Magnus Nord

Regulation of the Clara cell secretory protein/uteroglobin promoter in lung.

Abstract: Clara cell secretory protein/uteroglobin (CCSP/UG) is specifically expressed in the conducting airway epithelium of the lung in a differentiation‐dependent manner. The proximal promoter region of the rodent CCSP/UG gene directs Clara cell specificity. Previously, it was shown that the forkhead transcription factors HNF‐3α and β and the homeodomain factor TTF‐1 are important transcription factors acting through this region, suggesting that they contribute to cell specificity of the CCSP/UG gene. Members of the C/EBP family of transcription factors can also interact with elements of the proximal rat and mouse CCSP/UG promoters. The onset of C/EBPα expression in Clara cells correlates with the strong increase of CCSP/UG expression. Thus, C/EBPalpha; may play a crucial role for differentiation‐dependent CCSP/UG expression. Transfection studies demonstrate that C/EBPα and TTF‐1 can synergistically activate the murine CCSP/UG promoter. Altogether, these results suggest that C/EBPalpha;, TTF‐1, and HNF‐3 determine the Clara cell‐specific, differentiation‐dependent expression of the CCSP/UG gene in murine lung. The relative importance of these three transcription factors, however, differs in rabbits and humans.

Glucocorticoids regulate the CCSP and CYP2B1 promoters via C/EBPβ and δ in lung cells

Glucocorticoids have several important roles in the lung and play a key role in lung development and maturation. However, the specific molecular mechanisms of glucocorticoid action in lung are unclear. In this study, we have investigated two glucocorticoid-regulated genes expressed in the lung epithelium, the secretory protein CCSP, and the P450-enzyme CYP2B1. In transient transfections of lung epithelial cells, glucocorticoids increased expression from the CCSP and CYP2B1 promoters and we demonstrated that induction was dependent on the integrity of C/EBP-binding sites in both promoters. Electrophoretic mobility shift assays revealed increased DNA-binding of C/EBPβ and C/EBPδ after glucocorticoid treatment, which was not correlated to altered protein levels. The results of this study indicate a previously unknown role for C/EBP transcription factors in glucocorticoid signaling in the lung epithelium.

Decreased CCAAT/Enhancer Binding Protein Transcription Factor Activity in Chronic Bronchitis and COPD

BACKGROUNDnCCAAT/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.nnnMETHODSnGelshifts 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).nnnRESULTSnC/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.nnnCONCLUSIONSnWe 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.

Elevated Exhaled Nitric Oxide in Allergen-Provoked Asthma Is Associated with Airway Epithelial iNOS

Background Fractional exhaled nitric oxide is elevated in allergen-provoked asthma. The cellular and molecular source of the elevated fractional exhaled nitric oxide is, however, uncertain. Objective To investigate whether fractional exhaled nitric oxide is associated with increased airway epithelial inducible nitric oxide synthase (iNOS) in allergen-provoked asthma. Methods Fractional exhaled nitric oxide was measured in healthy controls (nu200a=u200a14) and allergic asthmatics (nu200a=u200a12), before and after bronchial provocation to birch pollen out of season. Bronchoscopy was performed before and 24 hours after allergen provocation. Bronchial biopsies and brush biopsies were processed for nitric oxide synthase activity staining with nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), iNOS immunostaining, or gene expression analysis of iNOS by real-time PCR. NADPH-d and iNOS staining were quantified using automated morphometric analysis. Results Fractional exhaled nitric oxide and expression of iNOS mRNA were significantly higher in un-provoked asthmatics, compared to healthy controls. Allergic asthmatics exhibited a significant elevation of fractional exhaled nitric oxide after allergen provocation, as well as an accumulation of airway eosinophils. Moreover, nitric oxide synthase activity and expression of iNOS was significantly increased in the bronchial epithelium of asthmatics following allergen provocation. Fractional exhaled nitric oxide correlated with eosinophils and iNOS expression. Conclusion Higher fractional exhaled nitric oxide concentration among asthmatics is associated with elevated iNOS mRNA in the bronchial epithelium. Furthermore, our data demonstrates for the first time increased expression and activity of iNOS in the bronchial epithelium after allergen provocation, and thus provide a mechanistic explanation for elevated fractional exhaled nitric oxide in allergen-provoked asthma.

Lung Epithelial CCAAT/Enhancer-binding Protein-β Is Necessary for the Integrity of Inflammatory Responses to Cigarette Smoke

RATIONALEnCigarette 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)-β is known as a key intracellular regulator of inflammatory signaling, its role in pulmonary inflammation has not been established.nnnOBJECTIVESnTo characterize the role of C/EBPβ in the airway epithelial response to cigarette smoke.nnnMETHODSnmRNA 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/EBPβ 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.nnnMEASUREMENTS AND MAIN RESULTSnC/EBPβ 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/EBPβ 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-1β, compared with smoke-exposed controls. Inhibition of C/EBPβ in human airway cells in vitro caused a similarly compromised response to smoke.nnnCONCLUSIONnOur data suggest a previously unknown role for C/EBPβ and the airway epithelium in mediating inflammatory and innate immune responses to cigarette smoke.

In vivo modulation of glucocorticoid receptor mRNA by inhaled fluticasone propionate in bronchial mucosa and blood lymphocytes in subjects with mild asthma

BACKGROUNDnIn vivo regulation of the glucocorticoid receptor (GR) by glucocorticoids provides a means of modulating sensitivity of targeted cells.nnnOBJECTIVEnWe sought to determine the in vivo modulation of GR mRNA expression by fluticasone propionate (FP) in subjects with mild asthma.nnnMETHODSnTen atopic asthmatic subjects were treated with FP 250 microg twice daily for 4 weeks. Before and after treatment, the patients underwent fiberoptic bronchoscopy with endobronchial biopsy and sampling of venous blood for measurements of GR mRNA levels. A solution hybridization assay was used for quantitative analysis of GR mRNA. In addition, a 24-hour urinary cortisol excretion and an adrenocorticotropic hormone test before and after treatment with FP were performed.nnnRESULTSnA high interindividual variation in GR mRNA expression was seen. However, we detected a significant reduction of the GR mRNA levels in the endobronchial biopsy specimens after FP treatment (36.6 +/- 23.1 and 25.0 +/- 10.9 amol GR mRNA/microg RNA, respectively; P <.01). In the peripheral blood lymphocytes an even more striking downregulation of the GR by its cognate ligand was documented (30.3 +/- 26.5 and 8.8 +/- 5 amol GR mRNA/microg RNA, respectively; P <.001), possibly reflecting differences in glucocorticoid sensitivity between tissues. A small but significant reduction of the 24-hour urinary cortisol excretion was observed (233 +/- 109 and 157 +/- 66 nmol/L, respectively; P <.01), whereas the feedback regulation of glucocorticoid synthesis by means of the hypothalamic-pituitary-adrenal axis as assessed by the adrenocorticotropic hormone test remained normal after treatment with FP.nnnCONCLUSIONnThe results in this study confirm the potency of the inhaled corticosteroid FP and provide evidence for a considerable tissue-specific interindividual variation in the expression of the GR.

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

PURPOSEnPrenatal 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.nnnMATERIAL AND METHODSnSprague-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.nnnRESULTSnIn 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.nnnDISCUSSIONnTracheal 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.

Rat lung polychlorinated biphenyl-binding protein: Effect of glucocorticoids on the expression of the clara cell-specific protein during fetal development☆

Abstract Certain metabolites of polychlorinated biphenyls (PCBs) are retained in the Clara cells and in the airway lumen of rodent lung due to their interaction with a secretory 13-kDa protein. The expression of this Clara cellspecific, PCB-binding protein (PCB-BP) during the fetal development of the rat lung was studied by means of ligand binding and a monospecific antiserum. The PCB-BP and specific 4,4′-bis([ 3 H]methylsulfonyl)-2,2′,5,5′-tetrachlorobiphenyl binding was first detected on gestational Day 19 and subsequently the levels of PCB-BP and specific ligand binding increased as a function of gestational age. The start site of transcription for the rat PCB-BP gene was determined by primer extension analysis and the information thus obtained was used to develop a quantitative assay for the corresponding mRNA based on solution hybridization and S1 nuclease mapping. The appearance of PCB-BP mRNA during fetal lung development preceded the detection of immunoreactive protein and ligand binding by 1 day. By Day 21, the level of PCB-BP mRNA was 15 ng/100 μg total lung RNA which is approximately 30–40% of adult levels. In utero exposure to the synthetic glucocorticoid betamethasone was shown to increase specific 4,4′-bis([ 3 H]methylsulfonyl) 2,2′,5,5′-tetrachlorobiphenyl binding, PCB-BP protein, and PCB-BP mRNA if administered from gestational Day 18 and onward. By Days 21–22, glucocorticoid treatment resulted in a two- to threefold increase in the levels of specific ligand binding, immunoreactivity, and mRNA, i.e., to approximately adult levels.

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

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/EBPβ‐deficient mice, although C/EBPβ trans‐activate pulmonary genes by binding to virtually identical DNA‐sequences as C/EBPα. Thus, the pulmonary phenotype of mice lacking C/EBPβ could be explained by functional replacement with C/EBPα. We investigated whether C/EBPα and C/EBPβ 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 (CebpaΔLE), C/EBPβ (CebpbΔLE), or both genes (CebpaΔLE; CebpbΔLE). Results: Both CebpaΔLE mice and CebpaΔLE; CebpbΔLE mice demonstrated severe pulmonary immaturity compared to wild‐type littermates, while no differences in lung histology or epithelial differentiation were observed in CebpbΔLE mice. In contrast to CebpaΔLE mice, CebpaΔLE; CebpbΔLE 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 CebpaΔLE; CebpbΔLE mice. Conclusions: Our findings demonstrate that C/EBPα and C/EBPβ play pivotal, and partly overlapping roles in determining airway epithelial differentiation, with possible implications for tissue regeneration in lung homeostasis and disease. Developmental Dynamics 241:911–923, 2012.

C/EBPα and TTF-1 Synergistically Transactivate the Clara Cell Secretory Protein Gene

In the lung, the Clara cell secretory protein/uteroglobin (CCSP/UG) is specifically expressed in the Clara cells of the small airway epithelium in a differentiationdependent manner. Although the physiological function of CCSP is unknown, the differentiation-dependent expression of this protein makes it a good model to study the mechanisms behind pulmonary epithelial gene expression during lung development and differentiation. In the rat developing lung, low levels of CCSP expression are first detected around embryonic day 16. As development proceeds, CCSP expression starts increasing at day 18–19 and further increases until birth.1 Previously, the homeodomain thyroid transcription factor-1 (TTF-1) and the forkhead hepatocyte nuclear factor-3 (HNF-3) have been identified as major regulators of pulmonary CCSP gene expression.2–4 Both transcription factors are expressed in the bronchiolar epithelium and in Clara cells. However, during lung development, the expression of HNF-3 factors and TTF-1 does not temporally reflect the expression pattern of CCSP. Therefore, the increase in CCSP expression levels seen late in lung development cannot be explained by the action of HNF-3 factors or TTF-1 alone. The CCAAT/enhancer binding protein alpha (C/EBPα) is expressed in the lung epithelium and is first detected in the rat lung at embryonic day 18–19,5 correlating to the increase in CCSP expression. Recently, we have characterized a compound C/EBPresponse element in the proximal CCSP promoter.6 C/EBPα as well as C/EBPδ, another C/EBP family member that is expressed in the bronchiolar epithelium, transactivate the CCSP promoter through interaction with the two C/EBP-binding sites that form the compound element.6 The C/EBP-binding sites are situated in close proximity to the previously characterized TTF-1 and HNF-3 binding sites. Together, this prompted us to investigate putative cooperative interactions between these transcription factors in the regulation of CCSP. To address the question of whether C/EBP, TTF-1, and HNF-3 factors interact in the regulation of CCSP, we used Drosophila SL-2 cells. These cells lack many mammalian transcription factors and are thus well suited for studies of cooperative inter-