RESUMEN
There is substantial evidence supporting a hypertrophic action of serotonin [5-hydroxytryptamine (5-HT)] in cardiomyocytes. However, little is known about the mechanisms involved. We previously demonstrated that 5-HT-induced hypertrophy depends, in part, on the generation of reactive oxygen species by monoamine oxidase-A (MAO-A) (see Ref. 3). Cardiomyocytes express 5-HT(2) receptors, which may also participate in hypertrophy. Here, we analyzed the respective contribution of 5-HT(2) receptors and MAO-A in H9C2 cardiomyoblast hypertrophy. 5-HT induced a dose-dependent increase in [(3)H]leucine incorporation and stimulation of two markers of cardiac hypertrophy, ANF-luc and alphaSK-actin-luc reporter genes. Experiments using 1 microM 5-HT showed that hypertrophic response occurred independently from MAO-A. Using pharmacological inhibitors (M100907 and ketanserin), we identified a novel mechanism of action involving 5-HT(2A) receptors and requiring Ca(2+)/calcineurin/nuclear factor of activated T-cell activation. The activation of this hypertrophic pathway was fully prevented by 5-HT(2A) inhibitors and was unaffected by MAO inhibition. When 10 microM 5-HT was used, an additional hypertrophic response, prevented by the MAO inhibitors pargyline and RO 41-1049, was observed. Unlike the 5-HT(2A)-receptor-mediated H9C2 cell hypertrophy, MAO-A-dependent hypertrophic response required activation of extracellular-regulated kinases. In conclusion, our results show the existence of a dose-dependent shift of activation of distinct intracellular pathways involved in 5-HT-mediated hypertrophy of cardiac cells.
Asunto(s)
Cardiomegalia/inducido químicamente , Cardiomegalia/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Serotoninérgicos/toxicidad , Serotonina/toxicidad , Animales , Calcineurina/metabolismo , Cardiomegalia/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Leucina/farmacocinética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Monoaminooxidasa/genética , Monoaminooxidasa/metabolismo , Miocitos Cardíacos/citología , Factores de Transcripción NFATC/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Receptor de Serotonina 5-HT2A/genética , Receptor de Serotonina 5-HT2A/metabolismo , Tritio , Remodelación Ventricular/efectos de los fármacos , Remodelación Ventricular/fisiologíaRESUMEN
The c-fos gene expression is rapidly induced by various mitogenic agents and protein synthesis inhibitors in many cell types. Estradiol-17 beta can induce c-fos gene expression in breast cancer cell lines and in the uterus in vivo, but not in cultured guinea-pig endometrial cells. Using this model, we investigated whether a protein synthesis inhibitor, cycloheximide, could induce the c-fos gene and permit a superinduction by estrogens. In the presence of cycloheximide (10 micrograms/ml), protein synthesis was inhibited at 95% within the first hour. From 190 min after the addition of estradiol-17 beta or diethylstilbestrol (10(-8) M) and cycloheximide (10 micrograms/ml), there was a significant increase (ranging from 3- to 5-fold) of the c-fos messenger RNA level (2.2 kilobase in size), compared with the level in cells treated with cycloheximide alone. Nonestrogenic steroid hormones and estradiol-17 alpha were unable to induce c-fos gene expression in the presence of cycloheximide. The effect of estradiol-17 beta observed in the presence of cycloheximide was completely abolished by 4-hydroxy-tamoxifen or by Ly 156758 or by ICI 164384 (10(-6) M). The c-fos mRNAs were rather stable in cells treated with cycloheximide for 2 h (half-life = 51 +/- 6 min) and there was no further increase in the c-fos messenger RNA stability after the addition of cycloheximide plus estradiol-17 beta (half-life = 40 +/- 3 min). The overall results suggest a response at the transcriptional level. In conclusion, cycloheximide transmits activating signals to the c-fos gene which act as priming elements to allow the estrogen action in cultured guinea-pig endometrial cells.
Asunto(s)
Cicloheximida/farmacología , Endometrio/fisiología , Estradiol/farmacología , Genes fos , Tamoxifeno/farmacología , Androstenodiona/farmacología , Animales , Células Cultivadas , Dietilestilbestrol/farmacología , Endometrio/efectos de los fármacos , Estriol/farmacología , Estrona/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Cobayas , Hidrocortisona/farmacología , Cinética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Testosterona/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
A cDNA library was prepared from quiescent guinea-pig endometrial glandular epithelial cells stimulated for 2 h with estradiol-17 beta (E2) in the presence of cycloheximide. It was screened by differential hybridization for estrogen-regulated sequences. Six recombinants containing E2-regulated sequences were identified. One of them, called gec1 was then characterized by Northern blot hybridization. The gec1 mRNA was 1,800 bases in size. A 2-fold increase in the gec1 mRNA level was achieved at 120 min after E2 treatment. The E2 action on gec1 gene required the presence of cycloheximide. The cloned gec1 cDNA was 1 kb in size. The sequence so far determined did not show similarity with well characterized genes. This is the first report on a cloned cDNA probe of early estrogen-induced mRNA in a primary culture of endometrial epithelial cells.
Asunto(s)
Endometrio/metabolismo , Estradiol/fisiología , Regulación de la Expresión Génica , ARN Mensajero/biosíntesis , Animales , Northern Blotting , Células Cultivadas , Clonación Molecular , Cicloheximida/farmacología , Endometrio/citología , Endometrio/efectos de los fármacos , Femenino , Biblioteca de Genes , Cobayas , ARN Mensajero/análisis , ARN Mensajero/aislamiento & purificación , Factores de TiempoRESUMEN
Glandular epithelial (GE) and stromal cells were isolated from guinea-pig endometrium, cultured and subcultured separately. At the end of subculture, the purity of each cell population was higher than 95% and cells displayed a high level of estrogen receptors. Calcium phosphate transfection conditions were defined using a control plasmid containing the bacterial CAT gene driven by viral promoter and enhancer sequences. Transfection experiments were performed with other plasmids in which CAT gene was linked to different estrogen response elements (EREs) derived from those of vitellogenin genes. CAT activity was significantly increased by estradiol-17 beta treatment only when GE or stromal cells were transfected with plasmids containing EREs previously reported as functional EREs in other cell types. This induction was abolished by ICI 164,384 diethylstilbestrol was as effective as estradiol-17 beta for CAT induction and estradiol-17 alpha was ineffective. Transiently transfected endometrial cells in subculture are a suitable system to study the estrogen effect on gene regulatory elements.
Asunto(s)
Endometrio/metabolismo , Estradiol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Animales , Secuencia de Bases , Células Cultivadas , Células del Tejido Conectivo , Endometrio/citología , Inducción Enzimática/efectos de los fármacos , Células Epiteliales , Estradiol/análogos & derivados , Femenino , Cobayas , Datos de Secuencia Molecular , Alcamidas Poliinsaturadas , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/fisiología , Proteínas Recombinantes de Fusión/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , TransfecciónRESUMEN
Biochemical and pharmacologic studies suggest that I2 imidazoline binding sites (I2BS) represent a heterogeneous family of membrane proteins. Indeed, the imidazoline binding sites located on monoamine oxidases (MAO) A and B display different pharmacologic properties. Recent results suggest that in liver and brain, I2BS may be located on proteins distinct from MAOs. The following observations indicate that in liver and brain, [3H]idazoxan binds exclusively to I2BS located on MAO-B: (1) size exclusion chromatography of digitonin-solubilized preparations from rabbit and human liver showed that [3H]idazoxan-specific binding eluted only in two peaks (approximately 175,000 and approximately 100,000 Da, corresponding to 90% and 10% of the recovered [3H]idazoxan binding) which also contained MAOs as determined by [14C]tyramine oxidation and Western blot analysis; (2) according to previous results obtained in various human and rat tissues, experiments performed in mice liver and brain showed that idazoxan was a potent inhibitor of [125I]-AZIPI photoincorporation to MAO-B but not to MAO-A; (3) in MAO-deficient transgenic mice, [3H]idazoxan binding to liver and brain membranes was completely abolished in MAO-B knockout mice and was not affected in MAO-A knockout mice. Together, these results show that in both liver and brain, I2BS are located exclusively on MAO-B. The imidazoline binding site on MAO-A, which photoincorporates [125I]-AZIPI and displays a low affinity for idazoxan, may not belong to the family of the I2 imidazoline binding sites.
Asunto(s)
Hígado/metabolismo , Monoaminooxidasa/metabolismo , Receptores de Droga/metabolismo , Animales , Sitios de Unión , Humanos , Idazoxan/metabolismo , Receptores de Imidazolina , Isoenzimas/metabolismo , Cinética , Ratones , Ratones Noqueados , Ratones Transgénicos , Monoaminooxidasa/deficiencia , Monoaminooxidasa/genética , Conejos , Ratas , Tiramina/metabolismoRESUMEN
The expression of the biogenic amine degrading enzyme monoamine oxidases-A and -B depends on several factors including regional distribution, development and hormonal environment. In the present study, we investigated the expression of monoamine oxidases in developing kidney and their regulation by dexamethasone treatment. Immunoblots and enzyme assays, performed using [14C]5-hydroxytriptamine and [14C]beta-phenylethylamine as substrates for monoamine oxidases-A and -B, respectively, showed that monoamine oxidase-A is the isoenzyme largely predominant in 9-day-old rats renal cortex. Experiments performed in 5-week-old rats showed an increase in monoamine oxidase-B activity and a decrease in monoamine oxidase-A activity and substrate affinity. The changes of monoamine oxidase-A activity and affinity were mimicked by dexamethasone treatment (0.60 mg/kg body weight injected subcutaneously three times at intervals of 24 h) of 9-day-old rats. In contrast, dexamethasone administration induced a modification of monoamine oxidase-B activity opposite to that found between 9-day- and 5-week-old rats. Dexamethasone treatment did not modify immunoreactivity and mRNA corresponding to monoamine oxidases-A and -B indicating that changes of enzyme activities were unrelated to regulation of protein synthesis and mRNA turnover. These results show that monoamine oxidases-A and -B are differently expressed in developing renal cortex and are regulated by dexamethasone treatment.
Asunto(s)
Corteza Renal/enzimología , Monoaminooxidasa/metabolismo , Animales , Antiinflamatorios/farmacología , Dexametasona/farmacología , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Immunoblotting , Corteza Renal/efectos de los fármacos , Corteza Renal/crecimiento & desarrollo , Masculino , Monoaminooxidasa/efectos de los fármacos , Monoaminooxidasa/genética , Fenetilaminas/metabolismo , Embarazo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serotonina/metabolismo , Especificidad por SustratoRESUMEN
Our aim was to analyze EGF action on nuclear protooncogenes in RL95-2 since it has not been documented so far. Synchronization and partial' growth arrest were obtained by maintaining cells for 15 hours in L-methionine-free medium. After this depletion, EGF transiently increased fos and jun mRNAs: the expression peaked at 45 minutes for c-fos (5.5 fold induction) and at 60 minutes for c-jun and jun-B (3 fold induction) and the mRNA levels returned to the basal value within 3 hours. Upon EGF addition, c-myc mRNAs peaked at 12 hours (7.6 fold induction) and surprisingly remained higher than the control up to 48 hours. Unlike fetal calf serum, EGF did not increase the cell number and this could be linked to steadily induced c-myc expression. These data provide evidence for a differential EGF action on fos/jun and c-myc in RL95-2 cells.
Asunto(s)
Neoplasias Endometriales/genética , Factor de Crecimiento Epidérmico/farmacología , Proto-Oncogenes/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , División Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/fisiología , Femenino , Expresión Génica/efectos de los fármacos , Genes fos/efectos de los fármacos , Genes jun/efectos de los fármacos , Genes myc/efectos de los fármacos , Humanos , Células Tumorales CultivadasAsunto(s)
Antagonistas Adrenérgicos alfa/farmacología , Receptores Adrenérgicos alfa 2/fisiología , Receptores de Droga/metabolismo , Yohimbina/farmacología , Animales , Sitios de Unión , Células COS , Humanos , Idazoxan/metabolismo , Imidazoles/farmacología , Receptores de Imidazolina , Ensayo de Unión Radioligante , Receptores Adrenérgicos alfa 2/efectos de los fármacos , Receptores Adrenérgicos alfa 2/genética , Receptores de Droga/efectos de los fármacos , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Guinea pig endometrial stromal cells were cultured in serum-free medium to assess the effects of growth factors and ovarian steroids on cell proliferation. When the cells were made quiescent by serum depletion, [3H]thymidine incorporation was increased by the addition of insulin plus epidermal growth factor (EGF), reaching a peak after 24 h of stimulation. This effect was dose-dependent. Both factors acted synergistically. Estradiol-17 beta (E2), either alone or with various concentrations of growth factors, had no mitogenic effect. Thus, cell proliferation appeared to be estrogen-insensitive, despite a high level of estrogen receptors (19,000 sites per cell). The integrity of these receptors was checked by transfecting cells with a plasmid containing an estrogen-responsive element linked to a CAT gene: E2-induced CAT activity was reduced by the antiestrogen ICI 164,384. Despite the presence of progesterone receptors, the cells, either primed with E2 or not, were not growth-stimulated by progesterone. E2 had no effect on cells cultured in the presence of dextran-coated charcoal-stripped serum. Thus, whatever the culture conditions, stromal cells with functional estrogen receptors were insensitive to the putative mitogenic effects of E2 and progesterone. However, they were highly responsive to the mitogenic effects of insulin and EGF.
Asunto(s)
Endometrio/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Insulina/farmacología , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo , Endometrio/citología , Endometrio/metabolismo , Factor de Crecimiento Epidérmico/administración & dosificación , Receptores ErbB/metabolismo , Estradiol/farmacología , Femenino , Cobayas , Insulina/administración & dosificación , Factor I del Crecimiento Similar a la Insulina/farmacología , Progesterona/farmacología , Receptores de Estrógenos/metabolismo , Timidina/metabolismoRESUMEN
Glandular epithelial cells (GEC) and stromal cells (SC) were isolated from guinea-pig endometrium and cultured separately. After the cells were made quiescent by serum depletion, cell proliferation and c-fos gene expression were investigated. Estradiol-17 beta (E2) alone had no effect on cell proliferation or c-fos gene expression. Cell proliferation was observed in SC and GEC after stimulation with insulin plus EGF and a supplementary effect was obtained when E2 was associated with these factors only in GEC. In GEC, insulin plus EGF had no effect on c-fos expression but an effect was observed when E2 was associated with these factors or with a protein synthesis inhibitor, cycloheximide (Chx) or puromycine. An E2 effect in the presence of Chx was also observed in SC. These data suggest the presence of a labile protein preventing the in vitro estrogenic action in endometrial cells.
Asunto(s)
Endometrio/citología , Factor de Crecimiento Epidérmico/farmacología , Estradiol/farmacología , Expresión Génica/efectos de los fármacos , Genes fos/genética , Factor I del Crecimiento Similar a la Insulina/farmacología , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Femenino , Cobayas , Técnicas In Vitro , Células del Estroma/citologíaRESUMEN
BACKGROUND: Serotonin is one of the factors regulating mesangial cell proliferation, and convergent evidence supports its involvement in the development of glomerulonephritis. In this study, we identified a serotonin transporter and the amine-degrading enzyme monoamine oxidases (MAOs) in mesangial cells, and we studied their involvement in serotonin degradation. METHODS: MAOs were characterized in membrane preparations and intact mesangial cells by enzyme assay using [14C]5-hydroxytryptamine and [14C]beta-phenylethylamine as specific substrates for MAO-A and MAO-B, respectively, and by Western blot analysis. The expression of a serotonin transporter was determined by [14C]5-hydroxytryptamine uptake experiments and Western blot. Mesangial cell proliferation was measured by BrdU incorporation. RESULTS: Quantitation of the MAO isoforms by enzyme assay and Western blot analysis showed that MAO-A was largely predominant in mesangial cells, accounting for approximately 90% of the total enzyme population. The MAO substrate [14C]serotonin was transported into mesangial cells by a saturable uptake system (Vmax 310 +/- 36 pmol/30 min/mg protein; Km 5.9 +/- 1.4 microM) displaying the pharmacological properties of a serotonin transporter. The expression of a serotonin transporter was confirmed by Western blot analysis. MAO activity measured in intact cells showed that after accumulation into mesangial cells, [14C]serotonin was metabolized by MAO-A. Finally, serotonin-mediated mesangial cell proliferation was significantly increased after irreversible MAO inhibition. CONCLUSIONS: Our results suggest that serotonin concentration and function in glomeruli may be regulated in part by its transport into mesangial cells and degradation by MAO-A.
Asunto(s)
Proteínas Portadoras/metabolismo , Mesangio Glomerular/química , Mesangio Glomerular/enzimología , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Monoaminooxidasa/metabolismo , Proteínas del Tejido Nervioso , Serotonina/metabolismo , Secuencia de Aminoácidos , Animales , Antimetabolitos/metabolismo , Antimetabolitos/farmacología , Transporte Biológico/fisiología , Western Blotting , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacología , Radioisótopos de Carbono , División Celular/fisiología , Células Cultivadas , Activación Enzimática/fisiología , Mesangio Glomerular/citología , Corteza Renal/química , Corteza Renal/citología , Corteza Renal/enzimología , Datos de Secuencia Molecular , Monoaminooxidasa/análisis , Inhibidores de la Monoaminooxidasa/farmacología , Pargilina/farmacología , Ratas , Serotonina/farmacocinética , Proteínas de Transporte de Serotonina en la Membrana PlasmáticaRESUMEN
The c-fos expression was investigated in primary culture of guinea pig glandular epithelial cells. These cells were made quiescent by serum deprivation and stimulated with fetal calf serum (FCS, 15%), 17 beta-estradiol (E2 10(-8) mol/l) alone or in combination with epidermal growth factor (EGF, 100 ng/ml) and insulin (I, 10 micrograms/ml). Low levels of c-fos mRNA were detectable in quiescent cells and were not increased in cells stimulated with either E2, EGF, I, or EGF plus I. On the contrary, the c-fos mRNA were early and transiently increased by FCS or E2 plus EGF plus I (4.5 and 9.5 fold induction, respectively). This effect was independent of de novo protein synthesis since it was not abolished in the presence of cycloheximide. It appears that E2 acts in a multiple step process including the stimulation by EGF plus insulin.
Asunto(s)
Endometrio/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Estradiol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Insulina/farmacología , Proteínas Proto-Oncogénicas/genética , Animales , Sangre , Células Cultivadas , Endometrio/efectos de los fármacos , Epitelio/metabolismo , Femenino , Cobayas , Proteínas Proto-Oncogénicas c-fos , ARN Mensajero/metabolismoRESUMEN
Using differential hybridization of a guinea pig endometrial cell cDNA library, a potentially negatively estrogen-regulated gene, SOX-3, was isolated. According to the nucleotide and protein sequence similarities, SOx-3 belonged to the FAD-linked sulfhydryl oxidase family containing the egg white sulfhydryl oxidase, the rat seminal vesicle sulfhydryl oxidase-2 SOx-2, the quiescence-inducible protein hQ6. The SOX-3 transcript in the guinea pig as well as 5 different mRNAs in human tissues appeared differentially expressed in the tissues studied. In secondary endometrial cell culture, the SOX-3 mRNA level increased during a serum depletion-induced quiescence, decreased when cells enter the G1 phase after serum stimulation, and was restored during the S and G2/M phases. Thus, SOX-3 could be implicated in the negative cell cycle control. The SOx-3 protein appeared to be specific of epithelial cells in the uterus. Its expression level varied during the estrus cycle in the guinea pig, suggesting a regulation by steroid hormones.
Asunto(s)
Estro/metabolismo , Oxidorreductasas/aislamiento & purificación , Útero/enzimología , Secuencia de Aminoácidos , Animales , Ciclo Celular/fisiología , ADN Complementario/análisis , Proteínas de Unión al ADN/química , Endometrio/metabolismo , Estrógenos/farmacología , Femenino , Regulación Enzimológica de la Expresión Génica , Biblioteca de Genes , Cobayas , Proteínas HMGB , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/química , Especificidad de Órganos , Oxidorreductasas/biosíntesis , Oxidorreductasas/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Factores de Transcripción SOXB1 , Homología de Secuencia de Aminoácido , Factores de Transcripción , Útero/citologíaRESUMEN
The I(2) subgroup of imidazoline-binding sites was identified as monoamine oxidases (MAOs), but it is unclear whether there are I(2)-binding sites located on proteins distinct from MAOs. To address this issue, we characterized I(2)-binding proteins in liver and brain of wild-type and MAO A- and MAO B-deficient mice. I(2)-binding sites were identified using [(3)H]idazoxan and the photoaffinity adduct 2-[3-azido-4-[(125)I]iodophenoxyl]methylimidazoline ([(125)I]AZIPI). [(3)H]Idazoxan labeled binding sites with ligand recognition properties typical of I(2) sites in both brain and liver of wild-type mice. High-affinity, specific [(3)H]idazoxan binding were not altered in MAO A knockout (KO) mice. In contrast, [(3)H]idazoxan binding was completely abolished in both liver and brain of MAO B KO mice. In wild-type mice, [(125)I]AZIPI photolabeled three proteins with apparent molecular masses of approximately 28 (liver), approximately 61 (brain), and approximately 55 kDa (liver and brain). The photolabeling of each protein was blocked by the imidazoline cirazoline (10 microM). Photolabeling of the approximately 61- and approximately 55-kDa proteins was not observed in MAO A and B KO mice, respectively. In contrast, photolabeling of the liver approximately 28-kDa protein was still observed in MAO-deficient mice, indicating that this protein is unrelated to MAOs. These data indicate that I(2) imidazoline-binding sites identified by [(3)H]idazoxan reside solely on MAO B. The binding sites on MAO A and the liver approximately 28-kDa protein may represent additional subtypes of the family of the imidazoline-binding sites.