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Published as doi: 10.1096/fj.07-8369com.
 (The FASEB Journal. 2008;22:1335-1342.)
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Hypoxia-mediated Na-K-ATPase degradation requires von Hippel Lindau protein

Guofei Zhou*, Laura A. Dada*, Navdeep S. Chandel*, Kazuhiro Iwai{dagger}, Emilia Lecuona*, Aaron Ciechanover{ddagger} and Jacob I. Sznajder*,1

* Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA;

{dagger} Department of Molecular Cell Biology, Graduate School of Medicine, Osaka City University, Abeno-Ku, Osaka, Japan; and

{ddagger} Vascular and Tumor Biology Research Center, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

1Correspondence: Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, 240 E. Huron, McGaw Pavilion M-300, Chicago, IL 60611, USA. E-mail: j-sznajder{at}northwestern.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypoxia inhibits Na-K-ATPase activity and leads to its degradation in mammalian cells. Von Hippel Lindau protein (pVHL) and hypoxia inducible factor (HIF) are key mediators in cellular adaptation to hypoxia; thus, we set out to investigate whether pVHL and HIF participate in the hypoxia-mediated degradation of plasma membrane Na-K-ATPase. We found that in the presence of pVHL hypoxia decreased Na-K-ATPase activity and promoted the degradation of plasma membrane Na-K-ATPase. In pVHL-deficient cells, hypoxia did not decrease the Na-K-ATPase activity and the degradation of plasma membrane Na-K-ATPase was prevented. pVHL-mediated degradation of Na-K-ATPase required the functional pVHL E3 ligase and Ubc5 since pVHL mutants and dominant-negative Ubc5 prevented Na-K-ATPase from degradation. The generation of reactive oxygen species was necessary for pVHL-mediated Na-K-ATPase degradation during hypoxia. Desferrioxamine, which stabilizes HIF1/2{alpha}, did not affect the half-life of plasma membrane Na-K-ATPase. In addition, stabilizing HIF1/2{alpha} by infecting mammalian cells with adenoviruses containing the oxygen-dependent degradation domain of HIF1{alpha} did not affect the plasma membrane Na-K-ATPase degradation. In cells with suppression of pVHL by short hairpin RNA, the Na-K-ATPase was not degraded during hypoxia, whereas cells with knockdown of HIF1/2{alpha} retained the ability to degrade plasma membrane Na-K-ATPase. These findings suggest that pVHL participates in the hypoxia-mediated degradation of plasma membrane Na-K-ATPase in a HIF-independent manner.—Zhou, G., Dada, L. A., Chandel, N. S., Iwai, K., Lecuona, E., Ciechanover, A., Sznajder, J. I. Hypoxia-mediated Na-K-ATPase degradation requires von Hippel Lindau protein.


Key Words: hypoxia inducible factor • pVHL


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MAMMALIAN CELLS ARE KNOWN to adapt to hypoxia by maintaining energy homeostasis (1) . The adaptation to hypoxia typically involves increasing ATP production primarily through anaerobic glycolysis and/or decreasing energy consumption by down-regulating protein synthesis and ATP-dependent ion pumping systems, such as Na-K-ATPase (1) . Na-K-ATPase is a membrane-bound protein consisting of {alpha} and β subunits (2 , 3) and has been reported to consume 20–80% of ATP in resting cells (1 , 4) .

Hypoxia inducible factor (HIF) is the major transcription factor controlling cellular adaptation to hypoxia (5) . HIF consists of two subunits, HIF{alpha} and HIFβ. HIF{alpha} is regulated by von Hippel Lindau protein (pVHL), which forms a ubiquitin E3 protein ligase complex with Elongin B, Elongin C, Cullin 2, and Rbx 1 (6 7 8) . pVHL protein contains two domains, an {alpha} and a β domain. The {alpha} domain of pVHL binds to Elongin C, which acts as a bridge between pVHL and other components, while the β domain is the substrate docking site (9 , 10) . In vitro studies have shown that Ubc5 acts as the upstream ubiquitin conjugating enzyme (E2) for pVHL E3 ligase (11 , 12) . Under normoxic conditions, prolines within the oxygen-dependent degradation domain (ODDD) of HIF{alpha} are hydroxylated (13) . pVHL recognizes and associates with these hydroxylated proline sites on HIF{alpha}, targeting it to the proteasome for degradation (14) . During hypoxia, prolyl hydroxylase activity is inhibited and pVHL is unable to bind to HIF{alpha}, resulting in stabilization of HIF{alpha} protein (6) . The stabilized HIF activates downstream gene expression to adapt to hypoxic conditions (5) .

Short-term hypoxia leads to inhibition of Na-K-ATPase activity by promoting its endocytosis, and more prolonged hypoxia causes the degradation of plasma membrane Na-K-ATPase as a mechanism of cellular adaptation (15 16 17 18 19) .

In this study, we provide evidence that pVHL participates in plasma membrane Na-K-ATPase degradation during hypoxia. In cells lacking pVHL, hypoxia-mediated degradation of plasma membrane Na-K-ATPase is prevented. The intact E3 ligase activity of pVHL appears to be critical since mutation of pVHL and dominant-negative Ubc5 prevent the degradation of Na-K-ATPase during hypoxia. Moreover, transient stabilization of HIF1/2{alpha} is neither sufficient nor required for the degradation of plasma membrane Na-K-ATPase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Desferrioxamine (DFO) was purchased from Sigma (St. Louis, MO, USA). HIF1{alpha}-ODDD-wt (amino acid 531–575) and HIF1{alpha}-ODDD-mut (P564A) were constructed into adenoviruses (13) . Plasmids pcDNA3-pVHL-wt, pVHL-L158P, pVHL-1–157 (8) , dominant-negative (DN) -His-Ubc5A, DN-His-Ubc5B, DN-His-Ubc5C, and DN-His-E2–25K (20) have been described previously. The following antibodies were used in this study: {alpha}-tubulin, actin (Sigma), HIF2{alpha} (NB 100–122, Novus Biologicals, Littleton, CO, USA), Glut-1 (Chemicon, Temecula, CA, USA), E-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), HIF1{alpha} (BD Transduction Laboratories, Franklin Lakes, NJ, USA), Na-K-ATPase {alpha}1 (Upstate, Chicago, IL, USA), HA (Covance, Berkeley, CA, USA), 6x His (Qiagen, Valencia, CA, USA), and pVHL (BD Pharmingen, San Diego, CA, USA). Euk-134 was purchased from Cayman Chemical (Ann Arbor, MI, USA)

Cell culture
The following cell lines were used in this study: human alveolar epithelial cell line A549, African green monkey kidney cell line COS-7, and pVHL-deficient human renal clear carcinoma cell lines RCC4 and 786-0, which have been described previously (21 , 22) . RCC4+VHL cells express reconstituted wild-type pVHL (21) . 786-0/pRC3 and 786-0/WT8 were derived from 786-0 cells by stable transfection with empty vector or a plasmid containing the wild-type pVHL, respectively (22) . Cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were incubated in a humidified atmosphere of 5% CO2/95% air at 37°C. All cell cultures were routinely split when they were 85–90% confluent. Hypoxic conditions (1.5% O2, 93.5% N2, and 5% CO2) were maintained in a humidified workstation (In vivo2, Ruskinn Technologies, Leeds, UK), which contains an oxygen sensor to continuously monitor the oxygen tension of the chamber.

Na-K-ATPase activity in cells
Na-K-ATPase activity was determined as described previously (23) . After exposure to the indicated conditions, cells were scraped into PBS and aliquots were transferred to the assay buffer containing 50 mM NaCl, 5 mM KCl, 10 mM MgCl2, 1 mM EGTA, 50 mM Tris-HCl, pH 7.4, 3 mM Tris-ATP and 62.5 nCi [{gamma}-32P]ATP. Reaction mixtures were kept at –20°C for 20 min to make the cell membranes permeable to ATP. Samples were then incubated at 37°C for 15 min, and the enzyme reactions were terminated by addition of trichloroacetic acid (TCA)-charcoal suspension. After centrifugation at 12,000 g for 5 min, the liberated 32P was counted and Na-K-ATPase activity was assessed as the difference between total ATPase activity and ouabain-insensitive ATPase activity.

Plasma membrane Na-K-ATPase protein degradation
The degradation of plasma membrane proteins was determined as described previously (19) . Briefly, cells were washed three times with PBS containing 0.5 mM MgCl2 and 0.9 mM CaCl2 and labeled with 1 mg/ml EZ-link NHS-SS-biotin (Pierce Chemical Co., Rockford, IL, USA) for 20 min. Cells were then replenished with fresh DMEM without FBS and incubated in a normoxic or hypoxic environment for the indicated times. After being washed with PBS, cells were lysed in modified radioimmunoprecipitation buffer (mRIPA; 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, and 1% sodium deoxycholate, and protease inhibitors), and aliquots containing equal amount of proteins were rotated overnight at 4°C in the presence of streptavidin beads (Pierce). Proteins were analyzed by SDS-PAGE and Western blotting. Since we labeled with biotin first and then conducted the experiments to track the fate of the proteins present at the cell surface before the experiment started and no additional membrane proteins are labeled during hypoxia, the decrease of the target protein (Na-K-ATPase) reflects its degradation during hypoxia.

Transient transfection of plasmids
Plasmids were transfected into COS7 cells with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) following manufacturer’s recommendation.

Establishment of stable cell lines with suppression of HIF1/2{alpha} and pVHL
The short hairpin RNAs (shRNAs) targeting Drosophila HIF (dHIF), human HIF1{alpha}, human HIF2{alpha}, and human pVHL have been previously described (24 , 25) and were constructed into pSIREN-RetroQ vector (Clontech, Mountain View, CA, USA). pSIREN-RetroQ constructs were transfected into pT67 packaging cells. The culture media were collected and used to infect A549 cells. Selection media containing 1 µg/ml puromycin were added 24 h after infection. Clonal selection was applied, and selected colonies were then screened and characterized for suppression of targeted proteins by Western blot analysis.

Western blotting
Cultured cells were washed three times with ice-cold PBS and lysed in 250 µl of mRIPA lysis buffer. The cell lysates were cleared by centrifugation at 13,000 g for 5 min, and protein concentrations were determined using a Bio-Rad Dc protein assay. Typically, 25–50 µg of protein was then separated by SDS-PAGE. The gel was transferred using Semi-Dry transfer cell (Bio-Rad, Hercules, CA, USA) to BA-S 85 nitrocellulose membrane (OPTITRAN, Middlesex, UK). The proteins were detected with Western Lightning Chemiluminescence reagent plus (Perkin Elmer, Wellesley, MA, USA).

Statistical analysis
Statistical analysis was done using GraphPad Prism 4 (GraphPad, San Diego, CA, USA) and Microsoft Excel (Microsoft, Redmond, WA, USA) when applicable; t tests were performed and values of P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
pVHL is necessary for hypoxia-mediated decrease of Na-K-ATPase activity
To investigate whether pVHL plays a role in the Na-K-ATPase inhibition during hypoxia (1.5% O2), we conducted studies with cells lacking pVHL (RCC4) and cells with reconstituted pVHL (RCC4+VHL; ref. 21 ). As shown in Fig. 1 A, in cells lacking pVHL, hypoxia did not decrease Na-K-ATPase activity, while in pVHL-reconstituted cells, hypoxia caused a decrease of Na-K-ATPase activity. To further confirm the effect of pVHL on Na-K-ATPase, we tested other renal carcinoma cell lines: 786-0/pRC3 (pVHL deficient) and 786-0/WT8 (pVHL reconstituted) from the same parental cell line (22) . As shown in Fig. 1B , reconstitution of pVHL again caused inhibition of Na-K-ATPase activity during hypoxia. RCC4 and RCC4+VHL cells had similar cellular ATP levels, and exposure to hypoxia for 2 h did not change ATP levels (see Supplemental Fig. 1).


Figure 1
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  		Figure 1. pVHL is essential for degradation of plasma membrane Na-K-ATPase during hypoxia. A) RCC4 and RCC4+VHL cells were exposed to normoxia (21% O2) or hypoxia (1.5% O2) for 2 h. Na-K-ATPase activity was measured as described in Materials and Methods. Graph represents the mean ± SE of 3 different experiments. B) Na-K-ATPase activity of 786-0/pRC3 cells (pVHL deficient) and 786-0/WT8 cells (pVHL reconstituted) was measured as in A. Graph represents the mean ± SE of 4 different experiments. C) RCC4 and RCC4+VHL cells were labeled with active biotin and exposed to normoxia or hypoxia for 2 h, then the degradation of plasma membrane (PM) Na-K-ATPase was measured as a decrease in protein abundance as described in Materials and Methods. Graph represents the mean ± SE of 3 different experiments. The representative Western blot for plasma membrane Na-K-ATPase is immediately below the graph. D) Degradation of plasma membrane Na-K-ATPase measured in 786-0/pRC3 and 786-0/WT8 cells as in C. Graph represents the mean ± SE of 4 different experiments; *P < 0.05. The representative Western blot for plasma membrane Na-K-ATPase is below the graph. For C and D, representative immunoblots of Na-K-ATPase, HIF1{alpha}, HIF2{alpha}, actin, or tubulin (respectively) in the whole cell lysates (WCL) from RCC4 or RCC4+VHL cells exposed to normoxic or hypoxic conditions are shown.

pVHL is necessary for plasma membrane Na-K-ATPase degradation during hypoxia
To further investigate whether the decreased Na-K-ATPase activity was due to its degradation, we assessed degradation of plasma membrane Na-K-ATPase protein in cells exposed to hypoxia for 2 h. In pVHL-deficient cells (RCC4 and 786-0/pRC3), the plasma membrane Na-K-ATPase protein abundance remained unchanged after exposure to hypoxia. However, in pVHL-reconstituted cells, plasma membrane Na-K-ATPase was degraded during hypoxia (Fig. 1C, D ), suggesting that pVHL plays a role in hypoxia-mediated degradation of plasma membrane Na-K-ATPase. As depicted in Fig. 1C, D , the total amount Na-K-ATPase in the whole-cell lysate remained unchanged regardless of the pVHL status. As expected, HIF{alpha} was constitutively stabilized in RCC4 and 786-0/pRC3 cells and hypoxia stabilized HIF1{alpha} and/or HIF2{alpha} in RCC4+VHL and 786-0/WT8 cells (Fig. 1C, D ).

pVHL E3 ligase activity is required for Na-K-ATPase degradation
Mutations of the Elongin C binding site in pVHL (pVHL-1-157 or pVHL-L158P) disrupt the formation of intact pVHL E3 ligase complex, leading to inhibition of its E3 activity (9 , 26) . To determine whether pVHL E3 ligase activity is required for Na-K-ATPase degradation during hypoxia, we transfected COS7 cells with plasmids containing wild-type or mutant pVHL (Fig. 2 A). As shown in Fig. 2B , overexpression of mutant pVHL prevented Na-K-ATPase degradation during hypoxia.


Figure 2
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  		Figure 2. Functional pVHL E3 ligase complex is required for hypoxia-mediated degradation of plasma membrane Na-K-ATPase. A) Schematic diagram of the pVHL constructs. B) COS7 cells were transfected with empty vector (Vec), pVHL-wt, pVHL-1–157, and pVHL-L-158 P constructs. After 48 h, the degradation of plasma membrane Na-K-ATPase was measured as in Fig. 1 . Graph represents the mean ± SE of 3 different experiments; *P < 0.05. The amount of Na-K-ATPase was normalized and is represented as a percentage of the amount found in COS7 cells transfected with vector and exposed to normoxia. A representative Western blot for plasma membrane Na-K-ATPase is below the graph. HA-pVHL proteins in the whole cell lysate were detected by immunoblotting.

Ubc5 activity is required for Na-K-ATPase degradation
Since Ubc5 has been shown to act as the upstream E2 for pVHL E3 ligase (11 , 12) , we set out to determine which isoform of Ubc5 contributes to the degradation of Na-K-ATPase during hypoxia. As shown in Fig. 3 A, B, overexpression of DN-Ubc5A, 5B, and 5C prevented the degradation of Na-K-ATPase in cells exposed to hypoxia. On the other hand, overexpression of DN-E2–25K, which is not an E2 for pVHL E3 ligase (11 , 12) , did not prevent Na-K-ATPase from degradation (Fig. 3C ).


Figure 3
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  		Figure 3. Functional Ubc5 is required for pVHL-dependent Na-K-ATPase degradation during hypoxia. A) COS7 cells were transfected with empty vector or DN-His-Ubc5A or His-Ubc5B constructs. After 48 h, the degradation of plasma membrane Na-K-ATPase was measured as in Fig. 1 . Graph represents the mean ± SE of 5 different experiments; *P < 0.05. A representative Western blot for plasma membrane Na-K-ATPase is below the graph. B) COS7 cells were transfected with an empty vector or a vector carrying a DN-His-Ubc5C construct. After 48 h, the degradation of plasma membrane Na-K-ATPase was measured as in Fig. 1 . Graph represents the mean ± SE of 7 different experiments; *P < 0.05. A representative Western blot for plasma membrane Na-K-ATPase is below the graph. C) COS7 cells were transfected with empty vector or a vector carrying a DN-His-E2–25K construct. After 48 h, the degradation of plasma membrane Na-K-ATPase was measured as in Fig. 1 . Graph represents the mean ± SE of 3 different experiments; *P < 0.05. A representative Western blot for plasma membrane Na-K-ATPase is below the graph. Glut-1 or E-cadherin was measured by immunoblotting as a loading control for plasma membrane protein. The amount of Na-K-ATPase was normalized to Glut-1 or E-cadherin and is represented as a percentage of the amount found in COS7 cells transfected with vector and exposed to normoxia. The His-tagged DN-His-Ubc5A, DN-His-Ubc5B, DN-His-Ubc5C, and DN-His-E2–25K were detected by anti-His antibody in WCL.

ROS are necessary for Na-K-ATPase degradation in presence of pVHL
We have previously reported that the degradation of plasma membrane Na-K-ATPase is dependent on mitochondrial ROS production (19) . Here we tested whether ROS plays a role in pVHL-mediated Na-K-ATPase degradation by pretreating RCC4+VHL cells for 2 h with Euk-134, a synthetic superoxide dismutase and catalase mimetic, and assessed plasma membrane Na-K-ATPase degradation. As shown in Fig. 4 , Euk-134 prevented Na-K-ATPase degradation during hypoxia in presence of pVHL.


Figure 4
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  		Figure 4. pVHL-mediated Na-K-ATPase degradation requires ROS production. RCC4+VHL cells were pretreated with 20 µM Euk-134, followed with labeling with active biotin and exposure to normoxic or hypoxic conditions for 2 h. The degradation of plasma membrane Na-K-ATPase was then measured as described in Fig. 1 . Graph represents the mean ± SE of 3 different experiments; *P < 0.05. A representative Western blot for plasma membrane Na-K-ATPase is below the graph. CTL = control.

HIF is not sufficient for Na-K-ATPase degradation
We studied the rate of Na-K-ATPase {alpha}1 protein degradation in normoxic conditions by measuring the amount of biotinylated Na-K-ATPase {alpha}1 at different times and expressing them as a percentage of the amount labeled at time 0. As depicted in Fig. 5 A, in A549 cells, 50% of the biotinylated plasma membrane Na-K-ATPase {alpha}1 is degraded by ~4 h in normoxic conditions. Since HIF1/2{alpha} is stabilized during hypoxia, we conducted experiments to determine whether transient stabilization of HIF1/2{alpha} participates in the hypoxia-mediated degradation of Na-K-ATPase. DFO, an iron chelator that inhibits prolyl hydroxylase activity (27) , stabilized HIF1/2{alpha} and induced Glut-1, but did not alter the rate of plasma membrane Na-K-ATPase degradation in normoxic conditions (Fig. 5A ).


Figure 5
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  		Figure 5. Transient stabilization of HIF1{alpha} is not sufficient for degradation of Na-K-ATPase. A) A549 cells were labeled with active biotin and then treated with 150 µM DFO for the indicated times. The degradation of plasma membrane Na-K-ATPase was measured as in Fig. 1 . Graph represents the mean ± SE of 3 different experiments. The amount of Na-K-ATPase was normalized and is represented as a percentage of the amount detected at time point 0. The representative Western blot for plasma membrane Na-K-ATPase is below the graph. Aliquots of the whole cell lysate with equal amounts of protein were subjected to Western blotting analysis for HIF1{alpha}, HIF2{alpha}, and Glut-1. B) A549 cells were infected with wild-type or mutated HIF1{alpha}-ODDD adenoviruses. Aliquots of the whole-cell lysate with equal amounts of protein were subjected to Western blotting analysis for total Na-K-ATPase, HIF1{alpha}, HIF2{alpha}, Glut-1, and actin. C) A549 cells were infected with empty adenovirus vector, wild-type or mutated HIF1{alpha}-ODDD adenoviruses. After 48 h, the degradation of plasma membrane Na-K-ATPase was measured as in Fig. 1 . Graph represents the mean ± SE of 5 different experiments. The amount of Na-K-ATPase was normalized and is represented as a percentage of the amount detected in A549 cells without virus infection. A representative Western blot for plasma membrane Na-K-ATPase is below the graph.

Another approach to stabilize HIF1/2{alpha} is to saturate pVHL binding by an exogenous peptide containing HIF1{alpha}-ODDD-wt (amino acids from 531 to 575). HIF1{alpha}-ODDD contains a proline 564 and competes with endogenous HIF1/2{alpha} for pVHL recognition, leading to the stabilization of endogenous HIF1/2{alpha} (13) , resulting in the up-regulation of Glut-1 during normoxia (Fig. 5B ). On the other hand, HIF1-ODDD-mut, which has a mutation of proline 564 to alanine, had no significant effect on stabilization of endogenous HIF1{alpha}. More important, the degradation of Na-K-ATPase did not differ in A549 cells infected with wild-type or mutated HIF1{alpha}-ODDD (Fig. 5C ). These data suggest that the transient stabilization of HIF1/2{alpha} is not sufficient to induce degradation of Na-K-ATPase in normoxic conditions.

HIF is not required for Na-K-ATPase degradation during hypoxia
We explored the role of HIF{alpha} during hypoxia in the Na-K-ATPase degradation, using shRNA to knock down HIF1{alpha} or 2{alpha} in A549 cells (Fig. 6 A). We assessed the degradation of plasma membrane Na-K-ATPase in these cell lines. As shown in Fig. 6C , cells with suppressed HIF1{alpha} or 2{alpha} expression retained the ability to degrade Na-K-ATPase during hypoxia, suggesting that hypoxia-mediated degradation of plasma membrane Na-K-ATPase does not require transient HIF. Knockdown of pVHL in A549 cells (Fig. 6B ) prevented the degradation of Na-K-ATPase during hypoxia (Fig. 6C ), which is consistent with the results obtained from experiments with renal carcinoma cells (Fig. 1) .


Figure 6
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  		Figure 6. HIF1/2{alpha} is not necessary for pVHL-mediated degradation of Na-K-ATPase during hypoxia. A, B) Stable infected A549 cells with shRNA for dHIF, HIF1{alpha} HIF2{alpha}, or pVHL were exposed to normoxia or hypoxia for 2 h and then lysed as described in Materials and Methods. Samples containing the same amount of proteins were analyzed by Western blot for HIF1{alpha}, HIF2{alpha}, pVHL, tubulin, or actin, respectively. C) The degradation of plasma membrane Na-K-ATPase was measured as in Fig. 1 . Graph represents the mean ± SE of 7 different experiments; *P < 0.05. The abundance of Na-K-ATPase was normalized and is represented as a percentage of the amount detected in the samples exposed to normoxia. Bottom panel depicts a representative Western blot.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
pVHL has been implicated in multiple cellular processes such as regulating hypoxia inducible genes, extracellular matrix assembly, cytoskeletal organization, cell cycle control, and differentiation (28) . In this study, we provide evidence that pVHL has a novel function in regulating Na-K-ATPase degradation during hypoxia. Previous studies (17 18 19) have demonstrated that plasma membrane Na-K-ATPase is endocytosed and degraded during hypoxia. During hypoxia, 50% of the plasma membrane Na-K-ATPase {alpha}1 is degraded within 2 h (19) , whereas in normoxic conditions it takes 4 h, as depicted in the Fig. 5A . Here, we found that in cells lacking pVHL the down-regulation of Na-K-ATPase activity during hypoxia is prevented (Fig. 1) . Since we have previously shown that the decrease of Na-K-ATPase activity parallels with the degradation of plasma membrane Na-K-ATPase (19) , we further studied whether pVHL is necessary for hypoxia-mediated degradation of plasma membrane Na-K-ATPase. In 786-0/pRC3 and RCC4 cells without pVHL expression and in A549 cells with knockdown of pVHL, hypoxia-mediated degradation of plasma membrane Na-K-ATPase was prevented. Overexpression of mutant pVHL disrupts the pVHL/Elongin/Cullin E3 ligase complex and prevented plasma membrane Na-K-ATPase degradation, suggesting that pVHL E3 ligase activity is necessary for Na-K-ATPase degradation (Fig. 2) . All of the three isoforms of Ubc5 act as E2 since DN-Ubc5A/5B/5C abrogated the degradation of Na-K-ATPase during hypoxia (Fig. 3) .

Although plasma membrane proteins are thought to be degraded in lysosomes, mounting evidence suggests that the ubiquitin system is involved in the degradation of some membrane proteins (29) . A previous study (19) reported that a defect in the ubiquitin-activating enzyme E1 prevented the degradation of the Na-K-ATPase {alpha}1 subunit, implicating a role for the ubiquitin system in the hypoxia-mediated degradation of Na-K-ATPase. Our study suggests that the ubiquitin-conjugating enzyme Ubc5 and the ubiquitin E3 ligase pVHL are required for the degradation of the plasma membrane Na-K-ATPase during hypoxia. However, we do not think that pVHL acts directly as an E3 for Na-K-ATPase because we have been unable to ubiquitinate Na-K-ATPase in an in vitro assay (data not shown) and neither {alpha} nor β subunit of Na-K-ATPase contains the conserved ODDD domain and LXXLAP motif, which are shared by HIF{alpha} isoforms (30) . Moreover, E3 ligase forms a complex with its substrate (31) , and our attempts to coimmunoprecipitate pVHL and Na-K-ATPase have been unsuccessful (data not shown). Thus, we reason that during hypoxia pVHL indirectly affects the degradation of Na-K-ATPase via a yet unknown intermediary protein. Besides HIF, other pVHL targets have been identified, including the RNA polymerase II subunit hsRPB7, a deubiquitinating enzyme 1, and an atypical protein kinase C-{lambda} (36 37 38) . Whether these proteins or other unidentified pVHL targets are involved in the degradation of Na-K-ATPase warrants further research.

Since HIF{alpha} is stabilized during hypoxia (34) , we studied whether pVHL affected Na-K-ATPase degradation in a HIF-dependent pathway. DFO treatment and exogenous ODDD peptides resulted in increased HIF1/2{alpha} but did not affect Na-K-ATPase degradation (Fig. 5) . Knockdown of HIF1/2{alpha} by shRNA did not affect hypoxia-mediated degradation of the Na-K-ATPase (Fig. 6) . Furthermore, while RCC4 cells express HIF1 and 2{alpha}, 786-0 cells only express HIF2{alpha} but they have the same response to hypoxia in terms of Na-K-ATPase degradation regardless of the difference in HIF{alpha} expression pattern in these cells (Fig. 1C, D ). Thus, we conclude that transient stabilization of HIF{alpha} is not required for plasma membrane Na-K-ATPase degradation. However, in pVHL-deficient cells, HIF is constitutively active. HIF{alpha} translocates to the nucleus and forms a dimer with HIFβ. The HIF heterodimer recruits other transcription cofactors and binds to a specific sequence (RCGTG) in HIF-responsive genes, therefore activating the transcription of multiple downstream genes (6 , 35) . Whether these downstream genes are involved in the degradation of Na-K-ATPase requires further research.

How cells sense decreases in oxygen to initiate signaling mechanisms resulting in the changes in the various biological outputs remains controversial. One model suggests that mitochondrial complex III increases the release of free radicals during low oxygen levels that is required for HIF-1 activation (33) . We (18) have also reported that mitochondrial ROS are required for the endocytosis of Na-K-ATPase during hypoxia. Thus, it is reasonable to suggest that mitochondrial ROS are likely key regulator of the pVHL-dependent degradation reported in this study. To test whether ROS play a role in pVHL-mediated degradation of Na-K-ATPase, we pretreated RCC4+VHL cells with the ROS scavenger Euk-134 and found that Na-K-ATPase degradation was prevented (Fig. 4) . It has been reported that pVHL-deficient 786-0 cells generate higher ROS levels than cells with reconstituted pVHL (32) , yet in RCC4 cells hypoxia-mediated Na-K-ATPase degradation did not occur (Fig. 1) . These data suggest that in the absence of pVHL ROS alone are not sufficient for Na-K-ATPase degradation. Thus, we reason that Na-K-ATPase degradation requires both ROS generation and the presence of pVHL.

Prolonged hypoxia has been shown to decrease cellular ATP levels (39) , and we have previously reported that hypoxia decreased O2 consumption levels (19) . To investigate whether down-regulation of Na-K-ATPase by pVHL during hypoxia contributes to the maintenance of energy homeostasis, we measured the cellular ATP levels in RCC4 and RCC4+VHL cells exposed to hypoxia for 2 h. As shown in Supplemental Fig. 1, RCC4 and RCC4+VHL cells had comparable ATP levels in normoxic and hypoxic conditions, which is consistent with a previous report (18) , suggesting that down-regulation of Na-K-ATPase may compensate for the reduced ATP generation to maintain energy balance during hypoxia. Zhang et al. (40) reported that these cells have low O2 consumption levels through a metabolic reprogramming by an HIF-dependent pathway. Thus, the cellular ATP levels may reflect the combined effects of HIF and Na-K-ATPase regulation.

We provide evidence that pVHL is required, independently of HIF, for the degradation of plasma membrane Na-K-ATPase during hypoxia as a mechanism of cellular adaptation to hypoxia. Recently, it has been reported that in a HIF-independent process, hypoxia promoted pVHL interaction with nucleolar rDNA and restricted ribosome production, another major energy-demanding process in the cell (41) . Thus, pVHL appears to play a dual role in cell adaptation to hypoxia. On the one hand, during hypoxia pVHL-mediated degradation of HIF1/2{alpha} is inhibited, resulting in the activation of the transcription of multiple downstream genes to help oxygen and glucose delivery and anaerobic ATP production (6 , 35) . On the other hand, pVHL participates in the degradation of plasma membrane Na-K-ATPase and inhibits ribosome synthesis to decrease ATP consumption in a HIF-independent mechanism.


   ACKNOWLEDGMENTS
 
We thank L. Welch, A. Kelly, and I. T. Helenius for technical assistance and critical review of this manuscript. This study was supported in part by National Institute of Heart, Lung, and Blood grant HL-071643 and a Parker B. Francis Foundation fellowship (to G.Z.).

Received for publication March 18, 2007. Accepted for publication November 8, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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