RESUMO
INTRODUCTION: The development of the human haemochorial placenta requires complex regulatory mechanisms to protect invasive trophoblast cells from cytotoxic responses elicited by maternal immune cells. Leptin, the adipocyte derived hormone encoded by the Lep gene, is synthesized by placental trophoblasts and exerts pleiotropic effects on the immune system, including the promotion of inflammation and the activation of T cell responses. METHODS: To address its possible involvement in the modulation of maternal immune responses during pregnancy, we investigated the effect of leptin on the expression of the class Ib histocompatibility antigen HLA-G as one of the chief immunosuppressive strategies used by trophoblast cells. RESULTS: In vitro incubation of the trophoblast derived Swan 71 and JEG-3 cell lines with 25-50 ng/ml recombinant leptin significantly boosted HLA-G mRNA and protein expression, and this effect was abrogated upon pharmacological inhibition of the PI3K-Akt and MEK-Erk signaling pathways. A similar stimulatory effect of leptin was observed in term placental tissue explants, though 10-fold higher doses were required for stimulation. Further, JEG-3 cells treated with a leptin antisense oligodeoxynucleotide displayed decreased HLA-G expression levels, which were partially recovered by addition of stimulating doses of exogenous hormone. Immunofluorescence and qPCR analysis confirmed leptin biosynthesis in placental tissue, further showing that invasive extravillous trophoblast cells were a main source of this hormone during the first trimester of normal pregnancies. DISCUSSION: Taken together, our results show that leptin acts as an autocrine/paracrine signal promoting HLA-G expression in placental trophoblasts suggesting an important role in the regulation of immune evasion mechanisms at the fetal maternal interface.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Antígenos HLA-G/metabolismo , Leptina/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Placentação , Transdução de Sinais , Trofoblastos/metabolismo , Adulto , Linhagem Celular , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Inativação Gênica , Antígenos HLA-G/química , Antígenos HLA-G/genética , Humanos , Leptina/antagonistas & inibidores , Leptina/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Oligodesoxirribonucleotídeos Antissenso , Placentação/efeitos dos fármacos , Gravidez , Primeiro Trimestre da Gravidez , Terceiro Trimestre da Gravidez , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Técnicas de Cultura de Tecidos , Trofoblastos/citologia , Trofoblastos/efeitos dos fármacos , Trofoblastos/imunologiaRESUMO
The steroid hormone 17ß-estradiol is an estrogen that influences multiple aspects of placental function and fetal development in humans. During early pregnancy it plays a role in the regulation of blastocyst implantation, trophoblast differentiation and invasiveness, remodeling of uterine arteries, immunology and trophoblast production of hormones such as leptin. Estradiol exerts some effects through the action of classical estrogen receptors ERα and ERß, which act as ligand-activated transcription factors and regulate gene expression. In addition, estradiol can elicit rapid responses from membrane-associated receptors, like activation of protein-kinase pathways. Thus, the cellular effects of estradiol will depend on the specific receptors expressed and the integration of their signaling events. Leptin, the 16,000MW protein product of the obese gene, was originally considered an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy. The leptin gene is expressed in placenta, where leptin promotes proliferation and survival of trophoblastic cells. Expression of leptin in placenta is highly regulated by key pregnancy molecules as hCG and estradiol. The aim of this paper is to review the molecular mechanisms underlying estrogen functions in trophoblastic cells; focusing on mechanisms involved in estradiol regulation of placental leptin expression.
Assuntos
Estrogênios/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Leptina/metabolismo , Proteínas da Gravidez/metabolismo , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Trofoblastos/metabolismo , Distinções e Prêmios , Endométrio/irrigação sanguínea , Endométrio/metabolismo , Estradiol/metabolismo , Feminino , História do Século XXI , Humanos , Leptina/genética , Obstetrícia/história , Circulação Placentária , Placentação , Gravidez , Proteínas da Gravidez/genéticaRESUMO
Leptin is a 16000 MW protein originally described as an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy. The leptin gene is expressed in placenta, where leptin promotes proliferation and survival of trophoblast cells. Study of the major signaling pathways known to be triggered by leptin receptor has revealed that leptin stimulates JAK/STAT, MAPK and PI3K pathways in placental cells. Leptin also exerts an antiapoptotic action in placenta and this effect is mediated by the MAPK pathway. Moreover, leptin stimulates protein synthesis by activating the translational machinery via both PI3K and MAPK pathways. Expression of leptin in placenta is highly regulated, suggesting that certain key pregnancy molecules participate in such regulation. An important hormone in reproduction, hCG, induces leptin expression in trophoblast cells and this effect involves the MAPK signal transduction pathway. Moreover, the cyclic nucleotide cAMP, which has profound actions upon human trophoblast function, also stimulates leptin expression and this effect seems to be mediated by crosstalk between the PKA and MAPK signaling pathways. Estrogens play a central role in reproduction. 17ß-estradiol upregulates leptin expression in placental cells through genomic and non-genomic actions, probably via crosstalk between estrogen receptor-α and the MAPK and PI3K signal transduction pathways. Taken together these findings give a better understanding of the function of leptin and the regulatory mechanisms of leptin expression in human placental trophoblast and further support the importance of leptin in the biology of reproduction.
Assuntos
Proliferação de Células , Leptina/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Animais , Sobrevivência Celular/fisiologia , Feminino , Humanos , Placenta/citologia , Gravidez , Transdução de Sinais/fisiologia , Trofoblastos/citologiaRESUMO
Insulin regulates the expression of several hepatic genes. Although the general definition of insulin signaling has progressed dramatically, the elucidation of the complete signaling pathway from insulin receptor to transcription factors involved in the regulation of a specific gene remains to be established. In fact, recent works suggest that multiple divergent insulin signaling pathways regulate the expression of distinct genes. 5-Aminolevulinate synthase (ALAS) is a mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of heme biosynthesis. It has been reported that insulin caused the rapid inhibition of housekeeping ALAS transcription, but the mechanism involved in this repression has not been explored. The present study investigates the role of phosphatidylinositol 3-kinase (PI3-kinase) and mitogen-activated protein kinase pathways in insulin signaling relevant to ALAS inhibition. To explore this, we combined the transient overexpression of regulatory proteins involved in these pathways and the use of small cell permeant inhibitors in rat hepatocytes and HepG2 cells. Wortmannin and LY294002, PI3-kinase inhibitors, as well as lovastatin and PD152440, Ras farnesylation inhibitors, and MEK inhibitor PD98059 abolished the insulin repression of ALAS transcription. The inhibitor of mTOR/p70(S6K) rapamycin had no effect whatsoever upon hormone action. The overexpression of vectors encoding constitutively active Ras, MEK, or p90(RSK) mimicked the inhibitory action of insulin. Conversely, negative mutants of PKB, Ras, or MEK impaired insulin inhibition of ALAS promoter activity. Furthermore, inhibition of one of the pathways blocks the inhibitory effect produced by the activation of the other. Our findings suggest that factors involved in two signaling pathways that are often considered to be functionally separate during insulin action, the Ras/ERK/p90(RSK) pathway and the PI3K/PKB pathway, are jointly required for insulin-mediated inhibition of ALAS gene expression in rat hepatocytes and human hepatoma cells.
Assuntos
5-Aminolevulinato Sintetase/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Hepatócitos/enzimologia , Insulina/metabolismo , Fígado/enzimologia , Sistema de Sinalização das MAP Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Androstadienos/farmacologia , Animais , Carcinoma Hepatocelular , Células Cultivadas , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Vetores Genéticos , Hepatócitos/efeitos dos fármacos , Humanos , Insulina/farmacologia , Fígado/efeitos dos fármacos , Masculino , Morfolinas/farmacologia , Regiões Promotoras Genéticas/fisiologia , Prenilação de Proteína/efeitos dos fármacos , Prenilação de Proteína/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos , Proteínas Quinases S6 Ribossômicas/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologia , Tubulina (Proteína)/genética , Células Tumorais Cultivadas , Wortmanina , Proteínas ras/metabolismoRESUMO
The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions -38 and -142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression.
Assuntos
5-Aminolevulinato Sintetase/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas Nucleares/farmacologia , Transativadores/farmacologia , 5-Aminolevulinato Sintetase/biossíntese , Animais , Sítios de Ligação , Proteína de Ligação a CREB , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica , Humanos , Mutação , Oligonucleotídeos Antissenso/farmacologia , Plasmídeos , Regiões Promotoras Genéticas , Ratos , Transdução de Sinais , Transcrição Gênica , Células Tumorais CultivadasRESUMO
5-Aminolevulinate synthase (ALA-S) is a mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of the heme biosynthesis. There are two ALA-S isozymes encoded by distinct genes. One gene encodes an isozyme that is expressed exclusively in erythroid cells, and the other gene encodes a housekeeping isozyme that is apparently expressed in all tissues. In this report we examine the mechanisms by which phenobarbital and cAMP regulate housekeeping ALA-S expression. We have determined that cAMP and phenobarbital effects are additive and the combined action is necessary to observe the cAMP effect on ALA-S mRNA in rat hepatocytes. The role of the cAMP-dependent protein kinase (PKA) has been examined. A synergism effect on ALA-S mRNA induction is observed in rat hepatocytes treated with pairs of selective analogs by each PKA cAMP binding sites. A 870-bp fragment of ALA-S 5'-flanking region is able to provide cAMP and phenobarbital stimulation to chloramphenicol O-acetyltranferase fusion vectors in transiently transfected HepG2 cells. ALA-S promoter activity is induced by cotransfection with an expression vector containing the catalytic subunit of PKA. Furthermore, cotransfection with a dominant negative mutant of the PKA regulatory subunit impairs the cAMP analog-mediated increase, but the phenobarbital-mediated induction is not modified. Our data suggest that the transcription factor cAMP-response element binding protein (CREB) is probably involved in PKA induction of ALA-S gene expression. Finally, heme addition greatly decreases the basal and phenobarbital or cAMP analog-mediated induction of ALA-S promoter activity. The present work provides evidence that cAMP, through PKA-mediated CREB phosphorylation, and phenobarbital induce ALA-S expression at the transcriptional level, while heme represses it.
Assuntos
5-Aminolevulinato Sintetase/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Indução Enzimática/efeitos dos fármacos , Fenobarbital/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Domínio Catalítico , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/genética , Sinergismo Farmacológico , Hemina/farmacologia , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Mutação/genética , Fenobarbital/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Transcrição Gênica/genética , Células Tumorais CultivadasRESUMO
Insulin has been known to regulate intracellular metabolism by modifying the activity or location of many enzymes but it is only in the past few years that the regulation of gene expression is recognized to be a major action of this hormone. The present work provides evidences that insulin inhibits delta-aminolevulinate synthase (ALA-S) gene expression, the enzyme which governs the rate-limiting step in heme biosynthesis. The addition of 5 nM insulin to hepatocytes culture led to a significant decrease of both basal and phenobarbital-induced ALA-S mRNA in a dose-dependent manner, as measured by Northern and slot-blot analysis. Several clues as to how insulin regulates ALA-S transcription were determined. The inhibitory effect is achieved at physiological concentrations but much higher proinsulin doses are needed. Insulin's effect is rapid, quite specific, and protein synthesis is not required. Moreover, ALA-S mRNA half-life is not modified by the presence of the peptidic hormone. Our results demonstrate that the insulin effect is dominant; it overrides 8-CPT-cAMP plus phenobarbital-mediated induction. Also, insulin requires the activation of protein kinase C to exert its full effect. On the other hand, a 870-bp fragment of the ALA-S promoter region is able to sustain the inhibition of CAT expression in plasmid-transfected HepG2 cells. Thus, these results indicate that insulin plays an important role in regulating ALA-S expression by inhibiting its transcription.
Assuntos
5-Aminolevulinato Sintetase/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/farmacologia , Neoplasias Hepáticas/enzimologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Regiões 5' não Traduzidas/genética , 5-Aminolevulinato Sintetase/biossíntese , 5-Aminolevulinato Sintetase/genética , Animais , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Meia-Vida , Humanos , Fígado/citologia , Neoplasias Hepáticas/genética , Masculino , Biossíntese de Proteínas , Proteína Quinase C/deficiência , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Tionucleotídeos/farmacologia , Células Tumorais CultivadasRESUMO
There are many factors that regulate the rate of synthesis of delta-aminolevulinate synthase (ALA-S), the enzyme which governs the rate-limiting step in heme biosynthesis. In rat hepatocytes, phenobarbital increases ALA-S gene transcription and dibutyryl cAMP potentiates this induction, whereas insulin and glucose have the opposite effect. The present report provides evidence that protein kinase C (PKC) activation negatively influences ALA-S mRNA levels, as measured by Northern and slot-blot analysis. The addition of 1,2-dioctanoyl-sn-glycerol (DOG) or 12-O-tetradecanoylphorbol 13-acetate (TPA), a PKC activator that mimics diacylglycerol function, to cultures led to a significant decrease of both basal and phenobarbital-induced ALA-S mRNA levels in a dose-dependent manner. This TPA effect depends on the specific activation of PKC because the analog 4 alpha-phorbol 12,13-diacetate, a nonstimulatory PKC phorbol ester, is unable to inhibit ALA-S mRNA. Furthermore, the effect of TPA is blocked by the PKC inhibitors staurosporine and calphostin C. Desensitization of the PKC pathway by prolonged exposure to TPA abolished the subsequent action of the phorbol ester. On the other hand, neither TPA nor DOG modified the half-life of ALA-S mRNA. The study of the combinatorial action of TPA and cAMP revealed that the inhibitory effect of TPA overcomes dibutyryl cAMP induction. Thus, these results indicate that PKC plays an essential role in regulating ALA-S expression, probably at a transcriptional level.
Assuntos
5-Aminolevulinato Sintetase/biossíntese , Fígado/enzimologia , Proteína Quinase C/fisiologia , 5-Aminolevulinato Sintetase/genética , Animais , Bucladesina/farmacologia , Células Cultivadas , AMP Cíclico/fisiologia , Diglicerídeos/farmacologia , Ativação Enzimática , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Masculino , Naftalenos/farmacologia , Fenobarbital/farmacologia , Proteína Quinase C/antagonistas & inibidores , RNA Mensageiro/biossíntese , Ratos , Sistemas do Segundo Mensageiro , Estaurosporina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Transcrição GênicaRESUMO
In the present work, we demonstrate the presence of a glucose inhibitory effect on the phenobarbital-mediated induction of the delta-aminolevulinate synthase mRNA in normal rat hepatocytes, consistent with the results obtained with the delta-aminolevulinate synthase activity previously reported. This "glucose effect" can be prevented by adding cAMP, adenylate cyclase activators, or a phosphodiesterase inhibitor. Delta-Aminolevulinate synthase mRNA half-life is not modified in the presence of phenobarbital or glucose. When the same experiments are performed using diabetic cells, no glucose effect is observed, even when the endogenous cAMP content is lowered to normal levels. The results obtained in this study suggest that glucose decreases delta-aminolevulinate synthase biosynthesis by acting at a pretranslational step. Assuming that the glucose effect operates by a repression mechanism exerted by metabolites derived from or related to glucose, the present results may reflect a derangement in the formation of these metabolites as a result of the abnormal metabolism operating in the diabetic state.
Assuntos
5-Aminolevulinato Sintetase/biossíntese , Diabetes Mellitus Experimental/enzimologia , Glucose/farmacologia , Fígado/efeitos dos fármacos , Fenobarbital/toxicidade , Porfirias/induzido quimicamente , 1-Metil-3-Isobutilxantina/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 5-Aminolevulinato Sintetase/genética , Adenilil Ciclases/metabolismo , Animais , Glicemia/fisiologia , Bucladesina/farmacologia , AMP Cíclico/farmacologia , Diabetes Mellitus Experimental/sangue , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Heme/biossíntese , Imunidade Inata , Fígado/enzimologia , Masculino , Fenobarbital/antagonistas & inibidores , Fenobarbital/farmacologia , Porfirias/etiologia , Ratos , Sistemas do Segundo Mensageiro/fisiologia , EstreptozocinaRESUMO
We examined the mechanism underlying the effect of cAMP on delta-aminolevulinate synthase mRNA biosynthesis in isolated hepatocytes from normal and experimental diabetic rats. We have demonstrated that the potentiation by dibutyryl cAMP of the phenobarbital-mediated induction of delta-aminolevulinate synthase enzyme activity, observed in our previously reported studies, reflects an increased amount of its mRNA. The inducing effect exerted by phenobarbital on the biosynthesis of delta-aminolevulinate synthase mRNA in diabetic hepatocytes is greater than that observed in normal cells. This enhanced response to the increased level of endogenous cAMP in diabetic hepatocytes is apparently sufficient for a maximum activation of the cAMP-dependent protein kinase. The present results suggest that in rat liver dibutyryl cAMP modulates delta-aminolevulinate synthase mRNA biosynthesis by acting predominantly, if not exclusively, at the level of gene transcription.