Hog barn dust slows airway epithelial cell migration in vitro through a PKCα-dependent mechanism

Rebecca E. Slager, Diane S. Allen-Gipson, Alexi Sammut, Art Heires, Jane DeVasure, Susanna Von Essen, Debra J. Romberger, Todd A. Wyatt

Abstract

Agricultural work and other occupational exposures are responsible for ∼15% of chronic obstructive pulmonary disease (COPD). COPD involves airway remodeling in response to chronic lung inflammatory events and altered airway repair mechanisms. However, the effect of agricultural dust exposure on signaling pathways that regulate airway injury and repair has not been well characterized. A key step in this process is migration of airway cells to restore epithelial integrity. We have previously shown that agents that activate the critical regulatory enzyme protein kinase C (PKC) slow cell migration during wound repair. Based on this observation and direct kinase measurements that demonstrate that dust extract from hog confinement barns (HDE) specifically activates the PKC isoforms PKCα and PKCε, we hypothesized that HDE would slow wound closure time in airway epithelial cells. We utilized the human bronchial epithelial cell line BEAS-2B and transfected BEAS-2B cell lines that express dominant negative (DN) forms of PKC isoforms to demonstrate that HDE slows wound closure in BEAS-2B and PKCε DN cell lines. However, in PKCα DN cells, wound closure following HDE treatment is not significantly different than media-treated cells. These results suggest that the PKCα isoform is an important regulator of cell migration in response to agricultural dust exposure.

  • protein kinase C
  • chronic obstructive pulmonary disease

agricultural work has been shown to be associated with high rates of respiratory symptoms. For example, workers in swine confinement and other concentrated animal feeding operations (CAFOs) often experience symptoms such as sinusitis, mucous membrane inflammation, and bronchitis (20). In addition, grain and CAFO workers exposed to inhalable dusts experience an asthma-like syndrome characterized by cough, wheezing, and airway obstruction (16, 18). Such airborne dusts may include microbial products such as endotoxin as well as pesticides, ammonia, hydrogen sulfide, and a variety of volatile organic compounds (9). Increased medical complaints are more common in populations living near some CAFOs (21). Symptoms of respiratory disease and lung function test abnormalities have been described in workers employed in CAFOs (20), but the mechanisms of CAFO-induced lung injury have not been well characterized.

Occupational exposures are responsible for ∼15% of chronic obstructive pulmonary disease (COPD) (4). COPD is a disease syndrome characterized by chronic bronchitis and emphysema. COPD pathogenesis involves airway remodeling in response to chronic lung inflammatory events and altered airway repair (14). We have previously shown that in vitro organic dust exposure can stimulate inflammatory pathways in the bronchial epithelium. Dust extract from hog confinement (HDE) stimulates a significant production and release of IL-6 and IL-8 in an endotoxin-independent manner (15), and this signaling process is regulated by protein kinase C (PKC). Cattle feed lot dust extract (FLDE) also stimulates significant interleukin release in airway epithelial cells, through the activation of the novel isoform PKCε (25). Additionally, HDE enhances the adhesion of lymphocytes to airway epithelial cells in a PKC-dependent manner (13).

To date, the role of hog barn dust exposure injury in the context of airway repair has not been investigated. A key step in the airway repair process is migration of cells to restore epithelial integrity (14). Our laboratory has characterized the role of protein kinases in the regulation of airway cell migration in vitro in response to various critical environmental insults. Using an in vitro model of wound repair based on cell migration into the wound (17, 19, 23), we have shown that agents that activate PKC, such as cigarette smoke extract and malondialdehyde-acetaldehyde adducts (23), slow cell migration in wound repair; however, agents that activate the cAMP-dependent protein kinase (PKA), such as adenosine (2) and isoproterenol (17), accelerate wound closure. Based on these previous observations, as well as direct kinase measurement, which demonstrate that HDE activates specific PKC isoforms, we hypothesized that HDE would slow the rate of wound closure time in airway epithelial cells. We utilized the human bronchial epithelial cell line (BEAS-2B) and stably transfected BEAS-2B cell lines that express dominant negative (DN) forms of PKCα and PKCε to demonstrate that HDE retards wound closure in BEAS-2B and PKCε DN cell lines, but not in PKCα DN cells. These results suggest that the PKCα isoform is a key regulator of cell migration in wound repair in response to hog barn dust exposure.

MATERIALS AND METHODS

Bronchial epithelial cell lines.

BEAS-2B, a simian virus 40-immortalized human bronchial epithelial cell line (American Type Culture Collection, Manassas, VA), were cultured under serum-free conditions using a 1:1 medium mixture of RPMI 1640 medium (Invitrogen, Carlsbad, CA) and LHC-9 (prepared from LHC basal; Biofluids, Rockville, MD) (12) on 1% type I collagen-coated 100-mm plastic petri plates as previously described (15). LHC-9 media contains LHC basal media (Biofluids), 0.5 μM phosphosethanolamine-ethanolamine (Sigma, St. Louis, MO), 0.11 mM calcium (Fisher, Springfield, NJ), 50 U/ml penicillin and streptomycin (Life Technologies, Grand Island, NY), 2 μg/ml fungizone (Life Technologies), trace elements, 5 μg/ml bovine insulin (Sigma), 5 ng/ml epidermal growth factor (Sigma), 10 μg/ml bovine transferrin (Sigma), 10 nM 3,3′,5-triiodothyronine (Biofluids), bovine pituitary extract (50 μg protein/ml; Pel Freeze, Rogers, AR), 0.2 μM hydrocortisone (Biofluids), 0.5 μg/ml epinephrine (Sigma), and 0.1 μg/ml retinoic acid (Sigma) (3). Cells were maintained in culture at 37°C in humidified 95% air/5% CO2 for 48–72 h before each experiment (1).

Stably transfected BEAS-2B cell lines expressing a constitutive DN PKCε:EGFP fusion (PKCε DN) or DN tetracycline-sensitive (tet-on) PKCα construct have been previously described and characterized (25). In the presence of the tetracycline analog doxycycline, the PKCα DN construct is expressed (tet-on); however, this construct is not expressed in media without doxycycline (PKCα transfection control). These cell lines were also cultured in 1:1 RPMI 1640 medium (Invitrogen) and LHC-9 (prepared from LHC basal; Biofluids) (12) on 1% type I collagen-coated 100-mm plastic petri plates at 37°C in humidified 95% air/5% CO2 in the appropriate antibiotic selection.

Confluent monolayers of BEAS-2B, PKCε DN, tet-on PKCα DN, or PKCα transfection control cells were treated with media or 5–10% hog barn dust extract for 1–24 h at 37°C, conditions that do not induce a loss in bronchial epithelial cell viability (15).

HDE preparation.

Settled dust from hog confinement facilities was utilized for these experiments similar to previously described methods (13). Briefly, the HDE was prepared by placing 1 g of dust in 10 ml of PBS, pH 7.4. The mixture was vortexed and allowed to stand at room temperature for 1 h. The mixture was centrifuged at 10,000 g for 10 min. The final supernatant was filter sterilized (0.22 μm) and either used immediately or aliquots were frozen at −20°C (13).

PKC isoform activity assay.

After media supernatants were removed from treated cells, cell monolayers were flash frozen in cell lysis buffer as described (24). The cells were scraped with a cell lifter, pulse sonicated for 6 s, and centrifuged at 10,000 g for 30 min at 4°C. The supernatant was removed (cytosolic fraction), and the pellet was resuspended in cell lysis buffer containing 0.01% Triton X-100 and sonicated again (particulate fraction). PKC isoform activity was determined in crude whole cell cytosolic and particulate fractions of BEAS-2B similar to methods described previously (22, 24). To measure PKC isozyme activity specifically, 24 μg/ml PMA, 30 mM dithiothreitol, 150 μM ATP, 45 mM Mg-acetate, a specific PKC isoform substrate peptide, and 10 μCi/ml [γ-32P]ATP were mixed in a Tris·HCl buffer (pH 7.5). To assay PKCα, 1 mM CaCl2 was included to the reaction mix. Chilled (4°C) cell lysate (cytosolic or particulate) samples (20 μl) were added to 40 μl of the reaction mix and incubated for 15 min at 30°C. This mixture (60 μl) was then spotted onto P-81 phosphocellulose papers (Whatman, Clinton, NJ) to halt incubation, and papers were subsequently washed five times in 75 mM phosphoric acid for 5 min, washed once in 100% ethanol for 1 min, dried, and counted in nonaqueous scintillant (National Diagnostics, Atlanta, GA). PKC activity was expressed in relation to the total amount of cellular protein assayed as picomoles of phosphate incorporated per minute per milligram.

Specific PKC isoform substrate peptides for PKCε (ERMRPRKRQGSVRRRV; Calbiochem, San Diego, CA), PKCα (VRKRTLRRL; Bachem, Torrance, CA), PKCδ (RFAVRDMRQTVAVGVIKAVDKK; Calbiochem), and PKCζ (SIYRRGSRRWRKL; Biosource, Camarillo, CA) were used in isoform activity measurements. All other reagents not specified were purchased from Sigma.

Cell migration wound repair assay.

BEAS-2B, PKCε DN, tet-on PKCα DN, and PKCα transfection control cells were grown to confluency in 12-well flat-bottomed plates or 60-mm tissue culture dishes. Cell monolayers were “wounded” in serum-free media (M199; GIBCO, Carlsbad, CA) using methods previously described by our laboratory (2). Briefly, a small sterile scraper removed a circular area of cells, ∼1,000 μm2. The progress of epithelial cell migration was monitored with a phase-contrast microscope outfitted with a video camera, and the camera output was captured with image analysis software (NIH ImageJ 1.30v). Each wound was photographed with the video camera and image analysis software at specified times, and the area of the wound was measured. As wound size can affect cell migration rate, only wounds of similar size were used. The dishes were returned to the 37°C incubator between measurements. All treatment conditions were assayed in triplicate.

Statistical analysis.

All quantitative experiments were performed in triplicate. All data were analyzed using GraphPad Prism (version 4.00 for Windows; GraphPad Software, San Diego, CA) and represented as means ± SE. Data were analyzed for statistical significance using ANOVA or Student's t-test, as appropriate. Significance was accepted at the 95% confidence interval.

RESULTS

HDE activates specific PKC isoform in BEAS-2B.

Because previous studies revealed that organic dust extract obtained from hog confinement facilities stimulated the release of IL-6 and IL-8 in a PKC-dependent manner (15), we used direct kinase activity measurements to determine whether specific PKC isoforms that are expressed in airway epithelial cells (PKCα, PKCδ, PKCε, and PKCζ) were activated by HDE. In the human bronchial epithelial cell line BEAS-2B, PKCα activity was significantly elevated after a 1-h exposure with 5% HDE (Fig. 1A) compared with media treatment alone; kinase activity returned to baseline levels at 6 h. There was a statistically significant elevation in kinase activity after 24 h of 5% HDE treatment; however, the activity increase was not as great. Additionally, activation of the novel PKC isoform PKCε was observed after a 6-h exposure to 5% HDE (Fig. 1B) and returned to media control levels by 24 h. No significant activation of PKCδ or PKCζ in bronchial epithelial cells was observed under these same treatment conditions (data not shown). To characterize the stably transfected BEAS-2B cell lines, we also performed direct kinase activity assays. As shown in Fig. 1C, in the PKCα transfection control cell line (no doxycycline added to induce expression of the PKCα DN construct), PKCα was activated by 5% HDE after 1 h of treatment, similar to nontransfected BEAS-2B cells (Fig. 1A). However, in the tet-on PKCα DN cells (Fig. 1D), HDE-dependent activation of PKCα was completely abrogated at 1, 6, and 24 h. In fact, the 5% HDE-treated PKCα DN cells had statistically significantly less activity than media controls at 6 and 24 h. However, these activity changes of less than twofold are unlikely to have significant biological relevance.

Fig. 1.

Hog barn dust extract (HDE) activates specific PKC isoforms in BEAS-2B. Direct kinase activity measurement was used to determine the effect of HDE on PKC isoforms within the human airway epithelial cell line BEAS-2B. A: PKCα kinase activity was induced after a 1-h exposure to 5% HDE, and kinase activity returned to baseline levels at 6 h of treatment with 5% HDE. There was also an elevation in kinase activity after 24 h of 5% HDE treatment. B: PKCε kinase activity in BEAS-2B was stimulated after a 6-h exposure to 5% HDE. C: in the BEAS-2B PKCα transfection control cell line, PKCα was activated by 5% HDE at 1 h, similar to nontransfected BEAS-2B cells. D: PKCα activity in the tet-on PKCα dominant negative (DN) cells was extremely low at 1, 6, and 24 h and was not stimulated by 5% HDE, demonstrating that the PKCα DN effectively blocks stimulated PKCα enzyme activity. The 5% HDE-treated PKCα DN cells had statistically significantly less activity than media controls at 6 and 24 h. Error bars represent SE of 3 separate experiments (n = 9; ***P < 0.001 compared with media control).

In the graph of kinase fold-activity change shown in Fig. 2, PKCε DN cells also effectively blocked HDE-stimulated PKCε activity at 6 h, similar to kinase activity levels of BEAS-2B in media alone. As previously shown, dust continues to stimulate PKCα in these PKCε DN cell lines (25). These control experiments demonstrate the ability of these DN BEAS-2B cell lines to functionally block the activation of their specific PKC by HDE, allowing specific PKCα- and PKCε-stimulated functional responses to be assayed.

Fig. 2.

HDE-stimulated PKCε activity is blocked by DN cell line. As shown in this fold-activity graph, PKCε DN BEAS-2B cells effectively blocked HDE-stimulated kinase activity at 6 h, similar to kinase activity levels of nontransfected BEAS-2B in media alone. NS, not significant.

HDE slows wound repair in BEAS-2B.

To determine the effect of HDE on cell migration in wound repair in BEAS-2B, cells were grown to confluency, wounded with a sterile cell rake, and then treated with serum-free media and 5% or 10% HDE. The wound closure was monitored for ∼24 h and measured at 0, 5, and 22 h. As shown in Fig. 3, cells in media alone closed completely within 22 h. However, HDE treatment significantly slowed cell migration within 5 h, in a concentration-dependent manner. At 22 h, as highlighted by the inset, the 5% HDE-treated wounds were still ∼55% open, and the 10% HDE-treated wounds were ∼65% open. In the subsequent figures, 5% HDE treatment is used as an effective agent to slow wound closure in bronchial epithelial cells.

Fig. 3.

HDE slows wound closure in BEAS-2B. BEAS-2B cells in serum-free media alone or media containing 5% and 10% HDE were wounded and measured at 0, 5, and 22 h. There was a significant slowing of wound closure in cells treated with 5% and 10% HDE at 5 and 22 h. BEAS-2B in media closed completely within 22 h, whereas cells exposed to 5% HDE were still ∼55% open, and the 10% HDE-treated wounds were ∼65% open (inset). Error bars represent SE of 3 separate experiments (n = 9; ***P < 0.001 compared with media).

HDE slows wound repair in a PKCε DN BEAS-2B cell line.

Because HDE activates the novel isoform PKCε (Fig. 1B), we utilized a stably transfected PKCε DN BEAS-2B cell line to determine if PKCε-dependent signaling regulates wound closure in HDE-treated cells. PKCε DN cells were wounded and then treated with media alone or 5% HDE and measured at 0, 2, 4, 8, and 21 h. As shown in Fig. 4, there was a significant difference in wound closure between the PKCε DN cells treated with 5% HDE compared with media at each time point. At 21 h, PKCε DN cells in media alone were nearly closed (∼25% of the original wound area); however, 5% HDE-treated cells were still ∼65% open (inset), a statistically significant difference. This wound closure time course resembles the closure of the nontransfected BEAS-2B in Fig. 3, suggesting that blocking of PKCε signaling in BEAS-2B does not significantly affect slowing of wound repair subsequent to HDE treatment.

Fig. 4.

HDE slows wound repair in PKCε DN BEAS-2B. PKCε DN cells in serum-free media and or 5% HDE were wounded and measured at 0, 2, 4, 8, and 21 h. There was a significant slowing of wound closure in cells treated with 5% HDE at 2, 4, 8, and 21 h. Similar to nontransfected BEAS-2B, PKCε DN cells in media closed to ∼25% of the original wound area within 22 h; however, 5% HDE-treated cells were still ∼65% open (inset). Error bars represent SE of 3 separate experiments (n = 9; *P < 0.05 compared with media, **P < 0.01 compared with media, ***P < 0.001 compared with media).

HDE does not slow wound repair in PKCα DN cell lines.

HDE causes an early activation (within 1 h) of the classic, calcium-dependent isoform PKCα (Fig. 1A), which is blocked in the stably transfected tet-on PKCα DN BEAS-2B cell line (Fig. 1D). Interestingly, this PKCα DN cell line restores wound repair subsequent to treatment with 5% HDE. As shown in Fig. 5, HDE does not cause a significant slowing of wound closure within 22 h in this cell line compared with PKCα DN cells treated with media alone. As a control, a PKCα cell line grown without doxycycline was also used in which the DN form of PKCα is not expressed. In these cells (Fig. 6), 5% HDE treatment causes significant slowing of wound repair within 24 h, similar to nontransfected BEAS-2B cells (Fig. 3), suggesting that the PKCα isoform regulates HDE-dependent cell migration in human bronchial epithelial cells.

Fig. 5.

Hog barn dust does not slow wound closure in PKCα DN bronchial epithelial cells. PKCα DN cells in media alone and or 5% HDE were wounded and measured at 0, 3, 6, 12, and 24 h. HDE treatment did not induce a significant slowing of wound repair at any time point within 24 h in this cell line compared with media controls. Error bars represent SE of 3 separate experiments (n = 9). NS, not significant.

Fig. 6.

Hog barn dust retards wound closure in a PKCα transfection control bronchial epithelial cell line. As a transfection control, PKCα cells without doxycycline grown in serum-free media or 5% HDE were also wounded and measured at 0, 3, 6, 12, and 24 h. In this cell line, which does not induce expression of the PKCα DN, 5% HDE treatment causes significant slowing of wound repair at 6, 12, and 24 h (inset), similar to nontransfected BEAS-2B cells (Fig. 2). Error bars represent SE of 3 separate experiments (n = 9; ***P < 0.001 compared with media).

DISCUSSION

Non-cigarette smoke-induced COPD is greatly understudied. As clinical treatment of occupational COPD becomes of greater importance to the general population, a better understanding of the underlying mechanisms of organic dust-induced airway inflammation and injury repair is needed. COPD involves airway remodeling in response to chronic lung inflammatory events and altered airway repair mechanisms (14). Our previous studies have shown that agricultural dusts, including dust extract from HDE and cattle FLDE, have a significant effect on the proinflammatory signaling pathways in the airway epithelium. Both HDE and FLDE stimulate a significant release of IL-6 and IL-8 (15, 25) via activation of PKC. Our current work demonstrates that HDE, similar to FLDE (25), activates both PKCα and PKCε, but not other PKC isozymes. In BEAS-2B, PKCα is activated by HDE at 1 h and PKCε is activated at 6 h (Fig. 1). There was a slight elevation in PKCα activity at 24 h, which is less likely to be of biological significance, but further investigation is warranted to determine if a feedback mechanism modulates a sustained signaling cascade in response to HDE treatment. Following treatment of airway epithelial cells with FLDE, PKCε appears to be the dominant PKC isoform involved in regulation of cytokine release (25). However, the effect of organic dusts on other crucial epithelial functions, such as wound repair to maintain epithelial integrity, has not been well studied.

This report provides insight into the mechanism of bronchial epithelial cell migration, a critical early step in airway repair (14), in response to organic dust exposure. Our results demonstrate that HDE treatment significantly slows wound repair in BEAS-2B cells compared with cells in media alone. PKCε DN cells, which block HDE-induced PKCε kinase activation, migrate in a similar fashion as nontransfected BEAS-2B after treatment with HDE. In contrast, HDE does not slow wound closure in PKCα DN cells. Based on these results and previously published studies, we propose a reciprocal kinase regulation model of wound repair in which environmental agents that activate PKC, such as HDE or combined cigarette smoke and ethanol-generated malondialdehyde-acetaldehyde adducts (23), slow bronchial epithelial cell migration in wound repair. In contrast, reagents that activate the PKA, such as adenosine (2) and isoproterenol (17), accelerate wound closure.

Specifically, the PKCα isoform seems to play a key regulatory role in cell migration, and when a chemical inhibitor of PKCα (23) or a PKCα DN cell line is used (this work), inhibition of wound closure by PKC-activating agents can be blocked. However, the specific phosphorylation signaling targets of PKCα that alter cell migration are currently unknown. A possible target is the vasodilator-stimulated phosphoprotein (VASP), which has been shown to mediate actin assembly and thereby enhance cytoskeletal rearrangement necessary for epithelial cell migration (10, 11). PKCα has been shown to directly phosphorylate VASP in vascular smooth muscle cells independently of PKA and the cGMP-dependent protein kinase (5, 6, 8). We speculate that in airway epithelial cells, PKCα signaling may change VASP phosphorylation state through activation of phosphatase, which ultimately results in an alteration of cell migration. Alternatively, PKCα may be involved in the negative regulation of the β-catenin pathway (7), which has been shown previously to be involved in wound closure in mammalian bronchial epithelial cells (26). Further investigation to determine the downstream signaling targets of PKCα in bronchial epithelial cells is warranted.

In summary, we have shown that HDE slows wound repair in airway epithelial cells through activation of PKCα. A growing body of work demonstrates that agricultural organic dusts have a profound impact on airway inflammatory and injury repair pathways in vitro; however, it is not yet known how these interrelated mechanisms lead to chronic lung disease. In the lung, repair mechanisms can be instigated by the inflammatory response, and the ability of epithelial cells to migrate can be altered by inflammatory molecules (14). We have just begun to understand how organic dust may alter inflammatory cell release as well as disrupt repair mechanisms. As agricultural workers who experience chronic agricultural dust exposure may be at risk for respiratory illness, there is a continued need to explore the underlying pathological mechanism of dust exposure on the airways and to identify new therapeutic targets for lung injury.

GRANTS

This work was supported by a Veterans Affairs Merit Review grant (T. A. Wyatt) and R01OH008539 (D. J. Romberger.)

Acknowledgments

The authors acknowledge Dr. Anthony A. Floreani (UNMC) for providing the PKCα dominant negative cells.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

REFERENCES

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