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in Lung Epithelial Cells following Exposure to Asbestos
From the Departments of Pharmacology*and Pathology,University of Vermont, Burlington, Vermont
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Abstract |
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and its substrate, phosphorylated-adducin (p-adducin), in cells of the lung. Moreover, we characterized PKC 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 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 were seen in log phase as compared to confluent C10 cells. Moreover, enhanced immunoreactivity of PKC 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 in pulmonary epithelial cells is a consequence of inhalation of asbestos and may be linked to the activation of cell proliferation.
Our laboratory has shown in pulmonary epithelial and mesothelial cells in vitro that asbestos activates protein kinase C (PKC) and that down-regulation or inhibition of PKC prevents asbestos-induced proto-oncogene expression.5,6 We have also identified the mitogen-activated protein kinase (MAPK) cascade as a signaling pathway governing initial cell injury and proliferation by asbestos.7-9 Recently, we have developed a murine model of asbestosis where we demonstrated increased immunoreactivity of activated (phosphorylated), extracellular signal-regulated kinases (ERKs1/2) in bronchiolar and ATII cells at sites of asbestos fiber deposition and development of fibrotic lesions.10 These studies suggest that signaling pathways such as PKC and MAPK are initiated in epithelial cells that first contact asbestos fibers after inhalation. Moreover, initial signaling events may be integral to subsequent repair processes or the pathogenesis of asbestos-related pulmonary diseases.
Several studies show that PKCs modulate MAPK pathways in a variety of cell types.11-13
Members of the PKC family, ubiquitous Ser/Thr kinases, regulate a wide variety of normal and pathological processes. However, identifying the mechanisms of PKC signaling and their repercussions is complicated by the large numbers of PKC isoforms expressed in individual cell types, the lack of substrate specificity among the isoforms in in vitro assays, and the large numbers of proteins that can serve as substrates in vitro.14
A group of PKC substrates named STICKs for substrates that interact with C kinase has been cloned based on their ability to bind PKC. Antisera were raised against the individual phosphopeptides corresponding to phosphorylation sequences, affinity purified, and shown to preferentially react with phosphorylated compared to unphosphorylated peptides and proteins.15,16
Adducin is a STICK that facilitates the binding of actin to spectrin to form the cortical membrane cytoskeleton, a primary location for transducing extracellular signals to the cytoplasm. Preliminary studies have revealed that the phosphorylation-state selective antibody, pSer660-adducin or p-adducin, is preferentially phosphorylated by PKC and localizes to focal adhesions actively involved in cytoskeletal remodeling after wounding of REF2 fibroblasts in vitro.14,17
Little is known about the PKC isoforms and substrates expressed in lung and alterations in their expression after physiologically relevant injury to pulmonary epithelial cells. In research reported here, we focused on PKC and p-adducin because of their links to the development of injury, proliferation, and migration.18-21
We hypothesized that they would be expressed in bronchiolar and ATII epithelial cells after exposure to asbestos fibers. Moreover, we used a non-transformed murine alveolar epithelial cell line (C10)22
to determine whether increased expression of PKC and p-adducin was observed at sites of asbestos fiber deposition or after wounding. Results show for the first time that immunoreactive PKC and p-adducin are expressed in pulmonary epithelial cells exposed to asbestos and localized to cell membranes of proliferating and migrating cells after wounding or damage by asbestos fibers. These studies implicate activation of PKC as a signaling pathway important in wounding of epithelium by asbestos fibers and the development of subsequent fibrogenesis and carcinogenesis.
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Materials and Methods |
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C57/BL6 mice (8 to 12 weeks of age) were exposed to ambient air or 7 mg/m3 air (6 hours/day; 5 days/wk) of the National Institute of Environmental Health Sciences (NIEHS) reference sample of crocidolite asbestos generated as described previously.23 Groups of mice (n = 6–8 per group per time point) were euthanized after 4 and 30 days of exposure, time points corresponding to initial increases in epithelial proliferation and the development of fibrotic lesions respectively. Mice were administered a lethal dose of sodium pentobarbital (Abbott Laboratories, Chicago, IL) before chest cavities were opened, a polyurethane catheter was inserted into the trachea, and the lung was instilled with phosphate-buffered saline (PBS) at a pressure of 25 cm water. Left and right lung lobes were separated by suturing and processed for immunohistochemistry and immunofluorescence, respectively.
Immunohistochemistry
Reagents were obtained from Sigma Chemical Co., St. Louis, MO, unless otherwise specified. Left lung lobes from sham- and asbestos-exposed animals were placed in a tissue cassette overnight in 4% paraformaldehyde before embedding in paraffin blocks. Lung sections were cut at a thickness of 4 µm and some sections were stained by the Masson’s trichrome technique.23
For immunohistochemistry, lung sections were deparaffinized in xylene 3 x 5 minutes, rehydrated through graded ethanols, and equilibrated in PBS. Antigen was retrieved by boiling slides in 0.1 mol/L citrate buffer in distilled water (pH 6.0) three x 3 minutes. Endogenous peroxidase was dampened by treatment with 3% hydrogen peroxide in methanol for 20 minutes, followed by a 10-minute wash in distilled water. Sections were encircled with a hydrophobic film (PAP PEN, Electron Microscopy Sciences, Ft. Washington, PA), and nonspecific protein was blocked with 2% normal goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS for 2 hours at room temperature (RT). Excess buffer was absorbed before overlaying with primary rabbit polyclonal antibodies (PKC, 1 µg/ml; pSer660-adducin, 1 µg/ml; clone 35H-
-adducin, 1 µg/ml23
) in PBS containing 2% normal goat serum overnight at 4°C in a humid chamber. Immunoreactivity was then detected using the anti-rabbit IgG Vectastain ABC Elite kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine (DAB) as a chromogen according to the manufacturer’s protocols. Following color development, sections were rinsed in distilled water, counterstained with hematoxylin, dehydrated, cleared, and mounted with VectaMount (Vector Laboratories).
Preparation of Lung Sections for Immunofluorescence
After suturing, right lung lobes from sham- and asbestos-treated animals were placed in OCT embedding compound (Tissue Tek, Torrance, CA) and frozen at -80°C before cryostat sectioning at 7–8 µm onto Superfrost +/+ slides. Slides were placed in fresh 4% paraformaldehyde in PBS for 30 minutes at RT. After washing in PBS, sections were permeabilized in -20°C methanol for 3 minutes at RT followed by two 10-minute washes in PBS. Nonspecific antibody activity was then blocked with 5% normal goat serum (Vectastain ABC Elite kit, Vector Laboratories) in PBS for 1 hour in a humid chamber at RT. Slides were then analyzed by immunofluorescence as described below.
Co-Localization of PKC with p-Adducin by Immunofluorescence
To simultaneously detect PKC and p-adducin, mouse anti-PKC antibody (Transduction Laboratories, Franklin Lakes, NJ; 3 µg/ml) and rabbit anti-pSer660-adducin antibody (1 µg/ml) were combined and applied to pre-blocked slides overnight at 4°C in a humid chamber. Detection of p-adducin was accomplished by incubating with rabbit biotinylated IgG (Vectastain ABC Elite kit, Vector Laboratories) for 1 hour at RT followed by incubation with strepavidin-Alexa 568 (Molecular Probes, Eugene, OR) at 1:200 dilution in 1% BSA/PBS for 1 hour at RT. Secondary antibody for PKC (allophycocyanin-goat-anti-mouse IgG, Molecular Probes), diluted 1:100 in 1% BSA/PBS, was applied to the slides together with the strepavidin-Alexa 568 antibody. After the incubations, slides were washed in PBS, mounted with AquaPolyMount (Polysciences, Inc, Warrington, PA) and stored at 4°C until future examination by confocal laser scanning microscopy (BioRad MRC1024ES, Hercules, CA).
Co-Localization of PKC with MAC-3 and CytoKeratin7 Antibodies by Immunofluorescence
Pre-blocked slides were incubated in blocking reagent from a mouse-to-mouse kit (Vector Laboratories) for 1 hour. Primary antibodies were mixed together and applied on sections overnight at 4°C in a humid chamber. Rabbit polyclonal PKC antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used at 3 µg/ml, MAC-3 antibody (Pharmigen, San Diego, CA) was used at 20 µg/ml, and CytoKeratin7 antibody (Dako, Carpinteria, CA) was used at 1:250 dilution. PKC antibody was detected using biotinylated anti-rabbit IgG (Vectastain ABC Elite kit, Vector Laboratories) for 1 hour at RT followed by strepavidin-Alexa 568 at 1:200 dilution in 1% BSA/PBS for 1 hour at RT. MAC-3 and CytoKeratin7 were detected using allophycocyanin-goat-anti-mouse IgG diluted 1:100 in 1% BSA/PBS for 1 hour at RT. After the incubations, slides were washed in PBS, mounted with AquaPolyMount and stored at 4°C until future examination by confocal laser scanning microscopy.
Cell Culture and Treatment Conditions
A contact-inhibited, non-transformed murine alveolar type II epithelial cell line (C10) was used for in vitro studies.22 C10 cells were propagated in CMRL 1066 medium supplemented with L-glutamine, penicillin/streptomycin, and 10% fetal bovine serum (GIBCO BRL, Rockville, MD). Cells were grown on glass coverslips for all of the experiments. To model epithelial wounding in vitro, cells were grown to confluence, and a wound was created by scraping a section of the glass coverslip with a rubber policeman. The medium was removed, and fresh complete medium was added. Cells were then allowed to repopulate the wounded area for 18 hours. In additional experiments, log phase cells were examined at 24 hours after plating. For all other groups, cells were grown to confluence, complete medium was removed, and serum-free medium was added 24 hours before exposure to asbestos or phorbol dibutyrate (PDBu). Crocidolite asbestos fibers (NIEHS reference sample) were suspended in Hanks’ balanced salt solution (HBSS) (1 mg/ml), triturated 10 times through a 22-gauge needle to obtain a homogeneous suspension, and added directly to the medium at a final concentration of 5 µg/cm2-area culture dish previously shown to induce apoptosis in C10 cells.8 PDBu (Sigma, St. Louis, MO) was added for 10 minutes at 37°C at a final concentration of 100 ng/ml. Control cultures received medium without agents and were treated identically. All experiments were performed in triplicate.
Localization of PKC and Adducins in C10 Cells by Immunofluorescence
After cell monolayers grown on glass coverslips were exposed to agents as described above, culture dishes were placed on ice, the medium was aspirated, and the cells were washed twice with PBS. Cells were fixed in 3.7% paraformaldehyde in PBS for 5 minutes at RT, then washed in PBS and permeabilized in -20°C acetone for 3 minutes at RT. Cells were washed in PBS, then incubated with a blocking solution containing 1% BSA/PBS for 1 hour at RT. After aspiration of blocking solution, primary antibody (rabbit polyclonal PKC antibody (Santa Cruz; 3 µg/ml) diluted in 1% BSA/PBS, was added and incubated for 1 hour at RT. Cells then were washed in PBS and secondary antibody (FITC goat-anti-rabbit IgG, Jackson ImmunoResearch Laboratories; 1:200) was applied. After washing in PBS, coverslips were mounted onto slides with AquaPolyMount. For each sample, confocal images were collected in fluorescence modes, followed by electronic merging of the images.
Detection of PKC in Subcellular Fractions of C10 Cells by Western Blot
After treatment, cells in 100-mm dishes (2 plates per condition) were rinsed sequentially with ice-cold PBS and hypotonic lysis buffer (HLB; 25 mmol/L Tris (pH 8), 2 mmol/L MgCl2, 5 mmol/L KCl, 10 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride) before collection in 200 µl of HLB. Samples were kept at 4°C for the remainder of the procedure. Cells were lysed by homogenization then centrifuged at 1000 x g for 5 minutes to collect the nuclear pellet fraction. The supernatant fraction was reserved, and the nuclear pellet fraction was washed with HLB then resuspended in 100 µl of HLB. To separate membrane from cytosol, the supernatant fraction was centrifuged at 15,000 x g for 30 minutes. The supernatant served as the cytosol fraction. The resulting membrane pellet fraction was washed with HLB then resuspended in 100 µl of HLB. All fractions were triturated through a 23-gauge needle, and equal proportions were combined with Laemmli sample buffer, boiled, and electrophoresed through a 7.5% SDS polyacrylamide gel. Separated proteins were electroblotted onto nitrocellulose and Western blotting was performed as previously described8
using anti-PKC primary antibody (Santa Cruz; 1:100 dilution) and horseradish peroxidase-conjugated anti-rabbit secondary antibody (Vector Laboratories; 1:5000 dilution). Antibody binding was visualized by enhanced chemiluminescence according to the manufacturer’s protocol (Kirkegaard and Perry Laboratories, Gaithersburg, MD).
Detection of Proliferating Cell Nuclear Antigen by Immunofluorescence
An antibody against proliferating cell nuclear antigen (PCNA) was used to identify cells undergoing DNA synthesis. Cell monolayers on coverslips were fixed in 100% methanol for 1 hour on ice, washed in PBS, and incubated in 0.1% Tween-20 in PBS for 30 minutes at RT. After incubation in blocking solution (2% dry milk, 0.1% Tween-20 in PBS) for 30 minutes at RT, cells were incubated with a mix of mouse anti-PCNA (Pharmagen; 1:1000) and rabbit anti-PKC (Santa Cruz; 3 µg/ml) for 1 hour at RT. Cells were washed twice for 20 minutes in blocking solution, and once for 10 minutes in PBS. PCNA was detected using FITC goat-anti-mouse IgG, diluted 1:200 in 10 µg/ml BSA/PBS. PKC was detected using allophycocyanin-goat-anti-rabbit IgG, diluted 1:200 in 10 µg/ml BSA/PBS. After washing in PBS, slides were mounted using AquaPolyMount, and examined using confocal scanning laser microscopy as described above.
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Results |
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and p-Adducin Immunoreactivity in Mouse Lung
Using a mouse inhalation model of asbestosis, the expression and histological localization of PKC was first determined in sham and asbestos-exposed mice by immunoperoxidase staining of lung sections. In addition, as a measure of endogenous PKC activity, sections were also stained with an antibody specific for p-adducin (the phosphorylated form of the PKC substrates, 
- and -adducin).17
In comparison to sham controls, asbestos exposure resulted in an empirical increase in both PKC and p-adducin immunoreactivity in lung sections of exposed animals (Figure 1)
. Expression of PKC and p-adducin was evident in both the bronchiolar epithelial cells and in cells lining alveolar regions. Immunoreactivity of both PKC and p-adducin was particularly marked in areas with peribronchiolar lesions. In addition, p-adducin was also observed in the basal lamina of blood vessels (Figure 1, D–F)
as described previously in renal tubules.24
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expression and activity after asbestos exposure, lung sections from mice exposed to asbestos for 4 days and 30 days were examined by immunofluorescence. As shown in Figure 2
, increases in PKC and p-adducin immunoreactivity were apparent by 4 days (Figure 2B)
and extensive after 30 days of exposure to asbestos (Figure 2E)
. In addition, the immunoreactivity for PKC and p-adducin exhibited marked overlap, suggesting that PKC and p-adducin co-localize within airway cells.
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Expression by Asbestos is Specific to Epithelial Cells
To establish whether PKC expression was specific to bronchiolar and alveolar epithelial cells versus macrophages that infiltrate after asbestos exposure,4
lung sections were co-stained with antibodies recognizing macrophages (MAC-3) or the epithelial cell marker, keratin7 (CytoKeratin7). Whereas there was little overlap in staining between PKC antibodies and MAC-3 (Figure 3, A and B)
, the overlap between PKC antibodies and CytoKeratin7 was extensive (Figure 3, C and D)
. Thus, the observed increase in PKC expression after asbestos exposure likely originates from airway and alveolar epithelial cells.
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is Highly Expressed in Membranes and Nuclei of Wounded and/or Dividing C10 Alveolar Epithelial Cells
To further explore the etiology of the observed increase in PKC seen in lung epithelial cells after asbestos exposure, we used cultured alveolar type II epithelial cells (C10 cells), as a model to measure responses specific to epithelial cells. To determine the effect of cell growth state on the localization and activity of PKC, immunofluorescence was used to detect PKC, p-adducin, and -adducin (unphosphorylated form) in log phase versus confluent cultures. As shown in Figure 4A–I
, when compared with confluent cells, log phase cells exhibited PKC and p-adducin staining that was more localized to the cell membrane and both perinuclear and intranuclear regions. In addition, treatment of confluent cultures with the PKC activator, PDBu, resulted in a similar pattern of localization to the membrane and nucleus. The observed translocation of PKC to the nuclear and membrane fraction of log phase cells and after PDBu treatment was confirmed using Western blot analysis (Figure 4J)
. No changes in unphosphorylated -adducin were observed with addition of PDBu suggesting that the increase in p-adducin is due to an increase in the activity of PKC, not an increase in -adducin levels (Figure 4, G–I)
. These data indicate that PKC is activated and translocated to the membranes of proliferating cells and cells stimulated with phorbol ester.
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, confluent cultures of C10 cells were wounded mechanically, allowed to recover for 18 hours, and then examined for PKC and p-adducin immunoreactivity. A striking accumulation of PKC and p-adducin was observed in the plasma membrane and nuclei of cells migrating into the wounded area (Figure 5)
. PKC immunoreactivity was also detected along intracellular fibrils extending toward the wound, possibly due to association with actin fiber projections.
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activity may have a role in both migration and proliferation, PKC localization was correlated with the proliferative status of alveolar epithelial cells. As shown in Figure 6, A–C
, confluent C10 cells show diffuse PKC staining and no PCNA incorporation. In contrast, cells after wounding have increased PCNA staining as well as more membrane and nuclear localization of PKC, suggesting that PKC is greater in cells that are dividing (Figure 6, D–F)
.
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and P-Adducin in C10 Alveolar Epithelial Cells
To test whether asbestos fibers stimulate patterns of membrane and nuclear localization of PKC similar to that observed after wounding, C10 cells were exposed to asbestos followed by immunofluorescence staining using antibodies to PKC and p-adducin. Asbestos fibers were detected by reflectance confocal microscopy. As shown in Figure 6, G–I
, cells with direct exposure to asbestos fibers exhibited an increase in membrane and nuclear localization of PKC that was evident in PCNA-positive cells. Figure 7
illustrates that exposure to asbestos increased the levels of both PKC and p-adducin, especially in areas of direct contact of epithelial cells with the asbestos fibers. These results suggest that, like wounding, asbestos induces localization and activation of PKC to the membranes and nuclei of dividing lung epithelial cells.
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Discussion |
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PKC constitutes a large family of kinases that regulates a wide variety of both normal and pathological processes. Thus, dissecting signaling pathways of distinct PKC isoforms is necessary to assess their importance in pathologies such as the fibroproliferative response to asbestos. Our findings presented here demonstrate for the first time that in vivo asbestos-mediated responses in lung epithelial cells include an increase in the level of PKC expressed specifically in bronchiolar and alveolar epithelial cells. Overexpression of dominant negative PKC or inhibition of PKC inhibits growth of mammary tumor cells in soft agar18
and prevents expression of proto-oncogenes induced by ionizing radiation in thyroid cells.28
Thus, these and our results support a possible role for PKC in the proliferative responses of lung epithelial cells following exposure to asbestos.
The changes in PKC correlate with an increase in both localization and phosphorylation of adducins, a PKC substrate family.21
Adducins are central to regulation of the subcortical membrane and are necessary for cell polarity and differentiated function.29
Phosphorylation of adducins by PKC has been associated with an increase in growth potential and dedifferentiation of tumor cells.24
Taken together with the findings of others, our results support the hypothesis that PKC and adducin are important players in the asbestos-induced responses of lung epithelial cells.
Our in vitro findings show that PKC translocates to both the cytoplasmic and nuclear membrane of C10 lung epithelial cells after wounding or activation by PDBu. This pattern also appears in cells exhibiting focal contact with asbestos fibers. Translocation to the nucleus has been described for PKC30;31
and PKC
32
with functional effects on signal transduction through calcium33
and MAPK,34
respectively. Association of PKC with the nucleus leads to the possibility that PKC may also have a role in regulation of nuclear events as well as plasma membrane events. The positive correlation between PKC activation and expression of PCNA suggests a link between PKC and proliferation in cells responding to asbestos. Because, in some systems, PKC has opposing effects on growth compared with PKC,35
and both isoforms translocate to the nuclear envelope after phorbol ester treatment in fibroblasts,36
the two isoforms likely balance the regulation of growth promotion depending on the cellular environment.
Together our findings represent the first demonstration that PKC activity increases in epithelial cells of intact lung during the progression of asbestos-induced lung fibrosis. The increase in PKC protein level in lungs exposed to asbestos also suggests that levels of PKC are inducible. Thus, the response of lung epithelial cells to asbestos may include both direct activation of PKC and either induction of transcription or increased stabilization of PKC. The correlation of these findings with our localization studies, using a culture model of epithelial cell responses, leads us to the proposal that PKC activation and membrane translocation has an integral role in the response of epithelial cells to asbestos. Furthermore, we hypothesize that PKC is necessary for the migratory and proliferative responses observed in lung epithelial cells following exposure to asbestos. Further analysis of PKC isoforms activated by asbestos in lung and their functional role in cell responses will reveal specific contributions of the diverse PKC signaling pathways that lead to fibroproliferative lung diseases.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grant PO1HL67004.
Current address for Susan Jaken: Lilly Corporate Center, Eli Lilly and Company, Building 88/408 Dock 1543, 940 S. East Street, Indianapolis, IN 46225.
Accepted for publication February 27, 2002.
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References |
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and phosphoinositide-dependent protein kinase-1 are required for insulin-induced activation of ERK in rat adipocytes. J Biol Chem 1999, 274:30495-30500 in mammary tumor cells: relationship to transformation and metastatic progression. Oncogene 1999, 18:6748-6757[Medline]
-mediated phosphorylation of 6ß4 is associated with reduced integrin localization to the hemidesmosome and decreased keratinocyte attachment. Cancer Res 2001, 61:4591-4598 and protein kinase C play opposite roles in the proliferation and apoptosis of glioma cells. Cancer Res 2001, 61:4612-4619 involvement in mammary tumor cell metastasis. Cancer Res 1999, 59:3230-3238- and -adducin during renal tumor progression. Cell Growth Differ 1998, 9:405-413[Abstract]
mediates ionizing radiation-induced activation of c-Jun NH(2)-terminal kinase through MKK7 in human thyroid cells. Oncogene 2001, 20:989-996[Medline]
using PKC-GFP fusion proteins. Exp Cell Res 2000, 258:204-214[Medline]
is accompanied by the loss of constitutive nuclear mitogen-activated protein kinase/extracellular signal-regulated kinase activity. J Biol Chem 1997, 272:11557-11565 targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca(2+)](i). FASEB J 2000, 14:1653-1663. J Biol Chem 2001, 276:13015-13024This article has been cited by other articles:
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