RESUMO
The Human Variome Project (HVP) is an international effort aiming systematically to collect and share information on all human genetic variants. It has been working for years in collaboration with local scientific societies by establishing systems to collect every genetic variant reported in a country and to store these variants within a database repository: LOVD (Argentinian chapter: ar.lovd.org). Formally established in 2017 in the Argentinian Node, up to June 2019 we collected more than 25,000 genetic variants deposited by 17 different laboratories. Nowadays the HVP country nodes represent more than 30 countries. In Latin America there are four country nodes: Argentina, Brazil, Mexico and Venezuela; the first two interacted recently launching the LatinGen database. In the present work we want to share our experience in applying the HVP project focusing on its organization, rules and nomenclature to reach the goal of sharing genetic variants and depositing them in the Leiden Open Variation Database. Contributing laboratories are seeking to share variant data to gain access all over the country. It is one of our goals to stimulate the highest quality by organizing courses, applying current nomenclature rules, sponsoring lectures in national congresses, distributing newsletter to serve the Argentinian genomics community and to stimulate the interaction among Latin America countries.
El Proyecto Varioma Humano (HVP) es un esfuerzo internacional que tiene como objetivo recopilar y compartir sistemáticamente información sobre todas las variantes genéticas humanas. Hemos estado trabajando durante tres años en colaboración con sociedades científicas locales, mediante el establecimiento de sistemas para recolectar todas las variantes genéticas reportadas en el país y almacenarlas dentro de la base de datos LOVD (capítulo argentino: ar.lovd.org). En el año 2017 fue establecido formalmente el Nodo Argentino del HVP, habiéndose recolectado más de 25.000 variantes genéticas depositadas por 17 laboratorios diferentes hasta junio de 2019. Hoy en día existen al menos 30 nodos del HVP, correspondientes a diferentes países. En América Latina hay cuatro nodos: Argentina, Brasil, México y Venezuela; Los dos primeros interactuaron recientemente lanzando la base de datos LatinGen. En el presente trabajo queremos compartir nuestra experiencia en la aplicación del proyecto HVP centrándonos en su organización, reglas y nomenclatura para alcanzar el objetivo de compartir variantes genéticas y depositarlas en la base de datos de variaciones abiertas de Leiden (LOVD). Es uno de nuestros objetivos estimular la más alta calidad mediante la organización de cursos, aplicación de las reglas de nomenclatura actuales, patrocinio de conferencias en congresos nacionales, distribución de boletines informativos para la comunidad de genómica argentina, y estimulación de la interacción entre los países de América Latina.
RESUMO
Several reports suggest putative interactions between valproic acid (VPA) treatment and the hypothalamus-pituitary-adrenal axis. Given that VPA alters mitochondrial functions, an action of this drug on a mitochondrial process such as steroid synthesis in adrenal cells should be expected. In order to disclose a putative action of VPA on the adrenocortical cell itself we evaluated VPA effects on regulatory steps of the acute stimulation of steroidogenesis in Y1 adrenocortical cells. This study demonstrates that VPA increases progesterone production in non-stimulated cells without inducing the levels of Steroidogenic Acute Regulatory (StAR) protein, which facilitates cholesterol transport. This result suggests that VPA increases mitochondrial cholesterol transport through a StAR-independent mechanism and is further supported by the fact that in isolated mitochondria VPA stimulates exogenous cholesterol metabolization to progesterone. VPA also reduces the cAMP-mediated increase of the StAR protein, mRNA levels, promoter activity and progesterone production. In summary, the present data show that VPA can alter steroid production in adrenal cells by a complex mechanism that mainly involves an action on cholesterol access to the inner mitochondrial membrane. The VPA-mediated increase of basal steroidogenesis could be linked to the increase of basal cortisolemia described in patients under VPA treatment.
Assuntos
Córtex Suprarrenal/efeitos dos fármacos , Anticonvulsivantes/farmacologia , Colesterol/metabolismo , Mitocôndrias/efeitos dos fármacos , Ácido Valproico/farmacologia , 3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Córtex Suprarrenal/metabolismo , Animais , Anticonvulsivantes/toxicidade , Transporte Biológico , Linhagem Celular , Enzima de Clivagem da Cadeia Lateral do Colesterol/antagonistas & inibidores , AMP Cíclico/agonistas , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Camundongos , Mitocôndrias/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Progesterona/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Ácido Valproico/toxicidadeRESUMO
Stimulation of receptors and subsequent signal transduction results in the activation of arachidonic acid (AA) release. Once AA is released from phospholipids or others esters, it may be metabolized via the cycloxygenase or the lipoxygenase pathways. How the cells drive AA to these pathways is not elucidated yet. It is reasonable to speculate that each pathway will have different sources of free AA triggered by different signal transduction pathways. Several reports have shown that AA and its lipoxygenase-catalyzed metabolites play essential roles in the regulation of steroidogenesis by influencing cholesterol transport from the outer to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Signals that stimulate steroidogenesis also cause the release of AA from phospholipids or other esters by mechanisms that are not fully understood. This review focuses on the enzymes of AA release that impact on steroidogenesis.
Assuntos
Glândulas Suprarrenais/enzimologia , Ácido Araquidônico/metabolismo , Células Intersticiais do Testículo/enzimologia , Tioléster Hidrolases/metabolismo , Acetil-CoA Hidrolase/metabolismo , Animais , Colesterol/metabolismo , Humanos , Masculino , Mitocôndrias/enzimologia , Esteroides/biossínteseRESUMO
Germline mutations in specific hot spot-codons of the RET proto-oncogene are associated with multiple endocrine neoplasia type 2 (MEN 2). Clinical RET gene testing has been routine for the last 10 years in some countries. In Argentina, RET testing excluding MEN 2B was always reported with a mutation at codon 634, with one exception: we described a novel mutation T > C transition at codon 630 (C630R), the family to which we extend the study in the present report. This family comprised 29 members in four generations including 6 individuals affected with medullary thyroid cancer (MTC), positive for the C630R mutation and normal adrenaline/ noradrenaline and ionized calcium/parathyroid hormone levels. Two asymptomatic mutation carriers aged 5 and 11 years underwent total thyroidectomy. The histopathologic examination showed C-cell hyperplasia and microcarcinoma foci, while preoperative basal calcitonins were normal for both. Our report emphasizes the importance of testing for non-hot spot RET mutations in apparently mutation negative MEN 2 families. Furthermore, it would appear that C630R mirrors C634R in penetrance (100% in this family) and in early age of onset of MTC, although paradoxically, no pheochromocytomas and hyperparathyroidism have developed. In addition to recommending RET testing before 5 years of age; we also can postulate that codon 630 may be the key point along the extracellular domain, important in the tissue-specific penetrance.
Assuntos
Carcinoma Medular/genética , Mutação em Linhagem Germinativa/genética , Proteínas Oncogênicas/genética , Penetrância , Receptores Proteína Tirosina Quinases/genética , Neoplasias da Glândula Tireoide/genética , Adulto , Idade de Início , Criança , Pré-Escolar , Saúde da Família , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-retRESUMO
Heme oxygenase (HO) catalyzes the first and rate-controlling step of heme catabolism into biliverdin, iron and carbon monoxide. Three isoforms of HO have been identified so far: the inducible HO-1 and the constitutive HO-2 and HO-3. Both HO-1 and HO-2 were expressed in zona fasciculata (ZF) adrenal cells and in a mouse adrenocortical cell line (Y1). HO-1 but not HO-2 expression was upregulated by adrenocorticotropic hormone (ACTH) and accumulation of HO-1 protein correlated with an increase in HO activity in Y1 cells. ACTH induced HO-1 expression in a time- and dose-dependent manner with a maximum after 5 h of treatment and a threshold concentration of 0.1 mIU/ml. Actinomycin D and cycloheximide completely blocked the effect of ACTH on HO-1 mRNA expression whereas mRNA stability was not affected by ACTH. Permeable analogs of cAMP mimicked the effect of ACTH on HO-1 expression and ACTH induction was prevented by the protein kinase A (PKA) inhibitor H89. Steroid production was significantly increased when both HO-1 and HO-2 activities were inhibited by Sn-protoporphyrin IX (SnPPIX). The lipid peroxidation and increase in carbonyl content triggered by hydrogen peroxide was prevented by treatment of Y1 cells with bilirubin and ACTH.
Assuntos
Córtex Suprarrenal/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Heme Oxigenase (Desciclizante)/biossíntese , Sulfonamidas , Córtex Suprarrenal/efeitos dos fármacos , Animais , Bilirrubina/farmacologia , Western Blotting/métodos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Heme Oxigenase (Desciclizante)/análise , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1 , Immunoblotting/métodos , Imuno-Histoquímica/métodos , Isoquinolinas/farmacologia , Proteínas de Membrana , Metaloporfirinas/farmacologia , Camundongos , Pregnenolona/análise , Protoporfirinas/farmacologia , RNA Mensageiro/análise , Estimulação Química , Substâncias Reativas com Ácido Tiobarbitúrico/análise , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Although the role of arachidonic acid (AA) in trophic hormone-stimulated steroid production in various steroidogenic cells is well documented, the mechanism responsible for AA release remains unknown. We have previously shown evidence of an alternative pathway of AA generation in steroidogenic tissues. Our results are consistent with the hypothesis that, in steroidogenic cells, AA is released by the action of a mitochondrial acyl-CoA thioesterase (MTE-I). We have shown that recombinant MTE-I hydrolyses arachidonoyl-CoA to release free AA. An acyl-CoA synthetase specific for AA, acyl-CoA synthetase 4, has also been described in steroidogenic tissues. In the present study we investigate the new concept in the regulation of intracellular levels of AA, in which trophic hormones can release AA by mechanisms different from the classical PLA2-mediated pathway. Inhibition of ACS4 and MTE-I activity by triacsin C and NDGA, respectively results in a reduction of StAR mRNA and protein abundance. When both inhibitors are added together there is a synergistic effect in the inhibition of StAR mRNA, StAR protein levels and ACTH-stimulated steroid synthesis. The inhibition of steroidogenesis produced by the NDGA and triacsin C can be overcome by the addition of exogenous AA. In summary, results shown here demonstrate a critical role of the acyl-CoA synthetase and the acyl-CoA thioesterase in the regulation of AA release, StAR induction, and steroidogenesis. This further suggests a new concept in the regulation of intracellular distribution of AA through a mechanism different from the classical PLA2-mediated pathway that involves a hormone-induced acyl-CoA synthetase and a hormone-regulated acyl-CoA thioesterase.
Assuntos
Ácido Araquidônico/fisiologia , Hormônios/metabolismo , Transdução de Sinais/fisiologia , Esteroides/biossíntese , Acil Coenzima A/antagonistas & inibidores , Animais , Ácido Araquidônico/metabolismo , Ácido Araquidônico/farmacologia , Linhagem Celular , Sinergismo Farmacológico , Membranas Intracelulares/metabolismo , Masoprocol/farmacologia , Mitocôndrias/enzimologia , Palmitoil-CoA Hidrolase/antagonistas & inibidores , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , RNA Mensageiro/antagonistas & inibidores , Triazenos/farmacologiaRESUMO
Several stimuli, including stress conditions, promote the activation of MAP kinases family members (ERK1/2, JNK, p38). In turn, these enzymes regulate several cellular functions. Given that MAPK activation requires the phosphorylation of these proteins, their inactivation depends on the activity of specific phosphatases. MAPK phosphatase-1 (MKP-1), a phosphatase specifically involved in the inactivation of MAPK family members, is induced by mitogenic stimuli and stress conditions. Here we describe the effect of heat shock (HS), 10 min, 45 degrees C, on MAPKs activities and MKP-1 mRNA and protein levels in Y1 adrenocortical cells. Western blot analysis performed with antibodies against the phosphorylated forms of ERK1/2 and JNK revealed that HS produced the rapid activation of these kinases. Their inactivation was also a rapid event and occurred together with the increase of MKP-1 protein levels detected by Western blot analysis. In addition, the effect of HS on MKP-1 protein levels seems to be exerted at the transcriptional level, since the amount of its mRNA in heat shocked cells was higher than in nonheated cells. Comparison of the temporal profiles of MKP-1 protein induction and MAPKs phospho-dephosphorylation suggests that MKP-1 induction could contribute to ERK1/2 and JNK inactivation after HS.
Assuntos
Córtex Suprarrenal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Transtornos de Estresse por Calor/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Córtex Suprarrenal/patologia , Animais , Western Blotting , Proteínas de Ciclo Celular/genética , Linhagem Celular , Fosfatase 1 de Especificidade Dupla , Ativação Enzimática , Proteínas Imediatamente Precoces/genética , Camundongos , Fosfoproteínas Fosfatases/genética , Fosforilação , Proteína Fosfatase 1 , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/metabolismo , Choque/metabolismo , Fatores de TempoRESUMO
The LH signal transduction pathway features the activation of protein tyrosine phosphatases (PTPs) as one of the components of a cascade that includes other well characterized events such as cAMP-dependent protein kinase A (PKA) activation. Moreover, the action of PTPs is required to increase the rate-limiting step in steroid biosynthesis, namely the cAMP-regulated transfer of cholesterol to the inner mitochondrial membrane. Since both PKA activity and steroidogenic acute regulatory (StAR) protein induction are obligatory steps in this transfer of cholesterol, the present study was performed to investigate the role of PTPs in the regulation of PKA activity and StAR expression in response to LH/chorionic gonadotropin (CG) and 8Br-cAMP in MA-10 cells. While the exposure of MA-10 cells to the PTP inhibitor, phenylarsine oxide (PAO), did not modify PKA activity, it partially inhibited the effect of human CG and cAMP analog on StAR protein levels. Time-course studies demonstrated that PAO inhibited cAMP induction of StAR protein and mRNA. At 30 min, the effect on cAMP-stimulated StAR protein levels was a 35% inhibition, progressing to up to 90% inhibition at 120 min of stimulation. The maximal inhibitory effect on cAMP-induced StAR mRNA level was obtained at 60 min (85%). In summary, these results demonstrate that inhibition of PTP activity affected both StAR protein and mRNA synthesis and suggest that the activity of hormone-regulated PTPs is a requirement in the LH signaling cascade that results in the up-regulation of StAR protein and, subsequently, increased steroid synthesis.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Androgênios/biossíntese , Gonadotropina Coriônica/farmacologia , Células Intersticiais do Testículo/metabolismo , Fosfoproteínas/biossíntese , Proteínas Tirosina Fosfatases/metabolismo , Animais , Arsenicais/farmacologia , Colesterol/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos , Fosfoproteínas/genética , Proteínas Tirosina Fosfatases/antagonistas & inibidores , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Transdução de Sinais , Fatores de Tempo , Células Tumorais CultivadasRESUMO
A key regulatory step in the steroidogenic hormones signaling pathway is the synthesis of steroidogenic acute regulatory protein (StAR). This protein facilitates the delivery of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis. ACTH and LH pathway also includes tyrosine dephosphorylation processes. Indeed, our previous studies have demonstrated that both hormones increase protein tyrosine phosphatase (PTP) activity by a PKA-dependent mechanism and that the action of PTPs is required for the stimulation of steroid biosynthesis in adrenal and Leydig cells. In order to test the putative relationship between PTP activity and StAR protein induction in adrenocortical cells, in the present study we evaluated steroid production and StAR protein level in Y1 adrenocortical cells under PTP inhibition. Phenylarsine oxide (PAO), a powerful cell permeable PTP inhibitor, reduced ACTH-stimulated steroidogenesis in a concentration-dependent fashion. A concentration of 2.5 microM of this compound inhibited steroid synthesis in a 56% (ACTH = 318 +/- 30, ACTH + PAO = 145 +/- 18 ng progesterone/mL, P < 0.001) and also abrogated StAR protein induction. Phenylarsine oxide reduced the protein level after 60 min and this effect still remained at 120 min. A second PTP inhibitor, benzyl phosphonic acid, acting by a different mechanism, reproduced PAO effects on both steroidogenesis and StAR protein. Taken together, these results indicate that PTP activity participates in StAR protein induction and led us to attribute to the PKA-mediated PTP activation in steroidogenic systems a functional role, as mediator of StAR protein induction.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Fosfoproteínas/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Esteroides/biossíntese , Animais , Arsenicais/administração & dosagem , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Camundongos , Organofosfonatos/farmacologia , Fosfoproteínas/antagonistas & inibidores , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Esteroides/antagonistas & inibidores , Fatores de Tempo , Células Tumorais CultivadasRESUMO
In isolated human umbilical vein (HUV), the contractile response to des-Arg9-bradykinin (des-Arg9-BK), selective BK B1 receptor agonist, increases as a function of the incubation time. Here, we evaluated whether cyclooxygenase (COX) pathway is involved in BK B1-sensitized response obtained in 5-h incubated HUV rings. The effect of different concentrations of indomethacin, sodium salicylate, ibuprofen, meloxicam, lysine clonixinate or NS-398 administrated 30 min before concentration-response curves (CRC) was studied. All treatments produced a significant rightward shift of the CRC to des-Arg9-BK in a concentration-dependent manner, which provides pharmacological evidence that COX pathway is involved in the BK B1 responses. Moreover, in this tissue, the NS-398 pKb (5.2) observed suggests that COX-2 pathway is the most relevant. The strong correlation between published pIC50 for COX-2 and the NSAIDs' pKbs estimated further supports the hypothesis that COX-2 metabolites are involved in BK B1 receptor-mediated responses. In other rings, indomethacin (30, 100 micromol/l) or NS-398 (10, 30 micromol/l) produced a significant rightward shift of the CRC to BK, selective BK B2 agonist, and its pKbs were similar to the values to inhibit BK B1 receptor responses, suggesting that COX-2 pathway also is involved in BK B2 receptor responses. Western blot analysis shows that COX-1 and COX-2 isoenzymes are present before and after 5-h in vitro incubation and apparently COX-2 does not suffer additional induction.