Bleomycin upregulates expression of gamma-glutamylcysteine synthetase in pulmonary artery endothelial cells.
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. gamma-Glutamylcysteine synthetase (gamma-GCS) controls the key regulatory step in GSH synthesis, and Northern blots indicate that the gamma-GCS catalytic subunit [gamma-GCS heavy chain (gamma-GCS(h))] is upregulated by bleomycin within 3 h. The promoter for human gamma-GCS(h) contains consensus sites for nuclear factor-kappaB (NF-kappaB) 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-kappaB 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-kappaB activation by bleomycin is inhibited by U-0126, but not by NAC. NAC and U-0126 both inhibit bleomycin-induced upregulation of gamma-GCS expression. These data suggest that bleomycin can activate oxidative stress response pathways and upregulate cellular GSH.
Day
Regina M
RM
Pulmonary and Critical Care Division, Tupper Research Institute, New England Medical Center, Boston, Massachusetts 02111, USA. rday@lifespan.org
Suzuki
Yuichiro J
YJ
Lum
Julie M
JM
White
Alexander C
AC
Fanburg
Barry L
BL
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001761:Bleomycin / Q000494:pharmacology*; D002417:Cattle; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D051268:GA-Binding Protein Transcription Factor; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D051265:NF-E2-Related Factor 1; D016328:NF-kappa B / Q000378:metabolism; D051264:Nuclear Respiratory Factors; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
D000964:Antimetabolites, Antineoplastic; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D051268:GA-Binding Protein Transcription Factor; D051265:NF-E2-Related Factor 1; D016328:NF-kappa B; D051264:Nuclear Respiratory Factors; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D015534:Trans-Activators; D014157:Transcription Factors; D001761:Bleomycin; D004247:DNA; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione