Induction of Heme Oxygenase 1 by Hyperoxia is Enhanced with Iron in Human Pulmonary Endothelial Cells † 1830

Abstract

High concentrations of oxygen are often a necessary and lifesaving therapy, however, these high concentrations are also toxic adding to the overall tissue injury. Early lung damage is associated with endothelial leakage with increased pulmonary edema, protein leak, and hemorrhage. Hyperoxia-induced damage is thought to be from an excess production of toxic reactive oxygen species. Heme oxygenase-1 (HO-1) is induced in response to a variety of stressess including oxidants. We and others have recently identified HO-1as a hyperoxia-inducible gene, however, the increase in response to hyperoxia is relatively low as compared to other stimulants for HO-1. In this study we evaluated the response of HO-1 in isolated cultures of human pulmonary artery endothelial cells (HPAEC) in response to hyperoxia alone and in the presence of iron. HPAEC isolated from segments of PA from heart transplant donors grown to confluence were loaded with iron by exposure to 1 μM FeSO4 and 1 μM 8-hyroxyquinolone. Iron loaded and non-loaded HPAEC were exposed to either room air or 95% oxygen for 4 or 24 h. Total RNA was isolated and evaluated by Northern analysis utilizing a radiolabeled human HO-1 cDNA. We observed a 2-3 fold increase in HO-1 mRNA levels upon exposure to hyperoxia, and 2-4 fold in the iron loaded cells as compared to control cells. Upon exposure to hyperoxia in the iron loaded cells, there was an increase in HO-1 mRNA levels of 10 to 15 fold. A 4.5 kb promoter region of the human HO-1 gene construct was generated using the promoterless human growth hormone plasmid (pOGH). Transient transfection of this construct in HPAEC confered basal expression of the promoter region. There was no significant increase in secreted human growth hormone (reporter gene expression) in response to hyperoxia, iron, or iron plus hyperoxia as compared to heme (a potent stimulus for HO-1). This study indicates that hyperoxia induction of HO-1 in human cells is enhanced by the presence of iron, and begins to determine the regulatory mechanisms of HO-1 induction in response to a hyperoxic insult in HPAEC. The greater induction with iron could result from the production of a stronger oxidant in the presence of iron or a synergistic effect on the HO-1 gene with increased expression. Funded by ALA-FL and CMNT