Downregulation of Peptide Transporters PEPT1 and PEPT2 by Oxidative Stress Responsive Kinase OSR1

The oxidative-stress-responsive kinase 1 (OSR1) is engaged in the regulation of epithelial transport [1,2,3,4]. OSR1 and the related SPAK (STE20-related proline/alanine-rich kinase) upregulate the NaCl (NCC) and Na⁺,K⁺,2Cl^- (NKCC2) cotransporters and thus impact on blood pressure [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17]. By stimulating cellular ion uptake SPAK and OSR1 contribute to cell volume regulation [9,18]. OSR1 is under the control of with-no-K(Lys) (WNK) kinases [5,6,7], which are similarly involved in the regulation of renal tubular ion transport and blood pressure [7,19,20,21,22]. Mutations of genes encoding WNK kinases may lead to Gordon's syndrome, a monogenic disease characterized by hypertension and hyperkalaemia [23,24,25,26].

OSR1 and/or SPAK participate in the regulation of further epithelial transport processes, such as Na⁺ coupled phosphate transport [27,28], and Na⁺/H⁺ exchanger [29].

The present study explored whether OSR1 modifies the peptide transporters 1 (PEPT1) and/or 2 (PEPT2), carriers accomplishing electrogenic cellular uptake of di- and tripeptides [30,31,32] including peptide-like drugs [30,31]. Regulators of peptide transporters include glucocorticoids [33], leptin [34] and growth hormone [35]. Signaling participating in the regulation of peptide transporters include phosphoinositide (PI) 3 kinase [36], phosphoinositide dependent kinase PDK1 [36], serum & glucocorticoid inducible kinase SGK1 [37] and AMP activated kinase [38].

In order to possibly disclose an effect of OSR1 on the peptide transporters, cRNA encoding PEPT1 and PEPT2 were injected into Xenopus oocytes with or without cRNA encoding OSR1. In those oocytes peptide transport was estimated from peptide induced current.

Constructs encoding PEPT1 or PEPT2 or PEPH2HA [39], wild-type OSR1, WNK1 insensitive inactive^T185AOSR1, constitutively active ^T185EOSR1, and catalytically inactive ^D164AOSR1 [5], were used for generation of cRNA as described previously [27,28,40,41].

Xenopus oocytes were prepared as previously described [42,43]. Where not indicated otherwise, 10 ng cRNA encoding PEPT1 or 20 ng cRNA encoding PEPT2 was injected on the first day and 10 ng cRNA encoding wild type OSR1, constitutively active ^T185EOSR1, catalytically inactive ^D164AOSR1 or WNK1 insensitive inactive ^T185AOSR1 were injected on the same day after preparation of the oocytes [44]. The oocytes were maintained at 17°C in ND96 solution containing (in mM): 88.5 NaCl, 2 KCl, 1 MgC1₂, 1.8 CaC1₂, 2.5 NaOH , 5 HEPES (pH 7.4), 5 sodium pyruvate (C₃H₃NaO₃), Gentamycin (100 mg/l), Tetracycline (50 mg/l), Ciprofloxacin (1.6 mg/l), Theophylline (90 mg/l). The voltage clamp experiments were performed at room temperature 3 days after injection of cRNA encoding PEPT1 or 4 days after injection of cRNA encoding PEPT2. Concentration of cRNA and time for expression have been chosen according to previous experiences and preliminary experiments utilizing 10 or 20 ng cRNA and 3 or 4 days of expression. Comparisons were always made between oocytes injected with the same amount of cRNA and after identical expression times. Two-electrode voltage-clamp recordings were performed at a holding potential of -70 mV. The data were filtered at 10 Hz and recorded with a Digidata A/D-D/A converter (1322 Axon Instruments) and Clampex 9.2 software for data acquisition and analysis (Axon Instruments) [45]. The control superfusate (ND96) contained (in mM): 93.5 NaCl, 2 KCl, 1.8 CaCl₂, 1 MgCl₂, 2.5 NaOH and 5 HEPES, pH 7.4. [46]. Glycine-glycine was added to the solutions at a concentration of 2 mM, unless otherwise stated [41]. The flow rate of the superfusion was approx. 20 ml/min, and a complete exchange of the bath solution was reached within about 10 s [40,47].

For detection of PEPT2-HA cell surface expression, the oocytes were first incubated with primary mouse monoclonal anti-HA antibody conjugated to horseradish peroxidase (1:1000, Miltenyi Biotec Inc, CA, USA). Individual oocytes were placed in 96 well plates with 20 µl of SuperSignal ELISA Femto Maximum Sensitivity Substrate (Pierce) and chemiluminescence of single oocytes was quantified in a luminometer (Walter Wallac 2 plate reader, Perkin Elmer) by integrating the signal over a period of 1 sec [44]. Results display normalized relative light units. Integrity of the measured oocytes was assessed by visual control after the measurement to avoid unspecific light signals from the cytosol [46].

Data are provided as means ± SEM, n represents the number of oocytes investigated. All voltage clamp experiments were repeated with at least 2-3 batches of oocytes; in all repetitions qualitatively similar data were obtained. Data were tested for significance using ANOVA (Tukey test or Kruskal-Wallis test) or t-test, as appropriate. Results with p < 0.05 were considered statistically significant.

The present study explored whether OSR1 contributes to the regulation of the peptide transporters PEPT1 and PEPT2. To this end, cRNA encoding PEPT1 or PEPT2 was injected into Xenopus oocytes with or without additional injection of cRNA encoding OSR1. Peptide transport was estimated from the inward current observed following addition of the dipeptide glycine-glycine (2 mM) to the bath solution (I_gly-gly).

No I_gly-gly was detectable in water-injected Xenopus oocytes (Fig. 1Aa) indicating that Xenopus oocytes did not express sizable endogenous electrogenic glycine-glycine transport. In Xenopus oocytes injected with cRNA encoding PEPT1, however, addition of glycine-glycine to the bath was followed by the appearance of I_gly-gly. The additional injection of cRNA encoding wild type OSR1 resulted in a significant decrease of I_gly-gly in PEPT1 expressing Xenopus oocytes (Fig. 1).

Further experiments explored whether OSR1 was effective by modifying maximal transport rate and/or affinity of PEPT1. To this end, the oocytes were exposed to glycine-glycine concentrations ranging from 0.01 to 5 mM. As illustrated in Fig. 2 the increase of peptide concentration was followed by an increase of I_gly-gly in both, Xenopus oocytes expressing PEPT1 alone and Xenopus oocytes expressing PEPT1 and OSR1. The increase of I_gly-gly was, however, larger in Xenopus oocytes expressing PEPT1 alone than in Xenopus oocytes expressing PEPT1 with OSR1. Calculation of maximal currents yielded values, which were significantly (p<0.05) higher in Xenopus oocytes expressing PEPT1 alone (78 ± 11 nA, n = 10-12) than in Xenopus oocytes expressing both, PEPT1 and OSR1 (48 ± 5 nA, n = 10-12). The glycine-glycine concentrations required for halfmaximal current (K_M) were not significantly different between Xenopus oocytes expressing PEPT1 alone (795 ± 95 µM, n = 10-12) and in Xenopus oocytes expressing PEPT1 together with OSR1 (684 ± 63 µM, n = 10-12).

Further experiments explored whether the effect of OSR1 was dependent on kinase activity. As illustrated in Fig. 3, similar to wild type OSR1, the constitutively active ^T185EOSR1 decreased I_gly-gly. In contrast, neither the catalytically inactive ^D164AOSR1 nor the WNK1 insensitive inactive ^T185AOSR1 significantly affected I_gly-gly in PEPT1 expressing Xenopus oocytes.

A further series of experiments tested whether OSR1 similarly influences the peptide transporter isofom PEPT2. Similar to what has been observed in Xenopus oocytes expressing PEPT1, in Xenopus oocytes injected with cRNA encoding PEPT2 addition of glycine-glycine to the bath was followed by the appearance of I_gly-gly, which was significantly decreased following additional injection of cRNA encoding wild type OSR1 (Fig. 4). The down-regulation of I_gly-gly in PEPT1 or PEPT2-expressing Xenopus laevis oocytes following coexpression of OSR1 could have resulted from a decline of protein abundance in the cell membrane. Thus, chemiluminescence was employed to quantify the carrier protein abundance in the cell membrane. As illustrated in Fig. 5, coexpression of wild-type OSR1 resulted in a significant decrease of the PEPT2 protein abundance in the cell membrane of PEPT2 expressing Xenopus laevis oocytes.

The present study discloses a novel function of the oxidative-stress-responsive kinase 1 (OSR1), i.e. the down-regulation of the peptide transporter isoforms PEPT1 and PEPT2. Electrogenic dipeptide transport in PEPT1 expressing Xenopus oocytes was significantly decreased by coexpression of OSR1, of constitutively active ^T185EOSR1, but not of kinase dead ^D164AOSR1 or WNK1 insensitive inactive ^T185AOSR1. The inefficacy of the mutants indicates that the effect of OSR1 may require kinase activity. According to chemiluminescence the effect is at least in part due to a decrease of (PEPT2) carrier protein abundance in the cell membrane. Along those lines, OSR1 decreases the maximal transport rate without appreciably influencing the affinity of the (PEPT1) carrier.

The present observations reveal the ability of OSR1 to modify PEPT1 and PEPT2 peptide transporter activities. To which extent those effects are relevant for the regulation of peptide transport in vivo, remains, however, to be shown. It must be kept in mind that the expression level of the kinase or the transporters as well as signaling molecules regulating OSR1 and/or the peptide transporters may be different in the oocyte expression system and given cells in mammalian organisms.

The peptide transporters accomplish cellular uptake of peptides into renal [48] and intestinal [31,49] epithelial cells. Moreover, the peptide transporters are expressed in pancreatic cancer cells [50] and prostate cancer cells [51]. Substrates of peptide transporters include several drugs, such as beta-lactam antibiotics, angiotensin-converting enzyme inhibitors, antiviral drugs, and anti-cancer agents [31,49,52,53,54,55]. The peptide transporters thus participate in cellular drug uptake [30,38,56,57] and the carriers may be potential targets for tumor therapy [51].

Regulators of OSR1 include dietary salt [58], vasopressin [59,60] and insulin [61,][62]. Whether or not regulation of OSR1 by dietary salt, vasopressin or insulin contributes to the regulation of peptide transporters remains to be shown.

OSR1 has the potency to downregulate the peptide transporters PEPT1 and PEPT2. Further experiments are needed to define the in vivo significance of OSR1 sensitive peptide transport.

The authors of this manuscript state that they do not have any conflict of interests and nothing to disclose.

The authors acknowledge the meticulous preparation of the manuscript by Lejla Subasic and technical support by Elfriede Faber. This study was supported by the Deutsche Forschungsgemeinschaft, GRK 1302, SFB 773 B4/A1, La 315/13-3 and the Open Access Publishing Fund of Tuebingen University.