Relative role and interdependence of urokinase and hepatocyte growth factor in mediating epithelial cell-protective effects in lung fibrosis
Final Report Abstract
Based on analysis from our initial in vivo data we were hypothesizing that a link between the urokinase plasminogen activator (uPA) system and hepatocyte growth factor (HGF) system exists, which may be of considerable importance for protection of the alveolar epithelium in lung fibrosis, although the underlying mechanisms are largely unsettled. Our aims were therefore to gain further insights into the mechanisms of HGF-mediated epithelial cell protection. The following results have been obtained: 1) We could show that uPA is capable of activating Pro-HGF by direct proteolytic cleavage. In the healthy lung, which is characterized by a high fibrinolytic state, this might be part of a protective mechanism to maintain cellular integrity and homeostasis. In the fibrotic lung, where the fibrinolytic activity in the alveolar space is greatly reduced due to the overexpression of PAI-1, this mechanism may at least partially contribute to a reduced activation of HGF. However, PAI-1 does not inhibit HGFA, the endogenous activator of Pro-HGF, indicating that other mechnaisms/factors are responsible for the defective Pro-HGF activation in IPF. 2) We hypothesized that HGF is capable of antagonizing TGF-β mediated pro-fibrotic effects and aimed to investigate the capacity of HGF to limit or reverse the TGF-β induced epithelialmesenchymal transition (EMT) of alveolar epithelial cells. However, in contrast to kidney fibrosis, where such interference has been demonstated, HGF and uPA did not limit or reverse TGF-β induced epithelial-mesenchymal transition (EMT) of alveolar epithelial cells. 3) We aimed to decipher downstream signaling events in cultured epithelial cells fibroblasts upon treatment with HGF. For our in vitro studies we employed three major stimuli which are implicated in the pathologic sequelae of a fibrotic lung: oxidative stress, ER stress and FasL-triggered apoptosis. Our data show that HGF has anti-apoptotic properties on alveolar epithelial cells under oxidative and ER stress conditions, but not in FasL-induced apoptosis. We identified Bcl-xL as a novel cytoprotective factor which is regulated by HGF/c-Met signaling in alveolar epithelial cells under conditions of oxidative and ER stress. 4) We studied the role of HGF signaling for the maintenance of a regular alveolar structure in vivo and generated triple transgenic mice with inducible knockout of HGF and c-Met, respectively. Knockout of HGF or lung epithelial cell-specific knockout of cMet in otherwise healthy adult mice did not forward a lung-specific or global phenotype, especially no lung fibrosis. Further studies with these mice are underway to examine the role of HGF or cMet knockout in injured animals. 5) With regard to therapeutic interventions, we focused on the non-inflammatory driven mouse model of amiodarone-induced lung fibrosis, recently developed by our group. Application of recombinant HGF is currently studied in this model to answer the question if HGF may be helpful to alleviate alveolar epithelial stress and therefore attenuate the extent of lung fibrosis.
