Lung function is critically dependent on the integrity of the pulmonary epithelial cell layer, which is largely comprised of bronchial, Clara, and type I and II alveolar epithelial cells (AEC). While primary AEC culture has increased our knowledge of AEC gene expression in vitro, a comprehensive analysis of epithelial cell gene transcription in vivo has not yet been attempted. Therefore, we sought to profile the epithelial cell gene expression in the murine lung in vivo. We established a method to obtain epithelial cell RNA-enriched fractions using a diluted guanidinium isothiocyanate solution, administered intratracheally. After lavaging the lungs twice with saline, the optimal dilution and retention time were optimised. This resulted in an enriched epithelial cell RNA fraction with low contamination by RNA from fibroblasts, smooth muscle, or endothelial cells, as assessed by marker gene expression. This novel methodology, named as epithelial lavage (EL), thus allows for the selective profiling of lung epithelial-specific gene expression patterns in vivo.
Furthermore, in order to mimic the high levels of transforming growth factor beta 1 (TGF-beta1), a cytokine expression of which is dramatically upregulated in the lungs of patients with idiopathic pulmonary fibrosis (IPF), the recombinant TGF-beta1 was instilled into the murine lung. It was demonstrated that the orotracheal (OT) administration of TGF-beta1 stimulated Smad-dependent signalling in pulmonary epithelial cells, and induced transcription of TGF-beta1-responsive genes. The activation of the downstream signalling pathway, assessed by Western blotting, microarray, and reverse transcriptase-polymerase chain reaction (RT-PCR), was achieved within short stimulation time-points and with either low or high concentrations of TGF-beta1.
TGF-beta1-induced gene transcription was studied in the lung in vivo after OT administration of TGF-beta1 in combination with the EL methodology. A single dose of TGF-beta1 stimulated the regulation in expression of some markers after 8 h, as assessed by quantitative RT-PCR. Therefore, TGF-beta1 induction of gene expression on the epithelium was demonstrated in vivo.
Therefore the EL represents a novel methodology to isolate RNA from the lung epithelium and study the gene expression profile of these cells under different conditions, like the TGF-beta1 effect assessed in this study.
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