Hypoxia-Mediated Degradation of Na,K-ATPase via
Mitochondrial Reactive Oxygen Species and the
Ubiquitin-Conjugating System
Alejandro P. Comellas, Laura A. Dada, Emilia Lecuona, Liuska M. Pesce, Navdeep S. Chandel,
Nancy Quesada, G.R. Scott Budinger, Ger J. Strous, Aaron Ciechanover, Jacob I. Sznajder
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Abstract—We set out to determine whether cellular hypoxia, via mitochondrial reactive oxygen species, promotes
Na,K-ATPase degradation via the ubiquitin-conjugating system. Cells exposed to 1.5% O2 had a decrease in
Na,K-ATPase activity and oxygen consumption. The total cell pool of ␣1 Na,K-ATPase protein decreased on exposure
to 1.5% O2 for 30 hours, whereas the plasma membrane Na,K-ATPase was 50% degraded after 2 hours of hypoxia,
which was prevented by lysosome and proteasome inhibitors. When Chinese hamster ovary cells that exhibit a
temperature-sensitive defect in E1 ubiquitin conjugation enzyme were incubated at 40°C and 1.5% O2, the degradation
of the ␣1 Na,K-ATPase was prevented. Exogenous reactive oxygen species increased the plasma membrane Na,K-ATPase
degradation, whereas, in mitochondrial DNA deficient ␳0 cells and in cells transfected with small interfering RNA against
Rieske iron sulfur protein, the hypoxia-mediated Na,K-ATPase degradation was prevented. The catalase/superoxide
dismutase (SOD) mimetic (EUK-134) and glutathione peroxidase overexpression prevented the hypoxia-mediated Na,KATPase degradation and overexpression of SOD1, but not SOD2, partially inhibited the Na⫹ pump degradation. Accordingly,
we provide evidence that during hypoxia, mitochondrial reactive oxygen species are necessary to degrade the plasma
membrane Na,K-ATPase via the ubiquitin-conjugating system. (Circ Res. 2006;98:1314-1322.)
Key Words: ATP 䡲 oxygen 䡲 proteasome 䡲 antioxidants 䡲 cell adaptation
A
regulating ATP-consuming processes.2 This regulation allows
ATP levels to remain constant, even while ATP turnover rates
greatly decline. The ATP requirements of ion pumping are
downregulated by generalized “channel” arrest in hepatocytes
and by the arrest of specific ion channels in neurons.7
The Na,K-ATPase, a membrane protein critically important for the maintenance of the ion gradients required for cell
homeostasis, consists of a catalytic ␣ subunit and a regulatory
␤ subunit.8 –10 Active Na⫹ and K⫹ transport by this protein is
responsible for ⬇20% to 80% of the resting metabolic rate of
the cell11 and approximately 30% of cellular ATP consumption.2,12–16 Hypoxia, within minutes, induces a decrease of the
Na,K-ATPase activity by promoting the endocytosis of the ␣
subunit from the plasma membrane into intracellular pools.17
Here we report that hypoxia via mROS induces the catalytic
Na,K-ATPase ␣ subunit to undergo ubiquitin-mediated degradation, downregulating the activity of a major metabolic protein.
daptation to hypoxia represents a well-defined means to
improve ischemic tolerance. Unfortunately, there is no
definitive understanding of the mechanisms associated with
these phenomena.1 As mammalian cells encounter lower
oxygen levels, they develop mechanisms to prevent depletion
of oxygen to anoxia that might result in cell death.2 Cells
respond to hypoxia through the stabilization of the transcription factor hypoxia-inducible factor (HIF)-1␣. In normoxic
conditions, prolyl hydroxylases hydroxylate conserved proline residues in HIF-1␣.3,4 This substrate modification is
recognized by a ubiquitin ligase enzyme (Von–Hippel–Lindau
protein [VHL]) that ubiquitinates and targets HIF-1␣ to the
proteasome. During hypoxia, VHL-mediated degradation of
HIF-1␣ is suppressed, allowing its transcriptional activation.4,5
The intracellular mechanisms by which cells sense hypoxia to
stabilize HIF-1␣ are not fully understood. The generation of
mitochondrial reactive oxygen species (mROS) during hypoxia
has been proposed as part of an oxygen sensing pathway for the
hypoxic stabilization of HIF-1␣.6
Another mechanism to prevent the depletion of oxygen
during hypoxia is to decrease the cellular demand for oxygen
by upregulating anaerobic ATP-producing pathways and down-
Materials and Methods
Materials
Na,K-ATPase ␣-1 subunit monoclonal antibody (clone 464.6) was
purchased from Upstate Biotechnology (Lake Placid, NY). GFP
Original received June 15, 2005; resubmission received February 7, 2006; revised resubmission received March 22, 2006; accepted April 4, 2006.
From the Division of Pulmonary and Critical Care Medicine (A.P.C., L.A.D., E.L., L.M.P., N.S.C., N.Q., G.R.S.B., A.C., J.I.S.), Feinberg School of
Medicine, Northwestern University, Chicago, Ill; The Vascular and Tumor Biology Research Center (A.C.), Faculty of Medicine, Technion-Israel
Institute of Technology, Haifa, Israel; and University Medical Center Utrecht and Institute of Biomembranes (G.J.S.), Utrecht, The Netherlands.
Correspondence to Jacob I. Sznajder, MD, Division of Pulmonary and Critical Care Medicine, Northwestern University, Feinberg School of Medicine,
240 E Huron, McGaw 2-2300, Chicago, IL 60611. E-mail [email protected]
© 2006 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/01.RES.0000222418.99976.1d
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Comellas et al
polyclonal antibody was purchased from Clontech (Palo Alto, Calif),
and anti-ubiquitin monoclonal antibody was purchased from Santa
Cruz Biotechnology (Santa Cruz, Calif). EUK-134 was provided to
us by Eukarion Inc (Bedford, Mass). Superoxide dismutase (SOD) 2,
SOD1, and glutathione peroxidase were gifts from Dr J. Engelhardt
(purchased through University of Iowa, Viral Core). T-butyl hydroperoxide (t-H2O2) and chloroquine from Sigma-Aldrich (St Louis,
Mo). EZ-link NHS-SS-biotin and streptavidin beads purchased from
Pierce Chemical Co (Rockford, Ill). Proteasome inhibitors MG-132
and lactacystin purchased from Calbiochem (San Diego, Calif). All
other reagents were commercial products of the highest
grade available.
Cell Culture
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A549 cells (no. CCL 185; American Type Culture Collection),
permanent cell lines of A549 cells expressing a GFP-tagged rat
␣1 subunit (GFP-A549) (a gift from Dr Bertorello, Department of
Medicine, Karolinska Institutet and Atherosclerosis Research
Unit, Karolinska University Hospital, Stockholm, Sweden), and
V5-tagged rat ␣1 subunit (␣1V5-A549) were generated in our
laboratory (Lecuona and colleagues), as described18; ␳0-A549
cells were generated as described previously.17,19 Cells were
incubated under normoxic (16% O2, 5% CO2, 79% N2) and
hypoxic conditions (1.5% O2, 93.5% N2, and 5% CO2) in a
humidified workstation (Invivo O2; Ruskinn Technology). The
Chinese hamster ovary cell lines (CHO), CHO-ts20 (thermosensitive), and CHO-E36 (wild type)20,21 were incubated in a
humidified atmosphere of 5% CO2 and were exposed to hypoxia
or normoxia in a humidified 1-L glass chamber at 30°C or 40°C.
Experiments were performed with ⬇80% confluent cells.
The use of animals for the present study was approved by the
Northwestern University Institutional Animal Care and Use Committee. Rat alveolar epithelial type II cells (ATII cells) were isolated
from pathogen-free male Sprague–Dawley rats (200 to 225 g; Harlan
Inc, Indianapolis, Ind), as previously described.22,23
GFP-␣1 Na,K-ATPase
Subunit Immunoprecipitation
After cells were treated for the desired times, immunoprecipitation
was done as previously reported.17
Hypoxia-Mediated Na,K-ATPase Degradation
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Cell Surface Labeling
Cells were labeled for 1 hour using 1 mg/mL EZ-link Sulfo-NHSSS-biotin and pulled down with streptavidin. Proteins were analyzed
by 10% SDS-PAGE and Western blot.17
Pulse Chase Labeling
Cells permanently transfected with V5␣1 Na,K-ATPase were incubated with methionine/cysteine-deficient DMEM for 60 minutes at
37°C. The cells were then pulse-labeled with 0.2 mCi/mL of
[ 35 S]methionine/cysteine (Amersham Biosciences, Arlington
Heights, Ill) in methionine/cysteine-deficient DMEM for 120 minutes at 37°C and chased (DMEM containing 5 mmol/L unlabeled
methionine and 5 mmol/L unlabeled cysteine) for 0 and 24 hours at
16% and 1.5% oxygen. After cells were treated for the desired times,
media were aspirated and cells were washed twice with cold PBS,
and A549-V5␣1 cell lysates preparation and immunoprecipitation
was done as previously reported by Lecuona (personal communication). Autoradiograph of 35S labeled proteins was obtained, and
membranes were analyzed by blotting against V5␣1.
Oxygen Consumption
Oxygen consumption was measured by using the Oxytherm respirometer (Eurosep Instruments, Cergy Pontoise, France) that has been
described previously.6,24
Determination of Na,K-ATPase Activity
Na,K-ATPase activity was determined by 2 methods. One using
ATP-32P (Amersham Biosciences) labeled as described previously.25,26 The second method was ouabain-sensitive 86Rb⫹ assay (Amersham Biosciences), as described previously.27
Small Interference RNA
A549 cells were stably infected with small interference RNA
(siRNA) for Rieske iron sulfur protein (RISP) generated with a
siRNA targeted against the iron sulfur (FeS) protein as previously
described.28
Adenoviral Infection
A549 cells (⬇70% confluent) were infected with 10 ␮L (106/␮L pfu)
of SOD1 (Ad5CMVCuZnSOD), SOD2 (Ad5CMVMnSOD), and
Figure 1. Hypoxia decreases the total pool of Na,K-ATPase ␣1 subunit. A, A549 cells were incubated at 16% O2 or 1.5% O2 for the
indicated times. Equal amounts of cell lysates (100 ␮g) were analyzed by Western blot, and Na,K-ATPase abundance was determined
using an antibody against Na,K-ATPase ␣-1 subunit. Data were normalized to values obtained from normoxic controls (16% O2). Representative Western blot of n⫽3. B, ATII cells were incubated at 16% O2 or 1.5% O2 for the indicated times. Equal amounts of cell
lysates (100 ␮g) were analyzed by Western blot, and Na,K-ATPase abundance was determined using an antibody against Na,K-ATPase
␣-1 subunit. Data were normalized to values obtained from normoxic controls (16% O2). Representative Western blot of n⫽3. C, V5␣1
Na,K-ATPase subunit in transfected A549 cells were incubated with methionine/cysteine-deficient DMEM for 60 minutes at 37°C. The
cells were then pulse-labeled with 0.2 mCi/mL of [35S]methionine/cysteine in methionine/cysteine-deficient DMEM for 120 minutes at 37°C
and chased (DMEM containing 5 mmol/L unlabeled methionine and 5 mmol/L unlabeled cysteine) for 0 and 24 hours at 16% and 1.5% oxygen. At the times indicated the cells were harvested, lysed, and fractionated. Autoradiograph of S35 labeled proteins was obtained and
membranes were analyzed by blotting against V5␣1. Data were normalized. Representative Western blot of n⫽3. *P⬍0.05.
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Figure 2. Hypoxia increases the degradation of plasma membrane Na,KATPase. A, A549 cells were surfaced
labeled with biotin and then exposed to
1.5% O2 for 2 hours. Equal amount of
cell lysate (150 ␮g of protein) were
pulled down with streptavidin beads and
Na,K-ATPase protein abundance was
determined by performing a Western blot
against Na,K-ATPase ␣-1 subunit. Representative Western blot of n⫽4. B, ATII
cells were surfaced labeled with biotin
and then exposed to 1.5% O2 for 2
hours. Equal amounts of cell lysate (150
␮g of protein) were pulled down with
streptavidin beads, and Na,K-ATPase
protein abundance was determined by
performing a Western blot against Na,KATPase ␣-1 subunit. Representative
Western blot of n⫽3. C, HIF-1 ␣ protein
levels were assessed in nuclear extracts
of A549 cells exposed to 16% O2 or
1.5% O2 for 2 hours. Representative
Western blot of n⫽2. *P⬍0.05.
glutathione peroxidase (Ad5CMVGpx). Na,K-ATPase degradation
was studied 24 hours after adenoviral vector infection by plasma
membrane biotin labeling as described above.
Statistical Analysis
Data are reported as mean⫾SE. Statistical analysis was performed
by 1-way ANOVA and Tukey correction. When 2 groups were
compared, analysis was performed using Student’s t test. Results
were considered significant when P⬍0.05.
Results
Hypoxia Decreases the Total Cell Pool and
Increases the Degradation of Plasma Membrane
Na,K-ATPase, Resulting in Decreased Activity and
Oxygen Consumption
As shown in Figure 1A and 1B, there was ⬇30% decrease in
Na,K-ATPase ␣1 subunit protein abundance in whole cell
lysates of A549 and ATII cells after 30 hours of hypoxia.
Next, we determined whether hypoxia would increase the
degradation of the total pool of Na,K-ATPase protein by
labeling cells with [35S]methionine/cysteine as described
above. As shown in Figure 1C, there was a 50% decrease in
Na,K-ATPase ␣1 subunit protein at 24 hours, which did not
differ from cells incubated under hypoxia. Subsequently we
determined whether hypoxia would increase the degradation
of the Na,K-ATPase at the plasma membrane by labeling the
cells previously with biotin. Figure 2A and 2B depicts that
hypoxia (within 2 hours) caused a 50% degradation of the
plasma membrane Na,K-ATPase ␣1 subunit in A549 and
ATII cells. These effects were specific and not the result of
bulk degradation, as HIF-1␣ was stabilized after 2 hours of
hypoxia (Figure 2C), and total actin, Glut-1, and E-cadherin
were not degraded; there was no decrease in ATP levels (data
not shown). Paralleling the degradation of the Na,K-ATPase
at the plasma membrane, we observed that in A549 and ATII
cells exposed to hypoxia, there was a decrease in Na,KATPase activity of ⬇50% at 2 hours (Figure 3A through 3C)
and a similar decrease in oxygen consumption under hypoxia
(Figure 3D).
Lysosome and Proteasome Participate in the
Hypoxia-Mediated Degradation of the Plasma
Membrane Na,K-ATPase
Pretreatment with the lysosome inhibitor chloroquine and the
proteasome inhibitor lactacystin prevented the degradation of
the plasma membrane Na,K-ATPase (Figure 4A). This correlated with Na,K-ATPase activity, where pretreatment with
proteasome inhibitor MG-132 and lysosome inhibitors chloroquine and E-64 prevented the hypoxia-mediated inhibition
of Na,K-ATPase activity (Figure 4B).
ROS Participate in the Hypoxia-Mediated
Degradation of the Na,K-ATPase
As shown in Figure 5A, incubation of A549 cells with
exogenous ROS (t-H2O2) revealed increased Na⫹ pump degradation at the plasma membrane. Furthermore, pretreatment
of A549 cells with the combined SOD/catalase mimetic
EUK-134 prevented the hypoxia-mediated plasma membrane
Na,K-ATPase degradation (Figure 5B).
Ubiquitin-Conjugating System Participates in the
Hypoxic-Mediated Degradation of
the Na,K-ATPase
We used a thermosensitive mutant of the ubiquitin-activating
enzyme (E1) (CHO-ts20) that becomes inactivated when
Comellas et al
Hypoxia-Mediated Na,K-ATPase Degradation
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Figure 3. Hypoxia decreases the Na,K-ATPase activity and oxygen consumption. A549 and ATII cells were incubated at 16% O2 or 1.5%
O2. A, Na,K-ATPase activity was determined in A549 cells by 32P-ATP hydrolysis and expressed as a percentage of normoxic controls (16%
O2) (n⫽3). B, Na,K-ATPase activity was determined in A549 cells by ouabain-sensitive 86Rb⫹ uptake (16% O2) (n⫽3). C, Na,K-ATPase activity
was determined in ATII cells by 32P-ATP hydrolysis and expressed as a percentage of normoxic controls (16% O2) (n⫽3). D, A549 cells were
placed in respirometer and oxygen consumption was measured and expressed as a percentage of normoxic controls (16% O2). n⫽3; *P⬍0.05.
incubated at 40°C. The progenitor cell line (CHO-E36),
with the wild-type E1, served as control. As shown in
Figure 6A, the Na,K-ATPase ␣1 subunit protein abundance in whole cell lysate was not degraded when CHOts20 cells were incubated in hypoxic conditions at 40°C.
Next, we determined whether the Na,K-ATPase is directly
ubiquitinated during hypoxia. A549 cells expressing GFP␣1–Na,K-ATPase were pretreated with the proteasome
inhibitor MG132 for 2 hours and then incubated under
hypoxia; cells were lysed and protein– ubiquitin conjugates
were immunoprecipitated with an anti-GFP–specific antibody. As shown in Figure 6B, the extent of ubiquitinconjugated Na,K-ATPase was noticeably increased after
hypoxia exposure. Also, when cells were incubated with
exogenous ROS (t-H2O2), ubiquitin-conjugated Na,KATPase was markedly increased (Figure 6C). Finally, to
determine whether hypoxia-generated mROS participated
in the increase Na,K-ATPase ubiquitination, we generated
␳0-A549 cells (mitochondrial DNA deficient) that are
unable to generate ROS during hypoxia (data not shown).
As shown in Figure 6D, ␳0-A549 cells, when incubated
under hypoxia for 2 hours, demonstrated a lack of increase
in ubiquitin conjugates associated with Na,K-ATPase ␣1
subunit. However, when these cells were incubated with
exogenous ROS, ubiquitin-conjugated Na,K-ATPase was
clearly increased.
Mitochondrial ROS Are Involved in the
Hypoxia-Mediated Degradation of
the Na,K-ATPase
To determine whether mROS participated in the hypoxiamediated degradation of the Na,K-ATPase, besides generating ␳0-A549 cells as described above, we generated siRNA
constructs against RISP, which is a ubiquitous component of
cytochrome bc1 complexes of the mitochondria. When ␳0A549 cells were exposed to hypoxia, the plasma membrane
Na,K-ATPase was not degraded (Figure 7B); however, exposure of these cells to exogenous ROS increased the
degradation of the Na,K-ATPase. In addition, A549 cells
permanently transfected with siRNA against RISP blocked
the hypoxia-mediated Na,K-ATPase degradation, with no
effect when Drosophila HIF (dHIF) siRNA was transfected,
as an internal control of the siRNA transfection, and exposed
to hypoxia. Iron sulfur protein expression was confirmed in
both cells transfected with siRNA, showing that there was an
almost complete knockdown of RISP as opposed to the
transfected with dHIF siRNA (Figure 7C), with no difference
in tubulin expression as loading control (data not shown), all
of which suggesting that during hypoxia, the mitochondria
(complex III) is the source of ROS. We then sought to
determine whether superoxide anion, H2O2, or both generated
within the mitochondrial matrix or within the mitochondrial
intermembrane space were required for hypoxia induced
degradation of the Na,K-ATPase ␣-1 subunit. Thus, we
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Figure 4. Lysosome and proteasome are involved in the hypoxia-mediated degradation of the Na,K-ATPase ␣-1 subunit. A, A549 cells
were surfaced labeled with biotin, pretreated for 1 hour in the presence or absence of 100 ␮mol/L of chloroquine (C) or 10 ␮mol/L of
lactacystin (L), and then exposed to 1.5% O2 for 2 hours. Equal amounts of cell lysate (150 ␮g of protein) were pulled down with
streptavidin beads and Na,K-ATPase protein abundance was determined by performing a Western blot against Na,K-ATPase ␣-1 subunit. Representative Western blot of n⫽5. B, Na,K-ATPase activity was determined in A549 cells pretreated for 1 hour in the presence
or absence of proteasome inhibitor MG-132 (20 ␮mol/L) or lysosome inhibitors chloroquine (100 ␮mol/L of) and E64 (10 ␮mol/L), and
then exposed to 1.5% O2 for 2 hours by ouabain-sensitive 86Rb⫹ uptake and expressed as a percentage of normoxic controls (16%
O2). n⫽3; *P⬍0.05, **P⬍0.01.
infected A549 cells with adenoviral vectors encoding SOD1,
SOD2, and glutathione peroxidase. As shown in Figure 8A,
the overexpression of SOD1 attenuated Na,K-ATPase degradation, whereas the overexpression of SOD2 had no effect.
Glutathione peroxidase overexpression prevented completely
the degradation of the Na,K-ATPase. All cells were confirmed for infection of the respective adenoviruses, as shown
in Figure 8B. Because SOD1 metabolizes superoxide anion
generated in the cytosol and mitochondrial intermembrane
space, whereas SOD2 metabolizes superoxide anion in the
mitochondrial matrix, these results suggest that H2O2 generated from superoxide anion released into the intermembrane
space is required for the hypoxia mediated degradation of the
Na,K-ATPase ␣-1 subunit.
Discussion
The mechanisms of hypoxia-induced pulmonary edema are
not well understood. Recent evidence suggests that hypoxia
significantly reduces the capacity for active Na⫹ transport
across the alveolar epithelium.29 –31 It has been described that
␤-2 agonists can overcome the depressant effects of hypoxia
on alveolar fluid clearance,31,32 which in part explains the
preventive effect of ␤ agonists in the development of high
altitude pulmonary edema (HAPE) and the observed reduction in extraalveolar water in patients with acute respiratory
distress syndrome (ARDS).33,34
Adaptation to hypoxia is key for cell survival. To accomplish this, there is a need for cellular energy reallocation
between essential and nonessential ATP demand processes.11
Figure 5. ROS are involved in the degradation of
the Na,K-ATPase under hypoxia. Cells were surfaced labeled with biotin before exposing to different experimental conditions. Equal amounts of
proteins (150 ␮g) were pulled down with streptavidin beads, and Na,K-ATPase protein abundance
was determined by performing Western blot
against Na,K-ATPase ␣-1 subunit. A, A549 cells
were exposed to either 16% O2 or 100 ␮mol/L
t-H2O2 every 40 minutes for 2 hours. Representative Western blot of n⫽3. B, A549 cells were preincubated overnight with EUK-134 (20 ␮mol/L) and
exposed to either 16% O2 or1.5% O2. Representative Western blot of n⫽2. *P⬍0.05.
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Figure 6. Ubiquitin-conjugating system is involved in the hypoxia-mediated degradation of the Na,K-ATPase ␣-1 subunit. A, CHO-ts20
and E36 cells were incubated under 16% O2 or 1.5% O2 for 24 hours at either 30°C or 40°C. Equal amount of cell lysates (150 ␮g)
were analyzed by Western blot against the Na,K-ATPase ␣1 subunit. Representative Western blot of n⫽3. B, GFP-␣1 A549 cells were
pretreated with 20 ␮mol/L of MG-132 and then incubated at 16% O2 or 1.5% O2 for 2 hours. The Na,K-ATPase ␣1 subunit was immunoprecipitated using a GFP antibody. Immunoprecipitates were immunoblotted against ubiquitin. The membranes were then stripped
and reblotted against GFP as a loading control. Representative Western blot of n⫽3. C, GFP-␣1 A549 cells were pretreated with
20 ␮mol/L MG-132 and then incubated at 16% O2 or t-H2O2 (100 ␮mol/L) for 2 hours. The Na,K-ATPase ␣1 subunit was immunoprecipitated using a GFP antibody. Immunoprecipitates were immunoblotted against ubiquitin. The membranes were then stripped and
reblotted against GFP as a loading control. Representative Western blot of n⫽3. D, GFP-␣1 A549 ␳0 cells were pretreated with
20 ␮mol/L MG-132 and then incubated at 16% O2, 1.5% O2, and t-H2O2 (100 ␮mol/L) for 2 hours. The Na,K-ATPase ␣1 subunit was
immunoprecipitated using a GFP antibody. Immunoprecipitates were immunoblotted against ubiquitin. The membranes were then
stripped and reblotted against GFP as a loading control. Representative Western blot of n⫽3. **P⬍0.01, ***P⬍0.001.
Consistent with previous reports,30,35,36 the total Na,KATPase ␣1 cell pool decreased on exposure to prolonged
hypoxia (Figure 1A and 1B). Its half-life is approximately 24
hours and is not shortened in hypoxic conditions (Figure 1C),
suggesting a decrease of the total pool protein synthesis and
not of the total pool degradation, as it has been suggested
previously.30 However, ⬇15% to 20% of the Na⫹ pumps are
present at the plasma membrane of A549 cells (E. Lecuona
and J.I. Sznajder, personal communication) and are enzymatically active as opposed to the 80% of Na,K-ATPase in the
intracellular pools.37 Therefore, we decided to determine
whether the plasma membrane Na⫹ pumps, which are consuming ATP, would be affected first during hypoxia. We
observed that within a short period of time, hypoxia increased
the plasma membrane Na,K-ATPase degradation (Figure 2A
and 2B). As shown in Figure 3A through 3D, A549 and ATII
cells had a similar decrease in Na,K-ATPase activity when
incubated in hypoxic conditions, being prevented by either a
lysosome or proteasome inhibitor (Figure 4A and 4B). During
hypoxia, the cells appear to adapt, as described in hepato-
cytes, by downregulating consumption of ATP in response to
a decreasing oxygen availability.24,38
The involvement of both the proteasome and the lysosome
in degrading the Na,K-ATPase ␣1 subunit is still puzzling.
Following ubiquitination, soluble and endoplasmic reticulum
proteins are usually targeted to the proteasome, whereas cell
surface membrane proteins are usually targeted to the lysosome.39 Exceptions to these rules have been observed in other
systems; for example, proteolytic cleavage of the growth
hormone receptor is necessary before the protein can be
delivered to the lysosome40 and the glutamate receptor
requires the activity of the proteasome for its endocytosis.41
Also, it has been reported that specific inhibitors of lysosomal
proteases and inhibitors of the proteasome are effective in
reducing the ligand-induced platelet-activating factor receptor downregulation, indicating the importance of receptor
targeting to both lysosomes and proteasomes.42
The ubiquitin-conjugating system plays a crucial role in
cell homeostasis, including the regulation of membrane
proteins such as the Na⫹ channel and the growth hormone
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Figure 7. Mitochondrial derived ROS are involved in hypoxia-mediated Na,K-ATPase degradation. A, Southern blot analysis of total cellular DNA from wild type (A549) and A549 ␳0 cells. Hybridization was performed with a cytochrome oxidase subunit II probe, spanning
bp 7757 to 8195, generated by RT-PCR. B, A549 ␳0 cells were exposed to 16% O2, 1.5% O2, or 100 ␮mol/L t-H2O2 every 40 minutes
for 2 hours. Representative Western blot of n⫽3. C, A549cells transfected with siRNA against dHIF and RISP exposed to 16% O2 and
1.5% O2 for 2 hours and Western blot showing expression of iron sulfur protein in dHIF and RISP siRNA-transfected cells. Representative Western blot of n⫽4. *P⬍0.05, ***P⬍0.001.
receptor.20,21,43– 49 Multiubiquitination of proteins leads to
their recognition and subsequent degradation by the proteasome,45,50,51 and for most cell surface membrane proteins,
ubiquitin-tagging triggers the endocytic machinery that direct
proteins to the lysosome wherein they are degraded.44,48,52
Our data provide evidence that hypoxia ubiquitinates the
Na,K-ATPase, targeting it for degradation (Figure 6A and
6B). This phenomenon contrasts with the regulation of
HIF-1␣ which is stabilized as a result of the inhibition of its
constitutive ubiquitin mediated degradation during hypoxia
by the E3 ligase VHL.4,5,53 These data suggest that signaling
events initiated by exposure to hypoxia activates distinct
mechanisms that regulate the stabilization or degradation of
proteins through the ubiquitin/proteasome system.
During hypoxia, the mitochondrial electron transport is
partially inhibited causing redox changes in the electron
carriers that result in the generation of superoxide anions (O)
and hydrogen peroxide (H2O2), which on entering the cytosol,
can act as second messengers.54 –56 The mitochondria generate
ROS in response to a change in redox of the electron transport
proteins, specifically from complex III.54,57 Recently, Dada et
al, reported that exposing cells to hypoxia for 60 minutes
decreased Na⫹ pump activity by promoting endocytosis of
plasma membrane Na,K-ATPase but without degradation.17
We provide new evidence that exposing cells to severe
hypoxia or exogenous ROS (H2O2) for 2 hours results in an
increased Na,K-ATPase ubiquitination and degradation (Figures 5A and 6C). When A549 cells were preincubated with
the combined superoxide dismutase/catalase mimetic EUK134,58 which blocks completely the generation of ROS,59 the
hypoxia-mediated degradation of the plasma membrane
Na,K-ATPase was prevented (Figure 5B).
Figure 8. SOD1 and GPX overexpression
protect against hypoxia-mediated Na,KATPase degradation. A, A549 cells were
infected with adenovirus vectors overexpressing SOD2, SOD1, and glutathione
peroxidase (Gpx) 24 hours before treatment; cells were then exposed to 16%
O2 or 1.5% O2 for 2 hours. The membranes were then stripped and reblotted
against tubulin. Data were normalized to
values obtained from normoxic controls
(16% O2). Representative Western blot of
n⫽3. B, Western blot stripped and
reblotted against SOD2 (MnSOD), SOD1
(CuZnSOD), and GPx (anti-myc) antibodies. *P⬍0.05, **P⬍0.01.
Comellas et al
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When A549 ␳0 cells and siRNA RISP cells, which cannot
generate mitochondrial ROS,17,60 were exposed to hypoxia,
ubiquitination and degradation of ␣1 Na,K-ATPase was
prevented, suggesting that ROS are generated at the mitochondria, specifically at complex III, playing a key role in the
degradation of the Na⫹ pump during hypoxia (Figures 6D and
7). Collectively, these results suggest that during hypoxia the
mitochondrial generation of ROS is required for the degradation of the Na,K-ATPase and H2O2 acts downstream of the
mitochondria.
The mitochondria possess antioxidant defenses against O2·⫺
and H2O2. These include SOD2 (MnSOD), which is transported to the mitochondrial matrix where it forms the active
homotetramer.61 Although the O2·⫺ generated in the matrix is
eliminated in that compartment, part of the O2·⫺ produced in
the intermembrane space may be either dismutated by a
different SOD isozyme (SOD1) that contains copper and zinc
instead of manganese (CuZnSOD) or carried to the cytoplasm
via voltage-dependent anion channels (VDAC), where it can
be eliminated via CuZnSOD, which is also found in the
cytoplasm of eukaryotic cells. Moreover, H2O2, the byproduct
of O2·⫺ dismutation, is eliminated by glutathione peroxidase
localized in the cytosol.61 We found that the overexpression
of glutathione peroxidase in A549 cells prevented the hypoxia-mediated plasma membrane Na,K-ATPase degradation,
whereas SOD1 overexpression partially blocked the Na,KATPase degradation. However, SOD2 overexpression was
not protective (Figure 8A and 8B), suggesting that during
hypoxia, mitochondria-intermembrane-space, but not
mitochondrial-matrix, generated ROS participate in the degradation of the Na,K-ATPase.
In summary, we provide evidence that hypoxia-generated
mROS increases Na,K-ATPase degradation via the ubiquitinconjugating system. Whether ROS posttranslationally modify
specific residues of the Na,K-ATPase targeting it for recognition by the ubiquitin pathway and degradation warrants
further studies. This report supports the hypothesis that
during hypoxia, mROS play a physiological role in the
cellular response to hypoxia by signaling in a regulated
fashion and triggers the ubiquitin-mediated degradation of
energy consuming proteins, such as the Na,K-ATPase, although it stabilizes others that are energy-producing reactions, such as HIF-1␣.61
Hypoxia-Mediated Na,K-ATPase Degradation
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Acknowledgments
This work was supported in part by grants from the NIH (PO1HL73641 and 5F32HL071421). We are grateful to Dr A. Bertorello
for the ␣1-Na,K-ATPase-GFP-A549 cells and to Dr K. M. Ridge for
valuable discussions.
23.
24.
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Hypoxia-Mediated Degradation of Na,K-ATPase via Mitochondrial Reactive Oxygen
Species and the Ubiquitin-Conjugating System
Alejandro P. Comellas, Laura A. Dada, Emilia Lecuona, Liuska M. Pesce, Navdeep S. Chandel,
Nancy Quesada, G. R. Scott Budinger, Ger J. Strous, Aaron Ciechanover and Jacob I. Sznajder
Circ Res. 2006;98:1314-1322; originally published online April 13, 2006;
doi: 10.1161/01.RES.0000222418.99976.1d
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