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2.
Mol Cell Endocrinol ; 566-567: 111911, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-36905979

RESUMO

Liver impact of prolonged GH-treatment given to non-GH-deficient growing mice between the third and eighth week of life was evaluated in both sexes. Tissues were collected 6 h after last dose or four weeks later. Somatometric, biochemical, histological, immunohistochemical, RT-qPCR and immunoblotting determinations were performed. Five-week GH intermittent administration induced body weight gain and body and bone length increase, augmented organ weight, higher hepatocellular size and proliferation, and increased liver IGF1 gene expression. Phosphorylation of signaling mediators and expression of GH-induced proliferation-related genes was decreased in GH-treated mice liver 6h after last injection, reflecting active sensitization/desensitization cycles. In females, GH elicited EGFR expression, associated to higher EGF-induced STAT3/5 phosphorylation. Four weeks after treatment, increased organ weight concomitant to body weight gain was still observed, whereas hepatocyte enlargement reverted. However, basal signaling for critical mediators was lower in GH-treated animals and in male controls compared to female ones, suggesting signaling declination.


Assuntos
Hormônio do Crescimento , Transdução de Sinais , Camundongos , Masculino , Feminino , Animais , Hormônio do Crescimento/metabolismo , Fosforilação , Fígado/metabolismo , Peso Corporal
3.
J Mol Endocrinol ; 69(2): 357-376, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35608964

RESUMO

Growth hormone (GH) exerts major actions in cardiac growth and metabolism. Considering the important role of insulin in the heart and the well-established anti-insulin effects of GH, cardiac insulin resistance may play a role in the cardiopathology observed in acromegalic patients. As conditions of prolonged exposure to GH are associated with a concomitant increase of circulating GH, IGF1 and insulin levels, to dissect the direct effects of GH, in this study, we evaluated the activation of insulin signaling in the heart using four different models: (i) transgenic mice overexpressing GH, with chronically elevated GH, IGF1 and insulin circulating levels; (ii) liver IGF1-deficient mice, with chronically elevated GH and insulin but decreased IGF1 circulating levels; (iii) mice treated with GH for a short period of time; (iv) primary culture of rat cardiomyocytes incubated with GH. Despite the differences in the development of cardiomegaly and in the metabolic alterations among the three experimental mouse models analyzed, exposure to GH was consistently associated with a decreased response to acute insulin stimulation in the heart at the receptor level and through the PI3K/AKT pathway. Moreover, a blunted response to insulin stimulation of this signaling pathway was also observed in cultured cardiomyocytes of neonatal rats incubated with GH. Therefore, the key novel finding of this work is that impairment of insulin signaling in the heart is a direct and early event observed as a consequence of exposure to GH, which may play a major role in the development of cardiac pathology.


Assuntos
Acromegalia , Hormônio do Crescimento Humano , Animais , Hormônio do Crescimento/metabolismo , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Transdução de Sinais
4.
Trends Endocrinol Metab ; 32(6): 403-414, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33838976

RESUMO

Epidermal growth factor receptor (EGFR) signaling has a central role in the regenerative response of the liver upon injury and is involved in cellular transformation linked to chronic damage. Hepatic EGFR expression, trafficking, and signaling are regulated by growth hormone (GH). Chronically elevated GH levels are associated with liver cancer development and progression in mice. Studies in different in vivo experimental models indicate that EGF and GH mutually crossregulate in a complex manner. Several factors, such as the extent of exposure to supraphysiological GH levels and the pattern of GH administration, are important variables to be considered in exploring the interplay between the two hormones in connection with the progression of hepatic tumors.


Assuntos
Fator de Crescimento Epidérmico , Receptores ErbB , Hormônio do Crescimento , Fígado , Animais , Fator de Crescimento Epidérmico/fisiologia , Receptores ErbB/fisiologia , Camundongos
5.
Mol Cell Endocrinol ; 509: 110802, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32259636

RESUMO

Continuously elevated levels of growth hormone (GH) during life in mice are associated with hepatomegaly due to hepatocytes hypertrophy and hyperplasia, chronic liver inflammation, elevated levels of arachidonic acid (AA) at young ages and liver tumors development at old ages. In this work, the hepatic expression of enzymes involved in AA metabolism, cPLA2α, COX1 and COX2 enzymes, was evaluated in young and old GH-transgenic mice. Mice overexpressing GH exhibited higher hepatic expression of cPLA2α, COX1 and COX2 in comparison to controls at young and old ages and in both sexes. In old mice, when tumoral and non-tumoral tissue were compared, elevated expression of COX2 was observed in tumors. In contrast, exposure to continuous lower levels of hormone for a short period affected COX1 expression only in males. Considering the role of inflammation during liver tumorigenesis, these findings support a role of alterations in AA metabolism in GH-driven liver tumorigenesis.


Assuntos
Fosfolipases A2 do Grupo IV/genética , Hormônio do Crescimento/metabolismo , Fígado/metabolismo , Prostaglandina-Endoperóxido Sintases/genética , Regulação para Cima/genética , Alanina Transaminase/sangue , Animais , Peso Corporal , Bovinos , Proliferação de Células , Feminino , Fosfolipases A2 do Grupo IV/metabolismo , Hepatócitos/citologia , Fígado/anatomia & histologia , Masculino , Camundongos Transgênicos , Tamanho do Órgão , Fosforilação , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Receptor IGF Tipo 1/metabolismo , Receptores da Somatotropina/metabolismo
6.
Mol Cell Endocrinol ; 498: 110587, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31539597

RESUMO

The renin-angiotensin system modulates insulin action. Pharmacological stimulation of angiotensin type 2 receptor (AT2R) was shown to have beneficial metabolic effects in various animal models of insulin resistance and type 2 diabetes and also to increase insulin sensitivity in wild type mice. In this study we further explored the role of the AT2R on insulin action and glucose homeostasis by investigating the glycemic profile and in vivo insulin signaling status in insulin-target tissues from both male and female AT2R knockout (KO) mice. When compared to the respective wild-type (WT) group, glycemia and insulinemia was unaltered in AT2RKO mice regardless of sex. However, female AT2RKO mice displayed decreased insulin sensitivity compared to their WT littermates. This was accompanied by a compensatory increase in adiponectinemia and with a specific attenuation of the activity of main insulin signaling components (insulin receptor, Akt and ERK1/2) in adipose tissue with no apparent alterations in insulin signaling in either liver or skeletal muscle. These parameters remained unaltered in male AT2RKO mice as compared to male WT mice. Present data show that the AT2R has a physiological role in the conservation of insulin action in female but not in male mice. Our results suggest a sexual dimorphism in the control of insulin action and glucose homeostasis by the AT2R and reinforce the notion that pharmacological modulation of the balance between the AT1R and AT2R receptor could be important for treatment of metabolic syndrome and type 2 diabetes.


Assuntos
Adiponectina/sangue , Biomarcadores/sangue , Glicemia/metabolismo , Resistência à Insulina , Insulina/sangue , Receptor Tipo 2 de Angiotensina/fisiologia , Caracteres Sexuais , Tecido Adiposo/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores Sexuais , Transdução de Sinais
7.
Endocr Connect ; 8(8): 1108-1117, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31272083

RESUMO

Transgenic mice overexpressing growth hormone (GH) spontaneously develop liver tumors, including hepatocellular carcinoma (HCC), within a year. The preneoplastic liver pathology in these mice recapitulates that observed in humans at high risk of developing hepatic cancer. Although increased expression of galectin 1 (GAL1) in liver tissue is associated with HCC aggressiveness, a link between this glycan-binding protein and hormone-related tumor development has not yet been explored. In this study, we investigated GAL1 expression during liver tumor progression in mice continuously exposed to high levels of GH. GAL1 expression was determined by Western blotting, RT-qPCR and immunohistochemistry in the liver of transgenic mice overexpressing GH. Animals of representative ages at different stages of liver pathology were studied. GAL1 expression was upregulated in the liver of GH-transgenic mice. This effect was observed at early ages, when animals displayed no signs of liver disease or minimal histopathological alterations and was also detected in young adults with preneoplastic liver pathology. Remarkably, GAL1 upregulation was sustained during aging and its expression was particularly enhanced in liver tumors. GH also induced hepatic GAL1 expression in mice that were treated with this hormone for a short period. Moreover, GH triggered a rapid increment in GAL1 protein expression in human HCC cells, denoting a direct effect of the hormone on hepatocytes. Therefore, our results indicate that GH upregulates GAL1 expression in mouse liver, which may have critical implications in tumorigenesis. These findings suggest that this lectin could be implicated in hormone-driven liver carcinogenesis.

8.
Biochem Mol Biol Educ ; 47(1): 93-99, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30576049

RESUMO

Enzyme kinetics is an essential topic in undergraduate Biochemistry courses. A laboratory work that covers the principal basic concepts of enzyme kinetics in steady state is presented. The alkaline phosphatase catalyzed reaction of phenyl-phosphate hydrolysis was studied as a model. The laboratory experience was designed to reinforce the concepts of initial velocity dependence on substrate and enzyme concentration, and to highlight the importance of the accurate determination of initial reaction rate. The laboratory work consists in two parts, in which students first determine the enzyme concentration and the time to be used in the following session to obtain the kinetic parameters (KM and Vmax ) by non-lineal fitting of the Michaelis-Menten equation to the initial velocity dependence with substrate concentration results. The experimental methodology is robust, the cost per student is low and the equipment and reagents used are of easy access. © 2018 International Union of Biochemistry and Molecular Biology, 47(1):93-99, 2018.


Assuntos
Fosfatase Alcalina/metabolismo , Bioquímica , Laboratórios , Modelos Biológicos , Universidades , Humanos , Cinética , Estudantes
9.
Int J Mol Sci ; 18(5)2017 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-28509880

RESUMO

The reliability of reverse transcription-quantitative PCR (RT-qPCR) results in gene expression studies depends on the approaches used to account for non-biological variations. In order to find a proper normalization strategy for the study of genes related to growth hormone signaling in skeletal muscle of growing mice, nine unrelated genes were evaluated as internal controls. According to the most used algorithms-geNorm, the Comparative ΔCq method, NormFinder and BestKeeper-GSK3B, YWHAZ, RPL13A and RN18S were found as the most stable. However, the relative expression levels of eight of the potential reference genes assessed decreased with age in cDNA samples obtained from the same amount of total RNA. In a different approach to analyze this apparent discrepancy, experiments were performed with cDNA obtained from equal amounts of purified mRNA. Since the decline was still observed, the hypothesis of an age-related change in mRNA to total RNA ratio that could account for the systematic decrease was rejected. Differences among experimental groups could be due to a substantial increase with age in highly expressed mRNAs, which would bias the quantitation of the remaining genes. Consequently, those reference genes reflecting this dilution effect, which would have been discarded considering their variable relative expression levels, arose as suitable internal controls.


Assuntos
Perfilação da Expressão Gênica , Marcadores Genéticos , Reação em Cadeia da Polimerase em Tempo Real , Animais , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Regulação da Expressão Gênica , Hormônio do Crescimento/genética , Camundongos , Músculo Esquelético/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
J Endocrinol ; 233(2): 175-186, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28223314

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/metabolismo
11.
Cell Cycle ; 15(5): 748-59, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27028000

RESUMO

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 Sinais
13.
J Mol Endocrinol ; 54(2): 171-84, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25691498

RESUMO

GH/STAT5 signaling is desensitized in the liver in adult transgenic mice overexpressing GH; however, these animals present greater body size. To assess whether the STAT5 pathway is active during the growth period in the liver in these animals, and how signaling modulators participate in this process, growing transgenic mice and normal siblings were evaluated. STAT5 does not respond to an acute GH-stimulus, but displays higher basal phosphorylation in the livers of growing GH-overexpressing mice. GH receptor and the positive modulators glucocorticoid receptor and HNF1 display greater abundance in transgenic animals, supporting the activity of STAT5. The negative modulators cytokine-induced suppressor and PTP1B are increased in GH-overexpressing mice. The suppressors SOCS2 and SOCS3 exhibit higher mRNA levels in transgenic mice but lower protein content, indicating that they are being actively degraded. Therefore, STAT5 signaling is increased in the liver in GH-transgenic mice during the growth period, with a balance between positive and negative effectors resulting in accelerated but controlled growth.


Assuntos
Hormônio do Crescimento/metabolismo , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Animais , Regulação da Expressão Gênica no Desenvolvimento , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Receptores da Somatotropina/genética , Receptores da Somatotropina/metabolismo , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo
14.
Biomed Res Int ; 2014: 687037, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24772432

RESUMO

Lecithins, mainly composed of the phospholipids phosphatidylcholines (PC), have many different uses in the pharmaceutical and clinical field. PC are involved in structural and biological functions as membrane trafficking processes and cellular signaling. Considering the increasing applications of lecithin-based nanosystems for the delivery of therapeutic agents, the aim of the present work was to determine the effects of phosphatidylcholine nanoparticles over breast cancer cellular proliferation and signaling. PC dispersions at 0.01 and 0.1% (w/v) prepared in buffer pH 7.0 and 5.0 were studied in the MCF-7 breast cancer cell line. Neutral 0.1% PC-derived nanoparticles induced the activation of the MEK-ERK1/2 pathway, increased cell viability and induced a 1.2 fold raise in proliferation. These biological effects correlated with the increase of epidermal growth factor receptor (EGFR) content and its altered cellular localization. Results suggest that nanoparticles derived from PC dispersion prepared in buffer pH 7.0 may induce physicochemical changes in the plasma membrane of cancer cells which may affect EGFR cellular localization and/or activity, increasing activation of the MEK-ERK1/2 pathway and inducing proliferation. Results from the present study suggest that possible biological effects of delivery systems based on lecithin nanoparticles should be taken into account in pharmaceutical formulation design.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mitógenos , Nanopartículas/química , Fosfatidilcolinas , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Feminino , Humanos , Mitógenos/química , Mitógenos/farmacologia , Proteínas de Neoplasias/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/farmacologia
15.
J Endocrinol ; 221(2): 309-23, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24623798

RESUMO

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/genética
16.
Cell Cycle ; 12(7): 1042-57, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23428905

RESUMO

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 Transcricional
17.
Growth Horm IGF Res ; 23(1-2): 19-28, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23245546

RESUMO

Postnatal growth exhibits two instances of rapid growth in mice: the first is perinatal and independent of growth hormone (GH), the second is peripuberal and GH-dependent. Signal transducer and activator of transcription 5b (STAT5b) is the main GH-signaling mediator and it is related to IGF1 synthesis and somatic growth. The aim of this work was to assess differential STAT5 sensitivity to GH during the growth period in mouse liver of both sexes. Three representative ages were selected: 1-week-old animals, in the GH-independent phase of growth; 2.5-week-old mice, at the onset of the GH-dependent phase of growth; and 9-week-old young adults. GH-signaling mediators were assessed by immunoblotting, quantitative RT-PCR and immunohistochemistry. GH-induced STAT5 phosphorylation is low at one-week and maximal at 2.5-weeks of age when compared to young adults, accompanied by higher protein content at the onset of growth. Suppressor CIS and phosphatase PTP1B exhibit high levels in one-week animals, which gradually decline, while SOCS2 and SOCS3 display higher levels at adulthood. Nuclear phosphorylated STAT5 is low in one-week animals while in 2.5-week animals it is similar to 9-week control; expression of SOCS3, an early response GH-target gene, mimics this pattern. STAT5 coactivators glucocorticoid receptor (GR) and hepatic nuclear factor 1 (HNF1) abundance is higher in adulthood. Therefore, GH-induced STAT5 signaling presents age-dependent activity in liver, with its maximum coinciding with the onset of GH-dependent phase of growth, accompanied by an age-dependent variation of modulating factors. This work contributes to elucidate the molecular mechanisms implicated in GH responsiveness during growth.


Assuntos
Hormônio do Crescimento/metabolismo , Crescimento e Desenvolvimento , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Fator de Transcrição STAT5/metabolismo , Fatores Etários , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Feminino , Hormônio do Crescimento/farmacologia , Crescimento e Desenvolvimento/efeitos dos fármacos , Crescimento e Desenvolvimento/genética , Crescimento e Desenvolvimento/fisiologia , Janus Quinase 2/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Receptores da Somatotropina/genética , Receptores da Somatotropina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
18.
Cell Signal ; 24(2): 514-523, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22019461

RESUMO

The epidermal growth factor (EGF) activates the phosphatidylinositol 3-kinase (PI3K)-Akt cascade among other signaling pathways. This route is involved in cell proliferation and survival, therefore, its dysregulation can promote cancer. Considering the relevance of the PI3K-Akt signaling in cell survival and in the pathogenesis of cancer, and that GH was reported to modulate EGFR expression and signaling, the objective of this study was to analyze the effects of increased GH levels on EGF-induced PI3K-Akt signaling. EGF-induced signaling was evaluated in the liver of GH-overexpressing transgenic mice and in their normal siblings. While Akt expression was increased in GH-overexpressing mice, EGF-induced phosphorylation of Akt, relative to its protein content, was diminished at Ser473 and inhibited at Thr308; consequently, mTOR, which is a substrate of Akt, was not activated by EGF. However, the activation of PDK1, a kinase involved in Akt phosphorylation at Thr308, was not reduced in transgenic mice. Kinetics studies of EGF-induced Akt phosphorylation showed that it is rapidly and transiently induced in GH-overexpressing mice compared with normal siblings. Thus, the expression and activity of phosphatases involved in the termination of the PI3K-Akt signaling were studied. In transgenic mice, neither PTEN nor PP2A were hyperactivated; however, EGF induced the rapid and transient association of SHP-2 to Gab1, which mediates association to EGFR and activation of PI3K. Rapid recruitment of SHP2, which would accelerate the termination of the proliferative signal induced, could be therefore contributing to the diminished EGF-induced activity of Akt in GH-overexpressing mice.


Assuntos
Fator de Crescimento Epidérmico/metabolismo , Expressão Gênica , Hormônio do Crescimento/metabolismo , Fígado/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Fator de Crescimento Epidérmico/genética , Hormônio do Crescimento/genética , Fígado/citologia , Camundongos , Camundongos Transgênicos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinase/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil , Serina/genética , Serina/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Treonina/genética , Treonina/metabolismo
19.
Clin Immunol ; 136(3): 432-41, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20542739

RESUMO

We have previously shown a decreased frequency and function of Tregs in women suffering from recurrent spontaneous abortions (RSA). In the current study, we first investigated the expression of FOXP3 after T-cell activation. We observed that expression of FOXP3 in activated PBMCs was already present above baseline before any cell division, indicating that it was induced in cells that were previously negative for this transcription factor. Because RSA women showed a more limited expansion of FOXP3-positive cells, we next assessed the role of IL-2 signaling through STAT5, which is known to be required for generation of inducible Tregs (iTregs). We demonstrated not only that TGF-beta and IL-2 were diminished but also that the IL-2-STAT-5 signaling axis was down regulated in RSA women. Finally, in addition to a limited FOXP3(+) cells expansion in vitro, iTregs from RSA women showed a strikingly lower suppressor activity.


Assuntos
Aborto Habitual/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Estudos de Casos e Controles , Feminino , Fase Folicular/imunologia , Fatores de Transcrição Forkhead/metabolismo , Humanos , Tolerância Imunológica , Técnicas In Vitro , Interleucina-2/metabolismo , Interleucina-6/metabolismo , Isoantígenos/administração & dosagem , Cinética , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Masculino , Pessoa de Meia-Idade , Gravidez , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Adulto Jovem
20.
Growth Horm IGF Res ; 20(2): 118-26, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20022531

RESUMO

CONTEXT: Growth hormone (GH) is an anabolic hormone that regulates growth and metabolism. Ames dwarf mice are natural mutants for Prop1, with impaired development of anterior pituitary and undetectable levels of circulating GH, prolactin and TSH. They constitute an endocrine model of life-long GH-deficiency. The main signaling cascades activated by GH binding to its receptor are the JAK2/STATs, PI-3K/Akt and the MAPK Erk1/2 pathways. OBJECTIVES: We have previously reported that GH-induced STAT5 activation was higher in Ames dwarf mice liver compared to non-dwarf controls. The aim of this study was to evaluate the principal components of the main GH-signaling pathways under GH-deficiency in liver and skeletal muscle, another GH-target tissue. METHODS: Ames dwarf mice and their non-dwarf siblings were assessed. Animals were injected i.p. with GH or saline 15min before tissue removal. Protein content and phosphorylation of signaling mediators were determined by immunoblotting of tissue solubilizates. RESULTS: GH was able to induce STAT5 and STAT3 tyrosine phosphorylation in both liver and muscle, but the response was higher for Ames dwarf mice than for non-dwarf controls. When Erk1/2 activation was assessed in liver, only dwarf mice showed GH-induced phosphorylation, while in muscle no response to the hormone was found in either genotype. GH-induced Akt phosphorylation at Ser473 in liver was only detected in dwarf mice. In skeletal muscle, both normal and dwarf mice responded to a GH stimulus, although dwarf mice presented higher GH activation levels. The phosphorylation of GSK-3, a substrate of Akt, increased upon hormone stimulation only in dwarf mice in both tissues. In contrast, no differences in the phosphorylation of mTOR, another substrate of Akt, were observed after GH stimulus, either in normal or dwarf mice in liver, while we were unable to determine mTOR in muscle. Protein content of GH-receptor and of the signaling mediators studied did not vary between normal and dwarf animals in the assessed tissues. CONCLUSION: These results show that several components of the main GH-signaling pathways exhibit enhanced sensitivity to the hormone in liver and muscle of Ames dwarf mice.


Assuntos
Nanismo Hipofisário/metabolismo , Hormônio do Crescimento/farmacologia , Proteínas de Homeodomínio/genética , Fígado/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/genética , Nanismo Hipofisário/genética , Nanismo Hipofisário/patologia , Quinase 3 da Glicogênio Sintase/metabolismo , Hormônio do Crescimento/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Transgênicos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores da Somatotropina/agonistas , Receptores da Somatotropina/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR
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