Leucine-rich repeat kinase 2-sensitive Na+/Ca2+ exchanger activity in dendritic cells.

Gene variants of the leucine-rich repeat kinase 2 (LRRK2) are associated with susceptibility to Parkinson's disease (PD). Besides brain and periphery, LRRK2 is expressed in various immune cells including dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. However, the function of LRRK2 in the immune system is still incompletely understood. Here, Ca(2+)-signaling was analyzed in DCs isolated from gene-targeted mice lacking lrrk2 (Lrrk2(-/-)) and their wild-type littermates (Lrrk2(+/+)). According to Western blotting, Lrrk2 was expressed in Lrrk2(+/+) DCs but not in Lrrk2(-/-)DCs. Cytosolic Ca(2+) levels ([Ca(2+)]i) were determined utilizing Fura-2 fluorescence and whole cell currents to decipher electrogenic transport. The increase of [Ca(2+)]i following inhibition of sarcoendoplasmatic Ca(2+)-ATPase with thapsigargin (1 µM) in the absence of extracellular Ca(2+) (Ca(2+)-release) and the increase of [Ca(2+)]i following subsequent readdition of extracellular Ca(2+) (SOCE) were both significantly larger in Lrrk2(-/-) than in Lrrk2(+/+) DCs. The augmented increase of [Ca(2+)]i could have been due to impaired Ca(2+) extrusion by K(+)-independent (NCX) and/or K(+)-dependent (NCKX) Na(+)/Ca(2+)-exchanger activity, which was thus determined from the increase of [Ca(2+)]i, (Δ[Ca(2+)]i), and current following abrupt replacement of Na(+) containing (130 mM) and Ca(2+) free (0 mM) extracellular perfusate by Na(+) free (0 mM) and Ca(2+) containing (2 mM) extracellular perfusate. As a result, both slope and peak of Δ[Ca(2+)]i as well as Na(+)/Ca(2+) exchanger-induced current were significantly lower in Lrrk2(-/-) than in Lrrk2(+/+) DCs. A 6 or 24 hour treatment with the LRRK2 inhibitor GSK2578215A (1 µM) significantly decreased NCX1 and NCKX1 transcript levels, significantly blunted Na(+)/Ca(2+)-exchanger activity, and significantly augmented the increase of [Ca(2+)]i following Ca(2+)-release and SOCE. In conclusion, the present observations disclose a completely novel functional significance of LRRK2, i.e., the up-regulation of Na(+)/Ca(2+) exchanger transcription and activity leading to attenuation of Ca(2+)-signals in DCs.

Impact of Janus Kinase 3 on Cellular Ca Release, Store Operated Ca(2+) Entry and Na(+)/Ca(2+) Exchanger Activity in Dendritic Cells.

BACKGROUND/AIMS: Janus kinase 3 (JAK3), a tyrosine kinase mainly expressed in hematopoietic cells, participates in the signaling stimulating cell proliferation. The kinase is expressed in dendritic cells (DCs), antigen presenting cells involved in the initiation and regulation of antigen-specific T-cell responses. Dendritic cell function is regulated by cytosolic Ca(2+) activity ([Ca(2+)]i). Mediators increasing [Ca(2+)]i in DCs include ATP and the chemokine receptor CXCR4 ligand CXCL12. The present study explored, whether JAK3 participates in the regulation of [Ca(2+)]i in DCs. METHODS: Fura-2 fluorescence was employed to determine [Ca(2+)]i, and whole cell patch clamp to decipher electrogenic transport in immature DCs isolated from bone marrow of JAK3-knockout (jak3(-/-)) or wild-type mice (jak3(+/+)). RESULTS: Without treatment, [Ca(2+)]i was similar in jak3(-/-) and jak3(+/+) DCs. Addition of ATP (100 µM) was followed by transient increase of [Ca(2+)]i reflecting Ca(2+) release from intracellular stores, an effect significantly less pronounced in jak3(-/-) DCs than in jak3(+/+) DCs. CXCL12 administration was followed by a sustained increase of [Ca(2+)]i reflecting receptor operated Ca(2+) entry, an effect significantly less rapid in jak3(-/-) DCs than in jak3(+/+) DCs. In addition, the Ca(2+) release-activated Ca(2+) channel (CRAC) current triggered by IP3-induced Ca(2+) store depletion and CXCL12 was significantly higher in DCs from jak3(+/+) mice than in jak3(-/-) mice. Inhibition of sarcoendoplasmatic reticulum Ca(2+)-ATPase (SERCA) by thapsigargin (1 µM) in the absence of extracellular Ca(2+) was followed by a transient increase of [Ca(2+)]i reflecting Ca(2+) release from intracellular stores, and subsequent readdition of extracellular Ca(2+) in the continued presence of thapsigargin was followed by a sustained increase of [Ca(2+)]i reflecting store operated Ca(2+) entry (SOCE). Both, Ca(2+) release from intracellular stores and SOCE were again significantly lower in jak3(-/-) DCs than in jak3(+/+) DCs. Pretreatment of jak3(+/+) DCs with JAK inhibitor WHI-P154 (22 µM, 10 minutes or 24 hours) significantly blunted both thapsigargin induced Ca(2+) release and subsequent SOCE. Abrupt replacement of Na(+) containing (130 mM) and Ca(2+) free (0 mM) extracellular bath by Na(+) free (0 mM) and Ca(2+) containing (2 mM) extracellular bath increased [Ca(2+)]i reflecting Na(+)/Ca(2+) exchanger activity, an effect again significantly less pronounced in jak3(-/-) DCs than in jak3(+/+) DCs. CONCLUSIONS: JAK3 deficiency is followed by down-regulation of cytosolic Ca(2+) release, receptor and store operated Ca(2+) entry and Na(+)/Ca(2+) exchanger activity in DCs.

The Role of Janus Kinase 3 in the Regulation of Na⁺/K⁺ ATPase under Energy Depletion.

BACKGROUND/AIMS: Janus kinase-3 (JAK3) is activated during energy depletion. Energy-consuming pumps include the Na(+)/K(+)-ATPase. The present study explored whether JAK3 regulates Na(+)/K(+)-ATPase in dendritic cells (DCs). METHODS: Ouabain (100 µM)-sensitive (Iouabain) and K(+)-induced (Ipump) outward currents were determined by utilizing whole cell patch-clamp, Na(+)/K(+)-ATPase α1-subunit mRNA levels by RT-PCR, Na(+)/K(+)-ATPase protein abundance by flow cytometry or immunofluorescence, and cellular ATP by luciferase-assay in DCs from bone marrow of JAK3-knockout (jak3(-/-)) or wild-type mice (jak3(+/+)). Ipump was further determined by voltage clamp in Xenopus oocytes expressing JAK3, active (A568V)JAK3 or inactive (K851A)JAK3. RESULTS: Na(+)/K(+)-ATPase α1-subunit mRNA and protein levels, as well as Ipump and Iouabain were significantly higher in jak3(-/-)DCs than in jak3(+/+)DCs. Energy depletion by 4h pre-treatment with 2,4-dinitro-phenol significantly decreased Ipump in jak3(+/+) DCs but not in jak3(-/-)DCs. Cellular ATP was significantly lower in jak3(-/-)DCs than in jak3(+/+)DCs and decreased in both genotypes by 2,4-dinitro-phenol, an effect significantly more pronounced in jak3(-/-)DCs than in jak3(+/+)DCs and strongly blunted by ouabain in both jak3(+/+) and jak3(-/-)DCs. Ipump and Iouabain in oocytes were decreased by expression of JAK3 and of (A568V)JAK3 but not of (K851A)JAK3. JAK3 inhibitor WHI-P154 (4-[(3'-bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 22 µM) enhanced Ipump and Iouabain in JAK3 expressing oocytes. The difference between (A568V)JAK3 and (K851A)JAK3 expressing oocytes was virtually abrogated by actinomycin D (50 nM). CONCLUSIONS: JAK3 down-regulates Na(+)/K(+)-ATPase activity, an effect involving gene expression and profoundly curtailing ATP consumption.

Effect of TGFß on calcium signaling in megakaryocytes.

TGFß is a powerful regulator of megakaryocyte maturation and platelet formation. As previously shown for other cell types, TGFß may up-regulate the expression of the serum & glucocorticoid inducible kinase SGK1, an effect requiring p38 kinase. SGK1 has in turn recently been shown to participate in the regulation of cytosolic Ca(2+) activity ([Ca(2+)]i) in megakaryocytes and platelets. SGK1 phosphorylates the IκB kinase (IKKα/ß), which in turn phosphorylates the inhibitor protein IκBα resulting in nuclear translocation of nuclear factor NFκB. Genes up-regulated by NFκB include Orai1, the pore forming ion channel subunit accomplishing store operated Ca(2+) entry (SOCE). The present study explored whether TGFß influences Ca(2+) signaling in megakaryocytes. [Ca(2+)]i was determined by Fura-2 fluorescence and SOCE from the increase of [Ca(2+)]i following re-addition of extracellular Ca(2+) after store depletion by removal of extracellular Ca(2+) and inhibition of the sarcoendoplasmatic Ca(2+) ATPase (SERCA) with thapsigargin (1 µM). As a result, TGFß (60 ng, 24 h) increased SOCE, an effect significantly blunted by p38 kinase inhibitor Skepinone-L (1 µM), SGK1 inhibitor EMD638683 (50 µM) and NFκB inhibitor wogonin (100 µM). In conclusion, TGFß is a powerful regulator of store operated Ca(2+) entry into megakaryocytes, an effect mediated by a signaling cascade involving p38 kinase, SGK1 and NFκB.

Up-regulation of megakaryocytic Na(+)/Ca(2+) exchange in klotho-deficient mice.

The active form of vitamin D, 1,25(OH)2D3, is a powerful regulator of cytosolic Ca(2+)-concentration ([Ca(2+)]i) in a variety of cell types. The formation of 1,25(OH)2D3 is inhibited by FGF23, an effect requiring presence of klotho. 1,25(OH)2D3 plasma levels are excessive in klotho-deficient mice (kl/kl). A previous study revealed that klotho-deficiency is followed by decreased activation of platelets, an effect at least in part due to blunted store operated Ca(2+) entry (SOCE). In other cell types 1,25(OH)2D3 has been shown to up-regulate the Na(+)/Ca(2+)-exchanger, which could, depending on cell membrane potential and cytosolic Na(+) concentration, either decrease or increase [Ca(2+)]i. The present study explored whether Na(+)/Ca(2+)-exchanger activity is different in megakaryocytes isolated from kl/kl mice than in megakaryocytes isolated from wild type mice. Na(+)/Ca(2+)-exchanger induced currents were determined by whole cell patch clamp and the Na(+)/Ca(2+)-exchanger induced alterations of [Ca(2+)]i by Fura-2 fluorescence. As a result, the inward current and the increase of [Ca(2+)]i following replacement of extracellular Na(+) by NMDG were higher in kl/kl megakaryocytes than in wild type megakaryocytes, a difference abrogated by treatment of the mice with low Vitamin D diet. Pretreatment of wild type megakaryocytes with 1,25(OH)2D3 (100 nM, 48 h) was followed by enhancement of both, inward current and increase of [Ca(2+)]i following replacement of extracellular Na(+) by NMDG. In conclusion, the present observations reveal a powerful stimulating effect of 1,25(OH)2D3 on Na(+)/Ca(2+)-exchanger activity in megakaryocytes.

Up-regulation of Kv1.3 channels by janus kinase 2.

The janus-activated kinase 2 JAK2 participates in the signalling of several hormones including interferon, a powerful regulator of lymphocyte function. Lymphocyte activity and survival depend on the activity of the voltage-gated K(+) channel KCNA3 (Kv1.3). The present study thus explored whether JAK2 modifies the activity of voltage-gated K(+) channel KCNA3. To this end, cRNA encoding KCNA3 was injected in Xenopus oocytes with or without additional injection of cRNA encoding wild-type human JAK2, human inactive (K882E)JAK2 mutant, or human gain-of-function (V617F)JAK2 mutant. KCNA3-dependent depolarization-induced current was determined utilizing dual-electrode voltage clamp, and protein KCNA3 abundance in the cell membrane was quantified by chemiluminescence. Moreover, the effect of interferon-γ on voltage-gated K(+) current was determined by patch clamp in mainly KCNA3-expressing Jurkat T cells with or without prior treatment with JAK2 inhibitor AG490 (40 µM). As a result, KCNA3 channel activity and protein abundance were up-regulated by coexpression of JAK2 or (V617F)JAK2 but not (K882E)JAK2. The effect of JAK2 coexpression was reversed by AG490 treatment. In human Jurkat T lymphoma cells, voltage-gated K(+) current was up-regulated by interferon-γ and down-regulated by AG490 (40 µM). In conclusion, JAK2 participates in the signalling, regulating the voltage-gated K(+) channel KCNA3.

1,25(OH)2 vitamin D3-dependent inhibition of platelet Ca2+ signaling and thrombus formation in klotho-deficient mice.

Platelets are activated by increased cytosolic Ca(2+) concentration ([Ca(2+)]i) following store-operated calcium entry (SOCE) accomplished by calcium-release-activated calcium (CRAC) channel moiety Orai1 and its regulator STIM1. In other cells, Ca(2+) transport is regulated by 1,25(OH)2 vitamin D3 [1,25(OH)2D3]. 1,25(OH)2D3 formation is inhibited by klotho and excessive in klotho-deficient mice (kl/kl). The present study explored the effect of klotho deficiency on platelet Ca(2+) signaling and activation. Platelets and megakaryocytes isolated from WT and kl/kl-mice were analyzed by RT-PCR, Western blotting, confocal microscopy, Fura-2-fluorescence, patch clamp, flow cytometry, aggregometry, and flow chamber. STIM1/Orai1 transcript and protein levels, SOCE, agonist-induced [Ca(2+)]i increase, activation-dependent degranulation, integrin αIIbß3 activation and aggregation, and thrombus formation were significantly blunted in kl/kl platelets (by 27-90%). STIM1/Orai1 transcript and protein levels, as well as CRAC currents, were significantly reduced in kl/kl megakaryocytes (by 38-73%) and 1,25(OH)2D3-treated WT megakaryocytes. Nuclear NF-κB subunit p50/p65 abundance was significantly reduced in kl/kl-megakaryocytes (by 51-76%). Transfection with p50/p65 significantly increased STIM1/Orai1 transcript and protein levels in megakaryocytic MEG-01 cells (by 46-97%). Low-vitamin D diet (LVD) of kl/kl mice normalized plasma 1,25(OH)2D3 concentration and function of platelets and megakaryocytes. Klotho deficiency inhibits platelet Ca(2+) signaling and activation, an effect at least partially due to 1,25(OH)2D3-dependent down-regulation of NF-κB activity and STIM1/Orai1 expression in megakaryocytes.

SPAK Sensitive Regulation of the Epithelial Na Channel ENaC.

BACKGROUND/AIMS: The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase SPAK participates in the regulation of NaCl and Na(+),K(+),2Cl(-) cotransport and thus renal salt excretion. The present study explored whether SPAK has similarly the potential to regulate the epithelial Na(+) channel (ENaC). METHODS: ENaC was expressed in Xenopus oocytes with or without additional expression of wild type SPAK, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK or catalytically inactive (D212A)SPAK, and ENaC activity estimated from amiloride (50 µM) sensitive current (Iamil) in dual electrode voltage clamp experiments. Moreover, Ussing chamber was employed to determine Iamil in colonic tissue from wild type mice (spak(wt/wt)) and from gene targeted mice carrying WNK insensitive SPAK (spak(tg/tg)). RESULTS: Iamil was observed in ENaC-expressing oocytes, but not in water-injected oocytes. In ENaC expressing oocytes Iamil was significantly increased following coexpression of wild-type SPAK and (T233E)SPAK, but not following coexpression of (T233A)SPAK or (D212A)SPAK. Colonic Iamil was significantly higher in spak(wt/wt) than in spak(tg/tg) mice. CONCLUSION: SPAK has the potential to up-regulate ENaC.

Upregulation of the large conductance voltage- and Ca2+-activated K+ channels by Janus kinase 2.

The iberiotoxin-sensitive large conductance voltage- and Ca(2+)-activated potassium (BK) channels (maxi-K(+)-channels) hyperpolarize the cell membrane thus supporting Ca(2+) entry through Ca(2+)-release activated Ca(2+) channels. Janus kinase-2 (JAK2) has been identified as novel regulator of ion transport. To explore whether JAK2 participates in the regulation of BK channels, cRNA encoding Ca(2+)-insensitive BK channels (BK(M513I+Δ899-903)) was injected into Xenopus oocytes with or without cRNA encoding wild-type JAK2, gain-of-function (V617F)JAK2, or inactive (K882E)JAK2. K(+) conductance was determined by dual electrode voltage clamp and BK-channel protein abundance by confocal microscopy. In A204 alveolar rhabdomyosarcoma cells, iberiotoxin-sensitive K(+) current was determined utilizing whole cell patch clamp. A204 cells were further transfected with JAK2 and BK-channel transcript, and protein abundance was quantified by RT-PCR and Western blotting, respectively. As a result, the K(+) current in BK(M513I+Δ899-903)-expressing oocytes was significantly increased following coexpression of JAK2 or (V617F)JAK2 but not (K882E)JAK2. Coexpression of the BK channel with (V617F)JAK2 but not (K882E)JAK2 enhanced BK-channel protein abundance in the oocyte cell membrane. Exposure of BK-channel and (V617F)JAK2-expressing oocytes to the JAK2 inhibitor AG490 (40 µM) significantly decreased K(+) current. Inhibition of channel insertion by brefeldin A (5 µM) decreased the K(+) current to a similar extent in oocytes expressing the BK channel alone and in oocytes expressing the BK channel and (V617F)JAK2. The iberiotoxin (50 nM)-sensitive K(+) current in rhabdomyosarcoma cells was significantly decreased by AG490 pretreatment (40 µM, 12 h). Moreover, overexpression of JAK2 in A204 cells significantly enhanced BK channel mRNA and protein abundance. In conclusion, JAK2 upregulates BK channels by increasing channel protein abundance in the cell membrane.

Energy-sensitive regulation of Na+/K+-ATPase by Janus kinase 2.

Janus kinase 2 (JAK2) contributes to intracellular signaling of leptin and erythropoietin, hormones protecting cells during energy depletion. The present study explores whether JAK2 is activated by energy depletion and regulates Na(+)/K(+)-ATPase, the major energy-consuming pump. In Jurkat cells, JAK2 activity was determined by radioactive kinase assay, phosphorylated JAK2 detected by Western blotting, ATP levels measured by luciferase assay, as well as Na(+)/K(+)-ATPase α1-subunit transcript and protein abundance determined by real-time PCR and Western blotting, respectively. Ouabain-sensitive K(+)-induced currents (Ipump) were measured by whole cell patch clamp. Ipump was further determined by dual-electrode voltage clamp in Xenopus oocytes injected with cRNA-encoding JAK2, active (V617F)JAK2, or inactive (K882E)JAK2. As a result, in Jurkat T cells, JAK2 activity significantly increased following energy depletion by sodium azide (NaN3) or 2,4- dinitro phenol (DNP). DNP- and NaN3-induced decrease of cellular ATP was significantly augmented by JAK2 inhibitor AG490 and blunted by Na(+)/K(+)-ATPase inhibitor ouabain. DNP decreased and AG490 enhanced Ipump as well as Na(+)/K(+)-ATPase α1-subunit transcript and protein abundance. The α1-subunit transcript levels were also enhanced by signal transducer and activator of transcription-5 inhibitor CAS 285986-31-4. In Xenopus oocytes, Ipump was significantly decreased by expression of JAK2 and (V617F)JAK2 but not of (K882E)JAK2, effects again reversed by AG490. In (V617F)JAK2-expressing Xenopus oocytes, neither DNP nor NaN3 resulted in further decline of Ipump. In Xenopus oocytes, the effect of (V617F)JAK2 on Ipump was not prevented by inhibition of transcription with actinomycin. In conclusion, JAK2 is a novel energy-sensing kinase that curtails energy consumption by downregulating Na(+)/K(+)-ATPase expression and activity.

Up-regulation of Na(+)-coupled glucose transporter SGLT1 by caveolin-1.

The Na(+)-coupled glucose transporter SGLT1 (SLC5A1) accomplishes concentrative cellular glucose uptake even at low extracellular glucose concentrations. The carrier is expressed in renal proximal tubules, small intestine and a variety of nonpolarized cells including several tumor cells. The present study explored whether SGLT1 activity is regulated by caveolin-1, which is known to regulate the insertion of several ion channels and carriers in the cell membrane. To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of caveolin-1 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes, but not in oocytes injected with water or caveolin-1 alone, the addition of glucose to the extracellular bath generated an inward current (Ig), which was increased following coexpression of caveolin-1. Kinetic analysis revealed that caveolin-1 increased maximal Ig without significantly modifying the glucose concentration required to trigger half maximal Ig (KM). According to chemiluminescence and confocal microscopy, caveolin-1 increased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5µM) resulted in a decline of Ig, which was similar in the absence and presence of caveolin-1. In conclusion, caveolin-1 up-regulates SGLT1 activity by increasing carrier protein abundance in the cell membrane, an effect presumably due to stimulation of carrier protein insertion into the cell membrane.

Up-regulation of Kir2.1 (KCNJ2) by the serum & glucocorticoid inducible SGK3.

BACKGROUND/AIMS: The serum & glucocorticoid inducible kinase SGK3, an ubiquitously expressed serine/threonine kinase, regulates a variety of ion channels. It has previously been shown that SGK3 upregulates the outwardly rectifying K(+) channel KV11.1, which is expressed in cardiomyocytes. Cardiomyocytes further express the inward rectifier K(+) channel K(ir)2.1, which contributes to maintenance of resting cell membrane potential. Loss-of-function mutations of KCNJ2 encoding K(ir)2.1 result in Andersen-Tawil syndrome with periodic paralysis, cardiac arrhythmia and dysmorphic features. The present study explored whether SGK3 participates in the regulation of K(ir)2.1. METHODS: cRNA encoding K(ir)2.1 was injected into Xenopus oocytes with and without additional injection of cRNA encoding wild type SGK3, constitutively active (S419D)SGK3 or inactive (K191N)SGK3. Kir2.1 activity was determined by two-electrode voltage-clamp and K(ir)2.1 protein abundance in the cell membrane by immunostaining and subsequent confocal imaging or by chemiluminescence. RESULTS: Injection of 10 ng cRNA encoding wild type SGK3 and (S419D)SGK3, but not (K191N)SGK3 significantly enhanced K(ir)2.1-mediated currents. SGK inhibitor EMD638683 (50 µM) abrogated (S419D)SGK3-induced up-regulation of K(ir)2.1. Moreover, wild type SGK3 enhanced the channel protein abundance in the cell membrane. The decay of K(ir)2.1-mediated currents following inhibition of channel insertion into the cell membrane by brefeldin A (5 µM) was similar in oocytes coexpressing K(ir)2.1 and SGK3 as in oocytes expressing K(ir)2.1 alone, suggesting that SGK3 influences channel insertion into rather than channel retrieval from the cell membrane. CONCLUSIONS: SGK3 is a novel regulator of K(ir)2.1.

Serum- and glucocorticoid-inducible kinase 1 sensitive NF-κB signaling in dendritic cells.

BACKGROUND/AIMS: Dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity, are required for initiation of specific T cell-driven immune responses. Phosphoinositide-3-kinase (PI3K) suppresses proinflammatory cytokine production in DCs, which limits T helper (Th1) polarization. PI3K is in part effective by downregulation of transcription factor NF-κB. Downstream signaling elements of PI3K include serum- and glucocorticoid-inducible kinase 1 (SGK1) and its phosphorylation target N-myc downstream regulated gene 1 (NDRG1). The present study explored whether SGK1 and NDRG1 play a role in the regulation of NF-κB and DC-maturation. METHODS: DCs were isolated from bone marrow (BMDCs) or spleen of mice lacking functional SGK1 (sgk1(-/-)) and corresponding wild type mice (sgk1(+/+)). Protein abundance was determined by Western blotting. Transcription was inhibited by siRNA. Abundance of maturation markers was quantified by flow cytometry. FITC-dextran uptake was determined to quantify phagocytosis. RESULTS: NDRG1 was similarly expressed in sgk1(+/+) and sgk1(-/-)BMDCs, but SGK1-dependent phosphorylation of NDRG-1 was decreased in sgk1(-/-)BMDCs. Silencing of NDRG1 in sgk1(+/+)BMDCs as compared to control empty vector-treated BMDCs enhanced nuclear abundance of NF-κB subunit p65. Moreover, the abundance of phosphorylated NF-κB inhibitor IκBα, of phosphorylated IκB kinase (IKKα/ß) and of nuclear p65 were significantly higher in sgk1(-/-)BMDCs than in sgk1(+/+)BMDCs. Expression of maturation markers, MHC II, and CD86, was significantly larger and phagocytic capacity was significantly lower in sgk1(-/-) than in sgk1(+/+)BMDCs. Expression of CD86 and MHCII was also significantly higher in DCs isolated from the spleen of sgk1(-/-) mice than those from sgk1(+/+)mice. CONCLUSION: SGK1 and NDRG1 participate in the regulation of NF-κB signaling in and maturation of DCs.

Akt2- and ETS1-dependent IP3 receptor 2 expression in dendritic cell migration.

BACKGROUND/AIMS: The protein kinase Akt2/PKBß is a known regulator of macrophage and dendritic cell (DC) migration. The mechanisms linking Akt2 activity to migration remained, however, elusive. DC migration is governed by Ca(2+) signaling. We thus explored whether Akt2 regulates DC Ca(2+) signaling. METHODS: DCs were derived from bone marrow of Akt2-deficient mice (akt2(-/-)) and their wild type littermates (akt2(+/+)). DC maturation was induced by lipopolysaccharides (LPS) and evaluated by flow cytometry. Cytosolic Ca(2+) concentration was determined by Fura-2 fluorescence, channel activity by whole cell recording, transcript levels by RT-PCR, migration utilizing transwells. RESULTS: Upon maturation, chemokine CCL21 stimulated migration of akt2(+/+) but not akt2(-/-) DCs. CCL21-induced increase in cytosolic Ca(2+) concentration, thapsigargin-induced release of Ca(2+) from intracellular stores with subsequent store-operated Ca(2+) entry (SOCE), ATP-induced inositol 1,4,5-trisphosphate (IP3)-dependent Ca(2+) release as well as Ca(2+) release-activated Ca(2+) (CRAC) channel activity were all significantly lower in mature akt2(-/-) than in mature akt2(+/+) DCs. Transcript levels of IP3 receptor IP3R2 and of IP3R2 regulating transcription factor ETS1 were significantly higher in akt2(+/+) than in akt2(-/-) DCs prior to maturation and were upregulated by LPS stimulation (1h) in akt2(+/+) and to a lower extent in akt2(-/-) DCs. Following maturation, protein abundance of IP3R2 and ETS1 were similarly higher in akt2(+/+) than in akt2(-/-) DCs. The IP3R inhibitor Xestospongin C significantly decreased CCL21-induced migration of akt2(+/+)DCs and abrogated the differences between genotypes. Finally, knock-down of ETS1 with siRNA decreased IP3R2 mRNA abundance, thapsigargin- and ATP-induced Ca(2+) release, SOCE and CRAC channel activation, as well as DC migration. CONCLUSION: Akt2 upregulates DC migration at least in part by ETS1-dependent stimulation of IP3R2 transcription.

Effect of TGFß on Na+/K+ ATPase activity in megakaryocytes.

The Na(+)/K(+) ATPase generates the Na(+) and K(+) concentration gradients across the plasma membrane and is thus essential for cellular electrolyte homeostasis, cell membrane potential and cell volume maintenance. A powerful regulator of Na(+)/K(+) ATPase is the serum- and glucocorticoid-inducible kinase 1 (SGK1). The most powerful known regulator of SGK1 expression is TGFß1, which is pivotal in the regulation of megakaryocyte maturation and platelet formation. Signaling involved in the upregulation of SGK1 by TGFß1 includes p38 mitogen activated protein (MAP) kinase. SGK1 in turn phosphorylates the IκB kinase (IKKα/ß), which phosphorylates the inhibitor protein IκBα thus triggering nuclear translocation of nuclear factor kappa B (NF-κB). The present study explored whether TGFß influences Na(+)/K(+) ATPase activity in megakaryocytes, and if so, whether the effect of TGß1 requires p38 MAP kinase, SGK1 and/or NF-κB. To this end, murine megakaryocytes were treated with TGFß1 and Na(+)/K(+) ATPase activity determined from K(+) induced current utilizing whole cell patch clamp. The pump current (Ipump) was determined in the absence and presence of Na(+)/K(+) ATPase inhibitor ouabain (100µM). TGFß1 (60ng/ml) was added in the absence or presence of p38 MAP kinase inhibitor skepinone-L (1µM), SGK1 inhibitor EMD638683 (50µM) or NF-κB inhibitor wogonin (50nM). As a result, the Ipump was significantly increased by pretreatment of the megakaryocytes with TGFß1, an effect reaching statistical significance within 16 and 24h and virtually abrogated in the presence of skepinone-L, EMD638683 or wogonin. In conclusion, TGFß1 is a powerful regulator of megakaryocytic Na(+)/K(+) ATPase activity. Signaling mediating the effect of TGFß1 on Na(+)/K(+) ATPase activity involves p38 MAP kinase, SGK1 and NF-κB.

Down-regulation of K⁺ channels by human parvovirus B19 capsid protein VP1.

Parvovirus B19 (B19V) can cause inflammatory cardiomyopathy and endothelial dysfunction. Pathophysiological mechanisms involved include lysophosphatidylcholine producing phospholipase A2 (PLA2) activity of the B19V capsid protein VP1. Most recently, VP1 and lysophosphatidylcholine have been shown to inhibit Na(+)/K(+) ATPase. The present study explored whether VP1 modifies the activity of Kv1.3 and Kv1.5 K(+) channels. cRNA encoding Kv1.3 or Kv1.5 was injected into Xenopus oocytes without or with cRNA encoding VP1 isolated from a patient suffering from fatal B19V-induced myocarditis. K(+) channel activity was determined by dual electrode voltage clamp. Injection of cRNA encoding Kv1.3 or Kv1.5 into Xenopus oocytes was followed by appearance of Kv K(+) channel activity, which was significantly decreased by additional injection of cRNA encoding VP1, but not by additional injection of cRNA encoding PLA2-negative VP1 mutant (H153A). The effect of VP1 on Kv current was not significantly modified by transcription inhibitor actinomycin (10 µM for 36 h) but was mimicked by lysophosphatidylcholine (1 µg/ml). The B19V capsid protein VP1 inhibits host cell Kv channels, an effect at least partially due to phospholipase A2 (PLA) dependent formation of lysophosphatidylcholine.

Downregulation of chloride channel ClC-2 by Janus kinase 3.

Janus kinase-3 (JAK3) fosters proliferation and counteracts apoptosis of lymphocytes and tumor cells. The gain of function mutation (A572V)JAK3 has been discovered in acute megakaryoplastic leukemia. JAK3 is inactivated by replacement of lysine by alanine in the catalytic subunit ((K855A)JAK3). Regulation of cell proliferation and apoptosis involves altered activity of Cl(-) channels. The present study, thus, explored whether JAK3 modifies the function of the small conductance Cl(-) channel ClC-2. To this end, ClC-2 was expressed in Xenopus oocytes with or without wild-type JAK3, (A568V)JAK3 or (K851A)JAK3, and the Cl(-) channel activity determined by dual-electrode voltage clamp. Channel protein abundance in the cell membrane was determined utilizing chemiluminescence. As a result, expression of ClC-2 was followed by a marked increase of cell membrane conductance. The conductance was significantly decreased following coexpression of JAK3 or (A568V)JAK3, but not by coexpression of (K851A)JAK3. Exposure of the oocytes expressing ClC-2 together with (A568V)JAK3 to the JAK3 inhibitor WHI-P154 (4-[(3'-bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 22 µM) increased the conductance. Coexpression of (A568V)JAK3 decreased the ClC-2 protein abundance in the cell membrane of ClC-2 expressing oocytes. The decline of conductance in ClC-2 and (A568V)JAK3 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 µM) was similar in oocytes expressing ClC-2 with (A568V)JAK3 and oocytes expressing ClC-2 alone, indicating that (A568V)JAK3 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. In conclusion, JAK3 downregulates ClC-2 activity and thus counteracts Cl(-) exit-an effect possibly influencing cell proliferation and apoptosis.

SPAK-sensitive regulation of glucose transporter SGLT1.

The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase SPAK is a powerful regulator of ion transport. The study explored whether SPAK similarly regulates nutrient transporters, such as the Na(+)-coupled glucose transporter SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of wild-type SPAK, constitutively active (T233E)SPAK, WNK-insensitive (T233A)SPAK or catalytically inactive (D212A)SPAK, and electrogenic glucose transport determined by dual-electrode voltage-clamp experiments. Moreover, Ussing chamber was employed to determine the electrogenic glucose transport in intestine from wild-type mice (spak(wt/wt)) and from gene-targeted mice carrying WNK-insensitive SPAK (spak(tg/tg)). In SGLT1-expressing oocytes, but not in water-injected oocytes, the glucose-dependent current (I(g)) was significantly decreased following coexpression of wild-type SPAK and (T233E)SPAK, but not by coexpression of (T233A)SPAK or (D212A)SPAK. Kinetic analysis revealed that SPAK decreased maximal I(g) without significantly modifying the glucose concentration required for halfmaximal I(g) (K(m)). According to the chemiluminescence experiments, wild-type SPAK but not (D212A)SPAK decreased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5 µM) resulted in a decline of I(g), which was similar in the absence and presence of SPAK, suggesting that SPAK did not accelerate the retrieval of SGLT1 protein from the cell membrane but rather down-regulated carrier insertion into the cell membrane. Intestinal electrogenic glucose transport was significantly lower in spak(wt/wt) than in spak(tg/tg) mice. In conclusion, SPAK is a powerful negative regulator of SGLT1 protein abundance in the cell membrane and thus of electrogenic glucose transport.

Upregulation of excitatory amino acid transporters by coexpression of Janus kinase 3.

Janus kinase 3 (JAK3) contributes to cytokine receptor signaling, confers cell survival and stimulates cell proliferation. The gain of function mutation JAK3(A572V) is found in acute megakaryoplastic leukemia. Replacement of ATP coordinating lysine by alanine yields inactive JAK3(K855A). Most recent observations revealed the capacity of JAK3 to regulate ion transport. This study thus explored whether JAK3 regulates glutamate transporters EAAT1-4, carriers accomplishing transport of glutamate and aspartate in a variety of cells including intestinal cells, renal cells, glial cells, and neurons. To this end, EAAT1, 2, 3, or 4 were expressed in Xenopus oocytes with or without additional expression of mouse wild-type JAK3, constitutively active JAK3(A568V) or inactive JAK3(K851A), and electrogenic glutamate transport was determined by dual electrode voltage clamp. Moreover, Ussing chamber was employed to determine electrogenic glutamate transport in intestine from mice lacking functional JAK3 (jak3(-/-)) and from corresponding wild-type mice (jak3(+/+)). As a result, in EAAT1, 2, 3, or 4 expressing oocytes, but not in oocytes injected with water, addition of glutamate to extracellular bath generated an inward current (Ig), which was significantly increased following coexpression of JAK3. Ig in oocytes expressing EAAT3 was further increased by JAK3(A568V) but not by JAK3(K851A). Ig in EAAT3 + JAK3 expressing oocytes was significantly decreased by JAK3 inhibitor WHI-P154 (22 µM). Kinetic analysis revealed that JAK3 increased maximal Ig and significantly reduced the glutamate concentration required for half maximal Ig (Km). Intestinal electrogenic glutamate transport was significantly lower in jak3(-/-) than in jak3(+/+) mice. In conclusion, JAK3 is a powerful regulator of excitatory amino acid transporter isoforms.

Regulation of ion channels by the serum- and glucocorticoid-inducible kinase SGK1.

The ubiquitously expressed serum- and glucocorticoid-inducible kinase-1 (SGK1) is genomically regulated by cell stress (including cell shrinkage) and several hormones (including gluco- and mineralocorticoids). SGK1 is activated by insulin and growth factors through PI3K and 3-phosphoinositide-dependent kinase PDK1. SGK1 activates a wide variety of ion channels (e.g., ENaC, SCN5A, TRPV4-6, ROMK, Kv1.3, Kv1.5, Kv4.3, KCNE1/KCNQ1, KCNQ4, ASIC1, GluR6, ClCKa/barttin, ClC2, CFTR, and Orai/STIM), which participate in the regulation of transport, hormone release, neuroexcitability, inflammation, cell proliferation, and apoptosis. SGK1-sensitive ion channels participate in the regulation of renal Na(+) retention and K(+) elimination, blood pressure, gastric acid secretion, cardiac action potential, hemostasis, and neuroexcitability. A common (∼3-5% prevalence in Caucasians and ∼10% in Africans) SGK1 gene variant is associated with increased blood pressure and body weight as well as increased prevalence of type II diabetes and stroke. SGK1 further contributes to the pathophysiology of allergy, peptic ulcer, fibrosing disease, ischemia, tumor growth, and neurodegeneration. The effect of SGK1 on channel activity is modest, and the channels do not require SGK1 for basic function. SGK1-dependent ion channel regulation may thus become pathophysiologically relevant primarily after excessive (pathological) expression. Therefore, SGK1 may be considered an attractive therapeutic target despite its broad range of functions.