scholarly journals Identification of hepatocyte nuclear factor-3 binding sites in the Clara cell secretory protein gene

1993 ◽  
Vol 295 (1) ◽  
pp. 227-232 ◽  
Author(s):  
C D Bingle ◽  
J D Gitlin
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Secretory Protein ◽  
Clara Cell ◽  
Mouse Lung ◽  
Specific Gene ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Mobility Shift ◽  
Clara Cell Secretory Protein

To determine the mechanisms of cell-specific gene expression in the developing pulmonary epithelium the Clara cell secretory protein (CCSP) gene promoter was analysed by DNAase I footprinting. A prominent site of protein-DNA interaction was detected from nucleotides -132 to -76 using nuclear extract from mouse lung and human H441 cells. Mobility shift analysis revealed that an oligonucleotide corresponding to this region interacted with multiple proteins from lung and H441 cell nuclear extracts. Analysis of the nucleotide sequence of this region identified two potential binding sites for hepatocyte nuclear factor 3 (HNF-3), and consistent with this finding binding to this CCSP oligonucleotide was specifically competed for by an oligonucleotide corresponding to the HNF-3-binding site from the mouse transthyretin gene. Mobility shift of the CCSP oligonucleotide was supershifted using antisera specific to HNF-3 alpha and HNF-3 beta, and HNF-3 alpha and HNF-3 beta translated in vitro were found to bind specifically to this same oligonucleotide. Co-transfection of HNF-3 alpha- and HNF-3 beta-expression plasmids increased cell-specific reporter gene activity in H441 cells transfected with a CCSP-CAT gene chimeric construct containing this -132 to -76 region. Taken together, these results suggest a role for HNF-3 in mediating cell-specific CCSP gene expression within the bronchiolar epithelium. These findings support the hypothesis that members of the HNF-3 ‘forkhead’ family of transcription factors determine gene expression and cell fate in multiple cell lineages derived from the primitive gut endoderm.

scholarly journals Role of hepatocyte nuclear factor-3α and hepatocyte nuclear factor-3β in Clara cell secretory protein gene expression in the bronchiolar epithelium

1995 ◽  
Vol 308 (1) ◽  
pp. 197-202 ◽  
Author(s):  
C D Bingle ◽  
B P Hackett ◽  
M Moxley ◽  
W Longmore ◽  
J D Gitlin
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Secretory Protein ◽  
Clara Cell ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Clara Cell Secretory Protein ◽  
Region I ◽  
Bronchiolar Epithelium

The 5′ flanking region of the Clara cell secretory protein (CCSP) gene contains two cis-acting elements which bind hepatocyte nuclear factor (HNF)-3 alpha and HNF-3 beta in vitro. To determine the role of these proteins in mediating CCSP gene expression in the bronchiolar epithelium, chimeric CCSP-reporter gene constructs containing various regions of the CCSP 5′ flanking region were co-transfected into H-441 cells with HNF-3 alpha or HNF-3 beta expression plasmids. These studies indicate that each of these transcription factors positively regulates CCSP gene expression and revealed that CCSP region I (-132 to -76) is sufficient to mediate this effect. Gel-mobility-shift assays with oligonucleotides corresponding to CCSP region I, nuclear extract from bronchiolar epithelial cells and HNF-3-specific antibodies indicate that HNF-3 alpha and HNF-3 beta are the only proteins in bronchiolar epithelial cells which directly interact with this region. Consistent with these observations, HNF-3 alpha and HNF-3 beta transcripts were found to be enriched in this cell population and in situ hybridization of adult lung revealed HNF-3 gene expression in non-ciliated bronchiolar epithelial cells expressing the CCSP gene. Finally, experiments with CCSP region I and a heterologous promoter indicate that this region acts in a promoter-specific context, suggesting that additional factors interacting via the minimal CCSP promoter region are essential in determining the effects of HNF-3 on cell-specific CCSP gene expression in the bronchiolar epithelium.

C/EBP α and C/EBP δ Activate the Clara Cell Secretory Protein Gene through Interaction with Two Adjacent C/EBP-Binding Sites

2000 ◽  
Vol 22 (4) ◽  
pp. 469-480 ◽  
Author(s):  
Tobias N. Cassel ◽  
Lena Nordlund-Möller ◽  
Olof Andersson ◽  
Jan-Åke Gustafsson ◽  
Magnus Nord
Keyword(s):  
Binding Sites ◽  
Protein Gene ◽  
Secretory Protein ◽  
Clara Cell ◽  
Clara Cell Secretory Protein

Interferon-γ stimulates human Clara cell secretory protein production by human airway epithelial cells

1998 ◽  
Vol 274 (5) ◽  
pp. L864-L869 ◽  
Author(s):  
X. L. Yao ◽  
T. Ikezono ◽  
M. Cowan ◽  
C. Logun ◽  
C. W. Angus ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Epithelial Cells ◽  
Secretory Protein ◽  
Airway Epithelial Cells ◽  
Clara Cell ◽  
Dependent Manner ◽  
Airway Epithelial ◽  
Clara Cell Secretory Protein ◽  
Ifn Γ

Clara cell secretory protein (CCSP) is an inhibitor of secretory phospholipase A2. It is produced by airway epithelial cells and is present in airway secretions. Because interferon (IFN)-γ can induce gene expression in airway epithelial cells and may modulate the inflammatory response in the airway, it was of interest to study the effect of this cytokine on epithelial cell CCSP mRNA expression and CCSP protein synthesis. A human bronchial epithelial cell line (BEAS-2B) was used for this study. CCSP mRNA was detected by ribonuclease protection assay. IFN-γ was found to increase CCSP mRNA expression in a time- and dose-dependent manner. The CCSP mRNA level increased after IFN-γ (300 U/ml) treatment for 8–36 h, with the peak increase at 18 h. Immunobloting of CCSP protein also demonstrated that IFN-γ induced the synthesis and secretion of CCSP protein in a time-dependent manner. Nuclear run-on, CCSP reporter gene activity assay, and CCSP mRNA half-life assay demonstrated that IFN-γ-induced increases in CCSP gene expression were mediated, at least in part, at the posttranscriptional level. The present study demonstrates that IFN-γ can induce increases in steady-state mRNA levels and protein synthesis of human CCSP protein in airway epithelial cells and may modulate airway inflammatory responses in this manner.

scholarly journals Inhibition of hepatocyte nuclear factor 4 transcriptional activity by the nuclear factor κB pathway

2006 ◽  
Vol 398 (3) ◽  
pp. 439-450 ◽  
Author(s):  
Varvara Nikolaidou-Neokosmidou ◽  
Vassilis I. Zannis ◽  
Dimitris Kardassis
Keyword(s):  
Gene Expression ◽  
Inflammatory Cytokine ◽  
Transcriptional Activity ◽  
Binding Protein ◽  
Nuclear Factor Κb ◽  
Specific Gene ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Response Element ◽  
Hepatocyte Nuclear Factor 4

HNF-4 (hepatocyte nuclear factor 4) is a key regulator of liver-specific gene expression in mammals. We have shown previously that the activity of the human APOC3 (apolipoprotein C-III) promoter is positively regulated by the anti-inflammatory cytokine TGFβ (transforming growth factor β) and its effectors Smad3 (similar to mothers against decapentaplegic 3) and Smad4 proteins via physical and functional interactions between Smads and HNF-4. We now show that the pro-inflammatory cytokine TNFα (tumour necrosis factor α) antagonizes TGFβ for the regulation of APOC3 gene expression in hepatocytes. TNFα was a strong inhibitor of the activity of apolipoprotein promoters that harbour HNF-4 binding sites and this inhibition required HNF-4. Using specific inhibitors of TNFα-induced signalling pathways, it was shown that inhibition of the APOC3 promoter by TNFα involved NF-κB (nuclear factor κB). Latent membrane protein 1 of the Epstein–Barr virus, which is an established potent activator of NF-κB as well as wild-type forms of various NF-κB signalling mediators, also inhibited strongly the APOC3 promoter and the transactivation function of HNF-4. TNFα had no effect on the stability or the nuclear localization of HNF-4 in HepG2 cells, but inhibited the binding of HNF-4 to the proximal APOC3 HRE (hormone response element). Using the yeast-transactivator-GAL4 system, we showed that both AF-1 and AF-2 (activation functions 1 and 2) of HNF-4 are inhibited by TNFα and that this inhibition was abolished by overexpression of different HNF-4 co-activators, including PGC-1 (peroxisome-proliferator-activated-receptor-γ co-activator 1), CBP [CREB (cAMP-response-element-binding protein) binding protein] and SRC3 (steroid receptor co-activator 3). In summary, our findings indicate that TNFα, or other factors that trigger an NF-κB response in hepatic cells, inhibit the transcriptional activity of the APOC3 and other HNF-4-dependent promoters and that this inhibition could be accounted for by a decrease in DNA binding and the down-regulation of the transactivation potential of the AF-1 and AF-2 domains of HNF-4.

scholarly journals Clara Cell Secretory Protein Is Reduced in Equine Recurrent Airway Obstruction

2009 ◽  
Vol 46 (4) ◽  
pp. 604-613 ◽  
Author(s):  
P. Katavolos ◽  
C. A. Ackerley ◽  
L. Viel ◽  
M. E. Clark ◽  
X. Wen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Airway Obstruction ◽  
Secretory Protein ◽  
Lung Lavage ◽  
Clara Cell ◽  
Immunogold Label ◽  
Recurrent Airway Obstruction ◽  
Clara Cell Secretory Protein ◽  
Before And After ◽  
Active Disease

Horses are prone to recurrent airway obstruction (RAO), an inflammatory lung disease induced by repeated exposure to environmental mold, dust, and bacterial components. Active disease manifests with mucus hyperproduction, neutrophilic inflammation, bronchoconstriction, and coughing. Chronically affected animals have lung remodeling characterized by smooth muscle hyperplasia, collagen deposition, lymphoid hyperplasia, and impaired aerobic performance. Clara cell secretory protein (CCSP) counters inflammation in the lung, hence we hypothesized that CCSP depletion is a key feature of RAO in horses. Recombinant equine CCSP and specific antiserum were produced, and percutaneous lung biopsies were obtained from 3 healthy horses and from 3 RAO-affected horses before and after induction of RAO. CCSP relative gene expression in tissue, as well as protein concentration in lung lavage fluid, was determined. Immunocytochemical analysis, using both light and immunogold ultrastructural methods, demonstrated reduced CCSP staining in lung tissue of animals with RAO. Immunogold label in Clara cell granules was less in animals with chronic RAO than in normal animals, and absent in animals that had active disease. Median lung lavage CCSP concentration was 132 and 129 ng/ml in healthy horses, and 62 and 24 ng/ml in RAO horses before and after challenge, respectively. CCSP lung gene expression was significantly higher in healthy animals than in animals with chronic RAO. Together, these preliminary findings suggest that reduced production of CCSP and subcellular changes in Clara cells are features of chronic environmentally induced lung inflammation in horses.

scholarly journals Transgenic hCFTR expression fails to correct β-ENaC mouse lung disease

2012 ◽  
Vol 302 (2) ◽  
pp. L238-L247 ◽  
Author(s):  
B. R. Grubb ◽  
W. K. O'Neal ◽  
L. E. Ostrowski ◽  
S. M. Kreda ◽  
B. Button ◽  
...  
Keyword(s):  
Lung Disease ◽  
Transgenic Mouse ◽  
Ussing Chamber ◽  
Airway Disease ◽  
Secretory Protein ◽  
Clara Cell ◽  
Mouse Lung ◽  
Airway Epithelial ◽  
Clara Cell Secretory Protein ◽  
Chamber Studies

The relationships between airway epithelial Cl− secretion-Na+ absorption balance, airway surface liquid (ASL) homeostasis, and lung disease were investigated in selected transgenic mice. 1) To determine if transgenic overexpression of wild-type (WT) human CFTR (hCFTR) accelerated Cl− secretion and regulated Na+ absorption in murine airways, we utilized a Clara cell secretory protein (CCSP)-specific promoter to generate mice expressing airway-specific hCFTR. Ussing chamber studies revealed significantly (∼2.5-fold) elevated basal Cl− secretory currents in CCSP-hCFTR transgenic mouse airways. Endogenous murine airway Na+ absorption was not regulated by hCFTR, and these mice exhibited no lung disease. 2) We tested whether hCFTR, transgenically expressed on a transgenic mouse background overexpressing the β-subunit of the epithelial Na+ channel (β-ENaC), restored ion transport balance and ASL volume homeostasis and ameliorated lung disease. Both transgenes were active in CCSP-hCFTR/β-ENaC transgenic mouse airways, which exhibited an elevated basal Cl− secretion and Na+ hyperabsorption. However, the airway disease characteristic of β-ENaC mice persisted. Confocal studies of ASL volume homeostasis in cultured tracheal cells revealed ASL autoregulation to a height of ∼6 μm in WT and CCSP-hCFTR cultures, whereas ASL was reduced to <4 μm in β-ENaC and CCSP-hCFTR/β-ENaC cultures. We conclude that 1) hCFTR overexpression increases basal Cl− secretion but does not regulate Na+ transport in WT mice and 2) transgenic hCFTR produces increased Cl− secretion, but not regulation of Na+ channels, in β-ENaC mouse airways and does not ameliorate β-ENaC mouse lung disease.

scholarly journals The lung enriched transcription factor TTF-1 and the ubiquitously expressed proteins Sp1 and Sp3 interact with elements located in the minimal promoter of the rat Clara cell secretory protein gene

1996 ◽  
Vol 316 (2) ◽  
pp. 467-473 ◽  
Author(s):  
Ruud F. G. TOONEN ◽  
Sharon GOWAN ◽  
Colin D. BINGLE
Keyword(s):  
Promoter Region ◽  
Secretory Protein ◽  
Minimal Promoter ◽  
Clara Cell ◽  
Start Site ◽  
Transcription Start ◽  
Mobility Shift ◽  
Clara Cell Secretory Protein ◽  
Gel Mobility Shift ◽  
Minimal Promoter Region

The mechanisms that direct expression of the Clara cell secretory protein (CCSP) gene to the bronchiolar epithelial cells of the lung remain to be elucidated. Previous studies have identified a number of proteins which bind to a functionally important region (Region 1) located -132 to -76 bp from the transcription start site in the rat CCSP gene. Subsequently we have shown that while Region 1 is an important positive regulator of CCSP gene expression, sequences 3′ of this region (-75 to +38) are sufficient to confer tissue-specific expression of a reporter gene. In the present study we have used transient transfections with a deletion series of CCSP–CAT reporter plasmids (where CAT is chloramphenicol acetyltransferase) and gel mobility shift assays with a series of overlapping oligonucleotides covering the whole minimal promoter region to study protein–DNA interactions within this region. These studies have identified a conserved functional binding site for the lung and thyroid enriched homeodomain transcription factor TTF-1, located between positions -51 and -42 from the transcription start site. CCSP–CAT chimaeric reporters containing this region are specifically activated by TTF-1 in co-transfection assays, and nuclear extracts from cells which express TTF-1 bind to this region, as does in vitro translated rat TTF-1. Three additional conserved regions were identified, and in further gel mobility shift studies with an oligonucleotide spanning the conserved region immediately 5′ to the TTF-1 site we identified a binding site for the ubiquitously expressed zinc-finger-containing proteins Sp1 and Sp3. These studies suggest that cell-type-restricted and ubiquitous nuclear proteins may play a combined role in the regulation of the CCSP gene within the bronchiolar epithelium by interacting with the minimal promoter region.

Clara cell secretory protein gene expression in bronchiolar epithelium

1992 ◽  
Vol 262 (4) ◽  
pp. L399-L404 ◽  
Author(s):  
B. P. Hackett ◽  
N. Shimizu ◽  
J. D. Gitlin
Keyword(s):  
Gene Expression ◽  
Lung Tissue ◽  
Prolonged Exposure ◽  
Secretory Protein ◽  
Clara Cell ◽  
Fetal Life ◽  
Clara Cells ◽  
Adult Rats ◽  
Clara Cell Secretory Protein ◽  
Bronchiolar Epithelium

To determine the mechanisms of Clara cell secretory protein (CCSP) gene expression, a cDNA clone was isolated and used in RNA blot analysis. A single 600 bp CCSP specific transcript was detected in the developing rat lung on fetal day 18. This transcript increased in abundance during late fetal life such that adult levels were attained within 2 wk postpartum. CCSP gene expression was tissue specific, being confined to lung and trachea at all developmental stages. The abundance of CCSP mRNA in lung tissue was unchanged after the induction of lung injury in adult rats either with lipopolysaccharide or prolonged exposure to hyperoxia. In situ hybridization of lung tissue revealed that CCSP gene expression is localized to the nonciliated epithelial (Clara) cells of the bronchiolar epithelium throughout fetal and postnatal development. Taken together the results indicate that the gene for CCSP is abundantly expressed in a cell-specific fashion in the lung and suggest that analysis of such expression will be useful in elucidating the role of Clara cells in the growth and development of the bronchiolar epithelium.

scholarly journals Exclusive homodimerization of the orphan receptor hepatocyte nuclear factor 4 defines a new subclass of nuclear receptors.

1995 ◽  
Vol 15 (9) ◽  
pp. 5131-5143 ◽  
Author(s):  
G Jiang ◽  
L Nepomuceno ◽  
K Hopkins ◽  
F M Sladek
Keyword(s):  
Nuclear Receptors ◽  
Hormone Receptor ◽  
Thyroid Hormone Receptor ◽  
Orphan Receptor ◽  
Specific Gene ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Mobility Shift ◽  
Receptor Alpha ◽  
Hepatocyte Nuclear Factor 4

Hepatocyte nuclear factor 4 (HNF-4), a highly conserved member of the steroid hormone receptor superfamily critical for development and liver-specific gene expression, is very similar to another superfamily member, retinoid X receptor alpha (RXR alpha), in overall amino acid sequence and DNA binding specificity. Since RXR alpha is known to heterodimerize with many other nuclear receptors, the formation of heterodimers between HNF-4 and RXR alpha was examined. With the electrophoretic mobility shift assay, coimmunoprecipitation, and transient transfection assays, it is shown that, unlike other nuclear receptors, HNF-4 does not form heterodimers with RXR alpha either in the presence or in the absence of DNA. We also show that in vitro-translated HNF-4 does not form heterodimeric complexes on DNA with a number of other receptors, including RXR beta, RXR gamma, retinoic acid receptor alpha, or thyroid hormone receptor alpha. To investigate the hypothesis that the lack of heterodimerization between HNF-4 and RXR alpha is due to a strong homodimerization activity of HNF-4, glycerol gradient sedimentation and kinetic analysis were used to show that HNF-4 is in fact a stable homodimer in solution. Finally, immunohistochemistry is used to show that the HNF-4 protein is found exclusively in the nuclei in both HepG2 cells, which express endogenous HNF-4, and transfected COS cells, which overexpress HNF-4. These findings lead us to propose that HNF-4 defines a new subclass of nuclear receptors which reside primarily in the nucleus and which bind DNA and regulate transcription as homodimers.

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