Inhaled asbestos exacerbates atherosclerosis in apolipoprotein E-deficient mice via CD4+ T cells.

BACKGROUND: Associations between air pollution and morbidity/mortality from cardiovascular disease are recognized in epidemiologic and clinical studies, but the mechanisms by which inhaled fibers or particles mediate the exacerbation of atherosclerosis are unclear. OBJECTIVE AND METHODS: To determine whether lung inflammation after inhalation of a well-characterized pathogenic particulate, chrysotile asbestos, is directly linked to exacerbation of atherosclerosis and the mechanisms involved, we exposed apolipoprotein E-deficient (ApoE(-/-)) mice and ApoE(-/-) mice crossed with CD4(-/-) mice to ambient air, NIEHS (National Institute of Environmental Health Sciences) reference sample of chrysotile asbestos, or fine titanium dioxide (TiO(2)), a nonpathogenic control particle, for 3, 9, or 30 days. RESULTS: ApoE(-/-) mice exposed to inhaled asbestos fibers had approximately 3-fold larger atherosclerotic lesions than did TiO(2)-exposed ApoE(-/-) mice or asbestos-exposed ApoE(-/-)/CD4(-/-) double-knockout (DKO) mice. Lung inflammation and the magnitude of lung fibrosis assessed histologically were similar in asbestos-exposed ApoE(-/-) and DKO mice. Monocyte chemoattractant protein-1 (MCP-1) levels were increased in bronchoalveolar lavage fluid and plasma, and plasma concentrations correlated with lesion size (p < 0.04) in asbestos-exposed ApoE(-/-) mice. At 9 days, activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB), transcription factors linked to inflammation and found in the promoter region of the MCP-1 gene, were increased in aortas of asbestos-exposed ApoE(-/-) but not DKO mice. CONCLUSION: Our findings show that the degree of lung inflammation and fibrosis does not correlate directly with cardiovascular effects of inhaled asbestos fibers and support a critical role of CD4(+) T cells in linking fiber-induced pulmonary signaling to consequent activation of AP-1- and NF-kappaB-regulated genes in atherogenesis.

Mesothelial cell transformation requires increased AP-1 binding activity and ERK-dependent Fra-1 expression.

Mesothelioma is a unique and insidious tumor associated historically with occupational exposure to asbestos. The transcription factor, activator protein-1 (AP-1) is a major target of asbestos-induced signaling pathways. Here, we demonstrate that asbestos-induced mesothelial cell transformation is linked to increases in AP-1 DNA binding complexes and the AP-1 component, Fra-1. AP-1 binding to DNA was increased dramatically in mesothelioma cell lines in comparison to isolated rat pleural mesothelial (RPM) cells. Elevated levels of AP-1 complexes, including significant increases in c-Jun, JunB and Fra-1, were found in asbestos-exposed RPM cells, but only Fra-1 expression was significantly increased and protracted in both asbestos-exposed RPM cells and mesothelioma cell lines. Asbestos-induced Fra-1 expression in RPM cells was dependent on stimulation of the extracellular signal-regulated kinases (ERKs 1/2). Inhibition of ERK phosphorylation or transfection with dominant-negative fra-1 constructs reversed the transformed phenotype of mesothelioma cells and anchorage-independent growth in soft agar. In summary, we demonstrate that ERK-dependent Fra-1 is elevated in AP-1 complexes in response to asbestos fibers and is critical to the transformation of mesothelial cells.

A mutant epidermal growth factor receptor targeted to lung epithelium inhibits asbestos-induced proliferation and proto-oncogene expression.

Asbestos is a ubiquitous naturally occurring fiber causing multiple cancers and fibroproliferativedisease. The mechanisms of epithelial cell hyperplasia, a hallmark of the initiation of lung cancers by asbestos, have been unclear. We demonstrate here that mice expressing a dominant-negative mutant epidermal growth factor receptor (EGFR) under the control of the human lung surfactant protein-C promoter exhibit decreased pulmonary epithelial cell proliferation without alterations in asbestos-induced inflammation. In contrast to transgene-negative littermates, inhalation of asbestos by mice expressing the mutant EGFR does not result in early and elevated expression of early response proto-oncogenes (fos/jun or activator protein 1 family members). Additionally, quantitative reverse transcriptase-PCR analysis for levels of c-jun and c-fos in bronchiolar epithelium isolated by laser capture microdissection demonstrates increases in expression of these genes in asbestos-exposed epithelial cells. Results show that the EGFR mediates both asbestos-induced proto-oncogene expression and epithelial cell proliferation, providing a rationale for modification of its phosphorylation in preventive and therapeutic approaches to lung cancers and mesothelioma.

Age affects ERK1/2 and NRF2 signaling in the regulation of GCLC expression.

We previously reported that activator protein-1 (AP-1) DNA binding activity was increased in vascular smooth muscle cells (VSMC) from old rats when exposed to high glucose or tumor necrosis factor (TNF-alpha) (Li et al., 2003. J Cell Physiol 197:418-425). We have now examined the relationship between the age-dependent activation of the ERK1/2-AP-1 pathway and modulation of constitutive gene expression of the catalytic subunit of glutamate-cysteine ligase (GCLC) in response to high glucose and TNF-alpha. GCLC mRNA levels were higher in VSMC from old rats compared to young, a pattern consistent with its protein levels. To determine whether age-related activation of ERK1/2-AP-1 signaling is responsible for the up-regulation of GCLC, the MEK inhibitors, PD98059 and U0126, were used to block ERK1/2 in VSMC from old rats. An increase in GCLC with inhibitors was observed, diminishing the likelihood of ERK1/2-AP-1 activation as the up-regulating signal for GCLC. However, the transcription factor Nrf2 was higher in nuclei and accompanied by increased Nrf2-ARE binding in VSMC from old rats. Furthermore, MEK inhibitors increased nuclear Nrf2 and Nrf2/ARE binding. These data suggest opposing effects of Nrf2 and ERK1/2 signaling in the modulation of GCLC expression in old animals.

In vitro and in vivo activation of extracellular signal-regulated kinases by coal dusts and quartz silica.

Alveolar type II epithelial cells are the main precursor cells that develop into carcinomas after inhalation of poorly soluble particles (PSP) at overload concentrations, but the mechanisms leading to initial proliferative events in these cells are unclear. In studies here, cell cycle kinetics, mitogen-activated protein kinase (MAPK) signaling events, and gene expression of activator protein-1 family members were investigated in murine alveolar type II epithelial cells (C10) or rats in vivo after exposure to several coal mine dusts (CMDs) of high or low quartz content. In contrast to results using unexposed C10 cells or cells exposed to the nonpathogenic particle glass beads, flow cytometry showed increased numbers of hypodiploid cells and cells in S phase after addition of DQ12 quartz or CMDs. Using a ribonuclease protection assay, increased mRNA levels of fos and jun family members were seen in response to DQ12 quartz and CMD with high quartz content. Increased phosphorylation of extracellular signal regulated kinases (ERKs)1/2 occurred in DQ12- and CMD-exposed cells by Western blot analysis. The use of the hydroxyl radical scavenger tetramethylthiourea blocked S-phase entry by DQ12 and CMDs as well as the phosphorylation of ERKs. Immunohistochemistry on lung sections of CMD-exposed rats showed chronic activation of phosphorylated ERKs in epithelial cells, supporting the possible role of this signal cascade in proliferation of pulmonary epithelium by PSP in vivo.

Activation of NF-kappaB-dependent gene expression by silica in lungs of luciferase reporter mice.

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)-kappaB-regulated genes encoding inflammatory cytokines. Exposure of NF-kappaB luciferase reporter mice intratracheally to silica or lipopolysaccharide (LPS), but not the nonfibrogenic particle titanium dioxide (TiO(2)), increased immunoreactivity of luciferase protein in bronchiolar epithelial cells and AMs. Ribonuclease protection assays revealed significant (P < or = 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-kappaB in situ by fibrogenic particles in pulmonary epithelial cells and AMs. Increased expression of NF-kappaB-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-kappaB in preventive and therapeutic strategies.

Ultrafine airborne particles cause increases in protooncogene expression and proliferation in alveolar epithelial cells.

Exposure to ambient particulate matter (PM) is linked to increases in respiratory morbidity and exacerbation of cardiopulmonary diseases. However, the important components of PM and their mechanisms of action in lung disease are unclear. We demonstrate the development of dose-related proliferation and apoptosis after exposure of an alveolar epithelial cell line (C10) to PM or to ultrafine carbon black (ufCB), a component of PM. Ribonuclease protection assays demonstrated that increases in mRNA levels of the early response protooncogenes c-jun, junB, fra-1, and fra-2 accompanied cell proliferation at low concentrations of PM whereas apoptotic concentrations of PM caused transient increases in expression of fos and jun family members and dose responsive increases in mRNA levels of receptor-interacting protein, Fas-associated death domain, and caspase-8. Significant increases in steady-state mRNA levels of protooncogenes and apoptosis-associated genes, TNFR-associated death domain, and Fas were also observed after exposure of epithelial cells to ufCB, but not fine carbon black or glass beads, respectively, suggesting that the ultrafine particulate component of PM is critical to its biological activity.

Age-related differences in MAP kinase activity in VSMC in response to glucose or TNF-alpha.

Aortic vascular smooth muscle cells (VSMC) were used to study the effect of age on responses to high glucose concentrations or the cytokine, tumor necrosis factor-alpha (TNF-alpha). Activator protein-1 (AP-1) binding to DNA increased more in VSMC from old versus young rats (P < 0.02) and was related to increased expression of its components, c-Fos, Fra-1, and JunD. The relationship to upstream signals, i.e., activities of mitogen-activated protein kinases (MAPK), was studied using antibodies to total and phosphorylated forms of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK) and p38. High glucose and TNF-alpha increased ERK phosphorylation more in old (P < 0.05); whereas only TNF-alpha induced JNK activation in young (P < 0.04). PD98059, a MEK inhibitor, attenuated AP-1 activation, lowered c-Fos and Fra-1 protein levels and reduced cell number and cells positive for proliferating cell nuclear antigen in old. We concluded that age differentially influenced activation of signaling pathways in VSMC exposed to high glucose or TNF-alpha. This may contribute to the increased risk for vascular disease associated with aging and diabetes mellitus (DM).