It is increasingly accepted that the alveolar epithelial cell plays a major role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a dismal disease with an average survival time of ~ 3 years and a progressive decline in lung function and exercise capacity. In IPF, chronic injury of alveolar type II cells (AECII) seems to cause disturbed alveolar re-epithelialization (1). It appears that profoundly increased epithelial apoptosis, which occurs due to epithelial injury, causes accelerated epithelial cell proliferation and further apoptosis (2, 3 and 4). The Notch signaling functions as a mediator of a cell-cell communication. The Notch pathway is known to be involved in proliferation, cell death, stem cell maintenance and differentiation during embryonic and adult development (5-8). In addition the Notch network has already been identified to play a role in some chronic lung diseases such as COPD or PAH (9, 10). Until now, the Notch signaling pathway has not been investigated in IPF. In particular, the impact of Notch activation on alveolar epithelial proliferation and apoptosis has yet not been analyzed. The present study was undertaken to evaluate the regulation and the potential role of Notch activation in repair processes in IPF lungs. We investigated the cellular regulation of the Notch signaling pathway on mRNA, protein and immunohistochemical level (IPF vs. donor lungs; bleomycin-treated vs. control mice lungs). Proliferation and survival of an AECII and AECII-like cell line (MLE 12) was investigated after in vitro transfection with Notch1 ICD, POFUT1 siRNA and DAPT (γ-secretase inhibitor) treatment. Our transcriptome data proved differential regulation of the Notch signaling pathway in microdisected septae from still normal appearing areas (representative of the early-stage of the disease) of IPF lungs compared to septae of healthy organ donors. On protein level, no significant changes in the expression of Notch pathway elements were observed with the exception of the intracellular domain of Notch1 receptor (NICD1), the ligand DLL1 and the downstream target Hes1, which were found to be significantly increased in IPF vs. donor lungs. We also observed increased protein levels of NICD1 and Dll1 in lungs of bleomycin-treated mice. Expression of these proteins was mainly restricted to AECII of fibrotic lungs. Moreover on the IHC level, expression of NICD1 and DLL1 proteins seems to be increased in AECII in IPF as compared to controls. Furthermore, we observed a characteristic staining pattern, where subpopulation of AECII cells expressed Notch1 in cytoplasm and neighboring AECII cell showed localization of this receptor in the nucleus. Most importantly, observations made after NICD1 overexpression or Notch pathway inhibition in the MLE 12 alveolar epithelial cell line and mouse primary AECII cells isolated from bleomycin-challenged mice indicate that Notch plays a major role in uncontrolled AECII proliferation in vitro. In addition, there was no influence of the Notch signaling pathway on epithelial apoptosis. Furthermore, genome wide mRNA microarray analysis of NICD1-overexpressing MLE 12 cells revealed differential regulation of the MAPK pathway. We found that NICD1 overexpression in MLE 12 cells induced phosphorylation of Erk5. Therefore, we can speculate that Erk5 may be a downstream effector of Notch1 activation, involved in increased alveolar epithelial cell proliferation. Our findings demonstrate for the first time a potential role of the Notch signaling pathway in the re-epithelialization process in the lung, which may indicate involvement of Notch on pathogenesis of pulmonary fibrosis.
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