression and stimulates I- transport via the A1 adenosine receptor-Gi/Go protein signal transduction pathway.
Assuntos
Adenosina/farmacologia , Proteínas de Transporte/genética , Proteínas de Membrana/genética , Simportadores , Glândula Tireoide/citologia , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica/efeitos dos fármacos , Iodo/metabolismo , Toxina Pertussis , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/efeitos dos fármacos , Ratos , Receptores Purinérgicos P1/metabolismo , Tireotropina/genética , Tireotropina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Fatores de Virulência de Bordetella/metabolismo , Fatores de Virulência de Bordetella/farmacologiaRESUMO
We investigated the relation between cyclic AMP (cAMP) and nitric oxide (NO) production, as well as the effect of NO on Na , K+-ATPase activity in the human neuroblastoma cell line SH-SY5Y. Two cAMP agonists, dibutyryl cAMP (DBC) and beraprost sodium (BPS), increased cAMP accumulation and NO production in a time and dose dependent manner at 50 mmol/l glucose. On the other hand, cellular sorbitol and myo-inositol contents and protein kinase C activity were not altered by DBC or BPS. A specific protein kinase A inhibitor, H-89, suppressed increases in nitrite/nitrate and cyclic GMP (cGMP) and protein kinase A activity stimulated by DBC or BPS. This finding suggests that cAMP stimulates NO production by activating protein kinase A via a pathway different from the sorbitol-myo-inositol-protein kinase C pathway. We observed that an NO donor, sodium nitroprusside, and an NO agonist, L-arginine, enhanced ouabain sensitive Na+, K+-ATPase activity at 50 mmol/l glucose. We also found that a nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), inhibited Na+, K+-ATPase activity at 5 mmol/l glucose, and partially suppressed the enzyme activity stimulated by DBC or BPS. The results of this study suggest that cAMP regulates protein kinase A activity, NO production and ouabain sensitive Na+, K+-ATPase activity in a cascade fashion. The results also suggest that protein kinase A at least partially regulates Na+, K+-ATPase activity without mediation by NO in SH-SY5Y cells. We speculate that cAMP and NO are two important regulatory factors in the pathogenesis of diabetic neuropathy.
Assuntos
AMP Cíclico/fisiologia , Neuroblastoma/metabolismo , Óxido Nítrico/biossíntese , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Sulfonamidas , Bucladesina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , GMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Epoprostenol/análogos & derivados , Epoprostenol/farmacologia , Humanos , Isoquinolinas/farmacologia , Cinética , Nitratos/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Nitritos/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
The expression of thyrotropin receptor (TSH-R) on various cells derived from bone, including osteoblast-like rat osteosarcoma cells (UMR106 cells), was investigated. TSH receptor mRNA was detected in UMR106 cells by Northern blot analysis. 125I-labeled TSH binding analysis revealed specific high- and low-affinity binding sites (association constants of 5.6 x 10(9) M(-1) and 3.0 x 10(7) M(-1), respectively) on UMR106 cells. Recombinant TSH, but not recombinant human chorionic gonadotropin, increased cyclic adenosine monophosphate (cAMP) production in a concentration-dependent manner in these cells. Furthermore, immunoglobulin Gs from patients with Graves' disease induced cAMP response in UMR106 cells, and the cAMP response index in this cell line correlated with thyroid-stimulating antibody (TSAb) activity detected by Chinese hamster ovary (CHO)-K1 cells transfected with rat TSH-R. We have also demonstrated that recombinant TSH increased cAMP production in human osteoblast-like osteosarcoma (MG63) cells and mouse primary osteoblastic cells. These results suggest that osteoblasts possess functional TSH-R and that abnormal bone metabolism in Graves' disease may be partly explained by the interaction of TSAb with TSH-R in osteoblasts in some patients.
Assuntos
Osteoblastos/metabolismo , Osteoblastos/patologia , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Receptores da Tireotropina/metabolismo , Animais , Células CHO , Linhagem Celular , Gonadotropina Coriônica/farmacologia , Células Clonais/metabolismo , Cricetinae , AMP Cíclico/biossíntese , Doença de Graves/metabolismo , Humanos , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Imunoglobulinas Estimuladoras da Glândula Tireoide/metabolismo , Camundongos , RNA Mensageiro/metabolismo , Ratos , Receptores da Tireotropina/genética , Proteínas Recombinantes , Tireotropina/farmacologia , TransfecçãoRESUMO
Rat adipose tissue, as well as differentiated 3T3-L1 cells, has been shown to express TSH receptor (TSHR) mRNA in amounts approaching those in the thyroid. We investigated the molecular mechanisms of TSHR gene expression in adipose cells. Primer extension and cloned cDNA sequences showed that transcription of the TSHR gene in rat adipose tissue was from multiple start sites clustered between -89 to -68 bp and almost identical to those in FRTL-5 thyroid cells. By transient expression analysis, we localized, between -146 and -90 bp, a positive regulatory element, the activity of which was markedly increased after the differentiation of 3T3-L1 cells. Deoxyribonuclease I protection showed that nuclear extracts from differentiated 3T3-L1 cells strongly protected two sequences, from -146 to -127 bp, including a cAMP response element-like sequence and from -112 to -106 bp containing a putative Ets-binding sequence. In differentiated 3T3-L1 cells, disruption or deletion of either sequence was found to result in the loss of enhancer activity, suggesting both elements may synergistically activate the TSHR promoter. Electrophoretic mobility shift analysis revealed the induction of new protein/DNA complexes formed either with the cAMP response element-like site or with putative Ets elements after the differentiation into adipocytes. In contrast, nuclear proteins, whose binding to DNA was diminished after the differentiation of 3T3-L1 cells, were found to interact with the site contiguous to the 5'-end of the putative Ets-binding sequence. Mutations of this binding site, which reduced the protein/DNA complex formation, increased TSHR promoter activity in undifferentiated cells. These observations suggested that differentiation-induced diminution of suppressor interactions may allow the enhancers to synergistically activate the transcription of TSHR gene in adipocytes.
Assuntos
Adipócitos/metabolismo , Diferenciação Celular/genética , Receptores da Tireotropina/genética , Receptores da Tireotropina/metabolismo , Tecido Adiposo/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ratos , Sequências Reguladoras de Ácido Nucleico , Elementos de Resposta , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Cultured rat preadipocytes express thyrotropin receptor (TSHR) during their differentiation. To evaluate the effects of inflammatory cytokines on the expression of TSHR in cultured rat preadipocytes, we cultured those cells in the presence of recombinant human tumor necrosis factor (rhTNF)-alpha, recombinant human interferon (rhIFN)-gamma, and human transforming growth factor (hTGF)-beta1. The effects on the level of TSHR mRNA and signal transduction were evaluated. Addition to the medium of 1 ng/mL TNF-alpha, 1 ng/mL rhIFN-gamma, and 1 ng/mL hTGF-beta1 during the differentiation of rat preadipocytes inhibited the expression of TSHR mRNA. The decrease in TSHR mRNA was accompanied by a decrease in TSH-stimulated cyclic adenosine monophosphate (cAMP) production. Histochemical analysis showed that these cytokines inhibited the morphological differentiation of the cells. These cytokines also decreased the expression of mRNA for such fat-specific proteins as lipoprotein lipase and aP2. Results indicate that the loss of expression and function of the TSHR is closely related to the inhibition of differentiation. This confirms the close relation between the expression of the TSHR and the differentiation of the rat preadipocytes.
Assuntos
Adipócitos/efeitos dos fármacos , Citocinas/farmacologia , RNA Mensageiro/metabolismo , Receptores da Tireotropina/metabolismo , Complexo 2 de Proteínas Adaptadoras , Subunidades alfa do Complexo de Proteínas Adaptadoras , Proteínas Adaptadoras de Transporte Vesicular , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Northern Blotting , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Primers do DNA/química , Regulação para Baixo , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores da Tireotropina/genética , Proteínas Recombinantes/farmacologia , Transdução de SinaisRESUMO
To clarify the relationship between platelet function and diabetic complications, we investigated spontaneous platelet aggregation (SPA) and agonist-induced platelet aggregation by a particle counting method using light scattering (LS) and by a conventional light transmission method (LT) in 23 age- and sex-matched control subjects and 74 patients with type II diabetes mellitus. We also observed platelets using the FIC-2 (TOA Medical Electronics, Kobe, Japan) flow cytometer and imaging device. Observation by the FIC-2 device showed microaggregates of platelets in samples with increased SPA-LS. SPA-LS was significantly elevated in patients with type II diabetes mellitus as a whole compared with control subjects. SPA-LS also showed significant differences between control subjects and three diabetic patient subgroups with a varying severity of retinopathy, nephropathy, or neuropathy, and the mean values increased along with the increasing severity of complications. On the other hand, although SPA-LT also showed significant differences between these groups, the absolute values were all less than 10%, which we believe does not warrant quantitative analysis. Adenosine-5'-diphosphate (ADP)-induced platelet aggregation failed to show significant differences between controls and subjects with a varying severity of retinopathy by either LS or LT, which indicates that SPA is more sensitive than agonist-induced platelet aggregation in relation to diabetic complications. We observed significant correlations between SPA-LS and the patients' age, hemoglobin A1c (HbA1c) level, plasma fibrinogen level, or 6-keto-PGF1alpha (6KF) to 11-dehydro-thromboxane B2 (TXB2) ratio. Our study demonstrated a close relationship between platelet hyperaggregability and diabetic complications, and a longitudinal prospective study of SPA-LS in diabetic patients is warranted to clarify cause-and-effect relationships.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Agregação Plaquetária/fisiologia , Estudos Transversais , Nefropatias Diabéticas/sangue , Neuropatias Diabéticas/sangue , Retinopatia Diabética/sangue , Feminino , Humanos , Luz , Masculino , Métodos , Pessoa de Meia-Idade , Valores de Referência , Espalhamento de RadiaçãoRESUMO
Iodide is concentrated to a much lesser extent by papillary thyroid carcinoma as compared with the normal gland. The Na+/I- symporter (NIS) is primarily responsible for the uptake of iodide into thyroid cells. Our objective was to compare NIS mRNA and protein expression in papillary carcinomas with those in specimens with normal thyroid. Northern blot analysis revealed a 2.8-fold increase in the level of NIS mRNA in specimens with papillary carcinoma versus specimens with normal thyroid. Immunoblot analysis using anti-human NIS antibody that was produced with a glutathione S-transferase fusion protein containing NIS protein (amino acids 466-522) showed the NIS protein at 77 kD. The NIS protein level was elevated in 7 of 17 cases of papillary carcinoma but was not elevated in the normal thyroid. Immunohistochemical staining revealed abundant NIS in 8 of 12 carcinomas, whereas NIS protein was barely detected in specimens with normal thyroid. Although considerable patient-to-patient variation was observed, our results indicate that NIS mRNA is elevated, and its protein tends to be more abundant, in a subset of papillary thyroid carcinomas than in normal thyroid tissue.
Assuntos
Carcinoma Papilar/metabolismo , Proteínas de Transporte/metabolismo , Iodetos/metabolismo , Proteínas de Membrana/metabolismo , Sódio/metabolismo , Simportadores , Neoplasias da Glândula Tireoide/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunológicas , Iodo/metabolismo , RNA Mensageiro/genética , RNA Neoplásico/genética , Glândula Tireoide/metabolismo , Células Tumorais Cultivadas/metabolismoRESUMO
Retinoid X receptors (RXRs) are recently characterized transcription factors that are members of the nuclear hormone receptor superfamily. However, it is not known whether the endogenous RXR complex requires its ligand for access to its hormone response element (HRE) of a target gene in vivo. Hence, dimethyl sulfate-based genomic footprinting was carried out to examine occupancy of HREs in the retinoic acid (RA) receptor beta2 (RARbeta2) gene promoter in the murine melanoma cell line S91 cultured in the absence or presence of T3, all-trans-RA (atRA), or CD2624, an RXR-selective retinoid. No footprint was observed at the RA-response element (betaRARE) in the absence of ligands. However, a footprint was detected at the betaRARE and other cis-acting elements after a 6 h incubation with CD2624 and atRA. Interestingly, only the betaRARE was footprinted after 60 min incubation with CD2624. These results suggest that the endogenous RXR complex can interact with an HRE of a target gene in the presence of ligand, and subsequently may initiate additional interactions between DNA and other transcription factors.
Assuntos
DNA/metabolismo , Regiões Promotoras Genéticas , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Animais , Benzoatos/farmacologia , Pegada de DNA , Dimerização , Regulação da Expressão Gênica/efeitos dos fármacos , Luciferases/genética , Melanoma Experimental/metabolismo , Camundongos , Naftalenos/farmacologia , Proteínas Recombinantes de Fusão , Receptores X de Retinoides , Transfecção , Tretinoína/farmacologia , Tri-Iodotironina/farmacologia , Células Tumorais CultivadasRESUMO
131I therapy is a widely accepted treatment for differentiated thyroid cancers which can accumulate iodide. We evaluated the efficiency of 131I therapy against tumors which are transfected with the Na+/I- symporter (NIS) gene. We transfected the rat NIS cDNA expression vector into malignantly transformed rat thyroid cells (FRTL-Tc) which do not concentrate iodide. The resultant cell line (Tc-rNIS) accumulated 125I 60-fold in vitro. The FRTL-Tc cells formed solid tumors after injection of cells into subcutaneous tissues of Fischer 344 rats. Tumors formed with Tc-rNIS cells accumulated up to 27.3% of total 125I administered, and concentrated 125I 11 to 27-fold in the tumors. Extracorporeal measurement of radioactivity in the tumors revealed that 125I accumulation peaked at 90 min, and decreased to half levels 6 h after the injections. To investigate the effect of 131I administration on the tumor growth, we injected Na131I 2 and 3 weeks after the transplantation of the cells. The Na131I did not change the tumor volume significantly in either the FRTL-Tc or the Tc-rNIS-induced tumors. The short (6 h) effective half life of 131I in the tumors diminished the radiation dose to the tumor cells. However, this approach may prove beneficial in the treatment of radiosensitive cancers, and could be employed diagnostically.
Assuntos
Proteínas de Transporte/genética , Iodetos/farmacocinética , Iodetos/uso terapêutico , Radioisótopos do Iodo/farmacocinética , Radioisótopos do Iodo/uso terapêutico , Proteínas de Membrana/genética , Sódio/farmacocinética , Simportadores , Neoplasias da Glândula Tireoide/radioterapia , Animais , Transporte Biológico Ativo , Western Blotting , Proteínas de Transporte/metabolismo , Linhagem Celular Transformada , Transformação Celular Neoplásica/patologia , DNA Complementar/análise , DNA Complementar/química , DNA Complementar/genética , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Meia-Vida , Técnicas In Vitro , Iodetos/metabolismo , Radioisótopos do Iodo/metabolismo , Transporte de Íons , Proteínas de Membrana/metabolismo , Transplante de Neoplasias , Ratos , Ratos Endogâmicos F344 , Glândula Tireoide/química , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/química , Neoplasias da Glândula Tireoide/patologia , Transfecção , Células Tumorais CultivadasRESUMO
To investigate the mechanism of I- transport stimulation by TSH, we studied the effects of TSH on Na+/I- symporter (NIS) messenger RNA (mRNA) and protein levels in FRTL-5 cells and correlated these with I- transport activity. When 1 mU/ml TSH was added to quiescent FRTL-5 cells, a 12-h latency was observed before the onset of increased I- transport activity, which reached a maximum [approximately 27 times basal (5H medium) levels] at 72 h. In contrast, Northern blot analysis, using rat NIS complementary DNA as a probe, revealed that addition of TSH to these cells significantly increased NIS mRNA at 3-6 h, reaching a maximum after 24 h (approximately 5.9 times basal levels). Forskolin and (Bu)2cAMP mimicked this stimulatory effect on both the I- transport activity and mRNA levels. D-ribofranosylbenzimidazole, a transcription inhibitor, almost completely blocked TSH-induced stimulation of I- transport and NIS mRNA levels. Western blot analysis demonstrated that TSH increased NIS protein levels at 36 h, reaching a maximum at 72 h, in parallel with the kinetics of TSH-induced I- transport activity. However, it also showed that the amount of NIS protein already present in FRTL-5 cell membranes before the addition of TSH was about one third of the maximum level induced by TSH. These results indicate that stimulation of I- transport activity by TSH in thyrocytes is partly due to a rapid increase in NIS gene expression, followed by a relatively slow NIS protein synthesis. However, the existence of an abundant amount of protein in quiescent FRTL-5 cells with very low I- transport activity also suggests that this activity is controlled by another TSH-regulated factor(s).
Assuntos
Proteínas de Transporte/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana/biossíntese , Simportadores , Glândula Tireoide/metabolismo , Tireotropina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Bucladesina/farmacologia , Células CHO , Linhagem Celular , Colforsina/farmacologia , Cricetinae , Iodetos/metabolismo , Cinética , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
We previously have demonstrated that rat adipose tissue expresses TSH receptor (TSHR) messenger RNAs (mRNAs) at levels approaching those detected in the thyroid. Furthermore, we recently reported that TSHR mRNA is detected in fibroblast-like 3T3-L1 cells after their hormone-induced differentiation into adipocytes. TSH induces cAMP formation and lipolysis in differentiated 3T3-L1 cells. We now show that, in Northern blot analyses, TSH-induced down-regulation of TSHR mRNA levels, which can be duplicated by forskolin and dibutylyl cAMP, i.e. which is cAMP-mediated. We also have demonstrated that a beta-adrenergic stimulant, which stimulates cAMP formation in adipocytes, induces a down-regulation of TSHR mRNA levels in 3T3-L1 adipocytes. Nuclear run-on assays show that the ability of TSH/cAMP to decrease TSHR mRNA levels in 3T3-L1 cells reflects transcriptional regulation. This report also demonstrates that TSHR gene expression in 3T3-L1 adipocytes is regulated in a manner distinct from that observed in thyroid cells. Thus, in fully differentiated 3T3-L1 adipocytes, TSH-induced down-regulation of TSHR mRNA levels is evident within 1 h and is near maximum within 4 h after addition of TSH. A transient increase of TSHR gene expression, which has been demonstrated in FRTL-5 thyroid cells, was not observed in 3T3-L1 adipocytes. The down-regulation of TSHR gene expression induced by TSH/cAMP in 3T3-L1 cells is cycloheximide-insensitive, suggesting that continuous protein synthesis is not required for this process. In contrast, the down-regulation of TSHR gene expression observed in FRTL-5 cells is sensitive to cycloheximide. In both FRTL-5 thyroid cells and 3T3-L1 adipocytes, insulin or serum increased TSHR mRNA levels. Although insulin or serum was required for the TSH-induced down-regulation of TSHR mRNA levels in FRTL-5 thyroid cells, neither insulin nor serum was required for TSHR down-regulation in 3T3-L1 adipocytes. These findings demonstrate that TSH/cAMP regulates TSHR mRNA levels in adipocytes via a regulatory system distinct from that used in FRTL-5 cells. This report further demonstrates that adipose cells do not express thyroid transcription factor-1, which interacts with the TSHR promoter region in FRTL-5 cells, and that 3T3-L1 nuclear extracts exhibit a different binding activity to the cAMP-response element-like element in the TSHR promoter region compared with extracts from FRTL-5 cells.