Regulation of intestinal phosphate cotransporter NaPi IIb by ubiquitin ligase Nedd4–2 and by serum- and glucocorticoid-dependent kinase 1

M. Palmada,1 M. Dieter,1 A. Speil,1 C. Böhmer,1 A. F. Mack,2 H. J. Wagner,2 K. Klingel,3 R. Kandolf,3 H. Murer,4 J. Biber,4 E. I. Closs,5 and F. Lang1

Departments of 1Physiology I, 2Anatomy, and 3Molecular Pathology, University of Tübingen, 72076 Tübingen, Germany; 4Department of Physiology, University of Zürich, 8057 Zürich, Switzerland; and 5Department of Pharmacology, University of Mainz, 55099 Mainz, Germany

Submitted 18 March 2003 ; accepted in final form 5 March 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
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Serum and glucocorticoid-inducible kinase 1 (SGK1) is highly expressed in enterocytes. The significance of the kinase in regulation of intestinal function has, however, remained elusive. In Xenopus laevis oocytes, SGK1 stimulates the epithelial Na+ channel by phosphorylating the ubiquitin ligase Nedd4–2, which regulates channels by ubiquitination leading to subsequent degradation of the channel protein. Thus the present study has been performed to explore whether SGK1 regulates transport systems expressed in intestinal epithelial cells, specifically type IIb sodium-phosphate (Na+-Pi) cotransporter (NaPi IIb). Immunohistochemistry in human small intestine revealed SGK1 colocalization with Nedd4–2 in villus enterocytes. For functional analysis cRNA encoding NaPi IIb, the SGK isoforms and/or the Nedd4–2 were injected into X. laevis oocytes, and transport activity was quantified as the substrate-induced current (IP). Exposure to 3 mM phosphate induces an IP in NaPi IIb-expressing oocytes. Coinjection of Nedd4–2, but not the catalytically inactive mutant C938SNedd4–2, significantly downregulates IP, whereas the coinjection of S422DSGK1 markedly stimulates IP and even fully reverses the effect of Nedd4–2 on IP. The effect of S422DSGK1 on NaPi IIb is mimicked by wild-type SGK3 but not by wild-type SGK2, constitutively active T308D,S473DPKB, or inactive K127NSGK1. Moreover, S422DSGK1 and SGK3 phosphorylate Nedd4–2. In conclusion, SGK1 stimulates the NaPi IIb, at least in part, by phosphorylating and thereby inhibiting Nedd4–2 binding to its target. Thus the present study reveals a novel signaling pathway in the regulation of intestinal phosphate transport, which may be important for regulation of phosphate balance.

transforming growth factor-{beta}; phosphatidylinositol 3-kinase; transporter; protein kinase B; protein kinases


SERUM AND GLUCOCORTICOID-INDUCIBLE kinase 1 (SGK1) has originally been cloned as a glucocorticoid-inducible gene from rat mammary tumor cells (16, 54). The human isoform has been identified as a cell volume-sensitive gene upregulated by cell shrinkage (30, 51). Subsequent studies revealed that the expression of SGK1 is stimulated by mineralocorticoids (10, 11, 35, 44), gonadotropins (3, 4, 19, 41), and a number of cytokines including transforming growth factor-{beta} (TGF-{beta}) (29). The kinase is activated by IGF1 and insulin through phosphatidylinositol 3-kinase and 3-phosphoinositide-dependent protein kinase-1 (PDK1) (27, 28, 38), an effect involving phosphorylation of the serine at position 422. Replacement of this serine by aspartate at this location (S422DSGK1) leads to a constitutively active kinase, and destruction of the catalytic subunit by replacement of the lysine at position 127 with asparagine leads to the inactive mutant K127NSGK1 (27).

In situ hybridization of human intestinal tissue disclosed a selective, strong expression of SGK1 in apical villus enterocytes, whereas crypt cells did not express SGK1 (52). The localization suggests the involvement of SGK1 in the regulation of intestinal transport systems. As a matter of fact, evidence for a role for SGK1 in transport regulation was obtained in experiments utilizing the Xenopus laevis oocytes expression system (49). It has been shown that coexpression of SGK1 increases Na+ channel activity in X. laevis oocytes expressing the renal epithelial Na+ channel (ENaC) (8, 11, 31, 35, 39, 44, 50) by increasing the abundance of ENaC protein within the cell membrane (5, 50, 53). More recent evidence revealed that the stimulation of ENaC activity was secondary to phosphorylation of Nedd4–2, a ubiquitin ligase involved in the clearance of the channel proteins from the cell membrane (14, 45, 47). SGK1 phosphorylates and thereby impairs Nedd4–2 binding to its target.

The present study has been performed to search for a role of SGK1 in the regulation of intestinal transport systems. To this end, the intestinal type IIb sodium-phosphate cotransporter (NaPi IIb) has been expressed in X. laevis oocytes with and without coexpression of SGK1 and/or Nedd4–2. As a result, Nedd4–2 downregulates NaPi IIb, an effect reversed by SGK1.

SGK1 and its isoforms SGK2 and SGK3 (28) are related to the PKB (2), which may also participate in the regulation of phosphate transport. Thus cRNA encoding NaPi IIb has been injected with or without cRNA encoding wild-type SGK2, SGK3, or constitutively active T308D,S473DPKB into X. laevis oocytes. Our results show that the effect of S422DSGK1 on NaPi IIb is mimicked by SGK3, but not by SGK2 or T308D,S473DPKB.


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In situ hybridization.

Biopsy specimens from ileum mucosa, removed for diagnostic reasons, were fixed in 4% formaldehyde in PBS (pH 7.2) overnight and subsequently embedded in paraffin. Dewaxed paraffin sections were hybridized as described previously (22, 26). The hybridization mixture contained the {alpha}-35S-labeled SGK1 antisense RNA probe (500 µg/l) in 10 mM Tris·HCl (pH 7.4), 500 ml/l deionized formamide, 600 mM NaCl, 1 mM EDTA, 0.2 g/l polyvinylpyrrolidone, 0.2 g/l Ficoll, 0.5 g/l BSA, 100 g/l dextrane sulfate, 10 mM dithiothreitol, 200 mg/l denatured salmon sperm DNA, and 100 mg/l rabbit liver transfer RNA. Hybridization was performed at 42°C for 18 h. Posthybridization procedures included digestion of nonhybridized single-stranded RNA probes by ribonuclease A (20 mg/l) in Tris·HCl (10 mM, pH 8.0) and NaCl (0.5 mM) for 30 min at 37°C. Tissue slide preparations were autoradiographed for 3 wk and stained with hematoxylin and eosin. Control hybridizations were performed on consecutive tissue sections applying the corresponding {alpha}-35S-labeled sense SGK1-RNA probe.

Immunohistochemistry.

Mucosal biopsy specimens of normal ileum removed for diagnostic reasons were used for this study. Tissue sections were fixed in 4% paraformaldehyde/0.1 M sodium phosphate buffer (pH 7.2) for 4 h and embedded in paraffin. Paraffin-embedded tissue was sectioned at ~5 µm. Sections were deparaffinized and rehydrated. For antigen retrieval, the sections were covered with target retrieval buffer (10 mM citrate, pH 6.0) and treated in a microwave oven at 720 W three times for 5 min each time period. After a brief rinse, the sections were incubated for 1 h with blocking serum (3% BSA, 0.25% Triton in PBS). All of the following washes and incubations were performed in PBS containing 0.3% Triton and 1% DMSO. Without rinsing, primary antibodies, goat anti-SGK1 antibody (diluted 1:50; Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit anti-Nedd4–2 [diluted 1:100; kindly provided by O. Staub (University of Lausanne, Lausanne, Switzerland) (see Ref. 23)] were applied together in a moist incubation chamber for 12 h at 4°C. After three washes of 10 min each, sections were incubated with donkey anti-goat Alexa Fluor 660 secondary antibody diluted 1:200 for 1.5 h, and after thorough rinses with goat anti-rabbit Alexa Fluor 488 secondary antibody diluted 1:400 (both Molecular Probes, Eugene, OR), were incubated for 1.5 h. Controls were performed by omitting either one or both primary antibodies. After being placed on a coverslip with Fluorosave (Calbiochem, La Jolla, CA), the slides were analyzed on a Zeiss LSM 510 confocal microscope equipped with an Axioplan 2 microscope using a 40x oil immersion lens (numerical aperture, 1.3). With the use of the multitrack function, individual fluorochromes were scanned with laser excitation at 488 and 633 nm separately with appropriate filter sets to avoid cross-talk. Controls were scanned with identical laser excitation and filter settings. In addition, transmitted light images were recorded.

Expression in X. laevis oocytes and voltage-clamp analysis.

cRNA encoding constitutively active human SGK1 (S422DSGK1) (27, 51), SGK2, SGK3 (28), and constitutively active PKB (T308DS473DPKB) (2), wild-type X. laevis Nedd4–2 [kindly provided by O. Staub (see Ref. 14)], catalytically inactive X. laevis Nedd4–2 (C938SNedd4–2), wild-type mouse NaPi IIb (21), and wild-type human cationic amino acid transporter 1 (CAT1) (12) were synthesized in vitro as previously described (49). Dissection of X. laevis ovaries and collection and handling of the oocytes have been described in detail elsewhere (49). Oocytes were injected with 30 ng of NaPi IIb or 25 ng of CAT1, 7.5 ng of the respective protein kinases, and/or 15 ng of Nedd4–2. Control oocytes were injected with H2O. All experiments were performed at room temperature 3 days after cRNA injection. In two-electrode voltage-clamp experiments, currents were recorded at –60 mV after the addition of 3 mM Na2HPO4 to the bath solution for NaPi IIb measurements or of 1 mM L-arginine for CAT1 measurements. The data were filtered at 10 Hz and recorded with MacLab digital-to-analog converter and software for data acquisition and analysis (ADInstruments, Castle Hill, Australia). The control bath solution (ND96) contained (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4. The final solutions were titrated to the pH indicated by using HCl or NaOH. The flow rate of the superfusion was 20 ml/min, and a complete exchange of the bath solution was reached within ~10 s.

Detection of Nedd4–2 phosphorylation.

For determination of Nedd4–2 phosphorylation, 30 oocytes of each group were homogenized in lysis buffer containing (in mM) 50 Tris (pH 7.5), 0.5 EDTA (pH 8.0), 0.5 EGTA, 100 NaCl, plus 1% Triton X-100, 100 µM sodium orthovanadate, and protein inhibitor cocktail (Roche, Mannheim, Germany) at the recommended concentration. Proteins were separated on a 7% polyacrylamide gel and transferred to nitrocellulose membranes. After blots were blocked with 5% nonfat dry milk in PBS/0.15% Tween 20 at 4°C overnight, they were incubated for 1 h at room temperature with a rabbit anti-phosphoserine 328 Nedd4–2 antibody (Pineda, Berlin, Germany) diluted 1:250 in PBS/0.15% Tween 20/5% nonfat dry milk or a rabbit anti-Nedd4–2 antibody diluted 1:1,000 in PBS/0.15% Tween 20/5% nonfat dry milk. Secondary peroxidase-conjugated sheep anti-rabbit IgG diluted 1:1,000 in PBS/0.15% Tween 20/5% nonfat dry milk was used for chemiluminescent detection with an enhanced chemiluminescent kit (Amersham, Freiburg, Germany).

Cell culture.

Intestine-407 cells (American Type Culture Collection CCL6) derived from human embryonic jejunum and ileum were maintained in an atmosphere containing 5% CO2 at 37°C in MEM supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 1% nonessential amino acids, 1% L-glutamine, and 5 mM glucose. All cell culture reagents were purchased from Invitrogen (Karlsruhe, Germany). Cells were seeded in six-well tissue culture plates at 5 x 105 cells per well. After 48 h, cells were serum-starved for 16 h and then treated with 10 ng/ml TGF-{beta} (Sigma, St. Louis, MO) for 24 h. Thereafter, cells were harvested, and 30 µg of total protein were used for determination of Nedd4–2 phosphorylation.

Immunostaining of Intestine-407 cells.

Intestine-407 cells were plated on glass coverslips at 5 x 105 cells per well and 24 h later were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were subsequently washed with PBS, blocked, and permeabilized for 1 h in PBS containing 5% (vol/vol) normal goat serum (Sigma) and 0.2% (vol/vol) Triton X-100. Cells were incubated with a primary antibody against Nedd4–2 (rabbit anti-mouse Nedd4–2) or NaPi IIb (rabbit anti-mouse NaPi IIb) and against SGK1 (sheep immunoaffinity purified IgG; Upstate, Waltham, MA) diluted 1:100 in blocking solution for 1 h at room temperature. After the cells were washed with PBS, secondary goat anti-rabbit Texas Red-coupled antibody (Molecular Probes) was added at a dilution of 1:200 for 1 h at room temperature to detect Nedd4–2 or NaPi IIb. To detect SGK1, secondary donkey anti-sheep Alexa Fluor 488-coupled antibody (Molecular Probes) was used at a dilution of 1:200 for 1 h at room temperature. Coverslips were then mounted onto glass slides using the antifade agent in Mobiglow (MoBiTec, Göttingen, Germany). Epifluorescence microscopy was used to detect antibody localization. With each staining process, control stains were performed by omitting the primary antibodies that were analyzed with the same gain and brightness settings to test for unspecific and background staining.

Phosphate uptake in intestine-407 cells.

Intestine-407 cells were plated on six-well plates at a density of 5 x 105 cells per well, and 2 days later were serum-deprived for 40 h. During the last 24 h, cells were incubated with 10 ng/ml TGF-{beta} or vehicle. Thereafter, cells were washed once with phosphate-free uptake medium (in mM: 137 NaCl, 5.4 KCl, 2.8 CaCl2, 1.2 MgSO4, 10 HEPES, pH 7.0) and incubated with 2 ml of the same buffer containing 100 µM K2HPO4 and [32Pi]K2HPO4 (1 µCi/ml). After 5 min, the uptake medium was removed, and cells were washed 4 times with 2 ml of ice-cold stop solution [137 mM choline chloride (ChCl), 14 mM Tris·HCl, pH 7]. Cells were lysed with 1% Triton X-100, and 200 µl were taken for scintillation counting and protein determination. To determine sodium-independent transport, NaCl was substituted by ChCl. All uptakes were determined per triplicate in three independent experiments.

Calculations.

Data are provided as means ± SE; n represents the number of oocytes investigated. All experiments were repeated with at least three batches of oocytes; in all repetitions, qualitatively similar data were obtained. All data were tested for significance using Student's t-test, and only results with P < 0.05 were considered statistically significant.


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Similar to earlier findings (52), SGK1 cRNA was mostly detected in villus enterocytes and in some cells was scattered within the interstitium. No transcript was detected in crypt cells of the ileum mucosa (Fig. 1A). Immunohistochemistry in human small intestine not only confirmed the expression of SGK1 close to the apical membrane of enterocytes but revealed as well SGK1 colocalization with Nedd4–2 (Fig. 1B). With each staining process, control stains were performed by omitting the primary antibodies to test for nonspecific and background staining.



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Fig. 1. Expression of serum and glucocorticoid-inducible kinase 1 (SGK1) and Nedd4–2 in human small intestine. A: localization of SGK1 mRNA in human ileum. In situ hybridization reveals selective expression of SGK1 mRNA in villus enterocytes. No signal is detected in crypt cells. Note the sharp transition between SGK1 expressing and nonexpressing cells. B: immunoreactivity of SGK1 and Nedd4–2 in sections from human small intestine. SGK1 protein is expressed in human enterocytes, most abundantly close to the apical membrane in which SGK1 colocalizes with Nedd4–2. No SGK1 or Nedd4–2 staining could be detected when primary antibodies were omitted. DIC, differential interference contrast.

 
Coexpression studies in X. laevis oocytes showed a powerful stimulating effect of SGK1 on NaPi IIb-induced currents. In oocytes expressing NaPi IIb alone, 3 mM Na2HPO4 induced an inward current of 19.2 ± 2.2 nA (n = 30). In water-injected oocytes, Na2HPO4 (3 mM) did not produce a comparable inward current (0.4 ± 0.2 nA, n = 4). When Nedd4–2 was coexpressed with NaPi IIb, the phosphate-induced inward current was significantly decreased to 11.6 ± 1.4 nA (n = 19). On the contrary, the coexpression of S422DSGK1 led to a marked upregulation (46.3 ± 6.3 nA, n = 12) of phosphate-induced currents in X. laevis oocytes expressing NaPi IIb. Moreover, in oocytes coexpressing both Nedd4–2 and S422DSGK1, the phosphate-induced current was again significantly larger (61.5 ± 9.8 nA, n = 6) than the current in oocytes expressing NaPi IIb alone (Fig. 2).



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Fig. 2. Regulation of the intestinal type IIb sodium-phosphate cotransporter (NaPi IIb) by Nedd4–2 and SGK1. Original tracings (A), arithmetic means ± SE (B and C). B: Xenopus laevis oocytes were injected with cRNA of NaPi IIb, Nedd4–2, and/or S422DSGK1 or K127NSGK1. Whereas Nedd4–2 markedly downregulates NaPi IIb-mediated currents, S422DSGK1 stimulates the currents and reverses the effect of Nedd4–2. *Significant difference between expression of NaPi IIb alone and coexpression of NaPi IIb with Nedd4–2 and/or S422DSGK1 (n = 4–30). C: X. laevis oocytes were injected with cRNA of NaPi IIb, C938SNedd4–2 with or without S422DSGK1. NaPi IIb-mediated currents are not downregulated by coexpression of inactive C938SNedd4–2 but are upregulated by additional expression of S422DSGK1. *Significant difference between expression of NaPi IIb alone and coexpression of NaPi IIb with C938SNedd4–2 and S422DSGK1 (n = 10–11 oocytes investigated).

 
In contrast to S422DSGK1, coexpression of the inactive mutant K127NSGK1 did not stimulate NaPi IIb-mediated transport. The current approached 22.7 ± 4.1 nA, n = 11 in NaPi IIb and K127NSGK1 expressing oocytes compared with 19.2 ± 2.2 nA (n = 30) in oocytes expressing NaPi IIb alone. When the catalytically inactive Nedd4–2 (C938SNedd4–2) was expressed in NaPi IIb-injected oocytes, no significant modulation of inward current was observed (115.2 ± 16.2% of control, n = 11, Fig. 2). Thus modulation of NaPi IIb by SGK1 and Nedd4–2 involves phosphorylation and ubiquitination processes.

To test for the specificity of the effects observed, Nedd4–2 and SGK1 were coexpressed with the cation transporter CAT1 in X. laevis oocytes. Under the given experimental conditions, coexpression of Nedd4–2 and/or SGK1 did not affect the transporter activity significantly [29.0 ± 1.6 nA, n = 38 in oocytes expressing CAT1 vs. 27.8 ± 3.6 nA; n = 38 in oocytes coexpressing CAT1 and Nedd4–2 or 27.9 ± 3.3 nA; n = 38 in oocytes coexpressing CAT1 and SGK1 (Fig. 3) ].



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Fig. 3. Nedd4–2 and SGK1 do not affect intestinal cationic amino acid transporter 1 (CAT1). X. laevis oocytes were injected with cRNA of CAT1, Nedd4–2, and/or S422DSGK1. CAT1-mediated currents are not significantly modified by coexpression of Nedd4–2 or SGK1 (n = 38).

 
As shown in Fig. 4 and Table 1, Nedd4–2 and SGK1 modulate NaPi IIb activity by modifying the maximal transport rate (Vmax) while leaving the affinity constant (Km) unaffected. Nedd4–2 coexpression decreased Vmax from 19.0 ± 3.2 nA (n = 12) in oocytes expressing NaPi IIb alone to 12.8 ± 2.1 nA (n = 11) in oocytes expressing NaPi IIb with Nedd4–2. SGK1 coexpression increased Vmax to 34.1 ± 6.1 nA (n = 12).



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Fig. 4. Regulation of NaPi IIb kinetic parameters on coexpression of Nedd4–2 or SGK1. X. laevis oocytes were injected with cRNA encoding NaPi IIb without ({bullet}) or with cRNA encoding Nedd4–2 ({blacktriangledown}) or S422DSGK1 ({circ}). Nedd4–2 and SGK1 modulate NaPi IIb activity by modifying the maximal transport rate while leaving the affinity constant unaffected. Each data point represents the mean of 3 X. laevis oocytes from 1 representative experiment.

 

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Table 1. Kinetic parameters of NaPi IIb upon coexpression of Nedd4–2 or S422DSGK1

 
The effect of S422DSGK1 on NaPi IIb was mimicked by SGK3, but not by SGK2 or T308D,S473DPKB (Fig. 5). Coexpression of SGK3 led to a marked upregulation of phosphate-induced currents (39.7 ± 5.3 nA, n = 18) in X. laevis oocytes expressing NaPi IIb. In oocytes coexpressing both Nedd4–2 and SGK3, the phosphate-induced current was again significantly larger (40.4 ± 5.5 nA, n = 12) than the current in oocytes expressing NaPi IIb alone (19.2 ± 2.2 nA, n = 30, Fig. 5).



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Fig. 5. Regulation of NaPi IIb-induced currents by SGK3 but not by SGK2 or T308D,S473DPKB. X. laevis oocytes were injected with cRNA encoding NaPi IIb with or without cRNA encoding Nedd4–2 and/or either SGK2, SGK3, or PKB. SGK3 but not SGK2 or PKB enhances the phosphate-induced currents and reverses the downregulation of those currents by Nedd4–2. *Significant difference between expression of NaPi IIb alone and coexpression of NaPi IIb with Nedd4–2 and/or SGK3 (n = 12–30 oocytes investigated).

 
Western blot analysis of oocytes expressing Nedd4–2 alone or with SGK1, SGK2, or SGK3 showed that SGK1 and SGK3 but not SGK2 phosphorylate Nedd4–2 (Fig. 6).



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Fig. 6. Phosphorylation of Nedd4–2 in X. laevis oocytes by SGK1 and SGK3 but not by SGK2. X. laevis oocytes (n = 30) were injected with cRNA encoding Nedd4–2 with or without SGK1, SGK2, or SGK3. Coexpression of SGK1 and SGK3 but not of SGK2 phosphorylates Nedd4–2. A: immunoblotting with anti-P-Ser328 Nedd4–2 antibody (pN42) or anti-Nedd4–2 antibody (N42) as indicated. B: ratio of intensity was compared with the intensity obtained in oocytes expressing Nedd4–2 alone. *Significant difference between oocytes injected with Nedd4–2 alone and those injected with Nedd4–2 and SGK1 or SGK3.

 
Immunostaining of Intestine-407 cells revealed endogenous expression of Nedd4–2, SGK1, and NaPi IIb (Fig. 7). Twenty-four hour treatment with 10 ng/ml TGF-{beta}, a particularly strong stimulator of SGK1 expression (15, 31, 48, 52), increased tracer phosphate uptake from 56.3 ± 6.8 to 76.2 ± 5.5 pmol Pi·mg protein–1·5 min–1 (n = 9, Fig. 8). The increased phosphate transport correlated with enhanced SGK1 expression and Nedd4–2 phosphorylation (Fig. 9).



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Fig. 7. Immunoreactivity of Nedd4–2, SGK1, and NaPi IIb in Intestine-407 cells. Cells were incubated with a primary antibody against Nedd4–2 (rabbit anti-mouse Nedd4–2) or NaPi IIb (rabbit anti-mouse NaPi IIb) and against SGK1 (sheep immunoaffinity purified IgG). Secondary goat anti-rabbit Texas Red-coupled antibodies were added to detect Nedd4–2 or NaPi IIb. To detect SGK1, secondary donkey anti-sheep Alexa Fluor 488-coupled antibody was used.

 


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Fig. 8. Stimulation of phosphate transport by transforming growth factor-{beta} (TGF-{beta}) in Intestine-407 cells. Cells were seeded in 6-well tissue culture plates at 5 x 105 cells per well. After 48 h, cells were serum-starved for 16 h and then treated with 10 ng/ml TGF-{beta} for 24 h. *Significant difference of tracer phosphate uptake compared with the respective value in the absence of TGF-{beta} (mean of 3 experiments performed per triplicate).

 


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Fig. 9. Stimulation of Nedd4–2 phosphorylation by TGF-{beta} in Intestine-407 cells. Cells were seeded in 6-well tissue culture plates at 5 x 105 cells per well. After 48 h, cells were serum starved for 16 h and then treated with 10 ng/ml TGF-{beta} for 24 h. Thereafter, cells were harvested, and 30 µg of total protein was used for determination of Nedd4–2 phosphorylation. A: immunoblotting with anti-P-Ser328 Nedd4–2 antibody (pN42) or anti-Nedd4–2 antibody (N42) as indicated. B: ratio of intensity was compared with the intensity obtained in cells treated with 10 ng/ml TGF-{beta}. *Significant difference in Nedd4–2 phosphorylation on treatment with TGF-{beta} (n = 3 independent experiments).

 

    DISCUSSION
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The Na+-coupled electrogenic transporter NaPi IIb determines intestinal phosphate absorption (25, 34, 54, 55). The activity of NaPi IIb is thus critically important for the maintenance of phosphate balance and thus bone mineralization. Accordingly, the function of NaPi IIb is under tight hormonal control. Intestinal phosphate transport is stimulated by a number of hormones including 1,25(OH)2D3 (18, 33, 40), glucocorticoids (13, 56), growth hormone (6, 9, 17), and IGF-1 (46). Very little is known about the signaling of NaPi IIb regulation. The present observations disclose a novel signaling mechanism that may well participate in the hormonal regulation of phosphate transport.

Nedd4–2 is an ubiquitin ligase, linking ubiquitin to the respective target proteins. The ubiquitinated proteins are then internalized and subject to proteolytic degradation (20 42). Thus the activity of Nedd4–2 determines the abundance of transport proteins in the cell membrane by triggering their clearance. Transport proteins hitherto disclosed to be regulated by Nedd4–2 include ENaC (14) and the cardiac Na+ channel SCN5A (1). As shown here, Nedd4–2 also downregulates NaPi IIb. In contrast, the catalytically inactive Nedd4–2 mutant (C938SNedd4–2) failed to appreciably affect NaPi IIb activity, indicating that specific enzyme activity of Nedd4–2 is required for the effect. Because NaPi IIb does not have a proline-tyrosine motif required for Nedd4–2 interaction, the ubiquitin ligase might interact with the transporter through an intermediate protein.

The effect of Nedd4–2 is reversed by SGK1 and SGK3 but not by SGK2. Accordingly, SGK1 and SGK3 but not SGK2 stimulate NaPi IIb activity. Similarly, SGK1 and SGK3 but not SGK2 phosphorylate Nedd4–2. Similar to what has been shown for ENaC regulation by SGK1 (14, 45, 47), SGK1 and SGK3 could regulate NaPi IIb through phosphorylation of Nedd4–2. This does not rule out further mechanisms in the regulation of NaPi IIb by the kinases SGK1 and SGK3.

A strong stimulator of SGK1 expression is TGF-{beta} (15, 31, 48, 52). TGF-{beta} and its receptors are expressed in intestinal cells (7, 36, 43, 53). As shown here, treatment of intestinal cells with TGF-{beta} enhances SGK1 expression and Nedd4–2 phosphorylation, which may explain the observed increase in NaPi IIb activity. TGF-{beta} also modulates Pi transport of the type IIa (NaPi IIa) and type III (Glvr-1) phosphate transporter. Whereas TGF-{beta} stimulates type III (Glvr-1) transporter (37), it inhibits NaPi IIa (32). Differential regulation of type IIa and type IIb NaPi cotransporters has been already reported (24), i.e., parathyroid hormone induces downregulation of NaPi IIa without directly affecting the activity of NaPi IIb.

In conclusion, SGK1 and SGK3 are both able to phosphorylate Nedd4–2. Among the targets of Nedd4–2 is the intestinal phosphate transporter NaPi IIb. SGK1 and SGK3 are both strong stimulators of NaPi IIb, an effect at least partially due to their inhibitory effects on Nedd4–2. The present experiments thus disclose a novel mechanism in the regulation of intestinal phosphate absorption.


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This study was supported by Deutsche Forschungsgemeinschaft Grants Nr La 315/4–4 and La 315/5–1 and the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Center for Interdisciplinary Clinical Research) Grant 01 KS 9602.


    ACKNOWLEDGMENTS
 
The authors are grateful to Sir Philip Cohen for kindly providing cDNA encoding SGK2, SGK3, and T308D,S473DPKB and to Dr. O. Staub for providing X. laevis Nedd4-2 cDNA and Nedd4-2 antibody. We acknowledge the technical assistance of B. Noll and the meticulous preparation of the manuscript by T. Loch.


    FOOTNOTES
 

Address for reprint requests and other correspondence: F. Lang, Physiologisches Institut, Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany (E-mail: florian.lang{at}uni-tuebingen.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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