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J Appl Physiol 101: 1127-1135, 2006. First published June 1, 2006; doi:10.1152/japplphysiol.01287.2005
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Polyamine-mediated reduction in human airway epithelial migration in response to wounding is PGE2 dependent through decreases in COX-2 and cPLA2 protein levels

Mark J. Cowan,1 Timothy Coll,1 and James H. Shelhamer2

1Department of Medicine, Division of Pulmonary and Critical Care Medicine, The University of Maryland School of Medicine, Baltimore, and 2Critical Care Medicine Department, The National Institutes of Health, Bethesda, Maryland

Submitted 6 October 2005 ; accepted in final form 29 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Both ornithine decarboxylase inhibition to deplete polyamines and cyclooxygenase inhibition diminish the migration response to injury of human airway epithelial cells in tissue culture monolayers by ~75%. Restoration of normal migration responses is achieved in the polyamine depleted system either by exogenous reconstitution of polyamines or the addition of prostaglandin E2 (PGE2). However, only PGE2 was able to restore migration in the cyclooxygenase-inhibited systems. Western blot for cyclooxygenase-2 and cytosolic phospholipase A2 protein levels and ELISAs for PGE2 secretion demonstrate dramatic increases over 24–48 h after monolayer wounding. These increases are completely abolished by polyamine depletion or cyclooxygenase inhibition. We conclude that polyamine inhibition decreases cellular migration in response to injury in airway epithelial cells at least in part through inhibiting normal PGE2 production in response to injury. This may be brought about by decreases in cytosolic phospholipase A2 and cyclooxygenase-2 protein levels.

ornithine decarboxylase; putrescine, spermidine; spermine; restitution; airway epithelial cell; prostaglandin E2; cytosolic phospholipase A2; cyclooxygenase; asthma; chronic obstructive pulmonary disease

IN HUMANS, THE LUNGS FORM the largest surface of contact with the external environment in the body, with an area of between 50 and 100 square meters (29). Although necessary for gas transfer, this exposure also places the lung at risk for injury from a wide variety of gaseous, infectious, allergic, and particulate insults. Protection from environmental agents is achieved in the lung through mucociliary clearance in the airways, humoral and cell-mediated immunity systems within the lung tissue, neurological and behavioral mechanisms, and, importantly, responses by the airway epithelium. Airway epithelial cells are generally the first cell type to respond to external insults. Although there are many widely differing insulting agents, the lung is constrained in its available responses in that its structural integrity must be maintained at all times to ensure that gas exchange is never interrupted (26). One stereotypic pattern seen in many different types of injuries to the airway epithelium is death and desquamation of the airway epithelium, with exposure of the basement membrane to the airway lumen. This is generally quickly followed by reepithelialization of the denuded area from neighboring uninjured epithelial cells, a process known as restitution (5). Desquamation has been most extensively described in asthma and chronic bronchitis (9) but also occurs in a wide variety of pulmonary diseases (1). A body of work has developed linking bronchial hyperresponsiveness, and potentially desquamation, with both respiratory symptoms and decline in lung function in asthma, chronic obstructive pulmonary disease, and fibrotic lung diseases (6, 10, 21). At any time, the area of denuded epithelium in the airways is a result of the balance between injury and repair mechanisms (15). Epithelial layer restitution after injury involves multiple processes, including detachment of the tight bonds between cells in areas adjacent to injury, dedifferentiation to a more mesenchymal phenotype, migration of epithelial cells to the site of injury, proliferation of neighboring epithelial cells, redifferentiation to an epithelial phenotype, and restoration of the normal epithelial cell tight junctions (8). Aspects of this process have been examined in gastrointestinal epithelium; in particular the critical importance of polyamine formation by ornithine decarboxylase (ODC) in gut epithelial cell migration in response to injury has been well characterized (13, 14, 28). However, the mechanism(s) by which polyamines support epithelial cell restitution remains unknown.

Some studies of the role of eicosanoids in epithelial restitution have been performed. Arachidonic acid has been shown to be essential in epithelial cell migration in a corneal epithelial cell model (16). The role of PGE2 in epithelial cell wound healing (via the EP1 and EP4 receptors) has been demonstrated in cat tracheal explants, 16HBE14o- cells (an SV-40 transformed human airway epithelial cell line), and normal human bronchial epithelial cells (24).

Although prostaglandins have been shown to modify ODC activity and polyamine generation in T lymphocytes, rat liver and colon cancer cells, whether the effects are stimulatory or inhibitory remains unclear and may be context and cell-type specific (12, 23, 27). To date there has been only one report of the effects of polyamines or polyamine depletion on prostaglandin synthesis. DL-{alpha}-Difluoromethylornithine (DFMO) can increase cyclooxygenase-2 (COX-2) mRNA half-life and protein expression through the spermidine-regulated eIF-5A protein in Caco-2 cells, a human colon adenocarcinoma cell line (18). Better understanding the interaction between polyamines and eicosanoid generation in airway epithelial cells may yield crucial insight into the mechanisms of airway epithelial cell restitution and chronic airway diseases.

On the basis of experiments done in gut (IEC-6) epithelial cells (14), we have developed an in vitro wounding model using human BEAS-2B cell monolayers (an SV-40 transformed human airway epithelial cell line) cultured on collagen-coated plastic plates in defined, serum-free media (LHC-8) that measures migration responses in these cells to injury. This system enables us to control polyamine and prostaglandin levels both pharmacologically and genetically and thus to determine the effect of polyamine and or prostaglandin manipulation on migration responses in wounded airway epithelial cell culture monolayers. With HPLC and high-sensitivity competitive ELISA, we can follow the cellular levels of polyamines and PGE2 release in response to wounding and system manipulation. Finally, using Western blot for cytosolic phospholipase A2 (cPLA2), ODC, and COX-2, we are able to begin to determine the mechanisms controlling PGE2 and polyamine levels in our wounding system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Standard notation.   Notation for time is D – 3 for plating day, D – 2 and D – 1 for the next 2 days, D0 for wounding day, D1 for first day after wounding, and D2 for second day after wounding. Treatment data are coded as follows: DFMO (DFMO, Sigma-Aldrich, St. Louis, MO), ibuprofen (ibu, Ortho McNeil-PPC, Fort Washington, NJ), NS-398 (Oxford Biomedical Research, Oxford, MI), valdecoxib (val, Pfizer, Morris Plains, NJ), putrescine (put, Sigma-Aldrich), PGE2 (PGE2, Cayman Chemical, Ann Arbor, MI), and small inhibitory RNA against ODC mRNA (anti-ODC siRNA) and cPLA2 mRNA (anti-cPLA2 siRNA). See ODC and cPLA2-targeted siRNA generation and transfection. All numeric data are presented as the average of n separate experiments ± SE.

General laboratory protocols.   Oligonucleotide synthesis was performed by Sigma Genosys, The Woodlands, TX. PCR was performed using the GeneAmp system (Perkin Elmer) on a Programmable Thermal Controller thermocycler (MJ Research, Watertown, MA), utilizing standard PCR protocols.

Wounding experiment.   BEAS-2B cells (American Type Culture Collection, Manassas, VA) were grown in LHC-8 (Biosource, Camarillo, CA) media, at 37°C and 5% CO2 on collagen I-coated six-well plastic culture dishes (Biocoat, Becton-Dickinson, Bedford, MA). Cells are tested for mycoplasma infection every 2 wk and as needed. Optimal plating density of cells was determined to be ~10 x 104 cells/cm2, which provides confluent monolayers after 3 days (i.e., D0). At confluence, cells are denuded with a small (1 cm wide) cell scraper (Fisherbrand disposable cell scraper, Fisher Scientific, Pittsburgh, PA) across one diameter of the plate and digitally photographed at x20 (Nikon Diaphot microscope, Nikon, Tokyo, Japan, Sony DKC 5000 digital photo camera, Sony, Tokyo, Japan, Dell Dimension XPS T450, Dell Computer, Round Rock, TX). The exact location of the photograph is marked on the plate, so that repeat photomicrographs of the identical location can be obtained at D1 and D2. The photographs for D0, D1, and D2 are collected onto one Photoshop CS document (Adobe Systems, San Jose, CA) on an Apple 17" G4 Powerbook computer (Apple Computer, Cupertino, CA) and aligned by using the plate marking. A grid was digitally placed over the photomicrographs at 16 boxes/in.2 (=0.635 cm/side), and each grid is trimmed to eight boxes (total height ~5.1 cm = 2.5 mm in culture) to standardize counting. The number of grid squares containing migrated cells in the denuded area can then be counted at D1 and D2 (see Fig. 1 for example). Results are expressed as percent of boxes with migrated cells compared with control wells. In experiments that involved harvesting cells or supernatants for PGE2 levels, polyamine levels or Western blotting, a modification of the wounding technique was employed. In these cases, multiple scrapes of ~2 mm wide were made horizontally and vertically throughout the entire plate, with the goal of no remaining cell further than 2–3 mm away from a wound edge.


Figure 1
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  		Fig. 1. Example of quantification of migration response after injury in BEAS-2B cell culture monolayers. Photomicrographs of injured BEAS-2B cell monolayers were taken on day of injury (day 0; D0), postinjury day 1 (D1), and day 2 (D2) and are imported into a single Photoshop CS document. A 0.635 cm/side grid is digitally overlaid, and the pictures are aligned by using the marks on the culture plates. The original picture is then cut down to 8 boxes in height (5.1 cm at x200 = 255 mm in culture), and the starting injury line is aligned to the grid in each picture. At days 1 and 2, boxes on the denuded side of the injury that contain any fraction of a new cell are summed. In this example, the results are: D1 control 52 = 100%, D1 DL-{alpha}-difluoromethylornithine (DFMO) 11 = 21.2%, D2 control 64 = 100%, D2 DFMO 18 = 28.1%.

 
In indicated wells, 5 mM DFMO, a specific ODC inhibitor, was added to and maintained in the media from D – 3 through the end of the experiment. This protocol is known to rapidly cause 95% ODC inhibition and complete depletion of putrescine and spermidine in gut epithelial (IEC-6) cells (20). In indicated experiments, polyamines were reconstituted by adding 10 µM putrescine to the cells at D – 3 and for the duration of the experiment. On D – 2, in indicated wells, 1.5 µg of ODC or cPLA2 targeted siRNA was transfected (see complete protocol below) into cells using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). One hour before injury of the cells, 5 µM ibuprofen, 5 µM NS-398, 150 ng/ml valecoxib, or 10–7 M PGE2 were added (as indicated) and maintained in the medium through the end of the experiment as indicated.

Wounding experiment in NHBE cells.   To extend our investigations of the role of polyamines and PGE2 into more biologically relevant systems, we performed our migration assay in normal human bronchial epithelial (NHBE) cells. The experiment was conducted in exactly the same manner as described for BEAS-2B cells including ibuprofen at 5 µM, DFMO at 5 mM, PGE2 at 10–7 M, and putrescine at 10 µM, except for the following: 1) NHBE cells (Cambrex) were grown on plastic in bovine epithelial growth media with SingleQuots supplements and growth factors (bovine pituitary extract, hydrocortisone, human epithelial growth factor, epinephrine, insulin, triiodothyronine, transferrin, gentamicin, amphotericin-B, retinoic acid, and bovine serum albumin-fatty acid free). 2) Normal cells grow and migrate more slowly than transformed cells; therefore plating was performed on D – 5, wounding on D0, and assay for migration on D5. 3) Results are the average of four independent experiments using two separate original aliquots of cells from Cambrex.

Dosing justification.   DFMO at a concentration of 5 mM has been shown to inhibit 95% of ODC activity in gut epithelial cell culture (IEC-9) without significant toxicity or other known metabolic consequences (14). Ibuprofen, at 5 µM, has been shown to inhibit both COX-1 and COX-2 in a variety of cell types (2), although the IC50 for COX-1 is slightly higher (2.6 µM) than for COX-2 (1.53 µM). Dosing of COX-2 selective inhibitors was designed to have minimal impact on COX-1 or cellular toxicity, but to fully inhibit COX-2. The concentration chosen for NS-398 was 5 µM and 150 ng/ml for valdecoxib (2–4).

Because of the paracrine nature of PGE2, local tissue level measurements are fraught with difficulty, and the data are unclear regarding local PGE2 concentrations in vivo. PGE2 has been shown to upregulate COX-2 gene expression in a dose-dependent fashion from 10–5 to 10–9M (22). In experiments looking at col X or cAMP response element-luciferase activity in chondrocytes, the optimum PGE2 concentration used was 10–6 M (11). A dose of 3 x 10–6 M was used in investigating the secretory response of acid in the murine duodenum (7). In addition, the Ki for EP receptors 1–4 varies from 1–20 nM, making 10–7 the lowest dose expected to cause maximal stimulation of the PGE2 receptors (17).

To determine the optimal PGE2 concentration to utilize in our system, migration inhibition experiments (utilizing ibuprofen) were performed, using PGE2 rescue in a dose escalation fashion from 10–9 to 10–4 M. DFMO treatment resulted in 14.5 ± 2.5% of migration in control cells. There was no PGE2 rescue effect seen in 10–9 or 10–8 M experiments. Migration in treated cells increased to 89.7 ± 21.9% at 10–7 M, and to 96.1 ± 11.3% at 10–6 M. Higher doses of PGE2 resulted in obvious cellular death of the BEAS-2B monolayers, although 10–5 M yielded a statistically significant restoration of migration from 55.5 ± 7.9%. Results are average of four separate experiments (data not displayed graphically). Similar data were obtained by use of ibuprofen-treated cells. Ibuprofen decreased migration to 32.1 ± 7.3%. At 10–7 and 10–6 M, PGE2 increased these to 110.3 ± 10.6 and 102.1 ± 29.4%. The minimum PGE2 concentration for full migration restoration was 10–7 M. Decreasing effect, possibly due to cellular toxicity, was seen at doses greater than 10–6 M; thus 10–7 M was the concentration of PGE2 chosen for our experiments.

Toxicity assessment (lactate dehydrogenase assay).   To assess nonspecific treatment toxicity, the standard experiment was performed using DFMO, ibuprofen and anti-ODC siRNA wells, n = 3 for each group. Medium was changed to fresh LHC-8 after injuring the cells on D0 and was not changed during the remainder of the experiment. Lactate dehydrogenase (LDH) amounts in the supernatant were measured with a colorimetric ELISA kit using company protocol (Cytotox 96 nonradioactive cytotoxicity assay, Promega, Madison, WI). Optical density was determined with a {nu}max kinetic microplate reader (Molecular Devices, Sunnyvale, CA) at 490 nm. Each well was assayed in duplicate and averaged after background subtraction.

ODC- and cPLA2-targeted siRNA generation and transfection.   The Genbank accession number NM002539 (human ornithine decarboxylase 1) was used as a query into the Dharmacon siDESIGN Center web site to design primers for use in generating an siRNA-producing plasmid. The primers picked to maximize the chances for successful mRNA silencing were 5' TACAGTTGGTGCAGAGTCT and 5' CGATCTACTATGTGATGTC, corresponding to positions 618 to 1563, a span within the coding region of the gene. D-siRNA was produced according to company protocol (BLOCK-iT dicer RNAi kit, Invitrogen). Total RNA from cultured BEAS-2B cells (RNeasy mini kit, Qiagen, Valencia, CA) was used as template along with the above primers in a one-step RT-PCR reaction (SuperScript One-Step RT-PCR with Platinum Taq, Invitrogen) to generate the first PCR product. This PCR product was cloned into BLOCK-iT RNAi TOPO vector (Invitrogen). T7 primers were then used to generate ODC-specific dsRNA. Both PCR reactions used the following parameters: 2 min 94° C, 20 s 94° x 40 cycles, 30 s 50° x 40 cycles, and 1 min 72° x 40 cycles. The sense and anti-sense DNA obtained was utilized in a transcription reaction to generate ~65 µg of each sense and anti-sense RNA. These were annealed and diced to generate the final ODC-targeted siRNA. cPLA2-targeted siRNA corresponding to bases 299–319 of the cPLA2 coding region (GenBank no. M68874) was used in prior experiments to knock down cPLA2 protein levels in the A549 cell line, another airway epithelial cell line (19).

The siRNA transfection was performed per company protocol on D – 2, when the cells were 50–70% confluent. Briefly, 1.5 µg of ODC-targeted, cPLA2-targeted or random mix siRNA in 250 µl of LHC-8 medium were mixed with 5 µl of Lipofectamine 2000 in 250 µl of LHC-8 and incubated at room temperature for 30 min. This mix was added to each experimental well containing 2 ml of fresh LHC-8. No further media changes were performed for the remainder of the experiment.

Western blotting.   Cells from the standard experiment (except for the anti-ODC siRNA with ODC antibody experiment, which used unwounded wells) were lysed in RIPA buffer and quantitated by the Bradford method. Ten micrograms of total protein per well were subjected to PAGE on a 4–20% Tris-glycine gel at 120 V for 2 h (Novex-Invitrogen). Gels were transferred to nitrocellulose membranes (Novex-Invitrogen) and blocked in 5% milk (Carnation instant milk, Nestlé, Vevey, Switzerland) with 1% Triton X-100 (Sigma-Aldrich). Initial antibodies were polyclonal rabbit anti-human cPLA2 and COX-2 and goat anti-human polyclonal anti-ODC, all used at 1:2,000. Second detection antibodies were, respectively, bovine anti-rabbit IgG horseradish peroxidase-conjugated and bovine anti-goat IgG horseradish peroxidase conjugated, both used at 1:5,000. All antibodies were from Santa Cruz Biotechnologies, Santa Cruz, CA. Detection of signal was using the chemiluminescence detection system (ECL, Amersham, Buckinghamshire, UK) onto Kodak Bio-Max MR film (Eastman Kodak, Rochester, NY). All experiments were repeated four times. To ensure equal loading of protein in each lane, after the primary study antibody detection was completed, the blots were all stripped by soaking in 0.1 M glycine HCl, pH 2.5–3.0 for 2 h. The stripped probes were blocked overnight, probed using mouse anti-human actin antibodies and developed as above.

HPLC for polyamine levels.   The polyamine extraction procedure was carried out under ice-chilled conditions. Derivation and HPLC analysis was based on the methods of Spragg and Hutchings (25) with minor modifications. All chemicals and reagents were from Sigma-Aldrich unless otherwise noted. Each well of experimental BEAS-2B cells was lysed in 10 volumes of ice-chilled 0.4 M perchloric acid (Sigma-Aldrich) containing 2 mM EDTA and 40 µM 1,8-diaminooctane (Sigma-Aldrich) as an internal standard. Lysates were centrifuged at 15,000 g for 10 min (4°C). The supernatants were dried under vacuum and dissolved in 100 µl of 1 M sodium carbonate. The samples were derived with 300 µl of 4-fluoro-3-nitrobenzo-trifluoride (FNBT) reagent at 60°C for 20 min. At the end of derivation, 40 µl of 1 M histidine in 1 M sodium carbonate was added to the reaction mixture, and the derivation continued for another 5 min to scavenge any excess FNBT. After the mixture was cooled on ice, the N-2-nitro-4-trifluoromethylphenyl derivatives of the polyamines were extracted twice with 2 ml of 2-methylbutane. After centrifugation at 3,000 g for 10 min, the organic phase was evaporated under air flow, and the residue was reconstituted with 1 ml of HPLC grade methanol. Twenty microliters of the solution were applied to the isocratic reversed phase HPLC system (System Gold, Beckman-Coulter, Fullerton, CA) using an Ultrasphere Octyl C8 column (5 M, 4.6 mm, 25 cm, Beckman-Coulter). The separation of NTP-polyamines was accomplished by elution of acetonitrile-water (80:20, vol/vol) mobile phase at the flow rate of 3 ml/min for 15 min. The eluant was monitored by ultraviolet/visible detector set at 242 nm. Integration of the curves was done with System Gold Software (Beckman-Coulter).

ELISA for PGE2 levels.   In the standard wound model, medium was aspirated at D0, D1, and D2 and assayed for PGE2 release by use of the PGE2 high-sensitivity ELISA kit (R&D Systems, Minneapolis, MN) per company protocol. Optical density was determined on a {nu}max kinetic microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nm, wavelength correction at 570 nm. Each well was assayed in duplicate and averaged. With the generation of a standard logarithmic curve, the direct concentration in nanograms per milliliter of PGE2 in the media could be determined and is reported as the average ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Migration assay: effect of ODC inhibition, ibuprofen, and polyamine or PGE2 reconstitution.   Results of the migration assay with BEAS-2B cells at D2 for the ODC and ibuprofen treatment groups is shown in Fig. 2A. Control results are shown in the first group, showing the lack of any significant effect on migration in response to treatment with putrescine or PGE2 alone. In the second group, treatment with the ODC inhibitor DMFO decreases migration to 32.7 ± 4.2% of normal levels, whereas cotreatment with either putrescine or PGE2 restores normal migration (78.4 ± 9.0 and 75.4 ± 13.8, respectively). Migration is decreased to 36.8 ± 8.0% with ODC-targeted siRNA, again restored to 94.2 ± 21.1 or 89.5 ± 13.4% with the addition of putrescine or PGE2. In the final block of data, it can be seen that the cyclooxygenase inhibitor ibuprofen decreases the migration of BEAS-2B cells in response to injury to 26.9 ± 6.2% of baseline. PGE2 restores migration response in ibuprofen-treated cells (90.1 ± 9.9%); however, putrescine fails to rescue migration in the COX-inhibited experiment (41.5 ± 4.2%). cPLA2 targeted siRNA failed to influence migration in BEAS-2B cells (data not shown). All results are n = 5.


Figure 2
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  		Fig. 2. A: relative migration of BEAS-2B monolayers in response to injury. Effect of ornithine decarboxylase (ODC) or nonselective cyclooxygenase (COX) inhibition, rescue with putrescine or PGE2. Migration response in BEAS-2B cells at day 2. Indicated cells are treated with ibuprofen (ibu), DFMO, ODC-targeted small inhibitory RNA agonist (siRNA), putrescine (put), and/or PGE2. Results are expressed as percent of control well growth, n = 5 experiments. *P < 0.05 on a 2-tailed, 2-sample t-test assuming unequal variances, error bars are SE. B: relative migration of BEAS-2B cell monolayers after injury. Effect of COX-2 selective inhibition and rescue with PGE2 or putrescine. Migration response in BEAS-2B cells at day 2. Indicated cells are treated with ibuprofen, NS-398, and valdecoxib, with and without putrescine or PGE2. Results are expressed as percent of control well growth, n = 4 experiments. *P < 0.05 on a 2-tailed, 2-sample t-test assuming unequal variances, error bars are SE. C: relative migration of normal human bronchial epithelial (NHBE) monolayers in response to injury. Effect of ODC or nonselective COX inhibition, rescue with putrescine or PGE2. Migration response in NHBE cells at day 5. Indicated cells are treated with ibuprofen, DFMO, putrescine, and/or PGE2. Results are expressed as percent of control well growth, n = 4 experiments. *P < 0.05 on a 2-tailed, 2-sample t-test assuming unequal variances; error bars are SE.

 
In Fig. 2B, the results of inhibition and reconstitution using the COX-2-selective inhibitors NS-398 and valdecoxib is displayed. Migration is significantly inhibited using either COX-2-selective inhibitor (11.9 ± 2.1% with valdecoxib and 34.1 ± 3.5% with NS-398) and is reconstituted with the addition of PGE2 (155.0 ± 29.8% for valdecoxib and 89.4 ± 16.3% for NS-398) but not putrescine (3.0 ± 2.9% for valdecoxib and 11.1 ± 6.6% for NS-398). These results are similar to those obtained with nonselective COX inhibition.

To determine the relevance of this system to a more biologically relevant model, our wounding experiment was carried out in normal human bronchial epithelial cell monolayers. As can be seen from Fig. 2C, the results are similar, although the cells grow much more slowly, requiring 4–5 days of growth to reach confluence after plating, and a similar 4–5 days of growth after injury to demonstrate differences in migration responses. Treatment with DFMO or ibuprofen reduced migration to 34.8 ± 7.7 and 29.6 ± 1.8%, respectively, on D5. Reconstitution with putrescine restored migration to 89.3 ± 9.5% in DFMO-treated cells by D5 after injury, while having no effect on ibuprofen-treated cells, 14.7 ± 5.3%, P = not significant. Addition of 10–7 M PGE2 restored migration in DFMO- and ibuprofen-treated cell layers to 124.9 ± 13.4 and 98.2 ± 24.9%, respectively.

Treatment toxicity assay.   To determine whether there was nonspecific toxicity of BEAS-2B cells to ibuprofen, DFMO, or siRNA transfection, we performed an LDH release assay of BEAS-2B cells in tissue culture without wounding in control, DFMO and ibuprofen-treated cells at D2. Average LDH optical density was 0.359 ± 0.008 units in control cells, 0.297 ± 0.01 in DFMO-treated cells, and 0.324 ± 0.009 in ibuprofen-treated cells. These data indicate lack of any significant cytotoxicity from our treatment doses of these two agents. LDH release into media from confluent BEAS-2B monolayers was measured daily after the ODC-targeted siRNA transfection was performed at D – 2. At D – 2 (pretreatment), average supernatant OD was 0.159 ± 0.1 units in the control wells vs. 0.095 ± 0.05 in the treated wells. At D – 1, increases in LDH release should reflect transfection reagent damage, and this value was thus subtracted from D0, D1, and D2 ODs. The control values were analyzed in the same way, with subtraction of D – 1 OD from the remaining values. Corrected OD values for control cells were: 0.311 ± 0.1, 0.433 ± 0.2, 0.251 ± 0.1, and 0.616 ± 0.2, whereas the corresponding siRNA transfected OD values were 0.905 ± 0.3, 0.797 ± 0.3, 0.301 ± 0.2, and 0.674 ± 0.3. All ODs are an average of n = 5 separate wells, ± SE. These data demonstrates that there is an expected early increase in LDH release with lipofectamine 2,000 application that is inherent in its mode of action. This difference does not worsen over the following 3 days compared with controls, consistent with the injury being a one-time occurrence with transfection and not a result of siRNA action.

Demonstration of polyamine depletion with DFMO and anti-ODC siRNA.   To confirm DFMO and ODC-targeted siRNA-induced depletion of polyamines, we measured polyamine levels by HPLC in cytosolic extracts from cells subjected to treatment with DFMO or anti-ODC siRNA. Figure 3 graphically displays the results for putrescine; n = 3 for each group. DMFO treatment decreases putrescine levels to ~90% below baseline throughout D0, D1, and D2. Spermidine levels are decreased by ~99% during the time period. Spermine levels are less affected, declining from 20% inhibition on D0 to ~50% on D2. ODC-targeted siRNA decreases putrescine and spermidine levels 30–70%, whereas its effect on spermine levels are inconsistent and are difficult to interpret.


Figure 3
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  		Fig. 3. Putrescine levels in BEAS-2B cells by HPLC. Effect of ODC inhibition. HPLC measurements of putrescine levels were performed on ODC-inhibited BEAS-2B cells. Cells were harvested in perchloric acid (0.5 N) and assayed by HPLC. Results are expressed as percent polyamine levels compared with control wells, n = 3 experiments. *P < 0.05 by 2-tailed, 2-sample t-test assuming unequal variances, error bars are SE.

 
Effect of ODC- and cPLA2-targeted siRNA on protein levels.   To demonstrate that the inhibition of ODC protein expression by ODC-targeted siRNA and that inhibition of cPLA2 protein by cPLA2-targeted siRNA is successful, cell lysates of BEAS-2B treated with ODC or cPLA2-targeted siRNA and injured as in our standard model were harvested on D – 2 through D2, and Western blotting was performed using ODC or cPLA2 commercially available antibody. Results are shown in Fig. 4. As can be seen in Fig. 4A, protein levels of ODC are significantly decreased by D – 0, and ODC is undetectable by D2 with siRNA treatment. There is no effect of wounding on ODC protein levels. In Fig. 4B, it can be seen that injury readily induces cPLA2 protein expression in cell lysates, and that pretreatment with cPLA2-targeted siRNA completely eliminates changes in cPLA2 protein levels through the course of the experiment.


Figure 4
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  		Fig. 4. Effect of siRNA on ODC and cytosolic phospholipase A2 (cPLA2) protein levels in BEAS-2B cells. A: effect of ODC-targeted siRNA on ODC protein levels. BEAS-2B cells were treated with ODC-targeted or random (control) siRNA 2 days before wounding (D – 2). Wells were injured on D0, harvested on D0, D1, and D2, and lysed with RIPA buffer. Protein was quantitated by the Bradford method, and 10 µg of total protein were loaded per well on a Novex 4–20% PAGE gel. This was separated at 120 V for 1 h, transferred, and probed with anti-ODC antibody and developed. Blots were then stripped and reprobed with anti-human actin antibody. Gel shown is a representative blot from 4 separate experiments. B: effect of cPLA2-targeted siRNA on cPLA2 protein levels. BEAS-2B cells were treated with cPLA2-targeted or random (control) siRNA on D – 2. Wells were injured on D0, harvested on D0, D1, and D2, and lysed with RIPA buffer. Protein was quantitated by the Bradford method, and 10 µg of total protein were loaded per well on a Novex 4–20% PAGE gel. This was separated at 120 V for 1 h, transferred and probed with anti-cPLA2 antibody, and developed. Blots were then stripped and reprobed using anti-human tubulin antibody. Gel shown is a representative blot from 2 separate experiments.

 
Effect of wounding and ODC inhibition on cPLA2 and COX-2 protein levels by Western blot.   The first three lanes of Figs. 5 and 6 demonstrate clearly and consistently that wounding BEAS-2B monolayers without any other treatment results in the upregulation of both cPLA2 and COX-2 protein levels within 24 h, which persists for at least 48 h. DFMO treatment results in undetectable cPLA2 (Fig. 5A) and COX-2 (Fig. 5B) protein levels. Similarly, ODC-targeted siRNA completely eliminates detectable cPLA2 (Fig. 6A) and COX-2 proteins (Fig. 6B) in our wounding model.


Figure 5
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  		Fig. 5. cPLA2 and COX-2 protein levels in BEAS-2B cell lysates after injury. Effect of DFMO. BEAS-2B cells were treated in a wounding experiment as above, utilizing DFMO as indicated. The cells were harvested at the indicated times and lysed with RIPA buffer. Protein was quantitated by the Bradford method, and 10 µg of total protein were loaded per well on a Novex 4–20% PAGE gel. This was separated at 120 V for 1 h, transferred, and probed with cPLA2 (A) or COX-2 (B) antibody as described above. Development was with horseradish peroxidase-conjugated goat anti-mouse IgG antibody using the ECL system (Pierce). Blots were then stripped and reprobed using-anti human actin antibody. Gels shown are representative blots from 4 separate experiments.

 

Figure 6
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  		Fig. 6. cPLA2 and COX-2 protein levels in BEAS-2B cell lysates after injury. Effect of ODC-targeted siRNA. BEAS-2B cells were treated in a wounding experiment as above, utilizing ODC-targeted siRNA as indicated. The cells were harvested at the indicated times and lysed with RIPA buffer. Protein was quantitated by the Bradford method, and 10 µg of total protein were loaded per well on a Novex 4–20% PAGE gel. This was separated at 120 V for 1 h, transferred, and probed with cPLA2 (A) or COX-2 (B) antibody as described above. Development was with horseradish peroxidase-conjugated goat anti-mouse IgG antibody using the ECL system (Pierce). Blots were then stripped and reprobed using-anti human actin antibody. Gels shown are representative blots from 4 separate experiments.

 
Effect of wounding, DMFO, ODC-targeted siRNA, and ibuprofen on PGE2 release from BEAS-2B cells.   PGE2 levels were measured by commercial ELISA on supernatants from our wounding experiment. The average levels of PGE2 measured in the media are between 3.1 and 4.0 ng/ml in all experiments on D0 (see Fig. 7). The concentration of PGE2 increases to 7.5 ± 2 ng/ml in untreated, uninjured cells at D2. Injured BEAS-2B cells have PGE2 levels of 32.4 ± 5 ng/ml on D2. DFMO, ODC-targeted siRNA, and ibuprofen treatment completely abolish this injury-mediated increase, with levels of 8.1 ± 2, 11.0 ± 3, and 6.0 ± 3 ng/ml, respectively. Putrescine replacement in DFMO or ODC-targeted siRNA treated and wounded cells restores PGE2 production with levels of 24.2 ± 5 and 28.2 ± 6 ng/ml, respectively. Therefore, wounding causes a fourfold increase in PGE2 production from BEAS-2B cells. This effect is eliminated by ODC inhibition and restored in ODC-inhibited cells by polyamine replacement.


Figure 7
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  		Fig. 7. PGE2 release in wounded BEAS-2B cells in response to COX or ODC inhibition. BEAS-2B cells were treated in a wounding experiment as above, utilizing DFMO, ODC-targeting siRNA, put, and/or ibu as indicated. Culture media was collected at the indicated times, and PGE2 levels were measured by use of a commercial PGE2 ELISA kit (PGE2 High Sensitivity, R&D Systems, Minneapolis, MN). Results are expressed as mean PGE2 concentration (ng/ml), n = 3 experiments. *P < 0.05 by 2-tailed, 2-sample t-test assuming unequal variances; error bars are SE.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
This study is an attempt to describe and characterize the precise mechanisms by which wounded airway epithelial cells heal. An in-depth understanding of this process may allow us to intervene early in the development of long-term debilitating lung diseases with precise therapies to stimulate wound healing, thus preventing the irreversible remodeling that is characteristic of these common and devastating diseases. Airway and gut epithelial cell lines in culture appear to be sufficient to perform at least some of the tasks required to heal epithelial wounds. They will migrate into the site of an injury and reestablish monolayer integrity in a fashion very similar to epithelial cells in vivo. Understanding these processes and their determinants in a system that is easy to manipulate and measure should give us valuable information to guide later experiments in more biologically relevant systems (i.e., normal human bronchial epithelial cells in air-liquid interface culture and animal models) and with more diverse mechanisms of injury (i.e., free radical stress, cytokines, cigarette smoke, or infectious agents).

Major findings.   We have studied a model of lung epithelial healing after wounding, using mechanical injury to monolayers of BEAS-2B and NHBE cells. There are three major finding in this study:

First, both ODC and COX inhibition downregulate the normal migration response of adjacent airway epithelial cells into an area of injury over a 2- to 5-day time period in cell culture. This effect can be completely reversed by the addition of polyamines or PGE2 to the media of the ODC-inhibited cells. Only the addition of PGE2 can normalize migration in the COX-inhibited system. This implies that the effect of polyamine depletion may be mediated through decreased PGE2 generation as a downstream effect. The polyamine effect is not due to nonspecific toxicity of DFMO, as shown by the results of our LDH release assay. ODC was inhibited in two different ways, pharmacologically with DFMO and at the molecular level with ODC-targeted siRNA, resulting in identical experimental findings. Western blot for ODC protein confirmed that ODC-targeted siRNA treatment of BEAS-2B cells results in loss of measurable ODC protein. HPLC measurements confirm that cellular putrescine and spermidine were significantly depleted by both methods of ODC inhibition. Similarly, COX-2 was inhibited with three different pharmacological inhibitors, the COX-1/2 inhibitor ibuprofen and the COX-2-specific inhibitors NS-398 and valdecoxib, again with nearly identical results. It is interesting to note here that suppression of cPLA2 with targeted siRNA had no effect on migration. Although this would certainly be expected to decrease the production of PGE2, this enzyme is further upstream from the production of PGE2 than is COX-2 and, importantly, also regulates the production of lipoxygenase pathway products of arachidonic acid. Any decrease in migration from decreased PGE2 levels might be offset by concomitant decreases in lipoxygenase pathway products, and perhaps it is the balance between COX and lipoxygenase pathway products that determines wound healing. Alternatively, siRNA treatment would have been expected to decrease cPLA2 production by 50% or more. This level of inhibition might not be sufficient to inhibit some downstream products and effects.

Second, both cPLA2 and COX-2 protein levels are upregulated in response to wounding in untreated BEAS-2B cells. ODC inhibition by any method completely eliminates this effect and in fact results in undetectable enzymes by Western blot.

Third, PGE2 release into media without wounding is limited, but increases dramatically with wounding. Ibuprofen is able to completely eliminate this wound-dependent PGE2 production, as measured by ELISA. ODC inhibition also eliminates this PGE2 response, while replacing polyamines in an ODC-inhibited system restores injury-responsive PGE2 production.

Model of polyamine/PGE2 action in airway epithelial cell restitution.   Taken together, our results point to a model of polyamine supported COX-2 and cPLA2 production, and, importantly, polyamine support for upregulation of these enzymes to increase production of the PGE2 necessary to support restitution. The precise mechanism by which polyamines signal cPLA2 and COX-2 production is as yet unclear. There are some data to suggest a posttranscriptional mechanism for spermidine to interact with eIF-5A and thereby impact COX-2 mRNA levels (18). However, it remains of interest to determine whether polyamines control cPLA2 and/or COX-2 at the transcriptional level. Our hypothesis is that polyamines support COX-2 and cPLA2 enzyme production in response to injury through the actions of specific DNA binding proteins that can be elucidated with molecular studies on our system. Specifically, we hypothesize that polyamines allow an enhancer that is insufficient itself to stimulate COX-2 gene expression but is required by a second, injury-induced transcription factor for transcriptional upregulation. The alternative hypothesis is that polyamine depletion results in the activity of a downregulatory transcription factor that does not allow binding or activity from an injury-induced upregulator.

Additional questions.   Other questions of interest include whether polyamines and PGE2 play a similar role in air-liquid interface culture of normal human bronchial epithelial cells, or in vivo. There are multiple enzymes in the eicosanoid production system that may be affected by polyamines (FLAP, 5-LO, PGES, LTC4S, LTD4S, etc.) Other aspects of restitution might also be affected by the polyamine-PGE2 axis, such as proliferation or tight junction formation. This model may have relevance to other injurious mechanisms in the airway, such as viral, oxidant, or cytokine-induced injury. There is evidence to suggest that some promoters in the eicosanoid pathway may be functionally interdependent. For example, COX-2 transcription is at least in part coregulated by cPLA2 through PPAR-{gamma} (19). The effect of polyamines on transcription regulation of COX-2 and/or cPLA2 may similarly be secondary to its effect on only one promoter, but as yet the precise mechanism behind the transcriptional control of these important enzymes by injury is unknown. Broadening our wounding model to examine transcription using reporter genes, EMSA and DNAase I footprinting, along with extending our system into other models (such as air-liquid interface culture) and other aspects of restitution should allow us to precisely determine the molecular mechanisms behind the critical role of polyamines and prostaglandins in support of airway epithelial restitution.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported by the University of Maryland, Other Tobacco-Related Diseases Research Grant through the Maryland Cigarette Restitution Fund Program.


   ACKNOWLEDGMENTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We also thank Dr. Jeffrey Hasday for encouragement, suggestions, and the kind use of laboratory space.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. J. Cowan, Dept. of Medicine, The Univ. of Maryland, 10 North Greene St., Rm. 3D122, Baltimore, MD 21201 (e-mail: mcowan{at}medicine.umaryland.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
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 ABSTRACT
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 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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