RESUMO
Important clues to how the mammalian cerebral cortex develops are provided by the analysis of genetic diseases that cause cortical malformations [1-5]. People with Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) have a hemizygous deletion or mutation in the LIS1 gene [3,6]; both conditions are characterized by a smooth cerebral surface, a thickened cortex with four abnormal layers, and misplaced neurons [7,8]. LIS1 is highly expressed in the ventricular zone and the cortical plate [9,10], and its product, Lis1, has seven WD repeats [3]; several proteins with such repeats have been shown to interact with other polypeptides, giving rise to multiprotein complexes [11]. Lis1 copurifies with platelet-activating factor acetylhydrolase subunits alpha 1 and alpha 2 [12], and with tubulin; it also reduces microtubule catastrophe events in vitro [13]. We used a yeast two-hybrid screen to isolate new Lis1-interacting proteins and found a mammalian ortholog of NudC, a protein required for nuclear movement in Aspergillus nidulans [14]. The specificity of the mammalian NudC-Lis1 interaction was demonstrated by protein-protein interaction assays in vitro and by co-immunoprecipitation from mouse brain extracts. In addition, the murine mNudC and mLis1 genes are coexpressed in the ventricular zone of the forebrain and in the cortical plate. The interaction of Lis1 with NudC, in conjunction with the MDS and ILS phenotypes, raises the possibility that nuclear movement in the ventricular zone is tied to the specification of neuronal fates and thus to cortical architecture.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas Associadas aos Microtúbulos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Proteínas/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase , Sequência de Aminoácidos , Animais , Movimento Celular/fisiologia , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The pituitary peptide hormone prolactin (Prl) is a potent inducer of Nb2 T lymphoma cell proliferation. To analyze the early genetic response to the mitogenic signals of Prl, a cDNA library was constructed from Nb2 T cells stimulated for 4 h with Prl and the protein synthesis inhibitor cycloheximide. Of 26 distinct clones isolated by differential screening, one clone, designated c25, exhibited extremely rapid but transient kinetics of induction by Prl and superinduction by Prl plus cycloheximide. Run-on transcription analysis indicated that c25 gene transcription was induced greater than 20-fold within 30 to 60 min of Prl stimulation. Surprisingly, DNA sequence analysis of c25 cDNA revealed that this Prl-inducible early-response gene is the rat homolog of the mouse transcription factor interferon-regulatory factor 1 (IRF-1), sharing 91% coding sequence similarity with mouse IRF-1. At the protein level, rat IRF-1 shares 97% and 92% homology with mouse IRF-1 and human IRF-1, respectively, suggesting that this molecule has been functionally conserved throughout evolution. Our studies show that the gene for IRF-1 is an immediate-early gene in Prl-stimulated T cells, which suggests that IRF-1 is a multifunctional molecule. In addition to its role in regulating growth-inhibitory interferon genes, IRF-1 may, therefore, also play a stimulatory role in cell proliferation. The gene for IRF-1 is one of the earliest genes known to be transcriptionally regulated by Prl.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fosfoproteínas , Prolactina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Clonagem Molecular , Cicloeximida/farmacologia , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Biblioteca Gênica , Genes , Humanos , Fator Regulador 1 de Interferon , Linfoma , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Ratos , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/genéticaRESUMO
NUDC is a highly conserved protein important for nuclear migration and viability in Aspergillus nidulans. Mammalian NudC interacts with Lis1, a neuronal migration protein important during neocorticogenesis, suggesting a conserved mechanism of nuclear movement in A. nidulans and neuronal migration in the developing mammalian brain (S. M. Morris et al., 1998). To further investigate this possibility, we show for the first time that NudC, Lis1, and cytoplasmic dynein intermediate chain (CDIC) colocalize at the microtubule organizing center (MTOC) around the nucleus in a polarized manner facing the leading pole of cerebellar granule cells with a migratory morphology. In neurons with stationary morphology, NudC is distributed throughout the soma and colocalizes with CDIC and tubulin in neurites as well as at the MTOC. At the subcellular level, NudC, CDIC, and p150 dynactin colocalize to the interphase microtubule array and the MTOC in fibroblasts. The observed colocalization is confirmed biochemically by coimmunoprecipitation of NudC with CDIC and cytoplasmic dynein heavy chain (CDHC) from mouse brain extracts. Consistent with its expression in individual neurons, a high level of NudC is detected in regions of the embryonic neocortex undergoing extensive neurogenesis as well as neuronal migration. These data suggest a biochemical and functional interaction of NudC with Lis1 and the dynein motor complex during neuronal migration in vivo.
Assuntos
Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Neurônios/metabolismo , Proteínas/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase , Animais , Química Encefálica , Células COS , Proteínas de Ciclo Celular , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Plexo Corióideo/citologia , Plexo Corióideo/embriologia , Plexo Corióideo/metabolismo , Epêndima/citologia , Epêndima/embriologia , Epêndima/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Ventrículos Laterais/citologia , Ventrículos Laterais/embriologia , Ventrículos Laterais/metabolismo , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neocórtex/citologia , Neocórtex/embriologia , Neocórtex/metabolismo , Neurônios/citologia , Proteínas Nucleares , Testes de PrecipitinaRESUMO
DNA-mediated gene transfection using an alpha-casein minigene cloned into a bovine papilloma virus (BPV)-based neomycin-selectable expression vector has been employed to study the mechanisms by which hormonal and cell-substratum interactions regulate milk protein gene expression. Permanently transformed clones and pooled populations of normal midpregnant mouse mammary epithelial cells (COMMA-D) containing the minigene express an authentic rat alpha-casein mRNA, as well as a series of larger cytoplasmic RNA transcripts. These transcripts are correctly initiated and spliced; however, a large proportion also contain additional sequences at the 3'-end. Constitutive expression of the minigene in the absence of PRL and glucocorticoids in COMMA-D cells grown on floating type I collagen gels is observed. Thus, the minigene-BPV construct apparently overrides the normal posttranscriptional regulatory mechanisms which are responsible for the expression of unstable casein gene transcripts in the absence of PRL and glucocorticoids. In contrast, this minigene-BPV construct is regulated appropriately by cell-substratum interactions in pooled transfectants. Minigene expression is undetectable when pooled transfectants are plated on a plastic substratum, and readily detectable when cells are grown on floating type I collagen gels. Thus, hormones and cell-substratum interactions may regulate different steps in the same differentiation pathway leading to increased casein gene expression.
Assuntos
Caseínas/genética , Comunicação Celular , Regulação da Expressão Gênica , Hormônios/fisiologia , Transcrição Gênica , Transfecção , Animais , Papillomavirus Bovino 1/genética , Caseínas/biossíntese , Linhagem Celular , Meios de Cultura , DNA/isolamento & purificação , Enzimas de Restrição do DNA , Epitélio/fisiologia , Feminino , Vetores Genéticos , Glândulas Mamárias Animais/fisiologia , Hibridização de Ácido Nucleico , RNA/isolamento & purificação , RNA Mensageiro/biossínteseRESUMO
The interferon regulatory factor-1 (IRF-1) gene is both an immediate-early G1 phase gene and an S phase gene inducible by PRL in rat Nb2 T lymphocytes. To understand the mechanism by which PRL regulates the biphasic expression of IRF-1, we cloned the rat IRF-1 gene and functionally characterized the IRF-1 promoter. Upon transfection into Nb2 T cells, 1.7 kilobases (kb) of IRF-1 5'-flanking DNA linked to a chloramphenicol acetyl transferase (CAT) reporter gene mediated a 30-fold induction of CAT enzyme activity in response to 24 h of PRL stimulation. Deletion mutants containing 1.3, 0.6, and 0.2 kb 5'-flanking DNA were incrementally less transcriptionally active, although 0.2 kb still mediated a 12-fold induction by PRL. The sequence between -1.7 and -0.2 kb linked to a heterologous thymidine kinase promoter failed to respond to PRL stimulation, suggesting that the activity of upstream PRL response elements may require an interaction with promoter-proximal elements. By assaying CAT enzyme activity across a 24-h PRL induction time course, we were able to assign G1 vs. S phase PRL responses of the IRF-1 gene to different regions of the IRF-1 5'-flanking and promoter DNA. The 0.2-kb IRF-CAT construct was induced by PRL stimulation during the G1 phase of the cell cycle. In contrast, the 1.7-kb IRF-CAT construct was inducible by PRL during both G1 and S phase of the cell cycle. Hence, the PRL-induced biphasic expression of the IRF-1 gene appears to be controlled by separate PRL-responsive elements: elements in the first 0.2 kb of the IRF-1 promoter region act during early activation, and elements between 0.2 and 1.7 kb act in concert with the proximal 0.2-kb region during S phase progression.
Assuntos
Proteínas de Ligação a DNA/genética , Fase G1 , Regulação da Expressão Gênica , Genes env/fisiologia , Fosfoproteínas/genética , Prolactina/genética , Fase S , Animais , Sequência de Bases , Ciclo Celular , Linhagem Celular , Cloranfenicol O-Acetiltransferase/análise , Cloranfenicol O-Acetiltransferase/metabolismo , DNA/análise , DNA/genética , Genes Precoces , Genes Reporter , Genes fos/genética , Genes myc/genética , Humanos , Fator Regulador 1 de Interferon , Camundongos , Dados de Sequência Molecular , Ornitina Descarboxilase/genética , Prolactina/fisiologia , Regiões Promotoras Genéticas/genética , Ratos , Linfócitos T/citologia , Fatores de Transcrição/genética , Transcrição Gênica , TransfecçãoRESUMO
PRL induces quiescent Nb2 rat T-lymphoma cells to undergo mitogenesis. Upon PRL stimulation, the transcription factor interferon regulatory factor-1 (IRF-1) is induced as a novel T-cell activation gene in Nb2 cells. Surprisingly, IRF-1 is expressed twice during a single PRL-induced growth cycle: first during the early G1 phase, in an immediate transient peak from 15 min to 2 h, and second during the G1/S phase transition, in a broader peak beginning at 8 h. The unusual biphasic expression of IRF-1 mRNA is accompanied both times by de novo IRF-1 protein synthesis. However, the rate of IRF-1 protein turnover appears to be different in G1 and S phases. IRF-1 protein expressed in G1 exhibits a half-life of about 25 min, whereas in the S phase, the half-life is about 60 min. By washing out PRL at various times during G1, we found a direct correlation among the length of PRL exposure, the second peak of IRF-1 mRNA expression, and DNA synthesis. Our data suggest that PRL and one putative nuclear mediator, IRF-1, may be important in two distinct phases of the cell cycle: first in cell cycle activation, and then in S phase progression.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Interfase/efeitos dos fármacos , Fosfoproteínas/biossíntese , Prolactina/farmacologia , Fase S/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Fatores de Transcrição/biossíntese , Animais , Divisão Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/biossíntese , Proteínas de Ligação a DNA/genética , Fator Regulador 1 de Interferon , Linfoma de Células T , Fosfoproteínas/genética , Ratos , Linfócitos T/metabolismo , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
PRL has been shown to induce a number of genes after the stimulation of quiescent Nb2 T-cells, including c-fos, c-myc, ornithine decarboxylase, interferon regulatory factor-1, and others. One of these genes, LHRH, has not previously been reported to respond in this manner, although we and others have reported its presence in rat and human T- and B-cells. Furthermore, recent evidence suggests that LHRH functions as an immunoregulator in a cytokine-like manner. Using the rat immature T-cell line Nb2, we present data showing for the first time that 1) the LHRH gene is regulated by PRL at various times during the cell cycle; 2) an alternatively spliced LHRH messenger RNA exists in Nb2 cells and may produce a new truncated GnRH-associated peptide (alternatively called PIF for PRL-inhibiting factor); 3) the LHRH receptor is expressed in lymphocytes in a manner similar to the LHRH gene after PRL addition, and its complementary DNA sequence is identical to that of the pituitary receptor; 5) the SH gene, found on the opposite strand of the LHRH gene, is expressed in lymphocytes at the same time and in the same manner as the LHRH gene; 6) the LHRH messenger RNA has a very short half-life in these cells; and 7) the lymphocyte LHRH transcription start site is essentially the same as the hypothalamic site. These data strengthen the relationship between PRL and LHRH expression in the immune system and further support our contention that LHRH is an important immunoregulator, on par with other known cytokines.
Assuntos
Regulação da Expressão Gênica , Hormônio Liberador de Gonadotropina/biossíntese , Fatores Inibidores da Liberação da Prolactina/biossíntese , Prolactina/farmacologia , Receptores LHRH/biossíntese , Linfócitos T/efeitos dos fármacos , Animais , Sequência de Bases , Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Hormônio Liberador de Gonadotropina/genética , Meia-Vida , Sistema Hipotálamo-Hipofisário/fisiologia , Linfoma de Células T , Modelos Genéticos , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neuroimunomodulação/genética , Fatores Inibidores da Liberação da Prolactina/genética , Splicing de RNA , RNA Antissenso/biossíntese , RNA Antissenso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Ratos , Receptores LHRH/genética , Linfócitos T/metabolismo , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Stimulation of quiescent Nb2 T cells by PRL leads to the rapid transcriptional activation of a T cell activation gene, interferon regulatory factor-1 (IRF-1). IRF-1 is induced twice by PRL in a single cell cycle, first during G1 at 30-60 min and again over early S phase at 10-12 h. By nuclear run-on transcription analysis of IRF-1 promoter-chloramphenicol acetyl transferase (CAT) constructs, the -1.7 kilobase (kb) 5'-flanking IRF-1 DNA was shown to contain elements that mediate both G1 and S phase expression. The -200 bp IRF-1 promoter DNA contains elements that respond to G1 PRL stimulation in a protein synthesis independent manner, suggesting the involvement of pre-existing factors. Further promoter deletion analysis delineated a minimal PRL responsive region between -112 and -205 bp. Within this region is a Gamma Interferon Activated Sequence or GAS, consisting of two inverted GAAA motifs (-123/-113), which confers PRL-inducible expression to a reporter gene, suggesting that GAS can function as a PRL responsive element. Further, GAS exhibits binding with nuclear proteins in a PRL-inducible, cell cycle-dependent manner. One of these proteins appears to be related to the emerging family of Signal Transducer and Activator of Transcription or Stat factors. These studies suggest that the GAS site and Stat-like proteins participate in PRL receptor signal transduction to regulate the biphasic expression of the IRF-1 gene in PRL-stimulated T cells.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Fosfoproteínas/genética , Prolactina/farmacologia , Transativadores/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Sequência de Bases , Proteínas de Ligação a DNA/biossíntese , Fase G1 , Fator Regulador 1 de Interferon , Dados de Sequência Molecular , Fosfoproteínas/biossíntese , Ratos , Sequências Reguladoras de Ácido Nucleico , Fase S , Fator de Transcrição STAT1 , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
To examine how PRL regulates lymphocyte proliferation, a number of PRL-activated genes were identified from a PRL-dependent rat T lymphoma cell line, Nb2. One of the downstream genes in the PRL signaling cascade was identified as clone 15 (c15). PRL stimulation of quiescent Nb2 T cells results in the expression of a 1.7-kilobase c15 mRNA, which reaches maximum levels between 8 and 10 h after stimulation. Corresponding [3H]thymidine incorporation experiments show that the maximum level of c15 mRNA expression correlates with the G1/S transition phase of the cell cycle. Sequencing of approximately 1.3-kilobase cDNA revealed one open reading frame that predicts a 332-amino acid protein. In vitro transcription/translation of c15 cDNA resulted in the production of a 45-kilodalton protein. Sequence analysis revealed that the c15 open reading frame contains a potential nuclear localization signal, a very acidic region, and a carboxy-terminal region of 94 amino acids which are 68% identical and 78% similar to the nuclear movement protein, NUDC, found in Aspergillus nidulans. Such a high degree of conservation suggests that the NUDC-like motif in c15 has been conserved through evolution for an important structure and/or function.
Assuntos
Proteínas Fúngicas , Expressão Gênica/efeitos dos fármacos , Proteínas Nucleares , Prolactina/farmacologia , Proteínas/genética , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Aspergillus nidulans , Sequência de Bases , Genisteína , Isoflavonas/farmacologia , Cinética , Linfoma de Células T , Dados de Sequência Molecular , Proteínas/química , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência , Transdução de Sinais , Células Tumorais Cultivadas , Vanadatos/farmacologiaRESUMO
PRL has been shown to stimulate mRNA expression of both ERalpha and ERbeta in the rat corpus luteum and decidua of pregnancy. To investigate whether PRL may stimulate ER expression at the level of transcription and which transcription factors may mediate this stimulation, we have cloned the 5'-flanking regions of both rat ER genes. A constitutively active PRL receptor (PRL-R(CA)) stimulated both ERalpha and ERbeta promoter activity, indicating that PRL is acting to stimulate ER transcription. Putative signal transducer and activator of transcription (Stat)5 response elements were identified at -189 in the ERalpha promoter and at -330 in the ERbeta promoter. Mutation of these response elements or overexpression of dominant negative Stat5 prevented stimulation of ERalpha and ERbeta promoter activity, indicating that PRL regulation of ER expression requires both intact Stat5 binding sites as well as functional Stat5. Interestingly, either Stat5a or Stat5b could stimulate ERalpha transcription while stimulation of ERbeta occurred only in the presence of Stat5b. Through mutational analysis, a single nucleotide difference between the ERalpha and ERbeta Stat5 response elements was shown to be responsible for the lack of Stat5a-mediated stimulation of ERbeta. These findings indicate that PRL stimulation of ER expression occurs at the level of transcription and that PRL regulation of ERalpha can be mediated by either Stat5a or Stat5b, while regulation of ERbeta appears to be mediated only by Stat5b.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas do Leite , Prolactina/fisiologia , Receptores de Estrogênio/genética , Transativadores/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Gravidez , Regiões Promotoras Genéticas/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de Estradiol/biossíntese , Receptores de Estrogênio/biossíntese , Fator de Transcrição STAT5 , Transdução de Sinais/fisiologia , Ativação Transcricional/fisiologiaRESUMO
To identify gene products important for gastrulation in the amphibian Pleurodeles waltl, a screen for regional differences in new protein expression at the early gastrula stage was performed. A 45 kDa protein whose synthesis was specific for progenitor endodermal cells was identified. Microsequencing and cDNA cloning showed that P45 is highly homologous to rat NUDC, a protein suggested to play a role in nuclear migration. Although PNUDC can be detected in all regions of the embryo, its de novo synthesis is tightly regulated spatially and temporally throughout oogenesis and embryonic development. New PNUDC synthesis in the progenitor endodermal cells depends on induction by the mesodermal cells in the gastrula. During development, PNUDC is localized in the egg cortical cytoplasm, at the cleavage furrow during the first embryonic division, around the nuclei and cortical regions of bottle cells in the gastrula, and at the basal region of polarized tissues in the developing embryo. These results show for the first time the expression and compartmentalization of PNUDC at distinct stages during amphibian development.
Assuntos
Embrião não Mamífero/citologia , Desenvolvimento Embrionário , Proteínas Fúngicas/metabolismo , Pleurodeles/embriologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Eletroforese em Gel Bidimensional , Embrião não Mamífero/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Gástrula/citologia , Gástrula/metabolismo , Técnicas Imunoenzimáticas , Testes Imunológicos , Dados de Sequência Molecular , Oogênese/fisiologia , Pleurodeles/genética , Pleurodeles/crescimento & desenvolvimento , Homologia de Sequência de AminoácidosRESUMO
GH is known to activate JAK2 tyrosine kinase and members of the Stat family of transcription factors, including Stats 1, 3, and 5. The recent observation that at least two Stat5 proteins (Stat5A and Stat5B) exist in mouse and human, raises the question of whether GH activates both Stat5A and Stat5B and, if so, whether the requirements for activation are the same. An initial report investigating this issue demonstrated GH-dependent activation of Stat5A but not Stat5B. In this paper, we demonstrate (in COS cells expressing rat GH receptor (rGHR) and either Stat5A or Stat5B, 3T3-F442A fibroblasts, and CHO cells expressing rGHR) that GH induces tyrosyl phosphorylation of both Stat5A and Stat5B. Similar time courses of phosphorylation were observed for the two proteins. Interestingly, the pattern of observed bands differs for the two forms of Stat5. Two closely migrating Stat5A bands can be detected in cells treated with or without GH. Both of these bands become tyrosyl phosphorylated in response to GH. Three species of Stat5B are observed in untreated cells. An additional, more slowly migrating Stat5B band, appears upon treatment with GH. The three more slower migrating Stat5B bands observed in response to GH contain phosphorylated tyrosyl residues. We further demonstrate that GH induces binding of Stat5A and Stat5B, as well as Stat1, to the GAS-like element in the beta-casein promoter. We and others have demonstrated previously that specific regions of GHR are required for GH-dependent activation of what is here identified as Stat5B. To gain insight into the mechanism by which GH promotes tyrosyl phosphorylation of Stat5A, GH-dependent tyrosyl phosphorylation of Stat5A was examined in CHO cells expressing truncated and mutated rGHR. The results indicate that Stat5A and Stat5B require the same regions of rGHR for maximal activation by GH: the C-terminal half of the cytoplasmic domain; tyrosines 333 and/or 338 in the N-terminal half of the cytoplasmic domain; and the regions required for JAK2 activation. To dissect further the mechanism by which GH activates Stat5A and B, the requirement for JAK2 in GH-dependent Stat5 tyrosyl phosphorylation was assessed using JAK2-deficient cells expressing GHR (gamma2A-GHR) and the wild-type parental cell line expressing GHR (2C4-GHR). GH-induced tyrosyl phosphorylation of Stat5B in 2C4-GHR cells but not in the JAK2 deficient, gamma2A-GHR cells, indicating that JAK2 is required for GH-dependent tyrosyl phosphorylation of Stat5B. Western blotting revealed that Stat5A is not expressed in this cell type. Taken together, these findings suggest that: 1) GH activates both Stat5A and Stat5B in several cell types; 2) the pattern of bands observed differs for Stat5A and Stat5B; 3) GH-dependent tyrosyl phosphorylation of Stat5A requires specific regions of GHR, and these requirements are the same as for Stat5B; and 4) JAK2 kinase is required for GH-dependent tyrosyl phosphorylation of Stat5B and, most likely, Stat5A.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Hormônio do Crescimento/farmacologia , Proteínas do Leite , Proteínas Proto-Oncogênicas , Transativadores/metabolismo , Tirosina/metabolismo , Animais , Anticorpos/análise , Anticorpos/imunologia , Especificidade de Anticorpos , Western Blotting , Células CHO/citologia , Células CHO/metabolismo , Células CHO/fisiologia , Células COS/citologia , Células COS/metabolismo , Células COS/fisiologia , Linhagem Celular , Cricetinae , Proteínas de Ligação a DNA/imunologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Janus Quinase 2 , Camundongos , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/fisiologia , Ratos , Receptores da Somatotropina/genética , Fator de Transcrição STAT5 , Transativadores/imunologia , TransfecçãoRESUMO
Interferon-tau (IFNtau), a type I IFN produced by sheep conceptus trophectoderm, is the signal for maternal recognition of pregnancy. Although it is clear that IFNtau suppresses transcription of the estrogen receptor alpha and oxytocin receptor genes and induces expression of various IFN-stimulated genes within the endometrial epithelium, little is known of the signal transduction pathway activated by the hormone. This study determined the effects of IFNtau on signal transducer and activator of transcription (STAT) activation, expression, DNA binding, and transcriptional activation using an ovine endometrial epithelial cell line. IFNtau induced persistent tyrosine phosphorylation and nuclear translocation of STAT1 and -2 (10 min to 48 h), but transient phosphorylation and nuclear translocation of STAT3, -5a/b, and -6 (10 to <60 min). IFNtau increased expression of STAT1 and -2, but not STAT3, -5a/b, and -6. IFN-stimulated gene factor-3 and STAT1 homodimers formed and bound an IFN-stimulated response element (ISRE) and gamma-activated sequence (GAS) element, respectively. IFNtau increased transcription of GAS-driven promoters at 3 h, but suppressed their activity at 24 h. In contrast, the activity of an ISRE-driven promoter was increased at 3 and 24 h. These results indicate that IFNtau activates multiple STATs and has differential effects on ISRE- and GAS-driven gene transcription.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endométrio/fisiologia , Células Epiteliais/fisiologia , Interferon Tipo I/farmacologia , Proteínas da Gravidez/farmacologia , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica/fisiologia , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Endométrio/citologia , Células Epiteliais/citologia , Feminino , Interferon Tipo I/fisiologia , Fator Gênico 3 Estimulado por Interferon , Luciferases , Fosforilação , Proteínas da Gravidez/fisiologia , Regiões Promotoras Genéticas , Transporte Proteico , Proteínas Recombinantes de Fusão/análise , Fator de Transcrição STAT1 , Fator de Transcrição STAT2 , Ovinos , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
PRL expression in the human uterus is up-regulated during the mid to late secretory phase of the menstrual cycle. This coincides with up-regulation of the expression of the PRL receptor, which is localized primarily to the endometrial glandular epithelial cells. Recent data have demonstrated activation of the Jak (Janus kinase)/Stat (signal transducer and activator of transcription) signaling pathway in the secretory endometrium after stimulation with exogenous PRL. However, the target genes for the action of PRL on the endometrial epithelial cells have not been elucidated. In this study we have investigated the pattern/site of expression of the transcription factor interferon regulatory factor-1 (IRF-1) as well as the effect of exogenous PRL on the transcription of IRF-1 in the human endometrium during the mid to late secretory phase of the menstrual cycle. Expression of the IRF-1 gene was confirmed by RNase protection assays using a 260-bp homologous [alpha-32P]UTP-labeled IRF-1 complementary ribonucleic acid (RNA) probe and 10 microg total RNA extracted from human endometrium (n = 5) collected between days 19 and 26 of the menstrual cycle. Northern and Western blot analyses were conducted on secretory phase human endometrium (n = 3) using human [alpha-32P]dCTP-labeled IRF-1 complementary DNA and antihuman IRF-1 antibody. Expression of the IRF-1 gene in the secretory phase endometrium was encoded by a RNA transcript of approximately 2.1 kb and a protein of 48 kDa. Furthermore, expression of the IRF-1 gene in the secretory phase endometrium was localized by immunohistochemistry predominantly to the glandular epithelial cells as has been shown previously for the PRL receptor. To investigate the effect of PRL on expression of IRF-1, human endometrial biopsies (n = 3) collected between days 24-26 of the menstrual cycle were cultured in the presence of cycloheximide with or without 100 ng/mL human PRL for 2 and 4 h. Culture of endometrial tissue with PRL for 2 and 4 h resulted in 2.9 +/- 0.3-fold (P < 0.01) and 1.7 +/- 0.1-fold induction of expression of the IRF-1 gene, respectively. These data demonstrate the expression of the transcription factor IRF-1 in the glandular epithelium of the endometrium and its regulation by PRL during the secretory phase of the menstrual cycle. Previous observations of the temporal up-regulation of expression of both PRL and PRL receptors in the secretory human endometrium and their localization to the stromal and glandular compartments, respectively, suggest that endometrial PRL mediates transcription of the IRF-1 gene in a paracrine fashion.
Assuntos
Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Endométrio/química , Regulação da Expressão Gênica/efeitos dos fármacos , Ciclo Menstrual/fisiologia , Fosfoproteínas/análise , Fosfoproteínas/genética , Prolactina/farmacologia , Northern Blotting , Western Blotting , Técnicas de Cultura , Endométrio/metabolismo , Epitélio/química , Epitélio/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Fator Regulador 1 de Interferon , Prolactina/análise , Sondas RNA , RNA Mensageiro/análise , Receptores da Prolactina/análise , Células Estromais/química , Células Estromais/metabolismo , Fatores de TranscriçãoRESUMO
The pituitary peptide hormone prolactin exerts a profound effect on various physiological processes involving both cellular proliferation and differentiation. The rat Nb2 T lymphoma cell line has been used as a model system for studying prolactin regulation of cell proliferation. Several genes associated with cell growth (c-myc, ornithine decarboxylase (ODC), heat shock protein 70 (hsp 70)-homologue, and beta-actin) are induced rapidly within 4 h after prolactin addition. Nuclear run-on transcription assays indicate that prolactin induction of these growth-related genes occurs primarily at the transcriptional level. According to the different kinetics of transcriptional response to prolactin, these growth-related genes can be divided into immediate-early (actin, c-myc), early (ODC) and mid-G1 (hsp 70-homologue) genes. Thus, prolactin may regulate Nb2 T cell-proliferative responses by modulating the transcriptional induction of various growth-related genes. These studies also represent a first report of a transcriptional cascade set off in rapid response to prolactin in cultured T cells.
Assuntos
Actinas/genética , Divisão Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Genes/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Ornitina Descarboxilase/genética , Prolactina/farmacologia , Proteínas Proto-Oncogênicas/genética , Transcrição Gênica/efeitos dos fármacos , Actinas/biossíntese , Animais , Northern Blotting , Proteínas de Choque Térmico/biossíntese , Linfoma , Camundongos , Ornitina Descarboxilase/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-myc , Ratos , Células Tumorais CultivadasRESUMO
Transcription of the interferon regulatory factor-1 gene (IRF-1) is induced in a biphasic manner (G1 and G1/S phase) in Nb2 T cells in response to prolactin (PRL) stimulation. Signal transducer and activator of transcription 1 (Stat1) is required for PRL activation of the IRF-1 promoter. Mutation of a -200 bp Sp1 site in the IRF-1 promoter results in a loss of G1 but not G1/S IRF-1 transcriptional activity in response to PRL. These studies illustrate that the temporal transcription of the IRF-1 gene is mediated by not only Stat1 but also Sp1 in response to PRL stimulation.
Assuntos
Proteínas de Ligação a DNA/genética , Fosfoproteínas/genética , Prolactina/farmacologia , Fator de Transcrição Sp1/fisiologia , Ativação Transcricional/efeitos dos fármacos , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Fase G1 , Fator Regulador 1 de Interferon , Mutação , Fosfoproteínas/efeitos dos fármacos , Prolactina/metabolismo , Prolactina/fisiologia , Regiões Promotoras Genéticas , Ratos , Fator de Transcrição STAT1 , Transdução de Sinais , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Transativadores/efeitos dos fármacos , Transativadores/metabolismo , Transativadores/fisiologia , Transfecção , Células Tumorais CultivadasRESUMO
Interferon regulatory factor-1 (IRF-1) is a major immediate early gene induced by prolactin (PRL) in a biphasic, cell cycle-dependent manner in Nb2 T cells. This biphasic expression (30 min and 10 h) is mediated in part by an interferon-gamma activation sequence (GAS) in the IRF-1 promoter which binds factors belonging to the Signal Transducers and Activators of Transcription (Stat) family. By electrophoretic mobility shift assays (EMSA), Stat1 alpha was found to be the major and Stat5a a minor component of the 30 min complex. At 10 h, Stat-like factors were again found at the IRF-1 GAS. Western blot analyses show that Stat5a was rapidly induced by PRL to enter the nucleus, but unexpectedly, Stat1 alpha and the alternatively-spliced Stat1 beta were already present in the uninduced nucleus. Further, Stat1 alpha but not Stat1 beta is preferentially tyrosine phosphorylated in response to PRL stimulation. Our studies suggest that multiple Stat complexes may contribute to the biphasic transcription of the IRF-1 gene in PRL-stimulated T cells.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas do Leite , Fosfoproteínas/genética , Prolactina/farmacologia , Regiões Promotoras Genéticas , Linfócitos T/metabolismo , Transativadores/metabolismo , Fator Regulador 1 de Interferon , Fosforilação , Fator de Transcrição STAT1 , Fator de Transcrição STAT5 , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Fatores de Tempo , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
To understand the role of pituitary prolactin (PRL) and its receptor (PRL-R) in the growth and differentiation of lymphoid cells, PRL-R gene expression was analyzed in various lymphoid tissues and in a rat T lymphoma cell line, Nb2, which requires PRL for growth. The technique of reverse transcription coupled to polymerase chain reaction (RT-PCR) was used to detect the low abundance PRL-R transcripts. Within 30 min to 1 h, PRL stimulates a rapid but transient increase in PRL-R mRNA levels in Nb2 T cells. By 4 h, PRL-R mRNA returned to near basal levels and then gradually declined to a new steady-state level by 12 h. Significant increases in receptor RNA levels were observed in the presence of protein synthesis inhibitors, which suggests that PRL-R mRNA levels are under negative regulation. PRL-R gene expression was also demonstrated in normal mouse thymocytes, splenocytes, and in several lymphoid cell lines. The expression of the PRL-R gene in stimulated lymphoid cells provides additional evidence for the role of PRL as an immunomodulatory molecule.
Assuntos
Sistema Linfático/metabolismo , Receptores da Prolactina/genética , Animais , Sequência de Bases , Diferenciação Celular , Divisão Celular , Técnicas de Cultura , DNA , Regulação da Expressão Gênica , Sistema Linfático/citologia , Linfoma , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Receptores da Prolactina/metabolismo , Baço/metabolismo , Timo/metabolismo , Células Tumorais CultivadasRESUMO
Interferon regulatory factor-1 (IRF-1) gene expression is rapidly upregulated in the prolactin (PRL)-activated Nb2 rat T lymphoma cell line. To further elucidate its role as a T cell activation molecule, IRF-1 gene expression in response to various T cell stimuli was examined. In Nb2 T cells, PRL induced two peaks of IRF-1 gene expression: a rapid, transient peak at 1 h and a sustained peak at 12 h. PRL subsequently induced interferon-gamma (IFN-gamma) gene expression at 3-6 h. However, the early induction of IRF-1 and IFN-gamma does not appear to be interdependent. Interleukin-2 (IL-2) also induced IRF-1 gene expression in Nb2 T cells but only one broad peak at 10 h was observed. In primary mouse splenocytes, concanavalin A induced rapid and transient expression of the IRF-1 gene; maximal expression occurred by 6 h, and then returned to basal levels by 12-15 h. These results provide additional evidence for the importance of IRF-1 in T cell activation.
Assuntos
Concanavalina A/farmacologia , Proteínas de Ligação a DNA/biossíntese , Interleucina-2/farmacologia , Fosfoproteínas/biossíntese , Prolactina/farmacologia , RNA Mensageiro/biossíntese , Linfócitos T/metabolismo , Animais , Células Cultivadas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator Regulador 1 de Interferon , Linfoma de Células T/patologia , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Ratos , Receptores de Superfície Celular/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Estimulação Química , Linfócitos T/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
Recent evidence suggests that lymphocytes produce prolactin (PRL). Here, we report the cDNA cloning and expression of PRL from normal human thymocytes. Sequence analysis showed that the thymocyte cDNA encodes a 23 kDa protein which is identical to pituitary PRL. RNA blot analysis showed that the thymocyte PRL mRNA is approximately 170 nucleotides larger than the pituitary PRL message. PRL message was also detected in several non-pituitary human cell lines including Jurkat T, HeLa, and JEG cells. Furthermore, PRL gene expression in JEG cells was inhibited by glucocorticoid treatment. Our data support the hypothesis that PRL is a T cell-derived cytokine.