Karen M. Ridge, PhD Assistant Professor Medicine Structure and function of the intermediate filament system in lung epithelial cells Curricula: Cell Biology E-mail:   kridge@northwestern.edu

PROJECT 1:            The role of intermediate filaments in epithelial injury and repair
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 cells that are damaged during the ALI.  It also requires the coordination of extracellular matrix turnover and regeneration of alveolar epithelial type II and type 1 cells. Orderly re-epithelialization suppresses fibroblast proliferation and matrix deposition. However, resolution of ALI can result in disordered repair of the alveolus, characterized by fibrocellular proliferation. 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.

Vimentin is a type III intermediate filament (IF) protein normally expressed in cells of mesenchymal origin.  However, vimentin expression has been described in epithelial cells involved in pathological or physiological processes which require epithelial cell migration.  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). 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.  One focus of the lab is to study the role of intermediate filaments during wound repair and remodeling following ALI.   We hypothesize that alveolar epithelial cells (AECs) transiently express 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 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 ATII cells 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 to identify the elements that regulate keratin and vimentin intermediate filaments (IFs) in the repair and remodeling of the alveolar epithelium.

PROJECT 2:            Alveolar Epithelial Type I cells: role and regulation of Na,K-ATPase
Cardiogenic pulmonary edema can result from increased hydrostatic pressures across the pulmonary circulation as seen in patients with congestive heart failure. Pulmonary edema causes acute hypoxemic respiratory failure where airspaces flood with liquid from pulmonary vessels and interferes with the transfer of oxygen from the airspaces into the blood. The VA patients are especially prone to respiratory failure because of their age and history of tobacco smoking.  Despite technological advances in supporting these patients, the mortality rate remains at >50%.  The importance of active sodium transport and lung edema clearance in the survival of patients with respiratory failure has been clearly established, but the role that alveolar epithelial type 1 cells play has not been defined.  The surface area available for active sodium transport and fluid reabsorption in the lung is large, ~100 to 150 m² in human lung. The alveolar epithelium is comprised of alveolar type 1 (AT1) and type 2 (AT2) cells.  AT2 cells, which cover 2-5% of the surface area, produce, secrete, and recycle pulmonary surfactant.   AT2 cells, which contain both Na,K-ATPase and amiloride-sensitive epithelial Na⁺ channels (ENaC), actively transport Na⁺ in culture. Very little is known about AT1 cells because this cell type has been very difficult to isolate.  Until recently ATI cells were considered to be "inert" cells that solely provide a barrier function, rather than having active functions. However, there is increasing evidence that the Na,K-ATPase is localized in the basolateral surface of ATI cell and plays an important role contributing to active Na+ transport and thus to alveolar fluid reabsorption. Consequently, the AT1 cells may be important in keeping the alveolar surface free of edema and available for normal gas exchange.  We hypothesize that AT1 cells do indeed express the Na,K-ATPase and are capable of active Na+ transport and fluid reabsorption.  This project will focus on the mechanisms by which the β-adrenergic agonists regulate Na,K-ATPase in alveolar epithelial type 1 cells and the role of AT1 cells in alveolar fluid reabsorption.

PROJECT 3:            Effects of Hypoxia on Alveolar Epithelial Cytoskeleton
Acute respiratory distress syndrome (ARDS) is characterized by the rapid and progressive deterioration of lung function, which can evolve to multi-organ dysfunction and death.  Hypoxia causes lung edema and has been associated with changes in the ultrastructure of alveolar epithelial cells.  The mechanisms contributing to the development of hypoxia-mediated dysfunction of the lung are incompletely understood.  The cytoskeleton is largely responsible for a cell’s structural integrity, and the fibrous system of intermediate filaments (IFs) is particularly important for the maintenance of epithelial cells.  In preliminary experiments, we are observing that K8-deficient mice have a profound impairment in their ability to clear edema fluid, suggesting the importance of an intact keratin IF network for normal alveolar function. This project is focused on determining whether hypoxia-induced changes in the keratin intermediate filament (IF) network lead to alveolar epithelial cell dysfunction.  We hypothesize that hypoxia generates mitochondrial reactive oxygen species (ROS) in alveolar epithelial cells, which activate protein kinases that phosphorylate keratin proteins and regulates the organization, disassembly and degradation of the keratin intermediate filaments.  Modifications to keratin IFs in the alveolar epithelial cell may contribute to impaired alveolar epithelial function.  The project will integrate studies examining the cellular mechanisms regulating the re-organization of keratin IFs and the resultant epithelial cell dysfunction caused by hypoxia.  Specifically, we will explore the role of reactive oxygen species (ROS), protein kinase C (PKC), and the ubiquitin-proteasome pathway in regulating the assembly state, phosphorylation, and degradation of keratin proteins.  In vitro studies will be complemented with studies performed in K8-deficient mice.  Completion of the proposed studies will provide novel insights on the role of keratin IF in the pathogenesis of hypoxia-induced dysfunction of the alveolar epithelium, which is of biological and clinical importance in patients with alveolar epithelial injury.

Selected Publications:

Litvan, J., Briva, A.,  Wilson, M.S., Budinger, G.S., Sznajder, J.I., and K.M. Ridge.  Low pO₂ tension impairs alveolar fluid reabsorption and Na,K-ATPase via reactive oxygen species.  J. Biol. Chem.  281:19892-8, 2006.

Sivaramakrishnan, S., J.V. DeGiulio, R.D. Goldman, K.M. Ridge, Micromechanical properties of keratin intermediate filament network under shear stress.  Proc. Natl. Acad. Sci., 105:889-94. 2008.  Accompanied by editorial review:  Proc. Natl. Acad. Sci. 105:1105-6, 2008. ·           

Adir, Y., Welch, L.C., Dumasius, V., Factor, P.H., Sznajder, J.I., and K.M. Ridge.  Overexpression of the Na,K-ATPase α2 subunit improves lung liquid clearance during ventilation-induced lung injury.  Am. J. Physiol., 294:L1233-1237, 2008.

Jaitovich, A., Mehta, S.B., Ciechanover, A., Goldman, R.D., and K.M. Ridge., Ubiquitin-proteasome mediated degradation of keratin intermediate filaments in mechanically stimulated alveolar epithelial cells.  J. Biol. Chem., 283: 25348-55, 2008.

Sivaramakrishnan, S., N. Ni, R. G. Goldman, K. M. Ridge.  PKCζ is required for the Shear Stress-Induced Structural Reorganization of Keratin Intermediate Filaments in Alveolar Epithelial Cells.  Mol. Biol. Cell., 2009, Epub April 8, 2009.

Ni,N, N.S. Chandel, J. Litvan, K.M. Ridge.  Mitochondrial reactive oxygen species are required for hypoxia-induced degradation of keratin intermediate filaments.  FASEB J., 2009, accepted.

View Publications by Karen Ridge listed in the National Library of Medicine (PubMed).

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