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Human Molecular Genetics Advance Access originally published online on October 18, 2007
Human Molecular Genetics 2008 17(2):190-200; doi:10.1093/hmg/ddm296
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Sodium-potassium ATPase β1 subunit is a molecular partner of Wolframin, an endoplasmic reticulum protein involved in ER stress

Malgorzata Zatyka1, Christopher Ricketts1, Gabriela da Silva Xavier2, Jayne Minton1, Sarah Fenton1, Sabine Hofmann-Thiel3, Guy A Rutter2 and Timothy G. Barrett1,*

1 Section of Medical and Molecular Genetics, The Medical School, University of Birmingham, Birmingham B15 2TT, UK 2 Division of Medicine, Department of Cell Biology, Sir Alexander Fleming Building, Imperial College, Exhibition Road, London SW7 2AZ, UK 3 Institute of Microbiology and Laboratory Diagnostics, Robert-Koch-Allee 2, D-82131 Gauting, Germany

* To whom correspondence should be addressed at: Diabetes Unit, Birmingham Children’s Hospital, Steelhouse Lane, Birmingham B4 6 NH, UK. Tel: +44 1213339267; Fax: +44 1213339272; Email: t.g.barrett{at}bham.ac.uk

Received April 20, 2007; Accepted September 30, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Wolfram syndrome, an autosomal recessive disorder characterized by diabetes mellitus and optic atrophy, is caused by mutations in the WFS1 gene encoding an endoplasmic reticulum (ER) membrane protein, Wolframin. Although its precise functions are unknown, Wolframin deficiency increases ER stress, impairs cell cycle progression and affects calcium homeostasis. To gain further insight into its function and identify molecular partners, we used the WFS1-C-terminal domain as bait in a yeast two-hybrid screen with a human brain cDNA library. Na+/K+ ATPase β1 subunit was identified as an interacting clone. We mapped the interaction to the WFS1 C-terminal and transmembrane domains, but not the N-terminal domain. Our mapping data suggest that the interaction most likely occurs in the ER. We confirmed the interaction by co-immunoprecipitation in mammalian cells and with endogenous proteins in JEG3 placental cells, neuroblastoma SKNAS and pancreatic MIN6 β cells. Na+/K+ ATPase β1 subunit expression was reduced in plasma membrane fractions of human WFS1 mutant fibroblasts and WFS1 knockdown MIN6 pancreatic β-cells compared with wild-type cells; Na+/K+ ATPase {alpha}1 subunit expression was also reduced in WFS-depleted MIN6 β cells. Induction of ER stress in wild-type cells only partly accounted for the reduced Na+/K+ ATPase β1 subunit expression observed. We conclude that the interaction may be important for Na+/K+ ATPase β1 subunit maturation; loss of this interaction may contribute to the pathology seen in Wolfram syndrome via reductions in sodium pump {alpha}1 and β1 subunit expression in pancreatic β-cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Diabetes mellitus is a heterogeneous disorder characterized by glucose intolerance and affects over 170 million people worldwide (1). The disease arises from a combination of absolute (type 1) or relative (type 2) insulin deficiency with a variable tissue insulin resistance (2). Recently, endoplasmic reticulum (ER) stress has been identified as a novel contributor to insulin resistance in type 2 diabetes (3) and may be involved in the loss of pancreatic β-cells that occurs in this disease (4).

Wolfram syndrome (5) is characterized by childhood-onset diabetes mellitus and neurodegeneration manifesting as progressive optic atrophy, diabetes insipidus, sensorineural deafness, neuropathic bladder and cerebellar ataxia (6). The syndrome is caused by loss of function mutations in the WFS1 gene (7,8), encoding Wolframin, an ER membrane protein (9). Mutations are distributed throughout the entire gene, but with one loss of function mutation at the C-terminal end (c.2648-2651delTCTT; F883fsX950) recurring in white European populations (7,10). Mice with a disrupted WFS1 gene show glucose intolerance and progressive pancreatic β-cell loss (11,12). This appears to be via activation of ER stress pathways, impaired cell cycle progression and apoptosis (12–14). Finally, this gene has recently become topical as common variants in WFS1 are strongly associated with risk for type 2 diabetes in man (15).

Pancreatic β-cells have a highly developed ER, reflecting their major function of secreting insulin (16). The ER has many roles, which include post-translational modification, folding and assembly of newly synthesized proteins such as insulin. Conditions that disturb ER function are collectively known as ‘ER stress’, and in the β-cell may include an increased demand for insulin synthesis subsequent to insulin resistance in obesity or type 2 diabetes. At least in other cell types, the ER stress response (17,18) involves upregulation of genes encoding ER chaperone proteins which increase protein folding activity and prevent protein aggregation; translational attenuation to reduce the synthesis of new protein and prevent the further accumulation of unfolded proteins; degradation of misfolded proteins and finally apoptosis, which occurs when severe and prolonged ER stress extensively impairs ER functions. Pancreatic β-cells are some of the most susceptible cells to ER stress, and ER stress-mediated apoptosis causes diabetes in Wolfram syndrome (14).

All three ER stress pathways (PERK, IRE1 ATF6) are activated by WFS1 deficiency in pancreatic β-cells (13). In addition, WFS1 expression increases in response to ER stress (19). ER stress pathways are activated by many stimuli which include inhibition of protein glycosylation, reduction in formation of disulphide bonds, calcium depletion from the ER lumen, impairment of protein transport from the ER to the Golgi and expression of malfolded proteins (16). There is also some evidence that Wolframin functions as an ion channel or regulator of existing channels (20) and is involved in intracellular calcium homeostasis by modulating the filling state of the ER calcium store (21). It is possible that in WFS1-deficient cells, impaired ER calcium homeostasis leads to ER stress and pancreatic β-cell apoptosis. It was also suggested that Wolframin may function in protein assembly, protein folding (e.g. pro-insulin folding and processing) and/or transport out of the ER (13).

To further investigate the function of Wolframin and the mechanism of pancreatic β-cell failure in Wolfram syndrome, we undertook a yeast two-hybrid screen using the WFS1 C-terminal domain as bait in a human cDNA library. Using this and complementary approaches, we identify the Na+/K+ ATPase β1 subunit as a novel interacting partner and show that the expression of Wolframin parallels that of Na+/K+ ATPase β1 subunit in a variety of settings. Decreases in Na+/K+ ATPase expression may thus contribute to changes in β-cell mass and function in Wolfram syndrome.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Identification of Na+/K+ ATPase β1 subunit as an interacting partner for WFS1
We used the yeast two-hybrid system to screen a pre-transformed human brain cDNA library (Clontech) with the C-terminal domain of the human WFS1 gene (amino acids 652–890) as bait. The C-terminal domain is located in the ER lumen and many Wolfram patients have mutations that result in its truncation or frame shift and an extended polypeptide tail (10). We chose a human brain library as neurodegeneration is a major component of Wolfram syndrome. Approximately 6 x 106 independent transformants were screened, of which eight clones were positive for {alpha}-galactosidase expression. One of the interacting clones was identified as the C- terminal domain of the Na+/K+ ATPase β1 subunit containing the coding region for amino acids 113–303 (of 303). We chose this clone for further study.

Confirmation of WFS1-Na+/K+ ATPase β1 interaction in mammalian cells
We confirmed the above interaction in mammalian Cos7 cells transiently co-transfected with full-length WFS1 (pCMV–c-myc-WFS1) and either the C-terminal domain of β1 subunit, corresponding to the clone identified in the library (pCMV-HA-β1-C), or with full-length Na+/K+ ATPase β1 subunit (pCMV-HA-β1) (Fig. 1C). Using rabbit polyclonal c-myc antibody (Sigma) for immunoprecipitation, and mouse monoclonal HA antibody for immunoblotting, we detected either a ~25 kDa protein, which corresponded to the C-terminal domain of Na+/K+ ATPase β1 subunit (Fig. 1A, lanes 9 and 10), or a 35–50 kDa protein corresponding to full-length Na+/K+ ATPase β1 subunit (Fig. 1A lanes 4, 5). The 35–50 kDa protein appeared to be migrating as multiple bands possibly due to different levels of glycosylation (22). The identity of these proteins was confirmed by reprobing the membrane with specific anti-Na+/K+ ATPase β1 subunit antibody (mouse, monoclonal, Sigma). In the reverse experiment, using mouse, monoclonal HA antibody (Sigma) for immunoprecipitation and polyclonal, rabbit c-myc antibody (Sigma) for immunoblotting, we detected a ~100 KDa protein corresponding to Wolframin (Fig. 1B, lanes 4–7). Its identity was confirmed by reprobing with specific anti-Wolframin antibody [rabbit, polyclonal (23)]. No co-immunoprecipitation was observed in control extracts co-transfected with either empty pCMV-Myc and pCMV-HA-β1 (Fig. 1A, lanes 6 and 7) or empty pCMV-HA and pCMV-Myc-WFS1 (Fig. 1A, lanes 2, 3, 11 and 12; Fig. 1B, lanes 2 and 3). No co-immunoprecipitation of Wolframin with unrelated proteins FLAG-RasF1 or GFP was observed in this mammalian system (data not shown). We conclude that the interaction between Wolframin and Na+/K+ ATPase β1 subunit is an interaction specific to Wolframin.


Figure 1
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  		Figure 1. Co-immunoprecipitation of WFS1 and Na+/K+ ATPase β1 subunit cotransfected to Cos7 cells. WFS1 interacts with both full length Na+/K+ ATPase β1 subunit (A, lanes 4 and 5; B, lanes 4 and 5) and with β1 C-terminal domain (A, lanes 9 and 10; B, lanes 6 and 7). (A) Co-immunoprecipitation with C-myc antibody (polyclonal, Sigma), detection with HA antibody (monoclonal, Sigma). Below, reprobing with anti-Na+/K+ ATPase β1 antibody (monoclonal, Sigma) to confirm identity of the clone and with anti-C-myc antibody (monoclonal, Sigma) to show the input. (B) Co-immunoprecipitation with anti-HA antibody (monoclonal, Sigma), detection with anti-C-myc antibody (polyclonal, Sigma). Below, reprobing with WFS1 antibody (polyclonal, S. Hofmann) to confirm identity of the clone and with anti-HA monoclonal antibody (Roche) to visualize the input. (C) Diagram showing plasmids used in this experiment. Black rectangle, Myc tag; –, negative control, immunoprecipitation with either rabbit serum fraction normal (A) or unrelated monoclonal antibody anti-GFP (CRUK) (B); +, immunoprecipitation with indicated antibody; I, input, 5% of lysate used for immunoprecipitation; W, C-myc tagged full-length WFS1 (pCMV-Myc-WFS1), HA-empty vector pCMV-HA; myc, empty vector pCMV-Myc; β1, HA-tagged, full-length Na+/K+ ATPase β1 subunit (pCMV-HA-β1); β1-C, HA-tagged C-terminal domain of Na+/K+ ATPase β1 subunit (pCMV-HA-β1-C).

 
The C-terminal and transmembrane domains of Wolframin interact with Na+/K+ ATPase β1 subunit
Next, we mapped the Wolframin domains interacting with Na+/K+ ATPase β1 subunit. We prepared a series of WFS1 deletion constructs in plasmid pCMV-Myc, tagged with c-Myc at the N-terminus and tested their expression in Cos7 cells (Fig. 2A). Wolframin (amino acids 1–652) lacking its C-terminus ({Delta}C) and C-terminal domain of Wolframin (C-; amino acids 652–890) were both well expressed and migrated in SDS–PAGE as single bands of the expected size. Wolframin lacking its N-terminus (amino acids 322–890; {Delta}N) was expressed very weakly, probably due to instability. The N-terminal domain of Wolframin (N-; amino acids 1–321) was always seen with a small degradation product (Fig. 2A). We also attempted to prepare a Wolframin transmembrane domain (TM; amino acids 322–652), but were never able to express a product of the expected size from the plasmid. We transiently co-transfected truncated WFS1 plasmids with full-length Na+/K+ ATPase β1 subunit to Cos7 cells and co-immunoprecipitated with anti-c-Myc antibody (Fig. 2B–E). In each experiment, we included a positive control (WFS1 wt co-transfected with β1 subunit: W x β1) and a negative control (empty vector pCMV-Myc co-transfected with β1 subunit: V x β1). The C-terminal domain (Fig. 2B), but not the N-terminal domain (Fig. 2C) of Wolframin co-immunoprecipitated with Na+/K+ ATPase β1 subunit as expected from our yeast two-hybrid experiment. Truncated Wolframin missing its N-terminus ({Delta}N) was able to co-immunoprecipitate with β1 subunit, as expected ({Delta}N includes the Wolframin C-terminal domain). This protein was poorly expressed (again, possibly due to inherent instability, Fig. 2A and D). Interestingly, the truncated Wolframin missing its C-terminus ({Delta}C) was also able to interact with Na+/K+ ATPase β1 subunit (Fig. 2E), which indicates that there may be an additional β1-binding domain located in the transmembrane region. In summary, we demonstrated two Wolframin domains interacting with Na+/K+ ATPase β1 subunit, the C-terminus (amino acids 652–890) and transmembrane domain (amino acids 322–652), and found that the N-terminal domain is not able to interact.


Figure 2
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  		Figure 2. Mapping of WFS1 domains interacting with Na+/K+ ATPase β1 subunit. (A) Expression of WFS1 deletions in Cos7 cells. Each deletion construct was transfected into Cos7 cells. Twenty micrograms of protein extract was loaded in each lane. Below, diagram illustrating amino acids content of deletion constructs. (B–E) Co-immunoprecipitation of WFS1 deletions and β1 subunit with c-myc antibody. For each experiment, a positive control (WFS1 full-length; W x β1) and a negative control (empty vector: V x β1) are included. (B) Co-IP of C-terminal WFS1 domain with Na+ K+ ATPase β1 subunit (C x β1). C-terminal domain co-immunoprecipitates with β1. (C) Co-IP of N-terminal WFS1 domain with Na+ K+ ATPase β1 subunit (N x β1). N-terminal domain is not able to co-immunoprecipitate β1 subunit. (D) Co-IP of {Delta}N (WFS1 with missing N-terminal domain) with β1 subunit ({Delta}N x β1). {Delta}N is able to interact with β1 subunit, although the protein is very unstable. (E) Co-IP of {Delta}C (WFS1 with missing its C-terminus) with β1 subunit. Interestingly, this fragment of WFS1 is able to co-immunoprecipitate with β1 subunit ({Delta}C x B1). +, immunoprecipitation with c-Myc antibody (polyclonal, Sigma); –, negative control; immunoprecipitation with rabbit serum fraction normal (negative control). Below every panel, the same membrane is shown reprobed with c-Myc to illustrate the input.

 
Demonstration of interaction between endogenous proteins
We further confirmed the interaction between Wolframin and Na+/K+ ATPase β1 subunit by co-immunoprecipitation of endogenous proteins. Antibody to Na+/K+ ATPase β1 subunit (mouse, monoclonal, Sigma) was used for immunoprecipitation, and anti-N-Wolframin affinity purified antibody (in house, polyclonal, sheep) was used for immunoblotting. A 100 kDa protein was detected in JEG-3 placental cells (Fig. 3A) and SKNAS neuroblastoma cell lines (Fig. 3B) (both expressing high levels of Wolframin). No immunoprecipitated protein was detected in negative controls, where unrelated, monoclonal anti-HA antibody (Sigma) was used for immunoprecipitation (Fig. 3A and B). Moreover, we demonstrated co-immunoprecipitation between endogenous proteins in MIN6 mouse pancreatic cells (using MIN6pSuper extract). Antibodies to Na+/K+ ATPase β1 subunit (goat, polyclonal, Santa Cruz) were used for immunoprecipitation and anti-C-Wolframin affinity-purified antibodies (in house, polyclonal, sheep) were used for immunoblotting (as the combination of antibodies used for co-immunoprecipitation from human cells was not detecting mouse proteins). We detected a 100 kDa band corresponding to mouse WFS1 (Fig. 3C); no protein was detected in the negative control, where unrelated c-myc antibody (Sigma) was used for immunoprecipitation.


Figure 3
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  		Figure 3. Co-immunoprecipitation of endogenous WFS1 and Na+/K+ ATPase β1 subunit. (A) Co-IP from JEG3 placental cell line. (B) Co-IP from SKNAS neuroblastoma cells. (C) Co-IP from mouse pancreatic MIN6 cells. I, input: 2%; C, control–mouse Wfs1 construct transfected into Cos7 cells; +, co-immunoprecipitation with either monoclonal anti-Na+/K+ ATPase β1 subunit antibody (Sigma) for (A) and (B) or polyclonal goat anti-Na+/K+ ATPase β1 subunit antibody (Santa Cruz) for (C). –, negative control; co-immunoprecipitation with monoclonal anti-HA antibody (Sigma) for (A) and (B) or monoclonal C-myc (Sigma) for (C). Detection with anti-N-WFS1, sheep polyclonal antibody for (A) and (B) or with C-WFS1 sheep polyclonal antibody for (C).

 
Expression levels of Na+/K+ ATPase β1 subunit are reduced with absent or reduced WFS1 expression
To address the question of the possible role of Wolframin/Na+/K+ ATPase β1 subunit interaction, we first tested whether there are differences in overall expression levels of Na+/K+ ATPase β1 subunit in cells expressing different levels of Wolframin. We compared the levels of Na+/K+ ATPase β1 subunit expression in wild-type (wt) fibroblasts with fibroblasts from a Wolfram patient carrying a homozygous W700X mutation (24). This mutation leads to truncation of the major portion of the hydrophilic C-terminus and results in almost undetectable levels of Wolframin in fibroblasts from this patient, caused probably by instability of this protein. We found that the expression level of Na+/K+ ATPase β1 subunit in this Wolfram patient was significantly lower than that in wild-type fibroblasts: on average, the decrease was 91 ± 2% and varied from 88 to 93% (n = 5; Fig. 4A). A similar decrease in Na+/K+ ATPase β1 expression was observed in fibroblasts from another patient carrying a homogenous C insertion (1029insC/1029insC) which results in a frame shift and introduces a premature stop at codon 395 (data not shown). This mutation also results in undetectable levels of Wolframin in fibroblasts (23).


Figure 4
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  		Figure 4. Expression of Na+/K+ ATPase β1 subunit in wild-type and Wolfram patient’s fibroblasts and in WFS1 knockdowns in MIN6 cells. (A) Expression in fibroblasts; C-control (wt WFS1 fibroblasts), MT- W700X patient carrying null mutation in WFS1. The mean decrease in β1 expression in mutant fibroblasts was 91% (SD 2, range 93–98%; n = 5) (LabWorks). The bar charts in Figures 4–6 show densitometric analyses of the mean changes in expression (SDs shown as solid lines). (B) Expression in MIN6 knockdowns, C-control (MINpSuper), KD1, MIN6 with WFS1 expression reduced ~50%; KD2, MIN6 with WFS1 expression reduced ~70% (12). The mean decrease in β1 expression in KD2 cells was 64% (SD 10, range 49–79%; n = 5). The mean decrease in β1 expression in KD1 cells was 43% (SD 6, range 20–65%; n = 5). β1-, Na+/K+ ATPase β1 subunit.

 
To determine whether this difference in Na+/K+ ATPase β1 subunit expression was a consequence of altered Wolframin expression, we studied this in another system: stable MIN6 cell lines with reduced Wolframin expression (a kind gift from Professor Alan Permutt). The MIN6 knockdowns were constructed using RNA interference in mouse MIN6 insulinoma cells as described previously, and either 50% (KD1) or 70% (KD2) reduction in Wolframin expression was demonstrated (12). We observed a significant reduction in Na+/K+ ATPase β1 subunit expression in both KD1 and KD2 cell lines compared with the control: KD2 (70% knockdown) mean (SD) 64 ± 10% decrease in Na+/K+ ATPase β1 subunit (range 49–79%, n=5; Fig. 4B); KD1 (50% knockdown) mean (SD) 43 ± 6% (range 20–65%, n = 5). We considered two possible explanations for our observations: first that it may be due to an indirect effect of induction of ER stress in WFS1 mutant cells; ER stress is induced in WFS1 mutant and knockdown cells (12,13), and part of the ER stress response is to generally attenuate protein translation. Secondly, we considered that Wolframin might be necessary for proper Na+/K+ ATPase β1 subunit folding or maturation.

To explore these possibilities, we examined the levels of Na+/K+ ATPase β1 subunit expression in wild-type fibroblasts and wild-type MIN6pSuper (12) cells after induction of ER stress with thapsigargin. We then compared the expression of Na+/K+ ATPase β1 subunit in untreated cells (T0) and cells treated with thapsigargin for 24 h (T24) or 48 h (T48). The ER stress response was measured by detecting the ER stress marker CHOP (Ddit3 for mice), barely detectable under physiological conditions but strongly induced in response to ER stress (16). In the wt fibroblasts and MIN6 cells, we detected CHOP expression after 24 hours of treatment with thapsigargin (Fig. 5A and B). This showed that an ER stress response was induced in both wild-type MIN6 cells and wild-type fibroblasts (Fig. 5A and B). In wild-type fibroblasts, after 24 h treatment with thapsigargin, we observed a decrease in Na+/K+ ATPase β1 subunit expression in comparison to T0 (mean decrease 33 ± 9%, n = 7; range 20–39%; Fig. 5A). A similar result with a slightly larger reduction in β1 expression was observed after prolonging the exposure to thapsigargin to 48 h (data not shown). However, in both cases, the decrease was smaller than that observed in WFS1 mutant fibroblasts (90%). In MIN6 pSuper cells treated with thapsigargin for 24 h, the levels of Na+/K+ ATPase β1 subunit expression were also decreased (mean decrease 50 ± 8%, n = 5; range 37–59%; Fig. 5B). When exposure to thapsigargin was prolonged to 48 h, the MIN6 cells died. The above decreases were comparable to that observed in MIN6 knockdown cells (43% decrease in KD1, 64% decrease in KD2). In summary, we observed the same or larger reductions in Na+/K+ ATPase β1 subunit expression in Wolframin-depleted cells compared with thapsigargin-treated wild-type cells. This suggests that induction of ER stress does not account for all the reduction in β1 subunit expression in Wolframin-depleted cells.


Figure 5
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  		Figure 5. Expression of Na+/K+ ATPase β1 subunit in WFS1-positive fibroblasts and MIN6 cells after thapsigargin induced ER stress response. The ER stress was induced as shown by induction of ER stress marker CHOP. (A) Induction of ER stress response in WFS1-positive fibroblasts. The mean decrease in β1 expression was 33% (SD 9, range 20–39%; n = 7). (B) Induction of ER stress response in Min6pSuper [used as the WFS1-positive control in published WFS1 knockdown experiments (12)]. The mean decrease in β1 expression in thapsigargin-treated cells was 50% (SD 8, range 37–59%; n = 5). Tg, thapsigargin. Serum-starved cells were treated with 1.5 µmol/l of thapsigargin dissolved in DMSO. T0, cells were harvested immediately after addition of thapsigargin; T24, cells were harvested after 24 h treatment with thapsigargin. The increased level of ER stress marker CHOP shows that ER stress was induced. β1, Na+/K+ ATPase β1 subunit.

 
Expression of Na+/K+ ATPase {alpha}1 and β1 subunits in plasma membrane protein extracts of WFS1 mutant fibroblasts and MIN6 WFS1 knockdowns
To demonstrate whether Na+/K+ ATPase expression is altered on the cell surface, we tested the expression of both the catalytic {alpha}1 and regulatory β1 subunits of the sodium pump in plasma membrane protein extracts (Biovision plasma membrane protein extraction kit as described in Materials and Methods). We found significant decreases in β1 subunit in both mutant fibroblasts and MIN6 knockdowns (Fig. 6) compared with the respective controls. In WFS1 W700X mutant fibroblasts, we observed a mean (SD) decrease of 93 ± 4% (range 94–99%, n = 3). In WFS1 KD1 knockdown in MIN6 cells, we observed a mean (SD) decrease of 64 ± 4% (range 59–69%, n = 3), and in KD2 a mean (SD) decrease of 69 ± 4% (range 64–78%, n = 3). These results are similar to our findings in total cell lysates (Fig. 4). The expression of Na+/K+ ATPase {alpha}1 subunit remained unchanged in W700X mutant fibroblasts (Fig. 6A) and KD1 Min6 knocked down cells (where WFS1 expression is decreased ~50%, Fig. 6B). However, we demonstrated mean (SD) decreases of 46 ± 8% (range 32–61%, n = 3) in KD2, where WFS1 expression was reduced by ~70%. We concluded that in WFS1 70% knockdown pancreatic β-cells, the decrease in Wolfram expression correlates with a significant decrease in expression of both subunits of sodium pump in plasma membranes.


Figure 6
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  		Figure 6. Expression of Na+/K+ ATPase {alpha}1 and β1 subunits in plasma membrane extracts. (A) Expression in fibroblasts; C-control (wt WFS1 fibroblasts), MT- W700X patient carrying null mutation in WFS1. The mean decrease in β1 expression in mutant fibroblasts was 93 ± 4% (range 84–99%, n = 3), whereas there was almost no change in {alpha}1 expression (mean decrease 12 ± 9%, range 2–23%, n = 3). (B) Expression in MIN6 knockdowns; C-control (MINpSuper); KD1, MIN6 with WFS1 expression reduced ~50%; KD2, MIN6 with WFS1 expression reduced ~70% (12). The mean decrease in β1 expression in KD2 cells was 69% (SD 4, range 64–78%; n = 3). The mean decrease in β1 expression in KD1 cells was 64% (SD 4, range 59–69%; n = 3). β1, Na+/K+ ATPase β1 subunit. Expression of {alpha}1 subunit was not significantly changed in KD1 (mean decrease 4%±29, range from 43% decrease to 27% increase, n = 3). Expression of {alpha}1 subunit in KD2 was reduced, mean decrease 46 ± 8%, range 32–61%, n=3.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
We identify here an interaction between Wolframin and Na+/K+ ATPase β1 subunit in transfected Cos7 cells, and between endogenous proteins in placental, neuroblastoma and MIN6 pancreatic β-cell lines. Furthermore, we demonstrate reduced Na+/K+ ATPase β1 subunit expression in whole cell and plasma membrane extracts of WFS1 mutant fibroblasts and WFS1 knockdown MIN6 pancreatic β-cells. In studies of plasma membrane fractions, we found normal levels of Na+/K+ ATPase {alpha}1 subunit in mutant fibroblasts, but reduced levels in WFS1 70% knockdown MIN6 pancreatic β-cells. We conclude that the reduction in β1 subunit expression in mutant fibroblasts and knockdown MIN6 cells is likely to be a direct result of reduced Wolframin expression. Further, the reduced plasma membrane expression of both Na+/K+ ATPase {alpha}1 (catalytic) and β1 (regulatory) subunits in 70% knockdown MIN6 cells suggests a reduction in function, but normal expression of Na+/K+ ATPase {alpha}1 subunit in mutant fibroblast plasma membranes suggests that Na+/K+ ATPase activity is likely to be normal in these cells.

We demonstrate that the Wolframin-Na+/K+ ATPase β1 interaction occurs with the Wolframin C-terminal domain (amino acids 652–890), and the transmembrane region (amino acids 322–655), but not the N-terminal domain (amino acids 1–322) (Fig. 2). The localization and membrane topology of Wolframin is known: it is an ER membrane protein (9) with the C-terminal domain located in the ER lumen and N-terminal domain in the cytoplasm with nine membrane spanning domains and loops facing both sides of the ER membrane (23,25). It assembles into higher molecular weight complexes of ~400 kDa in the ER membrane (23). The mature sodium pump ({alpha}1 and β1 subunits) is located in the plasma membrane; the N-terminal amino acids (1–34) of Na+/K+ ATPase β1 subunit are in the cytoplasm; amino acids 36–62 form the signal anchor and the C-terminal domain (amino acids 63–303) is located extracellularly (Fig. 7). However, the sodium pump is present transiently in the ER during maturation and assembly in common with other oligomeric membrane proteins (26). The ER membrane may be juxtaposed to the plasma membrane, allowing interactions between ER and plasma membrane proteins (e.g. the interaction of Na+/K+ ATPase β1 subunit with inositol 1,4,5-trisphosphate receptor (InsP3R) (27). However, our mapping data showed that the C-terminal domain of Wolframin (facing the ER lumen) is sufficient for interaction with Na+/K+ ATPase β1 subunit and the cytoplasmic N-terminal Wolframin domain did not interact; we therefore conclude that the ER lumen is the most likely site for this interaction (Fig. 7).


Figure 7
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  		Figure 7. Localization of WFS1 and its interacting partner Na+/K+ ATPase β1 subunit in the cell. The final destination for the sodium pump is the plasma membrane; however, it is transiently present in the ER during its maturation and assembly of its subunits ({alpha} and β). WFS1 is an ER membrane protein with cytoplasmic N-terminus and luminal C-terminus. The most likely compartment where the WFS1–Na+/K+ ATPase β subunit interaction can take place is the ER (see Discussion).

 
The reduction in Na+/K+ ATPase β1 subunit in WFS1 knockdown cells is unlikely to be explained by off-target effects of the WFS1siRNA, as a blast search revealed only WFS1-derived sequences. We first considered the possibility that the reduced expression is secondary to attenuation of protein translation as part of the ER stress response induced by the absence of Wolframin (12–14). The MIN6 knockdown cells we used are known to display ER stress (12). The induction of ER stress response in WFS1 knockouts and knockdowns (including KD1 and KD2 used in this study) was reported before (12–14), by demonstrating elevated levels of typical ER stress markers including Hspa5 (BIP), Ddit3 (CHOP), GRP94, GRP78 and p58IPK as well as the presence of phosphorylated PERK and eIF2{alpha} and increased XBP1 mRNA splicing. In particular, induction of CHOP in KD1 and KD2 was demonstrated at the mRNA level (12). Although we did not detect CHOP at the protein level in KD1 or KD2, we concluded that the ER stress was below the level detectable by western blots with our anti-CHOP antibody.

We therefore induced ER stress with thapsigargin in wild-type (wt) fibroblasts and wild-type MIN6 cells. In wt MIN6 pSuper, after 24 h treatment with thapsigargin, the levels of Na+/K+ ATPase β1 subunit expression were reduced by 50% in comparison to T0 (Fig. 5B). This was comparable to the reduced expression observed in KD2 WFS1 knockdown cells (64%, Fig. 4B). However, in thapsigargin-treated fibroblasts, the levels of Na+/K+ ATPase β1 subunit expression were reduced by 30%, compared with a 90% reduction in Na+/K+ ATPase β1 subunit in W700X null mutant fibroblasts (Figs 4A and 5A). We considered using additional ER stressors, dithiothreitol (DTT) and tunicamycin. However, DTT inhibits the formation of disulphide bonds in the ER and reduces the expression of numerous proteins, including β1 subunits of the P-ATPase family; tunicamycin inhibits glycosylation, and both β1 subunit and Wolframin are glycosylated. Consequently, these alternative agents would not allow the induction of ER stress independently of effects on Wolframin or β1 subunit expression. We then undertook an additional time course analysis of the effects of thapsigargin to 48 h: the MIN6 wild-type cells were dying or dead at 48 h; the fibroblasts were living and cell extracts showed a reduction in β1 subunit expression of ~50% compared with untreated cells, but still less than the ~90% reduction seen in WFS1 mutant fibroblasts. The smaller reduction in Na+/K+ ATPase β1 subunit expression in MIN6 knockdown cells may reflect the incomplete suppression of Wolframin expression. Our observations in WFS1 mutant and wt fibroblasts suggest that the reductions in Na+/K+ ATPase β1 subunit expression cannot be solely explained by a secondary effect of ER stress and may be a direct effect of reduced Wolframin expression.

Is loss of this interaction important to the pathology of Wolfram syndrome? The clinical features are due to loss of neurons and pancreatic β-cells, both of which express high levels of Wolframin, and are responsible for neurodegeneration and diabetes respectively. These cells are also protein-secreting cells and highly susceptible to ER stress (16,18). Wolframin has been proposed to be involved in the folding and processing of proteins in conditions of ER stress (13). If Wolframin is an additional facilitator of ER folding and assembly of β1 subunits with {alpha}1 subunits in cells susceptible to ER stress, then a reduction in Wolframin would likely result in less expression of both β1 and {alpha}1 subunits on the plasma membranes of affected cells. In contrast, fibroblasts have lower WFS1 expression, are not high protein secretors and are less susceptible to ER stress; any reduction in β1 subunit expression due to the absence of Wolframin could be mitigated by other protein-folding pathways (and other β subunits) that operate independently of ER stress. We believe that an alternative β subunit (β2 subunit, for instance) may be co-translationally associated with the {alpha}1 subunit on the cell surface of fibroblasts and that sodium pump activity is unlikely to be impaired in WFS1 mutant fibroblasts. However, the reduced expression of both β1 and {alpha}1 subunits (responsible for the catalytic activity of the sodium pump) in MIN6 β-cells is likely to result in reduced Na+/K+ ATPase activity. These data are consistent with the distribution of pathology to the pancreatic β-cells but not fibroblasts in Wolfram syndrome.

The activity of Na+/K+ ATPase is known to be reduced in many tissues of streptozotocin-induced diabetic animals, and in the red blood cell membranes of type 1 diabetic (insulin-deficient) humans (28). Na+/K+ ATPase deficiency has been identified as a contributor to apoptosis and neural degenerative disease, of which Wolfram syndrome is one example. To the best of our knowledge, reduction in Na+/K+ ATPase expression has not previously been reported in ER stress. Our findings suggest that Na+/K+ ATPase insufficiency may contribute to the pattern of cell loss reported in Wolfram syndrome.

We considered whether Wolframin is involved in the ER folding and assembly of subunits of other oligomeric proteins. Na+/K+ ATPase is a member of the P-type ATPases and shares with H+/K+ ATPase the requirement for a β subunit. ATPases, like other membrane proteins, are translocated into the ER during synthesis as individual subunits and integrated into the ER membrane for correct folding and assembly (29). In preliminary experiments, we have demonstrated co-immunoprecipitation of overexpressed WFS1 with the β1 subunit of the proton pump (H+/K+ ATPase). It is therefore possible that Wolframin has a general facilitatory role in the assembly of subunits of oligomeric proteins.

In summary, our findings suggest that Na+/K+ ATPase insufficiency may contribute to the known mechanisms of β-cell apoptosis, and possibly dysregulated insulin secretion, reported in Wolfram syndrome. The sodium pump offers a potential therapeutic target as its activity is modulated by several known drugs. ER stress is now recognized as a contributor to the apoptosis seen in both type 2 and also type 1 diabetes (30), and WFS1-depleted cells are useful models with which to study these pathways. It remains to be determined how much of the ER stress response observed in Wolfram syndrome is actually secondary to the loss of the WFS1- β1 subunit interaction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
FUNDING
 REFERENCES
 
Yeast two-hybrid system
Yeast two-hybrid screening was performed using MATCHMAKER system 3 (BD-Clontech) according to manufacturer's instructions. The C-terminal domain of WFS1 (amino acids 652–890) was used as a bait to screen a pre-transformed brain cDNA library (Clontech). Positive clones were retransformed to yeast to confirm a positive interaction and were sequenced using automated DNA sequence analysis (ABI) and homologies identified using National Center for Biotechnology Information BLASTN/BLASTX.

Plasmids
Yeast two-hybrid bait: the C-terminal WFS1 cDNA (amino acids 652–890) was amplified on the IMAGE 412765 with primers: 9 and 10 (discussed subsequently) and cloned into pGBKT7 (Clontech).

pCMV-Myc-WFS1: full-length WFS1 cDNA (amino acids 1–890) was amplified on IMAGE 412765 with primers 1 and 2 and cloned in pCMV-Myc (Clontech). Full-length Na+/K+ ATPase β1 subunit: the cDNA was amplified on IMAGE 3506311with primers 7 and 8 and cloned in pCMV-HA (Clontech). The C-terminal part of Na+/K+ ATPase β1 subunit was subcloned as an EcoRI–XhoI fragment to pCMV-HA from the yeast two-hybrid library prey clone (in pACT2). Truncated WFS1 clones were amplified on pCMV-Myc-WFS1 as a template as follows: N-: with primers 1 and 6; C- with primers 5 and 2; TM with primers 3 and 4; {Delta}C with primers 1 and 4 and {Delta}N with primers 3 and 2 and cloned in pCMV-Myc.

Primers

  1. 5'-GCAGG AATTC GGATG GACTC CAACA CTGCT CC
  2. 5'-GCAGG GTACC TCAGG CCGCC GACAG GAATG
  3. 5'-GAGAA TTCGG CACCA CATCA ACGCG CTCAT
  4. 5'-GCAGG GTACC CACAT AGAAC CAGCA GAACA
  5. 5'-GAGAA TTCGG TACCG CTCAG AGGGC ATGAA
  6. 5'-GACGG GTACC CGTGG GGATG ATGGT GGACA
  7. 5'-GGGAATTCCCATGGCCCGCGGGAAAGCCAA
  8. 5'-GGCTCGAGTGTGATCAGCTCTTAACTTC
  9. 5'-GCAGG AATTC CCGGG GTACC GCTCA GAGGG CATG
  10. 5'-GCAGG GATCC TCAGG CCGCC GACAG GAATG GGAAG

Cell lines
Cos7 cells, JEG3 placental cell line, neuroblastoma SKNAS cells and human fibroblasts were grown in DMEM (4500 mg/l L-glucose, L-glutamine and pyruvate, Invitrogen) with 10% FCS, penicillin (25 000 UI), streptomycin (25 000 µ/g), glutamine and non-essential amino acids. MIN6 cells (MINpSuper, WFS1KD1,WFS1KD2 (12) were grown in DMEM (4500 mg/l L-glucose, L-glutamine and pyruvate) with 15% FCS, penicillin (25 000 UI), streptomycin (25 000 µ/g), glutamine, non-essential amino acids, 5 µl/1 l of medium β-mercaptoethanol and geneticin 200 µg/ml. Transient transfections were performed using FUGENE transfection reagent (Roche) according to manufacturers’ instructions. ER stress was induced with thapsigargin. Cells were serum starved for 14 h before the experiments. Thapsigargin (1.5 µmol/l) in DMSO was added to the medium. Control T0 was harvested immediately; thapsigargin treated cells were harvested after 24 h in Laemelli buffer and resolved on SDS–PAGE gel.

Anti-WFS1 antibodies
Peptides corresponding to the N-terminal human WFS1 sequence: SLEQERSERPRAPGPQAGPGPGVRDB and C-terminal WFS1 sequence RCLYGEAYPACSPGNTSTAEEELCRB were used to generate polyclonal antibodies in sheep (The Binding Site, University of Birmingham). The antibody was subsequently affinity purified on controlled pore glass affinity columns (Alta Biosciences) and their specificity tested by western blots with our WFS1 human and mouse expression plasmids, mutants and knockdowns. N-WFS1 antibody does not recognize mouse Wfs1, whereas C-WFS1 does.

Immunoprecipitation
Transiently transfected Cos7 cells were harvested by scraping in RIPA buffer (50 mM Tris pH8, 150 mM NaCl, 0.1% SDS, 1 mM EDTA, 0.5% deoxycholate, 1% Igepal), sonicated2x 10 s, spun down at 4°C and supernatant collected. For co-immunoprecipitation, ~200 µg of each extract was pre-cleaned with Protein A agarose beads (Sigma).

Twenty microlitres of c-Myc rabbit polyclonal antibodies (Sigma) or HA mouse monoclonal antibodies (Sigma) was added to cleaned lysate and incubated overnight rotating at 4°C. The same amounts of either rabbit serum fraction normal (Dako) or monoclonal GFP antibody (CRUK UK) were used as negative controls. Twenty microlitres of pre-cleaned Protein A agarose beads were added next morning and extracts were incubated for 2 h rotating at 4°C. The complexes were precipitated by spinning for 2 min at 13 000 r.p.m. and washed three and four times with RIPA buffer. After a final spin, 30 µl of 2x Laemelli loading buffer was added and samples were run on 10% SDS–PAGE gel. For precipitation of endogenous proteins, 400–800 µg of either JEG3, SKNAS or MIN6pSuper cell extracts was used. Human proteins were precipitated with 40 µl of Na+/K+ ATPase β1 mouse monoclonal antibody (Sigma) or mouse monoclonal HA (Sigma) as a negative control (JEG3 and SKNAS). Mouse proteins (MIN6pSuper) were precipitated with 50 µl of Na+/K+ ATPase β1 goat polyclonal antibody (Santa Cruz) or monoclonal C-myc (Sigma) as a negative control.

Immunoblotting
Samples were resolved on 10% SDS–PAGE, at 120 V with Rainbow marker (GE Healthcare Life Sciences) run alongside to estimate the size. Samples were transferred to Hybond P membrane (Amersham Biosciences) by electroblotting at 80 V for 1 h in standard transfer buffer. Antibodies were used overnight at 4°C at the following concentrations: anti-C-myc mouse monoclonal (Sigma) 1:1000, C-myc rabbit polyclonal (Sigma) 1:1000, HA mouse monoclonal (Sigma) 1:1000, HA mouse monoclonal (Roche) 1:500, Na+/K+ ATPase β1 mouse monoclonal (Sigma) 1:5000; rabbit, polyclonal Na+/K+ ATPase β1 subunit (Upstate) 1:5000; mouse, monoclonal anti-{alpha}1 subunit (Upstate) 1:10 000; anti-WFS1 rabbit polyclonal (23) 1:5000; anti-N-WFS1or C-WFS1 (both sheep, polyclonal, affinity purified; The Binding Site, University of Birmingham) 1:5000; mouse, monoclonal anti-CHOP (Affinity Bioreagents) 1:250, mouse, monoclonal anti-flotillin-1 (abcam) 1:1000; mouse, monoclonal anti-β actin (Sigma) 1:15 000 in 5%milk in PBS/Tween. Secondary antibodies: anti-rabbit, sheep, goat or mouse (Dako) were used at 1:20 000 in 5% milk in PBS/Tween, incubated for 1 h at RT. Immunoblots were developed with Amersham ECL Plus western blotting detection system (GE Healthcare). Quantitative analysis of the altered expression of Na+/K+ ATPase β1 subunit was performed by measuring integrated optical density using the programme LabWorks.

Preparation of plasma membrane extracts
Plasma membrane extracts of fibroblasts and MIN6 cells were prepared using plasma membrane protein extraction kit (Biovision). Cells were harvested at 5–10 x 107 by scraping in cold PBS, spun down, washed once in cold PBS and frozen. Homogenization (with Dounce homogenizer) and plasma membrane protein extraction were performed according to manufacturer's instructions using buffers included in the kit. Plasma membrane fraction was tested for the presence of plasma membrane markers: cadherins (with rabbit, polyclonal pan-cadherins antibody from Abcam) and flotillin-1 (with mouse, monoclonal antibody, BD Biosciences).


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
REFERENCES
 
This study was supported by SPARKS, WellChild, Birmingham Children’s Hospital Research Foundation, Ferring Pharmaceuticals and Ipsen Pharmaceuticals. G.A.R. was supported by Programme Grants and a Research Leave Fellowship from the Wellcome Trust and Research and Project grants from the MRC (UK), Juvenile Diabetes Research Fund, NIH and the European Union.


   ACKNOWLEDGEMENTS
 
We are very grateful to Professor Alan Permutt from Washington University, St Louis, MO, USA, for kindly allowing us to use WFS1 suppressed MIN6 cells and appropriate controls. The Molecular Genetics Laboratory at University of Birmingham is supported by Wellchild. J.A.L. Minton was a Wellchild PhD student.

Conflict of Interest statement. The authors have no conflicts of interest to declare.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 

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