Cindy J. Bachurski

The Transcription Factor Gene Nfib Is Essential for both Lung Maturation and Brain Development

ABSTRACT 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.

Toll-Like Receptors and Agonist Responses in the Developing Fetal Sheep Lung

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.

Nuclear Factor I/Thyroid Transcription Factor 1 Interactions Modulate Surfactant Protein C Transcription

ABSTRACT 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.

Pulmonary and systemic induction of SAA3 after ventilation and endotoxin in preterm lambs.

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.

Mesenchymal nuclear factor I B regulates cell proliferation and epithelial differentiation during lung maturation.

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 Nfib⁻/⁻ 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 Nfib⁻/⁻ 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 Nfib⁻/⁻ 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.

Functional genomics of oxidant-induced lung injury.

In summary, acute lung injury is a severe (>40% mortality) respiratory disease associated with numerous precipitating factors. Despite extensive research since its initial description over 30 years ago, questions remain about the basic pathophysiological mechanisms and their relationship to therapeutic strategies. Histopathology reveals surfactant disruption, epithelial perturbation and sepsis, either as initiating factors or as secondary complications, which in turn increase the expression of cytokines that sequester and activate inflammatory cells, most notably, neutrophils. Concomitant release of reactive oxygen and nitrogen species subsequently modulates endothelial function. Together these events orchestrate the principal clinical manifestations of the syndrome, pulmonary edema and atelectasis. To better understand the gene-environmental interactions controlling this complex process, we examined the relative sensitivity of inbred mouse strains to acute lung injury induced by ozone, ultrafine PTFE, or fine particulate NiSO4 (0.2 microm MMAD, 15-150 microg/m3). Measuring survival time, protein and neutrophils in bronchoalveolar lavage, lung wet: dry weight, and histology, we found that these responses varied between inbred mouse strains, and susceptibility is heritable. To assess the molecular progression of NiSO4-induced acute lung injury, temporal relationships of 8734 genes and expressed sequence tags were assessed by cDNA microarray analysis. Clustering of co-regulated genes (displaying similar temporal expression patterns) revealed the altered expression of relatively few genes. Enhanced expression occurred mainly in genes associated with oxidative stress, anti-proteolytic function, and repair of the extracellular matrix. Concomitantly, surfactant proteins and Clara cell secretory protein mRNA expression decreased. Genome wide analysis of 307 mice generated from the backcross of resistant B6xA F1 with susceptible A strain identified significant linkage to a region on chromosome 6 (proposed as Aliq4) and suggestive linkages on chromosomes 1, 8, and 12. Combining of these QTLs with two additional possible modifying loci (chromosome 9 and 16) accounted for the difference in survival time noted in the A and B6 parental strains. Combining these findings with those of the microarray analysis has enabled prioritization of candidate genes. These candidates, in turn, can be directed to the lung epithelium in transgenic mice or abated in inducible and constitutive gene-targeted mice. Initial results are encouraging and suggest that several of these mice vary in their susceptibility to oxidant-induced lung injury. Thus, these combined approaches have led to new insights into functional genomics of lung injury and diseases.

Involvement of transcription factor NR2F2 in human trophoblast differentiation.

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-2α), 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.

Surfactant-Associated Protein B Is Critical to Survival in Nickel-Induced Injury in Mice

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.

NFIB overexpression cooperates with Rb/p53 deletion to promote small cell lung cancer.

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.

Functional Genomics of Particle-Induced Lung Injury

Abstract Currently, the biological mechanisms controlling adverse reactions to particulate matter are uncertain, but are likely to include oxidative lung injury, inflammation, infection, and preexisting pulmonary disease (e.g., chronic obstructive pulmonary diseaseJ. Each mechanism can be viewed as a complex trait controlled by interactions of host (genetic) and environmental factors. We propose that genetic factors play a major role in susceptibility to particulate matter because the number of individuals exposed (even in occupational settings) is often large, but relatively few people respond with increases in morbidity and even mortality. Previous clinical studies support this hypothesis, having discovered marked individual variation in diminished lung function following oxidant exposures. Advances in functional genomics have facilitated the examination of this hypothesis and have begun to provide valuable new insights into gene-environmental interactions. For example, genome-wide scans can be completed readily in mice that enable assessment of chromosomal regions with linkage to quantitative traits. Recently, we and others have identified linkage to oxidant-induced inflammation and mortality. Such linkage analysis can narrow and prioritize candidate gene(s) for further investigation, which, in turn, is aided by existing transgenic mouse models. In addition, differential expression (microarray) analysis enables simultaneous assessment of thousands of genes and expressed sequence tags. Combining genome-wide scan with microarray analysis permits a comprehensive assessment of adverse responses to environmental stimuli and will lead to progress in understanding the complex cellular mechanisms and genetic determinants of susceptibility to particulate matter.