Th1 cytokines in pediatric acute lymphoblastic leukemia.

Immune milieus play an important role in various types of cancer. The present study focuses on the effect of Th1 cytokines on pediatric acute lymphoblastic leukemia (ALL). The reaction of ALL cell lines and patient-derived xenografts (PDX) to the most important Th1 cytokines TNF-α (tumor necrosis factor alpha) and IFN-γ (interferon gamma) is analyzed and correlated with the respective cytokine receptors and the intracellular signaling molecules. ALL cell lines and ALL PDX display a great heterogeneity in cell death after incubation with TNF-α and IFN-γ. Several samples show a dose-dependent and additive induction of cell death by both cytokines; others do not react at all or even display an increased viability. Apoptosis is the main type of cell death induced by Th1 cytokines in ALL cells. Over all leukemia cells analyzed, IFN-γ receptor (IFNGR) shows a higher expression than both TNF-receptors, resulting in higher phosphorylation of STAT1 (signal transducer and activator of transcription) compared to phosphorylation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B-cells) in the TNF pathway. The activation of STAT1 correlates with the amount of cell death after stimulation with Th1 cytokines. TNF-α and IFN-γ lead to heterogeneous reactions in ALL cell lines and ALL PDX but are able to induce cell death by apoptosis in the majority of ALL blasts. The correlation of a high expression of IFNGR and following activation of STAT1 with cell death indicates an important role for IFN-γ signaling in this setting.

Epitope detection in monocytes (EDIM) for liquid biopsy including identification of GD2 in childhood neuroblastoma-a pilot study.

BACKGROUND: Neuroblastoma (NB) is the most common paediatric extracranial solid malignancy. We analysed the role of the epitope detection in monocytes (EDIM) technique for liquid biopsy in NB patients. METHODS: Tumour epitopes transketolase-like 1 (TKTL1), Apo10 (DNaseX) and GD2 were assessed: expression levels in seven NB tumour samples and five NB cell lines were analysed using RT-PCR and flow cytometry. LAN-1 cells were co-cultured with blood and assessed using EDIM. Peripheral blood macrophages of patients with neuroblastoma (n = 38) and healthy individuals (control group, n = 37) were labelled (CD14+/CD16+) and assessed for TKTL1, Apo10 and GD2 using the EDIM technology. RESULTS: mRNA expression of TKTL1 and DNaseX/Apo10 was elevated in 6/7 NB samples. Spike experiments showed upregulation of TKTL1, Apo10 and GD2 in LAN-1 cells following co-culturing with blood. TKTL1 and Apo10 were present in macrophages of 36/38 patients, and GD2 in 15/19 patients. The 37 control samples were all negative. EDIM expression scores of the three epitopes allowed differentiation between NB patients and healthy individuals. CONCLUSIONS: The EDIM test might serve as a non-invasive tool for liquid biopsy in children suffering from NB. Future studies are necessary for assessing risk stratification, tumour biology, treatment monitoring, and early detection of tumour relapses.

Extracellular vesicles released by myeloid-derived suppressor cells from pregnant women modulate adaptive immune responses.

Immunological pregnancy complications are a main challenge in reproductive medicine. Mechanisms regulating the adaptation of the maternal immune system to pregnancy are incompletely understood and therapeutic options limited. Myeloid derived suppressor cells (MDSC) are immune-modulatory cells expanding during healthy pregnancy and seem to play a crucial role for maternal-fetal tolerance. Recent studies showed that exosomes produced by MDSC have immune-modulatory effects corresponding to their parental cells under different pathological conditions. Here, we investigated immunological effects of exosomes of GR-MDSC during pregnancy. Isolated GR-MDSC exosomes from peripheral blood of pregnant women were tested for functionality in different in vitro assays. We show that GR-MDSC exosomes exhibited profound immune-modulatory effects such as suppression of T-cell proliferation, T helper 2 (Th2)-cell polarization, induction of regulatory T-cells and inhibition of lymphocyte cytotoxicity. Our results confirm that MDSC-derived exosomes functionally correspond to their parental cells and identify them as an interesting therapeutic target for immunological pregnancy complications.

Sildenafil triggers tumor lethality through altered expression of HSP90 and degradation of PKD2.

The repurposing of existing drugs has emerged as an attractive additional strategy to the development of novel compounds in the fight against cancerous diseases. Inhibition of phosphodiesterase 5 (PDE5) has been claimed as a potential approach to target various cancer subtypes in recent years. However, data on the treatment of tumors with PDE5 inhibitors as well as the underlying mechanisms are as yet very scarce. Here, we report that treatment of tumor cells with low concentrations of Sildenafil was associated with decreased cancer cell proliferation and augmented apoptosis in vitro and resulted in impaired tumor growth in vivo. Notably, incubation of cancer cells with Sildenafil was associated with altered expression of HSP90 chaperone followed by degradation of protein kinase D2, a client protein previously reported to be involved in tumor growth. Furthermore, the involvement of low doses of PU-H71, an HSP90 inhibitor currently under clinical evaluation, in combination with low concentrations of Sildenafil, synergistically and negatively impacted on the viability of cancer cells in vivo. Taken together, our study suggests that repurposing of already approved drugs, alone or in combination with oncology-dedicated compounds, may represent a novel cancer therapeutic strategy.

The epigenetic factor KDM2B regulates cell adhesion, small rho GTPases, actin cytoskeleton and migration in prostate cancer cells.

The histone demethylase KDM2B is an epigenetic factor with oncogenic properties that is regulated by the basic fibroblasts growth factor (FGF-2). It has recently been shown that KDM2B co-operates with Polycomb Group proteins to promote cell migration and angiogenesis in tumors. In the present study we addressed the role of KDM2B in regulating actin cytoskeleton signaling, cell-cell adhesion and migration of prostate tumor cells. We report here that KDM2B is functionally expressed in DU-145 prostate cancer cells, activated by FGF-2 and regulates EZH2. KDM2B knockdown induced potent up-regulation of gene transcription and protein expression of the epithelial markers E-cadherin and ZO-1, while KDM2B overexpression down-regulated the levels of both markers, suggesting control of cell adhesion by KDM2B. RhoA and RhoB protein expression and activity were diminished upon KDM2B-knockdown and upregulated in KDM2B-overexpressing cell clones. In accordance, actin reorganization with formation of stress fibers became evident in KDM2B-overexpressing cells and abolished in the presence of the Rho inhibitor C3 transferase. DU-145 cell migration was significantly enhanced in KDM2B overexpressing cells and abolished in C3-pretreated cells. Conversely, the retardation of cell migration observed in KDM2B knockdown cells was enhanced in C3-pretreated cells. These results establish a clear functional link between the epigenetic factor KDM2B and the regulation of cell adhesion and Rho-GTPases signaling that controls actin reorganization and cell migration.

The Epigenetic Factor KDM2B Regulates EMT and Small GTPases in Colon Tumor Cells.

BACKGROUND/AIMS: The epigenetic factor KDM2B is a histone demethylase expressed in various tumors. Recently, we have shown that KDM2B regulates actin cytoskeleton organization, small Rho GTPases signaling, cell-cell adhesion and migration of prostate tumor cells. In the present study, we addressed its role in regulating EMT and small GTPases expression in colon tumor cells. METHODS: We used RT-PCR for the transcriptional analysis of various genes, Western blotting for the assessment of protein expression and immunofluorescence microscopy for visualization of fluorescently labeled proteins. RESULTS: We report here that KDM2B regulates EZH2 and BMI1 in HCT116 colon tumor cells. Knockdown of this epigenetic factor induced potent up-regulation of the protein levels of the epithelial markers E-cadherin and ZO-1, while the mesenchymal marker N-cadherin was downregulated. On the other hand, KDM2B overexpression downregulated the levels of both epithelial markers and upregulated the mesenchymal marker, suggesting control of EMT by KDM2B. In addition, RhoA, RhoB and RhoC protein levels diminished upon KDM2B-knockdown, while all three small GTPases became upregulated in KDM2B-overexpressing HCT116 cell clones. Interestingly, Rac1 GTPase level increased upon KDM2B-knockdown and diminished in KDM2B-overexpressing HCT116 colon tumor- and DU-145 prostate cancer cells. CONCLUSIONS: These results establish a clear functional role of the epigenetic factor KDM2B in the regulation of EMT and small-GTPases expression in colon tumor cells and further support the recently postulated oncogenic role of this histone demethylase in various tumors.

Serum and Glucocorticoid Inducible Kinase 1-Sensitive Survival, Proliferation and Migration of Rhabdomyosarcoma Cells.

BACKGROUND/AIMS: Rhabdomyosarcoma, the most common pediatric soft tissue sarcoma, may show an intrinsic refractoriness to standard chemotherapy in advanced tumor stages, which is associated with poor prognosis. Cellular mechanisms conferring tumor cell survival and therapy resistance in many tumor types include the serum & glucocorticoid inducible kinase (SGK) 1 pathway, a kinase expressed ubiquitously with particularly strong expression in skeletal muscle and some tumor types. The present study explored whether SGK1 is expressed in rhabdomyosarcoma and, if so, whether this kinase impacts on tumor cell survival, proliferation and migration. Multiple in vitro techniques were used to study the role of SGK1 in rhabdomyosarcoma. METHODS: The Gene Chip® Human Genome U133 Plus 2.0 Array were employed to examine SGK1 transcriptional activity in healthy muscle and rhabdomyosarcoma tissue. SGK1 transcript levels were quantified in rhabdomyosarcoma cell lines RD (embryonal subtype) and RH30 (alveolar subtype) by RT-PCR, cell viability was measured using MTT assays. Clonal cell growth was assessed via colony forming assays and migration experiments were performed in a transwell system. RESULTS: SGK1 is expressed in embryonal and alveolar rhabdomyosarcoma tissue samples and in RD and RH30 rhabdomyosarcoma cell lines. Administration of EMD638683 - an inhibitor specific for SGK1 - decreased viability of RD and RH30 cells, enhanced the effects of the cytotoxic drug doxorubicin leading to reduced migration and decreased cell proliferation. CONCLUSIONS: SGK1 is expressed in rhabdomyosarcoma cells where it contributes to survival, therapy resistance, cell proliferation and migration. Thus, SGK1 inhibitors may be considered a therapeutic option for the treatment of therapy-resistant rhabdomyosarcoma.

Istaroxime Inhibits Motility and Down-Regulates Orai1 Expression, SOCE and FAK Phosphorylation in Prostate Cancer Cells.

BACKGROUND/AIMS: Istaroxime is a validated inotropic Na+/K+ ATPase inhibitor currently in development for the treatment of various cardiac conditions. Recent findings established that this steroidal drug exhibits potent apoptotic responses in prostate tumors in vitro and in vivo, by affecting key signaling orchestrating proliferation and apoptosis, such as c-Myc and caspase 3, Rho GTPases and actin cytoskeleton dynamics. In the present study we examined whether istaroxime is affecting cell motility and analyzed the underlying mechanism in prostate tumor cells. METHODS: Migration was assessed by transwell and wound healing assays, Orai1 and Stim1 abundance by RT-PCR and confocal immunofluorescence microscopy, Fura-2 fluorescence was utilized to determine intracellular Ca2+ and Western blotting for FAK/pFAK measurements. RESULTS: We observed strong inhibition of cell migration in istaroxime treated DU-145 prostate cancer cells. Istaroxime further decreased Orai1 and Stim1 transcript levels and downregulated Orai1 protein expression. Moreover, SOCE was significantly decreased upon istaroxime treatment. Furthermore, istaroxime strikingly diminished phosphorylated FAK levels. Interestingly, the efficacy of istaroxime on the inhibition of DU-145 cell migration was further enhanced by blocking Orai1 with 2-APB and FAK with the specific inhibitor PF-00562271. These results provide strong evidence that istaroxime prevents cell migration and motility of DU-145 prostate tumor cells, an effect at least partially attributed to Orai1 downregulation and FAK de-activation. CONCLUSION: Collectively our results indicate that this enzyme inhibitor, besides its pro-apoptotic action, affects motility of cancer cells, supporting its potential role as a strong candidate for further clinical cancer drug development.

Up-Regulation of Na+/Ca2+ Exchange in Megakaryocytes Following TGFß1 Treatment.

BACKGROUND: Blood platelets are activated by increase of cytosolic Ca2+ activity ([Ca2+]i). Ca2+ entry is accomplished in part by store operated Ca2+ entry (SOCE) involving Ca2+ release activated Ca2+-channel (CRAC) moiety Orai1 and its regulator STIM1, which are stimulated by depletion of intracellular Ca2+ stores. An increase of [Ca2+]i is terminated by Na+/Ca2+-exchange. The expression of both, Orai1 and STIM1 in megakaryocytes is up-regulated by tumor growth factor TGFß1, a powerful regulator of megakaryocyte differentiation. The present study explored whether TGFß1 similarly modifies megakaryocyte Na+/Ca2+-exchanger activity. METHODS: [Ca2+]i was determined utlizing Fura-2 fluorescence, SOCE from increase of [Ca2+]i, following readdition of extracellular Ca2+ after store depletion, and Na+/Ca2+-exchanger activity from increase of [Ca2+]i and whole cell currents following removal of extracellular Na+. RESULTS: TGFß1 treatment not only augments the increase of [Ca2+]i following store depletion and SOCE, but significantly up-regulates Na+/Ca2+-exchanger activity as apparent from [Ca2+]i measurements and whole cell currents. CONCLUSIONS: TGFß1 is a powerful stimulator of both, SOCE and Na+/Ca2+-exchanger activity in megakaryocytes.

Quantitative Determination of Ceramide Molecular Species in Dendritic Cells.

BACKGROUND/AIMS: The activation of acid sphingomyelinase by cellular stress or receptors or the de novo synthesis lead to the formation of ceramide (N-acylsphingosine), which in turn modifies the biophysical properties of cellular membrane and greatly amplifies the intensity of the initial signal. Ceramide, which acts by re-organizing a given signalosome rather than being a second messenger, has many functions in infection biology, cancer, cardiovascular syndromes, and immune regulation. Experimental studies on the infection of human cells with different bacterial agents demonstrated the activation of the acid sphingomyelinase/ceramide system. Moreover, the release of ceramide was found to be a requisite for the uptake of the pathogen. Considering the particular importance of the cellular role of ceramide, it was necessary to develop sensitive and accurate methods for its quantification. METHODS: Here, we describe a method quantifying ceramide in dendritic cells and defining the different fatty acids (FA) bound to sphingosine. The main steps of the method include extraction of total lipids, separation of the ceramide by thin-layer chromatography, derivatization of ceramide-fatty acids (Cer-FA), and quantitation of these acids in their methyl form by gas chromatography on polar capillary columns. The identification of FA was achieved by means of known standards and confirmed by mass spectrometry. RESULTS: FA ranging between C10 and C24 could be detected and quantified. The concentration of the sum of Cer-FA amounted to 14.88 ± 8.98 nmol/106 cells (n=10). Oleic acid, which accounted for approximately half of Cer-FA (7.73 ± 6.52 nmol/106 cells) was the predominant fatty acid followed by palmitic acid (3.47 ± 1.54 nmol/106 cells). CONCLUSION: This highly sensitive method allows the quantification of different molecular species of ceramides.

Chorein Sensitive Orai1 Expression and Store Operated Ca2+ Entry in Rhabdomyosarcoma Cells.

BACKGROUND: Chorein, a protein encoded by VPS13A (vacuolar protein sorting-associated protein 13A), is defective in chorea acanthocytosis, a rare disease characterized by acanthocytosis of red blood cells and neuronal cell death with progressive hyperkinetic movement disorder, cognitive dysfunction, behavioral abnormalities and chronic hyperkalemia. Chorein is highly expressed in ZF rhabdomyosarcoma cells and counteracts apoptosis of those cells. Chorein is effective in part by interacting with and fostering stimulation of phosphoinositide-3-kinase (PI3K)-p85-subunit. PI3K dependent signaling includes the serum and glucocorticoid inducible kinase SGK1. The kinase activates NFκB with subsequent up-regulation of the Ca2+ channel subunit Orai1, which accomplishes store operated Ca2+ entry (SOCE). Orai1 and SOCE have been shown to confer survival of tumor cells. The present study thus explored whether chorein impacts on Orai1 expression and SOCE. METHODS: In rhabdomyosarcoma cells chorein, Orai1, NFκB and SGK1 transcript levels were quantified by RT-PCR, Orai1 protein abundance by Western blotting, FACS analysis and confocal laser microscopy, [Ca2+]i utilizing Fura-2 fluorescence, and SOCE from the increase of [Ca2+]i following store depletion with extracellular Ca2+ removal and inhibition of the sarcoendoplasmatic reticular Ca2+ ATPase with thapsigargin. RESULTS: The mRNA coding for chorein was most abundant in drug resistant, poorly differentiated human ZF rhabdomyosarcoma cells. Chorein silencing significantly decreased Orai1 transcript levels and Orai1 protein expression, as well as SGK1 and NFκB transcript levels. SOCE in ZF rhabdomyosarcoma cells was significantly blunted by chorein silencing, Orai1 inhibitor 2-APB (50 µM), SGK1 inhibitor EMD638683 (50 µM, 10 h) and NFκB inhibitor wogonin (50 µM, 24 h). CONCLUSION: Chorein is a stimulator of Orai1 expression and thus of store operated Ca2+ entry. The effect may involve SGK1 and NFκB.

Store-operated Ca(2+) entry in rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, has an intrinsic or early-acquisition of resistance to chemo- and radiation therapy. Molecular determinants pivotal for RMS migration, metastatic invasion, cell proliferation, and survival are incompletely identified. Migration and cell proliferation were shown to correlate with cytosolic Ca(2+) activity ([Ca(2+)]i). Store-operated Ca(2+)-entry (SOCE) that increases intracellular [Ca(2+)] is accomplished by Orai1, a pore-forming ion channel unit, the expression of which is stimulated by the transcription factor NFκB. The present study explored the expression of Orai1 and its regulators STIM1 and NFκB in human rhabdomyosarcoma cell lines and analyzed their impact on cell proliferation and migration. For the study human rhabdomyosarcoma cell lines RD (embryonal) and RH30 (alveolar) were analyzed for Orai1, STIM1, and NFκB transcription by RT-PCR and their corresponding proteins in Western blot. [Ca(2+)]i was detected via Fura-2 fluorescence and SOCE - resulting from [Ca(2+)]i increase following store depletion with extracellular Ca(2+) removal and inhibition of the sarcoendoplasmatic reticular Ca(2+) ATPase - detected with thapsigargin. Cell migration was analyzed in transwell and mitotic cell death with the clonogenic assay. In summary, Orai1, STIM1, and NFκB are expressed in embryonal (RD) and alveolar (RH30) rhabdomyosarcoma. SOCE inhibitor BTP2, Orai1 inhibitor 2-APB, or NFκB inhibitor wogonin virtually abrogated (BTP2, 2-APB) or significantly reduced (wogonin) SOCE. Moreover, SOCE inhibitors 2-APB and BTP2 and wogonin significantly inhibited migration and proliferation of both, RD and RH30 cells. These results suggest that Orai1 signaling is involved in SOCE into rhabdomyosarcoma cells thus contributing to migration, invasion and proliferation.

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.

Pivotal role of serum- and glucocorticoid-inducible kinase 1 in vascular inflammation and atherogenesis.

OBJECTIVE: Atherosclerosis, an inflammatory disease of arterial vessel walls, requires migration and matrix metalloproteinase (MMP)-9-dependent invasion of monocytes/macrophages into the vascular wall. MMP-9 expression is stimulated by transcription factor nuclear factor-κB, which is regulated by inhibitor κB (IκB) and thus IκB kinase. Regulators of nuclear factor-κB include serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored involvement of SGK1 in vascular inflammation and atherogenesis. APPROACH AND RESULTS: Gene-targeted apolipoprotein E (ApoE)-deficient mice without (apoe(-/-)sgk1(+/+)) or with (apoe(-/-)sgk1(-/-)) additional SGK1 knockout received 16-week cholesterol-rich diet. According to immunohistochemistry atherosclerotic lesions in aorta and carotid artery, vascular CD45(+) leukocyte infiltration, Mac-3(+) macrophage infiltration, vascular smooth muscle cell content, MMP-2, and MMP-9 positive areas in atherosclerotic tissue were significantly less in apoe(-/-)sgk1(-/-)mice than in apoe(-/-)sgk1(+/+)mice. As determined by Boyden chamber, thioglycollate-induced peritonitis and air pouch model, migration of SGK1-deficient CD11b(+)F4/80(+) macrophages was significantly diminished in vitro and in vivo. Zymographic MMP-2 and MMP-9 production, MMP-9 activity and invasion through matrigel in vitro were significantly less in sgk1(-/-) than in sgk1(+/+)macrophages and in control plasmid-transfected or inactive (K127N)SGK1-transfected than in constitutively active (S422D)SGK1-transfected THP-1 cells. Confocal microscopy revealed reduced macrophage number and macrophage MMP-9 content in plaques of apoe(-/-)sgk1(-/-) mice. In THP-1 cells, MMP-inhibitor GM6001 (25 µmol/L) abrogated (S422D)SGK1-induced MMP-9 production and invasion. According to reverse transcription polymerase chain reaction, MMP-9 transcript levels were significantly reduced in sgk1(-/-)macrophages and strongly upregulated in (S422D)SGK1-transfected THP-1 cells compared with control plasmid-transfected or (K127N)SGK1-transfected THP-1 cells. According to immunoblotting and confocal microscopy, phosphorylation of IκB kinase and inhibitor κB and nuclear translocation of p50 were significantly lower in sgk1(-/-)macrophages than in sgk1(+/+)macrophages and significantly higher in (S422D)SGK1-transfected THP-1 cells than in control plasmid-transfected or (K127N)SGK1-transfected THP-1 cells. Treatment of (S422D)SGK1-transfected THP-1 cells with IκB kinase-inhibitor BMS-345541 (10 µmol/L) abolished (S422D)SGK1-induced increase of MMP-9 transcription and gelatinase activity. CONCLUSIONS: SGK1 plays a pivotal role in vascular inflammation during atherogenesis. SGK1 participates in the regulation of monocyte/macrophage migration and MMP-9 transcription via regulation of nuclear factor-κB.

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.

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.

Acid sphingomyelinase regulates platelet cell membrane scrambling, secretion, and thrombus formation.

OBJECTIVE: Platelet activation is essential for primary hemostasis and acute thrombotic vascular occlusions. On activation, platelets release their prothrombotic granules and expose phosphatidylserine, thus fostering thrombin generation and thrombus formation. In other cell types, both degranulation and phosphatidylserine exposure are modified by sphingomyelinase-dependent formation of ceramide. The present study thus explored whether acid sphingomyelinase participates in the regulation of platelet secretion, phosphatidylserine exposure, and thrombus formation. APPROACH AND RESULTS: Collagen-related peptide-induced or thrombin-induced ATP release and P-selectin exposure were significantly blunted in platelets from Asm-deficient mice (Smpd1(-/-)) when compared with platelets from wild-type mice (Smpd1(+/+)). Moreover, phosphatidylserine exposure and thrombin generation were significantly less pronounced in Smpd1(-/-) platelets than in Smpd1(+/+) platelets. In contrast, platelet integrin αIIbß3 activation and aggregation, as well as activation-dependent Ca(2+) flux, were not significantly different between Smpd1(-/-) and Smpd1(+/+) platelets. In vitro thrombus formation at shear rates of 1700 s(-1) and in vivo thrombus formation after FeCl3 injury were significantly blunted in Smpd1(-/-) mice while bleeding time was unaffected. Asm-deficient platelets showed significantly reduced activation-dependent ceramide formation, whereas exogenous ceramide rescued diminished platelet secretion and thrombus formation caused by Asm deficiency. Treatment of Smpd1(+/+) platelets with bacterial sphingomyelinase (0.01 U/mL) increased, whereas treatment with functional acid sphingomyelinase-inhibitors, amitriptyline or fluoxetine (5 µmol/L), blunted activation-dependent platelet degranulation, phosphatidylserine exposure, and thrombus formation. Impaired degranulation and thrombus formation of Smpd1(-/-) platelets were again overcome by exogenous bacterial sphingomyelinase. CONCLUSIONS: Acid sphingomyelinase is a completely novel element in the regulation of platelet plasma membrane properties, secretion, and thrombus formation.