RESUMO
Human pregnancy-specific glycoproteins (PSGs), encoded by eleven highly conserved genes, are the major placental polypeptides. Low PSG levels in maternal circulation have been associated with complicated pregnancies. However, expression of each PSG gene and their regulation during cytotrophoblast cell differentiation remain poorly explored. Herein, we analyze the expression of five PSG genes and demonstrate that they are almost undetectable in undifferentiated trophoblast, but are all transcribed in differentiated cells. Among them, PSG1, PSG3 and PSG5 genes achieve high mRNA levels while PSG7 and PSG9 are poorly expressed. In addition, total PSG proteins and transcripts markedly increase during trophoblast differentiation, preceding morphological syncytialization and betahCG expression. The 5' regulatory region contributes to the transcriptional control of PSG gene induction in trophoblast cells undergoing differentiation. This responsive region in PSG3 maps within a 130 bp promoter sequence, which overlaps the transcription start site and requires a functional Retinoic Acid Responsive Element (RARE) and a GA-binding protein (GABP) consensus site for basal and differentiation-dependent promoter activity, respectively. Present findings provide novel data for understanding the control of PSG gene expression and demonstrate that their proteins and transcripts represent early markers of trophoblast differentiation.
Assuntos
Diferenciação Celular/genética , Glicoproteínas/biossíntese , Glicoproteínas beta 1 Específicas da Gravidez/biossíntese , Trofoblastos/metabolismo , Linhagem Celular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Gravidez , RNA Mensageiro/metabolismo , Ativação Transcricional/fisiologiaRESUMO
Pregnancy-specific glycoprotein 5 gene (PSG-5) belongs to the human pregnancy-specific glycoprotein family, encoded by eleven highly similar and transcriptionally active genes. High levels of PSG biosynthesis are restricted to the placenta syncytiotrophoblast and are essential for the maintenance of normal gestation in mammalian species. We have investigated here the nature of the transcription factors that recognize the FP1 (-455/-433) and the CPE (-147/-140) regulatory sequences that significantly contribute to basal PSG-5 promoter activity. Both elements bear a similar GT-box motif; and DNA-protein complex formation, as well as promoter activity, is largely dependent on the integrity of these GT-box sequences. Gel shift, super gel shift and UV-crosslinking experiments clearly demonstrate that the ubiquitous specificity protein 1 (Sp1) is the major transcription factor involved in complex formation with both cis-acting elements in normal term placenta tissue and in PSG-non-expressing COS-7 cells. Furthermore, transfection experiments indicate that Sp1 activates PSG-5 promoter constructs. In addition, we show that Sp1 is indeed co-expressed with PSG genes in the syncytiotrophoblast cells, stressing its potential role in the in vivo regulation of PSG expression.
Assuntos
Regulação da Expressão Gênica/fisiologia , Glicoproteínas/genética , Glicoproteínas beta 1 Específicas da Gravidez/genética , Fator de Transcrição Sp1/genética , Animais , Sequência de Bases , Chlorocebus aethiops , Drosophila/genética , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Humanos , Dados de Sequência Molecular , Placenta/fisiologia , Gravidez , Regiões Promotoras Genéticas/genética , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Gênica/genética , Transfecção , Trofoblastos/metabolismoRESUMO
Gestational trophoblastic diseases, like the complete hydatidiform mole (CHM), are a group of human interrelated neoplasms whose etiology and progression is poorly understood at the molecular level. We have previously reported the cloning and expression of a new tumor necrosis factor receptor (TNF-R) related transcript, named CHMS-1 that encodes a potential death domain. Here we show that ectopic expression of the putative CHMS-1 death domain specifically induced apoptosis in a dose-dependent manner, in trophoblastic (JEG-3) and non-trophoblastic (COS-7) cells. We also investigated the expression of apoptosis-related molecules such as Bcl-2 and p53 and demonstrated that Bcl-2 is repressed in CHM while p53 is overexpressed in CHM compared with persistent gestational trophoblastic tumors. Altogether, these data indicate that the CHMS-1 death domain is able to trigger apoptosis, thus suggesting that this new entity might be an important inducer of molar regression mechanisms in women.
Assuntos
Apoptose , Proteínas de Neoplasias/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Trofoblastos/metabolismo , Animais , Northern Blotting , Feminino , Citometria de Fluxo , Expressão Gênica , Proteínas de Fluorescência Verde , Humanos , Técnicas Imunoenzimáticas , Proteínas Luminescentes/metabolismo , Proteínas de Neoplasias/genética , Gravidez , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Trofoblastos/patologia , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
The human pregnancy-specific glycoprotein (PSG) genes comprise a family of 11 highly conserved members whose expression is maximal in placental cells and marginal in other cell types. We have investigated here the molecular basis of PSG regulation by analysing a large regulatory region of the PSG-5 gene in cells that do and do not express these genes. The promoter region (-254 to -43), which does not contain a TATA-box, large GC-rich sequences or a classical initiator, was active in all cell types analysed. Additional upstream sequences up to position -3204 repressed promoter activity. Two independent repressor regions were identified and found to operate effectively in HeLa, COS-7 and HTR8/SVneo placental cells. More significantly, these negatively acting modules failed to repress a heterologous TATA-containing thymidine kinase promoter. Detailed transcriptional and DNA-protein analyses of the proximal repressor region (-605 to -254) revealed the presence of both negative and positive cis-acting elements. Disruption of the repressive functions resulted in an enhanced transcription of the reporter constructs. In conclusion, these results demonstrate that PSG-5 gene transcription is highly repressed by promoter-selective negative regulatory regions and the relief of repression allows enhanced PSG-5 gene transcription irrespective of the cell type. Furthermore, our findings suggest that PSG genes are expressed mainly through a derepression mechanism.
Assuntos
Glicoproteínas/metabolismo , Proteínas da Gravidez/genética , Glicoproteínas beta 1 Específicas da Gravidez , Transcrição Gênica , Animais , Células COS , Células Cultivadas , Cloranfenicol O-Acetiltransferase/metabolismo , Pegada de DNA , Desoxirribonuclease I/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Glicoproteínas/genética , Células HeLa , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Família Multigênica , Placenta/metabolismo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Timidina Quinase/genética , TransfecçãoRESUMO
Gestational trophoblastic diseases comprise a group of interrelated neoplasms, including complete hydatidiform mole (CHM), persistent gestational trophoblastic tumor (GTT), and choriocarcinoma. To better define the molecular features of these diseases, a CHM cDNA library was constructed and a novel cDNA sequence, named CHMS-1, was isolated by differential screening. The CHMS-1 sequence showed a 62% homology with the tumor necrosis factor receptor (TNF-R2) cDNA, and its amino acid deduced sequence shared a high level of homology with the "death domain" region found in various proteins, including two members of the TNF receptor superfamily, the TNF-R1 and Fas. We also determined the CHMS-1, TNF-R1, and TNF-R2 expression patterns among different CHM tissues and cell lines of trophoblastic (JEG-3) and nontrophoblastic (HeLa and COS-7) origin. Our results indicated that the CHMS-1 transcript is highly expressed in CHM in comparison with both normal early and term placenta and that it exhibits an expression profile identical to that of TNF-R1. Furthermore, the CHMS-1 transcript was undetectable in CHM-derived GTT and in the human choriocarcinoma-derived JEG-3 cells, suggesting that its expression is down-regulated in the malignant transformation of trophoblast. The presence of a potential "death domain" in CHMS-1, together with its high expression level in CHM, strongly suggests that the CHMS-1 gene encodes a protein that might be involved in tumor regression processes occurring at later stages of molar development.
Assuntos
Expressão Gênica , Proteínas de Neoplasias/genética , RNA Mensageiro/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Neoplasias Trofoblásticas/metabolismo , Neoplasias Uterinas/metabolismo , Sequência de Aminoácidos , Coriocarcinoma/metabolismo , Feminino , Humanos , Mola Hidatiforme/metabolismo , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Gravidez , Receptores do Fator de Necrose Tumoral/química , Alinhamento de Sequência , Células Tumorais CultivadasRESUMO
A group of eleven sesquiterpene lactones isolated from different Asteraceae species from north-western Argentina were investigated for their inhibitory action on the estrogen biosynthesis. Seven of them, of different skeleton types, were found to inhibit the aromatase enzyme activity in human placental microsomes, showing IC50 values ranging from 7 to 110 microM. The most active were the guaianolides 10-epi-8-deoxycumambrin B (compound 1), dehydroleucodin (compound 2) and ludartin (compound 3). These compounds were competitive inhibitors with an apparent Ki = 4 microM, Ki = 21 microM and Ki = 23 microM, respectively. Compounds 1 and 2 acted as type II ligands to the heme iron present in the active site of aromatase cytochrome P450 (P450arom). Besides, all of them failed to affect the cholesterol side-chain cleavage enzyme activity on human placental mitochondrias. This is the first report on the aromatase inhibitory activity of this group of natural compounds.
Assuntos
Inibidores da Aromatase , Lactonas/farmacologia , Sesquiterpenos/farmacologia , Antiulcerosos/farmacologia , Enzima de Clivagem da Cadeia Lateral do Colesterol/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Cinética , Lactonas/química , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Proteínas da Gravidez/antagonistas & inibidores , Sesquiterpenos/química , Relação Estrutura-AtividadeRESUMO
We describe a novel human cDNA isolated by target site screening of a placental expression library, using as a probe, an essential element of a TATA box-less promoter corresponding to a pregnancy-specific glycoprotein gene. The cDNA encoded a predicted protein of 290 amino acids, designated core promoter-binding protein (CPBP), which has three zinc fingers (type Cys2-His2) at the end of its C-terminal domain, a serine/threonine-rich central region and an acidic domain lying within the N-terminal region. Additional sequence analysis and data base searches revealed that only the zinc finger domains are conserved (60-80% identity) in other transcription factors. In cotransfection assays, CPBP increased the transcription from a minimal promoter containing its natural DNA-binding site. Moreover, a chimeric protein between CPBP and Gal4 DNA binding domain also increased the activity of an heterologous reporter gene containing Gal4 DNA binding sites. The tissue distribution analysis of CPBP mRNA revealed that it is differentially expressed with an apparent enrichment in placental cells. The DNA binding and transcriptional activity of CPBP, in conjunction with its expression pattern, strongly suggests that this protein may participate in the regulation and/or maintenance of the basal expression of PSG and possibly other TATA box-less genes.
Assuntos
Proteínas de Ligação a DNA/isolamento & purificação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas , TATA Box , Transativadores/genética , Dedos de Zinco , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/química , DNA Complementar/isolamento & purificação , Proteínas de Ligação a DNA/química , Biblioteca Gênica , Humanos , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Fases de Leitura Aberta , Placenta/metabolismo , Transativadores/químicaRESUMO
Pregnancy-specific beta 1 glycoprotein genes (PSG) are mainly expressed during human placental development, though their expression has been reported in other normal and pathological tissues, e.g. hydatidiform mole (HM), of distinct origins. However, the molecular components implicated in the regulation of PSG are not well understood. To identify some of the regulatory elements involved in the transcriptional control of PSG expression, the DNA-protein interactions and the basal activities of the TATA-box-less PSG5 promoter were determined in different tissues and cell types. In DNAse-I protection assays, DNA-binding proteins from human term placenta (HTP) protected a region of 27 bp located from nucleotides --150 to --124, overlapping the farthest 5' upstream cap site and resembling an initiator-like element. In electrophoretic mobility shift assays (EMSA), three complexes were detected using nuclear extracts from HTP and an oligonucleotide containing the 27-bp motif. In situ ultraviolet crosslinking analysis of the specific complexes revealed that two proteins of 78.0 kDa and 53.0 kDa are involved in such interactions, in accordance with the bands of 80.0 kDa and 57.5 kDa observed by Southwestern blotting. Competitive EMSA using mutant oligonucleotides with the substitution of 5'ACCCAT3' by 5'GATATC3' within the 27-bp motif revealed that this sequence is fundamental for the formation of the specific DNA-protein complexes. We show in transient transfection experiments performed in HeLa, COS-7 and JEG-3 cells, that such mutation completely abolished the transcriptional activity of the PSG5 promoter, independently of the cell type. Moreover, this mutation disrupted the formation of the specific DNA-protein complexes which were essentially the same as those displayed by HTP. We also determined the binding activities of nucleoproteins derived from placental tissues in earlier developmental and pathological stages, i.e. first trimester placenta (1-TRIM) and HM, respectively, showing that the DNA-binding patterns were different from each other and distinct from those elicited by HTP. Our results indicate that the cis-acting and trans-acting elements analyzed are indispensable to support PSG5 promoter activity in cell lines which do or do not produce PSG. In addition, these elements appear to play a role in the mechanisms involved in PSG basal expression during placental development and differentiation.
Assuntos
Proteínas Nucleares/metabolismo , Placenta/metabolismo , Proteínas da Gravidez/genética , Regiões Promotoras Genéticas , Sítios de Ligação , Pegada de DNA , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Glicoproteínas/genética , Humanos , RNA Mensageiro/genética , Trofoblastos/metabolismoRESUMO
The membrane-bound enzyme 3 beta-hydroxysteroid dehydrogenase delta 5-4 isomerase (3 beta-HSD) catalyzes the formation of delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids in placental, adrenal, testicular and ovarian tissues. In the present study was investigated the transverse-plane topography of 3 beta-HSD within the human placental microsome membranes employing immune-replica analysis in combination with surface specific proteolysis. The crucial domains of the enzyme for the dehydrogenase and isomerase reactions are inactivated by proteinase treatments under conditions where latency of hexose-6-phosphate dehydrogenase was 95%. The data indicate that these crucial domains face the cytosolic side of the endoplasmic reticulum membrane. Incubation of the intact microsomes with trypsin produces several immune reactive fragments ranging from 29 to 11 kDa in addition to 42 kDa native enzyme, one of them being shielded by the membrane structure and/or by other intrinsic and peripheral membrane proteins. Carboxypeptidase Y degraded the C terminus of the 42 kDa native 3 beta-HSD in intact and detergent-disrupted microsomes, preserving partially a fragment of 31 kDa. The results from the carboxypeptidase Y digestion indicate that the carboxy terminal end of the 3 beta-HSD enzyme is located on the cytoplasmic surface of the endoplasmic reticulum and that only a small fragment of approx. 11 kDa could be removed easily without affecting the enzyme activity. From these data and the predicted hydropathy analysis from the literature, we tried to assign a transmembrane arrangement to the human placental 3 beta-HSD. Our results support a topology model in which practically all the structural 3 beta-HSD enzyme is exposed to the cytoplasmic side of the membrane with one NH2-terminal-anchoring segment and all the 3 beta-HSD enzyme activity facing to the cytoplasmic side within the 31 kDa NH2-terminal peptide.
Assuntos
Complexos Multienzimáticos/química , Placenta/enzimologia , Progesterona Redutase/química , Esteroide Isomerases/química , Carboxipeptidases , Citoplasma/enzimologia , Endopeptidase K , Humanos , Immunoblotting , Membranas Intracelulares/enzimologia , Microssomos/enzimologia , Estrutura Molecular , Serina Endopeptidases , TripsinaRESUMO
The complete hydatidiform mole (CHM) is characterized by the presence of aberrant placenta, with hyperplasia of cyto- and syncytiotrophoblasts and the absence of maternal genetic information. Steroidogenesis in this condition is, thus, of special interest. In this study we investigated the kinetic parameters of aromatase in microsomes from CHM compared with those in normal early placenta (NEP). The enzyme activity was determined by measuring the conversion of [3H] testosterone to [3H]estradiol plus [3H]estrone. The Km value for testosterone was 33 nmol/L in CHM and 17 nmol/L in NEP of similar gestational ages. Aminoglutethimide, a nonsteroidal inhibitor, decreased in a dose-dependent manner and with the same potency the aromatization of testosterone in both tissues (ID50, 2 vs. 1 mumol/L in CHM and NEP, respectively). These results suggest that the enzymes from the two sources are kinetically similar. However, the enzyme efficiency, expressed as the maximum velocity/Km ratio, was 17-fold lower in CHM than in NEP tissue (1.22/33 vs. 10.68/17 min/mg.mL). These findings suggest that in molar pregnancy the decreased capacity of trophoblast tissue for the formation of estrogen could increase the testosterone concentration inside the molar vesicle, which, in turn, as we previously reported, inhibits progesterone formation. All of these data could provide an explanation for the low circulating level of progesterone, which may directly or indirectly affect the spontaneous expulsion of this aberrant tissue in the second trimester of pregnancy.