Sodium Arsenite Exposure Alters Cell Migration, Focal Adhesion Localization and Decreases Tyrosine Phosphorylation of Focal Adhesion Kinase
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《毒物学科学杂志》
Toxicology Department, School of Public Health, University of Michigan, Ann Arbor, Michigan
Department of Molecular Biosciences, Washington State University, Pullman, Washington
Department of Surgery, Medical School, University of Michigan, Ann Arbor, Michigan
Department of Cell and Developmental Biology, Medical School, University of Michigan, Ann Arbor, Michigan
ABSTRACT
Exposure to the environmental toxicant arsenic is reported to produce a variety of effects including disruption of signal transduction pathways, cell proliferation, and apoptosis. This suggests that arsenite may not have specific targets but rather extremely broad effects. The present study was designed to test the hypothesis that arsenite alters signaling involved in focal adhesion structure and function in cultured myoblasts. H9C2 cells were exposed to 1, 2.5, 5, or 10 μM sodium arsenite for 48 h. MTT metabolism and staining by neutral red, trypan blue, and propidium iodide showed that sodium arsenite treatments of 5 μM or less were not overtly cytotoxic. At these doses, sodium arsenite did not affect the amount of polymerized actin in cells, rate of protein synthesis, or amounts of vinculin, talin, paxillin, and focal adhesion kinase (FAK) in cells. However, sodium arsenite-treated cells contained fewer focal adhesions with an altered distribution pattern. Sodium arsenite exposure caused a dose-dependent reduction in cell migration and cell attachment rates. The average area of substrate covered by a cell was also reduced, although the average volume of cells was not significantly affected. Sodium arsenite exposure resulted in reduced tyrosine phosphorylation of FAK, its substrate paxillin and the FAK auto- phosphorylation site, Tyr397. Our results indicate that sodium arsenite can alter focal adhesion structure and function, thus affecting cell attachment and migration and possibly other aspects of focal adhesion function such as integrin signaling. These diverse consequences may be mediated by a relatively specific inhibition of FAK tyrosine phosphorylation, modifying scaffolding proteins.
Key Words: sodium arsenite; focal adhesions; FAK; paxillin; phosphotyrosine; cell migration.
INTRODUCTION
Human exposure to arsenic is a significant public health concern. Arsenic is a known human carcinogen and epidemiological studies have correlated arsenic exposure with increased risk for diabetes, spontaneous abortions, Blackfoot disease, and arteriosclerosis (Gebel, 2001; Hughes, 2002; Nickson et al., 1998; Tseng et al., 2002). Epidemiological studies have also shown an increased incidence of ischemic heart disease in places with high arsenic exposures (Chen et al., 1996). Exposure of cells to arsenite results in numerous effects including apoptosis, malignant cell transformation, cell cycle arrest, induction of the stress response, inhibition of cell proliferation, and cytoskeletal injury (Bode and Dong, 2002; Chou, 1989; Li and Chou, 1992; Liu et al., 2001).
Drinking water is the principle source of human exposure to arsenic, although occupational exposures also occur, particularly in the metal smelting and glass making industries (Bode and Dong, 2002; Yih et al., 2002). Arsenate, the oxidized form of arsenic, is the principal species of inorganic arsenic found in drinking water. Arsenate is reduced in vivo to arsenite by either glutathione or arsenate reductase. Arsenite may be further metabolized to monomethylarsonic acid and dimethylarsonic acid with S-adenosyl-methionine acting as the methyl-donating cofactor (Gebel, 2001). Arsenite is generally considered to be the more toxic species, possibly due to an increased reactivity of the trivalent form with biomacromolecules (Bode and Dong, 2002).
The exact mechanism of arsenite's toxicity is unknown. Several possibilities have been proposed including alterations in DNA repair and methylation and the generation of reactive oxygen species (ROS) (Harris and Shi, 2003; Shi et al., 2004). Arsenite also has the ability to bind to protein thiol groups. It is believed that the binding of thiol groups as well as ROS generation can alter many protein functions. Thus, the distinct and diverse effects of arsenite may result from the activation and/or inactivation of a variety of signal transduction pathways in cells (Qian et al., 2003).
The present study developed from our initial observation that treatment of cells with sub-lethal concentrations of sodium arsenite reduced cell migration rates. Cell migration is a complex and dynamic process that is dependent upon several series of events including the assembly and disassembly of focal adhesions. Focal adhesions are sites in cells that mediate a connection between the actin cytoskeleton and the extracellular matrix (ECM). Focal adhesions are also believed to be important sites of cell signaling events because several different protein kinases have been localized to these structures (Zamir and Geiger, 2001). Sodium arsenite has been shown to affect several of the signaling kinases (Ishrath et al., 2002; Porter et al., 1999; Trouba et al., 2000; Yih et al., 2002) localized in focal adhesions (Clark and Brugge, 1995; Fincham et al., 2000; Martin et al., 2002; Zamir and Geiger, 2001).
We hypothesized that the reduced cell migration rates we observed would be the result of disruption of focal adhesion function caused by arsenite inhibition of protein kinases involved in focal adhesion regulation. We report here that sodium arsenite at sublethal concentrations alters numbers and distribution of focal adhesions, reduces protein phosphor- ylation of focal adhesion proteins as well as cell adhesion to a substrate, while total polymerized actin and the expression of focal adhesion proteins are unaffected. In a larger context, altered structure and signaling of focal adhesion structures could be expected to have a variety of additional effects in cells that could account for many of the observed effects of arsenite including increased apoptosis, characteristics of malignancy, cell cycle arrest/inhibition of proliferation, and disruption of the cell cytoskeleton.
MATERIALS AND METHODS
Cell culture and arsenite exposure.
H9C2 cells, an immortalized cell line derived from fetal rat hearts (Hescheler et al., 1991), were purchased from the American Type Culture Collection (CRL-1446). Cells are cultured in Dulbecco's Minimal Essential Medium (Life Technologies) supplemented with 10% fetal bovine serum (JRH-Biosciences) and 1% penicillin and streptomycin (Life Technologies) and maintained in 95% air and 5% CO2 at 37°C. Cells were treated with 0, 1, 2.5, 5, or 10 μM sodium arsenite for all cytotoxicity experiments, while for all additional experiments cells were treated with sodium arsenite concentrations of 0, 1, 2.5, or 5 μM.
Cell cytotoxicity assays.
The cytotoxicity to H9C2 cells of sodium arsenite following 24 or 48 h exposure was assayed by the colorimetric conversion of tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as previously described (Morgan, 1998) or by propidium iodide (PI; Molecular Probes) staining as previously described (Bonham et al., 2003). For MTT assays 48 wells of a 96 well plate were prepared for each arsenite concentration and results combined for statistical analysis. A minimum of 50 cells were scored from each plate in three separate experiments to analyze PI staining.
Neutral red and trypan blue absorbance assays.
H9C2 cells were exposed to sodium arsenite for 24 or 48 h. Both assays were conducted as previously described (Bonham et al., 2003). Neutral red and trypan blue measurements from three independent experiments were combined for statistical analysis.
Tritiated leucine incorporation.
H9C2 cells were exposed to 1, 2.5, or 5 μM sodium arsenite for 42 h. The medium was changed to DMEM minus leucine supplemented with sodium arsenite and 5 mCi/ml 3H-leucine (170 Ci/mmol) and the cells were cultured an additional 6 h. Cell lysates were analyzed for 3H- leucine incorporation by TCA precipitation essentially as described (Bonifacino, 1998). For each experiment, three cultures were assayed for each arsenite concentration and the experiment was repeated three times.
Cell migration assay and time-lapse video microscopy.
H9C2 cells were grown in 35 mm tissue culture dishes until confluent. A wound was made using a cell scraper to provide a linear, cell-free area in the middle of the dish and cells were incubated in DMEM with 5 mg/l HEPES (pH 7.2) with or without sodium arsenite. Temperature was maintained at 37°C using an Airtherm Airstream Incubator and images were recorded at 3-min intervals for approximately 16 h using a cooled integrating CCD camera mounted on a Nikon light microscope using a 10X objective lens. Six videos were made for each treatment and the movement 10 cells were analyzed from each movie.
Immunofluorescence labeling.
H9C2 cells plated on glass coverslips were exposed to sodium arsenite for 48 h. After treatment, cells were fixed in 3.7% paraformaldehyde and permeabilized with PBS + 0.5% Triton X-100 for 10 min. The coverslips were incubated with a vinculin antibody (Sigma), in blocking buffer (2% gelatin (225 bloom), 0.2% saponin and 0.02% sodium azide (Sigma) overnight at 4°C. The coverslips were washed with PBS before being incubated with a rhodamine-conjugated antimouse secondary antibody in blocking buffer (Jackson Immunochemicals) for 2 h at room temperature. The coverslips were mounted using Prolong antifade mounting media (Molecular Probes). Images were obtained using a Nikon Eclipse TE 2000-U microscope with a 40X oil objective lens.
One-dimensional SDS-page gel electrophoresis and Western blotting.
Following sodium arsenite exposure, samples were solubilized in SDS-page sample buffer and equal amounts of protein were loaded and resolved on 7.5% polyacrylamide mini-gels and transferred to PVDF membranes (Millipore). Western blotting was performed as previously described (Wu and Welsh, 1996). The primary antibodies used were paxillin (BD Biosciences), vinculin (Sigma), talin (Sigma), FAK (BD Biosciences), and FAK-pY397 (Biosource International) while secondary antibodies were horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Jackson ImmunoResearch). Labeled protein was visualized by enhanced chemiluminescence (Amersham) and films were digitized with a flatbed scanner (Bio-Rad). This experiment was repeated three times and essentially identical results were obtained.
Measurement of cell F-actin content.
F-actin content in cells exposed to sodium arsenite for 48 h was measured as previously described (Singhal et al., 1992). Briefly, the cells were washed with warmed PBS and fixed for 30 min in 3.7% paraformaldehyde before being incubated in a phalloidin-Oregon Green 488 (Molecular Probes), solution (0.4 μM Oregon green 488 in PBS containing 0.1% saponin), for 30 min in the dark. The dishes were washed twice with PBS containing 0.1% saponin and the bound phalloidin was extracted using 1 ml of methanol for 30 min in the dark. The methanol-extracted phalloidin-Oregon green 488 was transferred to a microcentrifuge tube and centrifuged for 5 min at 13,000 x g after which 250 μl of the sample was diluted into 1.75 ml of methanol. Fluorescence was measured using a Perkin-Elmer LS-50B Luminescence Spectrometer with emission/excitation settings of 496 and 520 nm, respectively. Protein was determined by Bradford assay (Sigma) according to the manufacturer's directions. Four separate replicates were measured and the experiment repeated three times.
Cell attachment assay.
H9C2 cells were treated with sodium arsenite for 48 h. The cells were trypsinized, counted and 1 x 104 cells were plated in 96 well plates and allowed to attach for 15, 30, and 60 min. Next, the supernatant was removed and the cells washed twice with warm PBS before being fixed in 3.7% paraformaldehyde and allowed to air dry. The cells were stained with a 0.1% crystal violet solution for 20 min. The dye was aspirated and the wells washed three times with reagent grade water. The dye was extracted in 10% acetic acid and transferred to a new 96 well plate. The absorbance of each sample was read at 570 nm using a Bio-Rad plate reader. For each experiment, attachment of cells was measured in 48 wells of a 96 well plate and repeated three times.
Cell area analysis.
H9C2 cells in six-well tissue culture dishes were treated with sodium arsenite for 48 h. The cells were fixed with 3.7% paraformaldehyde, observed with a Zeiss Axiovert 135 microscope using a 10X objective lens and phase contrast microphotographs were collected of randomly selected cells by an investigator unaware of the treatments given each sample. Cell area was measured on 100 cells for each treatment using NIH Image 1.61 software, and the experiment was repeated three times.
Detection of focal adhesion protein tyrosine phosphorylation.
H9C2 cells were treated with sodium arsenite for 48 h. Cells were harvested and lysed in low-salt lysis buffer (1.0% NP-40, 50 mM Tris; pH 8.0) on ice for 30 min. The lysate was centrifuged for 15 min at 15,000 x g and the supernatant incubated with 2 μl paxillin or FAK antibodies (BD Biosciences) for 2 h on a rotating platform. Protein G Sepharose (Sigma) was added and the samples were incubated for an additional 2 h at 4°C. Samples were centrifuged at 3000 x g for 3 min and the beads were washed three times with lysis buffer. Samples were boiled at 100°C for 5 min and the supernatant fluid collected after centrifugation (15 min at 15,000 x g at 4°C). The samples were analyzed by one-dimensional gel electrophoresis, and Western blotting was conducted using a phosphotyrosine antibody (Transduction Labs). The Western blots were reprobed with the respective antibody used for the immunoprecipitation to ensure a similar amount of protein was precipitated from each sample. This experiment was repeated three times and results were virtually identical for each experiment.
Statistical analysis.
Results were analyzed for statistical significance by one-way ANOVA followed by Dunnett's post-test using Prism software (Graphpad Inc.). p values < 0.05 were considered to be significant.
RESULTS
Viability of H9C2Cells after Sodium Arsenite Treatment
Cell viability of sodium arsenite-treated cells was assessed with the MTT colorimetric assay, by neutral red and trypan blue staining, and by propidium iodide staining. In the MTT assay, mitochondrial dehydrogenases of viable cells convert MTT to purple formazan crystals. There was a significant decrease in MTT conversion in cells treated with 10 μM sodium arsenite for 24 and 48 h (Fig. 1). Total cell death was also measured using propidium iodide, which indicates loss of membrane integrity because viable cells are able to exclude the dye. Significant cell death was observed only after 10 μM sodium arsenite treatment for 24 or 48 h (Fig. 1). Neutral red, which indicates lysozyme activity in viable cells, and trypan blue staining, which allows estimation of total cell protein, was used to further determine the effects of sodium arsenite on H9C2 cell viability. Neutral red and trypan blue staining (Fig. 1) were significantly reduced only after 10 μM sodium arsenite. Leucine incorporation into proteins in control cells was also not significantly different from cells treated for 48 h with 1, 2.5, or 5 μM sodium arsenite (data not shown). Because only at the 10 μM sodium arsenite dose level was cell viability significantly affected, as indicated by several measures of cell viability, all subsequent experiments employed sodium arsenite concentrations of 1, 2.5, or 5 μM, doses which were considered to be sublethal.
Sodium Arsenite Effect on Cell Migration Rates
Time-lapse video microscopy was used to measure changes in cell migration rates caused by sodium arsenite treatment. H9C2 cell migration rates were analyzed during two days of sodium arsenite treatment for approximately 16 h each day. Migration rates are expressed in microns per hour. H9C2 cells were grown in 35 mm tissue culture dishes until confluent at which time a wound was made in the cell monolayer by scraping across the dish with a rubber cell scraper. Cell migration was measured by analyzing changes in cells at the edge of the wounded monolayer. During both days of sodium arsenite treatment, migration rates were decreased in comparison to control cells, with significant changes in migration rates occurring at sodium arsenite doses of 2.5 and 5 μM (Fig. 2).
Sodium Arsenite Effect on Distribution and Numbers of Focal Adhesions
During cell migration, focal adhesions are formed toward the leading edge of cells, while disassembly of adhesions occurs in the trailing edge of cells (Lauffenburger and Horwitz, 1996). This cycle of assembly and disassembly of focal adhesions is part of the process that allows the cell to move forward. To evaluate the effect of sodium arsenite on focal adhesion distributions and numbers, focal adhesions were detected by immunofluorescence localization of the structural protein vinculin after cells were treated with sodium arsenite for 48 h. Control cells and cells treated with 1 μM sodium arsenite exhibited focal adhesions both at the periphery as well as under more central regions of the cells (Fig. 3). Exposure to either 2.5 or 5 μM sodium arsenite resulted in changes in the distribution of adhesions, with adhesions being primarily at the cells' periphery (Fig. 3). The numbers of focal adhesions per cell was also reduced in sodium arsenite-treated cells (Fig. 4).
Sodium Arsenite Effect on Total F-actin Content
The polymerization state of actin in cells is another possible indicator of cell viability and is an important component related to focal adhesion formation, cell migration and adhesion. A phalloidin-binding assay was used to provide a measure of the total F-actin content in cells. F-actin content was standardized by normalizing measured F-actin to total cell protein. Total F-actin content was significantly decreased in cells only after 48 h exposure to 5 μM sodium arsenite, with lower doses having no significant effect on F-actin content of cells (Fig. 5).
Sodium Arsenite Effect on Cell Attachment and Area Covered by Spread Cells
We hypothesized that if sodium arsenite alters focal adhesion distribution and number then it could alter, in addition to migration, other cell activities in which focal adhesions are important. The ability of cells to adhere to a substrate depends on focal adhesions and is critical for cell motility as well as survival. Therefore, we analyzed the ability of sodium arsenite-treated cells to attach to a substrate. To determine if sodium arsenite exposure could affect cell attachment we treated H9C2 cells for 48 h with 1, 2.5, or 5 μM sodium arsenite. The cells were trypsinized and plated in 96 well plates for either 15, 30, or 60 min before being fixed and stained with crystal violet. Crystal violet absorbance was reduced with increasing concentrations of sodium arsenite indicating that the treated cells were adhering to the cell culture substrate at a slower rate (Fig. 6A).
Cell attachment leads to assembly of focal adhesions and spreading of the cell on the culture substrate. If cells are not attaching well, then the area covered by a spread cell may be reduced. To establish if cells were spreading when exposed to sodium arsenite, H9C2 myoblast cell area was measured. To determine cell area, digital phase contrast micrographs of 100 randomly selected control or sodium arsenite-exposed cells were analyzed. Cell area was measured by manually outlining the border of each cell and integrating the enclosed areas. There was no significant change in cell area in response to 1 μM sodium arsenite treatment. Following 48 h exposure to 2.5 or 5 μM sodium arsenite, the cells exhibited a significant reduction in average total area (Fig. 6B). If cells appeared to cover a smaller area, it is possible that the cells were spreading but covered less area because they were smaller in size. Flow cytometry was used to estimate the volume of trypsinized and rounded H9C2 cells. The mean forward scatter was used to determine if sodium arsenite had an effect on cell volume. The data indicated that there was a slight, but not statistically significant, increase in the volume of treated cells (data not shown).
Sodium Arsenite Effect on Expression of Focal Adhesion Proteins
To date over fifty different proteins have been localized to or suggested to be components of focal adhesions (Zamir and Geiger, 2001). We chose to measure the amounts of several major focal adhesion proteins, including FAK, paxillin, talin and vinculin, to determine if sodium arsenite affects focal adhesion protein expression. Cells were exposed to 1, 2.5, or 5 μM sodium arsenite for 48 h and then harvested for Western blot analysis. There was no apparent difference in the amounts of FAK, paxillin, talin, or vinculin (Fig. 7) in cells in response to any tested level of sodium arsenite exposure. These results indicate that sodium arsenite at the doses employed did not significantly affect focal adhesion protein expression.
Sodium Arsenite Effect on Focal Adhesion Protein Tyrosine Phosphorylation
We next sought to determine the effect of sodium arsenite exposure on the tyrosine phosphorylation of FAK. H9C2 cells were treated for 48 h with 0, 1, 2.5, or 5 μM sodium arsenite. Cell lysates were immunoprecipitated with anti-FAK antibody and further analyzed by western blotting with an anti- phosphotyrosine antibody. Sodium arsenite exposure caused a reduction in the tyrosine phosphorylation of FAK (Fig. 8A; panel 1) in a dose dependent manner. The Western blot membranes were stripped and reprobed with the FAK antibody to ensure that similar amounts of total FAK had been precipitated from each sample (Fig. 8A; panel 2). Western blot analysis confirmed that similar amounts of FAK were precipitated.
Reduced FAK tyrosine phosphorylation could be due to reduced phosphorylation of several tyrosine residues located within this protein. An important phosphorylation site for FAK function is the autophosphorylation site of tyrosine 397 (Tyr397) (Eide et al., 1995; Schaller et al., 1994). To test the hypothesis that sodium arsenite could inhibit FAK autophosphorylation at Tyr397, we examined the effect of sodium arsenite on this site by Western blot analysis using an antibody specific for this phosphorylated residue. The amount of Tyr397 showed a dose-dependent decrease after 48 h sodium arsenite exposure (Fig. 8B). These results suggest that the decrease in FAK tyrosine phosphorylation may be due to reduced phosphorylation of Tyr397, the major autophosphorylation site of FAK.
FAK, once phosphorylated at Tyr397, has the ability to phosphorylate additional focal adhesion proteins including paxillin (Bellis et al., 1995; Schaller and Parsons, 1994; Turner, 2000). If the decrease in Tyr397 phosphorylation of FAK were of functional significance, then it would be expected that phosphorylation of paxillin would also be decreased. We examined phosphorylation of paxillin by immunoprecipitation of paxillin followed by Western blot of tyrosine phosphorylation of the immunoprecipitated protein. Sodium arsenite exposure reduced paxillin's tyrosine phosphorylation in a dose-dependent manner (Fig. 8C; panel 1). Reprobing the membrane with paxillin antibody confirmed that similar amounts of paxillin had been precipitated from each sample (Fig. 8C; panel 2).
DISCUSSION
The genesis of these studies was an initial observation that a sublethal, 24–48 h exposure of cultured H9C2 cells to sodium arsenite reduced the migration rate of the cells. In an effort to better understand this phenomenon, we examined several aspects of cells that are believed to be involved in motility.
We first established what sodium arsenite treatments would not be significantly toxic to the cells. Treatments that were obviously toxic would likely affect many cell functions and would not reveal the pathways most sensitive to exposure to sodium arsenite. As measures of toxicity, we assayed the cells' ability to oxidize MTT, to take up neutral red or stain with trypan blue and to exclude propidium iodide. We also measured [H3] leucine incorporation into cell proteins (data not shown). For all of these measures, the lower doses of sodium arsenite employed in these studies (i.e., 1, 2.5, or 5 μM) caused no detectable toxicity to the cells, indicating that the results observed were not due to overt cytotoxicity.
We quantified cell motility in control and sodium arsenite- treated cells and found a dose-dependent decrease in motility. This observed decrease in motility correlated with a different distribution of focal adhesions, fewer numbers of focal adhesions, decreased attachment of cells to the substrate and less spreading of cells. However, the amounts of focal adhesion proteins (FAK, paxillin, talin, and vinculin) in the cells was not affected by sodium arsenite and at least at lower doses of sodium arsenite, the amount of polymerized actin in cells did not change significantly. That vinculin localization to focal adhesions decreased although total amount of vinculin and other focal adhesion proteins in cells was not changed indicated that sodium arsenite was affecting focal adhesion structure or formation and not turnover or amounts of focal adhesion proteins.
It has been shown by others that focal adhesion formation requires phosphorylation of FAK (Illic et al., 1997) and that FAK subsequently phosphorylates other focal adhesion proteins including paxillin (Bellis et al., 1995). Conversely, disassembly of focal adhesions correlates with dephosphorylation of these proteins (van de Water et al., 2001). Our results indicated that fewer focal adhesions were present, that they were possibly forming more slowly, and that cells were spreading less in the presence of sodium arsenite. Thus, we hypothesized that phosphorylation of focal adhesion proteins might be reduced in sodium arsenite treated cells. Increased FAK phosphorylation is initially due to the autophosphorylation of Tyr397 (Schaller et al., 1994). Therefore, we examined FAK phosphorylation, phosphorylation of its autophosphorylation site, Tyr397, as well as phosphorylation of paxillin, a FAK substrate. Reduced phosphorylation was observed for both FAK and Tyr397 of FAK. Tyrosine phosphorylation of paxillin was also reduced. A mechanism has not yet been determined for how decreased phosphorylation at Tyr397 is able to alter FAK localization and function in focal adhesions. However, FAK's decreased phosphotyrosine levels may have hindered its ability to phosphor- ylate other focal adhesion proteins, thus explaining the observed reduced tyrosine phosphorylation level of the FAK substrate paxillin.
The finding that inhibition of cell migration and decreased formation of focal adhesions is accompanied by reduction in FAK and paxillin tyrosine phosphorylation is consistent with other reports. For example, van de Water et al. (2001) demonstrated that reduced FAK tyrosine phosphorylation correlates with reduced paxillin tyrosine phosphorylation and with focal adhesion disruption. In a separate investigation, cerivastatin was seen to disrupt the actin cytoskeleton, which leads to the inhibition of glioblastoma cell migration. The process was shown to correlate with focal adhesion disruption and also with reduced FAK tyrosine phosphorylation (Obara et al., 2002). Conversely, lysophophatidic acid induces the formation of actin stress fibers, increases focal adhesions and increases the tyrosine phosphor- ylation of FAK and paxillin. This causes the enhancement of ovarian cancer cell migration in lysophophatidic acid treated cells (Sawada et al., 2002). The alteration of focal adhesion formation in sodium arsenite treated cells supports the conclusion that the toxicant decreases cell migration through an effect on focal adhesions and disruption of cell interactions with the extra-cellular matrix.
In contrast to the results we report here, sodium arsenite has also been shown to increase the tyrosine phosphorylation of proteins in murine T thymocytes and primary vascular cells in a dose dependent manner (Barchowsky et al., 1999; Hossain et al., 2000). The differences in our results from those of Barchowsky et al. and Hossain et al. may be due to differences in the cell types employed for experiments or in time of exposure to sodium arsenite. We treated cells for many hours while in the other studies the longest exposure time was 45 min. It is also possible that there is a biphasic effect and that acute responses by cells may involve pathways that are different from those that become involved after longer term exposure to sodium arsenite. These differences suggest that the complete series of events that lead to the inhibition of focal adhesions and phosphorylation of FAK and paxillin that we describe here are likely to be complex and to involve a changing succession of events over hours to days, and longer periods of time. Understanding the basis for these differences may be important in understanding the disease processes associated with chronic, long-term arsenic ingestion.
Focal adhesions are involved in integrin signaling and the fact that focal adhesions can be disrupted by sodium arsenite suggests that integrin signaling pathways may also be disrupted by arsenic exposure. Disruption of focal adhesions and cell interactions with the extra-cellular matrix can cause anoikis and reduced cell motility (Obara et al., 2002; van de Water et al., 2001). This has been observed in various pathologies including cancer, ischemia-repufusion injury, and cardiovascular diseases (Keely et al., 1998; Norman and Fine, 1999; van de Water et al., 1999, 2001). Focal adhesions are analogous to several structures in vivo including dense sarcolema plaques in smooth muscle and costameres observed in cardiac myocytes (Sharp et al., 1997). Focal adhesion proteins are localized in the Z-line of both cardiac and skeletal muscle and it is believed that contractile forces exerted at these structures are converted into chemical signaling by focal adhesions (Burridge and Chrzanowska-Wodnicka, 1996; Ross and Borg, 2001). Thus, sodium arsenite disruption of focal adhesion proteins may hinder their normal activity in muscle cells, possibly disrupting cell contraction and signaling.
Several different signaling molecules or pathways believed to be involved in cell migration have been shown previously to be affected by sodium arsenite including ERK, p21 activated kinase, PKC and the Rho family of small GTPases (Lauffenburger and Horwitz, 1996; Porter et al., 1999). The results we report here suggest sodium arsenite specifically affects FAK phosphorylation, phosphorylation of paxillin, and subsequent focal adhesion functions. Additional experiments should reveal how various signal transduction pathways that regulate focal adhesion assembly and function are affected by sodium arsenite. Understanding these pathways should lead to better insight into how the environmental toxicant arsenic leads to cancer and the other pathologies associated with arsenic exposure.
ACKNOWLEDGMENTS
This work was supported by NIH award F31HL68424 to S.L.Y. and NIH grant P01 ES 11188 to M.J.W.
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Department of Molecular Biosciences, Washington State University, Pullman, Washington
Department of Surgery, Medical School, University of Michigan, Ann Arbor, Michigan
Department of Cell and Developmental Biology, Medical School, University of Michigan, Ann Arbor, Michigan
ABSTRACT
Exposure to the environmental toxicant arsenic is reported to produce a variety of effects including disruption of signal transduction pathways, cell proliferation, and apoptosis. This suggests that arsenite may not have specific targets but rather extremely broad effects. The present study was designed to test the hypothesis that arsenite alters signaling involved in focal adhesion structure and function in cultured myoblasts. H9C2 cells were exposed to 1, 2.5, 5, or 10 μM sodium arsenite for 48 h. MTT metabolism and staining by neutral red, trypan blue, and propidium iodide showed that sodium arsenite treatments of 5 μM or less were not overtly cytotoxic. At these doses, sodium arsenite did not affect the amount of polymerized actin in cells, rate of protein synthesis, or amounts of vinculin, talin, paxillin, and focal adhesion kinase (FAK) in cells. However, sodium arsenite-treated cells contained fewer focal adhesions with an altered distribution pattern. Sodium arsenite exposure caused a dose-dependent reduction in cell migration and cell attachment rates. The average area of substrate covered by a cell was also reduced, although the average volume of cells was not significantly affected. Sodium arsenite exposure resulted in reduced tyrosine phosphorylation of FAK, its substrate paxillin and the FAK auto- phosphorylation site, Tyr397. Our results indicate that sodium arsenite can alter focal adhesion structure and function, thus affecting cell attachment and migration and possibly other aspects of focal adhesion function such as integrin signaling. These diverse consequences may be mediated by a relatively specific inhibition of FAK tyrosine phosphorylation, modifying scaffolding proteins.
Key Words: sodium arsenite; focal adhesions; FAK; paxillin; phosphotyrosine; cell migration.
INTRODUCTION
Human exposure to arsenic is a significant public health concern. Arsenic is a known human carcinogen and epidemiological studies have correlated arsenic exposure with increased risk for diabetes, spontaneous abortions, Blackfoot disease, and arteriosclerosis (Gebel, 2001; Hughes, 2002; Nickson et al., 1998; Tseng et al., 2002). Epidemiological studies have also shown an increased incidence of ischemic heart disease in places with high arsenic exposures (Chen et al., 1996). Exposure of cells to arsenite results in numerous effects including apoptosis, malignant cell transformation, cell cycle arrest, induction of the stress response, inhibition of cell proliferation, and cytoskeletal injury (Bode and Dong, 2002; Chou, 1989; Li and Chou, 1992; Liu et al., 2001).
Drinking water is the principle source of human exposure to arsenic, although occupational exposures also occur, particularly in the metal smelting and glass making industries (Bode and Dong, 2002; Yih et al., 2002). Arsenate, the oxidized form of arsenic, is the principal species of inorganic arsenic found in drinking water. Arsenate is reduced in vivo to arsenite by either glutathione or arsenate reductase. Arsenite may be further metabolized to monomethylarsonic acid and dimethylarsonic acid with S-adenosyl-methionine acting as the methyl-donating cofactor (Gebel, 2001). Arsenite is generally considered to be the more toxic species, possibly due to an increased reactivity of the trivalent form with biomacromolecules (Bode and Dong, 2002).
The exact mechanism of arsenite's toxicity is unknown. Several possibilities have been proposed including alterations in DNA repair and methylation and the generation of reactive oxygen species (ROS) (Harris and Shi, 2003; Shi et al., 2004). Arsenite also has the ability to bind to protein thiol groups. It is believed that the binding of thiol groups as well as ROS generation can alter many protein functions. Thus, the distinct and diverse effects of arsenite may result from the activation and/or inactivation of a variety of signal transduction pathways in cells (Qian et al., 2003).
The present study developed from our initial observation that treatment of cells with sub-lethal concentrations of sodium arsenite reduced cell migration rates. Cell migration is a complex and dynamic process that is dependent upon several series of events including the assembly and disassembly of focal adhesions. Focal adhesions are sites in cells that mediate a connection between the actin cytoskeleton and the extracellular matrix (ECM). Focal adhesions are also believed to be important sites of cell signaling events because several different protein kinases have been localized to these structures (Zamir and Geiger, 2001). Sodium arsenite has been shown to affect several of the signaling kinases (Ishrath et al., 2002; Porter et al., 1999; Trouba et al., 2000; Yih et al., 2002) localized in focal adhesions (Clark and Brugge, 1995; Fincham et al., 2000; Martin et al., 2002; Zamir and Geiger, 2001).
We hypothesized that the reduced cell migration rates we observed would be the result of disruption of focal adhesion function caused by arsenite inhibition of protein kinases involved in focal adhesion regulation. We report here that sodium arsenite at sublethal concentrations alters numbers and distribution of focal adhesions, reduces protein phosphor- ylation of focal adhesion proteins as well as cell adhesion to a substrate, while total polymerized actin and the expression of focal adhesion proteins are unaffected. In a larger context, altered structure and signaling of focal adhesion structures could be expected to have a variety of additional effects in cells that could account for many of the observed effects of arsenite including increased apoptosis, characteristics of malignancy, cell cycle arrest/inhibition of proliferation, and disruption of the cell cytoskeleton.
MATERIALS AND METHODS
Cell culture and arsenite exposure.
H9C2 cells, an immortalized cell line derived from fetal rat hearts (Hescheler et al., 1991), were purchased from the American Type Culture Collection (CRL-1446). Cells are cultured in Dulbecco's Minimal Essential Medium (Life Technologies) supplemented with 10% fetal bovine serum (JRH-Biosciences) and 1% penicillin and streptomycin (Life Technologies) and maintained in 95% air and 5% CO2 at 37°C. Cells were treated with 0, 1, 2.5, 5, or 10 μM sodium arsenite for all cytotoxicity experiments, while for all additional experiments cells were treated with sodium arsenite concentrations of 0, 1, 2.5, or 5 μM.
Cell cytotoxicity assays.
The cytotoxicity to H9C2 cells of sodium arsenite following 24 or 48 h exposure was assayed by the colorimetric conversion of tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as previously described (Morgan, 1998) or by propidium iodide (PI; Molecular Probes) staining as previously described (Bonham et al., 2003). For MTT assays 48 wells of a 96 well plate were prepared for each arsenite concentration and results combined for statistical analysis. A minimum of 50 cells were scored from each plate in three separate experiments to analyze PI staining.
Neutral red and trypan blue absorbance assays.
H9C2 cells were exposed to sodium arsenite for 24 or 48 h. Both assays were conducted as previously described (Bonham et al., 2003). Neutral red and trypan blue measurements from three independent experiments were combined for statistical analysis.
Tritiated leucine incorporation.
H9C2 cells were exposed to 1, 2.5, or 5 μM sodium arsenite for 42 h. The medium was changed to DMEM minus leucine supplemented with sodium arsenite and 5 mCi/ml 3H-leucine (170 Ci/mmol) and the cells were cultured an additional 6 h. Cell lysates were analyzed for 3H- leucine incorporation by TCA precipitation essentially as described (Bonifacino, 1998). For each experiment, three cultures were assayed for each arsenite concentration and the experiment was repeated three times.
Cell migration assay and time-lapse video microscopy.
H9C2 cells were grown in 35 mm tissue culture dishes until confluent. A wound was made using a cell scraper to provide a linear, cell-free area in the middle of the dish and cells were incubated in DMEM with 5 mg/l HEPES (pH 7.2) with or without sodium arsenite. Temperature was maintained at 37°C using an Airtherm Airstream Incubator and images were recorded at 3-min intervals for approximately 16 h using a cooled integrating CCD camera mounted on a Nikon light microscope using a 10X objective lens. Six videos were made for each treatment and the movement 10 cells were analyzed from each movie.
Immunofluorescence labeling.
H9C2 cells plated on glass coverslips were exposed to sodium arsenite for 48 h. After treatment, cells were fixed in 3.7% paraformaldehyde and permeabilized with PBS + 0.5% Triton X-100 for 10 min. The coverslips were incubated with a vinculin antibody (Sigma), in blocking buffer (2% gelatin (225 bloom), 0.2% saponin and 0.02% sodium azide (Sigma) overnight at 4°C. The coverslips were washed with PBS before being incubated with a rhodamine-conjugated antimouse secondary antibody in blocking buffer (Jackson Immunochemicals) for 2 h at room temperature. The coverslips were mounted using Prolong antifade mounting media (Molecular Probes). Images were obtained using a Nikon Eclipse TE 2000-U microscope with a 40X oil objective lens.
One-dimensional SDS-page gel electrophoresis and Western blotting.
Following sodium arsenite exposure, samples were solubilized in SDS-page sample buffer and equal amounts of protein were loaded and resolved on 7.5% polyacrylamide mini-gels and transferred to PVDF membranes (Millipore). Western blotting was performed as previously described (Wu and Welsh, 1996). The primary antibodies used were paxillin (BD Biosciences), vinculin (Sigma), talin (Sigma), FAK (BD Biosciences), and FAK-pY397 (Biosource International) while secondary antibodies were horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Jackson ImmunoResearch). Labeled protein was visualized by enhanced chemiluminescence (Amersham) and films were digitized with a flatbed scanner (Bio-Rad). This experiment was repeated three times and essentially identical results were obtained.
Measurement of cell F-actin content.
F-actin content in cells exposed to sodium arsenite for 48 h was measured as previously described (Singhal et al., 1992). Briefly, the cells were washed with warmed PBS and fixed for 30 min in 3.7% paraformaldehyde before being incubated in a phalloidin-Oregon Green 488 (Molecular Probes), solution (0.4 μM Oregon green 488 in PBS containing 0.1% saponin), for 30 min in the dark. The dishes were washed twice with PBS containing 0.1% saponin and the bound phalloidin was extracted using 1 ml of methanol for 30 min in the dark. The methanol-extracted phalloidin-Oregon green 488 was transferred to a microcentrifuge tube and centrifuged for 5 min at 13,000 x g after which 250 μl of the sample was diluted into 1.75 ml of methanol. Fluorescence was measured using a Perkin-Elmer LS-50B Luminescence Spectrometer with emission/excitation settings of 496 and 520 nm, respectively. Protein was determined by Bradford assay (Sigma) according to the manufacturer's directions. Four separate replicates were measured and the experiment repeated three times.
Cell attachment assay.
H9C2 cells were treated with sodium arsenite for 48 h. The cells were trypsinized, counted and 1 x 104 cells were plated in 96 well plates and allowed to attach for 15, 30, and 60 min. Next, the supernatant was removed and the cells washed twice with warm PBS before being fixed in 3.7% paraformaldehyde and allowed to air dry. The cells were stained with a 0.1% crystal violet solution for 20 min. The dye was aspirated and the wells washed three times with reagent grade water. The dye was extracted in 10% acetic acid and transferred to a new 96 well plate. The absorbance of each sample was read at 570 nm using a Bio-Rad plate reader. For each experiment, attachment of cells was measured in 48 wells of a 96 well plate and repeated three times.
Cell area analysis.
H9C2 cells in six-well tissue culture dishes were treated with sodium arsenite for 48 h. The cells were fixed with 3.7% paraformaldehyde, observed with a Zeiss Axiovert 135 microscope using a 10X objective lens and phase contrast microphotographs were collected of randomly selected cells by an investigator unaware of the treatments given each sample. Cell area was measured on 100 cells for each treatment using NIH Image 1.61 software, and the experiment was repeated three times.
Detection of focal adhesion protein tyrosine phosphorylation.
H9C2 cells were treated with sodium arsenite for 48 h. Cells were harvested and lysed in low-salt lysis buffer (1.0% NP-40, 50 mM Tris; pH 8.0) on ice for 30 min. The lysate was centrifuged for 15 min at 15,000 x g and the supernatant incubated with 2 μl paxillin or FAK antibodies (BD Biosciences) for 2 h on a rotating platform. Protein G Sepharose (Sigma) was added and the samples were incubated for an additional 2 h at 4°C. Samples were centrifuged at 3000 x g for 3 min and the beads were washed three times with lysis buffer. Samples were boiled at 100°C for 5 min and the supernatant fluid collected after centrifugation (15 min at 15,000 x g at 4°C). The samples were analyzed by one-dimensional gel electrophoresis, and Western blotting was conducted using a phosphotyrosine antibody (Transduction Labs). The Western blots were reprobed with the respective antibody used for the immunoprecipitation to ensure a similar amount of protein was precipitated from each sample. This experiment was repeated three times and results were virtually identical for each experiment.
Statistical analysis.
Results were analyzed for statistical significance by one-way ANOVA followed by Dunnett's post-test using Prism software (Graphpad Inc.). p values < 0.05 were considered to be significant.
RESULTS
Viability of H9C2Cells after Sodium Arsenite Treatment
Cell viability of sodium arsenite-treated cells was assessed with the MTT colorimetric assay, by neutral red and trypan blue staining, and by propidium iodide staining. In the MTT assay, mitochondrial dehydrogenases of viable cells convert MTT to purple formazan crystals. There was a significant decrease in MTT conversion in cells treated with 10 μM sodium arsenite for 24 and 48 h (Fig. 1). Total cell death was also measured using propidium iodide, which indicates loss of membrane integrity because viable cells are able to exclude the dye. Significant cell death was observed only after 10 μM sodium arsenite treatment for 24 or 48 h (Fig. 1). Neutral red, which indicates lysozyme activity in viable cells, and trypan blue staining, which allows estimation of total cell protein, was used to further determine the effects of sodium arsenite on H9C2 cell viability. Neutral red and trypan blue staining (Fig. 1) were significantly reduced only after 10 μM sodium arsenite. Leucine incorporation into proteins in control cells was also not significantly different from cells treated for 48 h with 1, 2.5, or 5 μM sodium arsenite (data not shown). Because only at the 10 μM sodium arsenite dose level was cell viability significantly affected, as indicated by several measures of cell viability, all subsequent experiments employed sodium arsenite concentrations of 1, 2.5, or 5 μM, doses which were considered to be sublethal.
Sodium Arsenite Effect on Cell Migration Rates
Time-lapse video microscopy was used to measure changes in cell migration rates caused by sodium arsenite treatment. H9C2 cell migration rates were analyzed during two days of sodium arsenite treatment for approximately 16 h each day. Migration rates are expressed in microns per hour. H9C2 cells were grown in 35 mm tissue culture dishes until confluent at which time a wound was made in the cell monolayer by scraping across the dish with a rubber cell scraper. Cell migration was measured by analyzing changes in cells at the edge of the wounded monolayer. During both days of sodium arsenite treatment, migration rates were decreased in comparison to control cells, with significant changes in migration rates occurring at sodium arsenite doses of 2.5 and 5 μM (Fig. 2).
Sodium Arsenite Effect on Distribution and Numbers of Focal Adhesions
During cell migration, focal adhesions are formed toward the leading edge of cells, while disassembly of adhesions occurs in the trailing edge of cells (Lauffenburger and Horwitz, 1996). This cycle of assembly and disassembly of focal adhesions is part of the process that allows the cell to move forward. To evaluate the effect of sodium arsenite on focal adhesion distributions and numbers, focal adhesions were detected by immunofluorescence localization of the structural protein vinculin after cells were treated with sodium arsenite for 48 h. Control cells and cells treated with 1 μM sodium arsenite exhibited focal adhesions both at the periphery as well as under more central regions of the cells (Fig. 3). Exposure to either 2.5 or 5 μM sodium arsenite resulted in changes in the distribution of adhesions, with adhesions being primarily at the cells' periphery (Fig. 3). The numbers of focal adhesions per cell was also reduced in sodium arsenite-treated cells (Fig. 4).
Sodium Arsenite Effect on Total F-actin Content
The polymerization state of actin in cells is another possible indicator of cell viability and is an important component related to focal adhesion formation, cell migration and adhesion. A phalloidin-binding assay was used to provide a measure of the total F-actin content in cells. F-actin content was standardized by normalizing measured F-actin to total cell protein. Total F-actin content was significantly decreased in cells only after 48 h exposure to 5 μM sodium arsenite, with lower doses having no significant effect on F-actin content of cells (Fig. 5).
Sodium Arsenite Effect on Cell Attachment and Area Covered by Spread Cells
We hypothesized that if sodium arsenite alters focal adhesion distribution and number then it could alter, in addition to migration, other cell activities in which focal adhesions are important. The ability of cells to adhere to a substrate depends on focal adhesions and is critical for cell motility as well as survival. Therefore, we analyzed the ability of sodium arsenite-treated cells to attach to a substrate. To determine if sodium arsenite exposure could affect cell attachment we treated H9C2 cells for 48 h with 1, 2.5, or 5 μM sodium arsenite. The cells were trypsinized and plated in 96 well plates for either 15, 30, or 60 min before being fixed and stained with crystal violet. Crystal violet absorbance was reduced with increasing concentrations of sodium arsenite indicating that the treated cells were adhering to the cell culture substrate at a slower rate (Fig. 6A).
Cell attachment leads to assembly of focal adhesions and spreading of the cell on the culture substrate. If cells are not attaching well, then the area covered by a spread cell may be reduced. To establish if cells were spreading when exposed to sodium arsenite, H9C2 myoblast cell area was measured. To determine cell area, digital phase contrast micrographs of 100 randomly selected control or sodium arsenite-exposed cells were analyzed. Cell area was measured by manually outlining the border of each cell and integrating the enclosed areas. There was no significant change in cell area in response to 1 μM sodium arsenite treatment. Following 48 h exposure to 2.5 or 5 μM sodium arsenite, the cells exhibited a significant reduction in average total area (Fig. 6B). If cells appeared to cover a smaller area, it is possible that the cells were spreading but covered less area because they were smaller in size. Flow cytometry was used to estimate the volume of trypsinized and rounded H9C2 cells. The mean forward scatter was used to determine if sodium arsenite had an effect on cell volume. The data indicated that there was a slight, but not statistically significant, increase in the volume of treated cells (data not shown).
Sodium Arsenite Effect on Expression of Focal Adhesion Proteins
To date over fifty different proteins have been localized to or suggested to be components of focal adhesions (Zamir and Geiger, 2001). We chose to measure the amounts of several major focal adhesion proteins, including FAK, paxillin, talin and vinculin, to determine if sodium arsenite affects focal adhesion protein expression. Cells were exposed to 1, 2.5, or 5 μM sodium arsenite for 48 h and then harvested for Western blot analysis. There was no apparent difference in the amounts of FAK, paxillin, talin, or vinculin (Fig. 7) in cells in response to any tested level of sodium arsenite exposure. These results indicate that sodium arsenite at the doses employed did not significantly affect focal adhesion protein expression.
Sodium Arsenite Effect on Focal Adhesion Protein Tyrosine Phosphorylation
We next sought to determine the effect of sodium arsenite exposure on the tyrosine phosphorylation of FAK. H9C2 cells were treated for 48 h with 0, 1, 2.5, or 5 μM sodium arsenite. Cell lysates were immunoprecipitated with anti-FAK antibody and further analyzed by western blotting with an anti- phosphotyrosine antibody. Sodium arsenite exposure caused a reduction in the tyrosine phosphorylation of FAK (Fig. 8A; panel 1) in a dose dependent manner. The Western blot membranes were stripped and reprobed with the FAK antibody to ensure that similar amounts of total FAK had been precipitated from each sample (Fig. 8A; panel 2). Western blot analysis confirmed that similar amounts of FAK were precipitated.
Reduced FAK tyrosine phosphorylation could be due to reduced phosphorylation of several tyrosine residues located within this protein. An important phosphorylation site for FAK function is the autophosphorylation site of tyrosine 397 (Tyr397) (Eide et al., 1995; Schaller et al., 1994). To test the hypothesis that sodium arsenite could inhibit FAK autophosphorylation at Tyr397, we examined the effect of sodium arsenite on this site by Western blot analysis using an antibody specific for this phosphorylated residue. The amount of Tyr397 showed a dose-dependent decrease after 48 h sodium arsenite exposure (Fig. 8B). These results suggest that the decrease in FAK tyrosine phosphorylation may be due to reduced phosphorylation of Tyr397, the major autophosphorylation site of FAK.
FAK, once phosphorylated at Tyr397, has the ability to phosphorylate additional focal adhesion proteins including paxillin (Bellis et al., 1995; Schaller and Parsons, 1994; Turner, 2000). If the decrease in Tyr397 phosphorylation of FAK were of functional significance, then it would be expected that phosphorylation of paxillin would also be decreased. We examined phosphorylation of paxillin by immunoprecipitation of paxillin followed by Western blot of tyrosine phosphorylation of the immunoprecipitated protein. Sodium arsenite exposure reduced paxillin's tyrosine phosphorylation in a dose-dependent manner (Fig. 8C; panel 1). Reprobing the membrane with paxillin antibody confirmed that similar amounts of paxillin had been precipitated from each sample (Fig. 8C; panel 2).
DISCUSSION
The genesis of these studies was an initial observation that a sublethal, 24–48 h exposure of cultured H9C2 cells to sodium arsenite reduced the migration rate of the cells. In an effort to better understand this phenomenon, we examined several aspects of cells that are believed to be involved in motility.
We first established what sodium arsenite treatments would not be significantly toxic to the cells. Treatments that were obviously toxic would likely affect many cell functions and would not reveal the pathways most sensitive to exposure to sodium arsenite. As measures of toxicity, we assayed the cells' ability to oxidize MTT, to take up neutral red or stain with trypan blue and to exclude propidium iodide. We also measured [H3] leucine incorporation into cell proteins (data not shown). For all of these measures, the lower doses of sodium arsenite employed in these studies (i.e., 1, 2.5, or 5 μM) caused no detectable toxicity to the cells, indicating that the results observed were not due to overt cytotoxicity.
We quantified cell motility in control and sodium arsenite- treated cells and found a dose-dependent decrease in motility. This observed decrease in motility correlated with a different distribution of focal adhesions, fewer numbers of focal adhesions, decreased attachment of cells to the substrate and less spreading of cells. However, the amounts of focal adhesion proteins (FAK, paxillin, talin, and vinculin) in the cells was not affected by sodium arsenite and at least at lower doses of sodium arsenite, the amount of polymerized actin in cells did not change significantly. That vinculin localization to focal adhesions decreased although total amount of vinculin and other focal adhesion proteins in cells was not changed indicated that sodium arsenite was affecting focal adhesion structure or formation and not turnover or amounts of focal adhesion proteins.
It has been shown by others that focal adhesion formation requires phosphorylation of FAK (Illic et al., 1997) and that FAK subsequently phosphorylates other focal adhesion proteins including paxillin (Bellis et al., 1995). Conversely, disassembly of focal adhesions correlates with dephosphorylation of these proteins (van de Water et al., 2001). Our results indicated that fewer focal adhesions were present, that they were possibly forming more slowly, and that cells were spreading less in the presence of sodium arsenite. Thus, we hypothesized that phosphorylation of focal adhesion proteins might be reduced in sodium arsenite treated cells. Increased FAK phosphorylation is initially due to the autophosphorylation of Tyr397 (Schaller et al., 1994). Therefore, we examined FAK phosphorylation, phosphorylation of its autophosphorylation site, Tyr397, as well as phosphorylation of paxillin, a FAK substrate. Reduced phosphorylation was observed for both FAK and Tyr397 of FAK. Tyrosine phosphorylation of paxillin was also reduced. A mechanism has not yet been determined for how decreased phosphorylation at Tyr397 is able to alter FAK localization and function in focal adhesions. However, FAK's decreased phosphotyrosine levels may have hindered its ability to phosphor- ylate other focal adhesion proteins, thus explaining the observed reduced tyrosine phosphorylation level of the FAK substrate paxillin.
The finding that inhibition of cell migration and decreased formation of focal adhesions is accompanied by reduction in FAK and paxillin tyrosine phosphorylation is consistent with other reports. For example, van de Water et al. (2001) demonstrated that reduced FAK tyrosine phosphorylation correlates with reduced paxillin tyrosine phosphorylation and with focal adhesion disruption. In a separate investigation, cerivastatin was seen to disrupt the actin cytoskeleton, which leads to the inhibition of glioblastoma cell migration. The process was shown to correlate with focal adhesion disruption and also with reduced FAK tyrosine phosphorylation (Obara et al., 2002). Conversely, lysophophatidic acid induces the formation of actin stress fibers, increases focal adhesions and increases the tyrosine phosphor- ylation of FAK and paxillin. This causes the enhancement of ovarian cancer cell migration in lysophophatidic acid treated cells (Sawada et al., 2002). The alteration of focal adhesion formation in sodium arsenite treated cells supports the conclusion that the toxicant decreases cell migration through an effect on focal adhesions and disruption of cell interactions with the extra-cellular matrix.
In contrast to the results we report here, sodium arsenite has also been shown to increase the tyrosine phosphorylation of proteins in murine T thymocytes and primary vascular cells in a dose dependent manner (Barchowsky et al., 1999; Hossain et al., 2000). The differences in our results from those of Barchowsky et al. and Hossain et al. may be due to differences in the cell types employed for experiments or in time of exposure to sodium arsenite. We treated cells for many hours while in the other studies the longest exposure time was 45 min. It is also possible that there is a biphasic effect and that acute responses by cells may involve pathways that are different from those that become involved after longer term exposure to sodium arsenite. These differences suggest that the complete series of events that lead to the inhibition of focal adhesions and phosphorylation of FAK and paxillin that we describe here are likely to be complex and to involve a changing succession of events over hours to days, and longer periods of time. Understanding the basis for these differences may be important in understanding the disease processes associated with chronic, long-term arsenic ingestion.
Focal adhesions are involved in integrin signaling and the fact that focal adhesions can be disrupted by sodium arsenite suggests that integrin signaling pathways may also be disrupted by arsenic exposure. Disruption of focal adhesions and cell interactions with the extra-cellular matrix can cause anoikis and reduced cell motility (Obara et al., 2002; van de Water et al., 2001). This has been observed in various pathologies including cancer, ischemia-repufusion injury, and cardiovascular diseases (Keely et al., 1998; Norman and Fine, 1999; van de Water et al., 1999, 2001). Focal adhesions are analogous to several structures in vivo including dense sarcolema plaques in smooth muscle and costameres observed in cardiac myocytes (Sharp et al., 1997). Focal adhesion proteins are localized in the Z-line of both cardiac and skeletal muscle and it is believed that contractile forces exerted at these structures are converted into chemical signaling by focal adhesions (Burridge and Chrzanowska-Wodnicka, 1996; Ross and Borg, 2001). Thus, sodium arsenite disruption of focal adhesion proteins may hinder their normal activity in muscle cells, possibly disrupting cell contraction and signaling.
Several different signaling molecules or pathways believed to be involved in cell migration have been shown previously to be affected by sodium arsenite including ERK, p21 activated kinase, PKC and the Rho family of small GTPases (Lauffenburger and Horwitz, 1996; Porter et al., 1999). The results we report here suggest sodium arsenite specifically affects FAK phosphorylation, phosphorylation of paxillin, and subsequent focal adhesion functions. Additional experiments should reveal how various signal transduction pathways that regulate focal adhesion assembly and function are affected by sodium arsenite. Understanding these pathways should lead to better insight into how the environmental toxicant arsenic leads to cancer and the other pathologies associated with arsenic exposure.
ACKNOWLEDGMENTS
This work was supported by NIH award F31HL68424 to S.L.Y. and NIH grant P01 ES 11188 to M.J.W.
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