The pathogenesis of fibrotic lung disease involves the inability of proliferating alveolar type II cells (AT2) to differentiate effectively into type I (AT1) cells, leading to faulty epithelial repair, irreversible damage, loss of function, and fibrosis. The mechanisms that normally control this process are not fully understood, so potential regulatory molecules or pathways that may be altered in fibrotic pulmonary diseases have not been elucidated. We propose that the key to normal alveolar cell differentiation is the relative sulfation of the extracellular matrix (ECM) microenvironment underlying alveolar cell types. This in turn controls expression of two important differentiation factors: a member of the forkhead (Fox) family of transcription factors and specific wingless (Wnt) signaling pathways. These factors act in conjunction with expression and signaling of transforming growth factor ? (TGF?), which enhances Wnt signaling targets, to collectively drive the cell differentiation process and establish stable alveolar phenotypes. In this proposal, we will show that following proliferative events associated with re-epithelialization in the alveolus, there is a critical, dynamic balance between alveolar epithelial cells and their ECM microenvironment. This is significantly modulated by both fixed and soluble sulfated ECMs, whose downstream effects are to specifically enhance Wnt7a and Foxa1 expression, which act together with TGF? to regulate the shift from the AT2 phenotype and control AT1 cell differentiation. The hypothesis to be addressed is: Following DNA synthesis and cytokinesis, exposure of the daughter AT2 cell to high levels of sulfated ECMs triggers enhanced expression of Foxa1 and Wnt7a in parallel with increased TGF? expression and signaling, which converge to effectively drive differentiation of AT2 to AT1 cells. To address this hypothesis, we will utilize isolated AT2 cells from humans and normal as well as conditional gene knockouts and overexpressors from rodents in traditional and modified co-culture with human and rodent fibroblasts - important regulators of the AT2 cell microenvironment. Cells and ECMs will be selectively modified with specific enhancers or inhibitors of Fox expression, and TGF? and Wnt expression and signaling, and ECM composition. These results will serve as a contextural backdrop for examination of targeted molecules by protein and/or gene expression methods in a whole animal model of alveolar injury and fibrosis. Results of these studies are expected to provide essential information needed to better understand basic cell-cell and cell-ECM relationships in alveolar epithelial homeostasis as well as the mechanisms that steer the pathogenesis of fibrogenic change in the lung as a consequence of alveolar injury and/or disease.

Public Health Relevance

Disease or environmentally-based toxic agents can damage or injure cells that line the internal surfaces of the lung, and how these cell repopulate themselves in large part determines whether the lung heals or not. To accomplish this, specific cells must divide and differentiate into other cells that carry out critically important lung-specific functions. Interruption of this process has serious consequences resulting in faulty repair and permanent damage to the lung and compromises function. This grant will define the specific mechanisms that control this process, and in doing so, enable the development of new and innovative ways to promote the healing process in injured lungs.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL044497-20
Application #
8449159
Study Section
Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
Program Officer
Eu, Jerry Pc
Project Start
1996-09-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
20
Fiscal Year
2013
Total Cost
$349,896
Indirect Cost
$114,276
Name
North Carolina State University Raleigh
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
042092122
City
Raleigh
State
NC
Country
United States
Zip Code
27695
Newman, Donna R; Sills, W Shane; Hanrahan, Katherine et al. (2016) Expression of WNT5A in Idiopathic Pulmonary Fibrosis and Its Control by TGF-β and WNT7B in Human Lung Fibroblasts. J Histochem Cytochem 64:99-111
Yi, Na Young; Newman, Donna R; Zhang, Huiying et al. (2015) Heparin and LPS-induced COX-2 expression in airway cells: a link between its anti-inflammatory effects and GAG sulfation. Exp Lung Res 41:499-513
Morales Johansson, Helena; Newman, Donna R; Sannes, Philip L (2014) Whole-genome analysis of temporal gene expression during early transdifferentiation of human lung alveolar epithelial type 2 cells in vitro. PLoS One 9:e93413
Coffey, Emily; Newman, Donna R; Sannes, Philip L (2013) Expression of fibroblast growth factor 9 in normal human lung and idiopathic pulmonary fibrosis. J Histochem Cytochem 61:671-9
Zhang, Huiying; Newman, Donna R; Bonner, James C et al. (2012) Over-expression of human endosulfatase-1 exacerbates cadmium-induced injury to transformed human lung cells in vitro. Toxicol Appl Pharmacol 265:27-42
Meuten, Travis; Hickey, Ariel; Franklin, Katherine et al. (2012) WNT7B in fibroblastic foci of idiopathic pulmonary fibrosis. Respir Res 13:62
Zhang, Huiying; Newman, Donna R; Sannes, Philip L (2012) HSULF-1 inhibits ERK and AKT signaling and decreases cell viability in vitro in human lung epithelial cells. Respir Res 13:69
Dush, Michael K; McIver, Andrew L; Parr, Meredith A et al. (2011) Heterotaxin: a TGF-ýý signaling inhibitor identified in a multi-phenotype profiling screen in Xenopus embryos. Chem Biol 18:252-63
Apparao, K B C; Newman, Donna R; Zhang, Huiying et al. (2010) Temporal changes in expression of FoxA1 and Wnt7A in isolated adult human alveolar epithelial cells enhanced by heparin. Anat Rec (Hoboken) 293:938-46
Newman, Donna R; Walsh, Eric; Apparao, K B C et al. (2007) Fibroblast growth factor-binding protein and N-deacetylase/N-sulfotransferase-1 expression in type II cells is modulated by heparin and extracellular matrix. Am J Physiol Lung Cell Mol Physiol 293:L1314-20

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