Our goal is to determine the role of the DNA damage response enzyme, poly(ADP-ribose) polymerase (PARP), in the anti- genotoxic effects of integrin cell adhesion receptor activation. Lung endothelial cells (LEC) are significant targets of environmental and therapeutic DNA-damaging agents including anticancer drugs, cigarette smoke, and reactive oxygen species. Inhibition of DNA damage and the cellular reaction to it could suppress lung injury by many agents. Two such agents are the DNA cleaving anti-tumor antibiotic, bleomycin (BLM), which causes pulmonary fibrosis, and bacterial endotoxin (LPS), a factor in Adult Respiratory Distress Syndrome. Both agents cause acute, reversible DNA breakage and pulmonary inflammation. Activation of integrin cell adhesion receptors by antibody clustering inhibits the DNA breakage in wildtype LEC, but not if PARP is inhibited or knocked out. Integrin effects on the LEC nucleus could reduce DNA damage or stimulate repair. PARP is known to modify histones and other nuclear proteins with the charged polymer, poly(ADP-ribose), altering DNA-protein interaction and enzyme activities. PARP knockout causes a defect in NfkappaB activity that suggests a potentially crucial role for PARP in inflammatory activation. Since beta1 integrin activation alters chromatin structure and histone (H1) immunostaining, it may change DNA-protein binding or conformation in a PARP-dependent manner, reducing DNA breakage and inflammatory activation of LEC. Specific elements of chromatin structure need to be investigated to explain the PARP-dependence of integrin-mediated protection and chromatin regulation. Our HYPOTHESIS is that integrin activation alters histone-DNA interactions, and that PARP regulates the histone response to integrin. We will activate beta1 integrins in wildtype and PARP knockout LEC (and inhibit PARP with 3-aminobenzamide ), and challenge with BLM or LPS to assess the dependence of integrin action on PARP.
Our SPECIFIC AIMS are to determine the effects of integrin clustering on 1) PARP activation, poly (ADP-ribosyl)ation and nuclear affinity of histone, 2) DNA breakage, and repair, and association of damaged and repairing DNA with histone, 3)Histone binding to promoter regions upregulated during EC Activation, and 4) Cell injury and lung disease. DNA-protein binding activity will be investigated in vitro by electromobility shift assays. DNA-protein interaction in the nuclear environment will be determined by chromatin immunoprecipitation from fixed cells.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL068054-03
Application #
6638812
Study Section
Lung Biology and Pathology Study Section (LBPA)
Program Officer
Gail, Dorothy
Project Start
2001-07-24
Project End
2005-06-30
Budget Start
2003-07-01
Budget End
2004-06-30
Support Year
3
Fiscal Year
2003
Total Cost
$331,875
Indirect Cost
Name
Ohio State University
Department
Type
Schools of Pharmacy
DUNS #
071650709
City
Columbus
State
OH
Country
United States
Zip Code
43210