RESUMO
Transgenic mice overexpressing growth hormone (GH) show increased hepatic protein content of the epidermal growth factor receptor (EGFR), which is broadly associated with cell proliferation and oncogenesis. However, chronically elevated levels of GH result in desensitization of STAT-mediated EGF signal and similar response of ERK1/2 and AKT signaling to EGF compared to normal mice. To ascertain the mechanisms involved in GH attenuation of EGF signaling and the consequences on cell cycle promotion, phosphorylation of signaling mediators was studied at different time points after EGF stimulation, and induction of proteins involved in cell cycle progression was assessed in normal and GH-overexpressing transgenic mice. Results from kinetic studies confirmed the absence of STAT3 and 5 activation and comparable levels of ERK1/2 phosphorylation upon EGF stimulation, which was associated with diminished or similar induction of c-MYC, c-FOS, c-JUN, CYCLIN D1 and CYCLIN E in transgenic compared to normal mice. Accordingly, kinetics of EGF-induced c-SRC and EGFR phosphorylation at activating residues demonstrated that activation of these proteins was lower in the transgenic mice with respect to normal animals. In turn, EGFR phosphorylation at serine 1046/1047, which is implicated in the negative regulation of the receptor, was increased in the liver of GH-overexpressing transgenic mice both in basal conditions and upon EGF stimulus. Increased basal phosphorylation and activation of the p38-mitogen-activated protein kinase might account for increased Ser 1046/1047 EGFR. Hyperphosphorylation of EGFR at serine residues would represent a compensatory mechanism triggered by chronically elevated levels of GH to mitigate the proliferative response induced by EGF.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Regulação da Expressão Gênica/fisiologia , Hormônio do Crescimento/metabolismo , Transdução de Sinais/fisiologia , Animais , Receptores ErbB/genética , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes src/genética , Genes src/fisiologia , Hormônio do Crescimento/genética , Humanos , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Growth hormone (GH) is a pleiotropic hormone that triggers STATs, ERK1/2 and Akt signaling, related to cell growth and proliferation. Transgenic mice overexpressing GH present increased body size, with a disproportionate liver enlargement due to hypertrophy and hyperplasia of the hepatocytes. We had described enhanced mitogenic signaling in liver of young adult transgenic mice. We now evaluate the activation of these signaling cascades during the growth period and relate them to the morphological alterations found. Signaling mediators, cell cycle regulators and transcription factors involved in cellular growth in the liver of GH-overexpressing growing mice were assessed by immunoblotting, RT-qPCR and immunohistochemistry. Hepatocyte enlargement can be seen as early as 2-weeks of age in GH-overexpressing animals, although it is more pronounced in young adults. Levels of cell cycle mediators PCNA and cyclin D1, and transcription factor c-Jun increase with age in transgenic mice with no changes in normal mice, whereas c-Myc levels are higher in 2-week-old transgenic animals and cyclin E levels decline with age for both genotypes. STAT3, Akt and GSK3 present higher activation in the adult transgenic mice than in the growing animals, while for c-Src and mTOR, phosphorylation in GH-overexpressing mice is higher than in control siblings at 4 and 9 weeks of age. No significant changes are observed for ERK1/2, neither by age or genotype. Thus, the majority of the mitogenic signaling pathways are gradually up-regulated in the liver of GH-transgenic mice, giving rise to the hepatic morphological changes these mice exhibit.
Assuntos
Hormônio do Crescimento/metabolismo , Fígado/metabolismo , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fígado/citologia , Fígado/crescimento & desenvolvimento , Masculino , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitose , Tamanho do Órgão , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
Aged testes undergo profound histological and morphological alterations leading to a reduced functionality. Here, we investigated whether variations in longevity affect the development of local inflammatory processes, the oxidative state and the occurrence of apoptotic events in the testis. To this aim, well-established mouse models with delayed (growth hormone releasing hormone-knockout and Ames dwarf mice) or accelerated (growth hormone-transgenic mice) aging were used. We hereby show that the testes of short-lived mice show a significant increase in cyclooxygenase 2 expression, PGD2 production, lipid peroxidation, antioxidant enzymes expression, local macrophages and TUNEL-positive germ cells numbers, and the levels of both pro-caspase-3 and cleaved caspase-3. In contrast, although the expression of antioxidant enzymes remained unchanged in testes of long-lived mice, the remainder of the parameters assessed showed a significant reduction. This study provides novel evidence that longevity confers anti-inflammatory, anti-oxidant and anti-apoptotic capacities to the adult testis. Oppositely, short-lived mice suffer testicular inflammatory, oxidative and apoptotic processes.
Assuntos
Envelhecimento/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Mediadores da Inflamação/metabolismo , Estresse Oxidativo , Testículo/metabolismo , Fatores Etários , Envelhecimento/genética , Envelhecimento/patologia , Animais , Biomarcadores/metabolismo , Caspase 3/metabolismo , Ciclo-Oxigenase 2/metabolismo , Genótipo , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento/deficiência , Hormônio Liberador de Hormônio do Crescimento/genética , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/patologia , Peroxidação de Lipídeos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fenótipo , Prostaglandina D2/metabolismo , Transdução de Sinais , Testículo/patologiaRESUMO
Current GH administration protocols imply frequent s.c. injections, resulting in suboptimal compliance. Therefore, there is interest in developing delivery systems for sustained release of the hormone. However, GH has different actions depending on its continuous or pulsatile plasma concentration pattern. GH levels and circulating concentration patterns could be involved in the regulation of epidermal growth factor receptor (EGFR) expression in liver. Aberrant expression of this receptor and/or its hyperactivation has been associated with the pathogenesis of different types of carcinoma. Considering that one of the adverse effects associated with GH overexpression and chronic use of GH is the increased incidence of malignancies, the aim of this study was to analyze the effects of GH plasma concentration patterns on EGFR expression and signaling in livers of mice. For this purpose, GH was administered by s.c. daily injections to produce an intermittent plasma pattern or by osmotic pumps to provoke a continuously elevated GH concentration. Intermittent injections of GH induced upregulation of liver EGFR content, augmented the response to EGF, and the induction of proteins involved in promotion of cell proliferation in female mice. In contrast, continuous GH delivery in male mice was associated with diminished EGFR in liver and decreased EGF-induced signaling and expression of early genes. The results indicate that sustained delivery systems that allow continuous GH plasma patterns would be beneficial in terms of treatment safety with regard to the actions of GH on EGFR signaling and its promitogenic activity.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Hormônio do Crescimento/administração & dosagem , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Esquema de Medicação , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/sangue , Bombas de Infusão , Injeções , Masculino , Camundongos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
The α1-adrenergic receptors (α1-ARs) are involved in preconditioning. Given that certain intracellular pathways seem to be shared by preconditioning and postconditioning, it is possible that postconditioning could also be mediated by α1-ARs. The objective was to evaluate, by analyzing infarct size, if α1-ARs activation could trigger postconditioning and also determine Akt and glycogen synthase kinase 3ß (GSK-3ß) phosphorylation. Langendorff-perfused rat hearts were subjected to 30 minutes of ischemia and 120 minutes of reperfusion (I/R; n = 8). After 30 minutes of global ischemia, we performed 6 cycles of reperfusion/ischemia of 10 seconds each, followed by 120 minutes of reperfusion [ischemic postconditioning group (postcon); n = 9]. In another postcon group, we administered prazosin during postcon protocol (postcon + prazosin; n = 7). Finally, we repeated the I/R group, but prazosin (prazosin; n = 7), phenylephrine (PE; n = 5) and clonidine (CL; n = 6) were administered during the first 2 minutes of reperfusion. Infarct size was measured using the triphenyltetrazolium chloride technique. Total and phosphorylated Akt and mitochondrial GSK-3ß expression were measured by Western blot. Infarct size was 58.1 ± 5.1% in I/R. Postcon and PE reduced infarct size to 40.1 ± 2.9% and 35.3 ± 5.5%, respectively (P < 0.05 vs. I/R). Postcon + prazosin administration abolished the beneficial effect on infarct size (61.6 ± 4.5%; P < 0.05 vs. postcon). Cytosolic Akt phosphorylation and mitochondrial GSK-3ß phosphorylation were higher in the postcon and PE groups compared with the I/R and postcon + prazosin groups. Prazosin or clonidine administration did not modify neither protein expression nor infarct size. Our data demonstrate that postconditioning decrease infarct size by activation of the α1-AR pathway through Akt and GSK-3ß phosphorylation.
Assuntos
Pós-Condicionamento Isquêmico/métodos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/terapia , Receptores Adrenérgicos alfa 1/metabolismo , Transdução de Sinais/fisiologia , Animais , Masculino , Ratos , Ratos WistarRESUMO
Growth hormone (GH) overexpression throughout life in transgenic mice is associated with the development of liver tumors at old ages. The preneoplastic pathology observed in the liver of young adult GH-overexpressing mice is similar to that present in humans at high risk of hepatic cancer. To elucidate the molecular pathogenesis underlying the pro-oncogenic liver pathology induced by prolonged exposure to elevated GH levels, the activation and expression of several components of signal transduction pathways that have been implicated in hepatocellular carcinogenesis were evaluated in the liver of young adult GH-transgenic mice. In addition, males and females were analyzed in parallel in order to evaluate sexual dimorphism. Transgenic mice from both sexes exhibited hepatocyte hypertrophy with enlarged nuclear size and exacerbated hepatocellular proliferation, which were higher in males. Dysregulation of several oncogenic pathways was observed in the liver of GH-overexpressing transgenic mice. Many signaling mediators and effectors were upregulated in transgenic mice compared with normal controls, including Akt2, NFκB, GSK3ß, ß-catenin, cyclin D1, cyclin E, c-myc, c-jun and c-fos. The molecular alterations described did not exhibit sexual dimorphism in transgenic mice except for higher gene expression and nuclear localization of cyclin D1 in males. We conclude that prolonged exposure to GH induces in the liver alterations in signaling pathways involved in cell growth, proliferation and survival that resemble those found in many human tumors.
Assuntos
Hormônio do Crescimento/metabolismo , Fígado/patologia , Animais , Núcleo Celular/metabolismo , Transformação Celular Neoplásica , Ciclina D1/metabolismo , Feminino , Expressão Gênica , Hormônio do Crescimento/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tamanho do Órgão , Fosforilação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Ativação TranscricionalRESUMO
OBJECTIVE: Growth hormone (GH) resistance leads to enhanced insulin sensitivity, decreased systolic blood pressure and increased lifespan. The aim of this study was to determine if there is a shift in the balance of the renin-angiotensin system (RAS) towards the ACE2/Ang-(1-7)/Mas receptor axis in the heart and the kidney of a model of GH resistance and retarded aging, the GH receptor knockout (GHR-/-) mouse. DESIGN: RAS components were evaluated in the heart and the kidney of GHR-/- and control mice by immunohistochemistry and Western blotting (n=12 for both groups). RESULTS: The immunostaining of Ang-(1-7) was increased in both the heart and the kidney of GHR-/- mice. These changes were concomitant with an increased immunostaining of the Mas receptor and ACE2 in both tissues. The immunostaining of AT1 receptor was reduced in heart and kidney of GHR-/- mice while that of AT2 receptor was increased in the heart and unaltered in the kidney. Ang II, ACE and angiotensinogen levels remained unaltered in the heart and the kidney of GH resistant mice. These results were confirmed by Western blotting and correlated with a significant increase in the abundance of the endothelial nitric oxide synthase in both tissues. CONCLUSIONS: The shift within the RAS towards an exacerbation of the ACE2/Ang-(1-7)/Mas receptor axis observed in GHR-/- mice could be related to a protective role in cardiac and renal function; and thus, possibly contribute to the decreased incidence of cardiovascular diseases displayed by this animal model of longevity.
Assuntos
Angiotensina I/genética , Rim/metabolismo , Miocárdio/metabolismo , Fragmentos de Peptídeos/genética , Peptidil Dipeptidase A/genética , Proteínas Proto-Oncogênicas/genética , Receptores Acoplados a Proteínas G/genética , Receptores da Somatotropina/genética , Regulação para Cima , Angiotensina I/biossíntese , Enzima de Conversão de Angiotensina 2 , Animais , Camundongos , Camundongos Knockout , Fragmentos de Peptídeos/biossíntese , Peptidil Dipeptidase A/biossíntese , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/biossíntese , Receptores Acoplados a Proteínas G/biossínteseRESUMO
The aim of the present work was to take advantage of lecithin's biocompatibility along with its physicochemical properties for the preparation of lecithin-based nanocarriers for small interfering RNA (siRNA) delivery. Water lecithin dispersions were prepared in different conditions, loaded with siRNA at different N/P ratios, and evaluated for loading capacity. The most appropriate ones were then assayed for cytotoxicity and characterized in terms of particle size distribution, zeta potential, and morphology. Results demonstrated that formulations prepared at pH 5.0 and 7.0 were able to load siRNA at broad N/P ratios, and cellular uptake assays showed an efficient delivery of oligos in MCF-7 human breast cancer cells; fluorescent-labeled dsRNA mainly located next to its target, near the nucleus of the cells. No signs of toxicity were observed for broad compositions of lecithin. The physicochemical characterization of the siRNA-loaded dispersions exhibited particles of nanometric sizes and pH-dependant shapes, which make them suitable for ex vivo and in vivo further evaluation.
RESUMO
Angiotensin (ANG)-(1-7) is known to attenuate diabetic nephropathy; however, its role in the modulation of renal inflammation and oxidative stress in type 2 diabetes is poorly understood. Thus in the present study we evaluated the renal effects of a chronic ANG-(1-7) treatment in Zucker diabetic fatty rats (ZDF), an animal model of type 2 diabetes and nephropathy. Sixteen-week-old male ZDF and their respective controls [lean Zucker rats (LZR)] were used for this study. The protocol involved three groups: 1) LZR + saline, 2) ZDF + saline, and 3) ZDF + ANG-(1-7). For 2 wk, animals were implanted with subcutaneous osmotic pumps that delivered either saline or ANG-(1-7) (100 ng·kg(-1)·min(-1)) (n = 4). Renal fibrosis and tissue parameters of oxidative stress were determined. Also, renal levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), ED-1, hypoxia-inducible factor-1α (HIF-1α), and neutrophil gelatinase-associated lipocalin (NGAL) were determined by immunohistochemistry and immunoblotting. ANG-(1-7) induced a reduction in triglyceridemia, proteinuria, and systolic blood pressure (SBP) together with a restoration of creatinine clearance in ZDF. Additionally, ANG-(1-7) reduced renal fibrosis, decreased thiobarbituric acid-reactive substances, and restored the activity of both renal superoxide dismutase and catalase in ZDF. This attenuation of renal oxidative stress proceeded with decreased renal immunostaining of IL-6, TNF-α, ED-1, HIF-1α, and NGAL to values similar to those displayed by LZR. Angiotensin-converting enzyme type 2 (ACE2) and ANG II levels remained unchanged after treatment with ANG-(1-7). Chronic ANG-(1-7) treatment exerts a renoprotective effect in ZDF associated with a reduction of SBP, oxidative stress, and inflammatory markers. Thus ANG-(1-7) emerges as a novel target for treatment of diabetic nephropathy.