RESUMO
It is very difficult to gain a better understanding of the events in human pregnancy that occur during and just after implantation because such pregnancies are not yet clinically detectable. Animal models of human placentation are inadequate. In vitro models that utilize immortalized cell lines and cells derived from trophoblast cancers have multiple limitations. Primary cell and tissue cultures often have limited lifespans and cannot be obtained from the peri-implantation period. We present here two contemporary models of human peri-implantation placental development: extended blastocyst culture and stem-cell derived trophoblast culture. We discuss current research efforts that employ these models and how such models might be used in the future to study the "black box" stage of human pregnancy.
Assuntos
Blastocisto/metabolismo , Implantação do Embrião , Placentação , Trofoblastos/metabolismo , Feminino , Humanos , Gravidez , Células-Tronco/metabolismoRESUMO
The development of human trophoblast stem cells (hTSC) and stem cell-derived trophoblast organoids has enabled investigation of placental physiology and disease and early maternal-fetal interactions during a stage of human pregnancy that previously had been severely restricted. A key shortcoming in existing trophoblast organoid methodologies is the non-physiologic position of the syncytiotrophoblast (STB) within the inner portion of the organoid, which neither recapitulates placental villous morphology in vivo nor allows for facile modeling of STB exposure to the endometrium or the contents of the intervillous space. Here we have successfully established properly-polarized human trophoblast stem cell (hTSC)-sourced organoids with STB forming on the surface of the organoid. These organoids can also be induced to give rise to the extravillous trophoblast (EVT) lineage with HLA-G + migratory cells that invade into an extracellular matrix-based hydrogel. Compared to previous hTSC organoid methods, organoids created by this method more closely mimic the architecture of the developing human placenta and provide a novel platform to study normal and abnormal human placental development and to model exposures to pharmaceuticals, pathogens and environmental insults. Motivation: Human placental organoids have been generated to mimic physiological cell-cell interactions. However, those published models derived from human trophoblast stem cells (hTSCs) or placental villi display a non-physiologic "inside-out" morphology. In vivo , the placental villi have an outer layer of syncytialized cells that are in direct contact with maternal blood, acting as a conduit for gas and nutrient exchange, and an inner layer of progenitor, single cytotrophoblast cells that fuse to create the syncytiotrophoblast layer. Existing "inside-out" models put the cytotrophoblast cells in contact with culture media and substrate, making physiologic interactions between syncytiotrophoblast and other cells/tissues and normal and pathogenic exposures coming from maternal blood difficult to model. The goal of this study was to develop an hTSC-derived 3-D human trophoblast organoid model that positions the syncytiotrophoblast layer on the outside of the multicellular organoid.
RESUMO
Robust embryonic stem cell (ESC) lines from livestock species have been difficult to derive and maintain, and unlike mouse ESC, have not contributed to our ability to understand directed differentiation in vitro. Nor have such cells yet provided a simpler means than pronuclear injection to manipulate the genomes of agriculturally important species, such as cattle, sheep and pigs. Induced pluripotent stem cells (iPSC) generated by reprogramming somatic cells, such as fibroblasts, with a set of stemness genes, most usually but not exclusively POU5F1, SOX2, KLF4 and c-MYC, offer an alternative to ESC in these regards, as they exhibit a pluripotent phenotype resembling that of ESC, yet are readily generated in the laboratory. Accordingly, such cells, in association with cloning technologies, may be useful for introducing complex genetic changes into livestock, although this potential has yet to be demonstrated. Porcine iPSC may be especially valuable because the pig is a prime biomedical model for tissue transplantation. In general, iPSC from livestock, like those from humans, are of the epiblast type and depend upon FGF2 and activin/nodal signalling systems to maintain their pluripotency and growth. Recent experiments, in which newly reprogrammed porcine and bovine cells were selected on a LIF-based medium in presence of specific protein kinase inhibitors, have allowed iPSC cells of the naïve type, resembling the more amenable blastocyst-derived mouse ESC and iPSC to be isolated. However, hurdles still remain if such cells are to achieve their biotechnological promise.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Suínos , Animais , Técnicas de Cultura de Células , Fator 4 Semelhante a Kruppel , Especificidade da EspécieRESUMO
Trophectoderm is specified from pluripotent blastomeres at some time prior to blastocyst formation. Proliferating cytotrophoblast derived from trophectoderm is the forerunner of the entire trophoblast component of the mature human placenta, including extravillous cytotrophoblast and syncytiotrophoblast. Recently human embryonic stem cells (hESC) have been employed to study these events in an in vitro situation. Here we review some of the work in this emerging area of trophoblast biology. We concentrate primarily on a model in which colonies of hESC are exposed to BMP4 in stem cell growth medium lacking FGF2. Under both low (4%) and high (20%) O(2) conditions, differentiation proceeds unidirectionally towards trophoblast from the outside of the colonies inwards, with the progression fastest under high O(2). Immunohistochemical observations performed on whole colonies combined with microarray analysis of mRNA can be employed to track developmental transitions as they occur over time and in two-dimensional space as the cells respond to BMP4.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Trofoblastos/citologia , Trofoblastos/fisiologia , Diferenciação Celular/fisiologia , Feminino , Humanos , GravidezRESUMO
Oct-4 is a POU family transcription factor associated with potentially totipotent cells. Genes expressed in the trophectoderm but not in embryos prior to blastocyst formation may be targets for silencing by Oct-4. Here, we have tested this hypothesis with the tau interferon genes (IFNT genes), which are expressed exclusively in the trophectoderm of bovine embryos. IFNT promoters contain an Ets-2 enhancer, located at -79 to -70, and are up-regulated about 20-fold by the overexpression of Ets-2 in human JAr choriocarcinoma cells, which are permissive for IFNT expression. This enhancement was reversed in a dose-dependent manner by coexpression of Oct-4 but not either Oct-1 or Oct-2. When cells were transfected with truncated bovine IFNT promoters designed to eliminate potential octamer sites sequentially, luciferase reporter expression from each construct was still silenced by Oct-4. Full repression required both the N-terminal and POU domains of Oct-4, but neither domain used alone was an effective silencer. Oct-4 and Ets-2 formed a complex in vitro in the absence of DNA through binding of the POU domain of Oct-4 to a site located between the "pointed" and DNA binding domains of Ets-2. The two transcription factors were also coimmunoprecipitated after being expressed together in JAr cells. Oct-4, therefore, silences IFNT promoters by quenching Ets-2 transactivation. The POU domain most probably binds to Ets-2 directly, while the N-terminal domain inhibits transcription. These findings provide further evidence that the developmental switch to the trophectoderm is accompanied by the loss of Oct-4 silencing of key genes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Interferon Tipo I/genética , Proteínas da Gravidez/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologia , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Ligação Competitiva/efeitos dos fármacos , Bovinos , Coriocarcinoma/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/farmacologia , Elementos Facilitadores Genéticos/fisiologia , Inativação Gênica/efeitos dos fármacos , Inativação Gênica/fisiologia , Genes Reporter/efeitos dos fármacos , Genes Reporter/fisiologia , Fator C1 de Célula Hospedeira , Humanos , Interferon Tipo I/metabolismo , Substâncias Macromoleculares , Mutagênese Sítio-Dirigida , Fator 1 de Transcrição de Octâmero , Fator 2 de Transcrição de Octâmero , Fator 3 de Transcrição de Octâmero , Testes de Precipitina , Proteínas da Gravidez/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologia , Proteína Proto-Oncogênica c-ets-2 , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/farmacologia , Especificidade por Substrato/genética , Transativadores/genética , Transativadores/farmacologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/farmacologia , Ativação Transcricional/efeitos dos fármacos , Transfecção , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Porcine growth hormone (PGH) precursor cDNAs were cloned from a pituitary cDNA library constructed in lambda gt11 by immunoscreening. One of the three clones characterized contained an entire nucleotide sequence for the 216-amino-acid precursor molecule. The deduced amino-acid sequence of PGH confirmed the sequence previously reported for that of the genomic DNA of PGH except for one base difference in the coding sequence. Expression of the full-length PGH cDNA was achieved in bacteria and mammalian cells. The mammalian cell line, COS-1, produced the GH molecule which processed the signal peptide and had the same molecular weight as standard PGH, in contrast to the higher molecular weight of the bacterial product. Radioimmunoassay of the recombinant PGH produced in COS-1 cells also revealed an inhibition curve similar to that of the standard PGH.
Assuntos
DNA/genética , Hormônio do Crescimento/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Biblioteca Gênica , Hormônio do Crescimento/isolamento & purificação , Dados de Sequência Molecular , Adeno-Hipófise/metabolismo , Proteínas Recombinantes/isolamento & purificação , Mapeamento por Restrição , SuínosRESUMO
We isolated genomic clones for bovine prostaglandin (PG) F2alpha receptor by the standard plaque hybridization method, using the cDNA fragments of bovine PGF2alpha receptor (PGF2alphaR) as probe DNAs. The coding regions of this receptor gene were interspersed by a large intron sequence (33 kb) at the splice junction in the sixth transmembrane domain. The 5'-RACE experiments revealed two alternative transcription start points (tsp), indicating the existence of two potential promoter regions. The major promoter, which was named promoter region A, was located upstream of exon 1 and lacked the typical TATA sequence and CAAT box but had three GC boxes with an overall high GC content. Another putative promoter, region B, was found upstream of exon 2 and had both a TATA-like sequence and a CAAT-like box with several potential binding sites for transcription factors. Southern blot analysis indicated that a single copy gene in the haploid genome encodes PGF2alphaR. Promoter activities of these two putative promoter regions were assayed in the bovine luteal cells, and one of them (promoter region A) was activated by phorbol 12-myristate 13-acetate (TPA) treatment.
Assuntos
Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Bovinos , Clonagem Molecular , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , TransfecçãoRESUMO
The gene for the common alpha subunit of the porcine anterior pituitary glycoprotein hormones was cloned from a genomic library constructed in EMBL3. The nucleotide sequence of the entire coding sequence of the porcine common alpha-subunit gene was determined in addition to one intron and 1059 and 160 bp of the 5'- and 3'-flanking regions respectively. Southern blot analysis of the porcine genomic DNA indicated that the common alpha-subunit gene is present as a single copy. The transcriptional unit of the porcine common alpha subunit spanned about 14 kb and contained four exons interrupted by three introns of about 11.5, 1.2 and 0.4 kb. The short untranslated sequence in the first exon and the location of the exon/intron junctions at amino acid residues +9/+10 and +71/+72 were highly conserved among the rat, human and bovine common alpha-subunit genes. In the proximal portion of the 5'-flanking region, one TATA box and one CCAAT box were present. A steroid-responsive element was not found up to 1059 bases upstream from the transcription start site. The potential AP-1 and AP-2 factor-responsive elements were present at three and one positions respectively in the 5'-flanking region. This feature suggests that hypothalamic gonadotrophin-releasing hormone stimulates the expression of the common alpha-subunit gene predominantly by a signal-transduction system, with the protein kinase C cascade and factors AP-1 and AP-2 as mediators. The cyclic AMP-responsive element was also present at two positions, but a single base substitution was found in each sequence compared with the consensus sequence.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Subunidade alfa de Hormônios Glicoproteicos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Éxons/fisiologia , Expressão Gênica , Íntrons/fisiologia , Dados de Sequência Molecular , Biossíntese de Proteínas , Suínos , TATA Box , Transcrição GênicaRESUMO
The gene for the beta subunit of porcine LH (LH-beta) was cloned from a genomic library constructed in EMBL3. The nucleotide sequence was determined for the entire gene transcriptional unit of porcine LH-beta in addition to 1277 and 372 bp of the 5'- and 3'-flanking regions respectively. Southern blot analysis of the porcine genomic DNA indicated that the LH-beta gene is present as a single copy. The transcriptional unit of porcine LH-beta spanned 1107 bp and contained three exons interrupted by two introns of 326 and 289 bp. The short untranslated sequence in the first exon and the location of the exon/intron junctions at amino acid residues -16/-15 and +41/+42 were highly conserved in the rat, human and bovine LH-beta genes. In the 5'-flanking region, one TATA box and two CCAAT boxes were present. The steroid-responsive element was not found up to 1277 bases upstream of the transcription start site. The potential AP-2 factor-responsive elements appeared nine times within the sequence that was determined, and four of them were located in the 5'-flanking region. Two distal AP-2 elements were arranged in an inverted repeat forming a 16 bp palindromic sequence. This feature suggested that hypothalamic gonadotrophin-releasing hormone stimulates expression of the LH-beta gene, predominantly by a signal-transduction system with the protein kinase C cascade and a mediator, the AP-2 factor. A further characteristic feature of the porcine LH-beta gene was the presence of clusters of GC boxes and CACCC elements in the 5'-flanking region and the downstream sequence. Co-existence of these regulatory elements with other elements, such as the AP-2 element or CCAAT box, was also found. The porcine LH-beta gene shows a structure distinct from the porcine FSH-beta and common alpha genes, which are counterparts of the LH-beta gene, reflecting differential control of their synthesis during gametogenesis.
Assuntos
Elementos Facilitadores Genéticos , Hormônio Luteinizante/genética , Suínos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Gonadotropina Coriônica/genética , Gonadotropina Coriônica Humana Subunidade beta , Regulação da Expressão Gênica , Genes , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Sequências Reguladoras de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais , Especificidade da EspécieRESUMO
The mechanisms responsible for prevention of corpus luteum regression during early pregnancy are diverse and appear to have arisen in concert with the evolutionary divergence of placental structure. That used by the sub-order Ruminantia is unique and involves the production of a Type I interferon (IFN), IFN-tau (tau). Although IFN-tau resembles other Type I IFNs (such as IFN-alpha, -beta, and -omega) in structure as well as in many of its biological properties, it is not virally inducible and is instead produced constitutively by embryonic trophectoderm during the period immediately prior to implantation. The transcription factor Ets-2 is a component of the regulatory mechanism involved in transcription of IFN-tau. These genes probably arose as the result of a duplication of an IFN-omega gene, 36 million years ago, at about the time the Ruminantia sub-order emerged. They have duplicated extensively since then and there may be 10 or more genes in some present-day species. The expression of different IFN-tau is unequal and they differ in biological potency. The rapid evolution of IFN-tau genes possibly reflects the placenta as a site of considerable genetic experimentation.
Assuntos
Interferon Tipo I , Interferons , Proteínas da Gravidez , Trofoblastos , Animais , Corpo Lúteo/fisiologia , Evolução Molecular , Feminino , Regulação da Expressão Gênica , Humanos , Interferon Tipo I/química , Interferon Tipo I/genética , Interferon Tipo I/fisiologia , Interferons/química , Interferons/fisiologia , Estrutura Molecular , Gravidez , Proteínas da Gravidez/química , Proteínas da Gravidez/genética , Proteínas da Gravidez/fisiologiaRESUMO
INTRODUCTION: Preeclampsia and other placental pathologies are characterized by a lack of spiral artery remodeling associated with insufficient invasion by extravillous trophoblast cells (EVT). Because trophoblast invasion occurs in early pregnancy when access to human placental tissue is limited, there is a need for model systems for the study of trophoblast differentiation and invasion. Human embryonic stem cells (hESC) treated with BMP4- differentiate to trophoblast, and express HLA-G, a marker of EVT. The goals of the present study were to further characterize the HLA-G(+) cells derived from BMP4-treated hESC, and determine their suitability as a model. METHODS: HESC were treated with BMP4 under 4% or 20% oxygen and tested in Matrigel invasion chambers. Both BMP4-treated hESC and primary human placental cells were separated into HLA-G(+) and HLA-G(-)/TACSTD2(+) populations with immunomagnetic beads and expression profiles analyzed by microarray. RESULTS: There was a 10-fold increase in invasion when hESC were BMP4-treated. There was also an independent, stimulatory effect of oxygen on this process. Invasive cells expressed trophoblast marker KRT7, and the majority were also HLA-G(+). Gene expression profiles revealed that HLA-G(+), BMP4-treated hESC were similar to, but distinct from, HLA-G(+) cells isolated from first trimester placentas. Whereas HLA-G(+) and HLA-G(-) cells from first trimester placentas had highly divergent gene expression profiles, HLA-G(+) and HLA-G(-) cells from BMP4-treated hESC had somewhat similar profiles, and both expressed genes characteristic of early trophoblast development. CONCLUSIONS: We conclude that hESC treated with BMP4 provide a model for studying transition to the EVT lineage.
Assuntos
Movimento Celular/fisiologia , Células-Tronco Embrionárias/citologia , Placenta/citologia , Trofoblastos/citologia , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular , Células-Tronco Embrionárias/efeitos dos fármacos , Feminino , Antígenos HLA-G/biossíntese , Humanos , Queratina-7/biossíntese , Oxigênio/farmacologia , Gravidez , Primeiro Trimestre da GravidezRESUMO
In situ binding assay with oligonucleotides is developed for detection of DNA-binding factors. Cryosections of porcine pituitary were examined by DNA binding with 35S-labeled oligonucleotides corresponding to the consensus cAMP-responsive element (CRE) and the activator protein-1 binding element (AP1). Specificity of the in situ binding was confirmed by inhibition with the unlabeled probe itself and inefficiency of binding with the activator protein-2 binding element (AP2). It was proved by treatment with proteinase K that the binding was dependent on the protein component. Immunocytochemical staining employed successively before in situ binding demonstrated that the binding proteins for CRE and AP1 are located in pituitary gonadotrophs as well as other cells. Grain counting showed that gonadotrophs were rich in these factors as compared with other pituitary cells. Taken together, in situ binding provides a convenient and rapid tool for investigation of regulatory sequences and binding factors as well for determining their specific cellular localization.
Assuntos
Proteínas de Ligação a DNA/análise , Oligodesoxirribonucleotídeos/metabolismo , Hipófise/metabolismo , Proteínas Repressoras , Animais , Sequência de Bases , Modulador de Elemento de Resposta do AMP Cíclico , Proteínas de Ligação a DNA/metabolismo , Endopeptidase K , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/metabolismo , Hipófise/citologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-jun/análise , Ribonuclease Pancreático , Serina Endopeptidases , SuínosRESUMO
Factors binding to consensus sequences of the cAMP-responsive element (CRE) and the AP1 factor binding site (AP1) were investigated using porcine anterior pituitary nuclear extracts. Each element showed specific gel mobility shifts. By reciprocal competition for the AP1 and CRE binding, CRE prevented AP1 binding completely. On the other hand, AP1 decreased the CRE binding considerably to 20%, suggesting that approximately 80% of the total CRE binding is due to factors which bind to a common site shared by both CRE and AP1, whereas proteins binding to AP1 alone are absent. Relative binding affinities of AP1 against CRE estimated from the reciprocal competition data were 0.17 for CRE binding and 0.56 for AP1 binding. UV cross-linking experiments showed that CRE and AP1 gave different patterns consisting of different molecular size. Inconsistency of the relative binding affinities and the multiple molecular size of binding factors, cannot be explained simply by the presence of two types of binding factor, common CRE/AP1-binding and specific CRE-binding factors. A more likely explanation is that the CRE/AP1-binding factors alter the dimer form by changing each respective partner to bind CRE and/or AP1.
Assuntos
Núcleo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , DNA/metabolismo , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Adeno-Hipófise/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Cinética , Dados de Sequência Molecular , Proteínas Nucleares/isolamento & purificação , Sondas de Oligonucleotídeos , Sequências Reguladoras de Ácido Nucleico , SuínosRESUMO
It is well established that the interferon tau (IFN-tau) family of proteins play a major role in preventing the regression of the corpus luteum during early pregnancy in ruminants, such as cattle, sheep and goats, but not in other mammals. These interferons, which are structurally and functionally related to type I interferon, such as IFN-alpha and -omega, arose from a duplication of an IFN-omega gene approximately 36 million years ago. The IFN-tau genes have continued to duplicate since that time and have acquired the ability to be transcribed uniquely in the trophectoderm. Low expression is first detectable at the blastocyst stage, but massive transcriptional upregulation occurs a few days later during the initial stages of conceptus elongation. Expression is finally terminated upon trophectoderm attachment to uterine endometrium. The major promoter element that controls expression is an Ets-2/AP-1 enhancer element. Growth factors and cytokines released by the maternal endometrium that, possibly in response to progesterone, act through Ras and the mitogen-activated protein kinase (MAP-kinase) signal transduction pathway have been implicated in controlling IFN-tau gene transcription by activating Ets-2. This timely expression of IFN-tau is not only required to rescue the corpus luteum of pregnancy but may also be an indicator of conceptus fitness, thereby serving as a critical factor that dictates the continuation of pregnancy in ruminants.
Assuntos
Manutenção do Corpo Lúteo/genética , Evolução Molecular , Interferon Tipo I/genética , Proteínas da Gravidez/genética , Regiões Promotoras Genéticas , Ruminantes/fisiologia , Trofoblastos/metabolismo , Animais , Bovinos , Sequência Conservada , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Cabras , Filogenia , Gravidez , Receptores de Interferon/metabolismo , Ovinos , Transdução de Sinais/fisiologiaRESUMO
Alternative splicing leads to the expression of multiple isoforms of the subunits (IFNAR1 and IFNAR2) of the type I IFN receptor. Here we describe two transcripts representing extracellular forms of ovine IFNAR1 and show that soluble extracellular forms of both IFNAR2 and IFNAR1, prepared in recombinant form in Escherichia coli, have antiviral (AV) activity in the absence of IFN. Exposure of Madin-Darby bovine kidney cells to the extracellular domain (R2E) of IFNAR2 at concentrations as low as 10 nM afforded complete protection against vesicular stomatitis virus and led to the rapid activation of the transcription factors ISGF3 and GAF. Although R2E can bind IFN (K(d) approximately 70 nM), activity was observed irrespective of whether or not ligand was present. R2E was inactive on mouse L929 cells but active on L929 cells expressing a membraneanchored, ovine/human chimeric IFNAR2 with an ovine extracellular domain. The data suggest that AV activity is conferred by the ability of soluble R2E to associate with the transfected IFNAR2 subunit rather than resident murine IFNAR1. Soluble extracellular forms of IFNAR1 have lower AV activity than R2E on Madin-Darby bovine kidney cells but are less species-specific and protect wild-type L929 cells as efficiently as the transfected cell line, presumably by interacting with one of the murine receptor subunits.
Assuntos
Antivirais/metabolismo , Receptores de Interferon/metabolismo , Vírus da Estomatite Vesicular Indiana/crescimento & desenvolvimento , Animais , Sequência de Bases , Bovinos , Linhagem Celular , Clonagem Molecular , Reagentes de Ligações Cruzadas , DNA Complementar , Proteínas de Ligação a DNA/metabolismo , Espaço Extracelular , Humanos , Fator Gênico 3 Estimulado por Interferon , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Proteínas de Membrana , Camundongos , Dados de Sequência Molecular , Receptor de Interferon alfa e beta , Receptores de Interferon/biossíntese , Receptores de Interferon/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Fator de Transcrição STAT1 , Fator de Transcrição STAT2 , Ovinos , Solubilidade , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
A cDNA clone of prostaglandin (PG) E receptor EP1 subtype (rEP1) was isolated from a rat uterus cDNA library. It encodes 405 amino acid residues with seven transmembrane-spanning domains and couples to Ca2+ mobilization. In addition, three cDNA clones encoding a variant form of rEP1 were isolated. The open reading frame can code a 366-amino acid protein carrying a specific change of 49 amino acids from the middle of transmembrane segment VI to COOH terminus; it possesses a transmembrane segment VII-like structure lacking an intracellular COOH-terminal tail. Southern blot analysis of rat genomic DNA and genomic polymerase chain reaction demonstrated that these cDNAs were derived from a single copy gene. Northern blot analysis and ribonuclease protection assay revealed that both rEP1 and rEP1-variant receptor mRNAs were highly expressed in the kidney. Immunoblot with an antibody directed toward the specific region of rEP1-variant receptor showed that rEP1-variant receptor protein was expressed in the membrane of the kidney and Chinese hamster ovary (CHO) cells transfected with rEP1-variant cDNA. Thus, the rEP1-variant receptor is translated from mRNA which is not spliced at nucleotide position 952 in the segment VI transmembrane region. rEP1-variant receptor retained the ligand binding activity with affinity and specificity similar to rEP1 receptor, but lost the coupling of signal transduction systems by itself. However, when rEP1-variant receptor was stably co-expressed with rEP1 receptor in CHO cells, the Ca2+ mobilization mediated by EP1 receptor was significantly suppressed. Furthermore, when rEP1-variant receptor was expressed in CHO cells, cAMP formation by activation of endogenous EP4 receptor was strongly blocked. These results suggest that the rEP1-variant receptor may affect the efficiency of signal coupling of PGE receptors and attenuate the action of PGE2 on tissues.
Assuntos
Receptores de Prostaglandina E/genética , Transdução de Sinais , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Cricetinae , DNA Complementar/química , Feminino , Biblioteca Gênica , Dados de Sequência Molecular , Ratos , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina E Subtipo EP1 , Mapeamento por Restrição , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Útero/químicaRESUMO
Expression of the multiple interferon-tau (IFN-tau) genes is restricted to embryonic trophectoderm of ruminant ungulate species for a few days in early pregnancy. The promoter regions of these genes are highly conserved. A proximal (bp -91 to -69) sequence has been implicated in controlling trophoblast-specific expression. Here it was used as a target for yeast one-hybrid screening of a day 13 conceptus cDNA library. Two transcription factors of the Ets family, Ets-2 and GABPalpha, were identified, consistent with the observation that active ovine IFN-tau genes contain a single 10-bp Ets motif (core: GGAA) in the proximal segment, whereas three known inactive ovine genes contain a mutated core motif (TGAA). Cotransfection of a promoter- (-126 to +50) luciferase reporter construct from an active gene (bovineIFN-tau1; boIFNT1) and an Ets-2 expression plasmid in human JAr cells provided up to a 30-fold increase in reporter expression, whereas promoters from inactive genes were not transactivated. GABPalpha alone was ineffective and had only a approximately 2-fold positive effect when coexpressed with its partner GABPbeta. Other Ets-related transcription factors, which were not detected in the genetic screen, also provided a range of lesser transactivation effects. Coexpression of Ets-2 and activated Ras failed to transactivate the IFNT promoter greater than Ets-2 alone in JAr cells. The presence of Ets-2 in nuclei of embryonic trophectoderm was confirmed immunocytochemically. Together, these data suggest that Ets-2 plays a role in the transient expression of the nonvirally inducible IFNT genes.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Interferon Tipo I/genética , Proteínas da Gravidez/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras , Transativadores/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , DNA Complementar/genética , Feminino , Fator de Transcrição de Proteínas de Ligação GA , Humanos , Dados de Sequência Molecular , Gravidez , Proteína Proto-Oncogênica c-ets-2 , Saccharomyces cerevisiae , Alinhamento de Sequência , Ovinos , TransfecçãoRESUMO
Capitalizing on the significant sequence homology comprising the transmembrane motif regions of known prostanoid receptor family, we targeted the cloning of a cDNA clone for prostaglandin (PG) F2 alpha receptor from a bovine corpus luteum cDNA library. By using several pairs of degenerated primers created from a common motif of transmembrane domains, polymerase chain reaction gave a clone SN463 carrying the homologous sequence, which covered transmembrane motif IV-VI of the thromboxane (TX) A2 receptor. This polymerase chain reaction product was used as a DNA probe for the following cross-hybridization, and a clone BC2211 carrying a 2.2-kilobase pair DNA insert was isolated. This clone encodes a protein of 362 amino acid residues (M(r) = 40,983) with seven potential transmembrane domains and represented significant overall sequence homology to human TXA2 receptor protein (34% in amino acid). Injection of the mRNA synthesized in vitro from the cloned cDNA into a Xenopus oocyte elicited electrophysiological response to PGF2 alpha. Ligand binding displacement in membranes of mammalian COS-7 cells transfected with the cDNA indicated the rank order of affinity of the receptor to PGs: PGF2 alpha > PGD2 > PGE2 > STA2, a TXA2 agonist. PGF2 alpha activated inositol phosphate formation in COS-7 cells transfected with receptor cDNA. Northern blot analysis and in situ hybridization indicated that the PGF2 alpha receptor mRNA is highly expressed and accumulated in corpus luteum. This is the first report on a successful cloning of functional receptor cDNA for PGF2 alpha.
Assuntos
Receptores de Prostaglandina/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Bovinos , Linhagem Celular , Clonagem Molecular/métodos , Corpo Lúteo/metabolismo , Primers do DNA , Dinoprosta/metabolismo , Dinoprosta/farmacologia , Feminino , Expressão Gênica , Humanos , Hibridização In Situ , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Poli A/isolamento & purificação , Poli A/metabolismo , Reação em Cadeia da Polimerase , RNA/isolamento & purificação , RNA/metabolismo , RNA Mensageiro , Receptores de Prostaglandina/efeitos dos fármacos , Receptores de Prostaglandina/fisiologia , Homologia de Sequência de Aminoácidos , Transfecção , XenopusRESUMO
The pregnancy-associated glycoproteins (PAG) are abundant secretory products of the placental trophectoderm of ungulate species. They are structurally related to pepsin, having the capability to bind peptides. However, many cannot function as enzymes due to amino acid substitutions in and around the catalytic site. Here, we demonstrate that pigs, like cattle and sheep, but unlike equids, have multiple PAG genes. One of the transcribed porcine PAG (poPAG) genes, the one for poPAG2, was cloned. It had a nine-exon organization similar to that of other mammalian aspartic proteinase genes with an atypical TATA sequence. A total of 1.2 kbp upstream from exon 1 was sequenced. This region shared identity (> 65%) with the promoter regions of the bovine (bo) PAG1, boPAG2 and equine (eq) PAG genes, but not with other aspartyl proteinase genes, including that of pepsinogen A. Nor were there clear similarities to the promoters of other genes with trophoblast-specific expression. Of the different poPAG2 promoter constructs tested in transfection experiments in two human (JAr and JEG3) and one rat (Rcho) choriocarcinoma cell lines, only the shortest (-149 bp) was required to provide full expression of a luciferase reporter. Although this short promoter was not active in Cos-1 and L-929 cells, it was active in CHO cells, a transformed non-trophoblast hamster ovarian cell line. Co-transfection of Ets2 elevated the activity of this short promoter approximately six-fold in JAr cells, but, disruption of the two putative Ets sites did not alter the ability of Ets2 to transactivate the promoter. In the non-trophoblast cell lines, Ets2 failed to elicit any response. Ets2 responsiveness may be a common feature of most or all trophoblast-expressed genes, although in the case of poPAG2, the effect may be indirect.
Assuntos
Ácido Aspártico Endopeptidases/genética , Proteínas de Ligação a DNA , Regiões Promotoras Genéticas , Proteínas Repressoras , Suínos/genética , Fatores de Transcrição , Regiões 5' não Traduzidas , Animais , Ácido Aspártico Endopeptidases/química , Sequência de Bases , Éxons , Feminino , Genes Reporter , Humanos , Íntrons , Dados de Sequência Molecular , Gravidez , Proteínas da Gravidez/química , Proteínas da Gravidez/genética , Proteína Proto-Oncogênica c-ets-2 , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional , Transfecção , Células Tumorais CultivadasRESUMO
We examined intracellular signal transduction of prostaglandin F2 alpha (PGF2 alpha) receptors by transfection and stable expression of the receptor cDNA in Chinese hamster ovary (CHO-PGF2 alpha) cells. The binding to the membrane was specific for PGF2 alpha, and the Scatchard plot analysis showed a single high-affinity binding site (Kd = 25.2 nM). PGF2 alpha gradually elevated intracellular Ca2+ and stimulated phosphoinositide metabolism in CHO-PGF2 alpha cells. In whole-cell clamp recordings, PGF2 alpha induced an outward current in the presence of external Ca2+, but it induced a long-lasting inward Ca2+ current in a Na(+)-free solution containing K+ channel blockers. Gq alpha antibody applied intracellularly blocked both outward and inward currents induced by 1 microM PGF2 alpha. These results demonstrate that the PGF2 alpha receptor is coupled to phosphoinositide metabolism in CHO-PGF2 alpha cells via Gq.