RESUMO
BACKGROUND: Similar to tumor cells, activated T-lymphocytes generate ATP mainly by glycolytic degradation of glucose. Lymphocyte glucose uptake involves non-concentrative glucose carriers of the GLUT family. In contrast to GLUT isoforms, Na+-coupled glucose-carrier SGLT1 accumulates glucose against glucose gradients and is effective at low extracellular glucose concentrations. The present study explored expression and regulation of SGLT1 in activated murine splenic cytotoxic T cells (CTLs) and human Jurkat T cells. METHODS: FACS analysis, immunofluorescence, confocal microscopy, chemiluminescence and Western blotting were employed to estimate SGLT1 expression, function and regulation in lymphocytes, as well as dual electrode voltage clamp in SGLT1 ± JAK3 expressing Xenopus oocytes to quantify the effect of janus kinase3 (JAK3) on SGLT1 function. RESULTS: SGLT1 is expressed in murine CTLs and also in human Jurkat T cells. 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose uptake was significantly decreased by SGLT1-blocker phloridzin (0.2 mM) and by pharmacological inhibition of JAK3 with WHI-P131 (156 µM), WHI-P154 (11.2 µM) and JAK3 inhibitor VI (0.5 µM). Electrogenic glucose transport (Iglucose) in Xenopus oocytes expressing human SGLT1 was increased by additional expression of human wild type JAK3, active A568VJAK3 but not inactive K851AJAK3. Coexpression of JAK3 enhanced the maximal transport rate without significantly modifying affinity of the carrier. Iglucose in SGLT1+JAK3 expressing oocytes was significantly decreased by WHI-P154 (11.2 µM). JAK3 increased the SGLT1 protein abundance in the cell membrane. Inhibition of carrier insertion by brefeldin A (5 µM) in SGLT1+JAK3 expressing oocytes resulted in a decline of Iglucose, which was similar in presence and absence of JAK3. CONCLUSIONS: SGLT1 is expressed in murine cytotoxic T cells and human Jurkat T cells and significantly contributes to glucose uptake in those cells post activation. JAK3 up-regulates SGLT1 activity by increasing the carrier protein abundance in the cell membrane, an effect enforcing cellular glucose uptake into activated lymphocytes and thus contributing to the immune response.
Assuntos
Glucose/imunologia , Janus Quinase 3/genética , Oócitos/metabolismo , Transportador 1 de Glucose-Sódio/genética , Linfócitos T Citotóxicos/imunologia , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacologia , Animais , Transporte Biológico , Brefeldina A/farmacologia , Células CACO-2 , Desoxiglucose/análogos & derivados , Desoxiglucose/farmacologia , Regulação da Expressão Gênica , Glucose/farmacologia , Humanos , Janus Quinase 3/imunologia , Células Jurkat , Ativação Linfocitária , Camundongos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Técnicas de Patch-Clamp , Florizina/farmacologia , Cultura Primária de Células , Quinazolinas/farmacologia , Transdução de Sinais , Transportador 1 de Glucose-Sódio/imunologia , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Transgenes , Xenopus laevisRESUMO
Calcitriol, a powerful regulator of phosphate metabolism and immune response, is generated by 25-hydroxyvitamin D 1α-hydroxylase in the kidney and macrophages. Renal 1α-hydroxylase expression is suppressed by Klotho and FGF23, the expression of which is stimulated by calcitriol. Interferon γ (INFγ) regulates 1α-hydroxylase expression in macrophages through transcription factor interferon regulatory factor-1. INFγ-signaling includes Janus kinase 3 (JAK3) but a role of JAK3 in the regulation of 1α-hydroxylase expression and mineral metabolism has not been shown. Thus, the impact of JAK3 deficiency on calcitriol formation and phosphate metabolism was measured. Renal interferon regulatory factor-1 and 1α-hydroxylase transcript levels, serum calcitriol and FGF23 levels, intestinal phosphate absorption as well as absolute and fractional renal phosphate excretion were significantly higher in jak3 knockout than in wild-type mice. Coexpression of JAK3 increased the phosphate-induced current in renal sodium-phosphate cotransporter-expressing Xenopus oocytes. Thus, JAK3 is a powerful regulator of 1α-hydroxylase expression and phosphate transport. Its deficiency leads to marked derangement of phosphate metabolism.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Calcitriol/sangue , Janus Quinase 3/metabolismo , Rim/enzimologia , Fosfatos/metabolismo , RNA Mensageiro/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/análise , Animais , Calbindinas/genética , Calcitriol/biossíntese , Fezes/química , Feminino , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Fator Regulador 1 de Interferon/análise , Fator Regulador 1 de Interferon/genética , Mucosa Intestinal/metabolismo , Janus Quinase 3/deficiência , Janus Quinase 3/genética , Rim/química , Masculino , Camundongos , Camundongos Knockout , Oócitos/enzimologia , Fosfatos/análise , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismo , Regulação para Cima , XenopusRESUMO
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.
Assuntos
Metabolismo Energético , Janus Quinase 2/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Adaptação Fisiológica , Trifosfato de Adenosina/metabolismo , Animais , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Células Jurkat , Potenciais da Membrana , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Fator de Transcrição STAT5/antagonistas & inibidores , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/genética , Fatores de Tempo , Xenopus laevisRESUMO
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.
Assuntos
Janus Quinase 2/biossíntese , Canais de Potássio Ativados por Cálcio de Condutância Alta/biossíntese , Regulação para Cima/fisiologia , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Feminino , Humanos , Camundongos , Canais de Potássio Cálcio-Ativados/biossíntese , Xenopus laevisRESUMO
Janus kinase-2 (JAK2), a signaling molecule mediating effects of various hormones including leptin and growth hormone, has previously been shown to modify the activity of several channels and carriers. Leptin is known to inhibit and growth hormone to stimulate epithelial Na(+) transport, effects at least partially involving regulation of the epithelial Na(+) channel ENaC. However, no published evidence is available regarding an influence of JAK2 on the activity of the epithelial Na(+) channel ENaC. In order to test whether JAK2 participates in the regulation of ENaC, cRNA encoding ENaC was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, gain-of-function (V617F)JAK2 or inactive (K882E)JAK2. Moreover, ENaC was expressed with or without the ENaC regulating ubiquitin ligase Nedd4-2 with or without JAK2, (V617F)JAK2 or (K882E)JAK2. ENaC was determined from amiloride (50 µM)-sensitive current (I(amil)) in dual electrode voltage clamp. Moreover, I(amil) was determined in colonic tissue utilizing Ussing chambers. As a result, the I(amil) in ENaC-expressing oocytes was significantly decreased following coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. Coexpression of JAK2 and Nedd4-2 decreased I(amil) in ENaC-expressing oocytes to a larger extent than coexpression of Nedd4-2 alone. Exposure of ENaC- and JAK2-expressing oocytes to JAK2 inhibitor AG490 (40 µM) significantly increased I(amil). In colonic epithelium, I(amil) was significantly enhanced by AG490 pretreatment (40 µM, 1 h). In conclusion, JAK2 is a powerful inhibitor of ENaC.
Assuntos
Regulação para Baixo , Canais Epiteliais de Sódio/metabolismo , Janus Quinase 2/fisiologia , Amilorida/farmacologia , Animais , Células Cultivadas , Colo/efeitos dos fármacos , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Feminino , Técnicas In Vitro , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/fisiologia , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Xenopus laevisRESUMO
BACKGROUND/AIMS: Janus-activated kinase-2 JAK2 participates in the signaling of several hormones including growth hormone, fosters tumor growth and modifies the activity of several Na(+) coupled nutrient transporters. Peptide uptake into intestinal and tumor cells is accomplished by electrogenic peptide transporters PEPT1 and PEPT2. The present study thus explored whether JAK2 contributes to the regulation of PEPT1 and PEPT2 activity. METHODS: cRNA encoding either PEPT1 or PEPT2 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, constitutively active (V617F)JAK2 or inactive (K882E)JAK2. The current created by the dipeptide glycine-glycine (Igly-gly) was determined by dual electrode voltage clamp and taken as measure for electrogenic peptide transport. RESULTS: No appreciable Igly-gly was observed in water injected oocytes. In PEPT1 or PEPT2 expressing oocytes Igly-gly was significantly increased by additional coexpression of JAK2. As shown in PEPT1 expressing oocytes, Igly-gly without significantly modifying the concentration required for halfmaximal Igly-gly (KM). Following disruption of carrier insertion with brefeldin A (5 µM) Igly-gly declined similarly fast in Xenopus oocytes expressing PEPT1 with JAK2 and in Xenopus oocytes expressing PEPT1 alone. In oocytes expressing both, PEPT1 and (V617F)JAK2, Igly-gly was gradually decreased by JAK2 inhibitor AG490 (40 µM). According to Ussing chamber experiments pharmacological JAK2 inhibition similarly decreased Igly-gly in mouse intestine. CONCLUSION: Regulation of the peptide transporters PEPT and PEPT2 does involve the Janus-activated kinase-2 JAK2.
Assuntos
Janus Quinase 2/metabolismo , Simportadores/metabolismo , Substituição de Aminoácidos , Animais , Brefeldina A/farmacologia , Humanos , Intestinos/fisiologia , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Transportador 1 de Peptídeos , RNA Complementar/metabolismo , Simportadores/genética , Tirfostinas/farmacologia , Regulação para Cima/efeitos dos fármacos , Xenopus/crescimento & desenvolvimentoRESUMO
BACKGROUND/AIMS: T-lymphocyte activation and function critically depends on Ca(2+) signaling, which is regulated by store operated Ca(2+) entry (SOCE). Human and mouse T lymphocytes express AMP activated kinase AMPKα1, which is rapidly activated following elevation of cytosolic Ca(2+) concentration ([Ca(2+)]i) by treatment of the cells with Ca(2+) ionophore or following inhibition of endosomal Ca(2+) ATPase with thapsigargin. AMPK is further activated by triggering of the T cell antigen receptor (TCR). The present study explored whether AMPK influences Ca(2+) entry and Ca(2+)-sensitive regulation of T-lymphocyte function. METHODS: T-lymphocytes were isolated and cultured from AMPKα1-deficient (ampk(-/-)) mice and from their wildtype (ampk(+/+)) littermates. The phenotype of the cells was analysed by flow cytometry, [Ca(2+)]i estimated from Fura-2 fluorescence, SOCE from increase of [Ca(2+)]i following thapsigargin treatment (1 µM), and cell function analysed by measuring cytokine secretion and western blotting. RESULTS: Expression of surface markers in CD4(+) and CD8(+) T-cells were similar in ampk(-/-) and ampk(+/+) T-lymphocyte blasts. Moreover, total STIM1 protein abundance was similar in ampk(-/-) and ampk(+/+) T-lymphocyte blasts. However, Orai1 cell membrane protein abundance was significantly higher in ampk(-/-) than in ampk(+/+) T-lymphocyte blasts. SOCE and increase of [Ca(2+)]i following TCR activation by triggering TCR with anti-CD3 and cross-linking secondary antibody were both significantly more pronounced in ampk(-/-) than in ampk(+/+) T-lymphocyte blasts. The difference of Ca(2+) entry between ampk(-/-) and ampk(+/+) T-lymphocytes was abrogated by Orai1 inhibitor 2-aminoethoxydiphenyl borate (2-APB, 50 µM). Proliferation of unstimulated ampk(-/-) lymphocytes was higher than proliferation of ampk(+/+) T-lymphocytes, a difference reversed by Orai1 silencing. CONCLUSIONS: AMPK downregulates Orai1 and thus SOCE in T-lymphocytes and thus participates in negative feed-back regulation of cytosolic Ca(2+) activity.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas Quinases Ativadas por AMP/deficiência , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Anticorpos/imunologia , Compostos de Boro/farmacologia , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Canais de Cálcio/química , Canais de Cálcio/genética , Proliferação de Células , Células Cultivadas , Fura-2/química , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteína ORAI1 , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Molécula 1 de Interação Estromal , Tapsigargina/farmacologiaRESUMO
BACKGROUND: Na(+) coupled phosphate transporter NaPiIIa is the main carrier accomplishing phosphate transport across the apical cell membrane of proximal renal tubules and thus renal tubular phosphate reabsorption. The carrier is regulated by a wide variety of hormones and cellular signaling molecules. Hormones stimulating renal tubular phosphate transport and thus leading to hyperphosphatemia include growth hormone. Signaling of growth hormone involves activation of janus-activated kinase-2 JAK2, which has previously been shown to participate in the regulation of several Na(+) coupled transporters. Experiments exploring the effect of JAK2 on phosphate transport have, however, never been reported. The present study thus addressed the effect of JAK2 on NaPiIIa. METHODS: cRNA encoding NaPiIIa was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, the gain of function mutant JAK2(V617F) or inactive JAK2(K882E). Phosphate-induced current (I(NaPi)) reflecting electrogenic phosphate transport was determined by two electrode voltage clamp. Moreover, NaPiIIa protein abundance in the cell membrane was determined by chemiluminescence. RESULTS: No appreciable I(NaPi) was observed in water injected oocytes or in oocytes expressing JAK2 alone. In NaPiIIa expressing oocytes I(NaPi) was significantly increased by additional expression of JAK2 or JAK2(V617F), but not by coexpression of JAK2(K882E). In oocytes expressing both, NaPiIIa and JAK2, I(NaPi) was gradually decreased by JAK2 inhibitor AG490 (40 µM). Coexpression of NaPiIIa and JAK2 or JAK2(V617F), but not of JAK2(K882E) increased NaPiIIa protein abundance in the cell membrane. Disruption of carrier protein insertion with Brefeldin A (5 µM) was followed by a decline of I(NaPi) to a similar extent in Xenopus oocytes expressing NaPiIIa with JAK2 and in Xenopus oocytes expressing NaPiIIa alone, suggesting that JAK2 did not affect carrier stability in the cell membrane. CONCLUSION: JAK2 contributes to the regulation of phosphate transporter NaPiIIa.
Assuntos
Janus Quinase 2/metabolismo , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/agonistas , Sódio/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Humanos , Transporte de Íons , Janus Quinase 2/genética , Oócitos , XenopusRESUMO
Janus kinase-2 (JAK2) participates in the signaling of several hormones, growth factors and cytokines. Further stimulators of JAK2 include osmotic cell shrinkage, and the kinase activates the cell volume regulatory Na(+)/H(+) exchanger. The kinase may thus participate in cell volume regulation. Cell shrinkage is known to inhibit K(+) channels. Volume-regulatory K(+) channels include the voltage-gated K(+) channel KCNQ4. The present study explored the effect of JAK2 on KCNQ4 channel activity. KCNQ4 was expressed in Xenopus oocytes with or without wild-type JAK2, constitutively active (V617F)JAK2 or inactive (K882E)JAK2; and cell membrane conductance was determined by dual-electrode voltage clamp. Expression of KCNQ4 was followed by the appearance of voltage-gated K(+) conductance. Coexpression of JAK2 or of (V617F)JAK2, but not of (K882E)JAK2, resulted in a significant decrease in conductance. Treatment of KCNQ4 and JAK2 coexpressing oocytes with the JAK2 inhibitor AG490 (40 µM) was followed by an increase in conductance. Treatment of KCNQ4 expressing oocytes with brefeldin A (5 µM) was followed by a decrease in conductance, which was similar in oocytes expressing KCNQ4 together with JAK2 as in oocytes expressing KCNQ4 alone. Thus, JAK2 apparently does not accelerate channel protein retrieval from the cell membrane. In conclusion, JAK2 downregulates KCNQ4 activity and thus counteracts K(+) exit, an effect which may contribute to cell volume regulation.
Assuntos
Janus Quinase 2/metabolismo , Canais de Potássio KCNQ/metabolismo , Animais , Brefeldina A/farmacologia , Tamanho Celular/efeitos dos fármacos , Eritropoetina/metabolismo , Feminino , Janus Quinase 2/antagonistas & inibidores , Canais de Potássio KCNQ/antagonistas & inibidores , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Tirfostinas/farmacologia , XenopusRESUMO
The tyrosine kinase Janus kinase 3 (JAK3) contributes to signaling regulating the proliferation and apoptosis of lymphocytes and tumor cells. Replacement of lysine by alanine in the catalytic subunit yields the inactive (K851A)JAK3 mutant that underlies severe combined immune deficiency. The gain-of-function mutation (A572V)JAK3 is found in acute megakaryoplastic leukemia and T cell lymphoma. The excessive nutrient demand of tumor cells requires upregulation of transporters in the cell membrane including peptide transporters PEPT1 and PEPT2. The carriers further accomplish intestinal peptide transport. Little is known about signaling regulating peptide transport. The present study explored whether PEPT1 and PEPT2 are upregulated by JAK3. PEPT1 or PEPT2 was expressed in Xenopus oocytes with or without additional expression of JAK3, and electrogenic peptide (glycine-glycine) transport was determined by dual-electrode voltage clamp. PEPT2-HA membrane protein abundance was analyzed by chemiluminescence. Intestinal electrogenic peptide transport was estimated from peptide-induced current in Ussing chamber experiments. In PEPT1- and PEPT2-expressing oocytes, but not in water-injected oocytes, the dipeptide gly-gly generated an inward current, which was significantly increased following coexpression of JAK3. The effect of JAK3 on PEPT1 was mimicked by (A568V)JAK3 but not by (K851A)JAK3. JAK3 increased maximal peptide-induced current in PEPT1-expressing oocytes but rather decreased apparent affinity of the carrier. Coexpression of JAK3 enhanced the PEPT2-HA protein abundance in the cell membrane. In JAK3- and PEPT1-expressing oocytes, peptide-induced current was blunted by the JAK3 inhibitor WHI-P154, 4-[(3'-bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline (22 µM). In intestinal segments gly-gly generated a current which was significantly smaller in JAK3-deficient mice (jak3â»/â») than in wild-type mice (jak3âº/âº). In conclusion, JAK3 is a powerful regulator of peptide transporters PEPT1 and PEPT2.
Assuntos
Janus Quinase 3/metabolismo , Simportadores/metabolismo , Animais , Feminino , Expressão Gênica , Humanos , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/genética , Masculino , Camundongos , Camundongos Knockout , Oócitos/metabolismo , Transportador 1 de Peptídeos , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Simportadores/genética , XenopusRESUMO
BACKGROUND/AIMS: The Janus kinase 3 JAK3 participates in the signaling of immune cells. Lack of JAK3 triggers inflammatory bowel disease, which in turn has been shown to affect intestinal activity of the epithelial Na(+) channel ENaC and thus colonic sodium absorption. At least in theory, inflammatory bowel disease in JAK3-deficient mice could lead to intestinal salt loss compromizing extracellular volume maintenance and blood pressure regulation. The present study thus explored whether JAK3 deficiency impacts on colonic ENaC activity, fecal Na(+) exretion, blood pressure and extracellular fluid volume regulation. METHODS: Experiments were performed in gene-targeted mice lacking functional JAK3 (jak3(-/-)) and in wild type mice (jak3(+/+)). Colonic ENaC activity was estimated from amiloride-sensitive current in Ussing chamber experiments, fecal, serum and urinary Na(+) concentration by flame photometry, blood pressure by the tail cuff method and serum aldosterone levels by immunoassay. RESULTS: The amiloride (50 µM)-induced deflection of the transepithelial potential difference was significantly lower and fecal Na(+) excretion significantly higher in jak3(-/-) mice than in jak3(+/+) mice. Moreover, systolic arterial blood pressure was significantly lower and serum aldosterone concentration significantly higher in jak3(-/-) mice than in jak3(+/+) mice. Both, absolute and fractional renal Na(+) excretion were significantly lower in jak3(-/-) mice than in jak3(+/+) mice. CONCLUSIONS: JAK3 deficiency leads to impairment of colonic ENaC activity with intestinal Na(+) loss, decrease of blood pressure, increased aldosterone release and subsequent stimulation of renal tubular Na(+) reabsorption.
Assuntos
Colo/metabolismo , Canais Epiteliais de Sódio/metabolismo , Motilidade Gastrointestinal/fisiologia , Hipovolemia/metabolismo , Janus Quinase 3/deficiência , Sódio/metabolismo , Animais , Feminino , Hipovolemia/diagnóstico , Masculino , Camundongos , Camundongos KnockoutRESUMO
JAK2 (Janus kinase-2) is activated by cell shrinkage and may thus participate in cell volume regulation. Cell volume regulatory ion channels include the small conductance Cl(-) channels ClC-2. The present study thus explored whether JAK2 influences ClC-2 activity. To this end, ClC-2 was expressed in Xenopus oocytes with or without wild type JAK2, active (V617F)JAK2 or inactive (K882E)JAK2 and the Cl(-) channel activity determined by dual electrode voltage clamp. Expression of ClC-2 was followed by a marked increase of cell membrane conductance. The conductance was significantly decreased following coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. Exposure of the oocytes expressing ClC-2 together with (V617F)JAK2 to the JAK2 inhibitor AG490 (40 µM) resulted in a gradual increase of the conductance. According to chemiluminescence JAK2 decreased the channel protein abundance in the cell membrane. The decline of conductance in ClC-2 and (V617F)JAK2 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 µM) was similar in oocytes expressing ClC-2 with (V617F)JAK2 and oocytes expressing ClC-2 alone, indicating that (V617F)JAK2 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. In conclusion, JAK2 down-regulates ClC-2 activity and thus counteracts Cl(-) exit, an effect which may impact on cell volume regulation.
Assuntos
Canais de Cloreto/fisiologia , Regulação para Baixo/fisiologia , Janus Quinase 2/fisiologia , Animais , Sequência de Bases , Canais de Cloro CLC-2 , Primers do DNA , Humanos , Janus Quinase 2/genética , Luminescência , Mutagênese Sítio-Dirigida , XenopusRESUMO
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 (V617F)JAK2 or inactive (K882E)JAK2. Inositol-induced current (I(SMIT)) was determined by dual electrode voltage clamp and taken as measure for electrogenic inositol transport. RESULTS: No appreciable I(SMIT) was observed in water injected oocytes. In SMIT expressing oocytes I(SMIT) was significantly decreased by additional coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. According to kinetic analysis coexpression of JAK2 decreased maximal I(SMIT) without significantly modifying the concentration required for halfmaximal I(SMIT) (K(M)). 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 I(SMIT) 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.
Assuntos
Proteínas de Choque Térmico/genética , Janus Quinase 2/fisiologia , Simportadores/genética , Animais , Transporte Biológico , Células Cultivadas , Regulação para Baixo , Proteínas de Choque Térmico/metabolismo , Humanos , Inositol/metabolismo , Janus Quinase 2/antagonistas & inibidores , Cinética , Potenciais da Membrana , Estabilidade Proteica , Simportadores/metabolismo , Tirfostinas/farmacologia , XenopusRESUMO
Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3α,ß. Lithium, a known unspecific GSK3 inhibitor protects against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3α,ß inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3(KI)). T cells from gsk3(KI) mice were compared to T cells from corresponding wild type mice (gsk3(WT)). As a result, in gsk3(KI) CD4(+) cells surface CD62L (L-selectin) was significantly less abundant than in gsk3(WT) CD4(+) cells. Upon activation in vitro T cells from gsk3(KI) mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3(KI) T cells, suggesting that GSK3 induces effector function in CD8(+) T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3α,ß is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.
Assuntos
Apoptose/imunologia , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Selectina L/biossíntese , Ativação Linfocitária , Perforina/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linfócitos T Citotóxicos/imunologia , Animais , Autoimunidade , Técnicas de Introdução de Genes , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Camundongos , Camundongos Mutantes , Fosforilação , Linfócitos T Citotóxicos/enzimologiaRESUMO
Janus-activated kinase-2 JAK2 is activated by hyperosmotic shock and modifies the activity of several Na(+) coupled transporters. Carriers up-regulated by osmotic shock include the Na(+) coupled osmolyte transporter BGT1 (betaine/GABA transporter 1), which accomplishes the concentrative cellular uptake of γ-amino-butyric acid (GABA). The present study thus explored whether JAK2 participates in the regulation of BGT1 activity. To this end, cRNA encoding BGT1 was injected into Xenopus oocytes with or without cRNA encoding wild type JAK2, constitutively active (V617F)JAK2 or inactive (K882E)JAK2, and electrogenic GABA transport determined by dual electrode voltage clamp. In oocytes injected with cRNA encoding BGT1 but not in oocytes injected with water or with cRNA encoding JAK2 alone, the addition of 1mM GABA to the extracellular fluid generated an inward current (I(BGT)). In BGT1 expressing oocytes I(BGT) was significantly increased by coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. According to kinetic analysis coexpression of JAK2 increased the maximal I(BGT) without significantly modifying the concentration required for halfmaximal I(BGT) (K(M)). In oocytes expressing BGT1 and (V617F)JAK2 I(BGT) was gradually decreased by JAK2 inhibitor AG490 (40 µM). The decline of I(BGT) following disruption of carrier insertion with brefeldin A (5 µM) was similar in the absence and presence of the JAK2 inhibitor AG490 (40 µM). In conclusion, JAK2 is a novel regulator of the GABA transporter BGT1. The kinase up-regulates the carrier presumably by enhancing the insertion of carrier protein into the cell membrane.
Assuntos
Proteínas de Transporte/biossíntese , Janus Quinase 2/metabolismo , Animais , Betaína/metabolismo , Proteínas de Transporte/genética , Proteínas da Membrana Plasmática de Transporte de GABA , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Mutação , Oócitos , Inibidores de Proteínas Quinases/farmacologia , RNA Complementar/genética , Tirfostinas/farmacologia , Regulação para Cima , Xenopus , Ácido gama-Aminobutírico/metabolismoRESUMO
Janus-activated kinase-2 (JAK2) participates in the regulation of the Naâº-coupled glucose transporter SGLT1 and the Naâº-coupled amino acid transporter SLC6A19. Concentrative cellular creatine uptake is similarly accomplished by Naâº-coupled transport. The carrier involved is SLC6A8 (CreaT). The present study thus explored whether JAK2 regulates the activity of SLC6A8. To this end, cRNA encoding SLC6A8 was injected into Xenopus oocytes with or without cRNA encoding wild-type JAK2, constitutively active (V617F)JAK2 or inactive (K882E)JAK2. Electrogenic creatine transport was determined in those oocytes by dual-electrode voltage-clamp experiments. In oocytes injected with cRNA encoding SLC6A8 but not in oocytes injected with water or with cRNA encoding JAK2 alone, addition of 1 mM creatine to the extracellular bath generated an inward current (I (crea)). In SLC6A8 expressing oocytes I (crea) was significantly decreased by coexpression of JAK2 or (V617F)JAK2 but not by coexpression of (K882E)JAK2. According to kinetic analysis, coexpression of JAK2 decreased the maximal transport rate without significantly modifying the affinity of the carrier. In oocytes expressing SLC6A8 and (V617F)JAK2 I (crea) was gradually increased by the JAK2 inhibitor AG490 (40 µM). In SLC6A8 and JAK2 coexpressing oocytes the decline of I (crea) following disruption of carrier insertion with brefeldin A (5 µM) was similar in the absence and presence of JAK2. In conclusion, JAK2 is a novel regulator of the creatine transporter SLC6A8, which downregulates the carrier, presumably by interference with carrier protein insertion into the cell membrane.
Assuntos
Regulação para Baixo/genética , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Animais , Transporte Biológico , Brefeldina A/farmacologia , Creatina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Humanos , Janus Quinase 2/antagonistas & inibidores , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Tirfostinas/farmacologia , Xenopus/genética , Xenopus/metabolismoRESUMO
Janus kinase 3, a tyrosine kinase expressed in haematopoetic tissues, plays a decisive role in T-lymphocyte survival. JAK3 deficiency leads to (Severe) Combined Immunodeficiency (SCID) resulting from enhanced lymphocyte apoptosis. JAK3 is activated by phosphorylation. Nothing is known about expression of JAK3 in erythrocytes, which may undergo apoptosis-like cell death (eryptosis) characterized by cell membrane scrambling with phosphatidylserine exposure and cell shrinkage. Triggers of eryptosis include energy depletion. The present study utilized immunohistochemistry and confocal microscopy to test for JAK3 expression and phosphorylation, and FACS analysis to determine phosphatidylserine exposure (annexin binding) and cell volume (forward scatter). As a result, JAK3 was expressed in erythrocytes and phosphorylated following 24h and 48h glucose depletion. Forward scatter was slightly but significantly smaller in erythrocytes from JAK3-deficient mice (jak3(-/-)) than in erythrocytes from wild type mice (jak3(+/+)). Annexin V binding was similarly low in both genotypes. The JAK3 inhibitors WHI-P131/JANEX-1 (4-(4'-Hydroxyphenyl)amino-6,7-dimethoxyquinazoline, 156µM) and WHI-P154 (4-[(3'-Bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 11.2µM) did not significantly modify annexin V binding or forward scatter. Glucose depletion increased annexin V binding, an effect significantly blunted in jak3(-/-) erythrocytes and in the presence of the JAK3 inhibitors. The observations disclose a completely novel role of Janus kinase 3, i.e. the triggering of cell membrane scrambling in energy depleted erythrocytes.
Assuntos
Eritrócitos/enzimologia , Glucose/farmacologia , Janus Quinase 3 , Inibidores de Proteínas Quinases/farmacologia , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/metabolismo , Animais , Anexina A5/análise , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Tamanho Celular , Contagem de Eritrócitos , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Feminino , Citometria de Fluxo , Deleção de Genes , Glucose/deficiência , Humanos , Imuno-Histoquímica , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/biossíntese , Janus Quinase 3/deficiência , Janus Quinase 3/genética , Masculino , Camundongos , Camundongos Knockout , Microscopia Confocal , Fosfatidilserinas/análise , Fosfatidilserinas/metabolismo , Fosforilação/efeitos dos fármacos , Quinazolinas/farmacologiaRESUMO
The Janus-activated kinase-2 JAK2 is involved in the signaling of leptin and erythropoietin receptors and mediates neuroprotective effects of the hormones. In theory, JAK2 could be effective through modulation of the glutamate transporters, carriers accounting for the clearance of glutamate released during neurotransmission. The present study thus elucidated the effect of JAK2 on the glutamate transporters EAAT1, EAAT2, EAAT3 and EAAT4. To this end, cRNA encoding the carriers was injected into Xenopus oocytes with or without cRNA encoding JAK2 and glutamate transport was estimated from glutamate induced current (I(glu)). I(glu) was observed in Xenopus oocytes expressing EAAT1 or EAAT2 or EAAT3 or EAAT4, but not in water injected oocytes. Coexpression of JAK2 resulted in an increase of I(glu) by 83% (EAAT1), 67% (EAAT2), 42% (EAAT3) and 126% (EAAT4). As shown for EAAT4 expressing Xenopus oocytes, the effect of JAK2 was mimicked by gain of function mutation (V617F)JAK2 but not by the inactive mutant (K882E)JAK2. Incubation with JAK2 inhibitor AG490 (40 µM) resulted in a gradual decrease of I(glu) by 53%, 79% and 92% within 3, 6 and 24 hours. Confocal microscopy and chemiluminescence analysis revealed that JAK2 coexpression increased EAAT4 protein abundance in the cell membrane. Disruption of transcription did not appreciably modify the up-regulation of I(glu) in EAAT4 expressing oocytes. The decay of I(glu) following inhibition of carrier insertion with brefeldin A was similar in oocytes expressing EAAT4 + JAK2 and oocytes expressing EAAT4 alone, indicating that JAK2 did not appreciably affect carrier retrieval from the membrane. In conclusion, JAK2 is a novel powerful regulator of glutamate transporters and thus participates in the protection against excitotoxicity.
Assuntos
Proteínas de Transporte de Glutamato da Membrana Plasmática/metabolismo , Janus Quinase 2/metabolismo , Substituição de Aminoácidos , Animais , Transportador 1 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Transportador 3 de Aminoácido Excitatório/metabolismo , Transportador 4 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Janus Quinase 2/genética , Oócitos/metabolismo , Técnicas de Patch-Clamp , Regulação para Cima , Xenopus laevis/genéticaRESUMO
JAK2 (Janus kinase-2) is expressed in a wide variety of cells including tumor cells and contributes to the proliferation and survival of those cells. The gain of function mutation (V617F)JAK2 mutant is found in the majority of myeloproliferative diseases. Cell proliferation depends on the availability of amino acids. Concentrative cellular amino acid uptake is in part accomplished by Na(+) coupled amino acid transport through SLC6A19 (B(0)AT). The present study thus explored whether JAK2 activates SLC6A19. To this end, SLC6A19 was expressed in Xenopus oocytes with or without wild type JAK2, (V617F)JAK2 or inactive (K882E)JAK2 and electrogenic amino acid transport determined by dual electrode voltage clamp. In SLC6A19-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of leucine (2mM) to the bath generated a current (I(le)), which was significantly increased following coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. Coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. Exposure of the oocytes to the JAK2 inhibitor AG490 (40µM) resulted in a gradual decline of I(le). According to chemiluminescence JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I(le) following inhibition of carrier insertion by brefeldin A (5µM) was similar in the absence and presence of JAK2 indicating that JAK2 stimulates carrier insertion into rather than inhibiting carrier retrival from the cell membrane. In conclusion, JAK2 up-regulates SLC6A19 activity which may foster amino acid uptake into JAK2 expressing cells.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Janus Quinase 2/metabolismo , Aminoácidos/metabolismo , Animais , Brefeldina A/farmacologia , Linhagem Celular Tumoral , Humanos , XenopusRESUMO
JAK2 (Janus kinase-2) overactivity contributes to survival of tumor cells and the (V617F)JAK2 mutant is found in the majority of myeloproliferative diseases. Tumor cell survival depends on availability of glucose. Concentrative cellular glucose uptake is accomplished by Na(+) coupled glucose transport through SGLT1 (SLC5A1), which may operate against a chemical glucose gradient and may thus be effective even at low extracellular glucose concentrations. The present study thus explored whether JAK2 activates SGLT1. To this end, SGLT1 was expressed in Xenopus oocytes with or without wild type JAK2, (V617F)JAK2 or inactive (K882E)JAK2 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of glucose to the extracellular bath generated a current (I(g)), which was significantly increased following coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. Kinetic analysis revealed that coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. The stimulating effect of JAK2 expression was abrogated by preincubation with the JAK2 inhibitor AG490. Chemiluminescence analysis revealed that JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I(g) during inhibition of carrier insertion by brefeldin A was similar in the absence and presence of JAK2. Thus, JAK2 fosters insertion rather than inhibiting retrieval of carrier protein into the cell membrane. In conclusion, JAK2 upregulates SGLT1 activity which may play a role in the effect of JAK2 during ischemia and malignancy.