1 research outputs found
P38 Kinase, SGK1 and NF-κB Dependent Up-Regulation of Na+/Ca2+ Exchanger Expression and Activity Following TGFß1 Treatment of Megakaryocytes
Background: 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. Methods: 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. Results: 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). Conclusions: 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