Introduction

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The phosphorylation of protein tyrosines is a pivotal event that initiates signaling in response to receptor activation by physiologic stimuli such as growth factors, or cellular stress presented by a diverse array of physicochemical agents such as radiation, heat, or metabolic inhibitors (1, 2). Tyrosine phosphorylation-dependent signaling culminates in a wide variety of cellular responses, including the activation of transcription factors (3) that modulate the expression of proteins involved in pulmonary inflammation induced by inhaled toxicants (4).

Levels of protein tyrosine phosphates (PTPs) are regulated by the opposing activities of kinases that phosphorylate specific tyrosine residues, and by activities of tyrosine phosphatases that specifically dephosphorylate tyrosines, thereby terminating the signal (7, 8). Although phosphorylation-dependent signaling is generally understood to be the result of kinase activation, inhibition of tyrosine phosphatases also activates intracellular signaling by permitting the accumulation of phosphotyrosines produced by basal levels of kinase activity (8). Moreover, transient inhibition of tyrosine phosphatases is believed to play a critical role in cell signaling activated by physiologic stimuli such as growth factors (9, 11).

The mammalian tyrosine phosphatases are a superfamily of enzymes with low homology and widely varying molecular weight, which contain a Cx5R motif at the active site (see Fauman and Saper [12] for a review). Four groups are presently recognized: the low molecular-weight tyrosine phosphatases; the cdc25-like enzymes; the VH1-like dual specificity enzymes, which also hydrolyze phosphoserine and phosphothreonine; and the tyrosine-specific (PTP) group, which is subdivided into receptor-like and non-receptor-like phosphatases. Shared characteristics of the activity of these enzymes include independence from metal ions (i.e., calcium); the ability to hydrolyze p-nitro phenyl phosphate, an absolute requirement for the active site cysteine; and sensitivity to inhibition by vanadate (12).

Metallic compounds are common occupational and ambient air pulmonary toxicants. Vanadium (V) compounds are industrial contaminants and constituents of ambient particulate matter in areas where oil is burned (13). V induces bronchitis and bronchopneumonia, and is the responsible agent in Boilermaker's bronchitis (14). Arsenic (As) is an occupational and ambient pollutant heavy metal released by incinerators, smelters, and chemical manufacturing plants (18) that causes upper respiratory irritation and is a known human lung carcinogen (19). Zinc (Zn) is a ubiquitous metal contaminant derived from metallurgic operations (18) and is the primary metal associated with metal fume fever (19).

The toxicologic mechanisms of action through which metals such as As, V, and Zn elicit untoward responses in the lung are poorly understood. However, V compounds are potent tyrosine phosphatase inhibitors (12, 20) and exposure to V ions results in marked activation of a variety of phosphorylation-dependent signaling pathways that are known to lead to inflammatory mediator synthesis (21, 22). Similarly, Zn has been shown to inhibit certain tyrosine phosphatases, such as PTP- (23), whereas As has been proposed to be a dual-specificity tyrosine phosphatase inhibitor (27).

We previously showed that exposure to a combustion-derived metallic air pollutant disrupts PTP homeostasis in the human airway epithelial cell line, BEAS (28). Recently, we reported that in vitro exposure to metallic compounds including As, V, or Zn ions activates elements of the mitogen-activated protein kinase (MAPK) pathways and leads to transcription factor activation and cytokine expression in BEAS cells (5). Given the reported inhibitory effect of As, V, and Zn on tyrosine phosphatase activity, it is possible that these metals activate MAPK by disregulating PTP metabolism in airway epithelial cells. In the present study, we have examined the effects of As, V, or Zn on protein phosphotyrosine catabolism in human airway epithelial cells (HAEC) using the BEAS cell line and normal human bronchial epithelial (NHBE) cell cultures. We report here that all three metals can induce PTP accumulation, and that V and Zn, but not As, are potent inhibitors of tyrosine phosphatase activity in HAEC.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Reagents

Tissue culture media, supplements, and supplies were obtained from Clonetics (San Diego, CA). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) supplies, such as molecular weight standards, polyacrylamide, and buffers were obtained from Bio-Rad (Richmond, CA). Metal salts, 2--mercaptoethanol, detergents, protease inhibitors, PolyGlu:Tyr(4:1), and other common laboratory chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). [³²P]--adenosine triphosphate (ATP) (7,000 Ci/mmol) was purchased from NEN Dupont (Wilmington, DE). Protein levels were quantified using a Coomassie blue reagent purchased from Bio-Rad. Stock solutions (100 mM) of sodium arsenite (As), vanadyl sulfate (V IV), and zinc sulfate (Zn II) were prepared in water and kept frozen until ready for use. Metal salts were obtained from Alfa (Ward Hill, MA) or Sigma. Specific antiphospho PTP1B antibodies and horseradish peroxidase (HRP)-conjugated goat antirabbit secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Tissue Culture

BEAS 2B (subclone S6) cells were obtained from Drs. Curtis Harris and John Lechner (National Institutes of Health, Washington, DC). The BEAS cell line was derived by transforming human bronchial epithelial cells with an ad12-SV40 adenovirus construct (29). BEAS cells were grown to 80 to 90% confluence on tissue culture-treated plastic dishes in keratinocyte growth medium supplemented with 30 µg/ml bovine pituitary extract, 5 ng/ml human epidermal growth factor, 500 ng/ml hydrocortisone, 0.1 mM ethanolamine, 0.1 mM phosphoethanolamine, and 5 ng/ml insulin, as described previously (6, 30). BEAS cells that had been passaged 80 to 100 times in our laboratory were used for the present studies. NHBE cells were obtained from normal adult human volunteers by brush biopsy of the mainstem bronchus using a cytology brush during bronchoscopy, conducted while following a protocol approved by the Committee on the Protection of the Rights of Human Subjects at the University of North Carolina at Chapel Hill. NHBE cells were initially plated in supplemented bronchial epithelial cell basal medium (0.5 ng/ml human epidermal growth factor, 0.5 µg/ml hydrocortisone, 5 µg/ml insulin, 10 µg/ml transferrin, 0.5 µg/ml epinephrine, 6.5 ng/ml triiodothyronine, 50 µg/ml gentamycin, 50 ng/ml amphotericin-B, 52 µg/ml bovine pituitary extract, and 0.1 ng/ml retinoic acid) (BEGM) on tissue culture plates coated with human collagen (Sigma), grown to confluence, and then passaged 2 or 3 times in BEGM on ordinary tissue culture plates.

Western Blotting

Phosphotyrosine Western blotting was performed essentially as described previously (28). Briefly, cells were extracted with a lysis buffer consisting of phosphate-buffered saline containing 1% Nonidet P40 (NP-40), 0.5% deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml aprotonin, and 1 µg/ml leupeptin. Protein extracts were mixed with one volume of SDS-PAGE loading buffer (0.125 M Tris [pH 6.8], 4% SDS, 20% glycerol, 10% -mercaptoethanol, and 0.05% bromophenol blue), boiled, and run on 11% SDS-PAGE gels. Prestained molecular weight markers (Sigma) were run on adjacent lanes. Samples were normalized for protein content before loading. Electrophoresed proteins were electroblotted onto nitrocellulose and the blots were blocked with 3% casein, washed briefly, and incubated overnight with the HRP-conjugated antiphosphotyrosine antibodies in 5% bovine serum albumin. PTP bands were detected using chemiluminescence reagents and film as per manufacturer's instructions (Amersham, Boston, MA).

Radiolabeling of [³²P]PolyGlu:Tyr(4:1)

A total of 500 µg PolyGlu:Tyr was radiolabeled using 10 µg of recombinant GST-FER kinase (a generous gift of Dr. Sheldon Earp, Lineberger Comprehensive Cancer Research Center, University of North Carolina) in the presence of 200 µCi [³²P]--ATP for 1 h at 37° C in 300 µl of a buffer consisting of 50 mM imidazole (pH 7.2), 10 mM dithiothreitol, 30 mM MgCl₂, 1 mM MnCl₂, 1 mM vanadate, 0.5% Triton-X 100, 0.2 mM ATP, and 0.1% -mercaptoethanol. The substrate was precipitated by adding a trichloroacetic acid (TCA) solution to 10% wt/vol, centrifuging at 12,000 × g for 5 min. The pellet was then washed three times in 10% TCA and the substrate was resuspended at 10 µg/ml in 2 M Tris, pH 8.0.

Tyrosine Phosphatase Activity Assays

Tyrosine phosphatase activity in treated cells was assayed by lysing the cells in 100 mM N-2-hydroxyethylpiperazine- N'-ethane sulfonic acid (Hepes), pH 7.3, containing 0.2% NP-40 and 20 µg/ml PMSF, followed by sonication (3 × 5 s). The assay was initiated by adding 30 µg of lysate protein to 300 µl of a reaction mixture containing 3 µg of [³²P]PolyGlu:Tyr substrate, 20 mM Hepes (pH 7.4), 350 mM sucrose, and 150 mM KCl. The reaction was sampled repeatedly by quenching 50 µl aliquots in 3 vol of 10% TCA. The substrate was then precipitated by centrifugation and the amount of radioactivity released into the supernatant was measured by liquid scintillation counting (28).

The in-gel phosphatase activity assays were carried out using a modification of a method described elsewhere (31). Protein extracts were prepared as for Western blots (described previously), except that samples were not boiled. The samples were then subjected to SDS-PAGE on 11% polyacrylamide gels containing [³²P]PolyGlu:Tyr (approximately 1.5 million cpm/40 ml gel). The proteins were then renatured in the gel by first removing the SDS with 20% isopropanol, followed by extensive washing of the gels with 0.04% Tween-40 in Tris, pH 8.0. In some experiments the wash buffer contained 200 µm As, V, or Zn. Clear bands indicative of tyrosine phosphatase activity were visualized by autoradiography using a Molecular Dynamics PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Immunocytochemistry

Primary HAEC cultured on glass coverslips were exposed to vehicle alone or to 100 µm As, V, or Zn ions for 15 min. The cells were washed and immediately fixed with 20°C methanol for 5 min, followed by air-drying. Phosphotyrosines were detected and stained using a commercially available antiphosphotyrosine immunohistochemistry kit (Santa Cruz Biotechnology) used as per manufacturer's instructions.

Fast Performance Liquid Chromatography (FPLC) Separation

Cell lysates prepared in 1% Triton X-100 were fractionated by ion-exchange chromatography on a 5-cm Mono Q column (Pharmacia, Uppsala, Sweden) connected to an LCC500 Plus Liquid Chromatography System (Pharmacia). Fractions collected by elution over a 20-min linear NaCl gradient were assayed for tyrosine phosphatase activity as described previously.

PTP1B Immunoprecipitation

Cell lysates were incubated with antihuman PTP1B antibody for 2 h, followed by 1 h with protein G agarose beads. The immunoprecipitated PTP1B was then analyzed for activity using the in-gel phosphatase assay as described previously.

Image Processing and Statistics

Films were digitized and band optical densities quantified using a Millipore Digital Bioimaging System (Millipore, Bedford, MA). Data are expressed as means ± standard error of the mean (SEM). Data comparisons were carried out using one-way analysis of variance followed by Dunnett's post hoc test for multigroup comparisons.

	Results

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We previously reported that exposure to metallic mixtures induces the accumulation of PTPs in human bronchial epithelial cells (28). We therefore evaluated the effect of an acute exposure to As, V, or Zn ions on levels of protein phosphotyrosines in HAEC. Unstimulated HAEC had detectable levels of phosphorylated tyrosines as assessed by immunocytochemical analysis of intact NHBE cells or by Western blotting of BEAS cell protein extracts using a specific antiphosphotyrosine antibody (Figure 1). In comparison to resting cells, HAEC treated with 100 µm As, V, or Zn for 15 min showed significantly elevated levels of PTPs when examined using either method. Maximal apparent increases in phosphotyrosines were evident in primary cells and BEAS cells exposed to V or As, with smaller elevations observed in cells treated with Zn (Figure 1). Similar apparent increases were observed in NHBE and BEAS cells following 60 min of exposure to 100 µm As, V, or Zn (data not shown).

View larger version (132K): [in this window] [in a new window]   View larger version (56K): [in this window] [in a new window]   Figure 1.   Effects of As, V, or Zn exposure on phosphotyrosine levels in HAEC. (A) Phosphotyrosine immunostaining of primary HAEC exposed to metals. HAEC were exposed to 100 µm As, V, or Zn for 15 min. The cells were washed and immediately fixed in methanol for 5 min at 20°C, endogenous peroxidase activity was quenched, and the slide was blocked with goat serum. The fixed cells were incubated with a monoclonal antiphosphotyrosine antibody for 2 h at room temperature. Protein phosphotyrosines were detected using a biotinylated goat-antimouse secondary antibody followed by HRP streptavidin complex and diaminobenzidine tetrahydrochloride staining. Magnification: ×400. (B) Phosphotyrosine Western blot of BEAS cells exposed to metals. BEAS cells were exposed to 500 µm As, V, or Zn for 15 or 60 min. Shown are data representative of three or more separate experiments.

We hypothesized that exposure to As, V, or Zn ions could induce PTP accumulation by inhibiting tyrosine phosphatase activities in HAEC. Therefore, we next examined the effect of an acute, noncytotoxic exposure to As, V, or Zn ions on the total tyrosine phosphatase activity present in BEAS cells using [³²P]PolyGlu:Tyr as a synthetic substrate. Exposure of BEAS cells to 500 µm V or Zn for 20 min effectively inhibited all tyrosine phosphatase activity detectable in sonicated cell preparations (Table 1). In marked contrast, there were no differences between levels of tyrosine phosphatase activities present in BEAS cells treated with 500 µm As for 20 min and untreated control cells.

To partially characterize PTPs present in HAEC and to characterize the effects of As, V, and Zn on isolated tyrosine phosphatases in a cell-free system, protein extracts from untreated NHBE cells were subjected to ion-exchange (Mono Q) chromatography followed by activity assay of individual fractions against [³²P]PolyGlu:Tyr. The bulk of the tyrosine phosphatase activity in the cell extracts appeared to elute from the FPLC column as several distinct but overlapping peaks of activity (Figure 2). Assay of the column fractions in the presence of 200 µm V or Zn ions significantly diminished the size of the activity peaks. As predicted by the activity assays of treated BEAS cells, the addition of 200 µm As to the assay mix did not affect NHBE tyrosine phosphatase activities in the FPLC fractions (Figure 2).

View larger version (24K): [in this window] [in a new window]   Figure 2.   Effects of As, V, and Zn ions on fractionated tyrosine phosphatase activities in HAEC. Cultured primary HAEC were lysed in 1% Triton X-100 and subjected to FPLC fractionation on a Mono Q ion exchange column. Eluted fractions were assayed for tyrosine phosphatase activity against [32P]- labeled PolyGlu:Tyr(4:1) in the presence of vehicle (Control), or 200 µm As, V, or Zn ions. Data are expressed as the amount of radioactivity released by the activity in each fraction. Shown are representative results from three separate experiments.

To further determine the number and size of the PTPs present in HAEC and to examine potential differential effects of As, V, and Zn on individual enzymes, column fractions corresponding to the peak of activity in NHBE extracts, shown in Figure 2, were analyzed using an in-gel phosphatase activity assay. In this assay, the proteins were separated according to mass in a substrate [³²P]PolyGlu: Tyr-impregnated polyacrylamide gel under denaturing electrophoresis conditions, and then renatured in situ to allow visualization of active phosphatases (31). As shown in Figure 3, the FPLC fractions contained numerous bands corresponding to PTPs of widely varying molecular masses (30 to 200 kD) present in NHBE. When the protein bands were renatured in the presence of 200 µm V or Zn, the number and intensity of the tyrosine phosphatase bands were greatly diminished. The inhibitory effects of V and Zn appeared to affect individual tyrosine phosphatases differentially, as evidenced by consistent differences in banding patterns and protein band intensities (Figure 3). In keeping with the findings obtained with activity assays of the metal-treated BEAS and the fractionated NHBE extracts, the presence of 200 µm As did not produce an appreciable change in the pattern or intensity of the PTP bands detected using the in-gel assay (Figure 3).

View larger version (41K): [in this window] [in a new window]   Figure 3.   Effects of As, V, and Zn ions on isolated tyrosine phosphatase activities. Fractions corresponding to the peak of tyrosine phosphatase activity in the FPLC run shown in Figure 2 (Fractions 18-23) were analyzed by in-gel phosphatase activity assay on SDS-PAGE gels containing [32P]- labeled PolyGlu:Tyr(4:1). Electrophoresed proteins were renatured in the presence of 200 µm As, V, or Zn ions. Shown are representative results from three separate experiments.

The large number of different bands observed using the in-gel phosphatase assay precluded the identification of individual PTPs affected by exposure to V or Zn. We therefore targeted a specific enzyme, which is known to be widely expressed in mammalian tissues, for additional investigation of the effects of V and Zn on PTPs in NHBE cells. PTP1B was immunoprecipitated from untreated NHBE cells using specific antibodies and subjected to in-gel phosphatase analysis. Assay of immunoprecipitated PTP1B was conducted using [³²P]PolyGlu:Tyr-impregnated gels in the presence of 200 µm As, V, or Zn. Immunoprecipitated PTP1B appeared as two closely migrating bands of molecular weight consistent with that of PTP1B (Figure 4), likely corresponding to the precursor and cleaved product of PTP1B (32, 33), although cross-reactivity of the antibody with another tyrosine phosphatase cannot be ruled out. Inclusion of V during the renaturation of PTP1B in the in-gel phosphatase assay caused a virtually complete loss of phosphatase activity, whereas the presence of Zn inhibited PTP1B to a lesser extent. As seen in the previous experiments, As had essentially no effect on NHBE PTP1B activity (Figure 4).

View larger version (63K): [in this window] [in a new window]   Figure 4.   V and Zn ions effectively inhibit the tyrosine phosphatase PTP1B in HAEC. Because ions have no discernible effect, PTP1B was immunoprecipitated from primary human airway epithelial cell lysates using a specific antibody. The immunoprecipitates were then analyzed for activity using an in-gel tyrosine phosphatase activity assay in the presence of metals (200 µm As, V, or Zn ions) or buffer alone (control, ct). Shown are the activities of the immunoprecipitated PTP1B (left lanes in each condition), next to the total activity remaining in the lysate after immunoprecipitation (right lanes in each condition). Note that the PTP1B bands are close in size to other phosphatases remaining in the lysate after immunoprecipitation. These data are representative of three separate experiments.

	Discussion

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PTP accumulation may be viewed as an intracellular marker of the activation of signal transduction pathways. Although kinase activation is studied more frequently, the role of phosphatase inhibition as a mechanism that leads to tyrosine phosphorylation-dependent signaling has been the subject of increased scrutiny in recent years (8). In this study we have characterized the effects of the As, V, and Zn ions on PTP catabolism in human bronchial epithelial cells. Our findings demonstrate that while acute, noncytotoxic exposure to either As, V, or Zn ions results in an intracellular accumulation of phosphorylated protein tyrosines, only V and Zn inhibit tyrosine phosphatases in BEAS and NHBE cells.

Our finding that V and Zn treatment induces accumulation of protein phosphotyrosines is consistent with the inhibitory effect that these metal ions have on PTPs in BEAS and NHBE cells. Moreover, V- and Zn-induced increases in phosphotyrosine concentrations may occur without the need for direct or indirect activation of kinases because unopposed basal levels of kinase activity may be sufficient to produce an accumulation of phosphotyrosines.

Inhibition by V ions is a defining characteristic of all tyrosine phosphatases (12), and V is known to activate multiple signaling pathways within the cell. The mechanism responsible for V-induced inhibition of tyrosine phosphatases is not clear. It has been proposed that the active species is a peroxovanadium compound produced through the reaction of V ions with cellular superoxide (34). Peroxovanadium compounds are potent, irreversible inhibitors of tyrosine phosphatases (35). The vanadate ion itself is a phosphate analog, which is believed to act as a competitive inhibitor of tyrosine phosphatases (36).

Formation of reactive oxygen species (ROS) is another potential mechanism for tyrosine phosphatase inhibition in cells exposed to Fenton-reactive metals such as V. Hydrogen peroxide (H₂O₂) is a known tyrosine phosphatase inhibitor, and the generation of H₂O₂ may be a pivotal event in signaling processes initiated by physiologic stimuli such as growth factors (9, 11, 37, 38). Unlike V, however, Zn (and for that matter, As) lacks the two neighboring stable valance states that would enable it to redox cycle at physiologically relevant potentials, and thereby generate ROS.

Tyrosine phosphatases are known to have strong affinity for Zn (39), and Wang and Pallen have shown that the catalytically active protein domain in HPTP interacts with Zn (24). However, it appears that not all tyrosine phosphatases are inhibited by Zn. The activity of the receptor-like CD45 is not diminished and may actually be activated by Zn ions (40). In this regard, it is worth noting that our in-gel phosphatase studies showed differences in the pattern of inhibition of tyrosine phosphatases in NHBE cells that may not simply reflect differences in the inhibitory potency of V and Zn, but may in fact be due to the selectivity of these metals for certain tyrosine phosphatases.

Significantly, the in-gel phosphatase activity assay used in this study was performed in the presence of mercaptoethanol, a reducing agent that would be expected to have afforded the opportunity to regenerate sulfhydryls oxidized by exposure to the metals. The fact that inhibition of tyrosine phosphatases was detected in the presence of mercaptoethanol, therefore, argues against the involvement of sulfhydryl oxidation by V and Zn ions, either directly by the metal ion itself or indirectly through the generation of ROS. The finding that the sulfhydryl-reactive heavy metal As did not affect tyrosine phosphatase activity may also be consistent with a mechanism of inhibition that does not involve alteration of sulfhydryls.

Interestingly, the arsenate ion, whose formation in vitro would necessitate the oxidation of arsenite, is also a structural analog of phosphate that might inhibit phosphatases. At least one previous report has implicated sodium arsenite as an inhibitor of dual-specificity phosphatases, a subgroup of the tyrosine phosphatase family susceptible to V inhibition (41). However, our data provide strong evidence against arsenite-mediated inhibition of tyrosine phosphatases in HAEC. As we show here, arsenite did not affect tyrosine phosphatase activity under a broad range of assay conditions, including those that permitted metal interactions with intact cells and direct access to gel- and FPLC-fractionated phosphatases.

From a toxicologic standpoint, it is simpler to envision mechanisms through which a toxicant might inhibit the function of a phosphatase than it is to conceive of a manner in which a kinase can be specifically activated upon interacting with a toxic compound. From this perspective, phosphatase inhibition might be thought of as representing a relatively common mechanism of signal transduction disregulation in cells exposed to toxicants. Moreover, the apparent similarity in the magnitude and kinetics of phosphotyrosine accumulation induced by As, V, and Zn exposure would suggest a common mechanism of action for these metals. In spite of this, the data shown in this study demonstrate that As exposure results in elevated phosphotyrosine levels without affecting tyrosine phosphatase activity, strongly implicating kinase activation as the responsible mechanism. The mechanism through which As ions might interact with a kinase(s) to effect their activation is not known. However, it is possible to speculate that As, a sulfhydryl-reactive heavy metal, activates kinases by altering the structural conformation of their regulatory domain in a manner that obviates the need for phosphorylation of the molecule. Alternatively, As could activate kinases by causing their translocation to a site of activation or by approximating accessory proteins or cofactors. It is also possible that exposure to As activates kinases by stimulating receptors, perhaps by forming receptor dimers by crosslinking sulfhydryls. The possibility also exists that As exposure inhibits serine/threonine phosphatases and that this results in an indirect activation of tyrosine kinases, which manifests itself as an increase in protein phosphotyrosines.

Although the experiments performed in this study produced remarkably consistent findings, a few caveats must be considered when interpreting these data. The first is that, because all of the assays performed in this study used PolyGlu:Tyr as a generic substrate, it is conceivable that tyrosine phosphatases with high sequence specificity requirements were not detected. Therefore, the effects of metals, including As, on those enzymes would have been missed. A second unavoidable limitation of these findings is the possibility that pivotal phosphatases that are normally expressed at low levels have gone unnoticed in the assays of total lysates or even in the fractionation experiments. The effect that As, V, and Zn ions might have on these important enzymes would not have been studied. Similarly, one group of tyrosine phosphatases that would not have been examined at all are those that are expressed only upon stimulation of the cell, an event that would presumably require exposure of intact cells for periods of longer duration than the ones used in these experiments. Finally, our data cannot exclude the possibility that, in addition to their inhibitory effects on tyrosine phosphatases, V and Zn activate kinases directly. Unfortunately, sorting the direct effects of these metals on kinases from the indirect activation of kinases that would be expected to result secondary to phosphatase inhibition would be very difficult, if not impossible, to do with existing methodologies. In spite of these limitations, the data we present in the current study provide clear evidence of V- and Zn-induced disregulation of PTP metabolism in HAEC through inhibition of tyrosine phosphatases.

Although As, V, and Zn are ambient and occupational air pollutants whose inhalation presents a toxic stress to the lung, it is difficult to ascertain the in vivo relevance of these mechanistic studies. Addressing the critical issue of dosimetry (i.e., the concentration of As, V, or Zn to which individual epithelial cells are exposed per inhaled mass of metal) requires extrapolations that are highly dependent on the choice of assumptions regarding, among other factors, the distribution, deposition, and solubility of inhaled metallic particles. Such calculations are important to the interpretation of mechanistic findings, such as those presented here, but they are beyond the scope of this study on the effects of As, V, and Zn on tyrosine phosphatases in HAEC.

The significance of this study is in the demonstration that normal human airway epithelium expresses multiple forms of PTPs, whose activities appear to be targets of exposure to metals found as ambient and occupational air contaminants. We were able to demonstrate clearly V- and Zn-induced inactivation of tyrosine phosphatases using a variety of assay conditions. Further, these data also support the argument that As-induced phosphotyrosine accumulation is not due to tyrosine phosphatase inhibition, implicating kinase activation. Thus, pulmonary toxicants such as As, V, and Zn may activate signaling pathways through multiple mechanisms. These alterations of intracellular signaling may ultimately be responsible for pulmonary responses to pollutant inhalation.