Resolution of acute lung injury (ALI) requires coordinated and effective tissue repair and remodeling to reestablish a functional alveolar epithelial barrier. In particular, restoration of the normal air space architecture requires reconstitution of denuded alveolar epithelial type I cells that undergo apoptotic and necrotic death following ALI. Coordination of extracellular matrix turnover and regeneration of alveolar epithelial type II cells is important in reestablishing surfactant production and ion transport. Orderly re-epithelialization suppresses fibroblast proliferation and matrix deposition after ALI. However, resolution of ALI can result in disordered repair of the alveolus, characterized by fibrocellular proliferation. The cellular regulatory mechanisms necessary for coordinating functional repopulation and reconstitution of the alveolar epithelial barrier remain unclear. Transdifferentiation of epithelial cells into matrix producing fibroblasts with enhanced matrix turnover has been suggested as one mechanism by which disordered epithelial remodeling promotes progression to fibroproliferation in ALI. This mesenchymal transdifferentiation has been characterized in part by expansion of vimentin positive cells, which are derived from alveolar epithelial cells following ALI [1,2]. Vimentin is a type III intermediate filament (IF) protein normally expressed in cells of mesenchymal origin [3]. However, vimentin expression has been described in epithelial cells involved in pathological or physiological processes which require epithelial cell migration [4-7]. Accumulating evidence now supports the model in which epithelial cell acquisition of migratory and/or invasive properties is associated with the loss of epithelial characteristics and the gain of mesenchymal properties, a phenomenon referred to as epithelial-to-mesenchymal transition (EMT) [1,2,8-10]. Several other groups of molecules (including cell-cell adhesion molecules, cell-substrate adhesion molecules, proteases, and transcription factors) have been implicated in EMT and are associated with different pathological processes, such as wound healing. This proposal will focus on the role of intermediate filaments during wound repair and remodeling following ALI. We hypothesize and provide preliminary results that alveolar epithelial cells (AECs) transiently induce vimentin IF protein expression and increase the rate of cell migration to promote wound closure. Further, the keratin IF network, which is required for maintaining alveolar epithelial cell integrity [11] is upregulated during wound healing. Upon wound closure, vimentin protein is rapidly degraded and the alveolar epithelial cell phenotype maintained. In the presence of TGF-β1, wounded AECs also induce vimentin protein expression, however the “native” keratin IF network is disassembled and degraded. Upon wound closure, in the continued presence of TGF-β1, the vimentin IF network continues to be expressed and the alveolar epithelial cell phenotype is not maintained. This hypothesis will be rigorously tested as outlined below. We have formulated three interrelated specific aims to study the regulation of keratin and vimentin intermediate filaments (IFs) in the repair and remodeling of the alveolar epithelium. Specific aim#1. To determine whether keratin and/or vimentin IFs are required for normal alveolar epithelial repair and remodeling in an in vivo and in vitro model of lung injury. Specific Aim #2. To determine whether TGF-β1/Smad, SIP1 and/or β-catenin/TCF transcriptionally regulate keratin and vimentin genes in alveolar epithelial cells to increase the rate of AEC migration and promote wound healing. Specific aim #3. To determine whether the degradation of keratin and vimentin intermediate filaments is mediated by the ubiquitin-proteasome pathway in alveolar epithelial cells during wound repair and remodeling. 
Figure A. Knock-down of vimentin expression by transient shRNAi transfections. (A) Agarose gel of vimentin mRNA expression in AECs transfected with either vimentin siRNA oligonucleotides or scrambled oligonucleotides. Knock-down efficiency was calculated from densitometric analysis of the RNA bands. 15S rRNA expression was used as a loading control. (B) Vimentin (red channel) and keratin (green channel) immunofluorescence staining of vimentin siRNA or scrambled siRNA transfected AEC monolayers that were scratch-wounded 24 h after transfection. (C) Representative phase contrast micrographs of scratch-wounded confluent cultures of control cells or vimentin siRNA transfected AECs at time points immediately after wounding and 24 h post wounding. |
