Role of extracellular signal-regulated protein kinases in apoptosis by asbestos and H2O2.
Jimenez-LA; Zanella-C; Fung-H; Janssen-YM; Vacek-P; Charland-C; Goldberg-J; Mossman-BT
Am J Physiol, Lung Cell Mol Physiol 1997 Nov; 273(5):L1012-L1035
The role of extracellular signal regulated protein kinases in cellular apoptosis induced by asbestos (1332214) and hydrogen- peroxide (7722841) was examined. Pleural mesothelial (RPM) cells harvested from Fischer-344-rats were incubated with 0 to 300 micromolar (microM) hydrogen-peroxide for 30 minutes (min) to 4 hours (hr) or to 5 micrograms per square centimeter (microg/cm2) crocidolite (12001284) for up to 72hr. The extent of activation of extracellular signal regulated protein-kinase-2 (ERK2) and c-jun-NH2- terminal-protein-kinase-1 (JNK1) was determined by immunoprecipitation assays. Hydrogen-peroxide caused a dose related increase in ERK2 activity. The 300microM dose significantly increased JNK1 activity. Maximum induction of ERK2 and JNK1 activity occurred at 30min and 1hr, respectively. Crocidolite significantly increased ERK2 activity at all time points, but had no effect on JNK1 activity. RPM cells were preincubated with 0, 50, or 500 units per milliliter (U/ml) catalase and then treated with 0 or 200microM hydrogen-peroxide or 5microg/cm2 crocidolite for 2 and 24hr, respectively. The extent of induction of ERK2 activity was determined. Similar experiments were performed with crocidolite fibers that had been pretreated with 2 millimolar (mM) desferrioxamine or ferrozine to chelate the iron or were untreated. Catalase inhibited the stimulatory effects of hydrogen-peroxide and crocidolite on ERK2 activity. Pretreatment with ferrozine, but not desferrioxamine, inhibited the level of ERK2 activity relative to that induced by untreated crocidolite fibers. RPM cells were pretreated for 18hr with 0, 1, or 10mMM N-acetylcysteine (616911) (NAC) followed by 2microg/cm2 crocidolite. The effects on ERK2 activity were determined. NAC at 10mM countered the stimulatory effect of crocidolite on ERK2 activity. RPM cells were pretreated with 0 or 10mM NAC or 35microM PD-98059, a synthetic inhibitor of the ERK pathway, followed by treatment with 5microg/cm2 crocidolite. Induction of apoptosis was assessed by the 4',6-diamidino-2- phenylindole staining assay. Crocidolite caused a three fold increase in apoptosis over the control value. NAC and PD-98059 both significantly reduced the level of crocidolite induced apoptosis. The authors conclude that activation of the ERK pathway plays a role in asbestos induced apoptosis. Distinct cell signaling cascades may be important in phenotypic responses induced by oxidative stresses.
NIOSH-Publication; NIOSH-Grant; Pulmonary-system-disorders; Enzyme-activity; Proteins; Asbestos-fibers; Peroxides; Lung-cells; In-vitro-studies; Oxidative-processes; Cell-damage; Bioactivation; Cellular-function; Dose-response;
Author Keywords: protein phosphorylation; oxidants; cell signaling
Luis Albert Jimenez, Departments of Pathology, Biostatistics, and Medicine, College of Medicine, University of Vermont, Burlington, Vermont 05405
1332-21-4; 7722-84-1; 12001-28-4; 616-91-1
American Journal of Physiology: Lung Cellular and Molecular Physiology
Pathology University of Vermont Medical Alumni Bldg a 249 Burlington, VT 05405-0068