Increased localization and substrate activation of protein kinase C delta in lung epithelial cells following exposure to asbestos - PubMed (original) (raw)

Increased localization and substrate activation of protein kinase C delta in lung epithelial cells following exposure to asbestos

Karen M Lounsbury et al. Am J Pathol. 2002 Jun.

Abstract

The protein kinase C (PKC) family consists of several isozymes whose substrates may be necessary for the regulation of key cellular events important in the pathogenesis of proliferative diseases. Asbestos is a carcinogen and fibroproliferative agent in lung that may cause cell signaling events through activation of PKC. Here we used a murine inhalation model of asbestos-induced inflammation and fibrosis to examine immunoreactivity of PKC delta and its substrate, phosphorylated-adducin (p-adducin), in cells of the lung. Moreover, we characterized PKC delta and p-adducin expression in a pulmonary epithelial cell line (C10) in both log versus confluent cells and in cells after mechanical wounding or crocidolite asbestos exposure. Both PKC delta and p-adducin were almost exclusively expressed in bronchiolar and alveolar type II (ATII) epithelial cells in lung sections and increased in these cell types after inhalation of asbestos by mice. Increases in membrane and nuclear localization of PKC delta were seen in log phase as compared to confluent C10 cells. Moreover, enhanced immunoreactivity of PKC delta was observed in epithelial cells expressing proliferating cell nuclear antigen (PCNA) after mechanical wounding or exposure to asbestos fibers. These studies show that activated PKC delta in pulmonary epithelial cells is a consequence of inhalation of asbestos and may be linked to the activation of cell proliferation.

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Figures

Figure 1.

Figure 1.

Increased expression of PKCδ and p-adducin in lungs treated with asbestos. PKCδ (A–C) and p-adducin (D–F) were detected in lung sections using immunoperoxidase staining of lung tissue sections. PKCδ: 30-day sham, A; 30-day asbestos exposure, B; 30-day asbestos exposure, C. p-adducin: 30-day sham, D; 30-day asbestos exposure, E; 30-day asbestos exposure, F. Note localization of PKCδ and p-adducin (arrows) in bronchiolar and alveolar epithelial cells and lesions (arrowheads) after 30 days of asbestos exposure; 30-day asbestos exposure (negative staining control using isotype control antibody), G; 30-day asbestos exposure (staining control omitting primary antibody), H. Original magnification, ×400 (A, B, D, E, G, H); magnification, ×200 (C and F).

Figure 2.

Figure 2.

PKCδ and p-adducin co-localize within bronchiolar epithelial cells after asbestos exposure. Representative images illustrating co-localization of PKCδ (green) and p-adducin (red) using immunofluorescence in lung tissue sections. A: 4-day sham exposure. B: 4-day asbestos exposure. C: 4-day asbestos exposure (staining control omitting primary antibodies). D: 30-day sham exposure. E: 30-day asbestos exposure. F: 30-day asbestos exposure (staining control omitting primary antibodies). Original magnification, ×400.

Figure 3.

Figure 3.

PKCδ is predominantly expressed in epithelial cells and not in macrophages. Representative images illustrating co-localization of PKCδ antibody (red) with MAC-3 (A, B) or CytoKeratin7 (C, D) antibodies (green) using immunofluorescence in lung tissue sections from sham control (A, C) or 30-day crocidolite asbestos exposed animals (B, D). Arrows in B show MAC-3 staining macrophages with no PKCδ reactivity Arrows in D show co-localization of PKCδ in bronchiolar epithelial cells stained with Cytokeratin7. Original magnification, ×400.

Figure 4.

Figure 4.

PKCδ and p-adducin are localized to the membrane and nuclei of dividing cells or confluent cells treated with PDBu. C10 cells were examined at low density (Log Phase), confluent density (Confluent) and after treatment with 100 nmol/L PDBu for 10 minutes (PDBu). A–I: Localization of proteins by immunofluorescence using antibodies to PKCδ (A–C); γ-adducin (D–F); and p-adducin (G–I). Original magnification, ×600. J: Localization of PKCδ in subcellular fractions of C10 cells by Western blot. Tot, total extract; M, membrane fraction; C, cytosolic fraction; N, nuclear fraction.

Figure 5.

Figure 5.

PKCδ and p-adducin are localized to the membrane and nuclei of cells migrating into a wound. Confluent C10 monolayers were wounded by a rubber policeman then allowed to respond for 24 hours. Cells were then fixed and immunostained as in Figure 4 ▶ using antibodies recognizing PKCδ (A), p-adducin (B), and γ-adducin (C). D: Staining control omitting primary antibody. Original magnification, ×400.

Figure 6.

Figure 6.

Cells exhibiting increased translocalization of PKCδ after wounding or asbestos exposure also exhibit an increase in PCNA. C10 cells were wounded as in Figure 5 ▶ (D–F) or exposed to asbestos (5 μg/cm2-area dish) for 24 hours (G–I). Cells were then co-stained by immunofluorescence using antibodies recognizing PKCδ (green) and PCNA (magenta). Asbestos fibers are shown in red. C and F represent overlay image of PKCδ and PCNA for wounded and asbestos exposed cells respectively. Original magnification, ×400.

Figure 7.

Figure 7.

Increased localization of PKCδ and p-adducin in focal regions of asbestos fiber deposition. Control C10 cells (A) and cells exposed to 5 μg/cm2 area dish of asbestos for 24 hours (B, C) were examined by immunofluorescence (green) for the localization of PKCδ (A, B) and p-adducin (C). Asbestos fibers are shown in red. Original magnification, ×400.

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