RESUMO
Cervical cancer (CC) is associated with alterations in immune system balance, which is primarily due to a shift from Th1 to Th2 and the unbalance of Th17/Treg cells. Using in silico DNA copy number analysis, we have demonstrated that ~20% of CC samples exhibit gain of 8q22.3 and 19q13.31; the regions of the genome that encodes the KLF10 and PSG genes, respectively. Gene expression studies demonstrated that there were no alterations in KLF10 mRNA expression, whilst the PSG2 and -5 genes were up-regulated by 1.76 and 3.97-fold respectively in CC compared to normal tissue controls. siRNA and ChIP experiments in SiHa cells have demonstrated that KLF10 participates in immune response through regulation of IL6, IL25 and PSG2 and PSG5 genes. Using cervical tissues from KLF10-/- mice, we have identified down-regulation of PSG17, -21 and -23 and IL11. These results suggest that KLF10 may regulate immune system response genes in cervical cancer among other functions. KLF10 and PSG copy number variations and alterations in mRNA expression levels could represent novel molecular markers in CC.
Assuntos
Fatores de Transcrição de Resposta de Crescimento Precoce/metabolismo , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/metabolismo , Glicoproteínas beta 1 Específicas da Gravidez/genética , Neoplasias do Colo do Útero/genética , Animais , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Feminino , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Glicoproteínas beta 1 Específicas da Gravidez/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias do Colo do Útero/imunologiaRESUMO
PROBLEM: The contribution of Pregnancy-specific glycoproteins (PSG), the major variant of PSG released into the circulation during pregnancy, to the pregnancy-dependent improvement of rheumatoid arthritis (RA) has still not been elucidated. METHOD OF STUDY: Collagen-induced arthritis (CIA) was used to test the hypothesis that PSG1a when released into circulation has a modulatory role on the Th1-pathogenic response, thus improving the CIA symptoms. In vivo expression of PSG1a was induced by injection of the vaccinia (Vac)-based expression vector harboring the complete open-reading frame of PSG1a cDNA. RESULTS: In vivo PSG1a expression during the induction of CIA ameliorated the clinical symptoms, thereby reducing the arthritis score and incidence. Significantly lower levels of IL-17, IL-6, and IFN-γ, but higher levels of TGF-ß and IL-10 were secreted by collagen type II-stimulated spleen mononuclear cells from Vac-PSG1a-treated mice compared with control mice. Moreover, Vac-PSG1a treatment promoted the increase in splenic CD4+CD25+Foxp3+ Treg cells. CONCLUSION: Pre-clinical Vac-PSG1a treatment suppressed the Th1- and Th17-type-specific responses, leading to an increase in splenic Treg cells as well as IL-10- and TGF-ß-secreting cells, with the CIA symptoms being ameliorated.
Assuntos
Artrite Experimental/terapia , Imunomodulação , Glicoproteínas beta 1 Específicas da Gravidez/imunologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Antígenos CD4/metabolismo , Células Cultivadas , Citocinas/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Vetores Genéticos/genética , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , Glicoproteínas beta 1 Específicas da Gravidez/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vaccinia virusRESUMO
BACKGROUND: Lysine acetylation is an important post-translational modification that plays a central role in eukaryotic transcriptional activation by modifying chromatin and transcription-related factors. Human pregnancy-specific glycoproteins (PSG) are the major secreted placental proteins expressed by the syncytiotrophoblast at the end of pregnancy and represent early markers of cytotrophoblast differentiation. Low PSG levels are associated with complicated pregnancies, thus highlighting the importance of studying the mechanisms that control their expression. Despite several transcription factors having been implicated as key regulators of PSG gene family expression; the role of protein acetylation has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: Here, we explored the role of acetylation on PSG gene expression in the human placental-derived JEG-3 cell line. Pharmacological inhibition of histone deacetylases (HDACs) up-regulated PSG protein and mRNA expression levels, and augmented the amount of acetylated histone H3 associated with PSG 5'regulatory regions. Moreover, PSG5 promoter activation mediated by Sp1 and KLF6, via the core promoter element motif (CPE, -147/-140), was markedly enhanced in the presence of the HDAC inhibitor trichostatin A (TSA). This effect correlated with an increase in Sp1 acetylation and KLF6 nuclear localization as revealed by immunoprecipitation and subcellular fractionation assays. The co-activators PCAF, p300, and CBP enhanced Sp1-dependent PSG5 promoter activation through their histone acetylase (HAT) function. Instead, p300 and CBP acetyltransferase domain was dispensable for sustaining co-activation of PSG5 promoter by KLF6. CONCLUSIONS/SIGNIFICANCE: Results are consistent with a regulatory role of lysine acetylation on PSG expression through a relaxed chromatin state and an increase in the transcriptional activity of Sp1 and KLF6 following an augmented Sp1 acetylation and KLF6 nuclear localization.
Assuntos
Histonas/genética , Placenta/metabolismo , Glicoproteínas beta 1 Específicas da Gravidez/genética , Regulação para Cima/fisiologia , Acetilação/efeitos dos fármacos , Linhagem Celular , Feminino , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Lisina/genética , Lisina/metabolismo , Placenta/efeitos dos fármacos , Gravidez , Glicoproteínas beta 1 Específicas da Gravidez/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Ativação Transcricional , Regulação para Cima/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
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
It has been proposed that pregnancy-specific factors induce the suppression of a specific arm of the maternal response accompanied by activation of the nonspecific, innate immune system. The aim of this study was to determine whether pregnancy-specific glycoprotein 1a (PSG1a), the major variant of PSG polypeptides, is able to modulate the monocyte/macrophage (Mo) metabolism to regulate T cell activation and proliferation. Using the recombinant form of this glycoprotein (rec-PSG1a), expressed in mammalian cells with a vaccinia-based expression vector, we have demonstrated that human PSG1a induces arginase activity in peripheral blood human Mo and human and murine Mo cell lines. In addition, rec-PSG1a is able to induce alternative activation because it up-regulates the arginase activity and inhibits the nitric oxide production in Mo activated by lipopolysaccharides. We also observed that rec-PSG1a is an important accessory cells-dependent T cell suppressor factor that causes partial growth arrest at the S/G2/M phase of the cell cycle. Additionally, an impaired T cell proliferative response induced by mitogens and specific antigen was observed in BALB/c mice upon in vivo expression of PSG1a. Our results suggest that PSG1a function contributes to the immunomodulation during pregnancy, having opposite effects on maternal innate and adaptative systems.
Assuntos
Ativação Linfocitária/fisiologia , Ativação de Macrófagos/fisiologia , Monócitos/citologia , Glicoproteínas beta 1 Específicas da Gravidez/fisiologia , Gravidez/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Arginase/biossíntese , Arginase/genética , Ciclo Celular , Divisão Celular , Linhagem Celular , Concanavalina A/farmacologia , Indução Enzimática/efeitos dos fármacos , Feminino , Glicosilação , Células HeLa/citologia , Humanos , Lipopolissacarídeos/farmacologia , Teste de Cultura Mista de Linfócitos , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Glicoproteínas beta 1 Específicas da Gravidez/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/fisiologiaRESUMO
The pregnancy-specific beta 1 glycoprotein (PSG) genes encode a group of heterogeneous proteins produced in large amounts by the human syncytiotrophoblast. Their expression seems to be regulated at the transcriptional level during normal pregnancy. In the present work, we isolated from a human placental library a 17 kb genomic fragment corresponding to a member of the PSG multigene family. DNA sequence analysis of 1190 nucleotides upstream of the translational start and of the first intron, revealed the presence of several putative regulatory sequences. In a transient chloramphenicol acetyltransferase expression assay, 5' flanking sequences within 123 nucleotides upstream to the first major transcription initiation site, functioned as a strong promoter in COS-7 cells. Meanwhile, sequences 5' further upstream had the ability to abolish this promoter activity. The sequence analyzed did not contain any obvious TATA-like boxes or G+C-rich regions, suggesting the existence of unique promoter elements implicated in transcription initiation and regulation of this PSG gene family member.