Cellular Physiology and Biochemistry (Jul 2015)
Impact of Janus Kinase 3 on Cellular Ca2+ Release, Store Operated Ca2+ Entry and Na+/Ca2+ Exchanger Activity in Dendritic Cells
Abstract
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 Ca2+ activity ([Ca2+]i). Mediators increasing [Ca2+]i in DCs include ATP and the chemokine receptor CXCR4 ligand CXCL12. The present study explored, whether JAK3 participates in the regulation of [Ca2+]i in DCs. Methods: Fura-2 fluorescence was employed to determine [Ca2+]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, [Ca2+]i was similar in jak3-/- and jak3+/+ DCs. Addition of ATP (100 µM) was followed by transient increase of [Ca2+]i reflecting Ca2+ 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 [Ca2+]i reflecting receptor operated Ca2+ entry, an effect significantly less rapid in jak3-/- DCs than in jak3+/+ DCs. In addition, the Ca2+ release-activated Ca2+ channel (CRAC) current triggered by IP3-induced Ca2+ store depletion and CXCL12 was significantly higher in DCs from jak3+/+ mice than in jak3-/- mice. Inhibition of sarcoendoplasmatic reticulum Ca2+-ATPase (SERCA) by thapsigargin (1 µM) in the absence of extracellular Ca2+ was followed by a transient increase of [Ca2+]i reflecting Ca2+ release from intracellular stores, and subsequent readdition of extracellular Ca2+ in the continued presence of thapsigargin was followed by a sustained increase of [Ca2+]i reflecting store operated Ca2+ entry (SOCE). Both, Ca2+ 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 Ca2+ release and subsequent SOCE. Abrupt replacement of Na+ containing (130 mM) and Ca2+ free (0 mM) extracellular bath by Na+ free (0 mM) and Ca2+ containing (2 mM) extracellular bath increased [Ca2+]i reflecting Na+/Ca2+ 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 Ca2+ release, receptor and store operated Ca2+ entry and Na+/Ca2+ exchanger activity in DCs.
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