1Department of Internal Medicine and Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8029; and 2Probiodrug AG, Halle (Saale) D-06120, Germany
Submitted 12 April 2004 ; accepted in final form 21 June 2004
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ABSTRACT |
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sodium/hydrogen exchange; diprotin A; P32/98; tyrosine kinase
To date, five Na+/H+ exchanger isoforms (NHE1, NHE2, NHE3, NHE4, and NHE8) have been identified to be expressed on the plasma membrane of renal tubular cells (5, 6, 9, 10, 19). Of these, NHE3 is expressed on the apical membrane of cells in the proximal tubule and the loop of Henle (3, 5, 7). Studies using inhibitors and knockout mice have demonstrated that NHE3 is responsible for the majority of apical membrane Na+/H+ exchange activity and transepithelial NaHCO3 and volume reabsorption in the proximal tubule (12, 30, 48, 51, 52, 55). The exquisite regulation of NHE3 activity is therefore essential for the maintenance of sodium, acid-base, and fluid homeostasis.
Coprecipitation experiments have indicated that NHE3 exists in physical complexes with dipeptidyl peptidase IV (DPPIV) in brush-border membranes isolated from proximal tubule cells (18). In contrast to the NHE3-megalin complex, which principally resides in the coated pit microdomain of the brush-border membrane, a microdomain in which NHE3 is inactive (4), the NHE3-DPPIV complex exists predominantly in the microvillar subdomain, in which NHE3 is active (18).
DPPIV (EC 3.4.14.5 [EC] ) is a highly specific serine protease that cleaves NH2-terminal dipeptides from peptides with a penultimate proline or alanine residue (16, 25). The kidney is a principal site of expression of DPPIV, where it is one of the major brush-border membrane proteins (25). It is also expressed on the cell surface of other types of epithelial cells, as well as endothelial cells and T lymphocytes, where it is known as CD26 (34, 42). Substrates of DPPIV include several regulatory peptides, neuropeptides, circulating hormones, and chemokines, so its activity is important for many different physiological processes in diverse tissues (33). In addition to its exopeptidase activity, DPPIV is known to interact with several proteins in the cell membrane. It has binding affinity for the extracellular matrix via fibronectin (11) and collagen (31). In T lymphocytes, DPPIV interacts with adenosine deaminase (35) and CD45 (44), a tyrosine phosphatase. The association with the latter permits its role in T cell signaling transduction. The physiological role of DPPIV in kidney other than as a peptidase for degrading filtered peptides remains unclear.
Interestingly, recent studies revealed that proteases are capable of regulating the activity of another major apical membrane sodium transport pathway expressed in the kidney, namely, the epithelial sodium channel (ENaC) (1, 47, 49). By analogy, these findings raise the possibility that DPPIV may play a similar role in regulating NHE3 activity. As an initial approach to test this hypothesis, we have evaluated whether highly specific inhibitors of DPPIV affect NHE3 activity in OKP cells, a line of opossum kidney cells that has transport properties very similar to those of the native proximal tubule (13). We report that DPPIV inhibitors decrease NHE3 activity in OKP cells, indicating that DPPIV plays an unexpected role in modulating Na+/H+ exchange mediated by NHE3 in proximal tubule cells.
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METHODS |
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Cell culture. OKP cells were maintained in 75-cm2 tissue culture flasks in DMEM containing 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cultures were incubated at 37°C in a humidified 5% CO2-95% air atmosphere. Cells were subcultured using Ca2+/Mg2+-free phosphate-buffered saline and 0.25% trypsin-EDTA. The medium was replaced every 2 days. For experiments, cells were seeded onto tissue culture plates and were used 2 days after reaching 100% confluence.
Immunoprecipitation. OKP cells grown in six-well plates were solubilized at 4°C in Tris-buffered saline (TBS) buffer (pH 7.4) containing 1% Triton X-100 and the protease inhibitors pepstatin A (0.7 µg/ml), leupeptin (0.5 µg/ml), and PMSF (40 µg/ml). The samples were subjected to centrifugation (15,000 g for 10 min) using a table-top centrifuge (Hermle model Z230M; National Labnet, Woodbridge, NJ). Primary antibodies (50 µg) were added to the supernatants, and the samples were incubated at 4°C for 1 h. Immune complexes were collected using 5 mg/sample of protein G-Sepharose 4B (Amersham Pharmacia Biotech, Piscataway, NJ). The beads were washed five times in solubilization buffer and then prepared for SDS-PAGE and immunoblotting.
SDS-PAGE and immunoblotting. Protein samples were solubilized in SDS sample buffer (2% SDS, 10% glycerol, 100 mM dithiothreitol, 0.1% bromphenol blue, and 50 mM Tris, pH 6.8), and proteins were separated by SDS-PAGE using 7.5% polyacrylamide gels according to the method reported by Laemmli (27). Immunoblotting was performed as described previously (18).
DPPIV enzymatic assay. DPPIV activity was assayed in OKP cells grown in 24-well plates by measuring the release of p-nitroaniline resulting from the hydrolysis of glycylprolyl-p-nitroanilide tosylate (21). Cells were incubated with 2 mM glycylprolyl-p-nitroanilide tosylate in phosphate-buffered saline (PBS) buffer (pH 7.4) for 30 min at 37°C. Reaction was terminated by the addition of 1 M acetate buffer (pH 4.2). Determination of p-nitroaniline liberated enzymatically was based on measuring absorbance at 380 nm. One unit of enzymatic activity was defined as the amount of enzyme catalyzing the formation of 1 µM p-nitroaniline per minute under the conditions described.
Sodium uptake assays. 22Na uptake assays were performed in 24-well plates in which OKP cells were preincubated for 20 min at room temperature in NH4+ loading buffer containing 30 mM NH4Cl, 90 mM choline chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, and 20 mM HEPES-Tris, pH 7.4. The NH4+ loading buffer was then removed, and cells were incubated for 5 min at room temperature with an NH4+-free solution containing 1 µCi/ml 22Na and 1 mM NaCl, 120 mM choline chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, and 20 mM HEPES-Tris, pH 7.4. Uptake was terminated by washing cells three times with ice-cold radionuclide-free NH4+-free buffer (pH 7.4). The cell monolayers were solubilized in 0.2 ml of 0.2 M NaOH and neutralized by adding 0.2 ml of 0.2 M HCl. Aliquots from each well were aspirated into a scintillation vial, and 22Na content was analyzed by liquid scintillation spectroscopy. Nonspecific retention (time 0 value) of 22Na uptake was determined and subtracted from the values for the incubated samples. All test compounds used in our studies were present during both the 20-min preincubation period and the 5-min Na uptake period. When the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) was used, it was preincubated with cells for an additional 30-min period before NH4+ loading.
Phosphate uptake assays. Sodium-dependent uptake of phosphate was measured in OKP cells grown to confluence on 24-well plates as described by Reshkin et al. (40). Briefly, cells were preincubated with a sodium-free medium (137 mM tetramethylammonium chloride, 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM MgSO4, 0.1 mM KH2PO4, and 10 mM HEPES-Tris, pH 7.4) at room temperature for 20 min. Measurement of phosphate uptake was initiated by adding transport medium containing 137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM MgSO4, 0.1 mM KH2PO4, 10 mM HEPES-Tris, pH 7.4, and [32P]phosphoric acid (2 µCi/ml) at room temperature. Uptake was terminated after 10 min by aspirating the transport medium and washing the cells with ice-cold sodium-free medium. Radioisotopic activity was determined by liquid scintillation spectroscopy. Nonspecific retention of 32PO43 was determined and subtracted from the values for the incubated samples.
Cell surface biotinylation. OKP cells grown to confluence in six-well plates were serum starved for 2448 h and subsequently incubated for 30 min at 37°C with either DPPIV inhibitors or diluent. All of the following manipulations were performed at 4°C. Cells were washed twice with PBS containing 0.1 mM CaCl2 and 1.0 mM MgCl2 (PBS-Ca-Mg). The surface membrane proteins were then biotinylated by incubating the cells twice for 25 min with 2 ml of biotinylation buffer (150 mM NaCl, 10 mM triethanolamine, 2 mM CaCl2, and 2 mg/ml EZ-Link sulfo-NHS-SS-biotin). The cells were washed twice for 20 min with a quenching buffer (PBS-Ca-Mg/100 mM glycine) and then solubilized for 1 h by adding a buffer containing 125 mM potassium acetate, 25 mM HEPES, 15 mM sodium pyrophosphate, 1% Triton X-100, 0.7 µg/ml pepstatin A, 0.5 µg/ml leupeptin, and 40 µg/ml PMSF, pH 7.4. The samples were centrifuged at 15,000 g for 10 min, and 50 µl of streptavidin-coupled agarose were added to the supernatants. After 1 h of incubation, the beads were washed five times in solubilization buffer and then prepared for SDS-PAGE and immunoblotting.
Analysis of protein tyrosine phosphorylation. Tyrosine kinase activity was measured in the OKP-DPPIV immune complex by Western blotting using an anti-phosphotyrosine antibody (17). OKP cells grown to confluence in six-well plates were incubated at 37°C for 20 min with NH4+ loading buffer. Cells were solubilized in 1 ml of ice-cold buffer containing 20 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, 100 mM sodium vanadate, 0.7 µg/ml pepstatin A, 0.5 µg/ml leupeptin, and 40 µg/ml PMSF. Immunoprecipitation with anti-DPPIV was then performed as described above. The immune complexes were solubilized by addition of 100 µl of hot Laemmli sample buffer and boiled for 5 min. Samples were resolved by SDS-PAGE using 10% polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and then blocked with TBS buffer containing 5% albumin. Immunoblotting was performed by an overnight incubation with anti-phosphotyrosine monoclonal antibody (4G10) diluted 1:2,000.
Statistical analysis. All results are reported as means ± SE. Comparisons between two groups were performed with unpaired t-tests. Differences among multiple groups were evaluated by analysis of variance with post hoc Tukey's test. A P value <0.05 was considered significant.
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RESULTS |
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For our experiments we used OKP cells, a line of opossum proximal tubule cells that has transport properties very similar to those of the native mammalian proximal tubule (13). We first verified whether NHE3 exists in protein complexes with DPPIV in OKP cells. Figure 1 shows the results of an experiment in which solubilized OKP cell proteins were immunoprecipitated with a commercial antibody to DPPIV (42). The immune complexes were prepared for immunoblotting and probed with antibodies against DPPIV, NHE3, and megalin. All three proteins were detected in the OKP cell lysate. DPPIV and NHE3 were coprecipitated by the anti-DPPIV antibody. In contrast, anti-DPPIV did not coprecipitate megalin, confirming the specificity of the NHE3-DPPIV interaction. This observation is in agreement with our previous studies performed in rabbit proximal tubule that revealed that the pools of NHE3 complexed with DPPIV and megalin are largely distinct (18).
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Role of protein kinases in mediating the effect of DPPIV inhibitors on NHE3 activity. The signal transduction cascade mediating the acute effect of NHE3 agonists and antagonists involves multiple pathways. Given that cAMP is one of the major intracellular messengers mediating inhibition of NHE3 in OKP cells (8, 54), we examined whether NHE3 modulation by DPPIV inhibitors would occur through activation of this second messenger system. The involvement of a cAMP/PKA-mediated pathway in the regulation of NHE3 by DPPIV inhibitors was examined by pretreating OKP cells for 30 min with the specific PKA inhibitor H-89. OKP cells were then incubated with 100 µM forskolin/1.0 mM IBMX, 10 µM P32/98, or vehicle. NHE3 transport activity was then measured. As expected, exposure to 10 µM H-89 completely blocked the inhibitory effect of forskolin/IBMX on Na+/H+ exchange (Fig. 7). However, 10 µM H-89 did not attenuate the effects of DPPIV inhibitors on NHE3 activity in OKP cells, indicating that PKA does not mediate the effect of DPPIV inhibitors.
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DISCUSSION |
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We now report that these two DPPIV inhibitors significantly decrease NHE3 activity in OKP cells, suggesting that the catalytic site of this peptidase has a role in modulating NHE3. Previously documented mechanisms that mediate the acute regulation of NHE3, including alteration of surface expression and phosphorylation by PKA, do not appear to be involved in the downregulation of NHE3 by DPPIV inhibitors. In addition, we could not detect any change in the apparent molecular weight of NHE3 resulting from possible DPPIV-mediated proteolysis of the transporter. However, we have found that the decrement in NHE3 activity induced by the DPPIV inhibitor P32/98 is not additive with that caused by the tyrosine kinase inhibitor genistein, suggesting that DPPIV inhibitors may affect NHE3 by a tyrosine kinase signaling pathway.
It is therefore of interest that DPPIV (CD26) regulation of cell proliferation and cytokine production in lymphocytes has been associated with changes in tyrosine kinase signaling (20, 2224, 36). Indeed, it has been specifically demonstrated that DPPIV inhibitors affect tyrosine phosphorylation of multiple proteins in lymphocytes (23, 24). Similarly, we found that the DPPIV inhibitor P32/98 decreased tyrosine phosphorylation of a high apparent molecular mass protein (>212 kDa) that coprecipitates with DPPIV, indicating a role for DPPIV in regulating tyrosine kinase signaling in OKP cells.
The molecular mechanisms by which DPPIV inhibitors affect protein tyrosine phosphorylation in lymphocytes or other cell types are not known. The cDNA sequence of DPPIV predicts a type II membrane protein, which is anchored to the cell surface by a single hydrophobic segment and has a short cytoplasmic region consisting of only six amino acids (42). Therefore, DPPIV is very unlikely to signal by itself. One possibility is that binding of DPPIV inhibitors to the catalytic site results in a conformational change that, in turn, affects tyrosine kinase signaling mediated by associated proteins. For instance, in T lymphocytes, DPPIV interacts with CD45 (22, 44), a tyrosine phosphatase that plays an important role in T cell activation. Another possibility is that DPPIV inhibitors affect cell signaling by blocking DPPIV catalytic activity. Known substrates for DPPIV include cytokines, chemokines, growth factors, and hormones (28). Processing by DPPIV activity can lead to either activation of proforms or peptide degradation. Thus DPPIV catalytic site inhibitors could alter cell signaling and protein phosphorylation by blocking DPPIV-mediated activation or inactivation of peptide ligands that bind to cell surface receptors.
In summary, we have found that DPPIV inhibitors significantly decrease NHE3 activity in OKP cells, most likely by inhibiting a tyrosine kinase signaling pathway. Thus our studies reveal an unexpected role for DPPIV in modulating NHE3 activity in proximal tubule cells.
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GRANTS |
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FOOTNOTES |
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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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