RFX3 modulation of FOXJ1 regulation of cilia genes in the human airway epithelium.

BACKGROUND: Ciliated cells play a central role in cleansing the airways of inhaled contaminants. They are derived from basal cells that include the airway stem/progenitor cells. In animal models, the transcription factor FOXJ1 has been shown to induce differentiation to the ciliated cell lineage, and the RFX transcription factor-family has been shown to be necessary for, but not sufficient to induce, correct cilia development. METHODS: To test the hypothesis that FOXJ1 and RFX3 cooperatively induce expression of ciliated genes in the differentiation process of basal progenitor cells toward a ciliated cell linage in the human airway epithelium, primary human airway basal cells were assessed under conditions of in vitro differentiation induced by plasmid-mediated gene transfer of FOXJ1 and/or RFX3. TaqMan PCR was used to quantify mRNA levels of basal, secretory, and cilia-associated genes. RESULTS: Basal cells, when cultured in air-liquid interface, differentiated into a ciliated epithelium, expressing FOXJ1 and RFX3. Transfection of FOXJ1 into resting basal cells activated promoters and induced expression of ciliated cell genes as well as both FOXJ1 and RFX3, but not basal cell genes. Transfection of RFX3 induced expression of RFX3 but not FOXJ1, nor the expression of cilia-related genes. The combination of FOXJ1 + RFX3 enhanced ciliated gene promoter activity and mRNA expression beyond that due to FOXJ1 alone. Corroborating immunoprecipitation studies demonstrated an interaction between FOXJ1 and RFX3. CONCLUSION: FOXJ1 is an important regulator of cilia gene expression during ciliated cell differentiation, with RFX3 as a transcriptional co-activator to FOXJ1, helping to induce the expression of cilia genes in the process of ciliated cell differentiation of basal/progenitor cells.

Airway epithelial cell differentiation during lung organogenesis requires C/EBPα and C/EBPß.

BACKGROUND: CCAAT/enhancer-binding protein (C/EBP)α is crucial for lung development and differentiation of the pulmonary epithelium. Conversely, no lung defects have been observed in C/EBPß-deficient mice, although C/EBPß trans-activate pulmonary genes by binding to virtually identical DNA-sequences as C/EBPα. Thus, the pulmonary phenotype of mice lacking C/EBPß could be explained by functional replacement with C/EBPα. We investigated whether C/EBPα and C/EBPß have overlapping functions in regulating lung epithelial differentiation during organogenesis. Epithelial differentiation was assessed in mice with a lung epithelial-specific (SFTPC-Cre-mediated) deletion of C/EBPα (Cebpa(ΔLE) ), C/EBPß (Cebpb(ΔLE) ), or both genes (Cebpa(ΔLE) ; Cebpb(ΔLE) ). RESULTS: Both Cebpa(ΔLE) mice and Cebpa(ΔLE) ; Cebpb(ΔLE) mice demonstrated severe pulmonary immaturity compared to wild-type littermates, while no differences in lung histology or epithelial differentiation were observed in Cebpb(ΔLE) mice. In contrast to Cebpa(ΔLE) mice, Cebpa(ΔLE) ; Cebpb(ΔLE) mice also displayed undifferentiated Clara cells with markedly impaired protein and mRNA expression of Clara cell secretory protein (SCGB1A1), compared to wild-type littermates. In addition, ectopic mucus-producing cells were observed in the conducting airways of Cebpa(ΔLE) ; Cebpb(ΔLE) mice. CONCLUSIONS: Our findings demonstrate that C/EBPα and C/EBPß play pivotal, and partly overlapping roles in determining airway epithelial differentiation, with possible implications for tissue regeneration in lung homeostasis and disease.

RNA-Seq quantification of the human small airway epithelium transcriptome.

BACKGROUND: The small airway epithelium (SAE), the cell population that covers the human airway surface from the 6th generation of airway branching to the alveoli, is the major site of lung disease caused by smoking. The focus of this study is to provide quantitative assessment of the SAE transcriptome in the resting state and in response to chronic cigarette smoking using massive parallel mRNA sequencing (RNA-Seq). RESULTS: The data demonstrate that 48% of SAE expressed genes are ubiquitous, shared with many tissues, with 52% enriched in this cell population. The most highly expressed gene, SCGB1A1, is characteristic of Clara cells, the cell type unique to the human SAE. Among other genes expressed by the SAE are those related to Clara cell differentiation, secretory mucosal defense, and mucociliary differentiation. The high sensitivity of RNA-Seq permitted quantification of gene expression related to infrequent cell populations such as neuroendocrine cells and epithelial stem/progenitor cells. Quantification of the absolute smoking-induced changes in SAE gene expression revealed that, compared to ubiquitous genes, more SAE-enriched genes responded to smoking with up-regulation, and those with the highest basal expression levels showed most dramatic changes. Smoking had no effect on SAE gene splicing, but was associated with a shift in molecular pattern from Clara cell-associated towards the mucus-secreting cell differentiation pathway with multiple features of cancer-associated molecular phenotype. CONCLUSIONS: These observations provide insights into the unique biology of human SAE by providing quantitative assessment of the global transcriptome under physiological conditions and in response to the stress of chronic cigarette smoking.

Lung epithelial-C/EBPß contributes to LPS-induced inflammation and its suppression by formoterol.

The inflammatory processes associated with pulmonary disorders remains incompletely understood. CCAAT/enhancer-binding protein (C/EBP)ß is implicated in inflammatory lung disorders as well as in ß(2)-adrenoceptor signaling. We hypothesized that C/EBPß in the lung epithelium contributes to lipopolysaccharide (LPS)-induced airway neutrophilia and expression of neutrophil chemoattractant chemokine (C-X-C) motif ligand (CXCL)1, as well as the suppressive effects of long-acting ß(2)-agonists (LABAs) and glucocorticoids (GCs). To investigate this, mice with a lung epithelial-specific deletion of C/EBPß (Cebpb(ΔLE)) and control littermates (Cebpb(fl/fl)) were pre-treated with a LABA, formoterol and/or a GC, budesonide, and challenged with LPS. Inflammatory cell recruitment in bronchoalveolar lavage (BAL) fluid and pulmonary expression of inflammatory mediators were investigated. In addition, the ability of formoterol to increase C/EBP transactivation was assessed in vitro. LPS-challenged Cebpb(ΔLE) mice exhibited fewer BAL neutrophils and lower pulmonary expression of CXCL1 versus Cebpb(fl/fl) mice. Suppression of LPS-induced neutrophilia by formoterol was impaired in Cebpb(ΔLE) mice and Cxcl1 expression was increased. However, suppression of the neutrophilia by budesonide with/without formoterol was preserved. Further studies indicated that C/EBP transactivation was increased by the cAMP elevating agent forskolin and formoterol in a ß(2)-adrenoceptor dependent manner. Thus, C/EBPß in the lung epithelium contributes to LPS-induced CXCL1 expression and airway neutrophilia as well as to the suppressive effects of formoterol. Reduced C/EBPß activity, observed in smokers with chronic obstructive pulmonary disease, may impair the responsiveness to LABAs when used without GCs.

Lung epithelial CCAAT/enhancer-binding protein-ß is necessary for the integrity of inflammatory responses to cigarette smoke.

RATIONALE: Cigarette smoke is the major cause of chronic obstructive pulmonary disease and lung cancer. The mechanisms by which smoking induces pulmonary dysfunction are complex, involving stress from toxic components and inflammatory responses. Although CCCAAT/enhancer-binding protein (C/EBP)-ß is known as a key intracellular regulator of inflammatory signaling, its role in pulmonary inflammation has not been established. OBJECTIVES: To characterize the role of C/EBPß in the airway epithelial response to cigarette smoke. METHODS: mRNA expression in the airway epithelium of current, former, and never-smokers, and in in vitro cigarette smoke extract-treated primary human airway epithelial cells, was analyzed by microarray and quantitative real-time polymerase chain reaction, respectively. Mice with lung epithelial-specific inactivation of C/EBPß were generated and exposed to cigarette smoke for 4 or 11 days. Lung histology, bronchoalveolar lavage cell differentials, and expression of inflammatory and innate immune mediators in the lungs were assessed. MEASUREMENTS AND MAIN RESULTS: C/EBPß was significantly down-regulated in the airway epithelium of both current and former smokers compared with never-smokers, and in cigarette smoke-treated primary human airway epithelial cells in vitro. Cigarette smoke-exposed mice with a lung epithelial-specific inactivation of C/EBPß displayed blunted respiratory neutrophil influx and compromised induction of neutrophil chemoattractants growth-regulated oncogene-α, macrophage inflammatory protein-1γ, granulocyte colony-stimulating factor, and serum amyloid A 3 and proinflammatory cytokines tumor necrosis factor-α and interleukin-1ß, compared with smoke-exposed controls. Inhibition of C/EBPß in human airway cells in vitro caused a similarly compromised response to smoke. CONCLUSION: Our data suggest a previously unknown role for C/EBPß and the airway epithelium in mediating inflammatory and innate immune responses to cigarette smoke.

Connective tissue growth factor expression pattern in lung development.

The aim of this study was to investigate the expression and distribution pattern of connective tissue growth factor (CTGF) in lung development during different stages, and to compare with a model of stimulated lung growth after tracheal ligation (TL) and with the teratogen model of induced congenital diaphragmatic hernia (CDH) and lung hypoplasia after nitrofen. Sprague-Dawley rat fetuses were obtained on gestational days 14, 17, and 21 (E14, E17, E21). For the experimental CDH group, pregnant rats were given 100 mg nitrofen on gestational day 9.5 (E9.5), and delivered E21. In another group, Sprague-Dawley rat fetuses were subjected to intrauterine tracheal ligation (TL) on gestational day 19 (E19), and delivered on day 21 (E21). All fetuses were delivered by cesarean section and lungs harvested. Lungs from 1-day-old newborn healthy, nonoperated rats were also obtained. Immunohistochemical (IHC) analysis for CTGF was performed on the different lung sections. CTGF mRNA expression levels in hyperplastic lungs after TL, hypoplastic lungs and CDH after nitrofen administration, and fetal controls at E21 were analyzed with real-time polymerase chain reaction (PCR). Immunohistochemical staining for CTGF at E14 showed that it was merely localized to the epithelium of terminal bronchiole, increasing during gestation, being more abundant at E17 and at E21. In the CDH group, lungs had an immature appearance and CTGF protein expression was decreased in the epithelium of the distal airways compared to the control group at E21, and was mainly observed in the lung mesenchyme. In the TL group, CTGF expression was more abundant compared to the control group at E21, especially in the epithelium of the terminal bronchioles, with a decreasing expression pattern distally. In the newborn lungs, CTGF had a pattern of expression in the epithelium of terminal bronchiole similar to TL lungs. At the mRNA level, CTGF expression was increased after TL, and decreased in the teratogen model of CDH and lung hypoplasia after nitrofen administration. This is, to the authors' knowledge, the first report of CTGF expression pattern during lung development, and of an impaired expression in CDH lungs after nitrofen. CTGF is suggested to enhance alveologenesis and microvascular development at late stages of lung development, and a decreased expression could lead to the impaired alveologenesis and abnormal microvascular pulmonary bed observed in CDH lungs. Increased understanding of the molecular mechanisms that control lung growth could provide a key to develop novel therapeutic techniques to stimulate pre- and/or postnatal lung growth in infants with impaired lung growth and development, such in congenital diaphragmatic hernia.

Decreased CCAAT/enhancer binding protein transcription factor activity in chronic bronchitis and COPD.

BACKGROUND: CCAAT/enhancer binding proteins (C/EBPs) are key regulators of cell differentiation and linked processes such as proliferation, apoptosis, and gene expression in several organs. C/EBPs are also central for inflammatory responses and infectious defenses, but so far little is known of their role in lung diseases. Chronic bronchitis (CB) and COPD are common smoking-associated lung diseases involving the airway epithelium. METHODS: Gelshifts were used to study C/EBP transcription factor activity in airway epithelial cells obtained by bronchial brush biopsy in four groups: healthy never-smokers (n = 10), asymptomatic smokers (n = 7), and smokers with CB and recurrent infectious exacerbations without COPD (n = 23) and with COPD (n = 13). RESULTS: C/EBP-binding activity was increased 4.6-fold in airway epithelial cells of healthy smokers compared with never-smokers. In contrast, C/EBP binding activity was not increased in the epithelium of smokers with CB or COPD. C/EBP-beta was the dominant C/EBP in the airway epithelium in all groups. CONCLUSIONS: We hypothesize that this lack of increase in C/EBP-beta activity renders the epithelium incompetent of efficient regeneration and more sensitive to infection, suggesting a previously unknown role for C/EBPs in the pathogenesis of CB and COPD.

Gene expression analysis after prenatal tracheal ligation in fetal rat as a model of stimulated lung growth.

PURPOSE: Prenatal tracheal occlusion or ligation (TL) has been proven to accelerate lung growth, but the mechanism of this is poorly understood. To increase understanding of the biological mechanisms involved in growth stimulation after TL in the fetal lung, we performed Global gene expression analysis using microarray technology. MATERIAL AND METHODS: Sprague-Dawley rats underwent surgery on gestational day 19. After a small hysterotomy, the trachea was mobilized and tied. As controls, we used littermates to manipulated fetuses. On day 21, fetuses were removed and lungs harvested. Global gene expression analysis was performed using Affymetrix Platform and the RAE 230 set arrays (Affymetrix Inc, Santa Clara, Calif). For validation of microarray data, we performed real time polymerase chain reaction (PCR) of the most significant upregulated or downregulated genes, combined with immunohistochemical (IHC) analysis of lung sections. RESULTS: In the group that underwent TL, several growth factors had an increased expression including connective tissue growth factor (CTGF), insulin-like growth factor 1 (IGF-1), and fibroblast growth factor 18 (FGF-18). Some of the genes that were downregulated in the group that underwent TL compared with controls were surfactant protein A (SP-A), apolipoprotein E (Apo-E), and phospholipase group II A2 (plg2a2). These results could be confirmed with real time PCR and IHC studies. DISCUSSION: Tracheal occlusion or ligation is a well-documented stimulator of fetal lung growth, and the present study provides novel insights into the underlying molecular mechanisms, with increased expression of genes and proteins with growth factor activity. One of these growth factors, CTGF, has never been previously described in this model. Also, decreased levels of genes involved in surfactant metabolism were observed, providing molecular insights into the decreased surfactant production that is known to occur in TL. Increased understanding of the molecular mechanisms that control lung growth may be the key to develop novel therapeutic techniques to stimulate prenatal and/or postnatal lung growth.

Ectopic expression of C/EBPalpha in the lung epithelium disrupts late lung development.

The lung develops from the endoderm through a process of branching morphogenesis. This process is highly active during the pseudoglandular stage of lung development and continues into the canalicular stage, resulting in the formation of terminal sacs. CCAAT/enhancer binding proteins (C/EBPs) are transcription factors regulating central aspects of differentiation and proliferation. We report here the developmental expression of C/EBPalpha, -beta, and -delta in the lung. C/EBPalpha exhibits a dynamic expression pattern and is first detected during the late pseudoglandular stage. At this stage, expression is observed in a subset of epithelial cells in the distal parts of the branching tubules. The expression of C/EBPalpha is confined to nonproliferating cells. To examine the role of C/EBPalpha in lung development, we generated transgenic mice ectopically expressing C/EBPalpha in the lung epithelium using the human surfactant protein C promoter. Lungs from these mice were of normal size but exhibited a phenotype characterized by fewer and larger developing epithelial tubules, indicating that the branching process was affected. No effects on overall proliferation or cellular differentiation were observed. When this phenotype was compared with that of mice carrying a targeted mutation of the Cebpa gene, the Cebpa-/- mice exhibited a similar developmental phenotype. In conclusion, our results show a role for C/EBPalpha in lung development and suggest a function in the later stages of lung branching morphogenesis.

Glucocorticoids increase C/EBPbeta activity in the lung epithelium via phosphorylation.

Glucocorticoids are widely prescribed anti-inflammatory drugs used for the treatment of many inflammatory lung disorders. However, much still remains unknown about their molecular mechanisms of action. We have previously shown that glucocorticoid-induced transcription in the lung epithelial cell line NCI-H441 is mediated via C/EBP sites in the promoters of target genes, and is likely to involve the transcription factors C/EBPbeta and C/EBPdelta. Here, we report that C/EBPbeta is the most active C/EBP-factor in both human and mouse lung epithelium and that glucocorticoids induce DNA binding of C/EBPbeta in cultured primary mouse lung epithelial cells. Mechanistic studies in H441 cells revealed that glucocorticoids, acting via the glucocorticoid receptor, increase C/EBPbeta binding starting 10 min after stimulation. The mechanism is independent of de novo protein synthesis and involves phosphorylation of C/EBPbeta at Thr(235). Together this shows that glucocorticoids increase DNA-binding activity of C/EBPbeta via post-translational mechanism(s) involving phosphorylation.