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Asbestos is a ubiquitous, naturally occurring fiber that has been linked to the development of malignant and fibrotic lung diseases. Asbestos exposure leads to apoptosis, followed by compensatory proliferation, yet many of the signaling cascades coupled to these outcomes are unclear. Because CREs ([Ca.sup.2+]/cAMP-response elements) are found in the promoters of many genes important for regulation of proliferation and apoptosis, CREB (CRE binding protein) is likely to play an important role in the development of asbestos-mediated lung injury. To explore this possibility, we tested the hypotheses that asbestos exposure leads to CREB phosphorylation in lung epithelial cells and that protein kinase A (PKA) and extracellular signal-regulated kinases 1/2 (ERK1/2) are central regulators of the CREB pathway. Persistent CREB phosphorylation was observed in lung sections from mice following inhalation of crocidolite asbestos. Exposure of C10 lung epithelial cells to crocidolite asbestos led to rapid CREB phosphorylation and apoptosis that was decreased by the inhibition of PKA or ERK1/2 using the specific inhibitors H89 and U0126, respectively. Furthermore, crocidolite asbestos selectively induced a sustained increase in MAP kinase phosphatase-1 mRNA and protein. Silencing CREB protein dramatically reduced asbestos-mediated ERK 1/2 phosphorylation, yet significantly increased the number of cells undergoing asbestos-induced apoptosis. These data reveal a novel and selective role for CREB in asbestos-mediated signaling through pathways regulated by PKA and ERK1/2, further providing evidence that CREB is an important regulator of apoptosis in asbestos-induced responses of lung epithelial cells. pulmonary fibrosis; signal transduction; small interfering RNA; mitogen-activated protein kinase phosphatase-1 doi:10.1152/ajplung.00279.2006.

Shukla, Arti, Michael Jung, Maria Stern, Naomi K. Fukagawa, Douglas J. Taatjes, Dennis Sawyer, Bennett Van Houten, and Brooke T. Mossman. Asbestos induces mitochondrial DNA damage and dysfunction linked to the development of apoptosis. Am J Physiol Lung Cell Mol Physiol 285: L1018-L1025, 2003. First published August 8, 2003; 10.1152/ajplung.00038.2003.--To test the hypothesis that asbestos-mediated cell injury is mediated through an oxidant-dependent mitochondrial pathway, isolated mesothelial cells were examined for mitochondrial DNA damage as determined by quantitative PCR. Mitochondrial DNA damage occurred at fourfold lower concentrations of crocidolite asbestos compared with concentrations required for nuclear DNA damage. DNA damage by asbestos was preceded by oxidant stress as shown by confocal scanning laser microscopy using MitoTracker Green FM and the oxidant probe Redox Sensor Red CC-1. These events were associated with dose-related decreases in steady-state mRNA levels of cytochrome c oxidase, subunit 3 (COIII), and NADH dehydrogenase 5. Subsequently, dose-dependent decreases in formazan production, an indication of mitochondrial dysfunction, increased mRNA expression of pro- and antiapoptotic genes, and increased numbers of apoptotic cells were observed in asbestos-exposed mesothelial cells. The possible contribution of mitochondrial-derived pathways to asbestos-induced apoptosis was confirmed by its significant reduction after pretreatment of cells with a caspase-9 inhibitor. Apoptosis was decreased in the presence of catalase. Last, use of HeLa cells transfected with a mitochondrial transport sequence targeting the human DNA repair enzyme 8-oxoguanine DNA glycosylase to mitochondria demonstrated that asbestos-induced apoptosis was ameliorated with increased cell survival. Studies collectively indicate that mitochondria are initial targets of asbestos-induced DNA damage and apoptosis via an oxidant-related mechanism. mitochondria; mesothelial cells; oxidants

Occupational exposure to crystalline silica is associated with the development of pulmonary inflammation and silicosis, yet how silica initiates pulmonary fibrosis and which cell types are involved are unclear. In studies here, we hypothesized that silica particles interact initially with pulmonary epithelial cells and alveolar macrophages (AMs) to cause transcriptional activation of nuclear factor (NF)-[kappa]B-regulated genes encoding inflammatory cytokines. Exposure of NF-[kappa]B luciferase reporter mice intratracheally to silica or lipopolysaccharide (LPS), but not the nonfibrogenic particle titanium dioxide (Ti[O.sub.2]), increased immunoreactivity of luciferase protein in bronchiolar epithelial cells and AMs. Ribonuclease protection assays revealed significant (P [less than or equal to] 0.05) increases in mRNA levels of inducible nitric oxide synthase, tumor necrosis factor-[alpha], macrophage inflammatory protein-2, macrophage chemotactic protein-1 (MCP-1), interferon-[gamma], interleukin (IL)-6, and IL-12 in lung homogenates of reporter mice after exposures to silica or LPS. Immunoreactivity of MCP-1 in these animals was localized to AMs and epithelial cells. These data are the first to show activation of NF-[kappa]B in situ by fibrogenic particles in pulmonary epithelial cells and AMs. Increased expression of NF-[kappa]B-related inflammatory cytokines by these cell types, which first encounter silica after inhalation, may be critical to the initiation of silica-associated lung diseases, thus providing a rationale for focusing on NF-[kappa]B in preventive and therapeutic strategies. silica; transgenic mice; nuclear factor-[kappa]B; silicosis; inflammation