Bleomycin upregulates expression of gamma -glutamylcysteine synthetase in pulmonary artery endothelial cells

• Published in: American journal of physiology. Lung cellular and molecular physiology 2002-06, Vol.282 (6), p.1349-L1357
• Main Authors: Day, Regina M, Suzuki, Yuichiro j, Lum, Julie M, White, Alexander C, Fanburg, Barry L
• Format: Article
• Language: English
• Subjects: Animals; Antimetabolites, Antineoplastic - pharmacology; Bleomycin - pharmacology; Cattle; Cells, Cultured; DNA - metabolism; DNA-Binding Proteins - metabolism; Electrophoretic Mobility Shift Assay; Endothelium, Vascular - cytology; Endothelium, Vascular - drug effects; Endothelium, Vascular - enzymology; Enzyme Inhibitors - pharmacology; Free Radical Scavengers - pharmacology; GA-Binding Protein Transcription Factor; Glutamate-Cysteine Ligase - genetics; Glutamate-Cysteine Ligase - metabolism; Glutathione - metabolism; NF-E2-Related Factor 1; NF-kappa B - metabolism; Nuclear Respiratory Factors; Proto-Oncogene Proteins c-fos - metabolism; Proto-Oncogene Proteins c-jun - metabolism; Pulmonary Artery - cytology; Pulmonary Artery - drug effects; Pulmonary Artery - enzymology; Reactive Oxygen Species - antagonists & inhibitors; Reactive Oxygen Species - metabolism; RNA, Messenger - metabolism; Trans-Activators - metabolism; Transcription Factors - metabolism; Up-Regulation - drug effects
• ISSN: 1040-0605; 1522-1504
• DOI: 10.1152/ajplung.00338.2001

1  Pulmonary and Critical Care Division, Tupper Research Institute, New England Medical Center; and 2  Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts 02111 The chemotherapeutic agent bleomycin induces pulmonary fibrosis through the generation of reactive oxygen species (ROS), which are thought to contribute to cellular damage and pulmonary injury. We hypothesized that bleomycin activates oxidative stress response pathways and regulates cellular glutathione (GSH). Bovine pulmonary artery endothelial cells exposed to bleomycin exhibit growth arrest and increased cellular GSH content. -Glutamylcysteine synthetase ( -GCS) controls the key regulatory step in GSH synthesis, and Northern blots indicate that the -GCS catalytic subunit [ -GCS heavy chain ( -GCS h )] is upregulated by bleomycin within 3 h. The promoter for human -GCS h contains consensus sites for nuclear factor- B (NF- B) and the antioxidant response element (ARE), both of which are activated in response to oxidative stress. Electrophoretic mobility shift assays show that bleomycin activates the transcription factor NF- B as well as the ARE-binding factors Nrf-1 and -2.   Nrf-1 and -2 activation by bleomycin is inhibited by the ROS quenching agent N -acetylcysteine (NAC), but not by U-0126, a MEK1/2 inhibitor that blocks bleomycin-induced MAPK activation. In contrast, NF- B activation by bleomycin is inhibited by U-0126, but not by NAC. NAC and U-0126 both inhibit bleomycin-induced upregulation of -GCS expression. These data suggest that bleomycin can activate oxidative stress response pathways and upregulate cellular GSH. reactive oxygen species; Nrf-1 and -2; nuclear factor- B; antioxidant response element; mitogen-activated protein kinase