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Epithelial cell PPAR contributes to normal lung maturation

Dawn M. Simon, Meltem C. Arikan^*, Sorachai Srisuma^,*, Soumyaroop Bhattacharya^*, Larry W. Tsai^*, Edward P. Ingenito^*, Frank Gonzalez, Steven D. Shapiro^* and Thomas J. Mariani^*,1

^* Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA;

Division of Respiratory Diseases, Children’s Hospital, Harvard Medical School, Massachusetts, USA;

Department of Physiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; and

Laboratory of Metabolism, National Cancer Institute, Bethesda, Maryland, USA

¹Correspondence: Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Thorn 908, 75 Francis St., Boston, MA 02115, USA. E-mail: tmariani{at}rics.bwh.harvard.edu

SPECIFIC AIMS

Peroxisome proliferator-activated receptor (PPAR)- has a complex influence on cellular differentiation, organ development, and the control of tissue homeostasis. This transcription factor is prominent in the conducting airway epithelium within the murine lung. We sought to understand the physiological role of epithelial cell PPAR and its potential contribution to lung development and homeostasis by conditionally disrupting the PPAR gene, specifically within the conducting airway epithelium, using a novel line of targeting mice.

PRINCIPAL FINDINGS

1. Generation of airway epithelium-specific PPAR-targeted mice
Immunostaining of normal mouse lungs revealed prominent localization of PPAR to the airway epithelium, with a majority of the staining consistent with the location of Clara cells. A novel line of mice useful for constitutive, airway epithelium-specific targeting (CCtCre) was generated using the 2.4 Rat kb CC10 promoter to drive expression of Cre recombinase. Using the ROSA26 Cre reporter (R26R) mouse, we observed airway epithelium specific targeting directed by the CCtCre transgene. Histological sections revealed targeting was specific to the conducting airway epithelium and not present within other cells, including the alveolar epithelium (Fig. 1 ). Lung epithelium-specific PPAR-targeted mice were generated by breeding the CCtCre transgenic line with mice harboring loxP sites flanking exon 2 of the PPAR gene (PPAR-floxed mice). To assess targeting efficiency and characterize the deficiency specifically within the epithelium, we directly assessed PPAR expression in freshly isolated airway epithelial cells (enriched in Clara and ciliated cells). Analysis of cells derived from the conditionally targeted mice revealed a 60% (P value<0.0001) reduction in steady-state PPAR mRNA expression by quantitative polymerase chain reaction (PCR) and a 50% (P value<0.001) reduction in PPAR-expressing cells by immunocytochemistry relative to littermate controls.

View larger version (85K): [in this window] [in a new window]   Figure 1. Airway epithelial-specific targeting in novel CCtCre line. Functional recombination activity in mice expressing Cre recombinase under control of the 2.4 kb Rat CC10 promoter was assessed using the ROSA26 Cre reporter mouse. All newborn (A and B) and mature (C and D) animals containing a copy of both transgenes showed staining within the lung. A and C) Whole mount of lungs reveal staining in the characteristic branching pattern of the airway. B and D) Histological sections show that staining is restricted to epithelial cells within the conducting airway. Further, only a minority of airway epithelial cells is stained in newborn lungs (B) while a majority of cells is stained in mature lungs (D).

2. Conditionally targeted mice develop enlarged airspaces
Histological inspection of the lungs from adult conditionally targeted animals revealed abnormal morphology. An increase in the size of the airspaces was noted, although significant numbers of normal-appearing alveoli existed. No overt signs of inflammation or tissue destruction were observed. Using quantitative morphometry, we found that both the mean chord length (29.3 µm vs. 25.9 µm, P value<0.001) and the mean airspace area (945 µm² vs. 712 µm², P value<0.001) were significantly increased in conditionally targeted mice at 8 wk of age (Fig. 2 ). No differences were observed in either the mean chord length (28.8 µm vs. 28 µm, P value>0.5) or mean airspace area (952 µm² vs. 889 µm², P value>0.4) at 2 wk of age in the conditionally targeted animals compared with their littermate controls. These data indicate that the phenotype results from an insufficiency in postnatal lung maturation in conditionally targeted animals. At 9 mo of age, conditionally targeted mice showed an increase in mean chord length (10%, P value=0.07) and mean airspace area (26%, P value=0.02) similar to that observed at 8 wk of age, indicating the airspace enlargement is not progressive.

View larger version (59K): [in this window] [in a new window]   Figure 2. Morphometric analysis of lungs from 8-wk-old animals. A and B) Histology of lung sections stained with modified Gill’s hematoxylin from 8-wk-old animals for conditionally targeted animals (B) and littermate controls (A). C and D) Computerized quantitative morphometric analysis of lung sections. Data are expressed as the mean chord length in micrometers (C) and airspace area in square micrometers (D) ± SD for conditionally targeted (solid bars) (n=12) and littermate control (open bars) (n=11) animals.

3. Conditionally targeted mice demonstrate altered lung physiology
Using the forced oscillation technique, respiratory impedance demonstrated reduced tissue resistance (G) in conditionally targeted animals (4.36 cmH₂O·s/ml vs. 5.86 cmH₂O·s/ml, P value=0.008 at PEEP 0 cmH₂O; 4.12 cmH₂O·s/ml vs. 5.34 cmH₂O·s/ml, P value=0.011 at PEEP 5 cmH₂O), except near total lung capacity (TLC), defined at a PEEP of 10 cmH₂O (4.9 cmH₂O·s/ml vs. 5.65 cmH₂O·s/ml, P value=0.123). Though not statistically significant, there was a trend toward reduced dynamic elastance and increased static lung compliance. Lung volumes were measured using whole body plethysmography and demonstrated an increase in total lung capacity (TLC) (1.16 ml vs. 0.99 ml, P value=0.017), expiratory reserve volume (ERV) (0.19 ml vs. 0.15 ml, P value=0.0007), inspiratory capacity (IC) (0.71 ml vs. 0.63 ml, P value=0.066) and vital capacity (VC) (0.9 ml vs. 0.78 ml, P value=0.016) in the targeted animals. These data are consistent with the observed airspace enlargement and confirm that the structural alterations resulting from airway epithelial cell PPAR targeting have functional consequences.

4. Reduced extracellular matrix gene expression in the conditionally targeted animals
To characterize the molecular defects associated with the abnormality in lung maturation seen in the airway-specific PPAR-targeted mice, we performed genome-wide expression profiling of lung tissue. Of the genes that were consistently decreased in the conditionally targeted mice, there was an overrepresentation of structural extracellular matrix (ECM) genes, including the elastic fiber proteins elastin (Eln) and fibrillin-1 (Fbn1), and the interstitial collagens, including procollagen type I alpha 1 (Col1a1) and procollagen type III alpha 1 (Col3a1). These findings are consistent with the observed increased airspace phenotype. However, as these proteins are expressed in the mesenchyme, and not by the targeted cells, they are secondary effects of airway-specific PPAR deficiency.

5. Characterization of gene expression in PPAR-targeted airway epithelial cells
To determine the direct effects of airway epithelial cell PPAR deficiency, we performed genome-wide expression profiling of freshly isolated airway epithelial cells from conditionally targeted and littermate control mice. As anticipated, conditionally targeted airway epithelial cells showed direct effects of decreased PPAR function. Two genes previously shown to be induced by PPAR in other cell types, ATP-binding cassette subfamily A member 1 (Abca1) and apolipoprotein E (Apoe), showed significantly decreased expression in conditionally targetted cells. Additionally, genes involved in lipid metabolism (lysosomal acid lipase 1 (Lip1), leukotriene C4 synthase (Ltc4s)) also showed decreased expression in conditionally targeted cells, suggesting that PPAR plays a role in promoting normal lipid metabolism and/or homeostasis in these cells. Of particular note, a number of genes involved in cellular differentiation (Kruppel-like factor 13 (Klf13) and transforming growth factor beta 1 (Tgfb1)) were also dysregulated in the conditionally targeted cells, suggesting alterations in the differentiation state of these cells.

CONCLUSIONS AND SIGNIFICANCE

PPAR is a known regulator of lung inflammation, lung epithelial cell gene expression, and markers of lung epithelial cell differentiation. We hypothesized that epithelial cell PPAR is necessary for the establishment and maintenance of normal lung structure through regulation of epithelial cell differentiation. Using a conditional targeting strategy to delete the PPAR gene specifically within conducting airway epithelial cells, we find PPAR is necessary for normal postnatal lung maturation. Targeted deletion of airway epithelial PPAR leads to a statistically significant change in lung structure at maturity that is not present at 2 wk of age. Although the control animals show the expected reduction in airspace size between 2 and 8 wk of age, coincident with alveogenesis, the conditionally targeted animals retain the same airspace size through maturation. These data suggest that the phenotype results from an insufficiency in postnatal lung maturation in conditionally targeted animals. This is not necessarily the result of a defect in alveogenesis, as numerous normal-sized alveoli exist in the conditionally targeted lungs. This phenotype is not progressive with aging and occurs in the absence of overt evidence of lung inflammation or tissue destruction.

We were interested in determining the functional consequences of the observed abnormality. Whole body plethysmography revealed increases in lung volumes of conditionally targeted mice and forced oscillation impedence measurements revealed alterations in lung mechanics consistent with airspace enlargement and similar to those observed in animal models of emphysema.

Molecular characterization of lung tissue from conditionally targeted mice using genome-wide expression profiling indicated a reduction in the expression of elastic and collagen fiber components, as might be expected from the observed morphology. Epithelial-mesenchymal interactions are appreciated as being essential for normal lung development, primarily during embryonic growth and differentiation. The data suggest that altered epithelial-mesenchymal interactions, secondary to epithelial PPAR deficiency, lead to changes in mesenchymal ECM gene expression and abnormal lung structure at maturity (Fig. 3 ). Analysis of gene expression from purified airway epithelial cells provides insight into the physiological functions of PPAR within this cell population. Some dysregulated genes, including Lip1, are involved in core PPAR gene family functions, such as cellular lipid trafficking and metabolism. Some genes, including the cholesterol transporter Abca1 and the chylomicron apoprotein Apoe, have been previously shown to be PPAR targets in other cell types. However, these genes have no clear, previously defined role within these cells or in the lung. The fact that a number of the genes are involved in cellular differentiation (Moesin, Ctsb, Klf13) supports our conclusion that PPAR deficiency leads to subtle alterations in airway cell phenotype that result in abnormal lung maturation.

View larger version (33K): [in this window] [in a new window]   Figure 3. Schematic showing the effects of conditional disruption of airway epithelial cell PPAR. Targeted deletion of PPAR in the conducting airway epithelial cell results in the dysregulation of gene expression in both epithelial and mesenchymal cells. This leads to alterations in both lung structure and function in conditionally targeted animals.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5410fje

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This Article Abstract Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.05-5410fjev1 20/9/1507    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Simon, D. M. Articles by Mariani, T. J. Search for Related Content PubMed PubMed Citation Articles by Simon, D. M. Articles by Mariani, T. J.