Hyperoxic lung injury is associated with widespread epithelial and endothelial injury and cell death together with airway inflammation, edema and hemorrhage. Using an inducible lung-specific transgenic system, we have shown that keratinocyte growth factor (KGF) has a profound protective effect on the lung epithelium and activates the pro-survival Akt pathway both in vivo and in vitro. We have identified intracellular molecules such as PAK4 and p90RSK that interact with the KGF receptor (KGFR) and induce pro-survival mechanisms in lung epithelial cells. We have also found that PAK4 interacts with Raf-1 in mammalian cells. Raf-1 has been recently shown to play a critical role in cell survival. We have identified an additional property of KGF, which is blockade of neutrophil transmigration into the alveolar space, another deleterious event during oxidative injury. In related studies, we have found that the soluble form of the receptor for advanced glycation end products (sRAGE) blocks neutrophil infiltration into the lung during hyperoxic conditions. These observations lead us to hypothesize that KGF and sRAGE inhibit oxidative lung injury through effects on cell survival and anti-inflammatory mechanisms. To test this hypothesis we will:
Aim I. Determine the functional importance of the signaling network induced by activated KGFR involving RSK, PAK4 and Raf-1 in epithelial cell responses to hyperoxia. a) Mouse lung epithelial (MLE-12) cells will be subjected to hyperoxia in the presence or absence of KGF and dominant-negative (DN) mutants for KGFR-interacting proteins. Effects on a) subcellular distribution and modification of pro- and anti-apoptotic proteins and on b) Akt and BAD phosphorylation will be followed.
Aim II. Investigate the role of KGFR-interacting molecules in Akt and BAD phosphorylation and epithelial protection in vivo, a) Mice with deletions in c-Raf-1 or PAK4 in a lung epithelial cell-specific fashion in the context of inducible KGF transgenic mice will be generated and will be subjected to hyperoxic injury. End-points will include epithelial protection and Akt and BAD phosphorylation.
Aim III. Investigate the mechanism by which KGF inhibits transepithelial neutrophil migration and study effects of KGF+sRAGE on protection from oxidative injury. Effects on neutrophil infiltration, chemokine secretion, matrix metalloproteinase activity, syndecan shedding, survival and edema will be followed.
Aim I V. Investigate the effect of KGF on cell survival and neutrophil transmigration using a human lung epithelial cell line and primary human alveolar epithelial cells. The epithelial protective effects of KGF and inhibition of transepithelial neutrophil migration will be studied using the human lung epithelial cell line Calu-3 and primary human lung alveolar epithelial cells that can form tight junctions in vitro. Effects on hyperoxia-induced cell death and transepithelial neutrophil migration will be followed. Collectively, these studies will address the mechanisms by which KGF induces anti-apoptotic and anti-inflammatory effects. ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL069810-05A1
Application #
7049249
Study Section
Lung Injury, Repair, and Remodeling Study Section (LIRR)
Program Officer
Harabin, Andrea L
Project Start
2001-07-01
Project End
2009-11-30
Budget Start
2005-12-23
Budget End
2006-11-30
Support Year
5
Fiscal Year
2006
Total Cost
$363,975
Indirect Cost
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Arora, Meenakshi; Poe, Stephanie L; Ray, Anuradha et al. (2011) LPS-induced CD11b+Gr1(int)F4/80+ regulatory myeloid cells suppress allergen-induced airway inflammation. Int Immunopharmacol 11:827-32
Ray, Prabir; Arora, Meenakshi; Poe, Stephanie L et al. (2011) Lung myeloid-derived suppressor cells and regulation of inflammation. Immunol Res 50:153-8
Arora, M; Poe, S L; Oriss, T B et al. (2010) TLR4/MyD88-induced CD11b+Gr-1 int F4/80+ non-migratory myeloid cells suppress Th2 effector function in the lung. Mucosal Immunol 3:578-93
Ray, Prabir; Krishnamoorthy, Nandini; Oriss, Timothy B et al. (2010) Signaling of c-kit in dendritic cells influences adaptive immunity. Ann N Y Acad Sci 1183:104-22
Shukla, Manasi N; Rose, Jane L; Ray, Rabindranath et al. (2009) Hepatocyte growth factor inhibits epithelial to myofibroblast transition in lung cells via Smad7. Am J Respir Cell Mol Biol 40:643-53
Krishnamoorthy, Nandini; Oriss, Timothy B; Paglia, Melissa et al. (2008) Activation of c-Kit in dendritic cells regulates T helper cell differentiation and allergic asthma. Nat Med 14:565-73
Ray, Prabir; Krishnamoorthy, Nandini; Ray, Anuradha (2008) Emerging functions of c-kit and its ligand stem cell factor in dendritic cells: regulators of T cell differentiation. Cell Cycle 7:2826-32
Arora, Meenakshi; Chen, Li; Paglia, Melissa et al. (2006) Simvastatin promotes Th2-type responses through the induction of the chitinase family member Ym1 in dendritic cells. Proc Natl Acad Sci U S A 103:7777-82
Ray, Prabir (2005) Protection of epithelial cells by keratinocyte growth factor signaling. Proc Am Thorac Soc 2:221-5
Pan, Zhong-Zong; Devaux, Yvan; Ray, Prabir (2004) Ribosomal S6 kinase as a mediator of keratinocyte growth factor-induced activation of Akt in epithelial cells. Mol Biol Cell 15:3106-13

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