RESUMO
The Nuclear factor I (NFI) transcription factor family consists of four genes (Nfia, Nfib, Nfic and Nfix) that regulate the development of multiple organ systems in mice and humans. Nfib is expressed in both lung mesenchyme and epithelium and mice lacking Nfib have severe lung maturation defects and die at birth. Here we continue our analysis of the phenotype of Nfibâ»/â» lungs and show that Nfib specifically in lung mesenchyme controls late epithelial and mesenchymal cell proliferation and differentiation. There are more PCNA, BrdU, PHH3 and Ki67 positive cells in Nfibâ»/â» lungs than in wild type lungs at E18.5 and this increase in proliferation marker expression is seen in both epithelial and mesenchymal cells. The loss of Nfib in all lung cells decreases the expression of markers for alveolar epithelial cells (Aqp5 and Sftpc), Clara cells (Scgb1a1) and ciliated cells (Foxj1) in E18.5 lungs. To test for a specific role of Nfib in lung mesenchyme we generated and analyzed Nfib(flox/flox), Dermo1-Cre mice. Loss of Nfib only in mesenchyme results in decreased Aqp5, Sftpc and Foxj1 expression, increased cell proliferation, and a defect in sacculation similar to that seen in Nfibâ»/â» mice. In contrast, mesenchyme specific loss of Nfib had no effect on the expression of Scgb1a1 in the airway. Microarray and QPCR analyses indicate that the loss of Nfib in lung mesenchyme affects the expression of genes associated with extracellular matrix, cell adhesion and FGF signaling which could affect distal lung maturation. Our data indicate that mesenchymal Nfib regulates both mesenchymal and epithelial cell proliferation through multiple pathways and that mesenchymal NFI-B-mediated signals are essential for the maturation of distal lung epithelium.
Assuntos
Diferenciação Celular , Proliferação de Células , Pulmão/crescimento & desenvolvimento , Fatores de Transcrição NFI/metabolismo , Mucosa Respiratória/citologia , Animais , Aquaporina 5/metabolismo , Bromodesoxiuridina/metabolismo , Epitélio/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular , Antígeno Ki-67/metabolismo , Pulmão/citologia , Pulmão/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Peptídeos/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína C Associada a Surfactante Pulmonar , Transdução de Sinais , Uteroglobina/metabolismoRESUMO
The etiology of acute lung injury is complex and associated with numerous, chemically diverse precipitating factors. During acute lung injury in mice, one key event is epithelial cell injury that leads to reduced surfactant biosynthesis. We have previously reported that transgenic mice that express transforming growth factor alpha (TGFA) in the lung were protected during nickel-induced lung injury. Here, we find that the mechanism by which TGFA imparts protection includes maintenance of surfactant-associated protein B (SFTPB) transcript levels and epidermal growth factor receptor-dependent signaling in distal pulmonary epithelial cells. This protection is complex and not accompanied by a diminution in inflammatory mediator transcripts or additional stimulation of antioxidant transcripts. In mouse lung epithelial (MLE-15) cells, microarray analysis demonstrated that nickel increased transcripts of genes enriched in MTF1, E2F-1, and AP-2 transcription factor-binding sites and decreased transcripts of genes enriched in AP-1-binding sites. Nickel also increased Jun transcript and DNA-binding activity, but decreased SFTPB transcript. Expression of SFTPB under the control of a doxycycline-sensitive promoter increased survival during nickel-induced injury as compared with control mice. Together, these findings support the idea that maintenance of SFTPB expression is critical to survival during acute lung injury.
Assuntos
Lesão Pulmonar Aguda/induzido quimicamente , Níquel/toxicidade , Proteína B Associada a Surfactante Pulmonar/metabolismo , Administração por Inalação , Aerossóis , Animais , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteína B Associada a Surfactante Pulmonar/genética , Mucosa Respiratória/citologia , Taxa de Sobrevida , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismoRESUMO
Forced overexpression of TEAD1 in human uterine fibroblast (HUF) and human endometrial stromal cells markedly inhibited prolactin promoter activity in both cell types in a dose-dependent manner, with maximal inhibition of greater than 90%. Conversely, the knockdown of TEAD1 expression in HUF cells with a TEAD1 siRNA resulted in a 75-80% increase in prolactin mRNA levels (p<0.01) compared to control cells exposed to a scrambled nonsense RNA. Mutagenesis of the putative TEAD site inhibited basal promoter activity by about 80%. However, mutagenesis of the TEAD site did not prevent TEAD1-induced inhibition of promoter activity; and the transcription activity of a minimal promoter fragment lacking a putative TEAD binding site was repressed by overexpression of TEAD1. Taken together, these findings suggest that the TEAD binding site on the prolactin promoter is important for the maintenance of basal prolactin promoter activity and that overexpression of TEAD1 has a dominant-negative effect on prolactin promoter activity, probably by interacting directly with other transcription factors.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Decídua/metabolismo , Proteínas Nucleares/metabolismo , Prolactina/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Sítios de Ligação , Células Cultivadas , Proteínas de Ligação a DNA/genética , Decídua/citologia , Regulação para Baixo , Feminino , Fibroblastos/metabolismo , Humanos , Mutação , Proteínas Nucleares/genética , Prolactina/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Células Estromais/metabolismo , Fatores de Transcrição de Domínio TEA , Fatores de Tempo , Fatores de Transcrição/genética , Transcrição Gênica , TransfecçãoRESUMO
The phylogenetically conserved nuclear factor I (NFI) gene family encodes site-specific transcription factors essential for the development of a number of organ systems. We showed previously that Nfia-deficient mice exhibit agenesis of the corpus callosum and other forebrain defects, whereas Nfic-deficient mice have agenesis of molar tooth roots and severe incisor defects. Here we show that Nfib-deficient mice possess unique defects in lung maturation and exhibit callosal agenesis and forebrain defects that are similar to, but more severe than, those seen in Nfia-deficient animals. In addition, loss of Nfib results in defects in basilar pons formation and hippocampus development that are not seen in Nfia-deficient mice. Heterozygous Nfib-deficient animals also exhibit callosal agenesis and delayed lung maturation, indicating haploinsufficiency at the Nfib locus. The similarity in brain defects in Nfia- and Nfib-deficient animals suggests that these two genes may cooperate in late fetal forebrain development, while Nfib is essential for late fetal lung maturation and development of the pons.
Assuntos
Encéfalo/crescimento & desenvolvimento , Pulmão/crescimento & desenvolvimento , Proteínas/metabolismo , Agenesia do Corpo Caloso , Animais , Biomarcadores , Encéfalo/anormalidades , Diferenciação Celular , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Feminino , Marcação de Genes , Idade Gestacional , Humanos , Pulmão/anormalidades , Pulmão/anatomia & histologia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFI , Gravidez , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Surfactant protein C (SP-C; Sftpc) gene expression is restricted to pulmonary type II epithelial cells. The proximal SP-C promoter region contains critical binding sites for nuclear factor I (NFI) and thyroid transcription factor 1 (TTF-1; also called Nkx2.1). To test the hypothesis that NFI isoforms interact with TTF-1 to differentially regulate SP-C transcription, we performed transient transfection assays in JEG-3 cells, a choriocarcinoma cell line with negligible endogenous NFI or TTF-1 activity. Cotransfection of NFI family members with TTF-1 induced synergistic activation of the SP-C promoter that was further enhanced by p300. TTF-1 directly interacts with the conserved DNA binding and dimerization domain of all NFI family members in coimmunoprecipitation and mammalian two-hybrid experiments. To determine whether SP-C expression is regulated by NFI in vivo, a chimeric fusion protein containing the DNA binding and dimerization domain of NFI-A and the Drosophila engrailed transcriptional repression domain (NFIen) was conditionally expressed in mice under control of a doxycycline-inducible transgene. Induction of NFIen in a subset of type II cells inhibited SP-C gene expression without affecting expression of TTF-1 in doxycycline-treated double-transgenic mice. Taken together, these findings support the hypothesis that NFI family members interact with TTF-1 to regulate type II cell function.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA , Proteínas Nucleares/metabolismo , Proteína C Associada a Surfactante Pulmonar/genética , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Linhagem Celular Tumoral , DNA/genética , Humanos , Camundongos , Camundongos Transgênicos , Fatores de Transcrição NFI , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/genética , Transcrição Gênica , Proteína 1 de Ligação a Y-BoxRESUMO
Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumor type that is typically metastatic upon diagnosis. We have a poor understanding of the factors that control SCLC progression and metastasis. TheNFIB transcription factor is frequently amplified in mouse models of SCLC, but clear evidence that NFIB promotes SCLC in vivo is lacking. We report that in mouse models, Nfib amplifications are far more frequent in liver metastases over primary SCLC, suggesting roles in tumor progression/metastasis. Overexpression of Nfib in a sensitized mouse model led to acceleration of SCLC, indicating that Nfib functions as a bona fide oncogene. Suppression of Nfib expression in cell lines derived from the doxycycline-inducible Rb/p53/TET-Nfib model led to increased apoptosis and suppression of proliferation. Transcriptional analysis revealed that Nfib regulates the expression of genes related to axon guidance, focal adhesion and extracellular matrix-receptor interactions. These data indicate that Nfib is a potent oncogene in SCLC, and the enrichment of Nfib amplifications in liver metastases over primary SCLC points to Nfib as a candidate driver of SCLC metastasis.
Assuntos
Neoplasias Pulmonares/metabolismo , Fatores de Transcrição NFI/metabolismo , Proteína do Retinoblastoma/metabolismo , Carcinoma de Pequenas Células do Pulmão/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Alelos , Animais , Apoptose , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Modelos Animais de Doenças , Progressão da Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Hepáticas/secundário , Camundongos , Fatores de Transcrição NFI/genética , Metástase Neoplásica , Oncogenes , Proteína do Retinoblastoma/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: During the in vitro differentiation of human villous cytotrophoblast (CTB) cells to a syncytiotrophoblast (STB) phenotype, mRNA levels for the nuclear hormone receptor NR2F2 (ARP-1, COUP-TFII) increase rapidly, reaching a peak at day 1 of differentiation that is 8.8-fold greater than that in undifferentiated CTB cells. To examine whether NR2F2 is involved in the regulation of villous CTB cell differentiation, studies were performed to determine whether NR2F2 regulates the expression of TFAP2A (AP-2alpha), a transcription factor that is critical for the terminal differentiation of these cells to a STB phenotype. METHODOLOGY/PRIMARY FINDINGS: Overexpression of NR2F2 in primary cultures of human CTB cells and JEG-3 human choriocarcinoma cells induced dose-dependent increases in TFAP2A promoter activity. Conversely, siRNA mediated silencing of the NR2F2 gene in villous CTB undergoing spontaneous differentiation blocked the induction of the mRNAs for TFAP2A and several STB cell specific marker genes, including human placental lactogen (hPL), pregnancy specific glycoprotein 1 (PSG1) and corticotropin releasing hormone (CRH) by 51-59%. The induction of TFAP2A promoter activity by NR2F2 was potentiated by the nuclear hormone receptors retinoic acid receptor alpha (RARA) and retinoid X receptor alpha (RXRA). CONCLUSIONS/SIGNIFICANCE: Taken together, these results strongly suggest that NR2F2 is involved in villous CTB cell differentiation and that NR2F2 acts, at least in part, by directly activating TFAP2A gene expression and by potentiating the transactivation of TFAP2A by RARA and RXRA.
Assuntos
Fator II de Transcrição COUP/genética , Diferenciação Celular/genética , Perfilação da Expressão Gênica , Trofoblastos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Receptor X Retinoide alfa/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fator de Transcrição AP-2/genética , Transfecção , Trofoblastos/citologiaRESUMO
Toll-like receptors (TLRs) are pattern recognition molecules that initiate innate immune responses. Intra-amniotic exposure of fetal sheep to pro-inflammatory stimuli causes pulmonary inflammation and induced lung maturation. We examined TLR ontogeny and fetal lung responsiveness to three different TLR agonists. We cloned ovine TLRs 2, 3, and 4 and found 83-88% homology between these ovine and human TLRs. Lung TLR2 and 4 mRNAs increased throughout late gestation to 50% of adult level in the term newborn lamb. Doses of 10 mg of PAMCysK4 (TLR2 agonist), poly I:C dsRNA (TLR3 agonist), or E. coli O55:B5 lipopoysaccharide (LPS) (TLR4 agonist) were given by intra-amniotic injection 2 d or 7 d before operative delivery of preterm lambs at 123 d (n = 4-7/group). The TLR4 agonist induced lung inflammation and maturation, whereas the TLR2 agonist gave less consistent responses. Intra-amniotic LPS increased TLR2 mRNA expression primarily in the inflammatory cells and TLR4 mRNA diffusely in multiple cell types. The TLR3 agonist had no effects, and TLR3 mRNA in the fetal lung did not change after LPS exposure. We conclude that TLR2 and TLR4 mRNAs increase through gestation and expression of TLR2 and TLR4 are induced by LPS in the fetal sheep lung.
Assuntos
Desenvolvimento Fetal/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Pulmão/embriologia , Pulmão/metabolismo , Receptores Toll-Like/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos/metabolismo , Citocinas/metabolismo , Feminino , Imunidade Inata/fisiologia , Lipopolissacarídeos/efeitos adversos , Pulmão/patologia , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Pneumonia/patologia , Gravidez , RNA Mensageiro/metabolismo , Ovinos , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/efeitos dos fármacos , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/agonistas , Receptor 3 Toll-Like/efeitos dos fármacos , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Receptores Toll-Like/agonistasRESUMO
Serum amyloid A (SAA), an acute phase reactant (APR) protein, is induced in liver during systemic inflammation. Serum amyloid A3 (SAA3), an isoform of SAA, is induced in both liver and extra hepatic sites in response to proinflammatory stimuli such as cytokines. Previously, we showed a modest increase in plasma cytokine levels in a preterm lamb model of lung injury. The study objective was to determine the relative contributions of lung and liver to the acute phase response during postnatal lung injury. Preterm (130d) and near term (141d) newborn lambs (term=150d) were randomized to either no ventilation (controls), ventilation+intratracheal (IT) endotoxin (endo) or ventilation+IT saline. A group of near term lambs were exposed to ventilation+IV endotoxin. In the lungs, ventilation alone increased SAA3 mRNA 3- and 13-fold while ventilation+IT endotoxin increased SAA3 mRNA 64 and 366-fold above controls in preterm and near term lambs, respectively. In the liver, SAA3 mRNA was induced by ventilation alone (three-fold) and ventilation+IT endotoxin (45-fold) above controls in both preterm and near term animals. Ventilation + IV endotoxin caused the highest increase in SAA3 mRNA (212-fold) in the liver of near term animals. A different isoform, identified as SAA-Liver inducible was maximally induced in liver by ventilation alone with minimal further response to endotoxin. Lung SAA3 mRNA expression was detected primarily in airway epithelium, bronchial glands, perichondrium of bronchial cartilage and vascular smooth muscle cells. Our experiments show rapid induction of an APR gene in lung in response to proinflammatory stimuli.
Assuntos
Pulmão/metabolismo , Pneumonia/metabolismo , Proteína Amiloide A Sérica/metabolismo , Doença Aguda , Animais , Animais Recém-Nascidos , Endotoxinas , Feminino , Fígado/química , Fígado/metabolismo , Pulmão/química , Pulmão/efeitos dos fármacos , Pneumonia/induzido quimicamente , Pneumonia/etiologia , Gravidez , Nascimento Prematuro , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ventilação Pulmonar , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Proteína Amiloide A Sérica/análise , Proteína Amiloide A Sérica/genética , OvinosRESUMO
Subtraction hybridization was performed to explore changes in gene expression in the fetal lung after 20 mg of intra-amniotic (IA) endotoxin. Interferon-gamma-inducible 10-kd protein (IP-10) and monokine induced by interferon-gamma (MIG) constituted 20% of 102 endotoxin-induced clones identified in the preterm lamb lung. IP-10 (CXCL10) and MIG (CXCL9) are T-cell chemoattractants that have angiostatic properties. Both IP-10 and MIG mRNA were induced 30- to 40-fold in the fetal lung at 1 to 2 days after IA endotoxin. Intense IP-10 mRNA expression was detected by in situ hybridization in the bronchiolar and peribronchiolar areas and the vascular endothelium after IA endotoxin at all time points tested. MIG mRNA expression was detected initially focally in infiltrating neutrophils (15 hours after IA endotoxin) and later in the bronchiolar and peribronchiolar areas and vascular endothelium (1 day after IA endotoxin). In contrast to endotoxin, IA tumor necrosis factor-alpha or interleukin-1 alpha did not induce IP-10 or MIG mRNA in the lung. IA endotoxin also caused a modest induction of IP-10 and MIG mRNA in the jejunum, liver, and spleen. The IP-10 and MIG receptor CXCR3 was detected in the bronchiolar epithelium of preterm lambs by immunostaining. IP-10 and MIG are potent angiostatic chemokines that may contribute to lung injury and altered pulmonary vascular development in the preterm exposed to chorioamnionitis.
Assuntos
Âmnio , Displasia Broncopulmonar , Quimiocinas CXC/fisiologia , Corioamnionite , Endotoxinas/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular , Pulmão/metabolismo , Animais , Quimiocina CXCL10 , Quimiocina CXCL9 , Quimiocinas CXC/genética , Interpretação Estatística de Dados , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Feminino , Humanos , Hibridização In Situ , Recém-Nascido , Interferon gama , Interleucina-1 , Pulmão/patologia , Neutrófilos/metabolismo , Gravidez , RNA Mensageiro/análise , Ovinos , Estatísticas não Paramétricas , Fatores de Tempo , Fator de Necrose Tumoral alfaRESUMO
Chorioamnionitis is associated with preterm delivery and bronchopulmonary dysplasia (BPD), characterized by impaired alveolar and pulmonary vascular development and vascular dysfunction. To study the vascular effects in a model of chorioamnionitis, preterm lambs were exposed to 20 mg of intra-amniotic endotoxin or saline for 1, 2, 4, or 7 days and delivered at 122 days gestational age (term = 150 days). This intra-amniotic endotoxin dose was previously shown to induce lung maturation. The effect of intra-amniotic endotoxin on expression of endothelial proteins was evaluated. Muscularization of the media and collagen deposition in adventitia of small pulmonary arteries was used to assess vascular remodeling. Compared with controls, bronchoalveolar lavage fluid protein content was increased 2 days after intra-amniotic endotoxin exposure. Vascular endothelial growth factor (VEGF) 165 isoform mRNA decreased 2-4 days after intra-amniotic endotoxin. VEGF, VEGF receptor-2, endothelial nitric oxide synthase (eNOS), platelet endothelial cell adhesion molecule-1, and Tie-2 protein expression in the lung coordinately decreased 1-7 days after intra-amniotic endotoxin. Intra-amniotic endotoxin appeared to selectively decrease eNOS expression in small pulmonary vessels compared with large vessels. Medial smooth muscle hypertrophy and increased adventitial fibrosis were observed 4 and 7 days after intra-amniotic endotoxin. These results demonstrate that, in the preterm lamb lung, antenatal inflammation inhibits endothelial cell protein expression followed by vascular remodeling changes in small pulmonary arteries. Exposure to antenatal inflammation may cause vascular remodeling and contribute to the development of BPD.
Assuntos
Endotoxinas/toxicidade , Âmnio , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/patologia , Líquido da Lavagem Broncoalveolar/química , Modelos Animais de Doenças , Endotoxinas/administração & dosagem , Escherichia coli , Feminino , Cinética , Óxido Nítrico Sintase/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III , Gravidez , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/patologia , OvinosRESUMO
Antenatal betamethasone (Beta) is widely used in women with asymptomatic chorioamnionitis at risk for preterm delivery, but its effects on fetal inflammation are unstudied. Groups of ewes at 109 +/- 1 days of gestation received the following treatments: intra-amniotic (IA) saline (control), 0.5 mg/kg intramuscular Beta, 10 mg IA endotoxin (Endo), and Beta + 2 h later Endo (Beta + Endo). Beta suppressed Endo-induced lung inflammation at 1 day. However, compared with Endo 5 days after treatment, Beta + Endo lambs had increased alveolar neutrophils, proinflammatory cytokine mRNA expression, and serum amyloid A3 (SAA3) mRNA expression. IL-1beta mRNA expression was localized to the inflammatory cells, whereas SAA3 mRNA expression was induced in the bronchial epithelium and the inflammatory cells. Compared with Endo, Beta + Endo lambs had increased lung inflammation but equivalent lung volumes 15 days after treatment. The late increase in inflammation in the Beta + Endo animals suggests that glucocorticoids impair the ability of the preterm lung to downregulate Endo-induced inflammation after fetal clearance of the glucocorticoids. These results have implications for lung inflammation and bronchopulmonary dysplasia in preterm infants exposed to chorioamnionitis and maternal glucocorticoids.
Assuntos
Betametasona/farmacologia , Glucocorticoides/farmacologia , Pulmão/embriologia , Pulmão/imunologia , Pneumonia/induzido quimicamente , Reação de Fase Aguda/imunologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Corioamnionite/tratamento farmacológico , Sinergismo Farmacológico , Endotoxinas , Feminino , Expressão Gênica/imunologia , Hidrocortisona/sangue , Interleucina-1/genética , Interleucina-6/genética , Interleucina-8/genética , Contagem de Linfócitos , Linfócitos/citologia , Monócitos/citologia , Neutrófilos/citologia , Pneumonia/fisiopatologia , Gravidez , Proteína Amiloide A Sérica/genética , Ovinos , Fator de Necrose Tumoral alfa/genéticaRESUMO
Thyroid transcription factor-1 (TTF-1) is a 43-kDa, phosphorylated member of the Nkx2 family of homeodomain-containing proteins expressed selectively in lung, thyroid, and the central nervous system. To assess the role of TTF-1 and its phosphorylation during lung morphogenesis, mice bearing a mutant allele, in which seven serine phosphorylation sites were mutated, Titf1PM/PM, were generated by homologous recombination. Although heterozygous Titf1PM/+ mice were unaffected, homozygous Titf1PM/PM mice died immediately following birth. In contrast to Titf1 null mutant mice, which lack peripheral lung tissues, bronchiolar and peripheral acinar components of the lung were present in the Titf1PM/PM mice. Although lobulation and early branching morphogenesis were maintained in the mutant mice, abnormalities in acinar tubules and pulmonary hypoplasia indicated defects in lung morphogenesis later in development. Although TTF-1PM protein was readily detected within the nuclei of pulmonary epithelial cells at sites and abundance consistent with that of endogenous TTF-1, expression of a number of known TTF-1 target genes, including surfactant proteins and secretoglobulin 1A, was variably decreased in the mutant mice. Vascular endothelial growth factor mRNA was decreased in association with decreased formation of peripheral pulmonary blood vessels. Genes mediating surfactant homeostasis, vasculogenesis, host defense, fluid homeostasis, and inflammation were highly represented among those regulated by TTF-1. Thus, in contrast to the null Titf1 mutation, the Titf1PM/PM mutant substantially restored lung morphogenesis. Direct and indirect transcriptional targets of TTF-1 were identified that are likely to play important roles in lung formation and function.
Assuntos
Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Pulmão/embriologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cesárea , Desenvolvimento Embrionário e Fetal/fisiologia , Fatores de Crescimento Endotelial/genética , Enzimas/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Vetores Genéticos , Biblioteca Genômica , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Pulmão/patologia , Linfocinas/genética , Camundongos , Camundongos Mutantes , Morfogênese , Proteínas Nucleares/genética , Fosforilação , Gravidez , Deleção de Sequência , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/genética , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
TNF-alpha has been associated with chorioamnionitis and the subsequent development of bronchopulmonary dysplasia in preterm infants. We asked whether bioactive recombinant ovine TNF-alpha could induce chorioamnionitis, lung inflammation, lung maturation, and systemic effects in fetal sheep. We compared the responses to IL-1alpha, a cytokine known to induce these responses in preterm sheep. Intra-amniotic TNF-alpha caused no chorioamnionitis, no lung maturation, and a very small increase in inflammatory cells in the fetal lung after 5 h, 2 days (d), and 7 d. In contrast, IL-1alpha induced inflammation and lung maturation. TNF-alpha given into the airways at birth increased granulocytes in the bronchoalveolar lavage fluid of ventilated preterm lungs and decreased the mRNA for surfactant protein C but did not adversely effect postnatal lung function. An intravascular injection of IL-1alpha caused a systemic inflammatory response in fetal sheep, whereas there was no fetal response to intravascular TNF-alpha. Fetal and newborn preterm sheep are minimally responsive to TNF-alpha. Therefore, the presence of a mediator such as TNF-alpha in a developing animal does not necessarily mean that it is causing the responses anticipated from previous results in adult animals.
Assuntos
Antineoplásicos/farmacologia , Corioamnionite/fisiopatologia , Pulmão/efeitos dos fármacos , Pneumonia/fisiopatologia , Fator de Necrose Tumoral alfa/farmacologia , Líquido Amniótico , Animais , Animais Recém-Nascidos , Corioamnionite/induzido quimicamente , Corioamnionite/imunologia , Feminino , Expressão Gênica/imunologia , Idade Gestacional , Injeções Espinhais , Interleucina-1/genética , Interleucina-1/farmacologia , Interleucina-6/genética , Interleucina-8/genética , Pulmão/embriologia , Pulmão/fisiologia , Pneumonia/induzido quimicamente , Pneumonia/imunologia , Gravidez , Proteínas Recombinantes/farmacologia , Respiração Artificial , OvinosRESUMO
The role of nitric oxide (NO) in acute lung injury remains controversial. Although inhaled NO increases oxygenation in clinical trials, inhibiting NO-synthase (NOS) can be protective. To examine the latter, nickel-exposed mice were treated with saline or NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME). Initial microarray analysis of nickel-induced gene expression of saline-treated mice revealed increased inflammatory mediator, matrix injury-repair, and hypoxia-induced factor-mediated sequences and decreased lung-specific (e.g., surfactant-associated protein B and C) sequences. Compared with saline control, L-NAME-treated mice had enhanced survival with attenuated serum nitrate/nitrite, endothelial NOS activity, and lavage neutrophils and protein. Although initial cytokine (i.e., interferon-gamma, interleukins-1beta and -6, macrophage inflammatory protein-2, monocyte chemotactic protein-1, and tumor necrosis factor-alpha) gene expression was similar between groups, subsequent larger cytokine increases only occurred in saline-treated mice. Similarly, surfactant protein gene expression decreased initially in both groups yet was restored subsequently with L-NAME treatment. Interestingly, the role of inducible NOS (iNOS) in these responses seems minimal. iNOS gene expression was unaltered, iNOS activity and nitrotyrosine residues were undetectable, and an iNOS antagonist, aminoguanidine, failed to increase survival. Rather, systemic L-NAME treatment appears to attenuate pulmonary endothelial NOS activity, subsequent cytokine expression, inflammation, and protein permeability, and thereby restores surfactant gene expression and increases survival.
Assuntos
Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Níquel/toxicidade , Óxido Nítrico/antagonistas & inibidores , Surfactantes Pulmonares/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/genética , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Lesão Pulmonar , Camundongos , Camundongos Endogâmicos A , NG-Nitroarginina Metil Éster/farmacologia , Neutrófilos/efeitos dos fármacos , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Proteína A Associada a Surfactante Pulmonar/genética , Proteína B Associada a Surfactante Pulmonar/genética , Proteína C Associada a Surfactante Pulmonar/genéticaRESUMO
Acute lung injury (ALI), a severe respiratory syndrome, develops in response to numerous insults and responds poorly to therapeutic intervention. Recently, cDNA microarray analyses were performed that indicated several pathogenic responses during nickel-induced ALI, including marked macrophage activation. Macrophage activation is mediated, in part, via the receptor tyrosine kinase Ron. To address the role of Ron in ALI, the response of mice deficient in the cytoplasmic domain of Ron (Ron tk-/-) were assessed in response to nickel exposure. Ron tk-/- mice succumb to nickel-induced ALI earlier, express larger, early increases in interleukin-6, monocyte chemoattractant protein-1, and macrophage inflammatory protein-2, display greater serum nitrite levels, and exhibit earlier onset of pulmonary pathology and augmented pulmonary tyrosine nitrosylation. Increases in cytokine expression and cellular nitration can lead to tissue damage and are consistent with the differences between genotypes in the early onset of pathology and mortality in Ron tk-/- mice. These analyses indicate a role for the tyrosine kinase receptor Ron in ALI.