Alveolar epithelial endoplasmic reticulum (ER)-stress in Idiopathic Pulmonary Fibrosis - identification of molecular trigger events and role in fibrotic repair
Final Report Abstract
Summary: We thank the DFG for funding of the project GU 405/8-1. We have initially been describing extensive ER stress and apoptosis in AECll, alongside with a profound alteration in the processing of the hydrophobic surfactant proteins SP-B and SP-C in patients with Idiopathic Pulmonary Fibrosis (IPF), a dreadful disease with no cure to date. Based on our initial in vitro data we were hypothesising that defective processing of SP-B would cause ER-stress, AECll apoptosis in vitro and lung fibrosis in vivo. Our aims were therefore to provide further evidence for this supposed pathomechanistic chain of events. The following results have been obtained throughout the three years of funding: 1) Transfection of murine or human alveolar type ll (AECll)-like epithelial cells with either murine or human, cleavage-resistant, mutant, FLAG-tagged surfactant protein B (SP-B) proteins resulted in reproducible transgene expression, increased protein load and induction of modest ER stress. However, ER stress - to a minor extent - was also seen in response to empty vector transfection and wt SP-B overexpression per se, suggesting that the in vitro approach was not perfectly suited to study the interdependence of defective SP-B processing, ER-stress and AECll apoptosis. 2) Stable transfection of murine AECIl-like epithelial cells (MLE12) with a shRNA directed against the cathepsin H {Ctsh) gene resulted in the identification of several clones with farreaching silencing of Ctsh, which was also proven by diminished cleavage of the Ctshsubstrate BMP4. However, expression of endogenous SP-B was down-regulated in response to Ctsh ShRNA overexpression for reasons still to be explored, and did therefore not result in a significant accumulation of uncleaved proSP-B. In parallel to our work, the almost normal phenotype of Ctsh knockout (KO) mice had been published, suggesting that Cathepsin H alone is not crucial for processing of proSP-B. 3) Generation of genetically engineered mice with deletion of the napsin A {Napsa) gene, another important enzyme involved in hydrophobic surfactant protein processing, did also not fonvard a lung-specific or global phenotype, especially no lung fibrosis, and molecular studies did suggest some minor alterations of the surfactant protein processing and some ER-stress (increased XBP-1 splicing), but these changes were modest and truly not significant enough to provoke substantial AECll cell stress and onset of lung fibrosis. 1 Report on DFG project GU 405/8-1 4) Further in silico and molecular studies provided evidence that lack of Napsa in the Napsa knockout (KO) mice was almost fully compensated by cathepsin D and E (Cfsd, Cfse). 5) Interestingly and in line with the in vitro data mentioned in 2), cross-breading of Ctsh- and Napsa knockout mice did also not provoke a serious phenotype, and disturbed processing of SP-B was not found to exceed the level of that found in the single Napsa KO mice. Crossbreading of double homozygous Napsa- / Ctsh KO mice with homozygeous Ctse mice is currently unden/vay and will probably result in a significant phenotype. 6) Our data therefore suggest that there is great redundancy in AECll to ascertain and guarantee proper processing of the hydrophobic surfactant proteins, especially proSP-B, and we believe that this is for good reason, as lack of mature SP-B seems to cause respiratory distress at birth, and defective processing with accumulation of proSP-B may cause AECll stress, death and fibrosis. 7) Within the studies undertaken in this proposal, we identified a novel murine Sftpb splicing variant with potential (and still to be proven) functional relevance, because the splicing variant encodes a truncated mature SP-B peptide. 8) In search for a reasonable explanation for the far-reaching disturbances of surfactant protein processing in IPF, we could not detect IPF-related changes in the regulation of the transcription factors identified in silico to play a role in the expression of napsin A and/or cathepsin H. An extension of our studies nevertheless provided evidence that aging per se affects surfactant metobolism/processing through downregulation of ER resident chaperones, surfactant-related proteases, components of the ER quality control apparatus, ERAD components, and is associated with induction of mild alveolar epithelial ER stress. Our current hypothesis is therefore that epigenetic alterations are largely responsible for the dysregulation ofthe proteases involved in surfactant protein processing. 9) With regard to therapeutic inten/entions, we focused on the generation of a Grp78 overexpressing mouse, which we do now have in our hands and which is currently studied in injury models to answer the question if overexpression of Bip/Grp78 may be helpful to alleviate ER-stress and AECll injury and therefore attenuate the extent of lung fibrosis.
