RESUMO
Preterm birth occurs in 9% to 13% of all human pregnancies and accounts for 80% of all neonatal morbidities and mortalities. Approximately 40% of all preterm births are idiopathic and about half are associated with infection and/or an activated inflammatory process. Further to studies showing anti-inflammatory effects of supernatant from the probiotic Lactobacillus rhamnosus GR-1 (GR-1), we tested its ability to modulate cytokine and chemokine production from amnion cells in response to stimulation by bacterial wall components, lipopolysaccharide (LPS), and lipoteichoic acid (LTA). Placentae were collected from women undergoing elective cesarean section at term. Amnion cells were cultured for 48 hours to confluence, serum starved for 12 hours, and then treated with GR-1 supernatant (1:20 dilution), followed after 12 hours by LPS (100 ng/mL) or LTA (10 ng/mL) for an additional 12 hours. Both LTA and LPS caused significant increases in the concentration of the pro-inflammatory cytokine, tumor necrosis factor α (TNF-α; 103.9 ± 67.5 pg/mL and 368.3 ± 65.7 pg/mL, respectively) in medium from cultured amnion cells compared to control (<4 pg/mL). There was no significant effect of GR-1 supernatant alone on TNF-α output, but there was significant reduction after LPS treatment. The basal output of the immunomodulatory cytokine, interleukin 6, was 613 ± 170 pg/mL and increased significantly after addition of GR-1 supernatant, LTA, LPS, and combinations of LTA/LPS with GR-1 supernatant. In conclusion, probiotic L rhamnosus GR-1 attenuates the effect of both LPS and LTA in stimulating the output of the pro-inflammatory cytokine TNF-α from mixed cultures of human amnion cells in keeping with previous findings in human trophoblast cells.
Assuntos
Âmnio/efeitos dos fármacos , Quimiocinas/metabolismo , Citocinas/metabolismo , Lacticaseibacillus rhamnosus , Lipopolissacarídeos/farmacologia , Probióticos/farmacologia , Ácidos Teicoicos/farmacologia , Âmnio/citologia , Âmnio/metabolismo , Células Cultivadas , Feminino , Humanos , Placenta/citologia , Placenta/efeitos dos fármacos , Placenta/metabolismo , GravidezRESUMO
OBJECTIVE: To determine the effects of betamethasone on fetal growth and neonatal outcomes. METHODS: A retrospective cohort study was performed of deliveries that occurred at Charité University Hospital Berlin, Germany, between January 1996 and December 2008. The betamethasone group included women with preterm labor and symptomatic contractions, cervical insufficiency, preterm premature rupture of membranes, or vaginal bleeding. Women in the control group were matched for gestational age at time of delivery and had not received betamethasone. Fetal growth changes and neonatal anthropometry were compared. RESULTS: Among 1799 newborns in the betamethasone group and 42 240 in the control group, betamethasone was associated with significantly lower birth weight (154 g lower on average) after adjusting for confounders (e.g. hypertension, smoking, and maternal weight), sex, and gestational age at delivery (P<0.05). The higher the dose, the greater the difference in mean birth weight versus controls in births before 34(+0)weeks (≤16 mg -444 g; 24 mg -523 g; >24 mg -811 g), without a detectable improvement in neonatal morbidity or mortality. There was a dose-dependent decline in expected fetal weight gain as estimated by serial ultrasonography examinations 6-8 weeks after betamethasone administration (P<0.05). CONCLUSION: Betamethasone exposure reduces fetal weight gain in a dose-dependent manner without improving neonatal morbidity or mortality.
Assuntos
Betametasona/administração & dosagem , Desenvolvimento Fetal/efeitos dos fármacos , Glucocorticoides/administração & dosagem , Resultado da Gravidez , Nascimento Prematuro/tratamento farmacológico , Nascimento a Termo/efeitos dos fármacos , Adulto , Peso ao Nascer , Parto Obstétrico , Feminino , Ruptura Prematura de Membranas Fetais/tratamento farmacológico , Alemanha , Idade Gestacional , Humanos , Recém-Nascido , Masculino , Gravidez , Síndrome do Desconforto Respiratório do Recém-Nascido/tratamento farmacológico , Estudos RetrospectivosRESUMO
Glucocorticoid treatment given in late pregnancy in sheep resulted in altered placental development and function. An imbalance of placental survival and apoptotic factors resulting in an increased rate of apoptosis may be involved. We have now investigated the effects of dexamethasone (DEX) in early pregnancy on binucleate cells (BNCs), placental apoptosis, and fetal sex as a determinant of these responses. Pregnant ewes carrying singleton fetuses (n = 105) were randomized to control (n = 56, 2 mL saline/ewe) or DEX treatment (n = 49, intramuscular injections of 0.14 mg/kg ewe weight per 12 hours over 48 hours) at 40 to 41 days of gestation (dG). Placentomes were collected at 50, 100, 125, and 140 dG. At 100 dG, DEX in females reduced BNC numbers, placental antiapoptotic (proliferating cell nuclear antigen), and increased proapoptotic factors (Bax, p53), associated with a temporarily decrease in fetal growth. At 125 dG, BNC numbers and apoptotic markers were restored to normal. In males, ovine placental lactogen-protein levels after DEX were increased at 50 dG, but at 100 and 140 dG significantly decreased compared to controls. In contrast to females, these changes were independent of altered BNC numbers or apoptotic markers. Early DEX was associated with sex-specific, transient alterations in BNC numbers, which may contribute to changes in placental and fetal development. Furthermore, in females, altered placental apoptosis markers may be involved.
Assuntos
Apoptose/efeitos dos fármacos , Dexametasona/toxicidade , Glucocorticoides/toxicidade , Placenta/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Feminino , Retardo do Crescimento Fetal/induzido quimicamente , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/patologia , Peso Fetal/efeitos dos fármacos , Idade Gestacional , Masculino , Placenta/metabolismo , Placenta/patologia , Gravidez , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores Sexuais , Ovinos , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
OBJECTIVE: The objective of this study was to determine the effect of probiotic Lactobacillus rhamnosus GR-1 supernatant (GR-1 SN) on lipopolysaccharide-induced preterm birth (PTB) and outputs of cytokines, chemokines, and progesterone in pregnant CD-1 mice. STUDY DESIGN: We compared PTB rates after intrauterine injection of lipopolysaccharide with and without previous GR-1 SN treatment. Cytokines and chemokines in the maternal plasma, myometrium, placenta, and amniotic fluid were examined with multiplex assay, and circulating maternal progesterone was measured with enzyme-linked immunoassay. Statistical significance was assessed with 2-tailed 1-way analysis of variance or analysis of variance on ranks. Fetal sex ratios in mice that delivered preterm were compared with those that delivered at term after lipopolysaccharide and GR-1 SN treatments. RESULTS: GR-1 SN reduced lipopolysaccharide-induced PTB by 43%. GR-1 SN significantly decreased the lipopolysaccharide-induced production of interleukin (IL)-1ß, -6, and -12p40, tumor necrosis factor-α, CCL4, and CCL5 in maternal plasma; IL-6, -12p70, -17, and -13 and tumor necrosis factor-α in myometrium; IL-6, -12p70, and -17 in placenta; and IL-6, tumor necrosis factor-α, CCL3, and CCL4 in amniotic fluid. Maternal plasma progesterone was reduced significantly after lipopolysaccharide injection with and without GR-1 SN pretreatment. There was no difference in fetal sex ratios between mice that delivered preterm and those that did not after lipopolysaccharide and GR-1 SN treatments. CONCLUSION: The supernatant of probiotic L rhamnosus GR-1 attenuated lipopolysaccharide-induced inflammation and PTB in vivo. GR-1 SN may confer therapeutic benefits in the prevention of infection-associated PTB by controlling systemic and intrauterine inflammation.
Assuntos
Inflamação/prevenção & controle , Lacticaseibacillus rhamnosus , Nascimento Prematuro/prevenção & controle , Probióticos/uso terapêutico , Animais , Biomarcadores/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Inflamação/diagnóstico , Inflamação/etiologia , Inflamação/metabolismo , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos ICR , Reação em Cadeia da Polimerase Multiplex , Gravidez , Complicações na Gravidez/diagnóstico , Complicações na Gravidez/etiologia , Complicações na Gravidez/metabolismo , Complicações na Gravidez/prevenção & controle , Nascimento Prematuro/etiologia , Progesterona/metabolismo , Distribuição Aleatória , Razão de MasculinidadeRESUMO
The aim of this study was to assess the effects of bacterial lipopolysaccharide (LPS) and Lactobacillus rhamnosus GR-1 supernatant (GR-1SN) on secretion profiles of cytokines, chemokines, and growth factors from primary cultures of human decidual cells. Lipopolysaccharide significantly increased the output of proinflammatory cytokines (interleukin [IL]-1B, IL-2, IL-6, IL-12p70, IL-15, IL-17A, interferon gamma [IFN-γ], and tumor necrosis factor [TNF]); anti-inflammatory cytokines (IL-1RN, IL-4, IL-9, and IL-10); chemokines (IL-8, eotaxin, IFN-inducible protein 10 [IP-10], monocyte chemoattractant protein 1 [MCP-1], macrophage inflammatory protein-1α [MIP-1α], macrophage inflammatory protein-1ß [MIP-1ß], and regulated on activation normal T cell expressed and secreted [RANTES]); and growth factors (granulocyte colony-stimulating factor [CSF] 3, CSF-2, and vascular endothelial growth factor A [VEGFA]). Lactobacillus rhamnosus GR-1SN alone significantly increased CSF-3, MIP-1α MIP-1ß, and RANTES but decreased IL-15 and IP-10 output. The GR-1SN also significantly or partially reduced LPS-induced proinflammatory cytokines TNF, IFN-γ, IL-1ß, IL-2 IL-6, IL-12p70, IL-15, IL-17, and IP-10; partially reduced LPS-induced anti-inflammatory cytokines IL-1RN, IL-4 and IL-10, and LPS-induced VEGFA output but did not affect CSF-3, MIP-1α, MIP-1ß, MCP-1, IL-8, and IL-9. Our results demonstrate that GR-1SN attenuates the inflammatory responses to LPS by human decidual cells, suggesting its potential role in ameliorating intrauterine infection.
RESUMO
CONTEXT: During pregnancy, the myometrium undergoes a phenotype programming starting from an early proliferative stage, to an intermediate synthetic stage, to a late contractile stage, after which the cells commit to labor. Steroid receptors play important roles in regulating myometrial cell phenotype during pregnancy, although detailed mechanisms are not fully defined. OBJECTIVE: The aim of the study was to investigate the expression and function of the androgen receptor (AR) in myometrial cells during pregnancy. DESIGN AND SETTING: Human primary myometrial cells, immortalized myometrial cells, rat pregnant and tubal ligation models were used. Immunohistochemistry, Western blot and real-time PCR, cell proliferation, and flow cytometry assays were applied. RESULTS: The AR is highly expressed in the proliferative stage of pregnancy, starts to decrease in the synthetic stage, and reaches the lowest levels in the contractile stage. Both the mechanical stretch by the growing fetus and the decreased ratio of progestin:estrogen are responsible for AR protein reduction. AR regulates myometrial cell proliferation ligand-independently. Decreased AR expression delays the G(1)-S phase transition of human myometrial cell cycling and reduces expression of several cyclins. These AR actions are mediated through reducing IGF-I receptor protein stability, thus weakening PI3K/Akt signal cascade downstream of IGF-I. AR is required for IGF-I receptor protein stability by preventing the IGF-I receptor from ubiquitylation and protein degradation through both proteosomal and lysosomal pathways. CONCLUSION: AR is a key regulator for myometrial cell proliferation, suggesting its critical role in myometrium phenotype programming during pregnancy.
Assuntos
Proliferação de Células , Desenvolvimento Embrionário/fisiologia , Miométrio/fisiologia , Receptores Androgênicos/fisiologia , Animais , Antineoplásicos Hormonais/farmacologia , Benzamidas , Células Cultivadas , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Idade Gestacional , Humanos , Metribolona/farmacologia , Miométrio/citologia , Miométrio/metabolismo , Nitrilas , Fenótipo , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Gravidez/genética , Gravidez/metabolismo , Ratos , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Congêneres da Testosterona/farmacologiaRESUMO
Progesterone (P4), acting through its receptor (PR), is essential for the maintenance of pregnancy. P4 acts by suppressing uterine contractility and the expression of contraction-associated proteins (CAP) such as connexin 43 (Cx43). P4 levels must be reduced or its actions blocked to allow the increased expression of CAP genes and the initiation of labor. Although the importance of progesterone in pregnancy has been known for about 80 yr, the fundamental mechanisms by which P4/PR maintains myometrial quiescence and by which this signaling is blocked at term labor remain to be determined. In this manuscript, we demonstrate that ligand-bound PR interacts with the Cx43 gene promoter through activator protein-1 transcription factors. We show that the ability of PR to repress Cx43 transcription is conferred through the recruitment of the PR coregulator, polypyrimidine tract binding protein-associated splicing factor (PSF), and the further recruitment of the yeast switch independent 3 homolog A/histone deacetylase corepressor complex. PSF expression is elevated during pregnancy but falls toward term as a result of increased mechanical stretch of the myometrium and a rise in the concentrations of circulating estrogen. These data together indicate that PSF is a critical regulator of P4/PR signaling and labor. We suggest that decreased PSF at term may result in a de-repression of PR transcriptional control of CAP genes and thereby contributes to a functional withdrawal of progesterone at term labor.
Assuntos
Trabalho de Parto/metabolismo , Miométrio/metabolismo , Progesterona/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Fenômenos Biomecânicos , Conexina 43/genética , Conexina 43/metabolismo , Estradiol/fisiologia , Feminino , Células HEK293 , Humanos , Masculino , Miométrio/citologia , Fator de Processamento Associado a PTB , Gravidez , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Ratos , Ratos Wistar , Receptores de Progesterona/metabolismo , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica , Ativação TranscricionalRESUMO
Originally identified as essential pre-mRNA splicing factors, non-POU-domain-containing, octamer binding protein (p54nrb) and PTB-associated RNA splicing factor (PSF) are also steroid receptor corepressors. The mechanisms by which p54nrb and PSF regulate gene transcription remain unclear. Both p54nrb and PSF contain protein phosphatase 1 (PP1) consensus binding RVxF motifs, suggesting that PP1 may regulate phosphorylation status of p54nrb and PSF and thus their function in gene transcription. In this report, we demonstrated that PP1 forms a protein complex with both p54nrb and PSF. PP1 interacts directly with the RVxF motif only in p54nrb, but not in PSF. Association with PP1 results in dephosphorylation of both p54nrb and PSF in vivo and the loss of their transcriptional corepressor activities. Using the CD44 minigene as a reporter, we showed that PP1 regulates p54nrb and PSF alternative splicing activities that determine exon skipping vs. inclusion in the final mature RNA for translation. In addition, changes in transcriptional corepression and RNA splicing activities of p54nrb and PSF are correlated with alterations in protein interactions of p54nrb and PSF with transcriptional corepressors such as Sin3A and histone deacetylase 1, and RNA splicing factors such as U1A and U2AF. Furthermore, we demonstrated a novel function of the RVxF motif within PSF that enhances its corepression and RNA splicing activities independent of PP1. We conclude that the RVxF motifs play an important role in controlling the multifunctional properties of p54nrb and PSF in the regulation of gene transcription.
Assuntos
Processamento Alternativo , Regulação da Expressão Gênica , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Proteína Fosfatase 1/química , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Sítios de Ligação , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Células HEK293 , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Imunoprecipitação , Fator de Processamento Associado a PTB , Fosforilação , Ligação Proteica , Proteína Fosfatase 1/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Bacterial vaginosis is associated with a 1.4-fold increased risk of preterm birth. We have shown previously that Lactobacillus rhamnosus GR-1 supernatant up-regulates interleukin 10 and down-regulates tumor necrosis factor-alpha output in lipopolysaccharide (LPS)-treated human primary placenta cultures in a fetal sex-dependent manner. We hypothesize that lactobacilli also exert their anti-inflammatory effect by up-regulation of colony-stimulating factor 3 (granulocyte) (CSF3), which is secreted from both immune and placental trophoblast cells, and that this activity is dependent on the sex of the fetus. Placental trophoblast cells were isolated from term elective cesarean section placentae using a Percoll gradient and separated from CD45(+) cells using magnetic purification. Cells were treated with LPS in the presence or absence of pretreatments with L. rhamnosus GR-1 supernatant or chemical inhibitors of the intracellular signaling pathways. Phosphorylations of mitogen-activated protein kinase 14 (MAPK14, previously known as p38) and signal transducer and activator of transcription (STAT) 3 were measured by Western blot analysis, and levels of CSF3 were determined by ELISA. CSF3 output was increased only in the placental trophoblast cells of female fetuses treated with LPS, GR-1 supernatant, and a combination of both treatments. The GR-1 supernatant up-regulated the phosphorylation of STAT3 and MAPK14. CSF3 output was inhibited by both Janus kinases (JAK) and MAPK14 inhibitors. None of the treatments was able to increase CSF3 output in either the pure trophoblast or the CD45(+) cell preparations alone. These results suggest an underlying mechanism for the sex difference in incidence of preterm birth and provide potential evidence for a therapeutic benefit of lactobacilli in reducing the risk of preterm labor.
Assuntos
Citocinas/metabolismo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Lacticaseibacillus rhamnosus/fisiologia , Placenta/citologia , Placenta/metabolismo , Trofoblastos/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Janus Quinases/genética , Janus Quinases/metabolismo , Lacticaseibacillus rhamnosus/classificação , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Gravidez , Probióticos/farmacologia , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Caracteres Sexuais , Transdução de Sinais , Trofoblastos/citologiaRESUMO
The identification of proinflammatory signal transduction pathways may suggest new therapeutic targets. In this study, we examine which signaling pathways are involved in tumor necrosis factor (TNF)-induced matrix metalloproteinase 9 (MMP9) secretion in human chorionic trophoblast (CT) cells. Purified CT cells were cultured in the presence of antibodies or chemical inhibitors that specifically block/inhibit distinct TNF receptors and kinase pathways. TNF-induced proMMP9 production, as measured by zymography, was significantly blocked/inhibited by TNF receptor 1 (TNFRSF1A) antibody, NFKB activation inhibitor (NFKBAI), and MAPK1/3 (ERK) inhibitor (U0126) (P < 0.01), but not by TNF receptor 2 (TNFRSF1B) antibody, MAPK14 (p38 MAPK) inhibitor (SB203580), and MAPK8/9/10 (JNK) inhibitor (SP600125). By Western blot analysis, we found that TNF rapidly and significantly increased phosphorylation of IKBKB, MAPK1/3, and MAPK8/9/10 and that the phosphorylation of these kinases by TNF was reduced significantly by TNFRSF1A neutralizing antibody, but not by TNFRSF1B neutralizing antibody. Moreover, we found that TNF increased TNF receptor-associated factor (TRAF) 1 and decreased TRAF2 protein expression through TNFRSF1A, but not TNFRSF1B. The CT cells that had increased TRAF1 and decreased TRAF2 after an initial TNF treatment demonstrated a dramatic deficiency in phosphorylation of the above protein kinases following a secondary TNF treatment. Localization of RELA subunit by immunocytochemistry was shifted to the nuclei after TNF treatment compared to cytosol in untreated controls. We also found cross-talk between the phosphoinositide 3-kinase pathway and ERK pathway. In summary, we have demonstrated that TNF stimulates proMMP9 production in CT cells through TNFRSF1A-TRAFs-IKBKB-NFKB and ERK signaling pathways, but not through TNFRSF1B and JNK/p38-AP-1 pathways.
Assuntos
Quinase I-kappa B/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trofoblastos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Análise de Variância , Anticorpos Neutralizantes , Western Blotting , Células Cultivadas , Humanos , Imuno-Histoquímica , Fosforilação/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trofoblastos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The progesterone receptor (PR) plays important roles in the establishment and maintenance of pregnancy. By dynamic interactions with coregulators, PR represses the expression of genes that increase the contractile activity of myometrium and contribute to the initiation of labor. We have previously shown that PTB-associated RNA splicing factor (PSF) can function as a PR corepressor. In this report, we demonstrated that the PSF heterodimer partner, p54nrb (non-POU-domain-containing, octamer binding protein), can also function as a transcription corepressor, independent of PSF. p54nrb Interacts directly with PR independent of progesterone. In contrast to PSF, p54nrb neither enhances PR protein degradation nor blocks PR binding to DNA. Rather, p54nrb recruits mSin3A through its N terminus to the PR-DNA complex, resulting in an inhibition of PR-mediated transactivation of the progesterone-response element-luciferase reporter gene. PR also repressed transcription of the connexin 43 gene (Gja1), an effect dependent on the presence of an activator protein 1 site within the proximal Gja1 promoter. Mutation of this site abolished PR-mediated repression and decreased the recruitment of PR and p54nrb onto the Gja1 promoter. Furthermore, knockdown p54nrb expression by small interfering RNA alleviated PR-mediated repression on Gja1 transcription, whereas overexpression of p54nrb enhanced it. In the physiological context of pregnancy, p54nrb protein levels decrease with the approach of labor in the rat myometrium. We conclude that p54nrb is a transcriptional corepressor of PR. Decreased expression of p54nrb at the time of labor may act to derepress PR-mediated inhibition on connexin 43 expression and contribute to the initiation of labor.
Assuntos
Conexina 43/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores de Progesterona/metabolismo , Animais , Linhagem Celular , Cricetinae , Proteínas de Ligação a DNA , Humanos , Modelos Biológicos , Progesterona/metabolismo , Regiões Promotoras Genéticas , RNA/metabolismo , Splicing de RNA , RNA Interferente Pequeno/metabolismo , Transcrição Gênica , Ativação TranscricionalRESUMO
Estrogens produced by the placenta are pivotal in human pregnancy and parturition. Several hormones are involved in regulating estrogen production. Recently, the corticotrophin releasing hormone family of peptides has expanded and among the new members, urocortin 2 is expressed from human placenta. The aim of the current study was to determine urocortin 2 effects on estradiol secretion and cytochrome P450 aromatase mRNA levels and protein expression from cultured human trophoblast cells. Trophoblast cell cultures were treated with urocortin 2 and for comparison, corticotrophin releasing hormone, or urocortin 1 in the presence of estrogen precursors dehydroepiandrosterone-sulfate, androstenedione, and testosterone. Estradiol output was measured using enzyme-linked immunosorbent assay. Cytochrome P450 aromatase mRNA levels and protein expression were evaluated using reverse transcriptase-polymerase chain reaction and Western blot. Trophoblast cell cultures treated with increasing amounts of corticotrophin releasing hormone and urocortin 1 showed increased secretion of E2 in the presence of androstenedione. In the presence of urocortin 2, E2 output in cultures treated with dehydroepiandrosterone, androstenedione, and testosterone was consistently raised in a time and dose-dependent manner to maximum values at 24 hours. P450 aromatase mRNA levels and protein expression were upregulated by urocortin 2 in the presence of C19 precursors. The addition of antisauvagine-30, a corticotrophin releasing hormone-receptor-2 antagonist, significantly reversed urocortin 2 effects on E2 secretion and P450 aromatase expression. Our results suggest that urocortin 2 may play a role in the regulation of E2 production throughout pregnancy, thereby contributing to the placental regulation of key reproductive events in pregnancy maintenance and parturition.
Assuntos
Estradiol/metabolismo , Proteínas da Gravidez/fisiologia , Receptores de Hormônio Liberador da Corticotropina/fisiologia , Urocortinas/fisiologia , Células Cultivadas , Feminino , Humanos , Gravidez , Trofoblastos/metabolismoRESUMO
OBJECTIVE: The objective of the study was to determine the effect of fetal sex on the output of cytokines and prostaglandin-regulating enzymes in lipopolysaccharide (LPS) and probiotic lactobacilli-treated placental trophoblast cells. STUDY DESIGN: We examined the effect of LPS and Lactobacillus rhamnosus GR-1 supernatant in placental trophoblast cells on tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-10 using enzyme-linked immunosorbent assay and on prostaglandin-endoperoxide synthase 2 (PTGS2), 15-hydroxy prostaglandin dehydrogenase (PGDH), and toll-like receptor-4 (TLR-4) using Western blotting. Comparisons were performed using one-way analysis of variance and Student t test. RESULTS: LPS increased the output of TNF-alpha, IL-10, and PTGS2 with a greater response in male placentae. L rhamnosus GR-1 supernatant inhibited the LPS-stimulated TNF-alpha and increased IL-10. It also up-regulated expression of PGDH in female placentae and partially reduced the LPS-stimulated PTGS2 in male placentae. There was no change in IL-1beta. Expression of TLR-4 was greater in placentae of male fetuses. CONCLUSION: These findings suggest an underlying mechanism for the sex difference in the incidence of preterm birth and provide potential evidence for a therapeutic benefit of lactobacilli in reducing preterm labor.
Assuntos
Citocinas/metabolismo , Lacticaseibacillus rhamnosus/imunologia , Trabalho de Parto Prematuro/prevenção & controle , Probióticos , Trofoblastos/microbiologia , Vaginose Bacteriana/terapia , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Feminino , Humanos , Hidroxiprostaglandina Desidrogenases/metabolismo , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Gravidez , Fatores Sexuais , Receptor 4 Toll-Like/metabolismo , Trofoblastos/citologia , Trofoblastos/enzimologia , Fator de Necrose Tumoral alfa/metabolismo , Vaginose Bacteriana/imunologia , Vaginose Bacteriana/metabolismoRESUMO
Prostaglandins (PGs) induce the mechanism of labor in humans. The enzymes responsible for PG synthesis and metabolism are prostaglandin-endoperoxide synthase 2 (PTGS2) and 15-hydroxyprostaglandin dehydrogenase (PGDH). In human chorion trophoblast cells, calcium ionophore A23187 upregulates PTGS2 and downregulates PGDH protein and mRNA. The authors hypothesize that this regulation requires activation of protein kinase C (PKC) and mitogen-activated protein kinases (MAPKs). Human chorion trophoblasts were incubated with A23187 or phorbol 12-myristate 13-acetate (PMA) in the absence or presence of inhibitors of PKC, c-Jun N-terminal kinase, p38, and MEK1/2. PTGS2 and PGDH mRNA were measured by real-time reverse-transcription polymerase chain reaction. PMA upregulated PTGS2 and downregulated PGDH. The PMA effect was reversed by the inhibition of PKC. The p38 inhibitor reduced the stimulatory effect of PMA and A23187 on PTGS2. MEK1/2 inhibitor reduced the effect of PMA on PTGS2. All MAPK inhibitors failed to reverse the effect of either A23187 or PMA on PGDH. The authors conclude that upon stimulation with the same upstream signals, different downstream intracellular pathways regulate PTGS2 and PGDH mRNA expression.
Assuntos
Carcinógenos/farmacologia , Ciclo-Oxigenase 2/genética , Hidroxiprostaglandina Desidrogenases/genética , Acetato de Tetradecanoilforbol/farmacologia , Trofoblastos/efeitos dos fármacos , Trofoblastos/fisiologia , Cálcio/metabolismo , Células Cultivadas , Córion/citologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/metabolismo , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Maleimidas/farmacologia , Gravidez , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Trofoblastos/citologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Placental hydroxysteroid 11-beta dehydrogenase 2 (HSD11B2) plays an important role in pregnancy maintenance and fetal maturation. In the event of intrauterine infection, lipoxygenase (LOX) metabolites are produced in the placenta and contribute to preterm labor and adverse fetal outcomes. On the other hand, LOX metabolites are involved in production of progesterone, which is required for pregnancy maintenance. In this study, we evaluated the interaction between the LOX pathway, progesterone, and HSD11B2. Specifically, we hypothesized that LOX metabolites would alter HSD11B2 and this effect would be mediated by progesterone. We cultured human term placental trophoblasts in the presence and absence of the LOX inhibitors Nordihydroguaiaretic acid (NDGA), AA861, and Baicalein; the LOX metabolites Leukotriene B(4) and 12(S)-Hydroxyeicosatetraenoate (12-HETE); and progesterone and progesterone receptor antagonist RU486. By radiometric conversion assay, real-time quantitative PCR, Western blot analysis, and ELISA, we examined HSD11B2 enzyme activity, HSD11B2 mRNA and HSD11B2 protein expression, and progesterone output. LOX metabolites down-regulated HSD11B2 activity and HSD11B2 expression. LOX inhibitors up-regulated HSD11B2 activity and HSD11B2 and HSD11B2 expression, and these effects were attenuated by addition of LOX metabolites. Net progesterone output was increased by LOX metabolites and decreased by LOX inhibitors. Progesterone down-regulated HSD11B2 activity and HSD11B2 and HSD11B2 expression, and these effects were blocked by RU486. Furthermore, the suppressive effect of 12-HETE on HSD11B2 activity was also reversed by RU486. We conclude that HSD11B2 in human placental trophoblasts is decreased by progesterone and increased by inhibition of endogenous LOX metabolites, and that a component of the effect of LOX metabolites on HSD11B2 is mediated by their stimulation of endogenous progesterone output.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , Regulação Enzimológica da Expressão Gênica , Lipoxigenase/fisiologia , Progesterona/metabolismo , Trofoblastos/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Benzoquinonas/farmacologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Feminino , Flavanonas/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Idade Gestacional , Humanos , Lipoxigenase/metabolismo , Inibidores de Lipoxigenase/farmacologia , Masoprocol/farmacologia , Modelos Biológicos , Placenta/enzimologia , Placenta/metabolismo , Gravidez , Progesterona/farmacologia , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Trofoblastos/efeitos dos fármacos , Trofoblastos/enzimologiaRESUMO
Nuclear receptors regulate gene activation or repression through dynamic interactions with coregulators. The interactions between nuclear receptors and RNA splicing factors link gene transcription initiation with pre-mRNA splicing, providing a coordinated control of the products of gene transcription. Here we report that two RNA splicing factors, PTB-associated splicing factor (PSF) and p54nrb, synergistically form protein complexes with the androgen receptor (AR) in a ligand-independent manner and inhibit its transcriptional activity. PSF does not affect AR protein stability, as in the case of the progesterone receptor, but impedes the interaction of AR with the androgen response element. Both splicing factors interact directly with mSin3A and attract mSin3A to the AR complex in a synergistic manner. The suppression of AR transcriptional activity by PSF and p54nrb is reversed by the inhibition of histone deacetylase activity. These data demonstrated that PSF and p54nrb complex with AR and play a key role in modulating AR-mediated gene transcription.
Assuntos
Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Splicing de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Androgênicos/genética , Ativação Transcricional , Linhagem Celular , Proteínas de Ligação a DNA , Histona Desacetilase 1 , Histona Desacetilases/metabolismo , Humanos , Antígeno Prostático Específico/genética , Ligação Proteica , Proteínas Repressoras/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3RESUMO
OBJECTIVE: 11Beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) is thought to act as a placental barrier protecting the fetus from high levels of maternal cortisol. On the other hand, intrauterine infection is one of the main causes of preterm birth and adverse fetal outcome, and pro-inflammatory cytokines may contribute to these adverse effects. However, the effect of pro-inflammatory cytokines on 11beta-HSD2 is still not clear. Therefore, we have evaluated the effect of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) on 11beta-HSD2 in cultured human placental trophoblast and in human choriocarcinoma JEG-3 cells. METHODS: Placental trophoblast cells were isolated from human term placenta. Placental trophoblast cells and JEG-3 cells were treated with TNF-alpha (0.1-10 ng/mL) or IL-1beta (0.1-10 ng/mL). Real-time reverse transcription polymerase chain reaction and Western blot were used to study the regulation of 11beta-HSD2 expression. 11beta-HSD2 activity was determined by measuring the rate of cortisol to cortisone conversion in the culture medium using thin-layer chromatography (TLC). RESULTS: In placental trophoblast, TNF-alpha and IL-1beta down-regulated 11beta-HSD2 mRNA expression and activity (both P <.05). The protein level was decreased only with IL-1beta (P <.05). In JEG-3 cells, 11beta-HSD2 mRNA was decreased by TNF-alpha but up-regulated by IL-1beta, with no significant change in protein expression and activity. CONCLUSION: Our results suggest caution in interpreting data using JEG-3 cells. However, our studies with primary trophoblast suggest that TNF-alpha and IL-1beta may increase the amount of cortisol crossing to the placenta and fetal circulation by attenuating 11beta-HSD2 activity, potentially contributing to preterm labor and altered fetal outcome in uterine infection.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 2/biossíntese , Gravidez/fisiologia , Técnicas de Cultura de Células , Coriocarcinoma/patologia , Regulação para Baixo , Feminino , Humanos , Hidrocortisona/farmacocinética , Infecções , Interleucina-1/fisiologia , Troca Materno-Fetal , Placenta/citologia , Nascimento Prematuro/fisiopatologia , RNA Mensageiro/biossíntese , Reprodutibilidade dos Testes , Trofoblastos , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/fisiologia , Neoplasias Uterinas/patologiaRESUMO
OBJECTIVE: Preterm birth is one of the major problems faced in perinatal medicine and is often associated with underlying clinical infection. Treatment with maternal betamethasone has helped to improve neonatal morbidity and mortality. We hypothesized that betamethasone treatment and chorioamnionitis would alter the bioavailability of placental glucocorticoids through the regulation of the 11beta hydroxysteroid dehydrogenase (11beta HSD) isozymes and the glucocorticoid receptor (GR). METHODS: Placental samples were obtained from three groups of women who delivered prematurely: (1) those who delivered in the absence of infection, (2) those who received betamethasone treatment before delivering without infection, and (3) those who had pregnancies complicated with chorioamnionitis. Western blotting was used to determine 11beta HSD-1, 11beta HSD-2, GRT, and GRalpha expression, and 11beta HSD-2 activity was determined in each group. JEG-3 cells were used to study the regulation of the 11beta HSD isozymes. RESULTS: In cases of chorioamnionitis where mothers had not been treated with betamethasone, placental 32-kd 11beta HSD-1 protein expression was increased. In cases of chorioamnionitis regardless of betamethasone treatment, placental 11beta HSD-2 expression and activity was decreased compared to controls. In these placental samples, the expression of GRT and GRalpha did not change significantly. In JEG-3 cells, 11beta HSD-1 32-kd expression was increased with interleukin (IL)-1beta and tumor necrosis factor alpha (TNF-alpha), while 11beta HSD-2 expression was unaffected. CONCLUSION: These data suggest that there could be an increased fetal exposure to maternal glucocorticoids in cases of chorioamnionitis as a result of changes in the expression and activity of the placental 11beta HSD isozymes.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Anti-Inflamatórios/farmacocinética , Betametasona/farmacocinética , Corioamnionite , Troca Materno-Fetal , Trabalho de Parto Prematuro/tratamento farmacológico , Gravidez/fisiologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/biossíntese , Adulto , Anti-Inflamatórios/uso terapêutico , Betametasona/uso terapêutico , Western Blotting , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica , Humanos , Isoenzimas , Placenta/química , Placenta/fisiologia , Segundo Trimestre da Gravidez , Terceiro Trimestre da Gravidez , Nascimento Prematuro , Receptores de Glucocorticoides/biossíntese , Receptores de Glucocorticoides/efeitos dos fármacos , Receptores de Glucocorticoides/fisiologia , Fatores de RiscoRESUMO
Progesterone is essential in all species for the maintenance of pregnancy, and its withdrawal is required to activate the myometrium and to initiate labor. However, unlike most other species, progesterone levels do not fall at term in humans, raising the paradox as to how labor can occur under the continued influence of progesterone. We hypothesized that an endogenous (myometrial) repressor of the progesterone receptor (PR) could induce a functional withdrawal of progesterone and hence lead to the initiation of labor. We used the human PR as bait in a protein pull-down assay and identified polypyrimidine tract-binding protein-associated splicing factor (PSF) as a PR-interacting protein. PSF functions as a potent inhibitor of PR (but not estrogen receptor) transcriptional activity in mammalian cells. It acts through two novel mechanisms, inducing degradation of the PR through the proteasomal pathway and also interfering with binding of PR to its DNA response element. Importantly, in vivo studies in rats demonstrated a dramatic increase in myometrial PSF expression at term that was temporally associated with reduced levels of the myometrial PR. Accordingly, we propose that PSF acts as a PR corepressor and contributes to the functional withdrawal of progesterone and the initiation of human labor.
Assuntos
Trabalho de Parto/fisiologia , Miométrio/metabolismo , Progesterona/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores de Progesterona/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Feminino , Humanos , Masculino , Fator de Processamento Associado a PTB , Gravidez , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Ratos , Ratos Wistar , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/genética , Proteínas Recombinantes de Fusão/genética , Elementos de Resposta , Ativação TranscricionalRESUMO
In sheep, parturition is initiated by increased fetal hypothalamic-pituitary-adrenal axis (HPAA) activity leading to PGE(2) and PGF(2alpha) production and a rise in the 17beta-estradiol-progesterone (E(2)/P(4)) ratio. Uteroplacental PG production can also increase fetal HPAA activity. Periconceptional maternal undernutrition accelerates fetal HPAA maturation resulting in preterm labor. We determined whether preterm labor was preceded by an increase in PG concentrations and E(2)/P(4) ratio and whether these increases preceded or followed the corresponding rise in cortisol concentrations. Singleton-bearing ewes were nourished ad libitum (N, n = 9) or undernourished (UN, n = 10) to reduce maternal weight by 15% from -61 days (d) to +30 d after mating with ad libitum intake thereafter. Paired maternal and fetal blood samples were collected from 126 d until delivery. Half the UN group delivered prematurely (>2 SD below mean gestation for the flock). PG and cortisol concentrations and E(2)/P(4) ratio increased before delivery in the same way in both groups. However, the increases occurred 7-10 d earlier in UN than in N animals. In both UN and N fetuses cortisol concentrations rose before fetal and maternal PG concentrations and maternal E(2)/P(4) ratio. Periconceptional maternal undernutrition induces preterm delivery in sheep by advancing the expected prepartum rise in cortisol and PG concentrations and E(2)/P(4) ratio. The rise in fetal cortisol concentration precedes the rise in fetal and maternal PG concentrations and maternal E(2)/P(4) ratio, suggesting that the underlying mechanism is likely to be acceleration of fetal HPAA maturation, resulting in initiation of the normal process of parturition.