Lenticular mitoprotection. Part B: GSK-3β and regulation of mitochondrial permeability transition for lens epithelial cells in atmospheric oxygen

Article (PDF Available)inMolecular vision 19:2451-2467 · November 2013with34 Reads
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Abstract
Loss of integrity of either the inner or outer mitochondrial membrane results in the dissipation of the mitochondrial electrochemical gradient that leads to mitochondrial membrane permeability transition (mMPT). This study emphasizes the role of glycogen synthase kinase 3beta (GSK-3β) in maintaining mitochondrial membrane potential, thus preventing mitochondrial depolarization (hereafter termed mitoprotection). Using 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763), an inhibitor of GSK-3β, and drawing a distinction between it and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (UO126), an inhibitor of extracellular-signal-regulated kinase (ERK) phosphorylation, the means by which GSK-3β influences mitoprotection in cultured human lens epithelial (HLE-B3) cells and normal, secondary cultures of bovine lens epithelial cells, maintained in atmospheric oxygen, was investigated. Virally transfected human lens epithelial cells (HLE-B3) and normal cultures of bovine lens epithelial cells were exposed to acute hypoxic conditions (about 1% O2) followed by exposure to atmospheric oxygen (about 21% O2). Specific antisera and western blot analysis was used to examine the state of phosphorylation of ERK and GSK-3β, as well as the phosphorylation of a downstream substrate of GSK-3β, glycogen synthase (GS, useful in monitoring GSK-3β activity). The potentiometric dye, 1H-benzimidazolium-5,6-dichloro-2-[3-(5,6-dichloro-1,3-diethyl-1,3-dihydro-2H-benzimidazol-2-ylidene)-1-propenyl]-1,3-diethyl-iodide (JC-1), was used to monitor mitochondrial depolarization upon exposure of inhibitor treatment relative to the control cells (mock inhibition) in atmospheric oxygen. Caspase-3 activation was scrutinized to determine whether mitochondrial depolarization inevitably leads to apoptosis. Treatment of HLE-B3 cells with SB216763 (12 µM) inactivated GSK-3β activity as verified by the enzyme's inability to phosphorylate its substrate, GS. SB216763-treated cells were not depolarized relative to the control cells as demonstrated with JC-1 fluorescent dye analysis. The HLE-B3 cells treated with UO126, which similarly blocked phosphorylation of GS, were nevertheless prone to mMPT relative to the control cells. Western blot analysis determined that Bcl-2-associated X (BAX) levels were unchanged for SB216763-treated or UO126-treated HLE-B3 cells when compared to their respective control cells. However, unlike the SB216763-treated cells, the UO126-treated cells showed a marked absence of Bcl-2, as well as phosphorylated Bcl-2 relative to the controls. UO126 treatment of bovine lens epithelial cells showed similar results with pBcl-2 levels, while the Bcl-2 content appeared unchanged relative to the control cells. HLE-B3 and normal bovine lens cell cultures showed susceptibility to mMPT associated with the loss of pBcl-2 by UO126 treatment. MITOCHONDRIAL DEPOLARIZATION MAY OCCUR BY ONE OF TWO KEY OCCURRENCES: interruption of the electrochemical gradient across the inner mitochondrial membrane resulting in mMPT or by disruption of the integrity of the inner or outer mitochondrial membrane. The latter scenario is generally tightly regulated by members of the Bcl-2 family of proteins. Inhibition of GSK-3β activity by SB216763 blocks mMPT by preventing the opening of the mitochondrial permeability transition pore. UO126, likewise, inhibits GSK-3β activity, but unlike SB216763, inhibition of ERK phosphorylation induces the loss of intracellular pBcl-2 levels under conditions where intracellular BAX levels remain constant. These results suggest that the lenticular mitoprotection normally afforded by the inactivation of GSK-3β activity may, however, be bypassed by a loss of pBcl-2, an anti-apoptotic member of the Bcl-2 family. Bcl-2 prevents the translocation of BAX to the mitochondrial outer membrane inhibiting depolarization by disrupting the normal electrochemical gradient leading to mMPT.
The dissipation of mitochondrial membrane potential
(Ψ) occurs in a process termed mitochondrial permeability
transition (mMPT) [1]. Lens epithelial cells represent an
ideal model for studying mMPT because the lens thrives
in a naturally hypoxic environment, and introducing atmo-
spheric oxygen increases the formation of reactive oxygen-
ated species (ROS), which, in turn, may cause a loss of ∆Ψ
[1,2]. The current literature suggests that mMPT is mediated
Molecular Vision 2013; 19:2451-2467 <http://www.molvis.org/molvis/v19/2451>
Received 2 April 2013 | Accepted 26 November 2013 | Published 29 November 2013
© 2013 Molecular Vision
2451
Lenticular mitoprotection. Part B: GSK-3β and regulation of
mitochondrial permeability transition for lens epithelial cells in
atmospheric oxygen
Morgan M. Brooks, Sudha Neelam, Patrick R. Cammarata
Department of Cell Biology and Anatomy, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX
Purpose: Loss of integrity of either the inner or outer mitochondrial membrane results in the dissipation of the mito-
chondrial electrochemical gradient that leads to mitochondrial membrane permeability transition (mMPT). This study
emphasizes the role of glycogen synthase kinase 3beta (GSK-3β) in maintaining mitochondrial membrane potential, thus
preventing mitochondrial depolarization (hereafter termed mitoprotection). Using 3-(2,4-dichlorophenyl)-4-(1-methyl-
1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763), an inhibitor of GSK-3β, and drawing a distinction between it and
1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (UO126), an inhibitor of extracellular-signal-regulated
kinase (ERK) phosphorylation, the means by which GSK-3β inuences mitoprotection in cultured human lens epithelial
(HLE-B3) cells and normal, secondary cultures of bovine lens epithelial cells, maintained in atmospheric oxygen, was
investigated.
Methods: Virally transfected human lens epithelial cells (HLE-B3) and normal cultures of bovine lens epithelial cells
were exposed to acute hypoxic conditions (about 1% O
2
) followed by exposure to atmospheric oxygen (about 21% O
2
).
Specic antisera and western blot analysis was used to examine the state of phosphorylation of ERK and GSK-, as
well as the phosphorylation of a downstream substrate of GSK-3β, glycogen synthase (GS, useful in monitoring GSK-3β
activity). The potentiometric dye, 1H-benzimidazolium-5,6-dichloro-2-[3-(5,6-dichloro-1,3-diethyl-1,3-dihydro-2H-
benzimidazol-2-ylidene)-1-propenyl]-1,3-diethyl-iodide (JC-1), was used to monitor mitochondrial depolarization upon
exposure of inhibitor treatment relative to the control cells (mock inhibition) in atmospheric oxygen. Caspase-3 activation
was scrutinized to determine whether mitochondrial depolarization inevitably leads to apoptosis.
Results: Treatment of HLE-B3 cells with SB216763 (12 µM) inactivated GSK- activity as veried by the enzymes
inability to phosphorylate its substrate, GS. SB216763-treated cells were not depolarized relative to the control cells
as demonstrated with JC-1 uorescent dye analysis. The HLE-B3 cells treated with UO126, which similarly blocked
phosphorylation of GS, were nevertheless prone to mMPT relative to the control cells. Western blot analysis determined
that Bcl-2-associated X (BAX) levels were unchanged for SB216763-treated or UO126-treated HLE-B3 cells when
compared to their respective control cells. However, unlike the SB216763-treated cells, the UO126-treated cells showed
a marked absence of Bcl-2, as well as phosphorylated Bcl-2 relative to the controls. UO126 treatment of bovine lens
epithelial cells showed similar results with pBcl-2 levels, while the Bcl-2 content appeared unchanged relative to the
control cells. HLE-B3 and normal bovine lens cell cultures showed susceptibility to mMPT associated with the loss of
pBcl-2 by UO126 treatment.
Conclusions: Mitochondrial depolarization may occur by one of two key occurrences: interruption of the electrochemi-
cal gradient across the inner mitochondrial membrane resulting in mMPT or by disruption of the integrity of the inner
or outer mitochondrial membrane. The latter scenario is generally tightly regulated by members of the Bcl-2 family of
proteins. Inhibition of GSK-3β activity by SB216763 blocks mMPT by preventing the opening of the mitochondrial per-
meability transition pore. UO126, likewise, inhibits GSK- activity, but unlike SB216763, inhibition of ERK phosphory-
lation induces the loss of intracellular pBcl-2 levels under conditions where intracellular BAX levels remain constant.
These results suggest that the lenticular mitoprotection normally afforded by the inactivation of GSK- activity may,
however, be bypassed by a loss of pBcl-2, an anti-apoptotic member of the Bcl-2 family. Bcl-2 prevents the transloca-
tion of BAX to the mitochondrial outer membrane inhibiting depolarization by disrupting the normal electrochemical
gradient leading to mMPT.
Correspondence to: Dr. Patrick R. Cammarata, Department of
Cell Biology and Anatomy, University of North Texas Health
Science Center at Fort Worth, 3500 Camp Bowie Boulevard, Fort
Worth, TX, 76107; Phone: (817) 735-2045; FAX: (817) 735-2610;
email: patrick.cammarata@unthsc.edu
Molecular Vision 2013; 19:2451-2467 <http://www.molvis.org/molvis/v19/2451> © 2013 Molecular Vision
2452
via the opening of the mitochondrial permeability transition
pore [3-5]. Studies have shown that glycogen synthase kinase
3beta (GSK-3β) is immediately proximal to the mitochondrial
permeability transition pore and acts as a point of integration
for many protective signals [6]. Thus, GSK-is a crucial
enzyme involved in preventing mMPT through regulating
the opening and closing of the mitochondrial permeability
transition pore [7,8]. Additional studies involving ischemic
reperfusion of cardiac myocytes have demonstrated that
inhibiting GSK-3β can prevent the dissipation of Ψ during
oxidative stress [9-11].
One of the multiple protective proteins that converge on
GSK-is the phosphorylated form of extracellular signal-
regulated kinase (ERK) [12]. Flynn et al. [1] has previously
demonstrated that after RNA suppresses ERK Ψ collapses
during oxidative stress in human lens epithelial cells (HLE-
B3) cells. Furthermore, studies conducted on metastatic
carcinoma cells have shown that phosphorylated ERK can
cause GSK-to become phosphorylated at its inhibitory
serine, thus inactivating the enzyme [12]. Combined, these
studies suggest that ERK can prevent the disruption of Ψ
by inactivating GSK-, presumably blocking the opening
of the mitochondrial transition pore. However, as will be
demonstrated in this study, inhibiting ERK phosphorylation
can, itself, cause mitochondrial depolarization regardless of
the activity of GSK-3β.
To date, the role that GSK-plays regarding preventing
mitochondrial depolarization has not been established in
an ocular system. In the current study, we demonstrate that
inactivating GSK-activity (as monitored by its failure to
phosphorylate glycogen synthase) using the pharmacological
inhibitor SB216763 has a regulatory function in preventing
mitochondrial depolarization. Furthermore, this study will
reveal that the mitoprotection normally afforded by GSK-
inactivation may be circumvented by inhibiting ERK phos-
phorylation, which culminates in inhibition of Bcl-2 phos-
phorylation, an anti-apoptotic member of the Bcl-2 family.
METHODS
Materials: The Mitogen Activated Protein Kinase-1/2
(MEK1/2) inhibitor 1,4-diamino-2,3-dicyano-1,4-bis[2-
aminophenylthio] butadiene (UO126) was purchased from
Cell Signaling Technology (Danvers, MA). The glycogen
synthase kinase inhibitor 3-(2,4-dichlorophenyl)-4-(1-
methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) was
purchased from Sigma-Aldrich (St. Louis, MO). The c-Jun
N-terminal kinase (JNK) inhibitors SP600125 (JNK Inhibitor
II) and AS601245 (JNK Inhibitor V) were purchased from
EMD Millipore Chemicals (Billerica, MA). Stock inhibitors
were prepared by adding to dimethyl sulfoxide (DMSO) as
follows: 20 mM for UO126, 16 mM for SB216763, 40 mM
for SP600125, and 40 mM for AS601245. The mitochondrial
dye 1H-benzimidazolium-5,6-dichloro-2-[3-(5,6-dichloro-
1,3-diethyl-1,3-dihydro-2H-benzimidazol-2-ylidene)-1-
propenyl]-1,3-diethyl-iodide (JC-1) was obtained from Life
Technologies (Grand Island, NY). All other reagents were
acquired from other commercially available sources as previ-
ously reported [1].
Cell cultures: HLE-B3 cells, a human lens epithelial cell line
immortalized by the SV-40 virus [13], were obtained from U.
Andley (Washington University School of Medicine, Depart-
ment of Ophthalmology, St. Louis, MO). Authentication of
the HLE-B3 cell line was verified with STR profile analysis
(American Type Culture Collection, Manassas, VA), which
confirmed that the cell line was human and of female origin,
as originally reported by Andley et al. [13]. A copy of the STR
profile is available upon request. All studies with HLE-B3
cells were performed with prefrozen stock cells (maintained
in liquid nitrogen) between passages 14 and 17. No experi-
ments exceeded five passages beyond the initial stock cell
passage. The cells were maintained in minimal essential
media (MEM) containing 5.5 mM glucose supplemented with
20% fetal bovine serum (Gemini Bio-Products, Sacramento,
CA), 2 mM L-glutamine, nonessential amino acids, and
0.02 g/l gentamycin solution (Sigma-Aldrich) and cultured at
37 °C and 5% CO
2
95% O
2
[1]. Cells were sub-cultured 4 to
5 days before the experiment and placed in MEM containing
20% fetal bovine serum (FBS). Twenty-four hours before the
day of the experiment, the cells were switched to serum-free
MEM. Unless otherwise specified, all experiments followed
a common protocol: Cells were maintained in atmospheric
O
2
(about 21%) for 90 min, then switched to hypoxic condi-
tions (about 1% O
2
) for 180 min, followed by reintroduction to
atmospheric O
2
. Each experiment was executed with control
DMSO-only cells (mock inhibitor treatment) and cells treated
with inhibitors. The DMSO concentration per experiment
never exceeded 0.05%.
Bovine eyes obtained from a local abattoir were trans
-
ported on ice to the laboratory, where the lenses were removed
aseptically. Bovine lens epithelial cells (BLECs) were isolated
and cultured in 20% bovine calf serum–supplemented Eagles
minimal essential medium. All studies with BLECs were
performed on cells of passage 2.
Western blot analysis: Whole cell lysates were collected from
HLE-B3 cultures using the hot protein extraction method as
described by Henrich et al. [14]. The cell cultures were rinsed
at room temperature in final concentration: 150 mM sodium
chloride, 10 mM sodium phosphate monobasic, 40 mM
Molecular Vision 2013; 19:2451-2467 <http://www.molvis.org/molvis/v19/2451> © 2013 Molecular Vision
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sodium phosphate dibasic), pH 7.4, before the monolayers
were lysed with hot lysis buffer (about 100 °C), subsequently
scraped into 1.7 ml micro centrifuge tubes, and immediately
sonicated. The lysis buffer consisted of 0.12 M Tris-HCl (pH
6.8), 4% sodium dodecyl sulfate (SDS), and 20% glycerol
[1]. Part of the lysate samples were removed and used to
determine the protein concentration. The protein concentra-
tion was calculated by using a DC protein assay kit from
Bio-Rad (Hercules, CA). The lysate samples contained 25 µg
of protein, 1X SDS laemmli buffer, and 1.5 µl 2-mercaptoeth-
anol (Sigma-Aldrich). The lysate samples were then boiled for
5 min and the proteins resolved on 12% SDS-polyacrylamide
gels. The proteins were then transferred to a nitrocellulose
membrane (Bio-Rad). The electrophoresis and western blot
apparatus were from Hoefer Scientific (Holliston, MA).
For western blot analysis, nitrocellulose membranes were
blocked with 0.1% BSA and 0.02% Tween-20 in Tris-buffered
saline (TTBS) for 60 min. These membranes were probed
overnight at 4 °C with primary antibodies at a 1:1,000 dilu-
tion. The blots were then rinsed in TTBS for 5 min 4X and
then incubated in either goat anti-rabbit horseradish peroxi-
dase conjugate or goat anti-mouse horseradish peroxidase
conjugate at 1:10,000 dilution (Santa Cruz Biotechnology,
Santa Cruz, CA) for 60 min at room temperature. Blots were
again rinsed in TTBS (4X 5 min washes), and proteins were
detected using a SuperSignal West Pico chemiluminescent
kit from Pierce (Rockford, IL) [1]. Probed membranes were
visualized on a Fluoro Chem TM 8900 imager (Alpha Inno-
tech, San Leandro, CA).
Primary antibodies were purchased from Cell Signaling
Technology (Danvers, MA). The antibodies used in this study
included rabbit anti-Bcl-2-associated X (BAX), rabbit anti-
glycogen synthase, rabbit anti-phosphoglycogen synthase
(Ser641), rabbit anti-phospho-GSK-(Ser9), rabbit anti-
GSK-, mouse anti-phospho-p44/42 mitogen-activated
protein kinase (Thr202/Tyr204), rabbit anti-p44/42 mitogen-
activated protein kinase, rabbit anti-phospho-Bcl-2 (Ser70),
rabbit anti-Bcl-2, and rabbit anti-phospho-c-Jun (Ser63).
Rabbit anti-actin was provided by Santa Cruz Biotechnology
(Santa Cruz, CA).
JC-1 fluorescence analysis and confocal microscopy: After
the HLE-B3 cells were subjected to inhibitor treatments, the
cells were stained with JC-1 to determine the mitochondrial
membrane potential. JC-1 is a membrane permeant lipophilic
dye that exists as J-aggregates in the mitochondrial matrix
(red fluorescence) and as monomers in the cytoplasm (green
fluorescence). During mitochondrial depolarization, the red
J-aggregates flow out of the mitochondria and accumulate in
the cytosol as green monomers [15]. Thus, depolarization can
be measured as an increasing green fluorescent/red fluores-
cent intensity ratio.
The JC-1 assay was performed as follows. HLE-B3 cell
monolayers were maintained in serum-free MEM with or
without inhibitor treatment, brought through atmospheric
oxygen into hypoxia, and then later switched back to atmo-
spheric oxygen as described above. At the end of the hypoxic
exposure, the hypoxic media on cells (oxygen depleted) were
poured off, and fresh (oxygen rich) serum-free MEM (with or
without an inhibitor) was added containing 5 µg/ml JC-1 for
30 min in a tissue culture incubator. The stained HLE-B3 cells
were then rinsed twice using serum-free MEM, and fresh
oxygenated serum-free MEM (with or without inhibitor, but
no JC-1 dye) was added. A random field of cells was imaged
every 2.5 min for 60 min using an X10 objective on a confocal
microscope (Ziess LSM410, LSM410, Thornwood, NY). The
excitation wavelength was 488 nm, and the microscope was
set to simultaneously detect green emission (540 nm) and red
emission (595 nm) channels using a dual bandpass filter [1].
Caspase-3 apoptosis detection assay: A caspase-3 enzyme-
linked immunosorbent assay (ELISA; Invitrogen, Camarillo,
CA) was used to determine apoptosis. HLE-B3 cell mono-
layers were maintained in serum-free MEM with or without
treatment and brought through our experimental protocol,
which included atmospheric oxygen into 3 h of hypoxia, and
then subsequently switched back to atmospheric oxygen.
Treatment included SB216763 (12 µM), UO126 (10 µM),
staurosporine (100 nM), or 0.05% DMSO vehicle maintained
throughout the course of the experiment. Sixty minutes after
atmospheric oxygen was reintroduced, all samples were
then lysed using our lysis buffer (0.12 M Tris-HCl [pH 6.8],
4% SDS, and 20% glycerol). Part of the lysate samples was
removed and used to determine the protein concentration. The
protein concentration was calculated by using a DC protein
assay kit (Bio-Rad). Apoptotic activity was determined
following the manufacturer’s protocol using 10 µg of protein.
Statistical analysis: Images from JC-1 confocal microscopy
were separated into individual red and green channels using
ImageJ (Baltimore, MD). The background fluorescence was
removed from each image before the intensity was measured.
The fluorescence intensity signal from each image was quan-
tified for the entire image and expressed as the ratio of green
fluorescent intensity over red fluorescent intensity. Western
blot densitometry was determined using ImageJ, and related
statistics was determined using GraphPad Prism 5 (La Jolla,
CA).
Molecular Vision 2013; 19:2451-2467 <http://www.molvis.org/molvis/v19/2451> © 2013 Molecular Vision
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RESULTS
Switching HLE-B3 cell cultures from hypoxia to atmospheric
oxygen or, conversely, from atmospheric oxygen to hypoxia,
does not induce mitochondrial depolarization: JC-1 analysis
of the DMSO mock-treated cells exposed to various cell
culture conditions was implemented to determine whether
environmental stress (i.e., hypoxia, atmospheric oxygen,
or reversal from one condition to the other) elicited mito-
chondrial depolarization. Four conditions were tested: cells
grown in constant atmospheric oxygen, cells switched from
atmospheric oxygen to hypoxia, cells switched from hypoxia
to atmospheric oxygen, and cells maintained in continuous
hypoxia. Under all tested conditions, the cells did not depo-
larize, indicating that continuous hypoxia, atmospheric
oxygen, and switching cell culture environments (hypoxia to
atmospheric oxygen or atmospheric oxygen to hypoxia) do
not induce depolarization (Figure 1).
SB216763 inhibits the enzymatic activity of GSK- and
prevents mitochondrial depolarization during oxidative
stress: To investigate the role GSK-plays in regulating
cellular mitoprotection with cells maintained in hypoxia and
subsequently exposed to atmospheric oxygen, HLE-B3 cells
were treated with the GSK-3β inhibitor, SB216763. Non-phos-
phorylated GSK-3β is the active form of the enzyme, and in
this form, the enzyme is capable of phosphorylating numerous
downstream substrates including glycogen synthase (GS)
[16]. The phosphorylation of GS is thus a useful parameter
for monitoring GSK-3β activity. Cultures of HLE-B3 cells
were grown on 100 mm dishes until >85% confluence. Cells
were treated with 12 µM SB216763 or mock-treated with
DMSO (control). After 90 min in ambient oxygen, the cells
were placed under hypoxic conditions (about 1% O
2
) for 3 h
and then switched back to atmospheric oxygen (about 21%
O
2
) for 3 h. Samples were collected from cells consistently
maintained in atmospheric oxygen (control), immediately
after hypoxic exposure and 1 h, 2 h, and 3 h of reexposure to
atmospheric oxygen. Western blot analysis showed that the
HLE-B3 cells subjected to SB216763 treatment had levels of
GSK-3β and phosphoglycogen synthase kinase-3beta (pGSK-
) similar to those of the controls (Figure 2). However, treat-
ment with SB216763 resulted in inhibition of phosphorylation
of GS. The failure to phosphorylate GS indicates the active
site of GSK-was inactivated. The continued presence
of pGSK-in the treated cells was because the autophos-
phorylation site of GSK-β was unaffected by SB216763 [17].
A key question that we wished to address in this study was
whether inhibiting GSK-activity positively correlated with
preventing mitochondrial depolarization. In a recent related
study [18], we observed that SB216763-treated cells monitored
for mMPT using emission spectroscopy displayed suppressed
mitochondrial depolarization relative to the control DMSO
mock-treated cells.
ERK1/2 inhibition prompts depolarization without affecting
pGSK-3β under oxidative stress conditions: A parallel
experiment, using the MEK1/2 inhibitor, UO126, was also
conducted on HLE-B3 cells to determine whether inhibiting
ERK1/2 phosphorylation had any impact on the phosphory-
lation of GSK-and GS. Lens cells were treated with 10
µM UO126 or DMSO and subsequently placed in hypoxic
conditions for 3 h. Following the hypoxic exposure, new
media were added to the culture dishes, and the cells were
exposed to atmospheric oxygen for up to 3 h in the continued
presence and absence of UO126. Lysates of these cells were
collected from cells maintained consistently in atmospheric
oxygen, after the hypoxic exposure and 1 h, 2 h, and 3 h
after atmospheric oxygen was reintroduced. Analysis of the
western blot membranes showed marked inhibition of p42
ERK1/2 and p44 ERK1/2 (Figure 3, upper panel, left) rela-
tive to the control cells. The loss of ERK1/2 phosphoryla-
tion did not affect the relative levels of GSK-3β or pGSK-
compared with the DMSO controls (Figure 3, upper panel,
right), whereas inhibiting ERK phosphorylation prevented the
downstream phosphorylation of GS. Therefore, the configu-
ration of the ratio of pGSK-/GSK-3β and pGS/GS appeared
identical irrespective of whether SB216763 or UO126 was
used (compare the bar graphs of Figure 2 and Figure 3). Given
the similarity in the profile of the ratio of pGSK-3β/GSK-
and pGS/GS, a critical question was whether UO126-treated
cells, similar to SB216763-treated cells, likewise prevented
mitochondrial depolarization relative to the control DMSO
mock-treated cells or whether UO126-treated cells were prone
to m MPT.
Parallel studies were conducted using JC-1 analysis.
HLE-B3 cells were treated with 10 µM UO126 or DMSO and
then placed under hypoxic conditions for 3 h. The cells were
exposed to the JC-1 dye for 30 min in atmospheric oxygen.
Following the JC-1 application, fresh media with U0126 or
DMSO were added to the culture plates. The cells were subse-
quently observed with confocal microscopy and the green and
red intensities recorded every 2.5 min for 60 min. The green
to red ratio of the UO126-treated cells markedly increased
over the time course relative to the control cells, indicating
the loss of ∆Ψ (Figure 3, bottom panel).
SB216763 versus UO126 treatment on BAX, Bcl-2, and Bcl-2
phosphorylation levels: As discussed above, comparison
of the western blots for pGSK-3β/GSK- and pGS/GS
between the two inhibitor treatments generated profiles
that were similar, if not, identical (compare Figure 2 and
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2455
Figure 3). However, whereas treatment with the GSK-
inhibitor, SB216763, likely blocked opening of the mitochon
-
drial membrane permeability transition pores, effectively
suppressing depolarization [18], treatment with UO126
elicited profound depolarization relative to the control cells
(Figure 3, bottom panel). We therefore examined in greater
detail the outcome of each inhibitor on the BAX, Bcl-2, and
pBcl-2 levels.
Lysates from all experimental treatments were evaluated
to determine the levels of Bcl-2, pBcl-2, and BAX. Western
blot membranes from the SB216763- and UO126-treated cells
relative to their respective controls indicated no alteration in
the levels of BAX for the treated and control cells (Figure 4,
Figure 1. JC-1 stained mock treated
(DMSO) HLE-B3 cells under
several adaptations of hypoxic
and atmospheric oxygen exposure.
Human lens epithelial (HLE-B3)
cells were stained with JC-1 (5 µg/
ml) and observed with confocal
microscopy with images captured
every 2.5 min over a 60 min period,
under the following conditions: (1)
continuous hypoxia, (2) continuous
atmospheric oxygen, (3) switching
from hypoxia to atmospheric
oxygen, or (4) switching from atmo-
spheric oxygen to hypoxia. Top
row: (continuous oxygen) HLE-B3
cells were maintained in atmo-
spheric oxygen and stained for 30
min in atmospheric oxygen. At the
end of the 30 min staining period,
fresh oxygenated media without
the JC-1 dye were added to the
dishes. A random eld of cells was
then imaged. Note to the reader:
The term random eld of cells”
here and with all successive JC-1
analyses is meant to infer an arbi-
trary eld of cells is selected, but
once chosen, the same eld of cells
is photographed throughout the 60
min image capture. Second row:
(atmospheric oxygen to hypoxia)
HLE-B3 cells were maintained in
atmospheric oxygen and stained for
30 min in atmospheric oxygen. At the end of the 30 min staining period, the cells were switched to hypoxic media (i.e., medium that had
been preincubated at 1% oxygen). A random eld of cells was immediately imaged. Third row: (hypoxia to atmospheric oxygen) HLE-B3
cells were placed in hypoxic conditions for 180 min. At the end of the hypoxic exposure, the media were removed and replaced with fresh
oxygenated media containing JC-1. The cells were stained for 30 min in atmospheric oxygen. After this 30 min period, the media were again
removed, and fresh oxygenated media were added without the JC-1 dye. A random eld of cells was then imaged. Fourth row: (continuous
hypoxia) HLE-B3 cells were stained with serum-free minimal essential media (MEM) cotaining JC-1 for 30 min in atmospheric oxygen. At
the end of this 30 min period, the media were removed, and fresh medium that had been preincubated at 1% oxygen was added without the
JC-1 dye. The cells were then switched into the hypoxic conditions for 180 min. The cells were imaged following the 3 h hypoxic exposure.
Under all experimental conditions, there was no evidence of loss of membrane potential throughout the 60 min image capture.
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2456
upper panel and Figure 5, upper panel). The relative levels of
pBcl-2 and Bcl-2 were not altered for the SB216763-treated
cells compared with the control cells (Figure 4, middle panel).
In contrast, the UO126-treated cells displayed significantly
diminished levels of pBcl-2 under all culture conditions,
including normoxic control, hypoxic exposure, and the rein-
troduction of oxygen subsequent to hypoxic exposure (Figure
5, middle panel); a significant loss of Bcl-2 was also observed
but, interestingly, only during the reintroduction of oxygen
phase (Figure 5, middle panel).
Of particular note, treatment with UO126 (Figure 5,
upper panel, left and bottom panels) but not with SB216763
(Figure 4, upper panel left and bottom panel) prevented the
phosphorylation of c-JUN, through all culture conditions.
Figure 2. Western blot analysis of glycogen synthase kinase 3β and glycogen synthase phosphorylation in HLE-B3 cells in the presence or
absence of SB216763. Total cell lysates were collected from >85% conuent human lens epithelial (HLE-B3) cell cultures that were incubated
for 90 min in serum-free minimal essential media (MEM), under conditions of atmospheric oxygen, containing either 12 µM SB216763
or 0.05% DMSO vehicle. Cells were then exposed to hypoxia for 3 h in the continued presence of SB216763. At the end of the hypoxic
incubation period, the hypoxic media were removed, and fresh, oxygenated serum-free MEM with SB216763 or DMSO vehicle were added
to the cultures. Cells were then placed in atmospheric oxygen for up to 3 h. Cultures were collected after (1) continuous normoxic exposure
(about 21% oxygen), (2) after the 3 h hypoxic exposure (about 1% oxygen), or (3) after reintroduction of atmospheric oxygen (about 21%)
for 1, 2, or 3 h subsequent to the 3 h hypoxic exposure. Total cell lysates were analyzed with immunoblots using 25 µg of protein per lane.
Anti-actin was used to normalize the bands to ensure equivalent lane loading. Three experiments, using independent cell populations, were
quantied using GraphPad Prism 5 and the relative densities plotted for pGSK-3β/GSK-3β and pGS/GS. No change was evident in the ratio
of pGSK-3β/GSK-3β while signicant inhibition of the phosphorylation of glycogen synthase by SB216763 was noted. Error bars represent
standard error. The asterisks (***) indicate p<0.001, Student t test.
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The phosphorylation of c-Jun is a useful parameter for moni-
toring JNK activity. That is, UO126, while effectively inhib-
iting ERK phosphorylation (refer to Figure 3, upper panel,
left), was nonetheless equally effective in inactivating JNK
activity. This observation imposed a burden of proof upon us
to determine whether the ERK pathway or the JNK pathway
negatively impacted the pBcl-2 levels.
JNK inhibition does not affect Bcl-2 phosphorylation:
HLE-B3 cells were treated with JNK inhibitors, SP600125
(5 µM, 10 µM, or 20 µM), AS601245 (5 µM, and 10 µM,
Figure 3. Western blot analysis
of GSK-and GS phosphoryla-
tion in HLE-B3 cells treated with
UO126 inhibitor. HLE-B3 cell
cultures were incubated for 90 min
in serum-free minimal essential
media (MEM), under conditions
of atmospheric oxygen, containing
either 10 µM UO126 or 0.05%
DMSO vehicle. Cells were then
exposed to hypoxia for 3 h. At
the end of the hypoxic incubation
period, the hypoxic media were
removed, and fresh, oxygenated
serum-free MEM with UO126 or
DMSO vehicle were added to the
cultures. Cells were then placed
in atmospheric oxygen for up to
3 h. Cultures were collected after
(1) continuous normoxic exposure
(about 21% oxygen), (2) after the
3 h hypoxic exposure (about 1%
oxygen), or (3) after reintroduction
of atmospheric oxygen (about 21%)
for 1, 2, or 3 h subsequent to the 3 h
hypoxic exposure. Total cell lysates
were analyzed with immunoblots
using 25 µg of protein per lane.
Anti actin was used to normalize
the bands to ensure equivalent lane
loading. The loss of phosphoryla-
tion of ERK by UO126 treatment
was noted (top, left panel), as was
the loss of phosphorylated glycogen
synthase by western blot analysis (top, right panel). Three experiments, using independent cell populations, were quantied using GraphPad
Prism 5, and the relative densities were plotted for pGSK-3β/GSK-3β and pGS/GS. No change was evident in the ratio of pGSK-3β/GSK-
while signicant inhibition of the phosphorylation of glycogen synthase by UO126 was indicated (middle panel). Error bars represent standard
error. The asterisks (***) indicate p<0.001, Student t test. (bottom panel, left) HLE-B3 cells were incubated for 90 min with serum-free MEM,
under atmospheric condition, containing 10 µM UO126 or 0.05% DMSO vehicle. Cells were switched to hypoxia for 3 h in the continued
presence of UO126 or DMSO vehicle. At the end of the hypoxic exposure, the cells had their media removed, and fresh, oxygenated serum-
free MEM containing 5 µg/ml JC-1 and either UO126 or DMSO added for 30 min in atmospheric oxygen. At the end of the 30 min incubation
period, the media were again switched with fresh serum-free MEM containing UO126 or DMSO in the absence of the JC-1 dye. The same
eld of cells was imaged every 150 s for 60 min. Serial confocal imaging of mitochondrial depolarization in HLE-B3 cells in the presence
of UO126 demonstrated signicant depolarization compared to control cells. (bottom panel, left) Images of the red and green intensity for
the UO126- and DMSO-treated cells at t=60 min (bar=20 µm). Note the marked intensity of the green channel with UO126-treated cells
relative to DMSO mock treatment at the completion of the 60 min analysis (bottom panel, right) indicating mitochondrial depolarization.
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Figure 4. Western blot analysis of BAX, Bcl-2, pBcl-2, and phospho-c-Jun in HLE-B3 cells in the presence or absence of SB216763. Total
cell lysates were collected from >85% conuent HLE-B3 cell cultures that were incubated for 90 min in serum-free minimal essential media
(MEM), under conditions of atmospheric oxygen, containing either 12 µM SB216763) or 0.05% DMSO vehicle. Cells were then exposed to
hypoxia for 3 h in the continued presence of SB216763 or DMSO vehicle. At the end of the hypoxic incubation period, the hypoxic media
were removed, and fresh, oxygenated serum-free MEM with SB216763 or DMSO were added to the cultures. Cells were then placed in
atmospheric oxygen for up to 3 h. Cultures were collected after (1) continuous normoxic exposure (about 21% oxygen), (2) after the 3 h
hypoxic exposure (about 1% oxygen), or (3) after reintroduction of atmospheric oxygen (about 21%) for 1, 2, or 3 h subsequent to the 3 h
hypoxic exposure. Total cell lysates were analyzed with immunoblots using 25 µg of protein per lane. Anti-actin was used to normalize the
bands to ensure equivalent lane loading. Three experiments, using independent cell populations, were quantied using GraphPad Prism 5
and the relative densities plotted for BAX/actin, Bcl-2/actin, pBcl-2/actin, and phospho-c-Jun/Actin. No change was evident in the ratio of
BAX/actin, Bcl-2/actin, pBcl-2/actin, or phospho-c-Jun/actin by treatment with SB216763. Error bars represent standard error, Student t test.
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2459
20 µM), or DMSO vehicle (control). Phosphorylated JNK is
the active form of the enzyme, and in this form, the enzyme is
capable of phosphorylating numerous downstream substrates,
including c-Jun. The phosphorylation of c-Jun is thus a useful
parameter for monitoring JNK activity. Treated and control
cells were placed in hypoxic conditions for 3 h and then
switched to atmospheric oxygen for 3 h. After the switch
to atmospheric oxygen, the hypoxic media were removed
from the cultures, and fresh oxygenated media containing
either inhibitor or vehicle were added. Lysates of all the cells
were collected after 3 h exposure in atmospheric oxygen.
SP600125 and AP601245 markedly reduced phospho-c-Jun
at all concentrations relative to the control cells (Figure 6).
Further analysis indicated no change in the levels of BAX,
Figure 5. Western blot analysis of
BAX, Bcl-2, pBcl-2, and phospho-
c-Jun in HLE-B3 cells in the pres-
ence or absence of UO126. Total cell
lysates were collected from >85%
confluent human lens epithelial
(HLE-B3) cell cultures that were
incubated for 90 min in serum-free
minimal essential media (MEM),
under conditions of atmospheric
oxygen, containing either 10 µM
UO126 or 0.05% DMSO vehicle.
Cells were then exposed to hypoxia
for 3 h in the continuous presence
of UO126 or DMSO vehicle. At
the end of the hypoxic incubation
period, the hypoxic media were
removed, and fresh, oxygenated
serum-free MEM with UO126 or
DMSO were added to the cultures.
Cells were then placed in atmo-
spheric oxygen for up to 3 h in the
presence or absence of SB216763.
Cultures were collected after (1)
continuous normoxic exposure
(about 21% oxygen), (2) after the
3 h hypoxic exposure (about 1%
oxygen), or (3) after reintroduction
of atmospheric oxygen (about 21%)
for 1, 2, or 3 h subsequent to the 3 h
hypoxic exposure. Total cell lysates
were analyzed with immunoblots
using 25 µg of protein per lane.
Anti-actin was used to normalize
the bands to ensure equivalent lane loading. Three experiments, using independent cell populations, were quantied using GraphPad Prism
5. (top panel, left) The phosphorylation of c-Jun, as well as that of Bcl-2, was blocked by treatment with UO126 under all conditions as
determined with western blot analysis. Interestingly, Bcl-2 levels were signicantly diminished with treatment by UO126 but only upon
reintroduction to atmospheric oxygen. (top panel, right) No change in relative density of the ratio of BAX/actin was evident by treatment
with UO126 under any condition. Error bars represent standard error. (middle panel, left) A signicant drop in Bcl-2 levels was noted but
only upon reintroduction of atmospheric oxygen for the 1, 2, and 3 h incubation periods. The asterisk (*) indicates p<0.05, Student t test.
(middle panel, right) A signicant loss of pBcl-2 was noted under all conditions (continuous atmospheric oxygen, continuous hypoxia,
and reintroduction from hypoxia to atmospheric oxygen). The asterisks (***) indicate p<0.001, Student t test. (bottom panel) Unlike with
SB216763 (refer to Figure 4), treatment with UO126 resulted in a marked decrease in the phosphorylation of c-Jun, indicating that UO126
adversely affects JNK activity. The asterisks (**) indicate p<0.01, Student t test.
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Bcl-2, or pBcl-2 for cells treated with SP600125 or AS601245
versus cells treated with DMSO vehicle (Figure 6). A slight
increase in the levels of pERK were observed with either
inhibitor treatment cells relative to the control cells, the
meaning of which is not immediately evident to us. However,
whereas UO126 negatively impacted JNK activity (Figure 5),
the JNK inhibitors did not cross-inactivate ERK phosphoryla-
tion (Figure 6). We therefore concluded that inhibition of the
ERK pathway, not the JNK pathway, elicited the inhibition of
phosphorylation of Bcl-2 (Figure 5).
Increased mitochondrial depolarization does not neces-
sarily affect cell viability: To determine if the inhibition of
ERK or GSK-activity influence the onset of apoptosis,
an active caspase-3 ELISA was implemented. As above, the
HLE-B3 cells were treated with either 12 µM SB216763,
10 µM UO126, or 0.05% DMSO. The 100 nM staurosporine
concentration was used as a positive activator of caspase-3.
Cells were incubated for 90 min and then placed in hypoxia
for 180 min. At the end of the hypoxic exposure, the media
were removed from the treated and untreated cells, and fresh
media were added. The cells were then placed in atmospheric
Figure 6. Western blot analysis of
phospho-c-Jun in the presence of
SP600125 or AS601245. HLE-B3
cell cultures were incubated for
90 min in serum-free minimal
essential media (MEM), under
conditions of atmospheric oxygen,
containing either SP600125 (5 µM,
10 µM, and 20 µM), AS601245 (5
µM, 10 µM, and 20 µM), or DMSO
(0.05%) vehicle. Cells were then
exposed to hypoxia for 3 h in the
continued presence of inhibitors.
At the end of the hypoxic exposure,
the media were removed, and fresh,
oxygenated serum-free MEM with
either inhibitor or DMSO were
added to the cultures. Cells were
subsequently placed in atmospheric
oxygen for 3 h. Whole cell lysates
were collected at the end of the 3
h reintroduction of atmospheric
oxygen. Lysates were analyzed with
western blot for phospho-c-Jun,
pERK, BAX, Bcl-2, and p-BCL-2
using 25 µg of protein per lane.
Anti-actin was used to normalize
the bands to ensure equivalent lane
loading. The inhibition of the phos-
phorylation of c-Jun by either JNK
inhibitor indicates the inactivation
of JNK activity, while no loss of
BAX, Bcl-2, or pBcl-2 was noted. Under this condition, the phosphorylation of ERK was unimpeded. This experiment was performed twice
with two independent cell populations with identical results. Treatment with either JNK inhibitor, SP600125 or AS601245, resulted in a
marked decrease in the phosphorylation of c-Jun, indicating that both inhibitors inhibited JNK activation. The asterisks (**) indicate p<0.01,
Student t test. BAX, Bcl-2, and pBcl-2 were not signicantly diminished relative to the DMSO control, indicating that the ERK pathway, but
not the JNK pathway, is involved in the loss of Bcl-2 and pBcl-2 (refer to Figure 5). Two experiments, using independent cell populations,
were quantied using GraphPad Prism 5. Reader note: The 20 µM AS601245 was run only once as reected in the western blot but not in
the densitometry plots because we cannot generate statistics on one run.
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oxygen for 60 min. SB216763 and U0126 were administered
throughout the duration of the experiment. At the end of this
60 min period, all samples were lysed with lysis buffer. The
DMSO mock-treated cells displayed minimal capase-3 activa-
tion relative to the cells treated with staurosporine (positive
control), which showed a significant increase in caspase-3
activity (Figure 7). The UO126- and SB216763-treated cells
demonstrated levels of capase-3 activity similar to the DMSO
mock-treated cells (Figure 7). The lack of caspase-3 activity
in the UO126- and SB216763-treated cells indicate that apop-
tosis did not occur in the presence of either inhibitor, although
the UO126 treatment elicited mitochondrial membrane
permeability transition (refer to Figure 3, bottom panel).
Bovine lens epithelium portray similar responses to
UO126-treatment as compared with HLE-B3 cells: To
determine that our results (and consequent interpretations)
had not been compromised by the viral transformation of the
HLE-B3 cells, we repeated the UO126 treatment, as described
above with HLE-B3 cells, with secondary cultures of normal
bovine lens epithelial cells. Inhibition of p42 ERK1/2 and
p44 ERK1/2 relative to the control cells was noted (Figure
8, upper panel). As with the HLE-B3 cells (Figure 3, upper
panel, right), neither GSK-3β nor pGSK-3β (Figure 8, middle
panel) was diminished, compared with the DMSO controls.
Likewise, inhibition of ERK phosphorylation prevented the
downstream phosphorylation of GS (compare Figure 3, upper
panel, right and Figure 8, middle panel).
We further investigated the effect of UO126 treatment
on BAX, Bcl-2, and pBcl-2 levels using secondary cultures
of bovine lens epithelial cells. Lysates were evaluated with
western blot analysis to determine the levels of BAX, Bcl-2,
and pBcl-2. Western blot membranes from the UO126-treated
cells relative to their respective controls indicated some lane
loading variability, but no obvious loss, in the levels of BAX
(Figure 8, bottom panel), similar to that of the HLE-B3
cells (Figure 5, upper left panel). As with the HLE-B3 cells
(Figure 5, upper left panel), the UO126-treated cells displayed
a significant reduction in pBcl-2 under all culture conditions:
normoxic control, hypoxic exposure, and the reintroduction
of oxygen (Figure 8, bottom panel). Of interest, unlike the
HLE-B3 cells (Figure 5, middle panel) where a significant
loss of Bcl-2 was noted (but only during the reintroduction
of the oxygen phase), there was no dramatic reduction in
the levels of Bcl-2 between the treated and untreated cells
under any condition (Figure 8, bottom panel). Finally, as first
observed with HLE-B3 cells (Figure 5, upper left panel),
UO126 elicited the inhibition of phosphorylation of c-JUN,
through all culture conditions (Figure 8, bottom panel).
Bovine lens epithelium depolarize in the absence of pBcl-2:
Parallel studies with UO126 were conducted using JC-1
analysis. Normal bovine cell cultures were treated with 10
Figure 7. Active caspase-3 ELISA
analysis of possible apoptosis
in HLE-B3 cells treated with
SB216763, UO126, staurosporine,
or DMSO. The possibility of the
onset of apoptosis was determined
using an active capase-3 ELISA
with HLE-B3 cells treated with
SB216763; 12 µM), UO126; 10
µM), staurosporine (100 nM), or
0.05% DMSO vehicle. Treated and
mock-treated HLE-B3 cells were
incubated with serum-free minimal
essential media (MEM) for 90 min
in atmospheric oxygen. The cells
were then switched to hypoxia for
180 min in the continued presence
of each individual treatment. At the
end of the hypoxic exposure, the media were removed and replaced with fresh, oxygenated media still containing SB216763, UO126,
staurosporine, or DMSO vehicle. The cells were placed in atmospheric oxygen for 60 min and subsequently lysed, and the quantity of protein
determined per treatment. Caspase-3 activity was determined using 10 µg of protein following the manufacturers instructions. Data are
based upon results from three independent cell populations and were analyzed using GraphPad Prism 5. The error bars represent the standard
error. Only treatment with staurosporine indicated a marked increase in activation of caspase-3.
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Figure 8. Western blot analysis
of GSK-and GS phosphoryla-
tion and BAX, Bcl-2, pBcl-2,
and phospho-c-Jun in secondary
cultures of normal bovine cells
treated with UO126 inhibitor.
Bovine cell cultures were incubated
for 90 min in serum-free minimal
essential media (MEM), under
conditions of atmospheric oxygen,
containing either 10 µM UO126 or
0.05% DMSO) vehicle. Cells were
then exposed to hypoxia for 3 h
in the presence of either UO126
or DMSO vehicle. At the end of
the hypoxic incubation period, the
hypoxic media were removed, and
fresh, oxygenated serum-free MEM
with UO126 or DMSO were added
to the cultures. Cells were then
placed in atmospheric oxygen for
up to 3 h. Cultures were collected
after (1) continuous normoxic expo-
sure (about 21% oxygen), (2) after
the 3 h hypoxic exposure (about 1%
oxygen), or (3) after reintroduction
of atmospheric oxygen (about 21%)
for 1, 2, or 3 h subsequent to the 3 h
hypoxic exposure. Total cell lysates
were analyzed with immunoblots
using 25 µg of protein per lane.
Anti-actin was used to normalize
the bands to ensure equivalent lane
loading. Prevention of phosphory-
lation of ERK with UO126 treat-
ment was noted (top panel), as was
the inhibition of phosphorylated
glycogen synthase with western
blot analysis (middle panel). The
levels of GSK-) and pGSK-3β
were consistent in the presence
and absence of UO126. The phos-
phorylation of c-Jun, as well as that
of Bcl-2, was blocked by treatment
with UO126 under all conditions as
determined with western blot anal-
ysis (bottom panel). Of particular
note, Bcl-2 levels were unaffected
by UO126 treatment compared to HLE-B3 cells (refer to Figure 5) where a signicant diminution of Bcl-2 was observed but only upon
reintroduction to atmospheric oxygen. No change in the relative levels of BAX was evident by treatment with UO126 under any condition.
The experiment with normal, secondary cultures of bovine cells was run once, to conrm that a similar pattern of biochemical modications
by treatment with UO126 was reproducible with normal bovine cell cultures as was observed with HLE-B3, afrming that the former results
were not inuenced by viral transformation.
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2463
µM UO126 or DMSO and then placed under hypoxic condi-
tions for 3 h. The cells were exposed to the JC-1 dye for 30
min in atmospheric oxygen. Following the JC-1 application,
fresh media with U0126 or DMSO were added to the culture
plates. The cells were subsequently observed with confocal
microscopy and the green and red intensities recorded every
2.5 min for 60 min. As with the UO126-treated HLE-B3 cells
(refer to Figure 3, bottom panel) the green to red ratio of the
UO126-treated bovine lens cells markedly increased over the
time course relative to the control cells, indicating the loss of
Ψ (Figure 9).
DISCUSSION
To establish our reference baseline, it was first necessary to
demonstrate that the experimental manipulation of switching
cells from hypoxia to atmospheric oxygen or vice versa, from
atmospheric oxygen to hypoxia, was not, of itself, suffi-
cient oxidative stress to elicit mitochondrial depolarization
(Figure 1). Once established that the physical manipulation of
switching cell cultures from one oxygen pressure to another
did not impose mitochondrial depolarization, we investigated
the regulatory function of GSK-insofar as its ability to
convey mitochondrial resistance to depolarization.
First, we sought to clarify whether blocking GSK-s
autophosphorylation site or inactivating its catalytic active
site conferred prevention of mitochondrial depolarization. To
monitor the enzyme’s active, catalytic site, we scrutinized
the phosphorylation of a downstream substrate of GSK-,
glycogen synthase (GS). Our data revealed that SB216763
did not block the autophosphorylation of GSK-3β relative to
control cells, but successfully eliminated the phosphoryla-
tion of GS, indicating that the catalytic site of GSK-3β was
inactivated (Figure 2). Stated another way, the failure to
phosphorylate GS, but not the autophosphorylation site of
GSK-3β, appears to be a better predictor of whether inhib-
iting GSK-s enzymatic activity positively correlates with
blocking mitochondrial membrane permeability transition. In
a recent report using the specific GSK- inhibitor, SB216763,
we demonstrated that “inhibition of GSK- activity by
SB216763 blocked mitochondrial membrane permeability
transition relative to the slow but consistent depolarization
observed with the control cells.” We concluded that inhibiting
GSK-activity with the GSK-inhibitor SB216763 provides
positive protection against mitochondrial depolarization [18].
The role of GSK- and how it may be influenced
by upstream signaling mechanisms has been the focus
of numerous studies. “There is evidence in different cell
types [19] that anti-apoptotic responses can be mediated
by phosphatidylinositol 3-kinase (PI3K) and the Akt/PKB
serine-threonine protein kinase, p42/p44 mitogen-activated
protein kinases or extracellular response kinases (ERKs),
Raf, and cyclic AMP-dependent protein kinase (PKA).To
further delve into this issue, using the lens cell model, we
compared and contrasted the pGSK-/GSK-3β, as well as
the pGS /GS western blot profiles of cells treated with an
inhibitor of ERK against the known catalytic site inhibitor
of GSK-3β, SB216763 (refer to above). We followed up with
a JC-1 evaluation to monitor whether ERK inhibitor treat-
ment prompted mitochondrial depolarization or whether,
like SB216763, inhibiting ERK phosphorylation imposed
resistance to mitochondrial depolarization [18].
Similar to the treatment with SB216763 (Figure 2),
treatment with UO126 had no effect on autophosphoryla-
tion of GSK-relative to the control cells, but successfully
eliminated the phosphorylation of GS (Figure 3, upper
panel, right). However, whereas treatment with SB216763
resulted in suppression of mitochondrial depolarization [18],
unexpectedly, the UO126-treated cells displayed profound
depolarization (Figure 3, bottom panel). We therefore sought
to explain the apparent discrepancy between the similarity
of the SB216763 and UO126 profiles of pGSK-3β/GSK-
and pGS/GS and the observation that the former [18] but not
the latter (Figure 3) conferred resistance to mitochondrial
depolarization.
Western blot analysis of HLE-B3 cells treated with
SB216763 or UO126 showed that the BAX levels were
unchanged relative to the untreated, control cells (compare
Figure 4 versus Figure 5). Moreover, the BAX content of the
UO126-treated normal bovine lens epithelial cells was similar
relative to the control cells (Figure 8, bottom panel). These
data support the notion that BAX does not play a direct role
in lens epithelial cell resistance to mitochondrial depolariza-
tion. We therefore directed our attention to answering the
question, “Is it the continuous expression of Bcl-2 or the phos-
phorylation of Bcl-2 that confers resistance to mitochondrial
depolarization?” The UO126-treated virally transformed
HLE-B3 cells, and the normal bovine lens epithelial cells
demonstrated a propensity to depolarize (compare Figure 3,
bottom panel and Figure 9). We took advantage of our obser-
vation that UO126 treatment with HLE-B3 cells instigated a
loss of Bcl-2 (but only under the condition of reintroduction
of oxygen (refer to Figure 5) whereas UO126-treatment with
normal bovine lens epithelial cells did not diminish the levels
of Bcl-2 (Figure 8, bottom panel), this although with both
types of cells, a profound loss of pBcl-2 was apparent under
all conditions (compare Figure 5, top panel, left with Figure 8,
bottom panel). Therefore, since the bovine cells depolarized
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2464
without the loss of Bcl-2, we conclude that pBcl-2 confers
anti-depolarization resistance in lens epithelial cells.
Lei et al. 2002 [20-24] stated, “this phosphorylation has
been reported to inhibit the pro-survival function of Bcl.
This conclusion, however, is controversial as other studies
have indicated that phosphorylation may enhance the anti-
apoptotic actions of Bcl-2” [25-27]. Data from our study
lend support to the latter statement in that the depolarization
observed with UO126 treatment is caused by a lack of pBcl-2
(compare Figure 3 and Figure 5 with Figure 8 and Figure
Figure 9. JC-1 analysis of bovine
lens epithelial cells treated with
UO126 inhibitor. Secondary
cultures of bovine lens epithelial
cells were incubated for 90 min
with serum-free minimal essential
media (MEM), under atmospheric
condition, containing 10 µM
UO126 or 0.05% DMSO vehicle.
Cells were switched to hypoxia
for 3h in the continued presence
or absence of UO126. At the end
of the hypoxic exposure, the cell
media were removed, and fresh,
oxygenated serum-free MEM
containing 5 µg/ml JC-1 and either
UO126 or DMSO added for 30 min
in atmospheric oxygen. At the end
of the 30 min incubation period,
the media were again switched to
fresh serum-free MEM containing
UO126 or DMSO in the absence
of the JC-1 dye. A random eld of
cells was chosen, and that eld of
cells was imaged every 150 s for
60 min. Serial confocal imaging
of mitochondrial depolari