AJP - Lung Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 293: L1469-L1474, 2007. First published October 12, 2007; doi:10.1152/ajplung.00274.2007
1040-0605/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/6/L1469    most recent
00274.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Slager, R. E.
Right arrow Articles by Wyatt, T. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Slager, R. E.
Right arrow Articles by Wyatt, T. A.

Hog barn dust slows airway epithelial cell migration in vitro through a PKC{alpha}-dependent mechanism

Rebecca E. Slager,1 Diane S. Allen-Gipson,1 Alexi Sammut,1 Art Heires,1 Jane DeVasure,1 Susanna Von Essen,1,2 Debra J. Romberger,1,3 and Todd A. Wyatt1,3

1Pulmonary and Critical Care Medicine Section, Department of Internal Medicine and 2College of Public Health, University of Nebraska Medical Center, Omaha; and 3Research Service, Department of Veterans Affairs Medical Center, Omaha, Nebraska

Submitted 16 July 2007 ; accepted in final form 7 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 PKC{alpha} and PKC{varepsilon}, 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 PKC{varepsilon} DN cell lines. However, in PKC{alpha} DN cells, wound closure following HDE treatment is not significantly different than media-treated cells. These results suggest that the PKC{alpha} 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 PKC{varepsilon} (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 PKC{alpha} and PKC{varepsilon} to demonstrate that HDE retards wound closure in BEAS-2B and PKC{varepsilon} DN cell lines, but not in PKC{alpha} DN cells. These results suggest that the PKC{alpha} isoform is a key regulator of cell migration in wound repair in response to hog barn dust exposure.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 PKC{varepsilon}:EGFP fusion (PKC{varepsilon} DN) or DN tetracycline-sensitive (tet-on) PKC{alpha} construct have been previously described and characterized (25). In the presence of the tetracycline analog doxycycline, the PKC{alpha} DN construct is expressed (tet-on); however, this construct is not expressed in media without doxycycline (PKC{alpha} 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, PKC{varepsilon} DN, tet-on PKC{alpha} DN, or PKC{alpha} 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 [{gamma}-32P]ATP were mixed in a Tris·HCl buffer (pH 7.5). To assay PKC{alpha}, 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 PKC{varepsilon} (ERMRPRKRQGSVRRRV; Calbiochem, San Diego, CA), PKC{alpha} (VRKRTLRRL; Bachem, Torrance, CA), PKC{delta} (RFAVRDMRQTVAVGVIKAVDKK; Calbiochem), and PKC{zeta} (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, PKC{varepsilon} DN, tet-on PKC{alpha} DN, and PKC{alpha} 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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
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 (PKC{alpha}, PKC{delta}, PKC{varepsilon}, and PKC{zeta}) were activated by HDE. In the human bronchial epithelial cell line BEAS-2B, PKC{alpha} 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 PKC{varepsilon} 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 PKC{delta} or PKC{zeta} 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 PKC{alpha} transfection control cell line (no doxycycline added to induce expression of the PKC{alpha} DN construct), PKC{alpha} was activated by 5% HDE after 1 h of treatment, similar to nontransfected BEAS-2B cells (Fig. 1A). However, in the tet-on PKC{alpha} DN cells (Fig. 1D), HDE-dependent activation of PKC{alpha} was completely abrogated at 1, 6, and 24 h. In fact, the 5% HDE-treated PKC{alpha} 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.


Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		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: PKC{alpha} 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: PKC{varepsilon} kinase activity in BEAS-2B was stimulated after a 6-h exposure to 5% HDE. C: in the BEAS-2B PKC{alpha} transfection control cell line, PKC{alpha} was activated by 5% HDE at 1 h, similar to nontransfected BEAS-2B cells. D: PKC{alpha} activity in the tet-on PKC{alpha} dominant negative (DN) cells was extremely low at 1, 6, and 24 h and was not stimulated by 5% HDE, demonstrating that the PKC{alpha} DN effectively blocks stimulated PKC{alpha} enzyme activity. The 5% HDE-treated PKC{alpha} 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, PKC{varepsilon} DN cells also effectively blocked HDE-stimulated PKC{varepsilon} activity at 6 h, similar to kinase activity levels of BEAS-2B in media alone. As previously shown, dust continues to stimulate PKC{alpha} in these PKC{varepsilon} 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 PKC{alpha}- and PKC{varepsilon}-stimulated functional responses to be assayed.


Figure 2
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 2. HDE-stimulated PKC{varepsilon} activity is blocked by DN cell line. As shown in this fold-activity graph, PKC{varepsilon} 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.


Figure 3
View larger version (21K):
[in this window]
[in a new window]

  		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 PKC{varepsilon} DN BEAS-2B cell line. Because HDE activates the novel isoform PKC{varepsilon} (Fig. 1B), we utilized a stably transfected PKC{varepsilon} DN BEAS-2B cell line to determine if PKC{varepsilon}-dependent signaling regulates wound closure in HDE-treated cells. PKC{varepsilon} 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 PKC{varepsilon} DN cells treated with 5% HDE compared with media at each time point. At 21 h, PKC{varepsilon} 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 PKC{varepsilon} signaling in BEAS-2B does not significantly affect slowing of wound repair subsequent to HDE treatment.


Figure 4
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 4. HDE slows wound repair in PKC{varepsilon} DN BEAS-2B. PKC{varepsilon} 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, PKC{varepsilon} 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 PKC{alpha} DN cell lines. HDE causes an early activation (within 1 h) of the classic, calcium-dependent isoform PKC{alpha} (Fig. 1A), which is blocked in the stably transfected tet-on PKC{alpha} DN BEAS-2B cell line (Fig. 1D). Interestingly, this PKC{alpha} 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 PKC{alpha} DN cells treated with media alone. As a control, a PKC{alpha} cell line grown without doxycycline was also used in which the DN form of PKC{alpha} 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 PKC{alpha} isoform regulates HDE-dependent cell migration in human bronchial epithelial cells.


Figure 5
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 5. Hog barn dust does not slow wound closure in PKC{alpha} DN bronchial epithelial cells. PKC{alpha} 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.

 

Figure 6
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 6. Hog barn dust retards wound closure in a PKC{alpha} transfection control bronchial epithelial cell line. As a transfection control, PKC{alpha} 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 PKC{alpha} 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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
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 PKC{alpha} and PKC{varepsilon}, but not other PKC isozymes. In BEAS-2B, PKC{alpha} is activated by HDE at 1 h and PKC{varepsilon} is activated at 6 h (Fig. 1). There was a slight elevation in PKC{alpha} 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, PKC{varepsilon} 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. PKC{varepsilon} DN cells, which block HDE-induced PKC{varepsilon} kinase activation, migrate in a similar fashion as nontransfected BEAS-2B after treatment with HDE. In contrast, HDE does not slow wound closure in PKC{alpha} 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 PKC{alpha} isoform seems to play a key regulatory role in cell migration, and when a chemical inhibitor of PKC{alpha} (23) or a PKC{alpha} 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 PKC{alpha} 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). PKC{alpha} 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, PKC{alpha} signaling may change VASP phosphorylation state through activation of phosphatase, which ultimately results in an alteration of cell migration. Alternatively, PKC{alpha} 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 PKC{alpha} in bronchial epithelial cells is warranted.

In summary, we have shown that HDE slows wound repair in airway epithelial cells through activation of PKC{alpha}. 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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
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 PKC{alpha} dominant negative cells.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. A. Wyatt, Dept. of Internal Medicine, Pulmonary and Critical Care Medicine Section, Univ. of Nebraska Medical Center, 985300 Nebraska Medical Center, Omaha, NE 68198-5300 (e-mail: twyatt{at}unmc.edu)

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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Allen-Gipson DS, Floreani AA, Heires AJ, Sanderson SD, MacDonald RG, Wyatt TA. Cigarette smoke extract increases C5a receptor expression in human bronchial epithelial cells. J Pharmacol Exp Ther 314: 476–482, 2005.[Abstract/Free Full Text]
  2. Allen-Gipson DS, Wong J, Spurzem JR, Sisson JH, Wyatt TA. Adenosine A2A receptors promote adenosine-stimulated wound healing in bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 290: L849–L855, 2006.[Abstract/Free Full Text]
  3. Beckmann JD, Takizawa H, Romberger D, Illig M, Claassen L, Rickard K, Rennard SI. Serum-free culture of fractionated bovine bronchial epithelial cells. In Vitro Cell Dev Biol 28A: 39–46, 1992.[CrossRef]
  4. Boschetto P, Quintavalle S, Miotto D, Lo Cascio N, Zeni E, Mapp CE. Chronic obstructive pulmonary disease (COPD) and occupational exposures. J Occup Med Toxicol 1: 11, 2006.[CrossRef][Medline]
  5. Butt E, Abel K, Krieger M, Palm D, Hoppe V, Hoppe J, Walter U. cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets. J Biol Chem 269: 14509–14517, 1994.[Abstract/Free Full Text]
  6. Chitaley K, Chen L, Galler A, Walter U, Daum G, Clowes AW. Vasodilator-stimulated phosphoprotein is a substrate for protein kinase C. FEBS Lett 556: 211–215, 2004.[CrossRef][ISI][Medline]
  7. Gwak J, Cho M, Gong SJ, Won J, Kim DE, Kim EY, Lee SS, Kim M, Kim TK, Shin JG, Oh S. Protein-kinase-C-mediated beta-catenin phosphorylation negatively regulates the Wnt/beta-catenin pathway. J Cell Sci 119: 4702–4709, 2006.[Abstract/Free Full Text]
  8. Harbeck B, Huttelmaier S, Schluter K, Jockusch BM, Illenberger S. Phosphorylation of the vasodilator-stimulated phosphoprotein regulates its interaction with actin. J Biol Chem 275: 30817–30825, 2000.[Abstract/Free Full Text]
  9. Kirkhorn SR, Garry VF. Agricultural lung diseases. Environ Health Perspect 108, Suppl 4: 705–712, 2000.[CrossRef][ISI][Medline]
  10. Krause M, Bear JE, Loureiro JJ, Gertler FB. The Ena/VASP enigma. J Cell Sci 115: 4721–4726, 2002.[CrossRef][ISI][Medline]
  11. Krause M, Dent EW, Bear JE, Loureiro JJ, Gertler FB. Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration. Annu Rev Cell Dev Biol 19: 541–564, 2003.[CrossRef][ISI][Medline]
  12. Lechner JF, LaVeck MA. A serum-free method for culturing normal human bronchial epithelial cells. J Tissue Cult Methods 9: 43–45, 1985.[Medline]
  13. Mathisen T, Von Essen SG, Wyatt TA, Romberger DJ. Hog barn dust extract augments lymphocyte adhesion to human airway epithelial cells. J Appl Physiol 96: 1738–1744, 2004.[Abstract/Free Full Text]
  14. Rennard SI. Inflammation and repair processes in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 160: S12–S16, 1999.[Abstract/Free Full Text]
  15. Romberger DJ, Bodlak V, Von Essen SG, Mathisen T, Wyatt TA. Hog barn dust extract stimulates IL-8 and IL-6 release in human bronchial epithelial cells via PKC activation. J Appl Physiol 93: 289–296, 2002.[Abstract/Free Full Text]
  16. Schenker MB. Respiratory health hazards in agriculture. Am J Respir Crit Care Med 158: S1–S76, 1998.[Free Full Text]
  17. Spurzem JR, Gupta J, Veys T, Kneifl KR, Rennard SI, Wyatt TA. Activation of protein kinase A accelerates bovine bronchial epithelial cell migration. Am J Physiol Lung Cell Mol Physiol 282: L1108–L1116, 2002.[Abstract/Free Full Text]
  18. Spurzem JR, Romberger DJ, Von Essen SG. Agricultural lung disease. Clin Chest Med 23: 795–810, 2002.[CrossRef][ISI][Medline]
  19. Spurzem JR, Veys T, Devasure J, Sisson JH, Wyatt TA. Ethanol treatment reduces bovine bronchial epithelial cell migration. Alcohol Clin Exp Res 29: 485–492, 2005.[CrossRef][ISI][Medline]
  20. Von Essen S, Romberger D. The respiratory inflammatory response to the swine confinement building environment: the adaptation to respiratory exposures in the chronically exposed worker. J Agric Saf Health 9: 185–196, 2003.[Medline]
  21. Von Essen SG, Auvermann BW. Health effects from breathing air near CAFOs for feeder cattle or hogs. J Agromedicine 10: 55–64, 2005.[Medline]
  22. Wyatt TA, Forget MA, Sisson JH. Ethanol stimulates ciliary beating by dual cyclic nucleotide kinase activation in bovine bronchial epithelial cells. Am J Pathol 163: 1157–1166, 2003.[Abstract/Free Full Text]
  23. Wyatt TA, Kharbanda KK, Tuma DJ, Sisson JH, Spurzem JR. Malondialdehyde-acetaldehyde adducts decrease bronchial epithelial wound repair. Alcohol 36: 31–40, 2005.[CrossRef][ISI][Medline]
  24. Wyatt TA, Schmidt SC, Rennard SI, Tuma DJ, Sisson JH. Acetaldehyde-stimulated PKC activity in airway epithelial cells treated with smoke extract from normal and smokeless cigarettes. Proc Soc Exp Biol Med 225: 91–97, 2000.[Abstract/Free Full Text]
  25. Wyatt TA, Slager RE, DeVasure J, Auvermann BW, Mulhern M, Von Essen SG, Mathisen T, Floreani AA, Romberger DJ. Feedlot dust stimulation of interleukin-6 and -8 requires protein kinase C-epsilon in human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol (August 24, 2007). doi:10.1152/ajplung.00103.2007.
  26. Zhu M, Tian D, Li J, Ma Y, Wang Y, Wu R. Glycogen synthase kinase 3beta and beta-catenin are involved in the injury and repair of bronchial epithelial cells induced by scratching. Exp Mol Pathol 83: 30–38, 2007.[CrossRef][ISI][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/6/L1469    most recent
00274.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Slager, R. E.
Right arrow Articles by Wyatt, T. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Slager, R. E.
Right arrow Articles by Wyatt, T. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.