Advisory Committee Chair
Anupam Agarwal
Advisory Committee Members
Shannon M Bailey
Bruce A Freeman
Rakesh Patel
Thomas M Ryan
C Roger White
Document Type
Dissertation
Date of Award
2009
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
Nitro-fatty acids (NO-FA) exert anti-inflammatory effects in the vasculature. Heme oxygenase-1 (HO-1) degrades heme into iron, biliverdin, and carbon monoxide and is up-regulated as an adaptive and protective response to inflammatory processes. This dissertation examined whether nitro-linoleic acid (LNO2) induced HO-1 gene expression and evaluated the molecular mechanism of induction in human aortic endothelial (HAEC), human embryonic kidney 293 (HEK 293) and mouse embryonic fibroblast (MEF) cells. LNO2 induced HO-1 mRNA and protein in HAEC at the level of transcription. NO scavenging by carboxy-PTIO partially reversed LNO2–mediated induction of HO-1, suggesting that LNO2 acts via NO-dependent and NO-independent mechanisms of HO-1 induction. Chemical or antagonist inhibition of PPARγ signaling did not attenuate induction of HO-1 protein, suggesting that LNO2 induces HO-1 by PPARγ-independent mechanisms. LNO2 binds to electrophile responsive proteins, including Keap-1, an inhibitor of Nrf2 signaling. Nrf2 +/+ and Nrf2 -/- MEF treated with LNO2 exhibited HO-1 protein induction in a dose dependent manner, suggesting that LNO2 induces HO-1 in the absence of Nrf2. LNO2 activated a -4.5Kb human HO-1 promoter reporter construct in HAEC, while a -4.0Kb construct with deletion of 500bp from the 5’ region was unresponsive. Double mutation of the cyclic AMP response element (CRE) and the NF-E2/AP-1 element in the -4.5Kb region abrogated LNO2 specific HO-1 promoter activation. A triple mutation of CRE, NF-E2/AP-1 and the proximal E-box abolished basal HO-1 promoter activity, showing that the CRE, NFE2/ AP-1, and E-box elements synergize for maximal activation of HO-1. Nuclear extracts from HAEC treated with LNO2 and co-incubated with an anti-CREB-1 antibody supershifted human HO-1 CRE and E-box oligonucleotides, suggesting that CREB binds to the CRE and the E-box. Dominant-negative CREB-1 expression inhibited HO-1 promoter activation in HEK 293 cells. Chromatin immunoprecipitation of the CRE and proximal E-box regions by an anti-CREB-1 antibody was enhanced in HAEC treated with LNO2. This dissertation elucidates the molecular control of human HO-1 by LNO2, which requires CRE, NF-E2/AP-1 and E-box sequence interaction within the HO-1 promoter, functions in the absence of Nrf2, and involves CREB-1. HO-1 induction by NO-FA represents a pathway for mediating anti-inflammatory responses in vascular disease.
Recommended Citation
Wright, Macienne M., "Regulation of the Human Heme Oxygenase-1 Gene By Nitro-Linoleic Acid" (2009). All ETDs from UAB. 95.
https://digitalcommons.library.uab.edu/etd-collection/95