Gene expression profiles reveal increased mClca3 (Gob5) expression and mucin production in a murine model of asbestos-induced fibrogenesis

Abstract

To elucidate genes important in development or repair of asbestos-induced lung diseases, gene expression was examined in mice after inhalation of chrysotile asbestos for 3, 9, and 40 days. We identified changes in the expression of genes linked to proliferation (cyclin B2, CDC20, and CDC28 protein kinase regulatory subunit 2), inflammation (CCL9, CCL6, complement component 1, chitinase3-like 3, TNF superfamily member 10, and IL-1B), and matrix remodeling (MMP12, MMP3, integrin alphaX, and cathepsins K, Z, B, and S). The most highly induced gene at all time points was mclca3 (gob5), a putative calcium-activated chloride channel involved in the regulation of mucus production and/or secretion. Using histochemistry, we demonstrated accumulation of mucus and increased mClca3 protein in the bronchiolar epithelium of asbestos-exposed mice at all time points but peaking at 9 days. Cytokine levels (interleukin-1beta, interleukin-4, interleukin-6) in bronchoalveolar lavage fluid also increased at 9 days, suggesting Th2-mediated immunity may play a role in asbestos-induced mucus production. In contrast, levels of cathepsin K, a potent elastase, increased between 3 and 40 days at both the mRNA and protein levels, localizing primarily in CD45-positive leukocytes and interstitial cells. Identification of genes involved in lung injury and remodeling after asbestos exposure could aid in defining mechanisms of airborne particulate-induced disease and in developing therapeutic strategies.

Figure 1

Bronchiolar epithelial hyperplasia, peribronchiolar fibrosis, and proliferation in mice exposed to chrysotile asbestos for 3, 9, and 40 days. A, top: H&E-stained lung sections showing hyperplasia of the epithelium at 3 and 9 days. In contrast to sham animals, the epithelium is more columnar and crowded. Middle: Lung sections showing peribronchiolar fibrosis at 40 days by increased staining of collagen with Masson’s trichrome stain. Bottom: Ki67-stained lung sections showing peak proliferation of bronchiolar epithelial cells at 3 days. B: Quantitation of Ki-67 in bronchiolar epithelial cells at 3, 9, and 40 days. All values are expressed as percent cells positive for Ki-67 labeling. *P ≤ 0.05 versus sham. Scale bar, 50 μmol/L.

Figure 2

A: Total number of significant (P < 0.05) gene changes ≥2× compared to air-exposed animals obtained in whole lung tissue by microarray analysis at 3, 9, and 40 days of chrysotile asbestos exposure. B: Total number of significant (P < 0.05) gene changes ≥2× in whole lung tissue that were categorized by biological process.

Figure 3

Individual profiles of gene changes obtained by microarray within various subgroups based on biological process in whole lung tissue of mice exposed to air (white bars), TiO₂ for 3 days (black bars), or chrysotile asbestos for 3, 9, and 40 days (cross-hatched bars). All values are expressed as fold change in comparison to air-exposed mice. *P < 0.05 and ≥2×. A: Proliferation; B: immune system; C: protein metabolism/matrix remodeling; D: signal transduction; E: oxidative stress.

Figure 4

Validation of microarray gene expression data for mclca3 (A), cathepsin K (B), and IL-1β (C) by QRT-PCR (mclca3 and cathepsin K) and RPA (IL-1β) analysis. All values were normalized to HPRT or L32 housekeeping genes and expressed as fold change compared to air controls. *P < 0.05 and ≥2×.

Figure 5

Cathepsin K is localized to the interstitial region of the lung and within CD45-positive cells. Lung sections of animals exposed to air and chrysotile asbestos for 3, 9, and 40 days were triple labeled with CCSP (blue), CD45 (green), and cathepsin K (red) using immunofluorescence and imaged by confocal scanning laser microscopy. Cathepsin K co-localizes with some CD45-positive cells (arrow), but only reacts minimally with the bronchiolar epithelium (CCSP). There is a significant increase in the quantity of cathepsin K immunostaining from 3 to 40 days in comparison to the air controls. Scale bar, 50 μmol/L.

Figure 6

Immunohistochemistry showing that asbestos induces mClca3 and mucus production in the bronchiolar epithelium in nonproliferating cells. Lung sections stained with Alcian blue (top) and an antibody to mClca3 (middle) showing increased mucus production and mClca3 in the bronchioles of mice exposed to asbestos for 3, 9, and 40 days, with peak levels observed at 9 days. Bottom: Dual labeling of Ki67 and mucus with Alcian blue/PAS showing the majority of mucus production in nonproliferating cells of the bronchiolar epithelium at all time points. Scale bars: 50 μmol/L (top, bottom); 100 μmol/L (middle).

Figure 7

Quantitation of the number of mucin-positive bronchioles (A) and the percentage of mucin-positive bronchiolar epithelial cells (B). All values are expressed as percentage of total. C: Scatter plot showing bronchiolar epithelial cells that are positive for Alcian blue/PAS and Ki-67. C, Sham control. Hatched bars represent asbestos-exposed groups. Each point represents the mean values for each animal (n = 3 per group per time point). *P < 0.05 compared to sham control.

Figure 8

Induction of cytokines in BALF of mice exposed to asbestos. Levels of the cytokines indicated above were measured in BALF of mice exposed to air (white bars) or asbestos (cross-hatched bars) for 9 days. Each bar represents the mean of six animals. *P < 0.05 compared to sham control.