RESUMEN
The response of granulosa cells to Luteinizing Hormone (LH) and Follicle- Stimulating Hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH- and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the Steroidogenic Acute Regulatory Protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.
RESUMEN
DNA cloning and protein engineering are basic methodologies employed for various applications in all life-science disciplines. Manipulations of DNA however, could be a lengthy process that slows down subsequent experiments. To facilitate both DNA cloning and protein engineering, we present Transfer-PCR (TPCR), a novel approach that integrates in a single tube, PCR amplification of the target DNA from an origin vector and its subsequent integration into the destination vector. TPCR can be applied for incorporation of DNA fragments into any desired position within a circular plasmid without the need for purification of the intermediate PCR product and without the use of any commercial kit. Using several examples, we demonstrate the applicability of the TPCR platform for both DNA cloning and for multiple-site targeted mutagenesis. In both cases, we show that the TPCR reaction is most efficient within a narrow range of primer concentrations. In mutagenesis, TPCR is primarily advantageous for generation of combinatorial libraries of targeted mutants but could be also applied to generation of variants with specific multiple mutations throughout the target gene. Adaptation of the TPCR platform should facilitate, simplify and significantly reduce time and costs for diverse protein structure and functional studies.
Asunto(s)
Clonación Molecular/métodos , Mutagénesis Sitio-Dirigida/métodos , Reacción en Cadena de la Polimerasa/métodos , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Bacterianas/genética , Calmodulina/genética , Simulación por Computador , Escherichia coli/genética , Vectores Genéticos , Modelos MolecularesRESUMEN
Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.
Asunto(s)
Clonación Molecular/métodos , Escherichia coli/genética , Vectores Genéticos/normas , Complejos Multiproteicos/biosíntesis , Proteínas Recombinantes/biosíntesis , Academias e Institutos , Factor de Unión a CCAAT/biosíntesis , Factor de Unión a CCAAT/genética , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Europa (Continente) , Geminina , Cooperación Internacional , Israel , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Factores de Transcripción TFII/biosíntesis , Factores de Transcripción TFII/genéticaRESUMEN
Molecular manipulations, including DNA cloning and mutagenesis are basic tools used on a routine basis in all life-science disciplines. Over the last decade new methodologies have emerged that facilitated and expanded the applications for DNA cloning and mutagenesis. Ligation-Independent Cloning (LIC) techniques were developed and replaced the classical Ligation Dependent Cloning (LDC) platform. Restriction Free (RF) cloning was originally developed for introduction of foreign DNA into a plasmid at any predetermined position. RF cloning is based on PCR amplification of a DNA fragment, which serves as a mega-primer for the linear amplification of the vector and insert. Here we present several novel applications of the Restriction Free (RF) cloning platform for DNA cloning and mutagenesis. The new applications include simultaneous cloning of several DNA fragments into distinct positions within an expression vector, simultaneous multi-component assembly, and parallel cloning of the same PCR product into a series of different vectors. In addition, we have expanded the application of the RF cloning platform for multiple alterations of the target DNA, including simultaneous multiple-site mutagenesis and simultaneous introduction of deletions and insertions at different positions. We further demonstrate the robustness of the new applications for facilitating recombinant protein expression in the Escherichia coli system.
Asunto(s)
Clonación Molecular/métodos , Mutagénesis Sitio-Dirigida/métodos , Proteínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN/genética , Escherichia coli/genética , Modelos Genéticos , Datos de Secuencia Molecular , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Proteínas/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
The gonadotrophins LH, FSH and human (h) CG are non-covalent heterodimers composed of a common alpha and the hormone-unique beta subunit. LH regulates the production of androgens and progestins in the follicle, and the levels of these steroids are critical for the ovarian function. Structural features of the gonadotrophins involved in the steroidogenic response of the ovary are not completely understood. As an approach to address how the topology of the ligand affects steroidogenesis we exploited the single-chain (SC) gonadotrophin methodology because manipulating the relative position of the tethered subunit domains in SC hCG analogs enabled to change in the conformation, secretion, receptor binding and adenylyl cyclase activity. We genetically engineered a SC bovine LH analog with a linker derived from the CTP domain of the hCGbeta subunit, NH2-alpha-CTP-LHbeta-COOH (denoted as alphaCTPLHbeta; AB configuration) and evaluated the secretion form transfected CHO cells and steroidogenesis in follicular derived cells in comparison to the variant NH2-LHbeta-CTP-alpha-COOH (LHbetaCTPalpha; BA configuration). The secretion of the analogs from CHO cells was quantitative, and that of alphaCTPLHbeta was more efficient than that of LHbetaCTPalpha The experiments suggested that both variants were N- and O- glycosylated, though the posttranslational modifications are likely to be non-identical in the AB and BA analogs. The analogs stimulated progesterone secretion by immortalized rat granulosa cells that express the rat LH receptor but the EC50 of alphaCTPLHbeta (AB orientation) was higher by 20 fold, as compared to LHbetaCTPalpha (BA). In primary cultures of bovine theca cells, alphaCTPLHbeta stimulated progesterone release with a reduced sensitivity (by at least 50 folds) and smaller magnitude over the basal levels (about 3 folds) relative to LHbetaCTPalpha. In contrast, the accumulation of androstenedione in the media of the same primary cultures appeared to be nearly identical. As a result, the androstenedione/progesterone ratio for the alphaCTPLHbeta analog was significantly increased relative to LHbetaCTPalpha (2-3 folds). This unequal response suggests a distinct regulation of progesterone and androstenedione biosynthesis. Our data demonstrate major differences in steroid balance following stimulation of the receptor with structural LH analogs and provide further insight into gonadotrophin regulation of ovarian steroid production.
Asunto(s)
Hormonas Glicoproteicas de Subunidad alfa/química , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Hormona Luteinizante de Subunidad beta/análogos & derivados , Hormona Luteinizante de Subunidad beta/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Androstenodiona/metabolismo , Animales , Bioensayo , Células CHO , Bovinos , Células Cultivadas , Cricetinae , Cricetulus , Femenino , Glicosilación/efectos de los fármacos , Células de la Granulosa , Humanos , Hormona Luteinizante de Subunidad beta/química , Proteínas Mutantes/metabolismo , Progesterona/metabolismo , Estructura Terciaria de Proteína , Ratas , Relación Estructura-Actividad , Células Tecales/efectos de los fármacos , Células Tecales/metabolismo , Transfección , Tunicamicina/farmacologíaRESUMEN
Follicle-stimulating hormone (FSH) controls the development of follicle-enclosed oocytes in the mammalian ovary by interacting with specific receptors located exclusively on granulosa cells. Its biological activity involves stimulation of intercellular communication, intracellular signaling, and up-regulation of steroidogenesis; the entire spectrum of genes regulated by FSH is not yet fully characterized. We have established monoclonal rat FSH-responsive granulosa cell lines that express FSH receptors at 20-fold higher rates than with primary cells, and thus increased the probability of yielding a distinct spectrum of genes modulated by FSH. Using Affymetrix DNA microarrays, we discovered 11 genes not reported earlier to be up-regulated by FSH and 9 genes not reported earlier to be down-regulated by FSH. Modulation of signal transduction associated with G-protein signaling, phosphorylation of proteins, and intracellular-extracellular ion balance was suggested by up-regulation of decay accelerating factor GPI-form precursor (DAF), membrane interacting protein RGS16, protein tyrosine phosphatase (PTPase), oxidative stress-inducible protein tyrosine phosphatase (OSIPTPase), and down-regulation of rat prostatic acid phosphatase (rPAP), Na+, K+-ATPase, and protein phosphatase 1beta. Elevation in granzyme-like proteins 1 and 3, and natural killer (NK) cell protease 1 (NKP-1) along with reduction in carboxypeptidase E indicates possible FSH-mediated preparation of the cells for apoptosis. Up-regulation of vascular endothelial growth factors indicates the ability of FSH to produce angiogenic factors upon their maturation; whereas, reduction in insulin-like growth factor binding protein (IGFBP3) indicates its increased potential to promote p53-induced apoptosis. Striking similarities in FSH modulation of gene expression were found in primary cultures of human granulosa cells obtained from IVF patients although these cells expressed only 1% of FSH receptor compared with immortalized rat cells, as indicated by microarray technique, which probably is in the normal range of expression of this receptor in nontransformed cells. These findings should increase our understanding of the mechanism of FSH action in stimulating development of the ovarian follicular cells, of intracellular and intercellular communication, and of increasing the potential of ovarian follicular cells to undergo apoptosis during the process of selection of the dominant follicle.
Asunto(s)
Hormona Folículo Estimulante/farmacología , Regulación de la Expresión Génica , Células de la Granulosa/metabolismo , Animales , Antioxidantes/metabolismo , Apoptosis , Comunicación Celular , Línea Celular Transformada , Células Cultivadas , Citocinas/biosíntesis , Citocinas/genética , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/fisiología , Sustancias de Crecimiento/biosíntesis , Sustancias de Crecimiento/genética , Humanos , Fenómenos Fisiológicos del Sistema Nervioso , Péptido Hidrolasas/biosíntesis , Péptido Hidrolasas/genética , Fosfoproteínas Fosfatasas/biosíntesis , Fosfoproteínas Fosfatasas/genética , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/genética , Ratas , Transducción de Señal , Esteroides/biosíntesisRESUMEN
Gonadotropins were recently demonstrated to be able to activate the MAPK cascade, but the physiological significance of this activation is still obscure. In the present work we demonstrate that highly luteinized human granulosa cells obtained from in vitro fertilization patients respond to human LH as well as to forskolin in phosphorylation of extracellular-signal regulated kinases 1 and 2 (ERK1 and -2). Moreover, the potent MAPK inhibitors, PD98059 and UO126, augment progesterone production in these cell cultures concomitantly with specific elevation of intracellular steroidogenic acute regulatory protein (StAR). Intracellular levels of the cytochrome P450 side-chain cleavage enzyme system do not seem to be affected. Similar observations were made with rat preovulatory or preantral granulosa cells stimulated with LH, FSH, or forskolin. Elevation of StAR expression by the MAPK inhibitors involved elevation of StAR mRNA, as demonstrated by RT-PCR in the human cells. Immunocytochemical studies using specific antibodies to StAR demonstrate a higher content of mitochondrial StAR in control as well as in gonadotropin-stimulated cells in the presence of PD98059 compared with cells not treated with PD98059. The cultured cells express the transcription factor steroidogenic factor-1 (SF-1), the phosphorylation of which is known to activate the expression of StAR, as well as dosage-sensitive sex reversal adrenal hypoplasia congenita, critical region on the X chromosome gene-1 (DAX-1), which is known to negate SF-1 activity. Intracellular levels of DAX-1 decreased significantly during 24 h of incubation of cells with or without LH in the presence of PD98059 or UO126 compared with those in cultures incubated in the absence of the MAPK inhibitors. The expression of SF-1 was suppressed by LH, whereas MAPK inhibitor could block this effect and further elevate SF-1 levels. Thus, activation of the MAPK cascade by gonadotropins may serve as a novel mechanism to down-regulate steroidogenesis via attenuation of StAR expression. Moreover, modulation of DAX-1 and SF-1 intracellular levels in these cells suggests that these transcription factors could be involved in MAPK suppression of StAR expression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Gonadotropinas/farmacología , Células de la Granulosa/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores de Ácido Retinoico/metabolismo , Proteínas Represoras , Esteroides/biosíntesis , Factores de Transcripción/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica , Animales , Núcleo Celular/química , Células Cultivadas , Gonadotropina Coriónica/farmacología , Colforsina/farmacología , Receptor Nuclear Huérfano DAX-1 , Proteínas de Unión al ADN/análisis , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Femenino , Flavonoides/farmacología , Hormona Folículo Estimulante/farmacología , Factores de Transcripción Fushi Tarazu , Expresión Génica , Células de la Granulosa/metabolismo , Proteínas de Homeodominio , Humanos , Hormona Luteinizante/farmacología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Ovulación , Fosfoproteínas/análisis , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares , Receptores de Ácido Retinoico/análisis , Factor Esteroidogénico 1 , Factores de Transcripción/análisisRESUMEN
It is well known that high intracellular levels of cAMP can effectively kill cancer cells in vitro. Unfortunately substances elevating cAMP such as forskolin, 8-bromo-cAMP, 8-chloro-cAMP, monobutiryl or dibutiryl cAMP are not recommended to be used as anti-cancer drugs because of their high cytotoxicity. In contrast blockers of phosphodieterases such as theophylline and aminophylline, which could elevate intracellular cAMP, are commonly used as anti-asthma drugs reaching concentrations in the blood of 10-20 microg/ml. We tested the effectiveness of theophylline and aminophylline to induce cell death alone or in combination with common anti-cancer drugs such as cisplatin and gemcitabine (gemzar). We examined such drug combinations in the induction of cell death in a variety of carcinoma cell lines derived from human ovarian, prostate and lung cancer and in granulosa cell line transformed by SV40 and Ras oncogene. While theophylline could induce moderate cell death alone, at 20-25 microg/ml concentrations, aminophylline was ineffective at this concentration. Theophylline (at 15-25 ng/ml) was found in all four representative cell lines to synergize with gemcitabine or cisplatin to induce programmed cell death, which permits a reduction in the effective doses of cisplatin and gemcitabine by 2-3-fold. The effect of theophylline in induction of apoptosis involved reduction of intracellular levels of Bcl2. Such a reduction was proportional to the extent of apoptosis induced by theophylline as well as by the combined drug treatments. Therefore, we propose that theophylline should be considered as a potential anti-cancer drug in combination with other chemotherapeutic drugs. Screening of other phosphodiesterase blockers, which are not severely toxic, could open a possibility to improved chemotherapeutic cancer treatments with reduced undesired side-effects. A clinical trial, using theophylline as an anti-cancer drug, is currently being conducted in lung cancer patients.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Inhibidores de Fosfodiesterasa/farmacología , Xantinas/farmacología , Animales , División Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Humanos , Neoplasias Pulmonares/patología , Masculino , Neoplasias Ováricas/patología , Teofilina/farmacología , Células Tumorales CultivadasRESUMEN
The gonadotropic hormones, FSH and LH exert a major effect on ovarian and testicular function through interaction with specific seven-transmembrane domain glycoprotein receptors. Desensitization to the hormones, which can occur both in vivo and in vitro, is essential for prevention of overstimulation of the gonadal cells. The long-term process of desensitization to the gonadotropic hormones is probably mediated, in part, by extensive clustering and internalization of the hormone-receptor complex. Short-term desensitization may occur as a result of phosphorylation of serine or threonine residues on the receptor molecules, although a specific receptor kinase has not yet been identified. Recently, we have discovered a novel mechanism of gonadotropin desensitization, which is exerted by down-regulation of StAR expression and steroidogenesis mediated by MAPK activation as a result of hormone-receptor interaction, cAMP accumulation and PKA activation. Thus, PKA not only mediates gonadotropin-induced steroidogenesis, it also activates the down-regulation mechanism that can silence steroidogenesis under certain conditions. Moreover, our findings raise the possibility that activation or inhibition of ERK by other pathways could be an important mechanism for diminution or amplification of gonadotropin-stimulated steroidogenesis. This could contribute to functional luteolysis, a process in which luteinized granulosa cells show reduced sensitivity to LH despite maintenance of LH receptors, or to up-regulation of the steroidogenic machinery during luteinization of granulosa cells.
Asunto(s)
Gonadotropinas/fisiología , Animales , Regulación hacia Abajo/fisiología , Femenino , Gonadotropinas/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Fosforilación , Receptores de HFE/metabolismo , Receptores de HFE/fisiología , Receptores de HL/metabolismo , Receptores de HL/fisiología , Sistemas de Mensajero SecundarioRESUMEN
Ovarian cell death is an essential process for the homeostasis of ovarian function in human and other mammalian species. It ensures the selection of the dominant follicle and the demise of excess follicles. In turn, this process minimizes the possibility of multiple embryo development during pregnancy and assures the development of few, but healthy embryos. Degeneration of the old corpora lutea in each estrous/menstrual cycle by programmed cell death is essential to maintain the normal cyclicity of ovarian steroidogenesis. Although there are multiple pathways that can determine cell death or survival, crosstalk among endocrine, paracrine and autocrine factors, as well as among protooncogenes, tumor suppressor genes, survival genes and death genes, plays an important role in determining the fate of ovarian somatic and germ cells. The establishment of immortalized rat and human steroidogenic granulosa cell lines and the investigation of pure populations of primary granulosa cells allows systematic studies of the mechanisms that control steroidogenesis and apoptosis in granulosa cells. We have discovered that during initial stages of granulosa cell apoptosis progesterone production does not decrease. In contrast, we found that it is elevated up to 24h following the onset of the apoptotic stimuli exerted by starvation, cAMP, p53 or TNF-alpha stimulation, before total cell collapse. These observations raise the possibility for an alternative unique apoptotic pathway, one not involving mitochondrial Cyt C release associated with the destruction of mitochondrial structure and steroidogenic function. Using mRNA from apoptotic cells and affymetrix DNA microarray technology we discovered that granzyme B, a protease that normally resides in T cytotoxic lymphocytes and natural killer cells of the immune system is expressed and activated in granulosa cells. Thus, the apoptotic signals could bypass mitochondrial signals for apoptosis, which can preserve their steroidogenic activity until complete cell destruction. This unique apoptotic pathway assures cyclicity of estradiol and progesterone release in the estrous/menstruous cycle even during the initial stages of apoptosis.
Asunto(s)
Apoptosis , Ovario/metabolismo , Ovario/patología , Esteroides/metabolismo , Animales , Muerte Celular , Línea Celular , Supervivencia Celular , AMP Cíclico/metabolismo , Femenino , Células de la Granulosa/metabolismo , Células de la Granulosa/patología , Granzimas , Humanos , Cinética , Mitocondrias/metabolismo , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Progesterona/metabolismo , Ratas , Serina Endopeptidasas/farmacología , Linfocitos T/metabolismo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Owing to the development of the DNA microarray technique, modulation of gene function can be studied systematically. Considerable attention has been focused on members of the growth factor family to elucidate the main regulators of oocyte maturation and ovarian follicle rupture. Among these growth factors, it was found both in rodents and in humans that amphiregulin (Ar) and epiregulin (Ep) of the epidermal growth factor (EGF) family were dramatically up-regulated by gonadotrophins in the intact ovary and in primary granulosa cells, respectively. Their role in cumulus expansion and oocyte maturation was established in rodents, and their formation under LH stimulation in granulosa cells was demonstrated in humans. To be activated, Ar and Ep must be cleaved by A Disintegrin And Metalloproteinases (ADAMs) family. However, the precise processing of Ar and Ep by the cumulus cells is still obscure. Future investigations using DNA microarray technique may reveal the repertoire of genes activated in Ar- and Ep-stimulated cumulus cells and may help elucidate the molecular basis of ovulation.
Asunto(s)
Factor de Crecimiento Epidérmico/genética , Glicoproteínas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Ovario/metabolismo , Anfirregulina , Familia de Proteínas EGF , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/fisiología , Epirregulina , Femenino , Expresión Génica/genética , Glicoproteínas/metabolismo , Glicoproteínas/fisiología , Células de la Granulosa/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/fisiología , Modelos Biológicos , Ovario/citologíaRESUMEN
Follicle-stimulating hormone (FSH) is a key stimulant for the development of the ovarian follicle, while luteinizing hormone (LH) plays a major role in triggering ovulation and luteinization. Both FSH and LH are glycoprotein hormones that share the same alpha subunit but bind to specific seven transmembrane-domain G coupled receptors located on the cell membrane of the granulosa cells, which comprise the main somatic population of the ovarian follicle. These hormone-receptor complexes may trigger different signaling cascades, but the entire repertoire of genes modulated by these hormones is far from being understood, in particular on the transcriptional level. The development of DNA micro-arrays technique, using the entire genome profile of some mammalian species, allows a global approach and screening of multiple signal transduction pathways. This method opened new insights into the cellular and molecular events that control ovulation and desensitization of the corpus luteum to hyperstimulation by gonadotropic hormones. In addition, this technique permitted the discovery of novel members of the EGF family, such as epiregulin and amphiregulin, that were found to be expressed in the gonadotropin-stimulated cells and were discovered to play a crucial role in the mechanism of ovulation. However, because of the pitfalls in interpreting the data other approaches, for example, Northern blots and RT-PCR must be conducted in parallel to verify the validity of the data.
Asunto(s)
Hormona Folículo Estimulante/fisiología , Regulación de la Expresión Génica/fisiología , Células de la Granulosa/metabolismo , Hormona Luteinizante/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN/análisis , Cuerpo Lúteo/fisiología , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Ovulación/fisiología , Transducción de Señal/fisiología , Transcripción Genética/fisiologíaRESUMEN
Gonadotropic hormone, luteinizing hormone, and follicle-stimulating hormone exert their effect via activation of G-coupled receptors, which activate the hormone sensitive adenylyl cyclase, protein kinase A, and cyclic AMP responsive elements. This activation leads to specific de novo synthesis of steroidogenic factors and steroidogenic enzymes. In normal cells and following activation of this signaling pathway, desensitization period will be followed. This down-regulation, which was studied in detail for the last three decays, was found to take place at various steps of these signal transduction pathways as well as at different kinetics. A common and diverse feature of the mechanism of desensitization in other G-coupled-7-transmembrane receptor system is also discussed.
Asunto(s)
Adenilil Ciclasas/metabolismo , Regulación hacia Abajo/fisiología , Gonadotropinas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Animales , Membrana Celular/metabolismo , Femenino , Hormona Folículo Estimulante/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Hormona Luteinizante/metabolismo , Masculino , Fosforilación , Estructura Terciaria de ProteínaRESUMEN
We have demonstrated previously that the synthesis of epiregulin and amphiregulin, of the EGF-like growth factor family, is stimulated by luteinizing hormone in human follicular (granulosa) cells obtained from in vitro fertilization program. In the present work, we demonstrate that H89, a PKA inhibitor, attenuated the expression of these growth factors both in the mRNA and the protein levels, suggesting PKA involvement in this signaling pathway. SV40-transformed human granulosa cells showed higher basal levels of epiregulin and amphiregulin than normal cells, which were still elevated following cAMP stimulation by Forskolin. Cleavage by a disintegrin and metalloproteinases (ADAMs) is essential for activation of these growth factors, allowing their interaction with EGF receptor. Expression of ADAMTS1 and ADAM12 was downregulated by cAMP in normal, but not in SV40-transformed cells, suggesting that in normal cells epiregulin and amphiregulin activity is downregulated by a feedback mechanism that may be lost in SV40-transformed cells and their loss of downregulation may be involved in the development of ovarian tumors.
Asunto(s)
Desintegrinas/genética , Factor de Crecimiento Epidérmico/genética , Hormona Folículo Estimulante/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/efectos de los fármacos , Hormona Luteinizante/farmacología , Metaloendopeptidasas/genética , Proteínas ADAM , Proteína ADAMTS1 , Secuencia de Bases , Línea Celular Transformada , Colforsina/farmacología , Cartilla de ADN , Femenino , Células de la Granulosa/metabolismo , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A substantial amount of highly purified, biologically active bovine FSH was isolated from pituitary extracts by immunoaffinity chromatography based on a novel anti-bovine FSH beta-subunit monoclonal antibody. The biological activity was assessed in vitro using a steroidogenic granulosa cell line constitutively expressing the FSH receptor. Amino acid analysis, N-terminal amino acid sequencing, and peptide mass mapping demonstrated that primary structure modifications do not contribute to the heterogeneity of bovine FSH. The monosaccharide composition of the N-linked oligosaccharides was quantified and remarkably two distinct forms of sialic acids, N-acetyl- and N-glycolyl-neuraminic acids were found. In conclusion, we showed that isoform differences in bovine FSH is likely due only to sugar chain heterogeneity, and we give the first evidence that two substituted sialic acids contribute to the diversity of mammalian glycoprotein hormone isoforms.