This proposal is in response to PAR-98-087, """"""""Small grant program for K08 recipients"""""""" and is linked to NIH K08 02446. The studies outlined in this proposal will evaluate the molecular mechanisms involved in the upregulation of heme oxygenase-1 (HO-1), a redox sensitive and cytoprotective gene, by oxidized low density lipoprotein (LDLox). LDLox is implicated in the pathogenesis of atherosclerosis and progression of renal disease. Previous studies have demonstrated that exposure to LDLox results in the induction of HO-1, in both renal epithelial and aortic endothelial cells. Induction of HO-1 is considered an adaptive response in cells and tissues exposed to oxidative stress of a diverse nature. LDLox is a complex structure consisting of several chemically distinct components including fatty acid hydroperoxides, modified apoB, oxidized sterols and phospholipids. These preliminary studies are the first to identify linoleyl hydroperoxide (LAox), an oxidized C:18 containing fatty acid, as the major component of LDLox responsible for HO-1 induction. Further, such induction is via increased HO-1 gene transcription through mechanisms different from known inducers of the gene. A approximately 4.5 kb HO-1 promoter which responds to heme and cadmium, known inducers of the gene, does not contain the cis-acting elements necessary for LAox-dependent gene induction. The goals of this proposal will be to delineate the region of the HO-1 gene that controls LDLox-mediated induction and will require further experiments using chromatin structure analysis and additional promoter transfections.
Specific Aim I will identify specific locations of DNA-protein interactions that alter chromatin structure of the human HO-1 gene under basal and LAox-stimulated conditions. These studies will identify DNA sequences that could exist anywhere outside of the 4.5 kb HO-1 promoter that has already been analyzed. The results from chromatin structure analysis will aid in promoter deletion studies (Specific Aim II) using a larger (approximately 18 kb XhoI/XhoI) 5' upstream region of the HO-1 gene that has been characterized from a human P1 phage clone. These two additional specific aims are crucial preliminary data for completion of Specific Aim III of the original K08 grant application that will evaluate DNA-protein interactions by in vivo genomic footprinting to identify binding sites for cis-acting regulatory sequences on the human HO-1 gene. These studies have a potential application to both renal and cardiovascular diseases, wherein LDLox is an important mediator and may aid in developing new therapeutic interventions for modifying the progression of renal disease and atherosclerosis. This grant will also enhance research productivity, fiscal independence and career development of the applicant towards becoming an independent investigator.
