Constitutively active phosphatidylinositol 3-kinase and AKT are sufficient to stimulate the epithelial Na+/H+ exchanger 3.

Phosphatidylinositol 3-kinase (PI 3-kinase) is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors and has been shown to be involved in regulation of stimulated exocytosis and endocytosis. One of the downstream signaling molecules activated by PI 3-kinase is the protein kinase Akt. Previous studies have indicated that PI 3-kinase is necessary for basal Na(+)/H(+) exchanger 3 (NHE3) transport and for fibroblast growth factor-stimulated NHE3 activity in PS120 fibroblasts. However, it is not known whether activation of PI 3-kinase is sufficient to stimulate NHE3 activity or whether Akt is involved in this PI 3-kinase effect. We used an adenoviral infection system to test the possibility that activation of PI 3-kinase or Akt alone is sufficient to stimulate NHE3 activity. This hypothesis was investigated in PS120 fibroblasts stably expressing NHE3 after somatic gene transfer using a replication-deficient recombinant adenovirus containing constitutively active catalytic subunit of PI 3-kinase or constitutively active Akt. The adenovirus construct used was engineered with an upstream ecdysone promoter to allow time-regulated expression. Adenoviral infection was nearly 100% at 48 h after infection. Forty-eight hours after infection (24 h after activation of the ecdysone promoter), PI 3-kinase and Akt amount and activity were increased. Increases in both PI 3-kinase activity and Akt activity stimulated NHE3 transport. In addition, a membrane-permeant synthetic 10-mer peptide that binds polyphosphoinositides and increases PI 3-kinase activity similarly enhanced NHE3 transport activity and also increased the percentage of NHE3 on the plasma membrane. The magnitudes of stimulation of NHE3 by constitutively active PI 3-kinase, PI 3-kinase peptide, and constitutively active Akt were similar to each other. These results demonstrate that activation of PI 3-kinase or Akt is sufficient to stimulate NHE3 transport activity in PS120/NHE3 cells.


is a component of neutral NaCl absorption.
This process explains basal ileal NaCl absorption and the increase in ileal sodium absorption that occurs after meals, and it is also the sodium absorptive process in ileal sodium absorbing cells that is inhibited in most diarrheal diseases. NHE3 is the component of neutral NaCl absorption that has been shown to be acutely stimulated and inhibited under these conditions. NHE3 activity is regulated by multiple growth factors and protein kinases, which mimic the changes associated with the digestive process (1). The mechanisms of inhibition and stimulation are only partially defined. For instance, EGF and clonidine stimulate ileal sodium absorbing cells by increasing the percentage of total NHE3 in the brush border (2). In PS120 fibroblasts stably expressing NHE3, FGF exposure increases NHE3 transport activity and the percentage of total NHE3 in the plasma membrane (3,4). The percent increase in Na ϩ /H ϩ exchange is quantitatively similar to the increase in percentage of NHE3 on the plasma membrane. In contrast, protein kinase C inhibition of NHE3 in Caco-2 cells is associated with a decrease in percentage of NHE3 on the plasma membrane, but in this case, the change in transport exceeds the change in percentage of surface NHE3, indicating change in turnover number in addition to change in NHE3 trafficking (5).
Insights are just beginning to be achieved in identifying the signal transduction processes involved in short-term regulation of NHE3. For instance, phosphatidylinositol 3-kinase (PI 3-kinase) has been shown to be necessary for 1) the basal level of plasma membrane NHE3 amount and NHE3 transport activity (3,6,7) and 2) EGF/FGF stimulation of NHE3 activity in PS120 fibroblasts and Caco-2 cells (3). PI 3-kinase is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors (8 -10). Activation of PI 3-kinase results in increased intracellular levels of 3Ј-phosphorylated inositol phospholipids and induction of signaling responses, including the activation of the protein kinase Akt, which is also known as protein kinase B (11,12). Activation of PI 3-kinase plays a role in growth factor signaling cascades, leading to metabolic and mitogenic cellular responses. In addition, PI 3-kinase activity has been implicated in regulated exocytosis and endocytosis. For instance, its activation is sufficient to stimulate glucose transporter 4 (GLUT4) translocation to the plasma membrane in 3T3-L1 adipocytes in the absence of insulin (13).
The conclusions of the above previous studies of PI 3-kinase and NHE3 were largely based on pharmacological approaches; inhibition of PI 3-kinase by wortmannin and LY294002 were utilized, although they were supported by biochemical evidence of activation of PI 3-kinase by growth factors. No previous studies have attempted to determine whether Akt has any role in regulation of NHE3. This is largely due to lack of Akt inhibitors.
In the current study, we tested the hypothesis that activation of PI 3-kinase or Akt alone is sufficient for the stimulation of NHE3 activity. This hypothesis was tested in PS120/NHE3 cells after somatic gene transfer using recombinant, replication-deficient adenovirus containing constitutively active PI 3-kinase or Akt. These studies demonstrate that an inducible adenoviral expression system can induce significant increases in PI 3-kinase and Akt activity and amount in PS120 cells, and activated PI 3-kinase and Akt were sufficient to stimulate NHE3. In addition, activating PI 3-kinase with a peptide that stimulates PI 3-kinase activity similarly stimulated NHE3 (14,15). This study demonstrates that activation of PI 3-kinase and Akt is sufficient to stimulate NHE3 in a fibroblast cell model.
Plasmid Vector Construction-Mammalian expression vectors directing the expression of Myc-tagged p110* (PI 3-kinase) and HA-tagged myr-Akt constructs, as well as these inserted constructs, were described (21)(22)(23)(24). The adenovirus shuttle vector used which contains an ecdysone-inducible promoter (pAdEcd), and pAdVgRXR encoding the ecdysone and RXR receptors were as described ( Fig. 1) (25). The p110* encodes for a constitutively active form of PI 3-kinase in which the inter-SH2 domain of the p85 regulatory subunit was ligated to the NH 2 terminus of the p110 catalytic subunit of PI 3-kinase. The p110* protein was tagged at the COOH terminus with the Myc epitope (22). In order to subclone the epitope-tagged p110*-Myc from a mammalian expression vector to pAdEcd, BamHI-digested p110*-Myc DNA was ligated into the multiple cloning site of pAdEcd. The plasmid containing the BamHI fragment in pAdEcd, the pAdEcd-BamHI plasmid, was digested with XbaI and NheI, and then the XbaI fragment from the p110*-Myc construct was subcloned into pAdEcd-BamHI. The Akt construct contains the HA epitope-tagged to its C terminus and is catalytically active due to its myristylation-related membrane location. The myr-Akt-HA plasmid was digested with HindIII and EcoRI, and the DNA fragment was subcloned into the multiple cloning site of pAdEcd for construction of pAdEmyr-Akt-HA (Fig. 1). The expression cassette of eGFP from pEGFP was subcloned into the multiple cloning site of pAdEcd, making vector pAdEGI (Fig. 1), which expressed enhanced green fluorescence protein and was used to assess adenovirus infection efficiency.
Adenoviral Infections-PS120/E3V cells were co-infected with Ad-VgRXR plus (i) AdEp110*-Myc, (ii) AdEmyr-Akt-HA, (iii) AdEGI, or (iv) empty virus (⌿5) (Fig. 1) in Dulbecco's modified Eagle's medium containing 2% fetal bovine serum and the appropriate amount of virus for 24 h. Then, expression was induced by addition of the ecdysone promoter ligand ponasterone A (3 M) for 24 h before further assays were performed. The dose of ponasterone A was selected to activate the ecdysone receptor maximally on the basis of previously reported doseresponse curves (28).
Confocal Microscopy-Images were taken after cells were fixed with 3% paraformaldehyde at 4°C for 20 min. Nuclei were stained with Hoechst 33342, washed with phosphate-buffered saline, and examined using a confocal fluorescent microscope (Zeiss LSC410). The excitation/ emission wavelengths were set at 488/510 nm and 351/364 nm for eGFP and Hoechst 33342, respectively.
Phosphatidylinositol 3-Kinase Activity Assay-PI 3-kinase activity was measured as described previously using TLC with phosphatidylinositol 4-phosphate, which co-migrates with PI-3P (Sigma mixture p9638), as a standard (29). Uninfected cells or adenovirus-infected PS120/NHE3V cells were lysed, and lysates were immunoprecipitated with a polyclonal anti-p110 antibody (Upstate Biotechnology Inc.). Immune complexes were precipitated from the supernatant with protein A-Sepharose (Upstate Biotechnology Inc.) and washed as described (29,30). The washed immune complexes were incubated with L-␣-phosphatidylinositol (Sigma) as substrate and [␥-32 P]ATP (3000 Ci/mmol) for 10 min at room temperature. Reaction was stopped with 20 l of 6 N HCl (final volume, 90 l), extracted with 160 l of chloroform: methanol (1:1), and centrifuged. The lower organic phase was removed and applied to a silica gel thin-layer chromatography plate (Merck) coated with 1% potassium oxalate. The TLC plates were developed in methanol:chloroform:water:ammonium hydroxide (60:47:11.3:2), dried, visualized by autoradiography with products identified as co-migrating with standard, and quantitated by scanning densitometry/Imagequant software.
Measurement of Na ϩ /H ϩ Exchange-Cellular Na ϩ /H ϩ exchange activity in PS120/E3V cells was determined fluorometrically using the intracellular pH-sensitive dye BCECF with cells grown to 60 -70% confluency on glass coverslips, as described previously (19,31). The effects of expressing p110* and Akt-myr in the absence or presence of ponasterone A on NHE3 were studied. The cells were loaded with the acetoxymethyl ester of 2Ј7Ј-bis(carboxyethyl) 5-6-carboxyfluorescein (BCECF-AM), 5 M) in Na ϩ medium (130 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgSO 4 , 1 mM NaH 2 PO 4 , 25 mM glucose, 20 mM HEPES, pH 7.4) for 20 min at 22°C and then washed with TMA ϩ medium (130 mM tetramethylammonium chloride, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgSO 4 , 1 mM NaH 2 PO 4 , 25 mM glucose, 20 mM HEPES, pH 7.4) to remove the extracellular dye; the coverslip was then mounted at an angle of 60 o in a 100-l fluorometer cuvette designed for perfusion and thermostated at 37°C. The cells were pulsed with 40 mM NH 4 Cl in TMA ϩ medium for 3 min, followed by TMA ϩ medium, which resulted in the acidification of the cells. Na ϩ medium was then added, which induced alkalinization of cells. Na ϩ /H ϩ exchange (H ϩ efflux) was calculated as described previously (19,20,31), as the product of sodium-dependent change in pH i times the buffering capacity at each pH i , and it was analyzed using a nonlinear regression data analysis program (Origin) that allowed fitting of data to a general allosteric model described by the Hill equation, with estimates for V max and KЈ[H ϩ ] i and their respective errors (S.E.).
Biotinylation and Immunoblotting-Plasma membrane NHE3 was measured by surface biotinylation, as described (7). Confluent PS120/ E3V cells were serum-starved for 5 h, and then 10 M PI 3-kinase peptide was incubated for 15 min. All subsequent manipulations were performed at 4°C. Cells were rinsed twice with ice-cold phosphatebuffered saline (150 mM NaCl and 20 mM Na 2 HPO 4 , pH 7.4) and once in borate buffer (154 mM NaCl, 1.0 mM boric acid, 7.2 mM KCl, and 1.8 mM CaCl 2 , pH 9.0). Plasma membrane surface was then exposed to 0.5 mg/ml sulfo-NHS-SS-biotin in borate buffer for 40 min with horizontal shaking. After labeling, cells were washed with the quenching buffer (20 mM Tris and 120 mM NaCl, pH 7.4) to scavenge the unreacted biotin. Cells were washed three times with ice-cold phosphate-buffered saline and lysed in 1 ml of N ϩ buffer (60 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM KCl, 5 mM Na 3 EDTA, 3 mM EGTA, and 1% Triton X-100). Cells were sonicated for 20 s and agitated on a rotating rocker for 30 min at 4°C. Insoluble cell debris was removed by centrifugation for 30 min at 12,000 ϫ g. Supernatant representing the total fraction was incubated with avidin-agarose for 2 h. After avidin precipitation, the supernatant was retained as the intracellular fraction. The avidin-agarose beads were washed five times in N ϩ buffer to remove the all of the nonspecifically bound proteins. The avidin-agarose bead bound proteins, representing plasma membrane NHE3, were solubilized in equivalent volumes of loading buffer (5 mM Tris-HCl, pH 6.8, 1% SDS, 10% glycerol, 1% 2-mercaptoethanol), boiled for 5 min, size-fractionated by SDSpolyacrylamide gel electrophoresis on 9% gels, and then electrophoretically transferred to nitrocellulose. After blocking with 5% nonfat milk, the blots were probed with a monoclonal anti-VSV-G antibody (P5D4 hybridoma supernatant) as the primary antibody and horseradish peroxidase-conjugated anti-mouse as the secondary antibody. Bands were visualized by enhanced chemiluminescence.

Standardization of Adenoviral Infection in PS120/E3V
Cells-The adenoviral infection efficiency in PS120/E3V cells was initially standardized to achieve nearly 100% infection 48 h after viral exposure and after activation of the ecdysone promoter with ponasterone A. The percentage of infection was evaluated with different mois of adenovirus expressing eGFP as a reporter protein. To estimate the percentage of infected cells, PS120/E3V cells were co-infected with various mois of ecdysone-driven receptors encoding eGFP (AdEGI) plus fixed ecdysone/retinoid X receptor virus (AdVgRXR). After 24 h, ponasterone A (3 M) was added to the media; 24 h later (48 h after infection), cells were fixed, and nuclei were stained with Hoechst 33342. As shown in Fig. 2, nuclear staining and cytosolic eGFP fluorescence were visible in PS120/E3V cells. Comparison was made between the number of cells with cytosolic eGFP fluorescence and the total number of PS120/E3V cells stained with Hoechst 33342 in the same field. Low basal levels of expression of eGFP occurred in the absence of ponasterone A at the different adenovirus mois indicating low level of "leakiness." eGFP expression increased in a moi-dependent manner after addition of ponasterone A (3 M) (Fig. 2), indicating that the infection percentages increased in a viral dose-dependent manner in the presence of ponasterone A. When the cells were co-infected with both AdEGI (moi ϭ 4.0) plus AdVgRXR (moi ϭ 0.8) for 48 h and incubated for the last 24 h in the presence of ponasterone A, ϳ90% of cells were eGFP-positive. These conditions were used for further experiments. PI 3-kinase and Akt activity and amount and NHE3 activity were measured under these conditions in the absence or presence of ponasterone A.
Expression of p110*-Myc and myr-Akt-HA Proteins-PS120/ E3V cells were co-infected with recombinant adenovirus encoding p110*-Myc (AdEp110*-Myc) or myr-Akt-HA (AdEmyr-Akt-HA) plus receptor virus AdVgRXR and were studied in the absence and presence of ponasterone A. Western blot analysis of total lysates were used to identify the presence of fusion proteins of p110*-Myc and myr-Akt-HA, using monoclonal antibodies directed against the Myc or HA epitope tags. Fig. 3 shows the results of an anti-Myc blot for p110* protein and an anti-HA blot for Akt protein. The level of expression of p110*-Myc and myr-Akt-HA were induced ϳ 3.6-and 4.0-fold in the presence of ponasterone A and were minimally above baseline in the absence of ponasterone A, indicating minimal but present leakiness of the ecdysone promoter.
Activities of p110* and myr-Akt-To assess the activities of p110* and myr-Akt, cells were infected with either p110* or myr-Akt encoding recombinant adenoviral construct in the absence and presence of ponasterone A. In the p110* studies, cells were lysed, the lysates were immunoprecipitated with anti-p110, and the immunoprecipitate was resolubilized and used to detect the total endogenous plus exogenous PI 3-kinase activity. For Akt activity, lysates were used, and the parameter to determine Akt activity was an antibody that detects phospho-rylated Ser 473 , which is activated Akt (phospho-Akt) (16,17). Thus, endogenous plus exogenous Akt was detected. Activity levels of PI 3-kinase (Fig. 4, A and B) and phospho-Akt (Fig. 4, C and D) were induced in the presence of ponasterone A compared with the absence of ponasterone A. As shown in Fig. 4, A and B, PI 3-kinase activity increased ϳ2.3-fold with ponasterone A (increased to 223 Ϯ 16% of control). As shown in Fig. 4, C and D, Akt activity increased ϳ3-fold with ponasterone A (increased to 310 Ϯ 31% of control).
Effects of Expression of Constitutively Active PI 3-Kinase on Akt Activity-To investigate whether activated PI 3-kinase is sufficient to stimulate Akt activity, the AdEp110*-Myc recombinant adenoviral construct was infected in PS120/NHE3V cells. The amount of phosphorylated Akt was detected by ani- phospho-Ser 473 Akt. Fig. 5A shows that endogenous Akt activity was stimulated by expression of p110* kinase activity without a change in amount of Akt expression (Fig. 5B).
Stimulatory Effects of Constitutively Active PI 3-Kinase and Akt on NHE3 Activity and Amount-Recently, we and others have reported that activation of PI 3-kinase is a necessary step in FGF stimulation of NHE3 in PS120/E3V and AP-1 cells (3,4,6,7,32). To investigate whether activation of PI 3-kinase or Akt is sufficient for activation of NHE3 transporter activity, NHE3 activity was determined in AdEp110*-Myc-infected and AdE-myr-Akt-HA-infected cells in the absence and in the presence of ponasterone A. The expression level of total cell NHE3 was very similar among the infected lines and uninfected control (Fig. 6A), despite changing the amount and activity of PI 3-kinase and Akt. The rates of Na ϩ /H ϩ exchange in acid-loaded cells were measured by the rate of pH i recovery with Na ϩ addition. A comparison of the transport with ponasterone A induction of p110* and Akt is shown in Fig. 6, B and C, respectively. NHE3 activity was stimulated by 21.5 Ϯ 4.0% above control after induction of p110* and 22.0 Ϯ 3.1% after induction of myr-Akt protein (Fig. 6D). There was no significant effect of viral infection with PI 3-kinase or AKT in the absence of ponasterone A compared with viral infection with empty control virus (⌿5) (Fig. 6, B and C). To further demonstrate the role of constitutively active PI 3-kinase in increasing the NHE3 activity, AdEp110*-Myc-infected PS120/E3V cells were preincubated with the PI 3-kinase inhibitor wortmannin for 30 min prior to study of Na ϩ /H ϩ exchange rate. Wortmannin (100 nM) pretreatment inhibited the NHE3 activity in both the presence and absence of ponasterone A in PS120/E3V cells (Fig. 6E). These results demonstrate that the expression of either constitutively active PI 3-kinase or Akt is sufficient to stimulate NHE3 transporter activity without changing the total amount of NHE3. Moreover, PI 3-kinase is involved in NHE3 stimulation under basal conditions. Stimulation of NHE3 Transport by PI 3-Kinase Peptide-induced Activation of PI 3-Kinase-A second method of increas-ing PI 3-kinase activity was studied for effects on NHE3 activity without plasma membrane receptor activation. We used a membrane-permeant synthetic peptide (a rhodamine-linked synthetic 10-mer) that is modeled on the polyphosphoinositide binding sequence of gelsolin and has been shown to increase PI 3-kinase in multiple cell types (14,15,33). PI 3-kinase peptide (10 M) exposed to PS120/NHE3 cells for 15 min stimulated PI 3-kinase activity ϳ2.3-fold (increased to 325 Ϯ 33% of control) (Fig. 7A). Incubation of synthetic peptide (10 M) for15 min increased NHE3 transport in PS120 cells and OK cells (Fig. 7,  B-D). The latter was studied to expand the findings to a polarized epithelial cell with brush border NHE3 (34). The magnitude of stimulation of NHE3 activity in PS120 cells was similar in magnitude to the stimulation by constitutively active PI 3-kinase (118 Ϯ 8% of control, p Ͻ 0.05). PI 3-kinase peptide also stimulated NHE3 activity in OK cells (132 Ϯ 12% of control, p Ͻ 0.05). To determine whether this increase in ac-

FIG. 4. Effects of overexpression of p110* on PI 3-kinase activity and
Myr-Akt on Akt activity. Cells were infected with AdEp110*-expressing or Ad-Emyr-Akt-expressing adenovirus at an moi of 4 in media containing 2% serum for 24 h and then incubated in the absence or presence of ponasterone A (3 M) for 24 h. A, AdEp110*-myr-infected cells were lysed and subjected to immunoprecipitation with p110 antibodies. The washed immunoprecipitates were assayed for PI 3-kinase activity with phosphatidylinositol as substrate, and the labeled PI 3-phosphate product (PI-3P) was resolved by thin layer chromatography and visualized by autoradiography. B, results of PI 3-kinase activity from three experiments similar to those in A. Results are expressed as the percentages of uninduced Ϯ S.E. PI 3-kinase activity. C, AdEmyr-Akt-HA cells were lysed and assayed for Akt activity. Activity level was measured with phosphospecific Akt antibody. D, results of amount of phospho-Akt from three experiments similar to those in C. Results are expressed as the percentages of uninduced Ϯ S.E. Akt kinase activity.
tivity is due to an increase in plasma membrane NHE3 protein, cell surface biotinylation was used. Plasma membrane protein were biotinylated by reaction with sulfo-NHS-SS biotin at 4°C and isolated by precipitation with streptavidin-bound agarose. Plasma membrane NHE3 was then identified by immunoblot. The addition of PI 3-kinase peptide (10 M) to PS120/E3V cells for 15 min increased plasma membrane NHE3 from 10.9% of total (without peptide) to 20.8% (with peptide) (Fig. 8), whereas total NHE3 abundance did not change. These results support the conclusion that the activation of PI 3-kinase is sufficient to stimulate NHE3 activity. DISCUSSION In this study, the question was asked whether the activation of PI 3-kinase or Akt was sufficient to stimulate NHE3 in PS120 cells. Previous studies demonstrated a role for PI 3-kinase in rapid stimulation of NHE3 (3,6,7,32), but there have been no previous studies implicating Akt in regulation of NHE3. PI 3-kinase activity has previously been shown to be necessary for basal NHE3 activity in the polarized epithelial cell lines Caco-2 and OK, as well as in the fibroblast cell line PS120 and in AP-1 cells, and also in EGF/FGF stimulation of NHE3 in fibroblasts and EGF stimulation of NHE3 in ileal brush border (3,6,32). Based on inhibitor studies in PS120, AP-1, OK, and Caco-2 cells, basal NHE3 activity is lowered by the PI 3-kinase inhibitor wortmannin (3,6,7). EGF/FGF stimulation of NHE3 is blocked in PS120 cells by the same inhibitors (1, 3, 7). FGF stimulated NHE3 in PS120 cells by 50%. 50% of this stimulation appears to be due to PI 3-kinase because wortmannin caused a 50% reduction of this stimulation (3). Thus, FGF stimulates NHE3 by both PI 3-kinase-dependent and PI 3-kinase-independent mechanisms (1, 3, 7).
Whether increased PI 3-kinase or AKT activity were sufficient to stimulate NHE3 had not been addressed before this study. However, this issue has begun to be studied in regards to the glucose transporter GLUT4 in adipocytes and smooth muscle cells. Insulin and growth factor stimulate glucose uptake and GLUT4 translocation to the plasma membrane by a process associated with activation and movement to the plasma membrane of PI 3-kinase (35)(36)(37). Both glucose uptake and GLUT4 translocation are inhibited by the PI 3-kinase inhibitors wortmannin and LY290042 (23, 28). Thus, PI 3-kinase activation is necessary for the insulin stimulation of glucose FIG. 6. Expression of constitutively active PI 3-kinase or Akt is sufficient to stimulate NHE3 activity without changing NHE3 amount and Inhibition of NHE3 Activity by Wortmannin. Cells were infected with AdEp110*-Myc-expressing or AdEmyr-Akt-HA-expressing recombinant adenovirus at an moi of 4 in media containing 2% serum for 24 h and incubated in the absence or presence of ponasterone A for 24 h. Empty adenovirus (⌿ 5) without the p110*-Myc or Akt-HA coding region was studied as a control. Following infection, PS120/NHE3 cells were acidified by NH 4 Cl and allowed to recover in the presence of sodium medium to a steady-state pH i . A, the effect of infection with AdEp110*-Mycexpressing or AdEmyr-Akt-HA-expressing adenovirus in the absence and presence of ponasterone A on amount of the NHE3 transporter was measured. The amount of expression of NHE3 was not altered by expression of constitutively active PI 3-kinase or Akt. The data are representative of three independent experiments. B and C, measurement of NHE3 activity was performed after expression of adenovirus (⌿5), AdE p110*-Myc or AdE myr-Akt-HA adenovirus, and results shown refer to in the absence or presence of ponasterone A. A single representative study is shown. D, mean Na ϩ /H ϩ exchange rates rate Ϯ S.E. are shown normalized to the minus ponasterone condition. NHE3 transporter activity was stimulated 21.5 Ϯ 4.0% above control by the expression of p110*-Myc and 22 Ϯ 3.1% above control by the expression of myr-Akt-HA. E, AdEp110*-Myc adenovirus-infected cells in the absence or presence of ponasterone A were incubated with 100 nM of wortmannin for 30 min prior to determination of the Na ϩ /H ϩ exchange rate. A single experiment is shown that was repeated twice with similar results. uptake and GLUT4 translocation. Initial studies demonstrated that constitutively active PI 3-kinase expression (p110) increased both glucose uptake and GLUT4 translocation in the absence of insulin, and dominant negative PI 3-kinase (p85) inhibited insulin-stimulated glucose uptake and GLUT4 translocation (38 -40). These data were contradictory, however, concerning whether the magnitude of the insulin stimulation of glucose uptake was duplicated by overexpression of constitutively active PI 3-kinase. Recent interpretation is that increasing PI 3-kinase activity alone is quantitatively less than the insulin stimulation of glucose uptake (38,41). In addition, increasing PI 3-kinase by separate mechanisms, including activation with platelet-derived growth factor and integrin receptor stimulation, did not increase glucose uptake at all (42,43). Moreover, exposure to membrane-permeant PIP3, which is converted intracellularly to end products of activated PI 3-kinase, failed to alter glucose transport in the absence of insulin (44). These results are currently interpreted as indicating that whereas activated PI 3-kinase is sufficient to induce a partial stimulation of GLUT4-related glucose uptake and membrane translocation, a second (or more) insulin-stimulated non-PI 3-kinase-dependent pathway(s) is necessary to reproduce the full insulin stimulation of glucose uptake (42,43,45). A candidate second pathway has been described with the recognition that insulin brings the adapter protein CAP to the insulin receptor, where it recruits Cbl and interacts with flotillin resulting in phosphorylation of Cbl and direction of the Cbl-CAP complex to lipid rafts (or caveolae) in the plasma membrane (47).
Our results demonstrate that stimulation of PI 3-kinase activity either by use of a peptide stimulator of PI 3-kinase activity (14) or transient infection with adenovirus containing constitutively active PI 3-kinase increases NHE3 activity. This stimulation is similar in magnitude to the PI 3-kinase-dependent component of rapid growth factor stimulation of NHE3 (3). The quantitative similarity suggests that an increase in PI 3-kinase activity is not only sufficient to increase NHE3 activity but that this component of the growth factor stimulation is entirely due to the increase in PI 3-kinase activity. The growth factor-stimulated messenger that initiates the PI 3-kinase-dependent growth factor stimulation of NHE3 remains unidentified.
Thus, similarities and differences between insulin stimulation of GLUT4 and growth factor stimulation of NHE3 have been identified. Both involve stimulation by trafficking and increases in exocytosis, with PI 3-kinase being necessary and sufficient for the stimulation. In addition, there is a second component of stimulation in addition to a pathway mediated through PI 3-kinase. The recently recognized pathway of insulin stimulation of GLUT4 that may include CAP-flotillin-Cbl FIG. 7. Effect of PI 3-kinase synthetic peptide on NHE3 transport and PI 3-kinase activity in PS120/E3V and OKE3V cells. A, PS120/E3V cells were preincubated for 15 min with PI 3-kinase peptide (10 M), and then PI 3-kinase was immunoprecipitated from lysates. Immune complexes were studied for PI 3-kinase activity. Results were normalized to no peptide condition and expressed as mean Ϯ S.E. of three separate studies. B, PS120/E3V cells were exposed to PI 3-kinase (10 M) peptide or control for 15 min, and then NHE3 transport was measured using BCECF fluorescence. A representative experiment is shown. C, OK/E3V cells were exposed to PI 3-kinase peptide (10 M) or control for 15 min and then NHE3 transport was measured using BCECF fluorescence. A representative experiment is shown. D, results of Na ϩ /H ϩ exchange activity in absence and presence of PI 3-kinase peptide are presented as the means Ϯ S.E. of three similar studies in PS120/E3V cells and OK/E3V cells, with results normalized to the no peptide condition, set at 100%.
has not been studied in regulation of NHE3, and no additional specific factor has been identified for the growth factor stimulation of NHE3. Concerning differences between growth factor stimulation of NHE3 and insulin stimulation of GLUT4, NHE3 trafficking under basal conditions is more than for GLUT4, which is nearly entirely intracellular. This basal stimulation of NHE3 is also PI 3-kinase-dependent. This difference is not surprising in that NHE3 is rapidly regulated by both stimulation and inhibition in the intestine and kidney, whereas regulation of glucose uptake is stimulated by insulin, and no inhibitory mechanism from basal rate has yet been identified. Importantly all GLUT4 mobilization is PI 3-kinase-dependent, while only part of NHE3 depends on PI 3-kinase. Another difference of the stimulatory mechanisms of GLUT4 and NHE3 is the suggestion that insulin mobilizes glucose from both the recycling endosomes and a special storage compartment, whereas growth factor mobilization of NHE3 has only been suggested as coming from the recycling compartment (48), with no specific storage compartment for NHE3 yet identified.
The current studies also represent the initial demonstration of involvement of Akt in growth factor stimulation of NHE3. Akt represents a major downstream signaling molecule in the PI 3-kinase cascade. In unpublished studies, we have shown that EGF stimulation of ileal NaCl absorption and brush border NHE3 activity is associated with a rapid increase in brush border Akt activity. 2 Due to lack of available Akt inhibitors, the functional significance of this stimulation could not be determined. These studies show that increasing Akt activity and amount stimulates NHE3. In addition, the similarity in mag-nitude of stimulation of NHE3 with transient transfection with constitutively active PI 3-kinase and Akt mutants and the fact that increasing PI 3-kinase activity stimulates Akt activity without altering Akt amount suggests that most, if not all, of the PI 3-kinase stimulation of NHE3 may be due to activation of Akt in PS120 cells.
Results assessing the contribution of AKT to insulin/PI 3-kinase stimulation of glucose uptake in adipocytes and muscle cells indicates that AKT activation is necessary and appears sufficient to stimulate glucose uptake. Constitutively active AKT (using several different constitutively active constructs different than that used in our studies consisting of Myr⌬-4 -129 Akt (in which the Akt plextin homology domain is deleted) and a v-Akt analogue (called Gag-protein kinase B) stimulated glucose uptake and GLUT4 plasma membrane translocation. The magnitude of the effects was between 69 and 100% of the insulin stimulation (49 -51). However, disagreement exists in that studies performed with several dominant negative AKT constructs either inhibited most of the insulin-stimulated glucose uptake or had inhibitory effects of only 0 -20% (52)(53)(54).
Other systems, in which the downstream signaling of PI 3-kinase involves Akt, have had additional downstream signals identified. In addition to Akt, atypical forms of protein kinase C (especially and ), but also the conventional isoforms ␤-2 and the novel isoform ␦ have been shown to represent downstream signaling molecules following PI 3-kinase activation in some cells (46). The current studies have not considered involvement of protein kinase C in the PI 3-kinase stimulation of NHE3. However, the similarity in magnitude of FGF stimulation of NHE3 and effects of constitutively active PI 3-kinase and Akt, as well as similarity in the stimulation of NHE3 with the 2 Li, X.-H., Shih, C., and Donowitz, M., unpublished observations. FIG. 8. Effect of PI 3-kinase peptide on surface NHE3: cell surface biotinylation of NHE3V. Intact PS120/E3V cells were exposed to PI 3-kinase peptide (10 M) or control for 15 min at 37°C after serum starvation for 5 h. Cells were then chilled to 4°C and biotinylated as described under "Materials and Methods." A, Western blots of PS120/E3V cells, with three dilutions of total, two of intracellular, and four of surface fractions, were probed with anti-VSV-G monoclonal antibody (P5D4) and visualized with ECL. B, quantitation of NHE3V is shown, with the density of bands determined by a scanning densitometer and Imagequant software and plotted against the sample volume. Quantitation was as described (7). increase in Akt activation when constitutively active PI 3-kinase is transfected, make it likely that the contribution of atypical forms of protein kinase C to PI 3-kinase stimulation of NHE3 is small, if it is present at all, in these cells.
Attempts were made to examine the contribution of PI 3-kinase and Akt in regulation of NHE3 by expressing dominant negative forms using the same adenoviral expression system used for these activation studies. The constructs used were described previously and consisted of p110* mutated in the kinase domain and Akt-AA (T308A/S473A) (12,53). In both cases, there was significant loss of cell viability, which we presumed, but did not demonstrate, was due to induction of apoptosis. The adenovirus infection system was used for transient infection in order to develop a method of obtaining transient expression in a high percentage of cells to allow correlation of biochemistry and functional data, in this case, NHE3 transport. The motivation for developing an inducible system was that in studying molecules that alter cell division and state of differentiation, we wanted to be able to select conditions in which changes in the differentiation status of cells studied could be minimized by controlling the time of expression of selected signaling molecules. Thus, after use of eGFP as a marker to determine conditions to infect nearly all cells, we selected a time window of expression of the signaling molecule of interest. The level of expression of PI 3-kinase due to the inducible expression was not compared with the endogenous level; however, the increase in activity due to ponasterone A was similar to that which occurred with growth factor exposure (17,32). In addition, our studies documented the low level of leakiness of this expression system in the absence of activation of the ecdysone receptor with ponasterone A (Fig. 3). Thus, we suggest the usefulness of this inducible adenovirus system for expressing signaling molecules that are to be activated at a relatively fixed time and studied over a limited period. In addition, the relatively small percentage of increase in amount of expressed protein may prove to be an advantage compared with other methods that lead to problems from large amounts of protein expression.