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Am J Physiol Lung Cell Mol Physiol 295: L293-L302, 2008. First published June 6, 2008; doi:10.1152/ajplung.00134.2007
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Arsenic upregulates MMP-9 and inhibits wound repair in human airway epithelial cells

Colin E. Olsen,1,2 Andrew E. Liguori,1,2 Yue Zong,1,2 R. Clark Lantz,2,3,4 Jefferey L. Burgess,2,5 and Scott Boitano1,2,3,4,6

1Arizona Respiratory Center; 2Southwest Environmental Health Sciences Center; 3Department of Cell Biology and Anatomy, School of Medicine; 4Bio5 Research Institute; 5Mel and Enid Zuckerman College of Public Health; and 6Department of Physiology, School of Medicine, Arizona Health Sciences Center, Tucson, Arizona

Submitted 3 April 2007 ; accepted in final form 3 June 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
As part of the innate immune defense, the polarized conducting lung epithelium acts as a barrier to keep particulates carried in respiration from underlying tissue. Arsenic is a metalloid toxicant that can affect the lung via inhalation or ingestion. We have recently shown that chronic exposure of mice or humans to arsenic (10–50 ppb) in drinking water alters bronchiolar lavage or sputum proteins consistent with reduced epithelial cell migration and wound repair in the airway. In this report, we used an in vitro model to examine effects of acute exposure of arsenic (15–290 ppb) on conducting airway lung epithelium. We found that arsenic at concentrations as low as 30 ppb inhibits reformation of the epithelial monolayer following scrape wounds of monolayer cultures. In an effort to understand functional contributions to epithelial wound repair altered by arsenic, we showed that acute arsenic exposure increases activity and expression of matrix metalloproteinase (MMP)-9, an important protease in lung function. Furthermore, inhibition of MMP-9 in arsenic-treated cells improved wound repair. We propose that arsenic in the airway can alter the airway epithelial barrier by restricting proper wound repair in part through the upregulation of MMP-9 by lung epithelial cells.

sodium arsenite; matrix metalloproteinase; cell migration; 16HBE14o- cells; airway epithelial barrier

ARSENIC IS A NATURALLY OCCURRING metalloid found in water, soil, and air. Exposure to inorganic arsenic occurs worldwide via environmental (e.g., contaminated drinking water, air, food, domestic fuel sources) and occupational exposures (e.g., smelting industries, pesticide production). In addition to its association with nonmalignant diseases, arsenic is of major worldwide health concern because of its carcinogenic potential in humans (5, 8, 22a, 43). More recently, there has been growing evidence that ingestion of arsenic also leads to noncarcinogenic lung disease (14, 33, 41, 44). Mouse models have provided evidence that single doses of ingested arsenic can lead to collection in the lung within 1 h, and chronic low-dose exposure leads to consistently elevated arsenic concentrations in the lung (21, 22, 24, 25). However, specific molecular and cellular mechanisms that lead to lung disease from low-dose arsenic exposure are not clearly elucidated.

Arsenic has been implicated in promoting alterations in growth and proliferation pathways, apoptotic pathways, DNA repair mechanisms, immunosurveillance, and stress-response pathways (1, 9, 31, 32, 40). Although chronic exposure to moderate and/or high levels of arsenic in drinking water may lead to the development of disease in humans, the effects at low-dose are inferred mostly from models of high-dose exposure. A variety of cellular signaling pathways have been implicated to be altered by arsenic exposure including reactive oxygen species production, cellular phosphorylation events, mitogen-activated protein kinase (MAPK) signaling, NF-{kappa}B activation, cellular proliferation, and apoptosis, among others (reviewed in Ref. 37). As in animal studies, direct effects in cellular studies are confounded by the wide range of arsenic used. However, an intriguing effect of arsenic exposure is the alteration of cellular migration (15, 52).

In high-throughput protein screening experiments with low-dose arsenic exposure, we found reduced expression of proteins associated with cellular migration in mouse lung tissue (26) and alteration of a specific wound repair protein marker in mouse bronchoalveolar fluid (27). Additional microarray experiments on lung tissue from mice fed low-dose arsenic revealed several changes in extracellular matrix (ECM) protein expression and a large increase in matrix metalloproteinase (MMP)-9 expression (26). In human studies for low-level arsenic exposure lung biomarkers, we found an increase in the ratio of MMP-9 to tissue inhibitor of matrix metalloproteinase (TIMP)-1 in collected sputum samples (23). MMPs are responsible for ECM degradation among other proteolyses. MMP-9 is the most prominently studied MMP in the lung and has been associated with a variety of lung diseases (4). The imbalance between MMP-9 and TIMP-1 is considered to contribute to the progression of airway remodeling in part due to changes in epithelial wound response (4, 6, 12, 29, 39, 47, 48).

In this study, we examined the effects of arsenic exposures on a human bronchial epithelial cell line (16HBE14o-). We found that as arsenic concentration increases, the ability for 16HBE14o- cells to repair monolayers in culture is inhibited. Furthermore, as arsenic concentration is increased, MMP-9 secretion and activity also increased, and this upregulation of MMP-9 is in part responsible for altered wound response. In conclusion, arsenic directly affects signaling pathways that contribute to cell migration, and remodeling of the airway, and in this manner may cause or exacerbate lung disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Minimum essential medium with Earle's salts (MEM), Hanks' balanced saline solution, L-glutamine, penicillin, streptomycin, amphotericin, FBS, LIVE/DEAD Viability/Cytotoxicity Kit, TRIzol reagent, Platinum SYBR Green qPCR SuperMix-UDG kit, Quant-iT and the OliGreen quantification kit were purchased from Invitrogen (Carlsbad, CA). Fibronectin and type I collagen were purchased from Becton-Dickinson (Franklin Lakes, NJ). Lechner and LaVeck (LHC) basal medium and BSA were purchased from Biosource International (Camarillo, CA). Antibodies to MMP-2, MMP-9, TIMP-1, and MMP inhibitor GM6001 were purchased from Calbiochem (La Jolla, CA). iScript cDNA synthesis kit was from Bio-Rad (Hercules, CA). Primers for real-time quantitative RT-PCR experiments were from Integrated DNA Technologies (Coralville, IA). All other chemicals were of the highest biochemical quality and purchased from Sigma-Aldrich (St. Louis, MO), VWR (West Chester, PA), or Fisher Scientific (Pittsburgh, PA).

16HBE14o- cell culture. 16HBE14o- cells are a SV40 transformed human bronchial epithelial cell line (18) and were obtained through the California Pacific Medical Center Research Institute (San Francisco, CA). 16HBE14o- cells were expanded in tissue culture flasks before culture on 15-mm glass coverslips. Flasks (2 ml) and coverslips (250 µl) were coated initially with matrix coating solution [consisting of: 88% LHC basal medium, 10% BSA (from 1 mg/ml stock), 1% bovine collagen type I (from 2.9 mg/ml stock), and 1% human fibronectin (from 1 mg/ml stock solution)] and incubated for 2 h at 37°C, after which the coating solution was removed and cultureware allowed to dry for at least 1 h. 16HBE14o- cells were plated onto the matrix-coated cultureware at a concentration of 1 x 105 cells/cm2. Cells were cultured in 250–350 µl of control growth medium (CGM; Eagle's MEM supplemented with 10% FBS, 2 mM L-glutamine, penicillin, streptomycin, and amphotericin) at 37°C in a 5% CO2 atmosphere. CGM was replaced every other day until the cells reached confluence.

Scrape wound repair assays. 16HBE14o- cells were grown to confluence on matrix-treated glass coverslips in CGM as described above. CGM was removed and replaced with either fresh CGM (0 ppb arsenic) or CGM supplemented with arsenic (30, 60, or 290 ppb as sodium arsenite; for comparison, 1 µM {approx} 75 ppb) arsenic for 24 h, after which growth medium was removed and replaced with new medium (with or without arsenic). A bent tip of a 21G syringe needle was sterilized and used to introduce a wound across the entire cell monolayer. Coverslips were mounted onto an Olympus IX70 microscope. Images of the wounds were captured on a Macintosh G4 computer using a CoolSNAP camera (Roper Scientific, Tucson, AZ) immediately following wounding (0 h) and every hour for 4 h. In between imaging, cells were returned to 37°C, 5% CO2 humidified incubator. Wound area in each image was measured using the NIH ImageJ freeware program and quantified by following the change in wound area over time compared with the original wound area. These values are expressed in graphs as percentage of original wound area ± SE.

For multiple scrape wounds, 16HBE14o- cells were grown to confluence, CGM was removed, and cells were incubated in either fresh CGM (0 ppb) or arsenic-supplemented CGM (60 or 290 ppb) for 24 h as above. After this time, the cell-conditioned medium (pre-wound) was collected and replaced with new medium. Monolayer cultures were wounded in a crosshatched pattern with two perpendicular strokes of a four-pronged comb to maintain consistent wound area. Wounded monolayers were washed with fresh medium and allowed to repair for 0, 4, or 12 h at 37°C, depending on the assay. Cell-conditioned medium was collected at the 12-h time point for post-wound analysis. All conditioned media were stored at –20°C until analysis. To visualize the wound area, coverslip cultures were washed with PBS and fixed/stained with 250 µl of crystal violet solution (1% formaldehyde, 0.5% crystal violet, and 20% methanol in PBS) for 30 min. Coverslips were washed with water three times to remove excess crystal violet stain and allowed to dry. After staining of 0-, 4-, and 12-h wounds, digital images of wound patterns were captured with Chemidoc XRS system under Quantity-One software control (Bio-Rad) with darkly stained areas representing cell density. Wound area was analyzed using NIH ImageJ freeware as described above.

For cell cytotoxicity assays, cell cultures were grown, treated with arsenic, and wounded as above. At the appropriate time, cells were washed with HBSS and treated with the Molecular Probes LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells per the manufacturer's instructions. Cultures were imaged on an Olympus IX 70 microscope in epifluorescence mode with appropriate filters. Images were captured by a CoolSnap Camera (Roper Scientific) onto a Macintosh G4 computer under Roper software (Tucson, AZ) control. Adobe Photoshop (San Jose, CA) was used to compile images into figures.

MMP assays. Real-time RT-PCR was used to assay MMP-9 or TIMP-1 mRNA expression in response to arsenic. 16HBE14o- cells were grown in T75 flasks as described above. At confluence, CGM was replaced with fresh or arsenic-supplemented CGM, with refeeding every 2 days. After 5 days, total RNA was isolated using TRIzol reagent according to the manufacturer's protocol. Total RNA was estimated spectrophotometrically at 260 nm. cDNA was synthesized using the iScript cDNA Synthesis kit and the Mastercycler EPgradient thermocycler (Eppendorf, Westbury, NY). cDNA from each isolation was quantified spectrofluorimetrically using a Quant-iT OliGreen quantification kit according to the manufacturer's instructions on a TBS-380 mini-fluorimeter (Turner BioSystems, Sunnyvale, CA). For quantitative PCR, 100 ng of total cDNA per reaction was amplified with a Platinum SYBR Green qPCR SuperMix-UDG kit according to the manufacturer's instructions in a Rotor-Gene 3000 real-time thermal cycler (Corbett Robotics, San Francisco, CA) under the following conditions: initial hold for 2 min at 50°C and hold for 2 min at 95°C followed by 35–45 cycles consisting of denature 15 s at 94°C; anneal 30 s at 60°C for MMP-9 or 54°C for TIMP-1; extension for 45 s at 72°C; and melted from 72°C-99°C (1°C/5 s). Human gene-specific primer pairs were designed using MacVector software. Primer pairs used in this study included: MMP-9 forward: 5'-CGG TGA TTG ACG ACG CCT TT-3'; MMP-9 backward: 5'-ACC AAA CTG GAT GAC GAT GTC TG-3; TIMP-1 forward: 5'-ACT GAT GGT GGG TGG ATG AGT AAT-3'; TIMP-1 backward: 5'-AGC AAC AAC AGG ATG CCA GAA G-3'. Individual analyses were performed in triplicate on cDNA samples obtained from at least three separate isolations for each experiment.

Immunoblot experiments were used to assess amounts of MMP-9 and TIMP-1 protein in conditioned medium. Conditioned media from above were thawed, and equal amounts of protein from experiments with 0, 60, or 290 ppb arsenic were run out on 7.5% (MMP-9) or 10% (TIMP-1) SDS-PAGE gels under nonreducing conditions. Proteins were transferred overnight to nitrocellulose and blotted with primary antibodies specific for human MMP-9 or TIMP-1, followed by washes and appropriate HRP-linked secondary antibodies. Blots were developed with the SuperSignal West Femto kit (Pierce, Rockford, IL) per the manufacturer's instructions. Band density was determined with the Chemidoc XRS system and Quantity-One software.

Gelatin zymography experiments to assess activity of MMP-9 were performed on conditioned media from multiple scrape wounds described above. Conditioned medium was thawed, and 50 µl was mixed with 2x sample buffer (to a final buffer concentration of: 0.125 M Tris, pH 6.8, 2% SDS, 10% glycerol, and 0.05% bromophenol blue) for 15 min at room temperature. Samples were electrophoresed on 10% SDS-polyacrylamide gels containing 0.1% gelatin under nonreducing conditions. Gels were washed in a 2.5% Triton-X 100 solution two times for 15 min each to remove SDS. Gels were then incubated in developing buffer (50 mM Tris, 0.2 M NaCl, 5 mM CaCl2, and 0.02% Brij 35, pH 7.5) for 30 min at room temperature, followed by 12 h at 37°C with shaking. Gels were incubated in Coomassie blue solution (0.25% wt/vol Coomassie blue R230 in 50% methanol, 10% acetic acid, and 40% H2O) and then a destaining solution (50:10:40 methanol:acetic acid:H2O) to visualize undigested gelatin. Gelatinase activity corresponded to areas of clearance of the gelatin (i.e., low Coomassie blue staining) from the native gel. Images of each gel were captured on the Chemidoc XRS system and analyzed with Quantity-One software as previously described.

To determine effects of the MMP inhibitor GM6001 on wound closure in the presence or absence of arsenic, cells were grown to confluence in CGM as described. CGM was then removed, and cells were treated with either fresh CGM or arsenic-supplemented CGM that included GM6001 (in DMSO) or a DMSO control. After 24 h, pre-wound medium was removed and replaced with new medium supplemented with GM6001 or DMSO. Multiple scrape wound assays were performed as described above.

Statistics. All statistical analyses were evaluated with GraphPad software (San Diego, CA). Multivariate comparisons were done with a one-way ANOVA with Tukey's multiple comparison posttest; pairwise comparisons were done with a two-tailed Student's t-test. A value of P < 0.05 was used to establish a significant difference between samples. Dose responses to increasing arsenic concentrations were evaluated using linear regression analysis; P values are given within the text. Figures are graphed ± SE unless otherwise noted.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Arsenic slows wound repair of human airway epithelial cells in vitro. Because in vivo biomarker data suggested that chronic arsenic exposure altered cell migrations and wound repair in the lung (23, 26, 27), we examined whether arsenic had an acute effect on wound healing of human airway epithelial cells in culture. In these experiments, 16HBE14o- cells were grown without arsenic until confluence. At that time, cultures were treated with growth medium supplemented with arsenic at 0, 30, 60, or 290 ppb. After 24 h, a single scratch wound was introduced into the culture, and repair of the wound was monitored over 4 h at 37°C (with or without arsenic). Under these conditions and in the absence of arsenic, the denuded area was largely repaired by migrating 16HBE14o- cells within 4 h (Fig. 1A). Increasing concentrations of arsenic from 30 to 290 ppb slowed wound repair in a dose-dependent manner. To quantify wound repair, experiments were repeated in fully supplemented medium (Fig. 1B). In the absence of arsenic in the growth medium, the area of the wound significantly decreased each hour until 80.8 ± 2.5% (n = 41) of the wound area was filled in at 4 h post-wounding. Similar to results observed in Fig. 1A, increased arsenic concentration displayed a dose-dependent inhibition of wound repair (P = 0.0003 at 3 h; P = 0.0007 at 4 h). In the presence of 30 ppb arsenic, the amount of wound closure at 4 h (48.3 ± 12.5%; n = 12) was significantly less than in the control. In the presence of 60 ppb arsenic, the amount of wound closure was only 24.3% ± 14.9% (n = 27) of the original wound after 2 h, a value significantly less than in control experiments, in which 58.5 ± 2.8% of the wound was covered at this point. Compared with the amount of wound healing in the arsenic-free medium, a significantly larger wound area persisted at 3 and 4 h in cultures incubated with 60 or 290 ppb. The delayed wound response was most dramatic at the highest arsenic concentration tested. In the presence of 290 ppb arsenic, wound area expanded to 109.2 ± 5.0% (n = 15) of the original area within 1 h and showed only 16.5 ± 18.5% healing by 4 h.


Figure 1
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  		Fig. 1. Effects of arsenic on lung epithelial wound closure in a single scrape wound model. Confluent 16HBE14o- monolayers grown for 24 h without (0 ppb) or with varying concentrations of arsenic (30, 60, 290 ppb) were subjected to a scrape wound and allowed to recover over time at 37°C and 5% CO2. A: representative phase contrast images of 16HBE14o- cells were captured at the time of wounding and 4 h following the wound to illustrate recovery from scrape wound. In this paradigm, arsenic-free cultures largely display wound recovery within 4 h. As arsenic is increased, a delay in wound healing was observed. B: the percentage of wound area closed at hourly intervals is graphed against time for cell monolayers maintained at 37°C, 5% CO2 and fully supplemented growth medium with or without arsenic. At 0 ppb arsenic, most wounds were healed within 4 h. As arsenic concentration increased, the ability to close the scrape wound was reduced. At the highest arsenic concentrations tested (290 ppb), wound area expanded before healing of the wound. Data in B are graphed ± SE; *significant difference in absorbance from 0 ppb samples in time-matched experiments (P < 0.05).

 
The observed inhibition of wound healing could be attributed to a reduction in cellular division, an increase in cellular death along the wound area, or a reduction in cellular spreading and migration. The short period of analysis and the slow duplication time of cells along the wound edge (data not shown) ruled out a prominent role for cellular division in these studies. To evaluate a role for cellular death, a live/dead assay was used to examine cells along the wound edge for up to 5 h following wounding (Fig. 2). At the time of wounding, a high number of dead cells were detected at the wound edge in the arsenic-free cultures (26.0 ± 4.2 cells/mm). This value was not significantly different from those in the 30 ppb (24.1 ± 2.2)-, 60 ppb (26.3 ± 3.6)-, or 290 ppb (24.6 ± 3.9)-treated cultures. The number of dead cells at the wound edge was reduced within 1 h of healing in the arsenic-free cultures (8.8 ± 1.3 cells/mm) as well as in the arsenic-treated cultures (11.5 ± 1.6 at 30 ppb; 12.5 ± 1.9 at 60 ppb; and 11.9 ± 1.6 at 290 ppb). Although these values were significantly different from those in the arsenic-matched cultures at the time of wounding, there were no significant differences among arsenic treatments at 1 h. At the 3- and 5-h time points, there were further reductions in the number of dead cells in all treatments, but no significant differences among the arsenic treatments at either of these times. In summary, the presence of arsenic for 24 h resulted in a significant reduction in the ability of human airway epithelial cells to repair scrape wounds in an in vitro model without an increase in cellular cytotoxicity; data suggested an arsenic-induced alteration in cellular migration.


Figure 2
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  		Fig. 2. Effects of arsenic exposure on cell death at the site of wounding. Cells were grown and subjected to scrape wounding as described in Fig. 1. At 0, 1, 3, and 5 h, individual cultures were evaluated for cytotoxicity. A: representative epifluorescent images of live (green) and dead (red) cells along the wound edge are shown for each time point and arsenic concentration. B: the number of dead cells along the wound edge after simple buffer wash is graphed at 0, 1, 3, and 5 h (10 < n < 22 wounds for each time point/arsenic concentration). At all arsenic concentrations tested, there was a significantly higher number of dead cells near the wound edge at the time of wounding compared with later time points. However, arsenic exposure did not significantly alter the number of dead cells at the wound edge at any time point. The inhibition of wound repair in the scrape wound model is not due to arsenic-induced cellular cytotoxicity. Data in B are graphed ± SE; *significant difference of dead cells per millimeter wound at time 0 compared with all other time points (P < 0.05).

 
MMP-9 mRNA expression, protein expression, and activity increase in human airway epithelial cells following exposure to arsenic. Because protease activity allows for degradation of extracellular matrix proteins and can contribute to cellular spreading and migration, we assayed whether arsenic exposure altered MMP-9 expression in 16HBE14o- cells. We used real-time RT-PCR to evaluate mRNA expression in confluent 16HBE14o- cells exposed to low-dose arsenic (Fig. 3). At 5 days of exposure, cells remained confluent at all arsenic concentrations tested (not shown) and displayed a dose-dependent increase in MMP-9 mRNA (Fig. 3A; P = 0.0002). Because the activity of MMP-9 is also dependent on inhibitors produced by the cell, we also evaluated the mRNA expression of TIMP-1, the major inhibitor of MMP-9. Unlike the MMP-9 expression, TIMP-1 mRNA expression was relatively stable at 60 ppb but displayed a sharp decrease after exposure to 290 ppb arsenic (Fig. 3B). Together, these changes in mRNA expression resulted in an effectively large increase in the MMP-9/TIMP-1 mRNA expression ratio in human airway epithelial cells in response to arsenic.


Figure 3
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  		Fig. 3. Effects of arsenic exposure on human lung epithelial matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 mRNA expression. 16HBE14o- cells were grown to confluence and treated with CGM supplemented with 0, 60, or 290 ppb arsenic. MMP-9 and TIMP-1 mRNA expression from each group was evaluated using real-time quantitative RT-PCR. A: MMP-9 mRNA expression showed a 2.8-fold increase at 60 ppb and a 3.4-fold increase at 290 ppb arsenic compared with 0 ppb controls. B: TIMP-1 mRNA expression showed an insignificant reduction at 60 ppb and a 2.8-fold reduction at 290 ppb arsenic compared with 0 ppb controls. These data are consistent with an increase in MMP-9/TIMP-1 mRNA ratio in response to low-level arsenic exposure of human lung epithelial cells. Data are graphed ± SE; *significant difference in expression from 0 ppb samples; ^significant difference in expression from 60 ppb samples (P < 0.05).

 
To better relate MMP-9/TIMP-1 expression patterns to cellular physiological changes observed in Fig. 1, we used the multiple scrape wounding method to assay specific protein changes in MMP-9 in response to arsenic during wounding. Similar to the single scrape model, the multiple scrape wounding assay resulted in a dose-dependent arsenic inhibition of wound repair (Fig. 4; P < 0.0001). We evaluated MMP-9 expression with immunocytochemistry of 16HBE14o- cells at the time of wound and continued throughout the monolayer repair process over 12 h (Fig. 5). At the time of wounding (i.e., after 24-h exposure to arsenic), a high percentage of cells exposed to 290 ppb arsenic displayed MMP-9 reactivity, whereas the 0- and 60-ppb-treated cells showed minimal staining. At 6 and 12 h following the wound, a pattern of MMP-9 reactivity in response to increasing arsenic concentrations developed, with highest expression near the wound sites associated with highest arsenic exposure. Although these results are not quantifiable, they suggest an increase in intracellular MMP-9 expression in response to acute arsenic exposure.


Figure 4
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  		Fig. 4. Effects of arsenic on lung epithelial wound closure in a multiple scrape wound model. 16HBE14o- cells were grown to confluent monolayers and treated for 24 h with fresh arsenic-free (0 ppb) or arsenic-supplemented (60 or 290 ppb) media. Coverslips were subjected to a multiple scrape assay and monitored for wound size at time 0 (A) and again at time 4 h (B). Representative crystal violet-stained coverslips from single experiments are shown above average wound area for 15-mm coverslips (7 < n < 10 for 0 h; 11 < n < 14 for 4 h; coverslip cultures were generated from at least 3 separate flask cultures for each paradigm). Although multiple scrape wounding did not result in significant differences of the initial wounds, increased arsenic concentrations resulted in a dose-dependent reduction in wound healing (P < 0.0001). At higher levels of arsenic (290 ppb), a consistent expansion of the wound area was observed at 4 h. Data in graphs are ± SE; *significant difference in wound area from 0 ppb; ^significant difference from 60 ppb experiments (P < 0.05).

 

Figure 5
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  		Fig. 5. Arsenic exposure increases MMP-9 production in lung epithelial cells. Confluent monolayers were subjected to 24 h of arsenic exposure, scrape wounded, and allowed to repair for 12 h. At 0, 6, and 12 h following the wound, individual coverslip cultures were fixed and stained for MMP-9. MMP-9 staining was most pronounced at the wound edge with higher immunofluorescence in cultures with growth medium supplemented with arsenic.

 
Because MMP-9 effects on cell migration are largely extracellular, we evaluated the effects of arsenic on MMP-9 protein release into the medium with immunoblots of conditioned media from 24-h pre-wound exposure (data not shown) and 12-h post-wound exposure to arsenic (Fig. 6). MMP-9 protein expression was increased almost 4-fold in conditioned medium that contained 60 ppb and more than 15-fold in conditioned medium that contained 290 ppb arsenic when compared with arsenic-free conditioned medium. Unlike the dose-dependent increase observed in the MMP-9 results (P = 0.007), immunoblots of conditioned media using TIMP-1 antibodies showed a similar expression of TIMP-1 protein concentration in 0, 60, or 290 ppb arsenic-supplemented conditioned media. These data are consistent with a dose-dependent, arsenic-induced increase in MMP-9/TIMP-1 protein ratio and a subsequent increase in extracellular MMP-9 activity.


Figure 6
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  		Fig. 6. Arsenic exposure increased MMP-9 protein expression. Monolayers of 16HBE14o- cells were grown to confluence and treated for 24 h with arsenic-free medium (0 ppb) or arsenic-supplemented media (60 or 290 ppb). At this time, multiple scrape wounds were applied to the culture, and conditioned medium collected at 12 h postwounding. The conditioned media were evaluated for MMP-9 and TIMP-1 protein via immunoblot. A: increased arsenic levels resulted in a 3.8-fold increase of MMP-9 protein expression in the 60-ppb samples and a 16.8-fold increase in protein expression in the 290-ppb samples. B: arsenic levels did not affect the protein levels of TIMP-1 in the conditioned medium. Together, the MMP-9/TIMP-1 ratio in the area surrounding the lung epithelial cells was increased as arsenic concentration was increased in the medium. Such an increase is consistent with an increase in MMP-9 activity. Data were graphed ± SE; *significant difference in protein expression compared with 0-ppb samples; ^significant difference in protein expression compared with 60 ppb samples (P < 0.05). As, arsenic.

 
Although protein evaluation of MMP-9 and TIMP-1 suggested increased MMP-9 function, MMP-9 protein is secreted from cells as a pro-enzyme before it is cleaved into an active form. To ensure that the detected MMP-9 protein changes represented an increase in the functional enzyme, we used gelatin zymography to directly evaluate MMP-9 activity in the conditioned media from above (Fig. 7). Conditioned media collected from 16HBE14o- cultures after 48-h exposure to arsenic and just before wounding showed a dose-dependent increase in MMP-9 activity (P < 0.0001; Fig. 7A). A slight increase in MMP-9 activity in conditioned media supplemented with 60 ppb arsenic and a significant increase in conditioned media supplemented with 290 ppb arsenic were also evident in the conditioned media collected after 12 h of wound repair, although collection at this time point was near the limit of detection (Fig. 7B). Unlike the activity of MMP-9, the activity of MMP-2 was stable across arsenic concentrations before and in response to wounding (data not shown).


Figure 7
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  		Fig. 7. Arsenic exposure increases MMP-9 activity in lung epithelial cells. Confluent monolayers of 16HBE14o- cells were grown and treated for 48 h with arsenic-free medium (0 ppb) or arsenic-supplemented media (60 or 290 ppb). A: representative gelatin zymograms of conditioned media from 0, 60, and 290 ppb and corresponding densitometry are shown. An arsenic-dependent dose-response of increased MMP-9 activity (light band at 92 kDa) was observed (P < 0.0001). B: representative gelatin zymograms of conditioned media from 16HBE14o- cells after multiple scrape wounding and 12-h recovery are shown. A slight increase in the 60-ppb sample and a significant increase in the 290-ppb sample were detected. Data are graphed ± SE; *significant difference in expression from 0-ppb samples; ^significant difference in expression from 60-ppb samples (P < 0.05).

 
To determine whether the observed increased MMP-9 activity in arsenic-treated 16HBE14o- cultures directly contributed to alteration of wound repair, we assayed wound healing response to multiple scrape wounded cultures in the presence of the MMP inhibitor GM6001. Individual monolayer cultures were treated for 24 h with growth medium supplemented with 290 ppb arsenic and either 10 µM GM6001 in DMSO or DMSO as a control. Monolayers were then subjected to multiple scrape wounds and provided fresh growth medium. As in Fig. 4, individual cultures were stained with crystal violet to evaluate the amount of wounding and wound repair at the time of wound (0 h) and following 4 or 12 h of recovery at 37°C (Fig. 8). Because the addition of DMSO alone reduced MMP-9 expression and activity (not shown), only the 290-ppb arsenic concentration was tested. By 4 h of recovery, the 290-ppb-treated cultures underwent a wound expansion (–39.2 ± 19.1% wound repair) that was partially repaired in the GM6001- treated samples (1.8 ± 5.9%). By 12 h of recovery, the DMSO controls subjected to 290 ppb arsenic had largely closed the wound expansion, but still had not progressed past the initial wound area (–8.1 ± 9.3%). In contrast, the GM6001-treated samples had repaired a significant portion of the original wound (31.3 ± 11.9%). The inhibition of MMP-9 was sufficient to significantly improve wound repair in the arsenic treatments tested in this assay.


Figure 8
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  		Fig. 8. MMP-9 inhibition improves wound repair in lung epithelial cultures. As in Fig. 4, confluent cultures were treated with 290 ppb arsenic for 24 h, this time in the presence of the MMP-9 inhibitor GM6001 or a vehicle control (DMSO). A: representative cultures after crystal violet staining to highlight cell (dark) and wound (light) areas at 0, 4, and 12 h following multiple scrape wound. B: the amount of repair at 4 and 12 h postwounding was graphed with and without MMP-9 inhibitor treatments. Wound repair in the 290-ppb samples is significantly improved by GM6001. Data in graphs are ± SE; *significant difference in wound area between GM6001 and DMSO controls in time-matched experiments; P < 0.05. For each sample, n > 14.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
It has long been accepted that high exposure to arsenic through ingestion can lead to tissue damage and cancer, including that of the lung. We have recently begun to evaluate the effects of chronic, low-dose arsenic (i.e., <100 ppb) exposure on lung tissue using protein and mRNA analyses from mouse models (26, 27) and sputum analyses from human populations (23, 27). Combined findings from these studies suggested that ingestion of arsenic may alter wound response and specifically, MMP-9/TIMP-1 ratios in the lung (23, 26, 27). In this report, we used acute arsenic exposure with an in vitro human cell model in an attempt to directly examine potential cellular mechanisms of arsenic exposure in the lung epithelium. We found that arsenic (30–290 ppb) inhibited reformation of the epithelial monolayer following scrape wounds of monolayer cultures. This alteration in wound repair was associated with increases in activity and expression of MMP-9 without increases of TIMP-1 protein expression. Furthermore, inhibition of MMP-9 resulted in improved epithelial cell wound repair response when cells were exposed to 290 ppb arsenic. Our results are consistent with the hypothesis that arsenic alters the airway epithelial barrier by restricting proper wound repair in part through the increased MMP-9/TIMP-1 ratios derived from lung epithelial cells.

We used 16HBE14o- cells to model the airway epithelium in part because they are one of the few human conducting airway cell lines capable of forming functional barriers (16, 17, 45). Within this study, we focused on the contribution of cell migration to the reestablishment of airway epithelial confluency in cell cultures using wound repair assays that limit the contributions of cell proliferation and were similar to those with 16HBE14o- in previous reports (20, 30, 34, 36, 49). Independent of the paradigm that included a single scrape wound or multiple scrape wounds, we found that arsenic significantly inhibited wound repair in a dose-dependent manner, and this inhibition could not be attributed to cytotoxicity. These previous studies with 16HBE14o- cells, in addition to other bronchial epithelial cell line or primary cultured cells (10, 20, 34–36), have established prominent roles for epidermal growth factor receptors and TGF-β in airway epithelial repair that may be altered in asthmatic-derived tissue and additionally have highlighted contributions of small GTPases, growth factors, and growth factor receptors to cellular migration, but have not implicated roles for proteases or evaluated the toxic effects of arsenic that lead to altered wound healing.

MMP-9 expression has been associated with airway epithelial wound repair in primary cultured cells and in vivo (6, 13, 29). Similar to results reported in primary cultured human respiratory epithelial cells (HRECs) (6) and human bronchial epithelial cells (HBECs) (29), MMP-9 immunoreactivity of 16HBE14o- cells reported herein (Fig. 5) was shown to be highest in cells at or near the wound edge during in vitro wound repair. To show that MMP-9 was important in cell migration during respiratory wound repair, migration of HRECs could be blocked by antibodies that neutralized MMP-9, and MMP-9 expression in HBECs directly coincided with the speed of migration. In a humanized xenograft model, MMP-9 is expressed throughout airway epithelial wound healing and cell differentiation, and blocking of this enzyme resulted in a dysregulated repair (13). Our results on 16HBE14o- cells in arsenic-free medium are consistent with findings from these studies, as MMP-9 is normally upregulated after wounding and is closely associated with cells near the wound edge. In contrast with these reports, we were able to observe dysregulated wound repair when MMP-9 is overexpressed in airway epithelial cells, i.e., in the presence of arsenic. Furthermore, we were able to test the effects of neutralizing MMP-9 overexpression in an effort to restore normal MMP-9 function and repair. The upregulation of MMP-9 activity and protein expression in 16HBE14o- cells contrasts with zymograph analysis of MMP-2, which stays at a low level of activity in arsenic-free cells or those treated with arsenic (data not shown). Together, these findings suggest that proper expression of MMP-9 is an important factor in airway epithelial wound repair in vitro.

There are few studies that relate arsenic (as arsenite) exposure to MMP-9 expression or cellular migration. In a human prostate epithelial cell line (RWPE-1), a 29-wk exposure to 375 ppb arsenite was shown to transform cells and concomitantly upregulate MMP-9 (2). The length of the exposure and transformation make comparisons with the present study difficult. In a keratinocyte cell line (HaCat cells), 4-h incubations with 2.25 ppm arsenite initiated EGF receptor signaling through MAPK pathways that resulted in increased MMP-9 production (11). These studies are interesting as previous studies on BEAS-2B cells, a bronchial epithelial cell line, have shown that a 1-h incubation with higher dose arsenite (7.5–37.5 ppm) can stimulate EGF signaling through MAPK pathways or through Ras/NF-{kappa}B pathways (50, 51). It should be noted that at these high concentrations, other metals had similar effects on BEAS-2B cells and thus may not be part of the mechanism that leads to MMP-9 production in the 16HBE14o- cells in this study. Such a conclusion is further supported by experiments in rat lung cells, which show a shift from ERK activation when exposed to 150 ppb arsenite for 24 h to JNK activation when exposed to 3 ppm arsenite over the same time period (28). This change in concentration effectively altered the cellular outcome of arsenite exposure from a proliferative effect to an apoptotic one. Arsenite exposure has been shown to alter cell migration independent of MMP-9 regulation in a fibroblast model (52). In the fibroblast model, a significant change in cellular migration was found near 200 ppb arsenite, and this change in migration was associated with alterations in focal adhesion kinases, without a significant effect on actin cytoskeleton rearrangements. These findings are in contrast with a report in an endothelial cell line that showed that 750 ppb arsenic (unknown form) alters actin cytoskeleton and can lead to superoxide production and limit cell migration (38).

In contrast with observations that arsenite can inhibit migration, there are also reports that show arsenic (37.5–375 ppb) in the form of arsenic trioxide (As2O3) can reduce carcinogenic cell invasiveness in carcinogenic cell lines in part by downregulating MMP-9 (15, 46, 53). In a nasopharyngeal cell line, As2O3 was shown to reduce MMP-9 expression, as measured by semiquantitative RT-PCR and immunoblot (15). In a second report using ovarian carcinoma cells, As2O3 exposure (37.5–150 ppb) was also shown to downregulate MMP-9 protein expression as measured by semiquantitative RT-PCR and ELISA (53). As in the previous report, the ovarian carcinoma cells displayed a reduced migration in a dose-dependent response to As2O3. As2O3 has also been used to prevent radiation-enhanced tumor invasions in cervical cancer cells (CaSki) (46). In addition to the obvious differences in cellular migration, machinery regulation among these carcinogenic cell lines, and ones used in our studies, the specific interaction of arsenic within the cell is influenced by the form of arsenic. Differences in cellular toxicity of trivalent and pentavalent inorganic and methylated arsenicals are well documented in both rat and human cells, including normal human bronchial epithelial cells (42). Although there are few direct comparison studies between cellular effects of arsenite and As2O3, it has been shown that low doses of these two arsenicals have differing effects on radiation-induced apoptosis in a human lymphoblastoid cell line (19). Thus, it is not unreasonable to believe that the chemistry or biochemistry of these compounds can lead to different cellular physiology.

Coordination of cellular migration involves an array of cellular processes that can be affected by arsenic on a variety of levels. Which process is altered by arsenic is dependent on time and concentration of arsenic exposure, as well as on the form of arsenic that is presented to the cell and the form that can be converted by the cell. We have concentrated on exposures in the range of 0 to 290 ppb to model potential damage that can result from concentrations similar to those that are encountered in chronic environmental exposures such as in drinking water. It is important to note that in January, 2006, the Environmental Protection Agency reduced the allowable arsenic level in drinking water for municipalities in the United States from 50 ppb down to 10 ppb. Yet, even at these low doses, we and others have shown distinct changes in mouse lung protein and mRNA expression (3, 26, 27). We have shown that mice chronically exposed to 10–50 ppb arsenic in drinking water increase mRNA for MMP-9 and a battery of proteins consistent with alterations in cell migration (26), as well as for altered bronchiolar lavage proteins (27) that included a biomarker for reduced wound healing [receptor for advanced glycation end products (RAGE)]. We have additionally reported that low-dose arsenic exposure (~20 ppb) in drinking water results in changes in specific wound repair proteins (e.g., RAGE and the MMP-9/TIMP-1 ratio) recovered from human sputum (23, 27). The work reported herein helps clarify initial cellular changes in human lung epithelial cells that, albeit on a more acute and at slightly higher concentrations than reported in the animal and human studies, may contribute to low-dose arsenic effects in the lung. Further work to examine specific pathways initiated by arsenic species and how they contribute to MMP-9 upregulation and inhibition of cell migration should allow insight into how arsenic contributes to compromise of lung function.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the United States Environmental Protection Agency (R832095), American Lung Association (CI-1350-N), and the National Institutes of Health (P30-ES-006694 and AI-061811).


   ACKNOWLEDGMENTS
 
We thank Brant E. Isakson for helpful suggestions and critical reading of the manuscript, Om Makwana and Raymond B. Runyan for help with and equipment used in the real-time RT-PCR experiments, and Barbara Liguori and Terri Boitano for manuscript editing.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Boitano, Arizona Respiratory Center, Arizona Health Sciences Center, 1501 N. Campbell Ave., Tucson, AZ 85724-5030 (e-mail: sboitano{at}email.arizona.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
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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