Zinc-induced PTEN Protein Degradation through the Proteasome Pathway in Human Airway Epithelial Cells*
Weidong Wu
,
Xinchao Wang
,
Wenli Zhang
,
William Reed
,
James M. Samet ¶,
Young E. Whang || and
Andrew J. Ghio ¶ **
From the
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.
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ABSTRACT
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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.
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INTRODUCTION
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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.
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EXPERIMENTAL PROCEDURES
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Cell Culture and in Vitro ExposureThe 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
7080) 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 2022 h before
treatment with zinc sulfate (Sigma).
Normal human bronchial epithelial cells (passages 23) 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 1216 h before
challenge with zinc sulfate.
ImmunoprecipitationBEAS-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).
ImmunoblottingCells 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
-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-PCRBEAS-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.
ImmunohistochemistryMale 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.
StatisticsData are presented as means ± S.E.
Unpaired Student's t tests with conferring correction were used for
pairwise comparison.
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RESULTS
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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.
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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).
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Zn2+-induced PI3K/Akt
Activation in Human Airway Epithelial CellsSince 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.
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PTEN mRNA Level Was Significantly Reduced by
Zn2+ ExposureReduction 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 4872 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.
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Proteasome-mediated PTEN Degradation in
Zn2+-exposed CellsThe 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.
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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."
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A PI3K Inhibitor, LY294002, Blocked
Zn2+-induced PTEN DegradationThe
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.
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DISCUSSION
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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
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-
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 4872 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. 
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. 
 |
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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