skepinone-l and wogonin

TGFβ1, a decisive regulator of megakaryocyte maturation and platelet formation, has previously been shown to up-regulate both, store operated Ca2+ entry (SOCE) and Ca2+ extrusion by Na+/Ca2+ exchange. The growth factor thus augments the increase of cytosolic Ca2+ activity ([Ca2+]i) following release of Ca2+ from intracellular stores and accelerates the subsequent decline of [Ca2+]i. The effect on SOCE is dependent on a signaling cascade including p38 kinase, serum & glucocorticoid inducible kinase SGK1, and nuclear factor NFκB. The specific Na+/Ca2+ exchanger isoforms involved and the signalling regulating the Na+/Ca2+ exchangers remained, however elusive. The present study explored, whether TGFβ1 influences the expression and function of K+ insensitive (NCX) and K+ sensitive (NCKX) Na+/Ca2+ exchangers, and aimed to shed light on the signalling involved.. In human megakaryocytic cells (MEG01) RT-PCR was performed to quantify NCX/NCKX isoform transcript levels, [Ca2+]i was determined by Fura-2 fluorescence, and Na+/Ca2+ exchanger activity was estimated from the increase of [Ca2+]i following switch from an extracellular solution with 130 or 90 mM Na+ and 0 mM Ca2+ to an extracellular solution with 0 Na+ and 2 mM Ca2+. K+ concentration was 0 mM for analysis of NCX and 40 mM for analysis of NCKX.. TGFβ1 (60 ng/ml, 24 h) significantly increased the transcript levels of NCX1, NCKX1, NCKX2 and NCKX5. Moreover, TGFβ1 (60 ng/ml, 24 h) significantly increased the activity of both, NCX and NCKX. The effect of TGFβ1 on NCX and NCKX transcript levels and activity was significantly blunted by p38 kinase inhibitor Skepinone-L (1 µM), the effect on NCX and NCKX activity further by SGK1 inhibitor GSK-650394 (10 µM) and NFκB inhibitor Wogonin (100 µM).. TGFβ1 markedly up-regulates transcription of NCX1, NCKX1, NCKX2, and NCKX5 and thus Na+/Ca2+ exchanger activity, an effect requiring p38 kinase, SGK1 and NFκB.

Topics: Benzoates; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cell Line; Dibenzocycloheptenes; Flavanones; Humans; Immediate-Early Proteins; Megakaryocytes; Microscopy, Fluorescence; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Protein Isoforms; Protein Serine-Threonine Kinases; Real-Time Polymerase Chain Reaction; Sodium-Calcium Exchanger; Transcription, Genetic; Transforming Growth Factor beta1; Up-Regulation

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/β), which phosphorylates the inhibitor protein IκBα thus triggering nuclear translocation of nuclear factor kappa B (NF-κB). The present study explored whether TGFβ 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.

Topics: Animals; Benzamides; Dibenzocycloheptenes; Enzyme Inhibitors; Female; Flavanones; Gene Expression Regulation; Hydrazines; Immediate-Early Proteins; Male; Megakaryocytes; Membrane Potentials; Mice; NF-kappa B; Ouabain; p38 Mitogen-Activated Protein Kinases; Patch-Clamp Techniques; Primary Cell Culture; Protein Serine-Threonine Kinases; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Transforming Growth Factor beta1