Decrease of Store-Operated Ca2+ Entry and Increase of Na+/Ca2+ Exchange by Pharmacological JAK2 Inhibition

Abstract

Background/Aims: Cell proliferation and migration are regulated by cytosolic Ca2+ activity ([Ca2+]i). Mechanisms modifying [Ca2+]i include store-operated Ca2+-entry (SOCE) accomplished by the pore-forming ion channel unit Orai1 and its regulator STIM1, as well as Ca2+ extrusion by Na+/Ca2+ 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+/Ca2+ 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, [Ca2+]i with Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and Na+/Ca2+ exchanger activity from increase of [Ca2+]i as well as Ca2+ 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 Ca2+ release from intracellular stores, decreased SOCE, increased Ca2+ entry as well as Ca2+-current following extracellular Na+-removal, and decreased migration. Similar effects on Ca2+ release, SOCE, and Ca2+-entry following extracellular Na+-removal were observed following treatment with AG490. Conclusion: The present observations disclose a novel powerful mechanism regulating intracellular Ca2+ release, cellular Ca2+ entry, cellular Ca2+ extrusion and cell migration.