Zinc-induced PTEN Protein Degradation through the Proteasome Pathway in Human Airway Epithelial Cells*

Weidong Wu {ddagger} §, Xinchao Wang {ddagger}, Wenli Zhang {ddagger}, William Reed {ddagger}, James M. Samet ¶, Young E. Whang || and Andrew J. Ghio ¶ **

From the {ddagger}Center for Environmental Medicine, Asthma, and Lung Biology and the ||Department of Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599 and the Human Studies Division, National Health Effects and Environmental Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Received for publication, March 31, 2003 , and in revised form, May 8, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The tumor suppressor PTEN is a putative negative regulator of the phosphatidylinositol 3-kinase/Akt pathway. Exposure to Zn2+ ions induces Akt activation, suggesting that PTEN may be modulated in this process. Therefore, the effects of Zn2+ on PTEN were studied in human airway epithelial cells and rat lungs. Treatment with Zn2+ resulted in a significant reduction in levels of PTEN protein in a dose- and time-dependent fashion in a human airway epithelial cell line. This effect of Zn2+was also observed in normal human airway epithelial cells in primary culture and in rat airway epithelium in vivo. Concomitantly, levels of PTEN mRNA were also significantly reduced by Zn2+ exposure. PTEN phosphatase activity evaluated by measuring Akt phosphorylation decreased after Zn2+ treatment. Pretreatment of the cells with a proteasome inhibitor significantly blocked zinc-induced reduction of PTEN protein as well as the increase in Akt phosphorylation, implicating the involvement of proteasome-mediated PTEN degradation. Further study revealed that Zn2+-induced ubiquitination of PTEN protein may mediate this process. A phosphatidylinositol 3-kinase inhibitor blocked PTEN degradation induced by Zn2+, suggesting that phosphatidylinositol 3-kinase may participate in the regulation of PTEN. However, both the proteasome inhibitor and phosphatidylinositol 3-kinase inhibitor failed to prevent significant down-regulation of PTEN mRNA expression in response to Zn2+. In summary, exposure to Zn2+ ions causes PTEN degradation and loss of function, which is mediated by an ubiquitin-associated proteolytic process in the airway epithelium.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
PTEN1 (phosphatase and tensin homolog deleted on chromosome 10) was discovered in 1997 as a new major tumor suppressor encoded by a gene localized to human chromosome 10q23 region (1, 2). Deletions or somatic mutations within the PTEN gene have been observed in many primary human cancers, tumor cell lines, and several familial cancer predisposition disorders (3, 4). It is now known that PTEN plays critical roles not only in suppressing cancer but also in embryonic development, cell migration, cell signaling, and apoptosis (512).

PTEN protein consists of 403 amino acids. It displays high homology in its NH2-terminal region to dual specificity phosphatases and also to tensin and auxilin (1, 2, 10, 13). The COOH terminus is most likely regulatory in nature and is composed of three subdomains: a C2 domain that has been implicated in phospholipid-binding; two tandem PEST domains that regulate PTEN protein stability; and a PDZ-binding domain that associates with PDZ-containing proteins (1418). Although PTEN has phosphatase activity in vitro against both lipid and protein substrates (5, 12, 19, 20), its primary physiological target appears to be a phospholipid rather than a phosphoprotein: specifically, the D3-phosphate group of phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) (13, 2124). PIP3 is specifically produced from phosphatidylinositol 4, 5-bisphosphate by phosphatidylinositol 3-kinase (PI3K), which is activated by cell receptors including various tyrosine kinase growth factor receptors and integrins (2527). Accumulation of PIP3 further results in the activation of the serine-threonine protein kinase B/Akt through the recruitment of PI3K-dependent serine/threonine kinases (2830), subsequently modulating both proliferative and apoptotic signals (10, 31). Thus, by dephosphorylating PIP3, PTEN antagonizes growth-promoting and antiapoptotic pathways mediated by PI3K/Akt signaling (3234).

Zinc is an essential micronutrient with multiple structural and regulatory cellular functions (35) but also a common airborne metallic contaminant that may contribute to the health effects of ambient and occupational air pollution (3639). There has been a recent increase in interest in zinc-induced intracellular signaling (40). Zn2+ ions have been shown to activate the signaling pathways involving the receptor or non-receptor tyrosine kinases, Ras/mitogen-activated protein kinases (MAPKs) (4143), and the PI3K/Akt/p70 S6 kinase pathway (44) and to inhibit the activity of protein tyrosine phosphatases (45). In contrast to the effects of Zn2+ on cell signaling, overexpression of PTEN has opposite effects, such as blocking downstream signaling of EGF receptor including the Ras/MEK/MAPK cascade and antagonizing PI3K/Akt signaling (5, 20). This suggested the possibility that Zn2+ exposure decreased PTEN activity. Our study investigated the effects of Zn2+ treatment on PTEN in human airway epithelial cells. We report here that Zn2+ exposure causes a significant decrease in PTEN protein levels and activity through a proteolytic mechanism that depends on PI3K and leads to Akt activation.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and in Vitro Exposure—The BEAS-2B (subclone S6) cell line was derived by transforming human bronchial cells with an adenovirus 12-simian virus 40 construct (46). BEAS-2B cells (passages 70–80) were grown to confluence on tissue culture-treated Costar 6- or 12-well plates in keratinocyte basal medium supplemented with 30 µg/ml bovine pituitary extract, 5 ng/ml human EGF, 500 ng/ml hydrocortisone, 0.1 mM ethanolamine, 0.1 mM phosphoethanolamine, and 5 ng/ml insulin, as described previously (43). Cells were placed in keratinocyte basal medium (without supplements) for 20–22 h before treatment with zinc sulfate (Sigma).

Normal human bronchial epithelial cells (passages 2–3) were obtained from normal adult volunteers by transbronchoscopic brush biopsy of mainstem bronchi, 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 (45). Normal human bronchial epithelial cells were 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) grown to confluence and then cultured with bronchial epithelial cell basal medium deprived of epidermal growth factor for 12–16 h before challenge with zinc sulfate.

Immunoprecipitation—BEAS-2B cells pretreated with MG132 were stimulated with Zn2+ for 1 h. Cells treated with different doses of Zn2+ were lysed with RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS in phosphate-buffered saline, pH 7.4) containing 0.1 mM vanadyl sulfate and protease inhibitors (0.5 mg/ml aprotinin, 0.5 mg/ml trans-epoxy succinyl-L-leucylamido-(4-guanidino)butane (E-64), 0.5 mg/ml pepstatin, 0.5 mg/ml bestatin, 10 mg/ml chymostatin, and 0.1 ng/ml leupeptin). Cell lysates (300 µg) were precleared with protein A-agarose and then incubated with agarose-conjugated ubiquitin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 °C overnight. The precipitates were washed with cold RIPA buffer before immunoblotting using a murine monoclonal anti-human PTEN antibody (Cascade Bioscience, Winchester, MA).

Immunoblotting—Cells with or without pretreatment of pharmacological inhibitors were treated with Zn2+ and then lysed in RIPA buffer. Cleared cell lysates or immunoprecipitates were subjected to SDS-PAGE, as described before (42). Proteins were transferred onto nitrocellulose membrane. Membrane was blocked with 5% non-fat milk, washed briefly, incubated with antibodies against human PTEN (Cascade Bioscience), phospho-specific Akt and Akt (Cell Signaling Technology, Beverly, MA), and {beta}-actin (USBiological, Swampscott, MA), respectively, at 4 °C overnight followed by incubating with corresponding horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Immunoblot images were detected using chemiluminescence reagents (Pierce) and the Gene Gynome Imaging System (Syngene, Frederick, MD).

Real-time Reverse Transcriptase-PCR—BEAS-2B cells grown to confluence were exposed to 50 µM Zn2+. Cells were lysed with 4 M guanidine thiocyanate (Roche Applied Science), 50 mM sodium citrate, 0.5% sarkoyl, and 0.01 M dithiothreitol. Total RNA (200 ng) was isolated using RNeasy kit (Qiagen Inc., Valencia, CA) and reverse-transcribed into cDNA. Quantitative PCR was performed using TaqMan Universal PCR Master Mix (Roche Applied Science) and an ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, CA) (47). PTEN mRNA levels were normalized using the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Relative amounts of PTEN and GAPDH mRNA were based on standard curves prepared by serial dilution of cDNA from human BEAS-2B cells. The following oligonucleotide primers and probes were used: PTEN, 5'-TGT TGT TTC ACA AGA TGA TGT TTG A-3' (sense), 5'-CGT CGT GTG GGT CCT GAA TT-3' (antisense), 5'-ACT ATT CCA ATG TTC AGT GGC GGA ACT TGC-3' (probe); GAPDH, 5'-GAA GGT GAA GGT CGG AGT C-3' (sense), 5'-GAA GAT GGT GAT GGG ATT TC-3' (antisense), 5'-CAA GCT TCC CGT TCT CAG CC-3' (probe). In addition, PTEN mRNA expression was also determined in cells pretreated with vehicle or inhibitors (MG132 or LY294002) prior to zinc exposure according to the same procedure.

Immunohistochemistry—Male Sprague-Dawley rats (60 days old) were anesthetized with halothane and instilled with either 0.5 ml of saline or 0.5 ml of 50 µM zinc sulfate in saline. 24 h after instillation, the lungs of euthanized rats were fixed with 10% formalin (35 ml/kg of body weight) (Fisher). The immunochemical staining was conducted as described before (48). Tissue sections were mounted on silane-treated slides (Fisher) and air-dried overnight. The slides were heat-fixed at 600 °C in a slide dryer (Shandon Lipshaw, Pittsburgh, PA) and then followed by deparaffinization and hydration to 95% alcohol (xylene for 10 min, absolute alcohol for 5 min, and 95% alcohol for 5 min). Endogenous peroxidase activity was blocked with H2O2 in absolute methanol (30% H2O2 in 30 ml of methanol) for 8 min. Slides were rinsed in 95% alcohol for 2 min, placed in deionized H2O, and washed in phosphate-buffered saline. After treatment with Cyto Q Background Buster (Innovex Biosciences) for 10 min, slides were incubated with mouse anti-human PTEN antibody (Cascade Bioscience, Winchester, MA) diluted 1:100 in 1% bovine serum albumin for 45 min at 37 °C followed by incubation with biotinylated linking secondary antibody from Stat-Q staining system (Innovex Biosciences) for 10 min at room temperature and washed with phosphate-buffered saline, and peroxidase enzyme label from Stat-Q Staining System was applied. Tissue sections were developed with 3,3'-diaminobenzidine tetrahydrochloride and counter-stained with hematoxylin. Coverslips were applied using a permanent mounting medium.

Statistics—Data are presented as means ± S.E. Unpaired Student's t tests with conferring correction were used for pairwise comparison.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reduction of PTEN Protein Levels in Human Airway Epithelial Cells Exposed to Zn2+Previous studies showed that Zn2+-induced activation of the PI3K/Akt signaling pathway (44) suggested that this metal may affect PTEN protein. To test this assumption, PTEN protein levels were measured in human airway epithelial cells exposed to Zn2+ using Western blotting. As shown in Fig. 1A, exposure to 50 µM Zn2+ for 4 and 8 h significantly decreased PTEN protein levels in BEAS-2B cells. Exposure of BEAS-2B cells to 50 µM Zn2+ for 8 h did not result in significant alterations in cell viability, as assessed by assay of lactate dehydrogenase activity released into the culture medium (data not shown). The magnitude of the Zn2+-induced reduction in PTEN content was proportional to the concentration of Zn2+ administered to the cells (Fig. 1B). PTEN protein level in untreated cells appeared constant over time (Fig. 1A). This finding was reproduced in normal human airway epithelial cells, excluding the possibility that this effect is an artifact of the BEAS-2B cell line (Fig. 1C). In comparison, 50 µM vanadyl sulfate, a potent tyrosine phosphatase inhibitor (45), produced only a minimal effect on PTEN protein levels in BEAS-2B cells (Fig. 1A). In addition, exposure of cells to 100 ng/ml EGF, a potent EGF receptor ligand (49), for 1, 2, 4, and 8 h did not show a significant effect on PTEN levels (data not shown). These data indicated that Zn2+ treatment specifically reduced PTEN protein content in human airway epithelial cells.



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FIG. 1.
Zn2+ reduced PTEN protein level in human airway epithelial cells. Confluent BEAS-2B cells were starved in keratinocyte basal medium overnight before treatment with zinc sulfate. Cells were exposed to 50 µM Zn2+ or vanadyl sulfate for 1, 4, and 8 h (A) or 0, 10, 25, and 50 µM Zn2+ for 4 h and lysed in RIPA buffer (B). Normal Human bronchial epithelial cells grown to confluence were starved in bronchial epithelial cell basal medium overnight and treated with 0, 10, 25, and 50 µM Zn2+ for 4 h and lysed in RIPA buffer (C). Cell lysates were subjected to SDS-PAGE and immunoblotting using anti-human PTEN antibody. PTEN protein bands were detected using enhanced chemiluminescence reagents. Data shown are representative of three separate experiments.

 

To determine whether this effect of Zn2+ also occurs in airway epithelium in vivo, Sprague-Dawley rats were intratracheally instilled with 50 µM Zn2+ or with saline as negative control. Both airway epithelium and alveolar macrophages in normal rat lung tissue were positively stained for PTEN protein that predominantly existed in cytoplasm (Fig. 2A). In contrast, PTEN protein immunostaining decreased markedly in airway epithelium exposed to Zn2+ (Fig. 2B), which was consistent with the in vitro observations. Interestingly, PTEN immunostaining in alveolar macrophages remained unchanged with Zn2+ exposure (Fig. 2B), suggesting that Zn2+-induced PTEN reduction may be cell type-specific.



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FIG. 2.
Zn2+ reduced PTEN protein level in airway epithelium of rat lung. Sprague-Dawley rats were instilled intratracheally with either 0.5 ml of saline (A) or 50 µM zinc sulfate in 0.5 ml saline (B). 24 h after instillation, rat lung was acquired after exsanguinations. After fixation in 10% formalin, sections were cut and stained with anti-PTEN antibody using immunohistochemistry techniques. Hematoxylin was used for counterstain. Immunostain for PTEN can be seen in airway epithelial cells (arrowheads) and alveolar macrophages (arrows).

 

Zn2+-induced PI3K/Akt Activation in Human Airway Epithelial Cells—Since PTEN antagonizes the PI3K/Akt pathway (13, 24, 27), reduction of PTEN protein levels induced by Zn2+ should result in Akt activation. To further characterize the inhibitory effect of Zn2+ on PTEN-associated signaling, we next studied the effect of Zn2+ treatment on the activation of Akt in BEAS-2B cells, as measured by the phosphorylation of Akt at serine 473 (50). Exposure to Zn2+ markedly induced Akt phosphorylation in BEAS-2B cells in a dose- and time-dependent fashion (Fig. 3, A and B). Interestingly, robust Akt phosphorylation was evident 1 h after Zn2+ exposure, when there was minimal effect on the total PTEN protein level. Next, a highly selective inhibitor of PI3K activity, LY294002 (51), was further used to ascertain the dependence of Zn2+-induced Akt phosphorylation on PI3K (52). Akt phosphorylation was clearly inhibited in BEAS-2B cells pretreated with LY294002 (Fig. 3C). As vehicle control, Me2SO appeared to elevate the baseline of Akt phosphorylation in BEAS-2B cells (Fig. 3C).



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FIG. 3.
Activation of Akt in BEAS-2B cells exposed to Zn2+. Cells grown to confluence were starved in keratinocyte basal medium overnight before treatment with zinc sulfate. Cells were exposed to 50 µM Zn2+ for 1, 4, and 8 h (A), exposed to 0, 10, 25, and 50 µM Zn2+ for 4 h (B), or pretreated with vehicle control (Ct) (0.1% Me2SO) or 25 µM LY294002 and challenged with 50 µM Zn2+ for 4 h (C). Cells were lysed in RIPA buffer, and the lysates were subjected to SDS-PAGE and immunoblotting using a phospho-specific Akt antibody. Data shown are representative of three separate experiments.

 

PTEN mRNA Level Was Significantly Reduced by Zn2+ Exposure—Reduction of PTEN protein level and function may be associated with decreased PTEN mRNA expression in several tumor cell lines (53). To identify the mechanisms responsible for PTEN protein reduction, PTEN mRNA expression in zinc-exposed cells was quantified using real-time reverse transcriptase-PCR. BEAS-2B cells were exposed to different doses of Zn2+ for different periods. As shown in Fig. 4A, reduction of PTEN mRNA expression (about 50%) was observed when cells were exposed to 50 µM Zn2+. Down-regulation of PTEN mRNA expression was only detected upon stimulation of BEAS-2B cells with 50 µM Zn2+ for 8 h (Fig. 4B). Thus, in contrast to PTEN protein levels in BEAS-2B cells, which declined as early as 4 h of exposure, the reduction of PTEN mRNA expression induced by Zn2+ occurred after PTEN protein levels had fallen. PTEN protein is a relatively stable protein with a half-life of 48–72 h (16). As shown in Fig. 1A, PTEN protein level in untreated BEAS-2B cells appeared constant within 8 h of exposure. Therefore, these data suggested that the down-regulation of PTEN mRNA levels might not play a major role in Zn2+-induced PTEN reduction.



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FIG. 4.
Zn2+ down-regulated PTEN mRNA expression in BEAS-2B cells. Cells grown to confluence were starved overnight and then exposed to 12.5, 25, and 50 µM Zn2+ for 8 h (A) or exposed to 50 µM Zn2+ for 1, 4, and 8 h (B). Total RNA was extracted and reverse-transcribed. Quantitative PCR was performed using the TaqMan method. PTEN mRNA abundance in panel A was normalized to the abundance of GAPDH mRNA (*, p < 0.05, as compared with control). The PTEN mRNA level in panel B was shown as fold over control.

 

Proteasome-mediated PTEN Degradation in Zn2+-exposed Cells—The involvement of the proteasome in the degradation and regulation of the function of short-lived proteins, including oncoproteins, tumor suppressors, and cell cycle proteins, has been extensively documented as a mechanism for post-translational regulation of protein levels (54). Therefore, we next investigated whether PTEN degradation in response to Zn2+ exposure was proteasome-dependent. BEAS-2B cells were pretreated with a specific proteasome inhibitor, MG132 (55), and PTEN protein levels were evaluated by Western blotting. As shown earlier, exposure of cells to 50 µM Zn2+ for 8 h caused significant decreases of PTEN protein levels and Akt activation. In comparison, pretreatment with the MG132 completely prevented both Zn2+-induced PTEN protein loss as well as Akt activation (Fig. 5, A and B). As seen before (Fig. 3C), Me2SO vehicle control appeared to elevate the baseline of Akt phosphorylation (Fig. 5B). To exclude the potential effect of MG132 on PTEN mRNA expression, PTEN mRNA expression induced by Zn2+ in BEAS-2B cells was examined using reverse transcriptase-PCR as described above. Using the same exposure conditions, MG132 had no significant effect on Zn2+-induced PTEN mRNA expression (Fig. 5C). These data strongly implied that the 26 S proteasome played a critical role in Zn2+-induced PTEN degradation.



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FIG. 5.
The proteasome inhibitor, MG132, blocked Zn2+-induced PTEN degradation and Akt phosphorylation with no significant effect on PTEN mRNA expression in BEAS-2B cells. Confluent BEAS-2B cells were starved overnight before pretreatment with vehicle control (Ct) (0.1% Me2SO) or 20 µM MG132 for 30 min followed by exposure to 50 µM Zn2+ for 8 h. Cells were lysed with RIPA buffer, and cell lysates were subjected to SDS-PAGE and immunoblotting using an anti-human PTEN antibody (A) and a phospho-specific Akt antibody (B). Cells were lysed with 4 M guanidine thiocyanate. Total RNA was extracted and reverse-transcribed. Quantitative PCR was performed as described above (C). Data shown are representative of three separate experiments.

 

One of the required steps in the proteasome degradation pathway is the formation of an ubiquitin-protein conjugate (56). The covalent addition of multiple ubiquitin molecules to the target protein is requisite for efficient recognition and degradation by the 26 S proteasome (18). Therefore, the state of ubiquitination of PTEN in Zn2+-exposed cells was determined using an immunoprecipitation assay. As shown in Fig. 6, treatment of BEAS-2B cells with Zn2+ induced PTEN ubiquitination in a dose-dependent manner. These data strongly suggested that Zn2+ treatment led to ubiquitination of PTEN protein, targeting PTEN protein to degradation by the proteasome in airway epithelial cells.



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FIG. 6.
Ubiquitination of PTEN in Zn2+-exposed BEAS-2B cells. Confluent cells were starved overnight and pretreated with 20 µM MG132 before exposure to Zn2+ for 4 h. Cell lysates were immunoprecipitated with agarose-conjugated ubiquitin antibody. The precipitates were subjected to SDS-PAGE and immunoblotting using anti-PTEN antibody. Ubiquitinated PTEN bands were detected as described under "Experimental Procedures."

 

A PI3K Inhibitor, LY294002, Blocked Zn2+-induced PTEN Degradation—The role of PTEN in antagonizing the PI3K/Akt pathway has been well recognized (4, 13, 24). However, the influence of the PI3K/Akt signaling pathway on PTEN has not been investigated. Therefore, we examined the possible role of PI3K in Zn2+-induced degradation of PTEN using a specific PI3K inhibitor, LY294002. Surprisingly, incubation with LY294002 significantly inhibited Zn2+-induced PTEN degradation (Fig. 7A). To exclude the possible effect of LY294002 on PTEN transcription, PTEN mRNA expression in cells with or without LY294002 pretreatment was measured using the procedure described above. As shown in Fig. 7B, LY294002 pretreatment produced a minimal effect on the Zn2+-induced reduction of PTEN mRNA expression.



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FIG. 7.
The PI3K inhibitor, LY294002, blocked Zn2+-induced PTEN degradation with no significant effect on PTEN mRNA expression in BEAS-2B cells. Confluent cells were starved and pretreated with 25 µM LY294002. Cells were then stimulated with 50 µM Zn2+ and lysed in RIPA buffer. As shown in A, cell lysates were subjected to SDS-PAGE and immunoblotting using anti-human PTEN antibody. PTEN protein bands were detected using enhanced chemiluminescence reagents. Ct, control. As shown in B, cells were lysed with 4 M guanidine thiocyanate. Total RNA was extracted and reverse-transcribed. Quantitative PCR was performed as described above. Data shown are representative of three separate experiments.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although the basic concepts of PTEN structure and function have been established and much work has been done on elucidating the PTEN activity, the regulation of PTEN protein levels via transcription, translation, and post-translational mechanisms remains to be fully characterized (7, 12, 57). In this study, regulation of PTEN protein was examined in transformed and normal human airway epithelial cells exposed to exogenous Zn2+ ions. Our findings revealed some novel features of PTEN regulation by Zn2+ exposure. First, PTEN protein degradation can be specifically induced by Zn2+ exposure at non-toxic concentrations, in addition to the reduction of PTEN mRNA expression; second, PTEN degradation is mainly mediated through protein ubiquitination and subsequent proteolytic degradation, although down-regulation of PTEN mRNA expression may contribute to this process, to a lesser extent; third, PI3K mediates the modulation of PTEN protein levels in response to Zn2+ treatment. Additionally, in comparison with previous work on PTEN regulation, which mostly employed malignant cell lines, this study shows an inducible regulation of PTEN protein in "benign" or normal primary human airway epithelial cells in culture by Zn2+ exposure and in rat airways in vivo.

The mechanisms that regulate PTEN protein have been studied at the gene and protein levels. Modulation of PTEN transcription was shown in different cell types to be associated with an alteration of PTEN protein levels (4, 13). For example, up-regulation of PTEN protein and mRNA expression induced by ultraviolet light was reported to occur through the induction of the early growth response-1 transcription factor, further leading to apoptosis (58). In addition, activation of nuclear receptor peroxisome proliferator-activated receptor {gamma} induced PTEN up-regulation by binding two response elements in the genomic sequence upstream of PTEN (59). However, a recent study using a breast cancer cell line showed that elevated PTEN protein levels induced by the bone morphogenetic protein-2 (BMP-2) were not due to an increase in new PTEN protein synthesis (57). Decreased PTEN expression both at the mRNA and protein levels was observed in tumor-derived cell lines and some primary tumor tissues (53). Thus, the modulation of PTEN expression is complex and may be stimulus and cell type specific. Factors that down-regulate PTEN transcription remain to be identified (57). Transforming growth factor-{beta} exposure was first found to decrease PTEN expression in human keratinocytes (60). In the present study, exogenous Zn2+ ions were shown to clearly down-regulate PTEN protein and mRNA levels in human airway epithelial cells. However, the contribution of decreased PTEN mRNA levels induced by Zn2+ exposure appears to be minimal in PTEN protein decline for the following reasons: first, the half-life of normal PTEN protein is in the range of 48–72 h (16), and the decrease in PTEN protein levels occurs earlier than the decrease in PTEN mRNA expression following Zn2+ exposure; second, proteasome inhibition completely blocks Zn2+-induced PTEN protein reduction with no significant effect on PTEN mRNA expression; and third, PI3K inhibition significantly suppresses Zn2+-induced PTEN protein degradation without affecting its mRNA expression.

PTEN turnover in cultured COS-7 cells was reported to depend mainly on proteasome-, not lysosome-mediated, degradation (55). The enhanced PTEN degradation was preceded by the ubiquitination of PTEN in 293T cells (18). The notion that PTEN ubiquitination played a critical role in its degradation was strongly supported by a recent study showing that BMP-2 stimulation inhibited PTEN protein degradation by decreasing the association of PTEN with two proteins in the degradative pathway, UbCH7 and UbC9 (57). Our observations in this study also suggested that ubiquitination of PTEN played a major role in Zn2+-induced PTEN degradation. Zinc induced PTEN ubiquitination in a dose-dependent fashion; moreover, a specific proteasome inhibitor MG132 could markedly block Zn2+-induced PTEN degradation. Therefore, ubiquitination of PTEN was required for Zn2+-induced protein degradation and resultant Akt activation. This effect of Zn2+ on PTEN degradation appeared to be specific since the potent phosphatase inhibitor vanadyl sulfate and the EGF receptor ligand EGF did not show significant effect on PTEN protein levels.

Previous studies have shown that the phosphorylation of the PTEN COOH terminus regulates protein stability by modulating PTEN binding to PDZ-containing proteins or by other possible mechanisms (13, 18, 31, 55, 61). Recent studies employing PTEN mutagenesis or biochemical analysis by mass spectroscopy have identified a number of serine and threonine residues as potential regulatory sites involved in protein stabilization (31, 55, 61). However, the identified residues are not completely consistent among the studies. In addition, the latest study using MCF-7 cells has demonstrated that BMP-2-stimulated increases in PTEN protein levels were independent of changes in PTEN phosphorylation state and protein synthesis (57). These data suggest that PTEN protein stability may be regulated by multiple distinct post-translational modifications. It remains to be determined whether Zn2+ destabilizes PTEN protein by reducing the phosphorylation of specific PTEN residues or by an alternative mechanism.

Given that the phosphorylation of PTEN regulates its stability, the identification of the responsible upstream protein kinases has been the subject of great investigative effort. To date, the protein kinase CK2 has been proposed as a PTEN kinase (30, 55, 61). Our data showing that LY294002 blocked Zn2+-induced PTEN degradation, as well as Akt activation, implicated the involvement of the PI3K/Akt pathway in PTEN regulation. A recent study suggested that Akt activation could promote degradation of tuberin and FOXO3a via the proteasome (62). Additional studies will investigate the possible link between Akt activation and Zn2+-induced PTEN degradation.

Apparently, the signaling downstream of PTEN was affected by Zn2+-induced PTEN degradation since pretreatment of cells with MG132 significantly inhibited Zn2+-induced Akt activation as well as PTEN degradation. However, the earlier phosphorylation of Akt (1 h after exposure) might result from the slight inhibition of PTEN phosphatase activity by Zn2+ (data not shown). Early studies of PTEN showed that its activity was able to promote cell cycle arrest and apoptosis and inhibit cell motility, but more recent investigations revealed other functional consequences of PTEN action, such as the effect on the regulation of angiogenesis, non-insulin-dependent diabetes, and inflammation (4, 13, 63). The fact that zinc ions could down-regulate PTEN may provide some information in understanding the mechanisms of zinc-related pathophysiological processes and disorders.


    FOOTNOTES
 
* This work was supported by United States Environmental Protection Agency Cooperative Agreement CR#829522 awarded to the Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina, National Institutes of Health Grant CA85772 (to Y. E. W.) and by United States Environmental Protection Agency Science to Achieve Results Grant R82921 [GenBank] 401-01 (to L. M. G. and W. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence may be addressed. Tel.: 919-843-2714; Fax: 919-966-9863; E-mail: Weidong_Wu{at}med.unc.edu.

** To whom correspondence may be addressed. Tel.: 919-966-0670; Fax: 919-966-6271; E-mail: Ghio.andy{at}epa.gov.

1 The abbreviations used are: PTEN, phosphatase and tensin homolog deleted on chromosome 10; PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol (3,4,5)-triphosphate; EGF, epidermal growth factor; MAPKs, mitogen-activated protein kinases; MEK, MAPK/ERK kinase; RIPA, radioimmune precipitation buffer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back


    ACKNOWLEDGMENTS
 
We greatly appreciate the advice and technical support from Dr. L. M. Graves, Department of Pharmacology, University of North Carolina at Chapel Hill and Drs. J. E. Dixon and G. S. Taylor, Department of Biological Chemistry, University of Michigan, Ann Arbor, MI. We are grateful to Dr. P. A. Bromberg, Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina, for review of this manuscript.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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