RESUMO
BACKGROUND AND PURPOSE: Lipoxidation-derived reactive carbonyl species (RCS) such as 4-hydroxy-2-nonenal (HNE) react with proteins to form advanced lipoxidation end products (ALEs), which have been implicated in both atherosclerosis and renal disease. L-carnosine acts as an endogenous HNE scavenger, but it is rapidly inactivated by carnosinase. This study aimed at assessing the effect of the carnosinase-resistant, D-carnosine, on HNE-induced cellular injury and of its bioavailable prodrug D-carnosine octylester on experimental atherosclerosis and renal disease. EXPERIMENTAL APPROACH: Vascular smooth muscle cells (VSMCs) were exposed to HNE or H2O2 plus D-carnosine. ApoE null mice fed a Western, pro-atherogenic diet were treated with D-carnosine octylester for 12 weeks. KEY RESULTS: In vitro, D-carnosine attenuated the effect of HNE, but not of H2O2, on VSMCs. In vivo, D-carnosine octylester-treated mice showed reduced lesion area and a more stable plaque phenotype compared with untreated animals, with reduced foam cell accumulation, inflammation and apoptosis and increased clearance of apoptotic bodies and collagen deposition, resulting in decreased necrotic core formation. Likewise, renal lesions were attenuated in D-carnosine octylester-treated versus untreated mice, with lower inflammation, apoptosis and fibrosis. This was associated with increased urinary levels of HNE-carnosine adducts and reduced protein carbonylation, circulating and tissue ALEs, expression of receptors for these products, and systemic and tissue oxidative stress. CONCLUSIONS AND IMPLICATIONS: These data indicate RCS quenching with a D-carnosine ester was highly effective in attenuating experimental atherosclerosis and renal disease by reducing carbonyl stress and inflammation and that this may represent a promising therapeutic strategy in humans.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Apolipoproteínas E/metabolismo , Aterosclerose/prevenção & controle , Carnosina/análogos & derivados , Sequestradores de Radicais Livres/uso terapêutico , Pró-Fármacos/uso terapêutico , Insuficiência Renal/prevenção & controle , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Aorta/efeitos dos fármacos , Aorta/imunologia , Aorta/metabolismo , Aorta/patologia , Apolipoproteínas E/genética , Aterosclerose/imunologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Carnosina/química , Carnosina/farmacologia , Carnosina/uso terapêutico , Linhagem Celular , Células Cultivadas , Dieta Aterogênica/efeitos adversos , Feminino , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/farmacologia , Rim/efeitos dos fármacos , Rim/imunologia , Rim/metabolismo , Rim/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/imunologia , Células Mesangiais/metabolismo , Células Mesangiais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/imunologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Estresse Oxidativo/efeitos dos fármacos , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Insuficiência Renal/imunologia , Insuficiência Renal/metabolismo , Insuficiência Renal/patologia , EstereoisomerismoRESUMO
Administration of radioiodine (I(131)) is currently exploited for both diagnostic and therapeutic treatment of thyroid cancer. Few data are available on the sodium/iodide symporter (NIS) expression in human testis, a particular important prerequisite to predict radioiodine accumulation in the gonads of males with thyroid cancer exposed to such a treatment. In this study, we analyzed the expression of NIS in mouse, rat and human normal testis in different stages of development. By using a quantitative RT-PCR, Western blot and immunohistochemical analysis, NIS mRNA and protein were measured in both fetal and adult testicular tissues. NIS transcript was detected in both fetal and adult testis, although its expression levels were approximately 10-fold less than in thyroid gland. Western blot analysis and immunohistochemistry showed the presence of NIS protein in germinal and Leydig cells, but not in Sertoli cells with prevalent expression in the cytosol compartment of the cells. Our study demonstrates that NIS transcript and protein are expressed in normal testis. Further studies will demonstrate whether it may act as the transporter of radioiodine in normal testis of male patients with thyroid cancer.
Assuntos
Simportadores/metabolismo , Testículo/citologia , Testículo/metabolismo , Animais , Humanos , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Modelos Animais , RNA Mensageiro/metabolismo , Ratos , Espermatozoides/citologia , Espermatozoides/metabolismo , Glândula Tireoide/citologia , Glândula Tireoide/metabolismoRESUMO
BACKGROUND & AIMS: Excess fatty acid oxidation and generation of reactive carbonyls with formation of advanced lipoxidation endproducts (ALEs) is involved in nonalcoholic steatohepatitis (NASH) by triggering inflammation, hepatocyte injury, and fibrosis. This study aimed at verifying the hypothesis that ablation of the ALE-receptor galectin-3 prevents experimental NASH by reducing receptor-mediated ALE clearance and downstream events. METHODS: Galectin-3-deficient (Lgals3(-/-)) and wild type (Lgals3(+/+)) mice received an atherogenic diet or standard chow for 8 months. Liver tissue was analyzed for morphology, inflammation, cell and matrix turnover, lipid metabolism, ALEs, and ALE-receptors. RESULTS: Steatosis was significantly less pronounced in Lgals3(-/-) than Lgals3(+/+) animals on atherogenic diet. NASH, invariably detected in Lgals3(+/+) mice, was observed, to a lower extent, only in 3/8 Lgals3(-/-) mice, showing less inflammatory, degenerative, and fibrotic phenomena than Lgals3(+/+) mice. This was associated with higher circulating ALE levels and lower tissue ALE accumulation and expression of other ALE-receptors. Up-regulation of hepatic fatty acid synthesis and oxidation, inflammatory cell infiltration, pro-inflammatory cytokines, endoplasmic reticulum stress, hepatocyte apoptosis, myofibroblast transdifferentiation, and impaired Akt phosphorylation were also significantly attenuated in Lgals3(-/-) animals. Galectin-3 silencing in liver endothelial cells resulted in reduced N(ε)-carboxymethyllysine-modified albumin uptake and ALE-receptor expression. CONCLUSIONS: Galectin-3 ablation protects from diet-induced NASH by decreasing hepatic ALE accumulation, with attenuation of inflammation, hepatocyte injury, and fibrosis. It also reduced up-regulation of lipid synthesis and oxidation causing less fat deposition, oxidative stress, and possibly insulin resistance. These data suggest that galectin-3 is a major receptor involved in ALE uptake by the liver.
Assuntos
Fígado Gorduroso , Galectina 3/genética , Galectina 3/metabolismo , Animais , Apoptose/fisiologia , Antígenos CD36/genética , Antígenos CD36/metabolismo , Dieta Aterogênica , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Feminino , Fibroblastos/patologia , Inativação Gênica , Leucócitos/metabolismo , Leucócitos/patologia , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica , Estresse Oxidativo/fisiologia , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Índice de Gravidade de DoençaRESUMO
In mammalian lung, at the transition to extrauterine life, NO/cGMP signal transduction system is known to play crucial roles in the regulation of vascular resistance and is supposed to act in angiogenesis. PDE5, which is the most abundant cGMP metabolizing enzyme within the lung, is highly expressed in the perinatal period, but its localisation in the different pulmonary cells is still poorly known. In our research, PDE5 immunohistochemical distribution was investigated in foetal and neonatal rat lung. The highest expression of PDE5 was found in cells randomly located in the stroma; in newborns, in particular, many cells in the intersaccular walls were heavily labelled, while much lower staining levels were shown by smooth myocytes belonging to vessels and airways. On the basis of their immunoreactivity for alpha-SM actin and/or desmin, most of the heavily PDE5-positive cells were identified as interstitial myofibroblasts and transitional pericytes, while only a few were interpreted as interstitial lipofibroblasts.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Pulmão/enzimologia , Actinas/metabolismo , Animais , Western Blotting , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/biossíntese , Proteínas do Citoesqueleto/metabolismo , Desmina/metabolismo , Embrião de Mamíferos , Feminino , Imuno-Histoquímica , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Masculino , Ratos , Ratos WistarRESUMO
Atherosclerosis and renal disease are related conditions, sharing several risk factors. This includes hyperlipidaemia, which may result in enhanced lipoprotein accumulation and chemical modification, particularly oxidation, with formation of advanced lipoxidation endproducts (ALEs). We investigated whether increased lipid peroxidation plays a major role in the pathogenesis of lipid-induced renal disease, via receptor-mediated mechanisms involving the scavenger and advanced glycation endproduct (AGE) receptors. Mice knocked out for galectin-3 (Gal3(-/-)), an AGE receptor previously shown to protect from AGE-induced renal injury, and the corresponding wild-type (Gal3(+/+)) animals, were fed an atherogenic high-fat diet (HFD; 15% fat, 1.25% cholesterol and 0.5% sodium cholate); mice fed a normal-fat diet (NFD; 4% fat) served as controls. Gal3(+/+) mice fed a HFD developed glomerular disease, as indicated by proteinuria, mesangial expansion and glomerular hypertrophy and sclerosis. Glomerular injury was associated with increased glomerular matrix protein expression, ALE and oxidized LDL content, oxidative stress, AGE and scavenger receptor expression and macrophage infiltration, with only modest renal/glomerular fat accumulation and changes in lipid metabolism. Fibrotic and inflammatory changes, together with accumulation of ALEs, such as 4-hydroxy-2-nonenal adducts and N(epsilon)-carboxymethyllysine, oxidative stress and expression of the receptor of AGEs (RAGE), were significantly more marked in Gal3(-/-) animals, whereas fat deposition and abnormalities in lipid metabolism remained modest. Thus, lipid-induced renal damage is mainly dependent on lipid peroxidation with formation of carbonyl reactive species and ALEs, which accumulate within the kidney tissue, thus triggering receptor-mediated pro-inflammatory signalling pathways, as in atherogenesis. Moreover, galectin-3 exerts a significant role in the uptake and effective removal of modified lipoproteins, with diversion of these products from RAGE-dependent pro-inflammatory pathways associated with downregulation of RAGE expression.
Assuntos
Dieta Aterogênica , Nefropatias/etiologia , Peroxidação de Lipídeos/fisiologia , Animais , Apoptose/fisiologia , Pressão Sanguínea/fisiologia , Matriz Extracelular/metabolismo , Feminino , Galectina 3/deficiência , Galectina 3/genética , Galectina 3/fisiologia , Produtos Finais de Glicação Avançada/metabolismo , Nefropatias/metabolismo , Nefropatias/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Metabolismo dos Lipídeos , Macrófagos/fisiologia , Camundongos , Camundongos Knockout , Estresse Oxidativo/fisiologia , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Receptores Depuradores/metabolismoRESUMO
OBJECTIVE: Modified lipoproteins, particularly oxidized LDLs, are believed to evoke an inflammatory response which participates in all stages of atherosclerosis. Disposal of these particles is mediated through receptors which may trigger proinflammatory signaling pathways leading to vascular injury. This study was aimed at assessing the role in atherogenesis of one of these receptors, galectin-3. METHODS AND RESULTS: Galectin-3-deficient and wild-type mice were fed an atherogenic diet or standard chow for 8 months. Lesion area and length were higher in galectin-3-deficient versus wild-type mice. At the level of the aortic sinus, wild-type animals showed only fatty streaks, whereas galectin-3-deficient mice developed complex lesions, associated with extensive inflammatory changes. This was indicated by the presence of T lymphocytes with activated Th1-phenotype and by more marked monocyte-macrophage infiltration, inflammatory mediator expression, vascular cell apoptosis, and proinflammatory transcription factor activation. Increased accumulation of oxidixed LDLs and lipoxidation products and upregulation of other receptors for these compounds, including the proinflammatory RAGE, were detected in galectin-3-deficient versus wild-type mice. CONCLUSIONS: These data suggest a unique protective role for galectin-3 in the uptake and effective removal of modified lipoproteins, with concurrent downregulation of proinflammatory pathways responsible for atherosclerosis initiation and progression.
Assuntos
Doenças da Aorta/metabolismo , Aortite/metabolismo , Aterosclerose/metabolismo , Galectina 3/deficiência , Peroxidação de Lipídeos , Transdução de Sinais , Animais , Aorta/imunologia , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/etiologia , Doenças da Aorta/imunologia , Doenças da Aorta/patologia , Aortite/etiologia , Aortite/imunologia , Aortite/patologia , Apoptose , Aterosclerose/etiologia , Aterosclerose/imunologia , Aterosclerose/patologia , Quimiotaxia de Leucócito , Dieta Aterogênica , Modelos Animais de Doenças , Progressão da Doença , Feminino , Galectina 3/genética , Mediadores da Inflamação/metabolismo , Lipoproteínas LDL/metabolismo , Ativação Linfocitária , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Estresse Oxidativo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Receptores Depuradores/metabolismo , Células Th1/imunologia , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVE: Nonfunctioning thyroid nodules (NFTNs) display a diminished iodide-concentrating ability, owing to defective expression and cell membrane targeting of the sodium-iodide symporter (NIS). Since NIS expression is primarily modulated by thyroid iodine content in vitro and in animal models, we attempted to determine whether iodine supply influences the expression and localization of human NIS (hNIS) in NFTNs. DESIGN: Using immunohistochemistry, we analyzed cold hyperplastic nodules and nonnodular thyroid samples (controls) from patients living in iodine-sufficient (n = 19) or severely iodine-deficient (n = 15) areas. MAIN OUTCOME: Nodules from the iodine-sufficient area exhibited weak or absent hNIS immunostaining whereas almost all nodules from the iodine-deficient area were hNIS positive. Heterogeneous hNIS staining was common among the iodine-deficient samples (p = 0.028). hNIS was localized on membrane in all nodular samples from the iodine-deficient area and in less than 40% in the iodine-sufficient area. CONCLUSIONS: hNIS is adequately expressed and appropriately localized in NFTNs cell membrane from iodine-deficient areas and its expression in vivo is modulated by iodine supply.
Assuntos
Iodo/farmacologia , Simportadores/metabolismo , Glândula Tireoide/metabolismo , Adulto , Idoso , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Bócio/patologia , Bócio/cirurgia , Humanos , Iodetos/metabolismo , Masculino , Pessoa de Meia-Idade , Simportadores/genética , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/patologia , Glândula Tireoide/cirurgia , Hormônios Tireóideos/sangue , Nódulo da Glândula Tireoide/metabolismoRESUMO
Expression of phosphodiesterase 5 (PDE5) in the rat testis at several pre and postnatal developmental stages was investigated by immunohistochemistry. The enzyme was localised in vascular smooth muscle cells, as well as in Leydig and peritubular cells. The latter were identified as myoid, based on their immunoreactivity to desmin and alpha-smooth muscle actin. The presence of PDE5 in myoid cells was confirmed by Western blot analysis and immunohistochemistry performed on highly purified cell fractions, obtained from 16-day-old rats. The expression of PDE5 in these somatic cells of rat testis is discussed in view of the roles played by cGMP signal transduction pathways in the mammalian male reproductive function.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Desenvolvimento Fetal/fisiologia , Células Intersticiais do Testículo/enzimologia , Músculo Liso Vascular/enzimologia , Testículo/embriologia , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Western Blotting , Fracionamento Celular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5 , Técnica Indireta de Fluorescência para Anticorpo , Idade Gestacional , Técnicas Imunoenzimáticas , Células Intersticiais do Testículo/citologia , Masculino , Músculo Liso Vascular/citologia , Ratos , Ratos Wistar , Túbulos Seminíferos/citologia , Túbulos Seminíferos/metabolismo , Testículo/enzimologiaRESUMO
CONTEXT: Evidence from in vitro studies or animal models has shown that TSH affects thyrocytes by thyroid-specific expression modulation. OBJECTIVE: The objective of our study was to analyze the role of TSH in human thyroid gene expression in vivo. DESIGN/SETTING: Thirty-nine normal thyroid tissues were collected at the same center. STUDY SUBJECTS: Patients were divided into two groups based on serum TSH levels: 17 with normal TSH levels (1-4 mU/liter; group 1) and 22 with TSH levels below 0.5 mU/liter (group 2). INTERVENTION: Group 2 underwent thyroidectomy after suppressive L-T4 therapy. MAIN OUTCOME MEASURES: mRNA levels of thyroid genes such as sodium/iodide symporter (NIS), apical iodide transporter, pendrin, thyroglobulin, thyroperoxidase, TSH receptor, paired box transcription factor 8, and thyroid transcription factor-1 were evaluated by quantitative PCR. RESULTS: The reduction of TSH stimulation causes decreases in NIS and apical iodide transporter gene expression in normal tissues and more limited reductions in thyroglobulin, thyroperoxidase, and paired box transcription factor 8, but it has no significant effect on TSH receptor, pendrin, or thyroid transcription factor-1. Comparison of NIS levels in normal and nodular tissues from the same patient confirmed that it is differentially expressed in nodules only in the presence of normal TSH (P < 0.01). In patients with suppressed TSH, nodular NIS levels were similar to those in normal tissues. CONCLUSIONS: Our data represent the first demonstration in human thyroid tissues that TSH contributes to the regulation of thyrocyte differentiation by modulating thyroid gene levels. It exerts a particularly important effect on the transcription of NIS, which becomes very low after prolonged TSH suppression.
Assuntos
Regulação da Expressão Gênica/fisiologia , Glândula Tireoide/metabolismo , Tireotropina/farmacologia , Adulto , Idoso , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Técnicas In Vitro , Iodeto Peroxidase/biossíntese , Iodeto Peroxidase/genética , Masculino , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados , RNA Mensageiro/biossíntese , Receptores da Tireotropina/biossíntese , Receptores da Tireotropina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transportadores de Sulfato , Simportadores/biossíntese , Simportadores/genética , Tireoglobulina/biossíntese , Tireoglobulina/genética , Glândula Tireoide/citologia , Fator Nuclear 1 de Tireoide , Tireoidectomia , Transativadores/biossíntese , Transativadores/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Recovery of iodide uptake in thyroid cancer cells by means of obtaining the functional expression of the sodium/iodide symporter (NIS) represents an innovative strategy for the treatment of poorly differentiated thyroid cancer. However, the NIS gene expression alone is not always sufficient to restore radioiodine concentration ability in these tumour cells. METHODS: In this study, the anaplastic thyroid carcinoma ARO cells were stably transfected with a Pax8 gene expression vector. A quantitative RT-PCR was performed to assess the thyroid specific gene expression in selected clones. The presence of NIS protein was detected by Western blot and localized by immunofluorescence. A iodide uptake assay was also performed to verify the functional effect of NIS induction and differentiation switch. RESULTS: The clones overexpressing Pax8 showed the re-activation of several thyroid specific genes including NIS, Pendrin, Thyroglobulin, TPO and TTF1. In ARO-Pax8 clones NIS protein was also localized both in cell cytoplasm and membrane. Thus, the ability to uptake the radioiodine was partially restored, associated to a high rate of efflux. In addition, ARO cells expressing Pax8 presented a lower rate of cell growth. CONCLUSION: These finding demonstrate that induction of Pax8 expression may determine a re-differentiation of thyroid cancer cells, including a partial recovery of iodide uptake, fundamental requisite for a radioiodine-based therapeutic approach for thyroid tumours.
Assuntos
Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Box Pareados/biossíntese , Simportadores/biossíntese , Neoplasias da Glândula Tireoide/metabolismo , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Citoplasma/metabolismo , Primers do DNA/química , Vetores Genéticos , Humanos , Iodetos/metabolismo , Radioisótopos do Iodo/metabolismo , Proteínas de Membrana Transportadoras/biossíntese , Microscopia de Fluorescência , Fator de Transcrição PAX8 , Plasmídeos/metabolismo , RNA/química , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transportadores de Sulfato , Timo/metabolismo , Tireoglobulina/biossíntese , Neoplasias da Glândula Tireoide/radioterapia , Transcrição Gênica , TransfecçãoRESUMO
Pax proteins are transcriptional regulators that control a variety of developmental decisions in vertebrates. During development, the paired-box gene 8 (PAX8) is expressed in the thyroid, kidney, and several areas of the central nervous system. It is also expressed in the adult thyroid gland, in which it mediates TSH-induced modulation of the expression of important genes, such as those encoding thyroglobulin, thyroperoxidase, and the sodium/iodide symporter (NIS). Thus far, placental expression of PAX8 has been described only in mice. In the present study, we show that PAX8 is also expressed in the human placenta at term. In an in vitro model of placental cancer, the JAR choriocarcinoma cell line, human chorionic gonadotropin (hCG) increased levels of PAX8 mRNA and protein, and gel retardation assays indicated that the up-regulation of PAX8 protein expression is associated with an increase in its DNA-binding activity. The effects of hCG were mimicked by forskolin, indicating that they are cAMP dependent. Levels of mRNA for the Wilms' tumor 1 (WT1) and NIS genes were increased in JAR cells by hCG treatment, whereas overexpression of PAX8 increased only levels of WT1 mRNA. In cells transfected with PAX8-specific small interfering RNA, the stimulatory effects of hCG on WT1 mRNA levels were abolished, but hormonal enhancement of NIS mRNA levels was unchanged. These findings indicate that, in JAR cells, hCG activates a cAMP-dependent pathway that can up-regulate WT1 expression through PAX8.
Assuntos
Coriocarcinoma/fisiopatologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Placenta/fisiologia , Transativadores/genética , Transativadores/metabolismo , Neoplasias Uterinas/fisiopatologia , Linhagem Celular Tumoral , Gonadotropina Coriônica/farmacologia , AMP Cíclico/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados , Placenta/citologia , Gravidez , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Simportadores/genética , Transcrição Gênica/fisiologia , Proteínas WT1/genéticaRESUMO
OBJECTIVE: Suppressor of cytokine signaling (SOCS) proteins negatively regulate signal transduction of several cytokines. Since cytokines participate in the pancreatic islet damage in type 1 diabetes, the aim of our study was to investigate the expression of SOCS-1, -2 and -3 in isolated human islets, in basal conditions and after exposure, in vitro, to a combination of interferon (IFN)-gamma, interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha cytokines and in control and in type 1 diabetic human pancreata, to establish (i) whether SOCS molecules are constitutively expressed in human pancreatic islets and (ii) whether their expression can be modulated in vitro by proinflammatory cytokines or ex vivo by an islet inflammatory process. METHODS: Gene expression of SOCS-1, -2 and -3 was evaluated by RT-PCR in untreated and cytokine-treated isolated human pancreatic islets and their protein expression by immunohistochemistry in control and in type 1 diabetic human pancreata paraffin-embedded sections. RESULTS: We found that SOCS-1, -2 and -3 mRNA is constitutively, although weakly, expressed in human pancreatic islets, similar to the expression observed in control pancreata by immunohistochemistry. SOCS-1, -2 and -3 mRNA expression was strongly increased in human islets after exposure, in vitro, to IFN-gamma, IL-1beta and TNF-alpha. Accordingly, an intense and islet-specific immunohistochemical staining for all three SOCS was detected in pancreata from type 1 diabetic patients. CONCLUSION: SOCS-1, -2 and -3 genes are constitutively expressed in human pancreatic islets; their expression increases after exposure to proinflammatory cytokines and during an autoimmune inflammatory process, raising the possibility that these molecules act as key regulators of cytokine signaling in pancreatic islets.
Assuntos
Citocinas/farmacologia , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Estudos de Casos e Controles , Proteínas de Ligação a DNA/genética , Diabetes Mellitus Tipo 2/metabolismo , Expressão Gênica , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Interferon gama/farmacologia , Interleucina-1/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ilhotas Pancreáticas/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina , Transativadores/genética , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Sodium/iodide symporter (NIS) expression has recently been described in human breast cancer, with emphasis on its potential exploitation for the treatment of these tumors with radioiodine. In this study, we analyzed the regulation of NIS expression and function in the MCF-7 human breast cancer cell line. Cell exposure to insulin, IGF-I, IGF-II, or prolactin induced significant increases in 125I uptake and the expression of both NIS mRNA and NIS protein. The latter increases were evident after 6 and 12 h of hormonal stimulation, respectively. In immunocytochemistry studies, NIS was detected mainly in the plasma membrane of MCF-7 cells. A low but significant increase in iodide uptake was produced by treatment with activators of the adenylyl cyclase (cAMP) or protein kinase C pathways. Our study demonstrates that: 1) MCF-7 breast cancer cells are capable of active iodide transport that can be stimulated by insulin, IGF-I, IGF-II, or prolactin; 2) both NIS transcript and protein are expressed in these cells, and this expression is also hormonally stimulated; and 3) MCF-7 iodide transport and NIS expression may be influenced by the activation of cAMP or protein kinase C-dependent signaling. These findings increase our understanding of the molecular mechanisms that regulate NIS expression in breast cancer cells, information that is fundamental for future research aimed at the development of targeted radioiodide treatment for this type of cancer.