Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive diffuse parenchymal lung disease of unknown etiology (Ley et al., 2011). Existing evidence strongly suggests that the alveolar epithelial cell (AEC) is the key player in the pathogenesis of IPF. It is believed that repetitive cycles of epithelial cell injury, followed by impaired wound healing, lead to an excessive apoptosis of AECs, accompanied by aberrant activation of fibroblasts/myofibroblasts, deregulated remodeling and, finally, irreversible restructuring of the lung parenchyma (Selman et al., 2002, Zoz et al., 2001). Hepatocyte growth factor (HGF) is a pleiotropic cytokine playing a major role in cellular repair processes, ensuring restoration of epithelial homeostasis in the damaged organ. Exogenous administration of HGF has been reported beneficial in experimental models of various organ fibrosis including the lung. Bcl-xL is an anti-apoptotic member of Bcl-2 family which consists of highly conserved proteins involved in the mitochondrial control of apoptosis. Since HGF signaling via c-Met receptor has been proposed to regulate the expression of Bcl-2 family members, the present study was performed to evaluate the potential role of Bcl-xL in HGF-mediated epithelial protection in IPF. We therefore aimed to characterize Bcl-xL expression and its cellular localization in lung tissues of IPF patients in comparison to donor lung tissues, to investigate if HGF mediates pro-survival effects on alveolar epithelial cells regardless of the kind of pro apoptotic stimulus and to assess the potential role of Bcl-xL in this context. Employing tissues from human IPF and donor lung resections, we observed that Bcl-xL protein was highly expressed in hyperplastic AECII found in regions of dense fibrosis in IPF. Donor lung tissues revealed a much weaker signal for Bcl-xL in the alveolar epithelium. These findings were confirmed by Western blot analysis which revealed a significant increase in the total Bcl-xL amount in IPF lung versus donor lung homogenates. Furthermore, staining for Bcl-xL in AECII in still regular imposing areas was less prominent than in hyperplastic AECII present in fibroblastic regions.In vitro studies were performed on mouse (MLE12, MLE15) and rat (RLE-6TN) alveolar epithelial cell lines. Since it has been reported that human IPF is characterized by permanent oxidative stress, enhanced activation of ER stress and up-regulation of Fas ligand (FasL), we chose hydrogen peroxide, thapsigargin and FasL as apoptosis-inducing factors in this study. We observed that simultaneous treatment with HGF and hydrogen peroxide or thapsigargin resulted in an improved survival of alveolar epithelial cells. In both cases, the HGF-mediated anti-apoptotic activity was associated with increased Bcl-xL expression and the beneficial effect of HGF could be abolished by using a c-Met specific inhibitor prior to HGF incubation. The siRNA-mediated knock-down of Bcl-xL caused an increased susceptibility of the epithelial cells to injury. However, although less efficient, HGF treatment still remained profitable and resulted in improved cell survival despite of the low level of Bcl-xL. Interestingly, FasL-triggered activation of Caspase 3 did not affect the expression level of Bcl-xL. In line with these results, we did not observe a beneficial effect of HGF on FasL-induced apoptotic cells. Altogether, our findings demonstrate that i) Bcl-xL is up-regulated in human IPF, predominantly in AECII and especially in areas with dense fibrosis, ii) knock down of Bcl-xL makes alveolar epithelial cells much more susceptible to injury and cell death, iii) Bcl-xL accounts at least in part for the HGF-elicited epithelial protection against oxidative as well as ER stress. Bcl-xL therefore offers as interesting candidate for epithelial-protective therapies in IPF and other forms of lung fibrosis associated with epithelial apoptosis.
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