Year of Award

2007

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Toxicology

Department or School/College

Department of Biomedical and Pharmaceutical Sciences

Committee Chair

J. Douglas Coffin

Committee Co-chair

Stephen M. Black

Commitee Members

David Poulsen, Mark Pershouse, J. B. Alexander Ross

Keywords

molecular biology

Abstract

Persistent pulmonary hypertension of the newborn (PPHN) is a very serious disease affecting nearly 5 in 1000 newborns each year. The development of PPHN has been linked to a decrease in the activity and expression of endothelial nitric oxide synthase (eNOS). Thus, it is critical to understand the mechanisms by which eNOS is regulated to identify new pathways and novel therapies for PPHN. eNOS is dynamically regulated at the transcriptional, post-transcriptional, post-translational, and developmental levels, however mechanisms of this regulation are unresolved. Our data indicates that increases in eNOS expression and activity correlate with increased cellular levels of labile zinc (Zn). In addition, our analysis of the eNOS promoter indicated the presence of a putative heavy metal response element (HRE) in the 5'-flanking sequence. We hypothesized that nitric oxide (NO) may be regulating eNOS expression and activity indirectly through its ability to regulate cellular levels of free Zn. Further, we hypothesized that Zn exerts an effect on eNOS at the transcriptional and post-translational levels. Our results suggested that both NO and hydrogen peroxide (H2O2) activate eNOS in a Zn-dependent manner by liberating cellular stores of Zn and activating the heavy metal-responsive transcription factor 1, MTF-1, which then would bind to the eNOS 5'-regulatory MRE. We found that basal zinc levels in endothelial cells (ECs) helped maintain eNOS promoter activity and total protein levels, but did not affect eNOS dimer levels. However, supplementing ECs with exogenous Zn increased eNOS total and dimer protein levels and activity, mostly through MTF-1. At higher, non-toxic doses of Zn, reactive oxygen species (ROS) production was much higher. eNOS was inhibited by high ROS levels through enzyme fragmentation. Lastly, an eNOS enzyme cofactor, tetrahydrobiopterin (BH4), was found to help maintain eNOS protein due to its high antioxidant potential. In conclusion, eNOS appears to be regulated by NO and ROS through an increase in intracellular Zn, thus activating MTF-1, which binds to the 5' flanking region of the eNOS promoter.

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© Copyright 2007 Jason Michael Wilham