The goal is to determine the effects of extracellular matrix and integrins on genotoxic signaling and apoptosis. Lung endothelial cells (LEC) are significant targets of environmental and therapeutic genotoxins. LEC may undergo apoptosis, whereas their survival is promoted by integrin receptor-matrix interaction. Defining the effect of integrin activation on genotoxic cell death will lead to novel methods prevention of genotoxic lung injury. DNA strand breaks induce and activate tumor suppressor protein, p53, which promotes apoptosis by inducing pro-apoptotic genes and repressing anti-apoptotic genes. The hypothesis is that integrin activation reduces DNA strand breakage, induces DNA repair, and/or inhibits p53 expression or function.
The Specific Aims are to treat LEC from wild-type C57B1/6 and homozygous C57B1/6 p53 knockout mice with bacterial endotoxin (LPS) or BLM and determine the ability of integrin ligand matrices, GRGDSP peptide, and anti-integrin antibodies (AIB) to: 1) activate integrin receptor signaling, 2) inhibit cell death, 3) inhibit DNA breakage and stimulate DNA repair, 4) inhibit expression of function of p53. Matrices will be used as coatings on the substrate for cells. AIB and GRGDSP will be used as coatings, and in medium, separately, and in combination to assess synergistic interaction. Activation of integrin signaling (aim 1) will be correlated with protection in aims 2-4. Integrin signaling will be indicated by activation of focal adhesion kinase (FAK) and mitogen activated protein kinase (MAPK). Cytoskeletal signaling by integrins will be indicated by MAPK and will be disrupted with cytochalasin D.
In Aim 1 FAK and MAPK will be studied by immunoprecipitation and western blotting for phosphotyrosine.
In Aim 2 cell death will be characterized by cell permeability, cell detachment, nuclear morphology and DNA fragmentation.
In Aim 3 DNA breakage will be measured by in situ end labeling of DNA breaks, and repair will be determined from bromodeoxyuridine incorporation and by the ability of nuclear extracts to repair oligonucleotide substrates.
In Aim 4 p53 expression will be obtained from northern and western blots. p53 function will be indicated by gel shift of a DNA binding site oligomer, and by measuring mRNA levels of p21 (waf1), BAX and BCL-2.
| Jones, C B; McIntosh, J; Huang, H et al. (2001) Regulation of bleomycin-induced DNA breakage and chromatin structure in lung endothelial cells by integrins and poly(ADP-ribose) polymerase. Mol Pharmacol 59:69-75 |
| Hoyt, D G; Rizzo, M; Gerritsen, M E et al. (1997) Integrin activation protects pulmonary endothelial cells from the genotoxic effects of bleomycin. Am J Physiol 273:L612-7 |
