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K+ ATPase activity and Na+ coupled glucose transport by β-catenin
Biochemical and Biophysical Research Communications 402 (2010) 467–470
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Stimulation of Na+/K+ ATPase activity and Na+ coupled glucose transport by b-catenin Mentor Sopjani a,c, Ioana Alesutan a, Jan Wilmes a, Miribane Dërmaku-Sopjani a,b, Rebecca S. Lam a,d, Evgenia Koutsouki a, Muharrem Jakupi b, Michael Föller a, Florian Lang a,⇑ a
Department of Physiology, University of Tübingen, Germany Faculty of Medicine, University of Prishtina, Kosova Department of Chemistry, University of Prishtina, Kosova d Department of Molecular Neurogenetics, Max Planck Institute of Biophysics, Frankfurt/Main, Germany b c
a r t i c l e
i n f o
Article history: Received 4 October 2010 Available online 14 October 2010 Keywords: Glucose transport Na+-pump Ouabain SGLT1 Actinomycin D Cell membrane
a b s t r a c t b-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. b-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that b-catenin influences membrane transport. To this end, b-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of b-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na+/K+-ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of b-catenin on the endogenous Na+/K+-ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of b-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of b-catenin expression. The stimulating effect of b-catenin on both Na+/K+ ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of b-catenin, i.e. the regulation of transport. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction The multifunctional protein b-catenin may enter the nucleus and stimulate the expression of several genes important for cell proliferation [1,2]. b-Catenin is phosphorylated by glycogen synthase GSK3, which triggers the proteasomal degradation of b-catenin [3–5]. GSK3 and b-catenin are bound to the adenomatous polyposis coli (APC) protein, which fosters its phosphorylation by GSK3 [6–9]. At least partially due to impaired degradation of bcatenin, mice carrying a truncating mutation in the apc gene (apcMin/+ ) develop multiple intestinal tumors [10]. b-Catenin-sensitive genes include the serum- and glucocorticoid-inducible kinase SGK1 [11,12]. SGK1 may phosphorylate and thus inactivate GSK3 [13], which is expected to counteract b-catenin degradation. Moreover, SGK1 is a stimulator of a wide variety of epithelial transport proteins [14] including the Na+-coupled glucose transporter SGLT1 [15] and the Na+/K+ ATPase [16–18]. The present study explored the role of b-catenin in the regulation of transport. To this end, voltage clamp experiments were ⇑ Corresponding author. Address: Department of Physiology, University of Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany. Fax: +49 7071 295618. E-mail address: fl[email protected] (F. Lang). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.10.049
performed to determine the influence of b-catenin on the activity of the Na+/K+ ATPase and the Na+-coupled transporter SGLT1. 2. Materials and methods 2.1. Voltage clamp experiments For generation of cRNA, constructs were used encoding wild type b-catenin (Deutsches Ressourcenzentrum für Genomforschung) and wild type SGLT1 [15]. The cRNA was generated as described previously [19]. For voltage clamp analysis, Xenopus oocytes were prepared as previously described [19,20]. Oocytes were injected with 7.5 ng cRNA encoding b-catenin and with 5 ng of SGLT1 cRNA on the first day after preparation of the Xenopus oocytes. All experiments were performed at room temperature (about 22 °C) 3 days after the injection. Two-electrode voltageclamp recordings were performed at a holding potential of 70 mV for analysis of SGLT1 or of 30 mV for analysis of endogeneous Na+/K+-ATPase. The data were filtered at 10 Hz and recorded with a GeneClamp 500 amplifier, a DigiData 1300 A/D-D/A converter and the pClamp 9.0 software packages for data acquisition and analysis (Axon Instruments, USA). The control bath solution (superfusate/ND96) contained 96 mM NaCl, 2 mM KCl, 1.8 mM
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M. Sopjani et al. / Biochemical and Biophysical Research Communications 402 (2010) 467–470
CaCl2, 1 mM MgCl2 and 5 mM HEPES, pH 7.4. The final solutions were titrated to pH 7.4 using NaOH. The flow rate of the superfusion was 20 ml/min, and a complete exchange of the bath solution was reached within about 10 s. For determination of SGLT1 activity, 10 mM glucose were added to the bath solution. To determine electrogenic transport by the Na+/K+-ATPase, the oocytes were incubated for 4 h in a potassium-free solution containing 96 mM NaCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES and 25 mM sucrose titrated to the pH indicated using NaOH. Subsequently, the oocytes were exposed to the same solution containing, in addition, 5 mM BaCl2 (replacing 15 mM sucrose) for inhibition of K+ channels. Then, 5 mM KCl (replacing 10 mM sucrose) was added in the continuous presence of BaCl2. Where indicated, ouabain (1 mM Calbiochem, Bad Soden, Germany), actinomycin D (10 lM; Calbiochem) or brefeldin A (5 lM; Sigma, Schnelldorf, Germany) were added. 2.2. Statistical analysis Data are provided as means ± SEM, n represents the number of independent experiments. All data were tested for significance using ANOVA or paired Student t-test, and results with P < 0.05 were considered statistically significant. 3. Results 3.1. Stimulation of Na+/K+-ATPase activity by b-catenin K+-induced pump currents were taken as a measure of Na+/K+ATPase activity. To this end, the Xenopus oocytes were first preincubated for 4 h in K+-free solution, thereafter superfused for a few minutes with K+-free bath solution and subsequently the K+channel blocker Ba2+ (5 mM) added to abrogate K+ fluxes through K+ channels. The readdition of K+ was followed by an outwardly directed pump current due to electrogenic extrusion of 3 Na+ in exchange for 2 K+ (Fig. 1A). The pump current was abrogated in the presence of the Na+/K+-ATPase inhibitor ouabain (1 mM). Conversely, application of ouabain (1 mM) was followed by an inward current due to inhibition of the pump current. Both the pump current and the ouabain-induced current were significantly higher in Xenopus oocytes injected with cRNA encoding b-catenin than in oocytes injected with DEPC water (Fig. 1A). Thus, b-catenin stimulated the endogenous Na+/K+-ATPase. To discriminate between an effect of b-catenin as a transcription factor and a more direct effect of b-catenin, b-catenin cRNA was injected in the presence and absence of the transcription inhibitor actinomycin D (10 lM). As illustrated in Fig. 1B, actinomycin D did not disrupt the stimulation of the Na+/K+ ATPase by expression of b-catenin. In theory, b-catenin could stimulate Na+/K+ ATPase-mediated currents by inhibiting the removal of the pump from the cell membrane. To explore this possibility, the ouabain-sensitive current was measured in the absence and presence of 5 lM brefeldin A for 24 h. Brefeldin A inhibits the formation of vesicles in the Golgi apparatus and thereby prevents the insertion of proteins into the cell membrane. As illustrated in Fig. 2, b-catenin did not prevent the decay of the ouabain-sensitive current in the presence of brefeldin A, which approached 65.2 ± 13.2% (n = 3) in oocytes expressing b-catenin and 65.0 ± 10.3% (n = 3) in oocytes injected with water. Thus, b-catenin did not significantly modify the retrieval of the Na+/K+ ATPase from the cell membrane. 3.2. Stimulation of SGLT1 activity by b-catenin A next series of experiments explored whether b-catenin enhances the activity of the Na+-coupled glucose transporter SGLT1. To this end, the glucose-induced currents were determined in
Fig. 1. Enhancement of Na+/K+-ATPase activity in Xenopus oocytes by b-catenin. (A) Original tracings (upper panels) recorded in oocytes injected with water (left tracing, b-catenin) and with b-catenin cRNA (right, + b-catenin). The arrows indicate the addition of the respective solution. Arithmetic means ± SEM (lower panels, n = 13–20) of the K+-induced current (left bars) and ouabain-induced current (right bars) measured in Xenopus oocytes injected with water (white bars) or with cRNA encoding b-catenin (black bars). *,**Indicate significant difference from water-injected Xenopus oocytes (ANOVA; P < 0.05, P < 0.01). (B) Arithmetic means ± SEM (lower panels, n = 9–10) of the ouabain-induced current measured in Xenopus oocytes injected with water (white bars) or with cRNA encoding b-catenin (black bars) in the absence ( actinomycin D) and presence (+ actinomycin D) of 10 lM actinomycin D for 3 days. **,***Indicate significant difference from waterinjected Xenopus oocytes (ANOVA; P < 0.01, P < 0.001).
Xenopus oocytes expressing SGLT1 without or with additional coexpression of b-catenin. As shown in Fig. 3, the glucose-induced current was significantly higher in SGLT1-expressing Xenopus oocytes injected with cRNA encoding b-catenin than in oocytes injected with water (Fig. 3). The expression of b-catenin without SGLT1 was not followed by a significant increase in the glucoseinduced current (Fig. 3). Again, b-catenin was expressed in the
M. Sopjani et al. / Biochemical and Biophysical Research Communications 402 (2010) 467–470
Fig. 2. Expression of b-catenin does not prevent the decline of Na+/K+-ATPase activity in Xenopus oocytes following exposure to brefeldin A. Arithmetic means ± SEM (n = 9–11) of the ouabain-induced current measured in Xenopus oocytes injected with water (white bars) or with cRNA encoding b-catenin (black bars) in the absence ( brefeldin A) and presence (+ brefeldin A) of 5 lM brefeldin A for 24 h. ***Indicates significant difference from water-injected Xenopus oocytes (ANOVA; P < 0.001). ###Indicates significant difference from absence of brefeldin A (t-test; P < 0.001).
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Fig. 4. Stimulation of SGLT1 activity by b-catenin in the presence of Na+/K+ ATPase inhibitor ouabain. Arithmetic means ± SEM (n = 13–43) of normalized glucoseinduced current measured in Xenopus oocytes injected with water (control), with cRNA encoding SGLT1 or with both, cRNA encoding b-catenin and SGLT1. The measurement was performed in oocytes superfused without (white bars) or with 1 mM ouabain (black bars) for 5–10 min prior to and during the measurement. , * ***Indicate significant difference from values obtained in oocytes expressing SGLT1 alone (ANOVA; P <0.05, P < 0.001).
presence and absence of actinomycin D (10 lM) to estimate the contribution of altered transcription to the effect of b-catenin on SGLT1 activity. As shown in Fig. 3, actinomycin D did not prevent the increase in glucose-induced current following expression of b-catenin. To test whether the b-catenin effect on SGLT1 currents was due to b-catenin-dependent enhancement of the Na+/K+ ATPase activity (Fig. 1), glucose-induced currents were determined in the presence and absence of ouabain (1 mM). As shown in Fig. 4, oubain blunted the b-catenin effect. The absolute b-catenin-stimulated SGLT1current was 83.6 ± 12.9 nA (n = 7 oocyte batches) in the absence and 78.1 ± 12.4 nA (n = 7 oocyte batches) in the presence of 1 mM ouabain (P < 0.05; paired t-test). Thus, the enhancement of SGLT1 currents by b-catenin is partially but not fully due to the b-catenin effect on the Na+/K+ ATPase.
ent observations b-catenin is a powerful stimulator of the Na+/K+ATPase and of the Na+-coupled glucose transport SGLT1. At least in theory b-catenin could stimulate transport by upregulation of the serum- and glucocorticoid-inducible kinase SGK1 [11,12], which regulates a wide variety of channels [21–23] including SGLT1 [15] and Na+/K+-ATPase [16–18]. SGK1 contributes to renal Na+ retention [24,25] and intestinal glucose transport [26]. However, as b-catenin is effective even in the presence of actinomycin D, its effect on epithelial transport does apparently include posttranscriptional mechanisms and does, therefore, not rely on transcriptional upregulation of SGK1. The effect of b-catenin on electrogenic glucose transport could at least in part have been due to stimulation of the Na+/K+-ATPase, which leads to lowering of the intracellular Na+ concentration and thus to an increase in the electrochemical driving force for Na+coupled transport. However, b-catenin is still effective in the presence of the Na+/K+-ATPase inhibitor ouabain, indicating that some other mechanisms must contribute to the stimulation of glucose transport by SGLT1. b-Catenin is regulated by the Wnt pathway [27], which has been implicated in the pathogenesis of polycystic kidney disease, a disorder involving deranged cell proliferation, epithelial polarity and transport [28]. The disease is paralleled by excessive formation of b-catenin [29,30], which presumably contributes to untoward stimulation of cell proliferation [31]. The present observations raise the possibility that b-catenin similarly contributes to the derangement of transepithelial transport. In conclusion, b-catenin stimulates the Na+/K+-ATPase and electrogenic glucose transport. The effect may in part be due to direct interaction of b-catenin with the pump protein, and is at least partially independent of genomic upregulation of SGK1.
4. Discussion
Acknowledgments
The present observations disclose a completely novel function of b-catenin, i.e. the regulation of transport. According to the pres-
This work was supported by grants from DFG and BMBF (F.L.) and from the IZKF of the Medical Faculty, University of Tübingen
Fig. 3. Enhancement of glucose-dependent SGLT1 currents in Xenopus oocytes by bcatenin. Arithmetic means ± SEM (n = 21–28) of glucose-induced current measured in Xenopus oocytes injected with water (control), with cRNA encoding b-catenin, with cRNA encoding SGLT1 or with both, cRNA encoding b-catenin and SGLT1. The respective mRNA was injected in the absence (white bars) or presence (black bars) of the transcription inhibitor actinomycin D for 3 days. *,***Indicate significant difference from values obtained in oocytes expressing SGLT1 alone (ANOVA; P < 0.05, P < 0.001).
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(M.F/J.W.). The authors gratefully acknowledge the expert technical assistance by Efi Faber and the meticulous preparation of the manuscript by Lejla Subasic. References [1] T.C. He, A.B. Sparks, C. Rago, H. Hermeking, L. Zawel, L.T. da Costa, P.J. Morin, B. Vogelstein, K.W. Kinzler, Identification of c-MYC as a target of the APC pathway, Science 281 (1998) 1509–1512. [2] O. Tetsu, F. McCormick, Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells, Nature 398 (1999) 422–426. [3] K.M. Cadigan, Y.I. Liu, Wnt signaling: complexity at the surface, J. Cell Sci. 119 (2006) 395–402. [4] M. van Noort, J. Meeldijk, Z.R. van der, O. Destree, H. Clevers, Wnt signaling controls the phosphorylation status of beta-catenin, J. Biol. Chem. 277 (2002) 17901–17905. [5] J. Zumbrunn, K. Kinoshita, A.A. Hyman, I.S. Nathke, Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3 beta phosphorylation, Curr. Biol. 11 (2001) 44–49. [6] R.H. Giles, J.H. van Es, H. Clevers, Caught up in a Wnt storm: Wnt signaling in cancer, Biochim. Biophys. Acta 1653 (2003) 1–24. [7] V. Korinek, N. Barker, P.J. Morin, D. van Wichen, R. de Weger, K.W. Kinzler, B. Vogelstein, H. Clevers, Constitutive transcriptional activation by a beta-cateninTcf complex in APC / colon carcinoma, Science 275 (1997) 1784–1787. [8] P.J. Morin, A.B. Sparks, V. Korinek, N. Barker, H. Clevers, B. Vogelstein, K.W. Kinzler, Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC, Science 275 (1997) 1787–1790. [9] P. Polakis, Wnt signaling and cancer, Genes Dev. 14 (2000) 1837–1851. [10] A.R. Moser, H.C. Pitot, W.F. Dove, A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse, Science 247 (1990) 322–324. [11] M. Dehner, M. Hadjihannas, J. Weiske, O. Huber, J. Behrens, Wnt signaling inhibits forkhead box O3a-induced transcription and apoptosis through upregulation of serum- and glucocorticoid-inducible kinase 1, J. Biol. Chem. 283 (2008) 19201–19210. [12] Y. Naishiro, T. Yamada, M. Idogawa, K. Honda, M. Takada, T. Kondo, K. Imai, S. Hirohashi, Morphological and transcriptional responses of untransformed intestinal epithelial cells to an oncogenic beta-catenin protein, Oncogene 24 (2005) 3141–3153. [13] H. Sakoda, Y. Gotoh, H. Katagiri, M. Kurokawa, H. Ono, Y. Onishi, M. Anai, T. Ogihara, M. Fujishiro, Y. Fukushima, M. Abe, N. Shojima, M. Kikuchi, Y. Oka, H. Hirai, T. Asano, Differing roles of Akt and serum- and glucocorticoid-regulated kinase in glucose metabolism, DNA synthesis, and oncogenic activity, J. Biol. Chem. 278 (2003) 25802–25807. [14] F. Lang, C. Bohmer, M. Palmada, G. Seebohm, N. Strutz-Seebohm, V. Vallon, (Patho)physiological significance of the serum- and glucocorticoid-inducible kinase isoforms, Physiol. Rev. 86 (2006) 1151–1178. [15] M. Dieter, M. Palmada, J. Rajamanickam, A. Aydin, A. Busjahn, C. Boehmer, F.C. Luft, F. Lang, Regulation of glucose transporter SGLT1 by ubiquitin ligase Nedd4-2 and kinases SGK1, SGK3, and PKB, Obes. Res. 12 (2004) 862–870.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Stimulation of Na+/K+ ATPase activity and Na+ coupled glucose transport by b-catenin Mentor Sopjani a,c, Ioana Alesutan a, Jan Wilmes a, Miribane Dërmaku-Sopjani a,b, Rebecca S. Lam a,d, Evgenia Koutsouki a, Muharrem Jakupi b, Michael Föller a, Florian Lang a,⇑ a
Department of Physiology, University of Tübingen, Germany Faculty of Medicine, University of Prishtina, Kosova Department of Chemistry, University of Prishtina, Kosova d Department of Molecular Neurogenetics, Max Planck Institute of Biophysics, Frankfurt/Main, Germany b c
a r t i c l e
i n f o
Article history: Received 4 October 2010 Available online 14 October 2010 Keywords: Glucose transport Na+-pump Ouabain SGLT1 Actinomycin D Cell membrane
a b s t r a c t b-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. b-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that b-catenin influences membrane transport. To this end, b-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of b-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na+/K+-ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of b-catenin on the endogenous Na+/K+-ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of b-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of b-catenin expression. The stimulating effect of b-catenin on both Na+/K+ ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of b-catenin, i.e. the regulation of transport. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction The multifunctional protein b-catenin may enter the nucleus and stimulate the expression of several genes important for cell proliferation [1,2]. b-Catenin is phosphorylated by glycogen synthase GSK3, which triggers the proteasomal degradation of b-catenin [3–5]. GSK3 and b-catenin are bound to the adenomatous polyposis coli (APC) protein, which fosters its phosphorylation by GSK3 [6–9]. At least partially due to impaired degradation of bcatenin, mice carrying a truncating mutation in the apc gene (apcMin/+ ) develop multiple intestinal tumors [10]. b-Catenin-sensitive genes include the serum- and glucocorticoid-inducible kinase SGK1 [11,12]. SGK1 may phosphorylate and thus inactivate GSK3 [13], which is expected to counteract b-catenin degradation. Moreover, SGK1 is a stimulator of a wide variety of epithelial transport proteins [14] including the Na+-coupled glucose transporter SGLT1 [15] and the Na+/K+ ATPase [16–18]. The present study explored the role of b-catenin in the regulation of transport. To this end, voltage clamp experiments were ⇑ Corresponding author. Address: Department of Physiology, University of Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany. Fax: +49 7071 295618. E-mail address: fl[email protected] (F. Lang). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.10.049
performed to determine the influence of b-catenin on the activity of the Na+/K+ ATPase and the Na+-coupled transporter SGLT1. 2. Materials and methods 2.1. Voltage clamp experiments For generation of cRNA, constructs were used encoding wild type b-catenin (Deutsches Ressourcenzentrum für Genomforschung) and wild type SGLT1 [15]. The cRNA was generated as described previously [19]. For voltage clamp analysis, Xenopus oocytes were prepared as previously described [19,20]. Oocytes were injected with 7.5 ng cRNA encoding b-catenin and with 5 ng of SGLT1 cRNA on the first day after preparation of the Xenopus oocytes. All experiments were performed at room temperature (about 22 °C) 3 days after the injection. Two-electrode voltageclamp recordings were performed at a holding potential of 70 mV for analysis of SGLT1 or of 30 mV for analysis of endogeneous Na+/K+-ATPase. The data were filtered at 10 Hz and recorded with a GeneClamp 500 amplifier, a DigiData 1300 A/D-D/A converter and the pClamp 9.0 software packages for data acquisition and analysis (Axon Instruments, USA). The control bath solution (superfusate/ND96) contained 96 mM NaCl, 2 mM KCl, 1.8 mM
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CaCl2, 1 mM MgCl2 and 5 mM HEPES, pH 7.4. The final solutions were titrated to pH 7.4 using NaOH. The flow rate of the superfusion was 20 ml/min, and a complete exchange of the bath solution was reached within about 10 s. For determination of SGLT1 activity, 10 mM glucose were added to the bath solution. To determine electrogenic transport by the Na+/K+-ATPase, the oocytes were incubated for 4 h in a potassium-free solution containing 96 mM NaCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES and 25 mM sucrose titrated to the pH indicated using NaOH. Subsequently, the oocytes were exposed to the same solution containing, in addition, 5 mM BaCl2 (replacing 15 mM sucrose) for inhibition of K+ channels. Then, 5 mM KCl (replacing 10 mM sucrose) was added in the continuous presence of BaCl2. Where indicated, ouabain (1 mM Calbiochem, Bad Soden, Germany), actinomycin D (10 lM; Calbiochem) or brefeldin A (5 lM; Sigma, Schnelldorf, Germany) were added. 2.2. Statistical analysis Data are provided as means ± SEM, n represents the number of independent experiments. All data were tested for significance using ANOVA or paired Student t-test, and results with P < 0.05 were considered statistically significant. 3. Results 3.1. Stimulation of Na+/K+-ATPase activity by b-catenin K+-induced pump currents were taken as a measure of Na+/K+ATPase activity. To this end, the Xenopus oocytes were first preincubated for 4 h in K+-free solution, thereafter superfused for a few minutes with K+-free bath solution and subsequently the K+channel blocker Ba2+ (5 mM) added to abrogate K+ fluxes through K+ channels. The readdition of K+ was followed by an outwardly directed pump current due to electrogenic extrusion of 3 Na+ in exchange for 2 K+ (Fig. 1A). The pump current was abrogated in the presence of the Na+/K+-ATPase inhibitor ouabain (1 mM). Conversely, application of ouabain (1 mM) was followed by an inward current due to inhibition of the pump current. Both the pump current and the ouabain-induced current were significantly higher in Xenopus oocytes injected with cRNA encoding b-catenin than in oocytes injected with DEPC water (Fig. 1A). Thus, b-catenin stimulated the endogenous Na+/K+-ATPase. To discriminate between an effect of b-catenin as a transcription factor and a more direct effect of b-catenin, b-catenin cRNA was injected in the presence and absence of the transcription inhibitor actinomycin D (10 lM). As illustrated in Fig. 1B, actinomycin D did not disrupt the stimulation of the Na+/K+ ATPase by expression of b-catenin. In theory, b-catenin could stimulate Na+/K+ ATPase-mediated currents by inhibiting the removal of the pump from the cell membrane. To explore this possibility, the ouabain-sensitive current was measured in the absence and presence of 5 lM brefeldin A for 24 h. Brefeldin A inhibits the formation of vesicles in the Golgi apparatus and thereby prevents the insertion of proteins into the cell membrane. As illustrated in Fig. 2, b-catenin did not prevent the decay of the ouabain-sensitive current in the presence of brefeldin A, which approached 65.2 ± 13.2% (n = 3) in oocytes expressing b-catenin and 65.0 ± 10.3% (n = 3) in oocytes injected with water. Thus, b-catenin did not significantly modify the retrieval of the Na+/K+ ATPase from the cell membrane. 3.2. Stimulation of SGLT1 activity by b-catenin A next series of experiments explored whether b-catenin enhances the activity of the Na+-coupled glucose transporter SGLT1. To this end, the glucose-induced currents were determined in
Fig. 1. Enhancement of Na+/K+-ATPase activity in Xenopus oocytes by b-catenin. (A) Original tracings (upper panels) recorded in oocytes injected with water (left tracing, b-catenin) and with b-catenin cRNA (right, + b-catenin). The arrows indicate the addition of the respective solution. Arithmetic means ± SEM (lower panels, n = 13–20) of the K+-induced current (left bars) and ouabain-induced current (right bars) measured in Xenopus oocytes injected with water (white bars) or with cRNA encoding b-catenin (black bars). *,**Indicate significant difference from water-injected Xenopus oocytes (ANOVA; P < 0.05, P < 0.01). (B) Arithmetic means ± SEM (lower panels, n = 9–10) of the ouabain-induced current measured in Xenopus oocytes injected with water (white bars) or with cRNA encoding b-catenin (black bars) in the absence ( actinomycin D) and presence (+ actinomycin D) of 10 lM actinomycin D for 3 days. **,***Indicate significant difference from waterinjected Xenopus oocytes (ANOVA; P < 0.01, P < 0.001).
Xenopus oocytes expressing SGLT1 without or with additional coexpression of b-catenin. As shown in Fig. 3, the glucose-induced current was significantly higher in SGLT1-expressing Xenopus oocytes injected with cRNA encoding b-catenin than in oocytes injected with water (Fig. 3). The expression of b-catenin without SGLT1 was not followed by a significant increase in the glucoseinduced current (Fig. 3). Again, b-catenin was expressed in the
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Fig. 2. Expression of b-catenin does not prevent the decline of Na+/K+-ATPase activity in Xenopus oocytes following exposure to brefeldin A. Arithmetic means ± SEM (n = 9–11) of the ouabain-induced current measured in Xenopus oocytes injected with water (white bars) or with cRNA encoding b-catenin (black bars) in the absence ( brefeldin A) and presence (+ brefeldin A) of 5 lM brefeldin A for 24 h. ***Indicates significant difference from water-injected Xenopus oocytes (ANOVA; P < 0.001). ###Indicates significant difference from absence of brefeldin A (t-test; P < 0.001).
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Fig. 4. Stimulation of SGLT1 activity by b-catenin in the presence of Na+/K+ ATPase inhibitor ouabain. Arithmetic means ± SEM (n = 13–43) of normalized glucoseinduced current measured in Xenopus oocytes injected with water (control), with cRNA encoding SGLT1 or with both, cRNA encoding b-catenin and SGLT1. The measurement was performed in oocytes superfused without (white bars) or with 1 mM ouabain (black bars) for 5–10 min prior to and during the measurement. , * ***Indicate significant difference from values obtained in oocytes expressing SGLT1 alone (ANOVA; P <0.05, P < 0.001).
presence and absence of actinomycin D (10 lM) to estimate the contribution of altered transcription to the effect of b-catenin on SGLT1 activity. As shown in Fig. 3, actinomycin D did not prevent the increase in glucose-induced current following expression of b-catenin. To test whether the b-catenin effect on SGLT1 currents was due to b-catenin-dependent enhancement of the Na+/K+ ATPase activity (Fig. 1), glucose-induced currents were determined in the presence and absence of ouabain (1 mM). As shown in Fig. 4, oubain blunted the b-catenin effect. The absolute b-catenin-stimulated SGLT1current was 83.6 ± 12.9 nA (n = 7 oocyte batches) in the absence and 78.1 ± 12.4 nA (n = 7 oocyte batches) in the presence of 1 mM ouabain (P < 0.05; paired t-test). Thus, the enhancement of SGLT1 currents by b-catenin is partially but not fully due to the b-catenin effect on the Na+/K+ ATPase.
ent observations b-catenin is a powerful stimulator of the Na+/K+ATPase and of the Na+-coupled glucose transport SGLT1. At least in theory b-catenin could stimulate transport by upregulation of the serum- and glucocorticoid-inducible kinase SGK1 [11,12], which regulates a wide variety of channels [21–23] including SGLT1 [15] and Na+/K+-ATPase [16–18]. SGK1 contributes to renal Na+ retention [24,25] and intestinal glucose transport [26]. However, as b-catenin is effective even in the presence of actinomycin D, its effect on epithelial transport does apparently include posttranscriptional mechanisms and does, therefore, not rely on transcriptional upregulation of SGK1. The effect of b-catenin on electrogenic glucose transport could at least in part have been due to stimulation of the Na+/K+-ATPase, which leads to lowering of the intracellular Na+ concentration and thus to an increase in the electrochemical driving force for Na+coupled transport. However, b-catenin is still effective in the presence of the Na+/K+-ATPase inhibitor ouabain, indicating that some other mechanisms must contribute to the stimulation of glucose transport by SGLT1. b-Catenin is regulated by the Wnt pathway [27], which has been implicated in the pathogenesis of polycystic kidney disease, a disorder involving deranged cell proliferation, epithelial polarity and transport [28]. The disease is paralleled by excessive formation of b-catenin [29,30], which presumably contributes to untoward stimulation of cell proliferation [31]. The present observations raise the possibility that b-catenin similarly contributes to the derangement of transepithelial transport. In conclusion, b-catenin stimulates the Na+/K+-ATPase and electrogenic glucose transport. The effect may in part be due to direct interaction of b-catenin with the pump protein, and is at least partially independent of genomic upregulation of SGK1.
4. Discussion
Acknowledgments
The present observations disclose a completely novel function of b-catenin, i.e. the regulation of transport. According to the pres-
This work was supported by grants from DFG and BMBF (F.L.) and from the IZKF of the Medical Faculty, University of Tübingen
Fig. 3. Enhancement of glucose-dependent SGLT1 currents in Xenopus oocytes by bcatenin. Arithmetic means ± SEM (n = 21–28) of glucose-induced current measured in Xenopus oocytes injected with water (control), with cRNA encoding b-catenin, with cRNA encoding SGLT1 or with both, cRNA encoding b-catenin and SGLT1. The respective mRNA was injected in the absence (white bars) or presence (black bars) of the transcription inhibitor actinomycin D for 3 days. *,***Indicate significant difference from values obtained in oocytes expressing SGLT1 alone (ANOVA; P < 0.05, P < 0.001).
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