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 (H(2)O(2)) (100 to 300 microM), an inducer of apoptosis; and crocidolite asbestos (5 microg/cm(2) 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 < or = 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 G(2)/M and S phases of the cell cycle. All stimuli caused increased proportions of cells in G(2)/M at 24 h that were inhibited by PD98059 (30 microM). Increases in G(2)/M cells by H(2)O(2) and asbestos also were decreased at 48 h by the MEK1 inhibitor. In addition, PD98059 abrogated elevations in S-phase cells by EGF and H(2)O(2) 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.

Activation of extracellular signal-regulated kinases (ERK) has been associated with the advent of asbestos-associated apoptosis and proliferation in me-3 Proliferation within pulmonary interstitial cells, increases in lung hydroxyproline content, a biomarker of collagen synthesis, and morphological evidence of pulmonary fibrosis become apparent with prolonged high-dose exposure (Ն14 days) to crocidolite asbestos.

Inhalation of asbestos and oxidant-generating pollutants causes injury and compensatory proliferation of lung epithelium, but the signaling mechanisms that lead to these responses are unclear. We hypothesized that a protein kinase (PK)C␦-dependent PKD pathway was able to regulate downstream mitogen-activated protein kinases, affecting pro-and anti-apoptotic responses to asbestos. Elevated levels of phosphorylated PKD (p-PKD) were observed in distal bronchiolar epithelial cells of mice inhaling asbestos. In contrast, PKC␦؊/؊ mice showed significantly lower levels of p-PKD in lung homogenates and in situ after asbestos inhalation. In a murine lung epithelial cell line, asbestos caused significant increases in the phosphorylation of PKC␦-dependent PKD, ERK1/2, and JNK1/2/c-Jun that occurred with decreases in the BH3-only pro-apoptotic protein, Bim. Silencing of PKC␦, PKD, and use of small molecule inhibitors linked the ERK1/2 pathway to the prevention of Bimassociated apoptosis as well as the JNK1/2/c-Jun pathway to the induction of apoptosis. Our studies are the first to show that asbestos induces PKD phosphorylation in lung epithelial cells both in vivo and in vitro. PKC␦-dependent PKD phosphorylation by asbestos is causally linked to a cellular pathway that involves the phosphorylation of both ERK1/2 and JNK1/2, which play opposing roles in the apoptotic response induced by asbestos.

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