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Down-Regulation of the Myoinositol Transporter SMIT by JAK2

Background/Aims: Janus-activated kinase-2 JAK2 is activated by energy depletion and hyperosmotic shock and modifies the activity of several Na+ coupled transporters. The Na+ coupled osmolyte transporter SMIT (myoinositol transporter) is upregulated by osmotic shock and downregulated by energy depletion. The present study thus explored whether JAK2 contributes to the regulation of SMIT activity. Methods: To this end, cRNA encoding SMIT was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, constitutively active V617FJAK2 or inactive K882EJAK2. Inositol-induced current (ISMIT) was determined by dual electrode voltage clamp and taken as measure for electrogenic inositol transport. Results: No appreciable ISMIT was observed in water injected oocytes. In SMIT expressing oocytes ISMIT was significantly decreased by additional coexpression of JAK2 or V617FJAK2, but not by coexpression of K882EJAK2. According to kinetic analysis coexpression of JAK2 decreased maximal ISMIT without significantly modifying the concentration required for halfmaximal ISMIT (KM). In oocytes expressing both, SMIT and JAK2, ISMIT was gradually increased by JAK2 inhibitor AG490 (40 µM). Disruption of carrier insertion with brefeldin A (5 µM) was followed by a decline of ISMIT to a similar extent in Xenopus oocytes expressing SMIT with JAK2 and in Xenopus oocytes expressing SMIT alone, suggesting that JAK2 did not affect carrier stability in the cell membrane. Conclusion: JAK2 contributes to the regulation of the inositol transporter SMIT.

The present study explored whether JAK2 regulates the activity of SMIT. To this end, SMIT was expressed in Xenopus oocytes with or without wild type JAK2, constitutively active V617F JAK2 or inactive K882E JAK2 and inositol transport estimated from inositol induced current.

Voltage clamp in Xenopus oocytes
Xenopus oocytes were prepared as described [45] Where not indicated otherwise, 25 ng SMIT cRNA were injected on the first day and 10 ng of wild type JAK2 cRNA on the second day or at the same day after preparation of the oocytes [46]. The oocytes were maintained at 17°C in a solution containing (in mM): 96 NaCl, 4 KCl, 1.8 MgC1 2 , 0.1 CaC1 2 , 5 HEPES, pH7.4, gentamycin (50 mg/l), tetracycline (50 mg/l), ciprofloxacin (1.6 mg/l), refobacin (100 mg/l), and theophiline (90 mg/l). Where indicated, the JAK2 inhibitor AG490 (40 µM), brefeldin A (5 µM) or actinomycin D (10 µM) were added to the respective solutions. The voltage clamp experiments were performed at room temperature 7-10 days after injection [47]. Two-electrode voltage-clamp recordings were performed at a holding potential of -60 mV. The data were filtered at 10 Hz and recorded with a Digidata A/D-D/A converter and Clampex 9.2 software for data acquisition and analysis (Axon Instruments) [48]. The control superfusate contained 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 and 5 mM HEPES, pH 7.4. Inositol was added to the solutions at a concentration of 1 mM, unless otherwise stated. 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.

Statistical analysis
Data are provided as means ± SEM, n represents the number of oocytes investigated. All 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 or t-test, as appropriate. Results with p < 0.05 were considered statistically significant.

Results
To explore, whether Janus kinase-2 (JAK2) modifies the function of the myoinositol transporter (SMIT), cRNA encoding SMIT was injected into Xenopus oocytes with or without additional injection of cRNA encoding JAK2. Electrogenic transport of inositol was apparent from inositol (1 mM) induced inward current (I SMIT ). No appreciable current was observed following exposure of non-injected or water-injected Xenopus oocytes to 1 mM inositol. Thus, Xenopus oocytes do not express appreciable endogenous electrogenic inositol transport (Fig.  1A,B). A sizable I SMIT was, however, observed in Xenopus oocytes injected with cRNA encoding SMIT. The additional injection of cRNA encoding wild type JAK2 was followed by a significant decrease of I SMIT in SMIT expressing Xenopus oocytes (Fig. 1A,B). The effect of wild type JAK2 was mimicked by the gain of function mutant V617F JAK2 but not by the inactive mutant K882E JAK2 (Fig. 1A,B). Accordingly, I SMIT was significantly lower in Xenopus oocytes expressing SMIT together with V617F JAK2 than in Xenopus oocytes expressing SMIT alone. In contrast, I SMIT was similar in Xenopus oocytes expressing SMIT together with K882E JAK2 and in Xenopus oocytes expressing SMIT alone. The effect of V617F JAK2 coexpression tended to be lower than that of wild type JAK2 coexpression.
Kinetic analysis was performed to determine, whether JAK2 influenced the maximal transport rate or the affinity of the carrier. As illustrated in Fig. 2, inositol-induced currents  The currents allowed calculating a maximal current, which approached 6.30 ± 0.03 nA (n = 9) in Xenopus oocytes expressing SMIT alone, and 3.84 ± 0.02 (n = 9) in Xenopus oocytes expressing SMIT together with JAK2. Accordingly, the maximal transport rate was significantly lower in Xenopus oocytes expressing SMIT together with JAK2 than in Xenopus oocytes expressing SMIT alone. The inositol concentration required for halfmaximal current (K M ) approached 0.06 ± 0.01 µM (n = 9) in Xenopus oocytes expressing SMIT alone, and 0.12    (Fig. 2). Treatment of Xenopus oocytes expressing both, SMIT and JAK2, with the JAK2 inhibitor AG490 (40µM) was followed by an increase of I SMIT (Fig. 3). The effect of the inhibitor on I SMIT reached statistical significance within 8 and 24 hours of preincubation with AG490.
The decrease of I SMIT in SMIT expressing Xenopus oocytes following coexpression of JAK2 could have been due to accelerated clearance of carrier protein from the cell membrane. In order to estimate the stability of I SMIT , the SMIT-expressing Xenopus oocytes were treated with 5 µM brefeldin A, a substance blocking the insertion of new carrier protein into the cell membrane. As shown in Fig. 4A, in the presence of brefeldin A I SMIT declined within 8 and 24 hours to a similar extent in Xenopus oocytes expressing SMIT together with JAK2 and in Xenopus oocytes expressing SMIT alone. The observation suggests that JAK2 decreases I SMIT by a mechanism other than accelerating carrier clearance from the cell membrane.
Inhibition of transcription by incubation (24-48 hours) with actinomycin D (10 µM) did not significantly modify I SMIT , which remained significantly lower in Xenopus oocytes expressing SMIT together with JAK2 than in Xenopus oocytes expressing SMIT alone (Fig.  4B). Thus, the effect of JAK2 did not involve altered transcription.

Discussion
The present study discloses that JAK2 contributes to the regulation of the myoinositol transporter SMIT. Coexpression of the Janus Kinase 2 (JAK2) significantly decreased the electrogenic transport of inositol in SMIT expressing Xenopus oocytes. Down-regulation of SMIT was similarly observed following coexpression of the gain of function mutant V617F JAK2 but not following coexpression of inactive K882E JAK2. Thus, JAK2 is effective as kinase. Coexpression of the kinase significantly decreased the maximal current. Thus, JAK2 is at least partially effective by decreasing the maximal transport rate of the carrier. JAK2 expression did not significantly affect K M . However, the large scatter of calculated K M values precludes safe conclusions on alterations of substrate affinity.
Inhibition of carrier insertion into the cell membrane by brefeldin A leads to a decline of SMIT activity, which is similar in Xenopus oocytes expressing SMIT together with JAK2 and Xenopus oocytes expressing SMIT alone. Thus, the clearance of carrier from the cell membrane appears not to be sensitive to JAK2 expression. Alternatively, JAK2 may be effective by fostering carrier insertion into the cell membrane or by activating the carrier within the cell membrane.
Exposure of Xenopus oocytes expressing SMIT together with JAK2 to the JAK2 inhibitor AG490 was followedd by a gradual increase of SMIT induced current indicating that the inhibitor counteracts the suppression of the carrier by the kinase. The effect appears to be slow, which may point to indirect regulation of the carrier. The observations in the presence of actinomycin D suggest that the observed effect is not due to an effect of JAK2 on gene transcription.
In conclusion, the present paper reveals that JAK2 is a powerful regulator of the Na + , Clcoupled myoinositol transporter SMIT. JAK2 down-regulates the transporter and thus limits the cellular uptake of Na + , Cland myoinositol.