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Am. J. Respir. Cell Mol. Biol., Volume 24, Number 4, April, 2001 405-413

Different Accumulation of Activated Extracellular Signal-Regulated Kinases (ERK 1/2) and Role in Cell-Cycle Alterations by Epidermal Growth Factor, Hydrogen Peroxide, or Asbestos in Pulmonary Epithelial Cells

Sylke Buder-Hoffmann, Cathy Palmer, Pamela Vacek, Douglas Taatjes, and Brooke Mossman

Departments of Pathology and Medical Biostatistics, University of Vermont College of Medicine, Burlington, Vermont

The extracellular signal-regulated kinase (ERK) pathway is induced by cytokines and oxidative stress. In this study we examined the patterns of localization of phosphorylated ERK proteins in relationship to subsequent phenotypic responses by the mitogenic agent epidermal growth factor (EGF) (5 ng/ ml); hydrogen peroxide (H2O2) (100 to 300 µM), an inducer of apoptosis; and crocidolite asbestos (5 µg/cm2 dish) in a nontransformed murine alveolar type II epithelial cell line (C10). Laser scanning cytometry and flow cytometry were used to determine: (1) whether expression of phosphorylated ERKs was cell cycle-related; and (2) whether cell-cycle alterations by agents could be modified after addition of the mitogen-activated protein kinase/ERK kinase (MEK) 1 inhibitor PD98059. In contrast to other stimuli which induced transient increases in phosphorylated ERKs, asbestos caused fiber-associated localization of phosphorylated ERKs that were elevated from 1 to 24 h (P =< 0.05), and striking apoptosis followed by increased numbers of cells in the S phase at 72 h. In both control and asbestos-exposed cells, the percentage of phosphorylated ERK-positive cells was greatest in cells in the G2/M and S phases of the cell cycle. All stimuli caused increased proportions of cells in G2/M at 24 h that were inhibited by PD98059 (30 µM). Increases in G2/M cells by H2O2 and asbestos also were decreased at 48 h by the MEK1 inhibitor. In addition, PD98059 abrogated elevations in S-phase cells by EGF and H2O2 at 24 h and by asbestos at 72 h. Our results suggest that ERKs mediate cell-cycle alterations during the development of epithelial cell apoptosis or proliferation by diverse ERK stimuli.

Abbreviations: bovine serum albumin, BSA; confocal scanning laser microscopy, CSLM; epidermal growth factor, EGF; EGF receptor, EGFR; extracellular signal-regulated kinase, ERK; hydrogen peroxide, H2O2; immunoglobulin, Ig; laser scanning cytometry, LSC; mitogen-activated protein kinase, MAPK; MAPK/ERK kinase, MEK; phosphate-buffered saline, PBS; proliferating cell nuclear antigen, PCNA; phosphorylated ERK, pERK; propidium iodide, PI; room temperature, RT; standard error of the mean, SEM; ultraviolet B, UVB.




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Copyright © 2001 American Thoracic Society.