Decrease of store-operated Ca 2+ entry and increase of Na + /Ca 2+ exchange by pharmacological JAK2 inhibition

Abstract

Background/Aims: Cell proliferation and migration are regulated by cytosolic Ca 2+ activity ([Ca 2+ ] i ). Mechanisms modifying [Ca 2+ ] i include store-operated Ca 2+ -entry (SOCE) accomplished by the pore-forming ion channel unit Orai1 and its regulator STIM1, as well as Ca 2+ extrusion by Na + /Ca 2+ exchanger NCX1. Kinases participating in the orchestration of cell proliferation include the Janus activated kinase JAK2. The present study explored the impact of pharmacological JAK2 inhibition on SOCE and Na + /Ca 2+ exchange. Methods: MCF-7 breast carcinoma cells were studied in the absence and presence of the JAK2 inhibitors TG101348 (250 nM - 1 μM) or of AG490 (20 - 40 μM). Transcript levels were quantified with RT-PCR, protein abundance with immunoblotting, [Ca 2+ ] i with Fura-2-fluorescence, SOCE from increase of [Ca 2+ ] i following Ca 2+ re-addition after Ca 2+ -store depletion with sarcoendoplasmatic Ca 2+ -ATPase (SERCA) inhibitor thapsigargin (1 μM), and Na + /Ca 2+ exchanger activity from increase of [Ca 2+ ] i as well as Ca 2+ current in whole cell patch clamp following extracellular Na + removal. Migratory activity was determined by a wound healing assay. Results: JAK2 inhibitor TG101348 (1 μM) decreased Orai1 and STIM1 protein abundance, increased NCX1 transcript levels, decreased Ca 2+ release from intracellular stores, decreased SOCE, increased Ca 2+ entry as well as Ca 2+ -current following extracellular Na + -removal, and decreased migration. Similar effects on Ca 2+ release, SOCE, and Ca 2+ -entry following extracellular Na + -removal were observed following treatment with AG490. Conclusion: The present observations disclose a novel powerful mechanism regulating intracellular Ca 2+ release, cellular Ca 2+ entry, cellular Ca 2+ extrusion and cell migration.